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A H I STO CHEMI CAL STUDY O F  BOV I NE SAL I VARY GLAND 
SECRETORY P RODUCTS AND AN I NVEST I GAT I ON O F  
I NT RAEP I THE L I AL GRANU LAR DUCT CELLS 
O F  THE PAROT I D  G LAND 
A the s i s p r e s e n t e d  in p a r t i a l  fu l f i l lment 
o f  the requi remen t s  f o r  the d e g r e e  
o f  Mas t e r  o f  P h i l o sophy i n  
Phys i o l o gy and Ana t omy a t  
Ma s s ey Un i ve r s i ty . 
Chandan Jayar a j  G urus i nghe 
1 9 8 3  
"The co-exis tance of the most  wonderful success 
with the most profound ignorance is one of 
the characteristic features of present day biology ."  
A. SZENT-GYORGYI. 
ABSTRACT 
P a r a f fin wax e mb e dd e d  h i s t o l o gica l  tis s ue s amp l e s  o f  
bovine s a l ivary g l ands w e r e  examine d  by s t aining , his t o­
chemi c a l  and immunohistochemic a l  metho d s . The cha r act e r ­
i s tic a l l y  tubul a r  s e cre t o ry e ndpi e c e s  we r e  compo s e d  o f  
e i ther p r o t e o s e rous c e l l s  o r  muc ous c e l l s  and d emilun e s . 
i i i  
P a ro t i d  g l ands and the his to l o g i c a l ly identic a l  vent r a l  
b ucca l g l ands we r e  comp o s e d  e n t i r e l y o f  p r o t e o s e rous c e l l s  
which o c c a s iona l l y cont ain e d  dia s t a s e  r e si s t an t  PAS p o sit i v e  
neut r al g l y cop ro t eins , addit i on a l l y  co nfirme d by a c e ty l ­
a t io n  and s aponific a tion . I mmuno his t o chemical s tud i e s  
e s t ab l i s h e d  that mo s t  p r o t eo s e rous c e l l s  a l s o  con t ained 
e i th e r  p r o t e i n b an d  4 or b and 1 0 .  An examina tion o f  s h e ep 
and c a t t l e  p a r o tid g l ands r ev e a l e d  that b a s a l  s t riations 
were ab s en t  from int r a l o bu l a r  duc t s  o f  c at t l e  but w e r e  
abundant i n  tho s e  o f  s h e ep . Some duc t  c e l l s  cont ained 
d ia s t a s e  l ab i l e  and d i a s t a s e  r e sis t an t  PAS po sitive mat e r i a l  
and ap i c a l  b l eb s . A g ranu l a t e d  int r a epithe lia l c e l l  typ e , 
wh ich was u l t ras tructur a l ly e xamin e d  and found t o  b e  simi l a r  
t o  a g l o b u l e  l euc o cy t e , w a s  s p e c i fic t o  int r a l obu l ar du c t  
wal l s  o f  t h e  p a r o tid g l ands ; t h e i r  p r e ci s e  func t i on w a s  no t 
e s t ab l i s h e d .  The main e x c r e t o ry du c t  o f  the p a ro tid g l and 
c ont ain e d  s eve r a l  g ob l e t  c e l l s . 
The mandibul a r  g l and muc o u s  c e l l s  cont aine d a c i d i c  
and neut r a l  muc o s ub s t anc e s .  T h e  p r e s ence o f  a c i dic g ro up s  
was confirmed by m e t hy l a t ion , s aponific ation and neuram i n ­
idas e d i g e s tio n .  T h e  con spicuous demilune s cont a i ned 
a cidic and neu t r a l  mucosub s t an c e s  and a cidoph i l  g r anul e s  
wh ich cont aine d p ro t ein b and 8 .  I nt r a l obul a r  duc t s  w i th 
t a l l  co l umnar c e l l s  were b as a l ly s t r i a t e d .  G ob l e t  c e l l s  
w e r e n o t  i d entifi e d  in the main e xc r e t o ry duc t . 
The s ub lingua l g l and muc o u s  c e l l s  cont a ined neut r a l  and 
a cid i c  mu c o sub s t ance s ; the l a t t e r w e r e  no t neu raminidas e  
l ab i l e . Un l i ke the mand ibul a r  g l ands , the sub lingu a l  mucous 
i v  
c e l l s  s t aine d  f o r  s u lphat e g o up s , a t t ribut ed t o  sulphat e d  o r  
sia l o - sulpha t e d  g l y c o p ro t eins , s ince hya luronida s e  dig e s tion 
did no t e l imin a t e  b a s ophilia a t  l ow pH . The demilun e  c e l l s  
w e r e  mos t l y  p ro t e o s e rous and c o n t aine d p ro tein b and 9 .  The 
" s t riat ed" in t r a l o b u l a r  duc t s  w e r e  identical to tho s e  o f  
t h e  mandibu l a r  g l an d . 
I nt e rm e di a t e  buc c a l ,  do rs a l  buc c a l , p a l a tine , p o s t e rior 
t o n gue and p h a ryng e a l  g l ands muc o u s  c e l l  his t o chemic a l  c om ­
p o sition w a s  simil a r  t o  tho s e  o f  t h e  s ub lingua l g l and s . 
The d emilune cont ent s o f  the m i n o r  g l ands were main l y  p ro t e o ­
s e r ous ; howev e r  tho s e  o f  the p h a ryng e a l  and p o s t e rio r t o ng u e  
o c c asiona l ly containe d  a cidic a n d  neut r a l  muco s ub s t ance s . 
Two unu s u a l  f e a t ure s o f  t h e  mino r  g l and s w e r e  t h e  
p r e s ence o f  g o b l e t  c e l l s  i n  int r a l obul a r  duc t s  o f  t h e  
p h a ryng e a l  g l and s and the app e arance o f  an atypic a l  s e c r e t ­
o ry mechani s m  in do r s a l  bucca l ,  int e rme diat e buc c a l  and 
p a l atine g l ands , t h e  s e c r e tions o f  which frequen t ly c o n ­
t aine d  c e l l u l a r  deb ris mixe d  wit h  mu cus . 
Humo r a l  immunit y  in bovine s a livary g l and s was me dia t e d  
b y  sub ling ua l , man dibul a r  and p h a ryng e a l  g l ands , thr e e  
g l ands whic h  c ont ained abundant sub epithe lia l p l a sma c e l l s . 
The p a ro tid and ven t r a l  buc c a l  g l ands notic e ab ly l ac k e d  
p l asma c e l l s  b u t  c o n t aine d  in t r a epithe lial g r anul a r  duc t  
c e l l s . I t  was p r op o s e d that t h e s e  c e l l s  may p r o vide c e l l ­
m e dia t e d  immunopro t e c tion a g ain s t  b l o at since inc r e a s e d 
numb e r s  o f  the s e  c e l l s  have b e e n  repo r t e d  in anima l s  with 
l ow b l o a t  s c o r e s . 
Sa liva ry p r o t ein b and 4 f r o m  p a ro tid s a liva h a s  b e en 
c o r r e l at e d  with b l o at s us c ep tibility in �a t t l e , but wa s 
equa l ly dis t rib ut e d  in p a rotid tis s ue s  of both l o w  and hig h  
b l o at s u s c ep tib l e  anima l s , sug g e s ting that b and 4 is 
s ynthe sis e d  but no t s e c r e t e d  by l ow b l o at s t r ain s . 
Acknowl e dg emen t s  
I w i s h  t o  e x t en d  my s i n c e r e  thanks t o  Mr . M . J .  B i r t l e s 
who s up e rv i s ed the p r eparat i o n  o f  t h i s  thes i s  and p ro v i d e d  
inval uab l e  a dv i c e  a n d  gui dance thro ughout t h e  c o u r s e .  
My thanks a r e  a l s o due t o  P r o f e s s o r  R . E .  Mun fo r d  who 
made ava i l ab l e  dep a r tment a l  f a c i l i t i e s fo r th i s  s tudy , 
and c r i t i c a l ly a s s e s s ed the d r a ft c opy o f  the t ex t . 
I am a l s o  g r a t e ful t o  Dr . W . T .  J o ne s , (App l i e d  B i o ­
c h emi s t ry D i v i s i on , D . S . I . R . ,  P a lme r s ton No r t h )  w i t hout 
whos e s k i l ful a s s i s t ance and s t imu l a t i ng d i s cu s s i on the 
i mmunoh i s t o chem i c a l  s tud i e s  w i t h  s a l iv a ry p ro t e i n s  wo u l d  
no t have b e e n p o s s i b l e .  
I am v e ry much i ndeb t e d  t o  Mr . Roy Sparksman who was 
r e spons ib l e  for t a k i ng the ex c e l l ent pho tomi c ro g r aphs 
p r es en t e d  in the t h e s i s , and to the s t a f f  o f  the E l e c t r o n  
M i c r o s copy Labor a t o ry , D . S . I . R . ,  P a lm e r s t on N o r t h , fo r 
i n s t ruc t i o n s  on us e o f  the e l e c t ron m i c r o s co p e  and fo r 
p r o c e s s ing the e l e c t ron m i c r o g r aphs . 
I wo u l d a l s o  w i s h  t o  thank P ro f e s s o r  J o hn B i enens t o ck , 
D ept . o f  P a tho logy , McMa s t e r Un ive r s i ty , Ont a r i o , C anad a , 
f o r  h i s  num e rous i l l uminat ing commun i c a t i ons p e r t a ining t o  
immuno l o g i c al fun c t i ons o f  mu c o s a l  c e l l  typ e s . 
S in c e r e  thanks a r e  a l s o  due t o  Mr s . Pam S l a c k  and 
M r . Roy S p a r k sman , for the i r  e xp e r t  t e chn i c a l  a dv i c e  and 
a s s i s t an c e at var i ou s  t im e s  throughout the cour s e .  
F i na l ly ,  I wou l d  w i s h  t o  t hank Mr s . E .  B ax t e r  for h e r  
s e rv i c e s  i n  typ ing the d r a f t  a n d  f i n a l  copy o f  t h i s  th e s i s . 
V 
Ab s t ract 
A cknow l e d g ements 
C HAPTER 1 
1 . 1 
1 . 1 . 1  
1 . 1 .  2 
1 . 1 .  3 
1 . 1 .  4 
1 . 1 .  5 
1 . 1 .  6 
1 . 1 . 7 
1 . 1 .  8 
1 . 2  
1 .  2 . 1  
1 .  2 .  2 
1 .  2 .  3 
1 .  2 .  4 
1 .  2 .  5 
1 .  2 .  6 
1 .  2 .  7 
1 .  2 .  8 
1 . 3  
1 .  3 . 1 
1 .  3 .  2 
1 .  3 .  3 
CONT ENT S 
I nt ro duc tion 
G r o s s  Anatomy o f  the Salivary 
Gl ands 
P a r o tid G l and 
Mandibular G l and 
Sub lingua l G l and s 
Buccal G l ands 
Labial G l ands 
Lingual G l ands 
P a l atine G l an d s  
Pha ryng e a l  G l ands 
His t o l ogy and Cyt o l ogy of 
Sa liva ry G l ands 
P ro t eo s e ro us C e l l s  
Mucous C e l l s  
D emilune C e l l s  
Myo epith e lial C e l l s 
The Duc t a l  Sy s t em 
I nt e r c a l a t e d  Duct s 
I nt ra l o b u l a r  Duc t s  
E x c r e t o ry Duc t s  
Bovine S a liva ry Compo sition 
S e cr e tion o f  wa t e r  and 
e l e c t ro l y t e s  
P rimary s a liv a  
Final s a liva 
P a g e  
iii 
V 
1 
5 
7 
7 
8 
9 
1 0  
1 0  
1 0  
1 0  
1 2  
1 4  
1 6  
1 7  
1 7  
1 8  
1 8  
1 9  
2 1  
2 3  
2 3  
2 4  
2 6  
1 .  3 .  4 
1 .  3 .  5 
1 .  3 .  6 
1 . 4  
1 . 5 
1 . 6  
CHAPTER 2 
2 .  1 
2 .  2 
2 .  2 .  1 
2 .  2 .  2 
2 .  2 .  3 
2 .  2 .  4 
2 .  2 .  5 
2 . 3  
2 .  3 .  1 
2 .  3 .  2 
2 .  3 .  3 
2 .  3 .  4 
O r g an i c  s e c r e t o ry p r o ducts 
Synthe s i s and s e c r e t i on of 
pro t e i n s  and muc o ­
s ub s t anc e s  
Compo s i t i o n o f  Bovine s a l ivary 
muco s ub s t anc e s  
Cont r o l  o f  S a l i v a ry S ec r e t i on 
Muc o s a l  I mmunity 
S a l ivary S e c r e t ions and B l o a t  
Sus c ept i b i l i ty 
Bovine S a liva ry G l and H i s t o l ogy 
and H i s t o chemi s t ry 
I n t r o duc t i o n  
Mat e r i a l s  and Metho ds 
T i s sue s amp l i n g  and f ixa t i o n  
p ro c e du r e s  
P a r a f fin wax p r o c e s s ing and 
micro t omy 
S t a ining and h i s to chemi cal 
me thods 
I mmunoh i s t o c h em i s t ry 
Pho t omi c ro g raphy 
R e s u l t s  
P a ro t i d G l and 
Mandibu l a r  G l and 
S ub l ingual G l and 
Buccal G l ands 
P a g e  
3 0  
3 1  
34 
3 8  
4 2  
4 7  
4 9  
49 
5 3  
5 3  
54 
54 
ss 
5 7  
5 8  
5 8  
5 9  
6 1  
6 2  
2 .  3 .  5 
2 .  3 .  6 
2 .  3 .  7 
2 .  3 .  8 
2 . 4  
CHAPT E R  3 
3 . 1 
3 . 2 
3 . 3 
3 .  3 . 1 
3 .  3 .  2 
3 .  3 .  3 
3 . 4  
CHAPTER 4 
CHAPTER 5 
App endix I 
App e ndix I I  
Bib lio g raphy 
P a l a tine G l and s 
P o s t e rio r Tongue G l ands 
Ph aryng e a l  G l ands 
I mmunohis t o c h emis t ry 
Dis cus sion 
P a ro tid G l and I n t r a epithelial 
Duc t  Ce l l s  
I nt roduction 
Mat e ria� and M e t h o d s  
Re s u l t s  
His t o l o gy 
His to chemis t r y  
U l t ras t ru c t u r e  
Dis c u s s ion 
G eneral Dis cu s sion 
C on c lusion 
The princip l e s  of his t o chemi c a l  
m e t ho ds us e d  f o r  t h e  dete ction 
o f  muc o s ub s t anc e s . 
S t aining and his t o c h emic a l  
t e c hniq u e s  
P a g e  
6 3  
6 4  
6 4  
6 5  
6 7  
7 6  
7 6  
7 8  
8 1  
8 1  
8 2  
8 3  
84 
91 
1 09 
1 1 4 
1 1 6  
1 2 8  
1 . 1  
1 . 2  
1 . 3  
2 . 1  
3 . 1  
5 . 1  
L I ST OF TABLES 
B o vine Sa livary P r o t eins I 
B o vine Salivary P r o t ein s I I  
I mmuno g l obulin Comp o si tion o f  
Adu l t  Ruminant Sa liva 
His t o chemical Re s ul t s  o f  B o vin e 
Majo r and Mino r Saliv ar y  G l ands 
His t o chemical Re s u l t s  o f  lEG Duc t 
C e l l s  and Ma s t  C e l l s  
His t o chemical C l a s sification o f  
B o vine Salivary G l ands 
Fo l l owing Page 
3 0  
3 6  
4 4  
5 8  
8 2  
1 09 
Fig u r e  
2 .  1 
2 .  2 
2 . 3  
L I ST O F  F I GURE S 
Fo l l owing Pag e 
P a ro tid and Ven t r a l  B u c c a l  
G l ands 
P a r o tid and Ven t r a l  B u c c a l  
G l and I nt ra lobu l a r  Du c t s  
I mmunohis t o chemis t ry : Dis t ribu tion 
of P a r o tid and Vent r a l  Buc c a l  G l and 
S a livary P r o t ein Bands 4 and 1 0  
6 6  
6 6  
6 6  
2 . 4  Mandibul a r  G l and 6 6  
2 . 5  S ub l ingua l G l and 6 6  
2 . 6  I nt e rmediat e Buc c a l  G l and 66 
2 . 7  D o r s a l  ( Upp e r )  Buc c a l  G l and 6 6  
2 . 8  P a l a tine G l ands 6 6  
2 . 9  P o s t e rio r Tongue G l ands 6 6  
2 . 1 0 Pha ryng e a l G l ands 6 6  
2 . 1 1 Hya luroni da s e  Ac tivit y  6 6  
3 . 1  Mo rpho l o gy and Dis t ribution o f  I nt r a - 8 3  
3 .  2 
epit h e l i a l  Granu l a r  ( I EG ) Duc t C e l l s 
o f  the P ar o tid and V e n t r a l  Buc c a l  
G l ands 
Mo rpho l o gy and Di s t ribution of Ma s t  
C e l l s  in Bovine S a liv a ry G l ands 
8 3  
Fig u r e  
3 . 3  
3 . •  4 
His to l ogic a l  and His t o chemic a l  
F e atur e s  o f  Cy t op l a s mic G r anul e s  
in l E G duct c e l l s  
Ul t ra s t ru c t u r a l  F e a t u r e s  o f  l EG 
Duc t C e l l s  
F o l l owing Pag e 
8 3  
8 3  
1 
CHAPTER 1 I NTRODUCT I ON 
S a l iva i s  p ro duced by s p e c i a l i s e d g l ands a s s o c i a t e d  
w i th th e o ra l  c av i ty . I n  mamma l s  the three majo r p a i r e d  
s a l i va ry g l ands a r e  t h e  p a ro t i d ,  mandibular a n d  sub l ingua l . 
Addi t i o na l l y th e r e  a r e  s ev e ra l  minor s a l ivary g l ands emb e d ­
d e d  i n  the s u b e p i th e l i a l  t i s su e s  o f  t h e  mou t h  a n d  o ro ­
p ha rynx . 
Mamma l i an s a l iva g en e r a l l y c o n s i s t s o f  w a t e r , e l e c t ro ­
l y t e s  a nd p ro t e i na c i ous m a t e r i a l . The vo l um e  and c omp o s ­
i t i o n  o f  s e c r e t ion from i n d i v i dua l g l ands v a r i e s w i th i n  
s p e c i e s  a n d  b e tw e en d i f f e r ent s p e c i e s . 
I n  many a n ima l s , inc l u d ing man , s a l iva c o n t a i n s  s i gni f ­
i c ant amoun ts o f  en zyme s s uch a s  s a l iva ry amy l a s e , l ip a s e ,  
m a l t a s e ,  p e rox i da s e  and l y s o zyme (Young and van L enne p , 
197 8 ) . Re cent ly s ev e r a l  b i o l o g i c a l ly a c t i v e  p ep t i d e s , 
w i th w i de rang ing func t i o n s , have b e en i s o l a t e d  f r om s a l ­
i v a ry g l and t i s s ue ( B a r k a , 198 0 ) . Th i s  may b e  s ug g e s t ive 
o f  a y e t  p o o r l y  unde r s t o o d  endo c r i ne ro l e  for s a l i v a ry 
g l an d s  in a dd i t i on to t h e i r  we l l  e s t ab l i s h e d  exo c r ine 
funct i on .  
F unct i o na l l y , s a l i v a r y  s e c r e t i o n  provi d e s  p r o t e c t i on 
f o r  t h e  o r a l  muc o s a  a g a i n s t abra s i on and d e s s i c a t i on , 
f a c i l i t a t e s  m a s t i c at i on , t a s t e , swa l l owing and e f f e c t iv e  
s uc k i n g  and s uc k l ing . S a l iva a l s o  exhib i t s  a n t i b a c t e r i a l  
p r op e r t i e s  and a l s o  ma int a i ns t h e  c a l c ium i o n  equ i l i b r ium 
n e c e s s ary fo r the maint enance o f  t e e t h .  
S a l iv ary s e c r e t ions a r e  u s ua l ly div i d e d  i nto two categ ­
o r i e s , s er o u s  and muco u s . S e rous s e c r e t i o n  i s  thin and 
wat e r y  and cons i s ts of w a t e r , inorganic i o n s  and s ome 
pro t e i na c i ou s  mater i a l , whi l e  muc ous s e c r e t i o n  i s  mo r e  
v i s c o us and cont ains a h i gh e r  conc ent ra t i on o f  o rg an i c  
p ro t e i na c i o u s  mat ter i n c l ud i ng neut r a l  and a c i d i c  g l yc o -
p r o t e i ns ( S chn eyer and S c hney e r , 196 7 ) . I n  g en e r a l , the 
p a ro t i d  g l and p r o duc e s  a s e rous s e c re t i on whi l e  the m an ­
d i b u l a r  and s ub l ingua l g l ands p r o du c e  a m i x e d  s e rous and 
mucous s e cr e t i on .  
Ruminant s ,  s uch a s  s h e e p  and c a t t l e , in cont ra s t  t o  
mono g as t r i c  s p e c i e s  s e c r e t e  l a r g e  quant i t i e s o f  s a l i v a  
owing to t h e  g enera l ly c o a r s e n a t u r e  o f  the i r  d i e t  a n d  the 
n e c e s s i ty to ma intain f l u i d  i n  a c ap a c i ous non- s e c re t o ry 
f o r e s t omach . S a l i va ry s e c r e t i ons i n  the r e t i cu l o rumen 
fun c t i on as a nut r i en t  m e d i um f o r  f e rmenta t i v e  m i c r o ­
o rg a n i sms wh i ch p ro du c e  e n zymes requ i r e d  f o r  b r e akdown o f  
p l an t  mat e r i a l  ( Church , 1 97 6 ) . 
B ovine s a l iva i s  comp o s e d  o f  ino rgan i c  and o rg an i c  
c o mp on ents . T h e  fo rmer cons i s t s  o f  e l e c t r o l y t e s  s u ch as 
+ + ++ ++ -- - - . . N a� , K , C a  , Mg , P04 , HC03 , and Cl . The 1no r g an 1 c  
an i on s  funct i on p r imar i ly a s  s a l i vary buf f e rs wh i l e  the 
2 
. + + . c a t i ons part 1 cu l a r l y  N a  and K , a r e  r e s pons 1 b l e  fo r e s t ab -
l i s h ing a concentra t i o n  g r a d i ent f o r  pas s i v e  t r ans p o r t  o f  
w a t e r . The org an i c  comp onent s ,  whi ch are c h i e fly muc o s ub ­
s t anc e s , p r o v i de phys i c a l  p ro t e c t i on for t h e  o r a l  and f o r e ­
s to m a ch muco s a  and a r e  a l s o a s o u r c e  o f  nut r i en t s  f o r  rumen 
b a c t e r i a .  S a l ivary i mmun o g l o b u l i n s  s uch as s e c r e t o ry I gA ,  
p ro b ab l y prov i d e  immuno p r o t e c t i o n  f o r  the e n t i r e  rum inant 
f o r e s t omach , s ince p l a s ma c e l l s  a r e  us ual l y  no t found in 
the muc o s a  of thes e r e g i o ns . In a d d i t ion , to s u ff i c i en t l y  
mo i s ten dry f e e d ,  a c o p i ous amo unt o f  wat e r  i s  s e c r e t e d  
during feeding and rum i n a t i on ( Kay , 196 0 ; Chur ch , 1 9 7 6 ) . 
The p a ro t i d  g l ands o f  c a t t l e  s e c r e t e  l a r g e  quant i t i e s 
o f  s a l iva wi th o r  wi tho u t  n e rvous s t imul a t i o n  comp a r ed t o  
t h e  mandibul a r  g l ands wh i ch s e c r e t e  l i t t l e  e xc ep t  during 
f e e d i ng p e r i o ds ( Kay , 1 96 0 ) . The s ub l ing u a l  g l ands , 
a l though con t i nuous ly s e c r e t ing , contr ibu t e  n e g l i g i b l e  
q uan t i t i e s  o f  s a l iva c o mp a r e d  t o  b o th the mand ib u l a r  and 
the p a ro t i d  ( Kay , 196 0 ) .  The vo l ume o f  s a l i va supp l i e d  by 
the minor g l ands des c r i b e d  by Kay ( 1 96 0 )  as the r e s i dual 
s a l iva , i s  approxima t e l y equa l to tha t s e c r e ted from b o th 
the p a ro t i d  g l ands . B a i l ey and Ba l c h ( 196 1 )  e s t ima t e d  the 
t o t a l  vo l ume of s a l iva s e c r e t e d  by ca t t l e  to b e  a s  h i gh 
a s  9 8-190 l i t re s  p e r  day . 
The rumen f l u i d  vo l um e  and comp o s i t i on i s  a f f e c t e d  by 
the b al an c e  b e tween i n f l ow o f  i ons from s a l i va and f o o d  
and t h e  n e t  out f l ow b y  a b s o rp t i on o r  p a s s a g e  t o  the omasum 
( K ay , 196 6 ) . The t on i c i ty b e tween rumen c o n t en t s  and the 
b l o o d  is th e r e fo r e  ma i n t a ine d  a t  an equ i l i b r i um wh i ch 
3 
l im i t s  the movement o f  wa t e r  a c r o s s  the rumi na! ep i th e l ium . 
F o r f e rmen t a t i v e  d i g e s t i on t o  func t i on op t im al ly the ma i n ­
t en a n c e  o f  t h e  rumen f l u i d  l eve l i s  e s s enti a l  ( B a i l ey , 196 1 ). 
The m i crob i a l  popu l a t i on o f  the r e t i c u l o rumen i s  
h e a v i ly dep endent o n  t h e  cons t i tuent s o f  s a l iva for the i r  
g r owth , p a r t i cu l a r l y  n i t rogen and mine r a l s  ( Church , 197 6 ) . 
U r e a  p r e s ent in ruminant s a l iva p r o v i d e s  a b o ut 7 5 - 8 5 %  o f  
t h e  n i t ro g en r e qu i r emen t s  ( Ph i l ip s on and Ma gnan , 1959) , 
wh i l e  the s a l ivary buf f e r s , b i c arbona t e  and pho spha t e  ions , 
m a i n t a in the pH a t  an o p t imum f o r  f e rment a t i v e  d i g e s t i on 
( Kay , 196 6 ) . The rumen pH i s  a l s o  inf luenc e d  by the nature 
and dryn e s s  of the f e e d , wh i ch in turn mo d i f i e s  s a l ivary 
s e c r e t i on ( Church , 1 9 7 6 ) . 
S e c r e t i ons from t h e  par o t i d , vent r a l  b u c c a l , p a l at i ne , 
p h a ryng e a l  and buc c a l  g l ands a r e  s t rong ly b u f f e r e d  w i th 
b i c a rbona t e  and pho sp h a t e  i o ns compa r e d  w i t h  the mandibu l a r  
a n d  s ub l ingua l g l and s e c r e t i o n s  wh i c h  a r e  w e akly b u f f e r e d . 
( Kay , 196 0 ) . Al thou gh p re s ent in l a r g e  q u ant i t i e s , b i ca r ­
b on a t e  and phospha t e  i ons a r e  c a p ab l e  o f  n e u t ra l i z ing only 
a f rac t i on o f  the vo l a t i l e  fa t ty ac i ds p r o du c e d  dur i ng 
m i c r ob i a l  f e rment a t i on b e caus e a c i d i ty o f  rumen contents 
f r equen t l y  exceeds t h e  bu f f e r ing c ap a c ity o f  the i ons 
( B a i l ey ,  1 961) . Sa l i va ry b u ff e r s  the r e fo r e , mainly p r ov i de 
a f i r s t l ine o f  d e f e n c e  a g a ins t s ha rp d e c l ines i n  rumina! 
pH by coun t e r a c t ing p e a k s  of vo l a t i l e  fa t ty a c i d  p r o duc t i on 
s o o n  a f t e r  fee ding . F a t ty a c i ds not neut r a l i z e d by s a l ivary 
b u f f e r s  a r e  absorbed th rough the rum ina! e p i th e l ium and 
n e u t ra l i z e d  by buffe r s  of the b l o od ( Kay , 1 96 6 ; Bar t l ey ,  
1 97 6 ) . 
Ruminant s a l iva , p ar t i cu l a r l y  o f  c a t t l e , has a l s o  b e en 
imp l i ca t e d  w i t h  s u s c ep t i b i l i ty t o  b l o a t , a d i s e a s e  o f  
c o ns i d e r ab l e  e conom i c  i mp o r tanc e .  S a l iva and i t s  a s s o c ­
i a t i on w i th b l o a t  w i l l  b e  d i s cus s e d in s e c t i o n 1 . 6 .  
4 
5 
1 . 1 G ro s s  Ana tomy o f  t h e  S a l ivary G l ands . 
The fo l l owing o ut l i ne i s  d e s c r i p t ive o f  s a l i vary g l and 
ar c h i t e c t u r e  ( s ource r e f e r ence : Young and van Lennep , 1 97 8 ). 
The majo r s a l iva ry g l ands a r e  l a r g e  d i s t i nct s t ructur e s  
l o c a t e d i n  t h e  head and neck r e g i on . Sa l iva fo rme d by 
th e s e  g land s  is de l ive r e d  to the o ra l  c a v i t y  by r e l a t ive l y  
l o n g  exc r e t o ry duc t s . E ach g l and i s  enc l o s e d  b y  a f i b rous 
t i s s ue cap s u l e  and the s e c r e t o ry t i s s ue i s  s ub d i v i ded into 
l o b e s  and s ma l l e r  l o bu l e s  by c o nne c t i v e  t i s sue s ep t a  f rom 
t h e  cap sul e .  The g l an d  p a r enchyma cons i s t s o f  c l o s e l y  
p a c ke d  s e c r e t o ry a c i n i  ( o r  endp i e c e s ) and d r a i na g e  duc t s . 
T h e  s t roma i s  comp o s e d  o f  l o o s e a r e o l ar conn e c t ive t i s s ue , 
b l o o d ve s s e l s , n e rve b und l e s  and excr e t o ry duc t s . The 
h i l us o f  the g l and c o n t a ins a r t e r i e s , v e ins , n e rve s , 
l ympha t i cs and the m a i n  e xc r e t o ry duc t  ( o r  duc t s ) .  Add i t ­
i o na l  to t h e  va s cu l a r  s up p l y  ent e r ing the h i l u s  sma l l  
a r t e r i e s  e n t e r  through the c ap s u l e  a t  s ev e r a l  p o ints . 
W h i l e  the mandibular and s ub l i ngua l g l ands d i s p l ay the 
a b o v e  f e a t u r e s , the p a ro t i d  has no t rue h i lus s i nce the 
a r t e r i a l  s upp ly and v e nous d r a inage are r andom ly s c a t t e r e d 
o v e r  the g l and sur fac e .  
The d i s t r ibut i on o f  b l o o d  v e s s e l s  w i t h i n  s a l ivary 
g l ands us u a l ly p a r a l l e l s  tha t o f  the b r anching duc t sy s t em .  
A r t e r i e s  a c c omp any t h e  duc t s  w i t h in l obul e s  and t e rmina t e  
i n  ex t ens ive cap i l l a ry p l exus e s  a round endp i e c e s  and int r a ­
l o b u l a r  ducts . The v enous d r a ina g e  fo l l ows a s im i l ar p a t ­
t e rn a l though s ome v e ins dra i n  d i r e c t l y  t o  the g l and p e r ­
i p h e ry . The p r e s en c e  o f  a r t e r i ovenous ana s t omo s e s  i n  s ome 
g l ands p r ovide s  a r e g u l a t o ry me chan i s m  wh ich influences the 
r a t e  of s a l ivary s e c r e t i o n . 
The n e rvous s upp ly t o  the t h r e e  majo r s a l ivary g l ands 
i s  p r o v i ded by b o th p a r a symp a the t i c  and s ymp a the t i c  d i v i s ion 
o f  the autonom i c  ne rvous s y s t em .  P r e g ang l i o n i c  p a r a s ymp ­
a th e t i c  f i b r e s  o r i g i na t e  in the ro s t ra l  and caud a l  s a l ivary 
nucl e i  in the l a t e r a l  r e t i c u l a r  f o rma t i o n  o f  the l ower 
6 
b r a in s t em , med i a l  t o  the s p ina l t e gmental t r a c t . P o s t ­
g a ng l i o n i c  p a ra s ymp a t h e t i c  f i b r e s  t o  indiv i du a l  g l ands a r e  
l o c a t e d  i n  V I I and I X  c r an i a l  nerv e s . Aft e r  ent e r ing t h e  
s t r oma o f  t h e  g l and t h ey f o rm s ma l l  b ranc h e s  tha t p en e t r a t e  
t h e  b a s a l  l am i na o f  t h e  s e cr e t o ry endp i e c e s  a n d  t e rmina t e  
i n  sma l l  b ud l i k e  th i ck en i n g s  o n  the b a s a l  s ur fa c e s  o f  
s e c r e t o ry c e l l s . P r e g ang l i o n i c  symp a the t i c  f i b r e s  o r i g i na t e  
i n  the f i r s t  two tho r a c i c  s egments o f  the s p i n a l  c o r d  and 
a s c ent i n  th e v a g o s ymp a th e t i c  t runk t o  synap s e  in t h e  
c ra n i a l  c e rv i c a l  g a ng l i on . T e rmina t ions o f  p o s t g ang l i on i c  
s ympa t he t i c  f i b r e s  t h a t  r e a ch the g l and a r e  d i s t r ib ut e d  in 
t h e  wa l l s  of b l o o d  v e s s e l s  and the b a s a l  p l a s ma l emma o f  
s e c r e t o ry c e l l s . P a r a symp a th e t i c  and s ymp a th e t i c  b r an ch e s  
o f  n e rv e s  t o  s ec r e t o ry c e l l s  a l s o  s up p l y  the s a l i v a ry 
g l and duc t c e l l s . 
I n  c o n t r a s t t o  t h e  maj o r  s a l ivary g l ands wh i ch a r e  
d i s t in c t  o r g ans , the mino r g l ands a r e  b a s i c a l ly l obul e s  
o f  g l andu l a r  t i s s ue emb e dd e d  i n  the s ubmuc o s a l  c onne c t iv e  
t i s s u e s  o f  t h e  mouth and o r opharynx . The m i no r  g l ands a r e  
n o t  s u r r o unded b y  a d i s t i n c t  conn e c t ive t i s s ue c ap s u l e  but 
a r e  a r r a n g e d  in g r o up s  of l obul e s , wh i c h  are mo s t l y  d i f fus e . 
S e c r e t i o n s  f rom th e s e g l ands a r e  d e l i v e r e d  into the o r a l  
c av i t y  v i a short duc t s  tha t open through the ep i the l i a l  
s ur fac e . 
I n  c a t t l e ,  the p a i r e d  m i n o r  s a l i v a ry g l and s a r e  t h e  
v en t r a l  b uc c a l , int e rmedi a t e  buc c a l , do r s a l  b uc ca l , l a b i a l  
a n d  pharyng e a l  g l an d s . The two non - p a i re d  m inor s al i v a ry 
g l ands a r e  the p a l a t ine and l i ngua l g l ands ( B i r t l e s , 1 9 8 1 ) .  
Kay ( 1 96 0 ) , s t a t e d  t h a t  the comb ine d t o t a l  we i gh t  o f  t h e  
m inor g l ands i n  c a t t l e  i s  ha l f  t h a t  o f  the thr e e  maj o r  
g l ands . The mino r g l ands the r e fo r e  undoub t e d l y  cons t i tut e 
an imp o r t ant mas s  o f  s a l ivary g l and t i s s ue i n  rum i nan t s . 
The Bov i n e  S a l iva ry G l ands . 
The fo l lowing de s c r i p t ions have b e en b a s e d  on a c c o unt s 
p re s ent e d  in t e x t s  o f  Ve t e r inary Ana t omy by Hab e l  ( 1 9 7 0 ) , 
G e t ty ( 1 9 7 5 )  and N i c ke l , Schumme r , S e i f e r l e  and S a e k  
( 19 7 3 ) . 
1 . 1 . 1  P a ro t i d  G l and 
The p a r o t i d  g l an d  i s  p i n k i s h  b rown in c o l o ur w i th an 
a ve rage we i ght of 1 1 5  g r ams . I t  i s  l o n g , nar row and t r i ­
angul a r  in shape w i t h  a w i d e  t h i c k  do r s a l  end that r e a che s 
the r e g i o n  o f  the t emp e r omandibul a r  j o i nt . The g l and l i e s  
a l ong t h e  cauda l b o r de r  o f  t h e  ma s s e t e r  mus c l e  and ext ends 
from the zygoma t i c  a rch t o  the ramus of the mand i b l e . The 
vent r a l  a s p e c t  fo l l ows the caudal bo r d e r o f  the mand i b l e  
and i s  r e l a te d  d e ep l y  t o  t h e  mand i bul a r  g l and . The d e ep 
s u r f a c e  i s  r e l a t e d  t o  the a ng l e  o f  the s ty l ohyo i d  bone and 
the o c c i p i tohyo i de u s  and d i ag a s t r i c  mus c l e s . 
The p a r o t i d  duc t l e a v e s  the g l and v entr a l l y w i th the 
f a c i a l  a r t ery and v e in and a s c ends o n  the l a t e r a l  sur f a c e  
o f  t h e  ma s s e t e r  mus c l e  t o  o p en near t h e  p o s t e r i o r  upp e r  
mo l a r  t e e th in the b uc c a l  c a v i ty . 
The h i ghly va s c u l a r  p a r o t i d  g l and r e c e i v e s  i t s  b l o o d  
s upp ly f rom a l l the unde r l y ing ar t e r i e s  p a r t i c u l ar l y the 
e x t e rna l car o t i d ; t h e  v enous draina g e  i s  by the max i l l a ry 
and e x t e rna l j ug u l a r  v e ins . 
The p aro t i d  g l ands a r e  i nnervat e d  by the s e c r e t omo t o r  
p a r o t i d  n e rve wh i c h  i s  a b r anch o f  the bucc a l  n e rv e  from 
the t r i g emina l n e rv e . The p ar o t i d  n e rv e  i s  l o c a t e d  do r s a l  
t o  the p a ro t i d  duc t  and a c c ompan i e s  i t  t o  the g l and . Sym ­
pathe t i c  f i b r e s  r e a ch the g l and in the va s c u l a r  p l exus e s  
and synap s e  with a and a r e c e p t o r s  o n  s e cr e t o ry c e l l s . 
1 . 1 .  2 Mandi bul a r  G l and 
The mandibul a r  g l and is l a r g e r  t h an the p a ro t i d  and 
w e i g hs about 1 4 0  g r ams . I t  is p a l e  y e l l ow in c o l o ur , 
d i s t inc t l y lobul a t e d  and e x t ends in a curve me d i a l  t o  the 
ang l e  of the mand i b l e  from t h e  a t l an t a l  f o s s a  t o  the 
7 
b a s i hyo i d  mus c l e . C auda l l y  the g l and i s  p a r t ly cove r e d  by 
t h e  p a ro t i d  g l and . 
The mandibul a r  duct l e ave s the g l an d  f rom the m i dd l e 
o f  i t s  r o s t r a! b o r d e r  and e x t ends l a t e r a l l y  a l o ng the 
d i g a s t r i c  mus c l e ,  then p a s s e s  fo rwar d  on t h e  d e ep s u r f a c e  
o f  t h e  my l ohyo i d  a n d  o p e n s  l a t e r a l  t o  t h e  s ub l ingual 
c a runc l e .  
8 
The v a s cu l a r  s up p ly t o  t h e  mand i b u l ar g l and i s  from 
b ranc h e s  o f  t h e  f a c i a l  and l i ngual a r t e r i e s ; venous d r a i na g e  
r e a ch e s  the l i nguo f a c i a l  and fac i a l  v e i ns . P r e g ang l i on i c  
p a r a symp a the t i c  f i b r e s  from t h e  r o s t r a! s a l i va ry nuc l eus 
l eave i n  the fa c i a l  nerve i n  n e rve t runks known a s  cho r da 
tymp an i , t o  j o i n  t h e  l ingua l n e rve . Synap t i c  jun c t i o n s  
a re u s ua l l y  found w i th i n  g ang l i a l o c a t e d  i n  the h i l u s  o f  
the g l and and p o s t g ang l i oni c f i b r e s  p a s s  i nt o  l obul e s  o f  
the g l and by fo l l ow i ng t h e  b r anch ing d r a ina g e  duc t  sys t em . 
1 . 1 . 3  Sub l ingua l G l ands 
The s ub l i ngua l g l ands are d i v i d e d  i nto two p a r t s , the 
mo r e  vent r a l  mono s t omat i c  and the d o r s a l  p o lys toma t i c  
p o r t ion . Th e p o l y s t oma t i c  p a r t  i s  c omp o s e d  o f  a cha i n  o f  
l ob u l e s  about 1 5 - 1 8  cm i n  l en g th , p a l e  ye l l ow i n  co l o u r  and 
ly ing under the f l o o r  of t h e  mout h . Th is g r oup extends 
f rom the inc i s iv e  a r e a  of t h e  man d i b l e  to the p a l at o ­
g l o s s a l  a r ch and i s  d r a i n e d  b y  many s m a l l  duc t s  wh i c h  o p en 
a l ong rows o f  l ong p ap i l l a e  f ound in t he l a t e r a l  s ub l ingua l 
r e c e s s  o f  the mou t h . 
The mo no s toma t i c  g l and i s  1 0 - 1 2  cm in l ength and 2 - 3  cm 
in width and e x t e n d s  from t h e  inc i s iv e  area o f  the mand ib l e  
t o  the midl ine o f  t h e  p o l y s t oma t i c  g l and wh i ch l i e s  d or s a l !� 
A s ing l e  ex c r e t o ry duc t p a s s e s  fo rward m e d i a l  to the g l a n d  
and accomp an i e s  t h e  mand i b u l a r  duc t t o  the subl ingu a l  
ca runc l e  l o c a t e d  o n  t h e  f l o o r  o f  t h e  mouth beh ind t h e  
inc i s o r  t e eth . 
C o l l e c t iv e l y , t h e  s ub l ingua l g l ands a r e  r e l a t e d on 
t h e i r  l a t e ra l  a sp ec t s to the my lohyo i d  mus c l e  and the s ub ­
l i ngu a l  n e rve , me d i a l ly to the hyo g l o s s us , s ty l o g l o s s u s  
and g en i o g l o s s u s  mus c l e s and ven t r a l ly t o  the g en i o -
hyo i d  mus c l e . 
9 
The b l o o d  s upp l y  and venous dra i na g e  a r e  v i a  the s ub ­
l ingu a l  a r t e ry and v e in r e s p e c t i ve ly . The n e rv e  s upp ly i s  
s im i l a r  t o  the man d i b u l a r  g l and i n  tha t s e c r e t omo tor f i b r e s  
f r om t h e  chorda tymp ani jo i n  t h e  l i ngua l nerve t o  s upp l y  
t h e  g l a n d . 
1 . 1 . 4  Buc c a l  G l ands 
The s e  g l an ds a r e  we l l  deve lop e d  and arrang e d  into 
t h r e e  g ro up s : do r s a l , i nt e rme d i a t e  and vent ra l buc c a l  
g l ands . 
The do rs a l  g r o up a r e  ye l l ow in c o l o u r ,  d i s t inctly 
l ob ul a t e d  and e x t e n d  from the ang l e  o f  the mouth to the 
m ax i l l a ry tub e ro s i ty .  Th in s up e rf i c i a l  l aye r s  of the 
b ucc inat o r  and mas s e t e r  mus c l e s  enve l op e  t h e  d o r s a l  buc c a l  
g l ands . Sho r t  duc t s  dra in the g l an d  through the p ap i l l a t e d  
a r e a  o f  the ep i th e l i um t o  the buc c a l  c avi ty . 
Th e vent r a l  b uc c a l  g l and i s  a comp a c t , brown - c o l o u r e d  
m a s s  w e i g hing about 1 5  g rams . The g l and l i e s  a dja c e n t  t o  
the l o w e r  mo l a r  t e e t h and ext ends f r o m  the ang l e  o f  the 
mand ib l e  t o  the r o s t r a l  border o f  t h e  mas s e t e r  mus c l e . 
The g l and i s  dra i n e d v i a  s h o r t  duc t s  t o  the b uc c a l  c av i ty . 
The v e n t r a l  buc c a l  g l and i s  s im i l ar i n  cons i s t ency and 
func t i o n to the p a r o t i d  g l and . 
The interme d i a t e  b uc c a l  g l ands a r e  di f fu s e  l o o s e l y ­
a r r ang e d  l obul e s  y e l l ow i n  c o l our , and ext end a l ong t h e  
dors a l  b o rder o f  t h e  vent r a l  buc c a l  g l and . L o bu l e s  o f  
g l andu l a r t i s s ue a r e  s c a t t e red w i thin conne c t i ve t i s s u e s , 
adipo s e  t i s s ue and p o r t i ons o f  the buc c i na t o r  mus c l e . The 
g l and i s  dra ined via sho r t  du c t s  tha t open into the buc c a l  
v e s t ibu l e . 
Co l l e c t iv e l y , t h e  t h r e e  bucc a l  g l ands a r e  inne rva t e d  
b y  the buc c a l n e rv e , a b ranch o f  t h e  mandibular nerve . 
1 . 1 . 5  L ab i a l  G l an d s  
1 0  
The s e  g l ands a r e  comp o s e d  o f  sma l l  s c a t t e r e d  y e l l ow ­
c o l o ur e d  l ob u l e s  o f  s e c r e t o ry t i s sue found n e a r  the l a b i a l  
commi s s ur e  o f  t h e  upp e r  l ip emb e dd e d  b e twe en bundl e s  o f  
l ab i a l  mus c l e s . Short duc t s  open a t  t h e  s umm i t s  o f  t i ny 
p ap i l l ae l o c a t e d  o n  the int e rnal s u r f a c e  o f  the l ip .  
1 . 1 . 6  Lingua l  G l ands 
The l i ngua l g l ands are c omp o s e d  o f  sma l l  ye l l ow - c o l ou r e d  
l obul e s  s c a t t e r e d  b e twe en s k e l e t a l  mus c l e s  a n d  conn e c t i v e  
t i s s u e s  o f  t h e  p o s t e r i o r  t h i rd o f  the tongue a n d  a l s o  
ext end ro s t ra l ly a l o ng the marg ins o f  the tongue . The 
g l ands are dr a in e d  by s eve r a l  sma l l  ducts wh ich op en onto 
the l ingual s ur f a c e  through r ows of p ap i l l a e . The g roup 
of g l ands a s s o c i a t e d w i t h the val l a t e  p ap i l l a e  a r e  c a l l e d  
gus t a t o ry o r  von Ebner ' s  g l ands . 
1 . 1 . 7  P a l a t ine G l ands 
Th i s  is a l a r g e  ma s s  o f  y e l l ow - c o l our e d ,  l obul a t e d  
g l andu l a r  t i s s ue embedded i n  t h e  s ub ep i the l i a l  c onne c t iv e  
t i s s ue s o f  t h e  s o f t  p a l a t e  a n d  d i s t a l  p o r t i on o f  the hard 
p a l ate . The g l ands a r e  d r a ined by s e veral s ma l l  duc t s  
wh i c h  open onto t h e  o r a l  c av i ty . 
1 . 1 . 8  Pharynge a l  G l and s 
Th i s  group a r e  ye l l ow i n  co l o ur and d i s t r i but e d  in 
the s ubmuco s al c o nne ct i v e  t i s s u e s  of the o r a l  and l a ryng e a l  
reg i ons o f  t h e  p h a rynx . T h e  g l ands a r e  d r a ined v i a  s ma l l  
duct s that o p en o n t o  the ep i t he l i um o f  the pharynx . G l an ­
dul a r  t i s s ue found i n  the r o o t  and l a t e ral marg ins o f  t h e  
1 1  
t ongue , in the a r e a  between tongue a n d  ep i g l o t t i s  a re a l s o  
i n c l ud e d  in the p h a ryng e a l  g rcup . 
1 . 2  Hi s to l ogy and Cyt o logy of S a l ivary G l ands 
H i s to l o g i c a l ly s a l i v a ry g l ands are c l a s s i f i e d  as e xo ­
c r i n e  g l ands , whe r e  p r o duc t s  o f  s e c re t o ry c e l l s  a r e  d i s ­
cha r g e d  t o  a n  e x t e rna l s ur fa c e  v i a  a sys tem o f  duct s .  
Four fa c t o r s  d i s t ingu i s h  e xo c r ine g l ands : -
1 2  
1 .  The numb e r  o f  c e l l s  ( un i c e l l u l a r  o r  mul t i c e l l u l a r ) . 
2 .  The s e c r e t o ry e n dp i e c e  ( tubul a r , a c inar o r  t ub u l a r ­
a c i na r )  and t h e  duc t s ys t em ( s imp l e  o r  c omp o und ) .  
3 .  Mo d e  o f  s e c r e t i on ( ap o c r i n e , me r o c r ine o r  ho l o ­
c r i ne ) . 
4 .  The n a t u r e  o f  s e c r e t i o n  (mucous o r  s e r o us ) . 
S a l ivary g l ands a r e  mul t i c e l l u l a r  and a r i s e  a s  t ub u l a r  
invag inat i o n s  o f  a n  ep i the l i a l  s h e e t  that e x t en d s  i n t o  the 
und e r l y i ng c o nn e c t ive t i s s ue . 
f in e d  t o  the t e rminal p o r t i o ns 
( Young and van Lennep , 1 97 8 ) . 
The s e c r e t o ry c e l l s  a r e  c o n ­
o f  t h e  tubu l a r  inva g in a t i on s  
The t e rminal s e c r e t o ry end -
p i e c e s  w e r e  c l a s s i c a l l y r e f e r r e d  t o  a s  a c i n i  owing t o  the 
b e l i e f  that s e c r e t o ry endp i e c e s  w e r e  sphe r i ca l  t e rm i n a l  
e xp ans ions . H oweve r  t h i s  may 
s e c r e t o ry e ndp i ec e  morpho l o gy 
on the g l and and the s p e c i e s . 
b e  an·overs imp l i f i c a t i o n  a s  
va r i e s cons i d e rab l y  dep ending 
T h e  mo rpho l ogy o f  mamm a l i an 
s a l i vary g l a n d s , w i th p a r t i cu l a r  r e f e r enc e t o  sp e c i e s  
v a r i at i o n , h a s  b e en e x t ens i v e l y  r ev i ewed b y  Young and 
van Lennep ( 1 9 7 8 )  and P i nks t a f f  ( 198 0 ) . 
Sal ivary g l ands a r e  c ompound exo c r ine g l ands w i th a 
b r anched s y s t em o f  duc t s  s inc e s imp l e  exo c r ine g l an d s  w i th 
unb r anched duc t s  have not b e en r e p o r t e d  f o r  s a l ivary g l and� 
The mo d e  o f  s ec r e t ion of s a l i vary g l ands can b e  d e s c ­
r i b e d  a s  m e r o c r i ne s inc e the s e c r e t o ry p r o duct e l a b o r a t e d  
by endp i e c e  c e l l s  i s  r e l ea s e d  t h r ough the ap i c a l  c e l l  
membran e  l ea v i ng the c e l l i n t a c t  . 
• 
An ent i r e  s e c r e t o ry endp i e c e , r e g ard l e s s  o f  i t s morp h -
o l o gy ,  i s  c o mp o s e d  o f  a co l l e c t i o n  o f  s e c r e t o ry c e l l s . The 
1 3  
p r e c i s e  i dent i f i c a t i o n  o f  a l l  t h e  c e l l  typ e s  in an end ­
p i e c e  i s  howeve r ,  s t i l l  c ontrove r s i a l . Trad i t i ona l ly four 
c e l l  typ e s ; s erous , muc o u s , s er omucous and s p e c i a l  s e rous 
have b e en imp l i c a t e d  i n  the format i on of s a l iva ( S ha c k l e fo r d  
and W i lborn , 196 8 ) . The mo rpho l o gy and s e c r e t o ry p ro duc t s  
o f  typ i c a l  s e rous and muc ous c e l l s  a r e  w e l l  document e d  
(Young and van Lennep , 1 9 7 8 ) . Howeve r , the p re c i s e  nature 
of s e c r e t o ry c e l l s  wi th charac t e r i s t i c s  c ommon t o  b o th 
s e r o u s  and mucous c e l l s , i s  open t o  deb a t e .  
I n  gene r a l , s e rous c e l l s  s e c r e t e  wa t e r , e l e c t r o l y t e s  
and p r o t e i na c i ous mat e r i a l  t h a t  m a y  b e  e i t h e r  b a s i c  p ro t e i n s  
o r  n e u t r a l  g l ycopro t e i n s  o r  b o th . Muc ous c e l l s  s ynthe s i s e  
and s e c r e t e  muc�sub s t an c e s  wh i l e  t h e  s e romu c o u s  c e l l s  syn­
t h e s i s e  and s e cr e t e  s ig n i f i c ant amoun t s  o f  muc o s ub s t ance 
in a d d i t ion t o  wa t e r  and e l e c t r o l y t e s  (Mung e r ,  1 96 4 ; 
Sh a c k l e fo r d  and W i l b o rn , 1 96 8 ) . The " s p e c i a l  s e r ou s "  c e l l  
de s c r i b e d  by Shac k l e f o r d  and Wi l b o rn ( 1 96 8 )  had u l t r a s t ruc ­
tu r a l  featur e s  found i n  s e rous c e l l s  but the s e c r e t o ry g r a n ­
ul e s , a l though s im i l ar t o  mucous g r a nu l e s , d i f f e r e d  h i s to ­
c h em i c a l ly . T and l e r  and Poul s e n  ( 197 7 )  r ep o r t e d  an i d ent i c al 
c e l l typ e but c a l l ed i t  s e romuc ou s . A s o l ut i on to the 
c l a s s i f i ca t i o n prob l em s ug g e s t e d  b y  Young and van L ennep 
( 1 97 8 )  was to adop t the t e rms s e r o u s , mucous and s e r omuc ous 
( s p e c i a l  s e r ous wa s no t ment i o n e d )  to d e s c r i b e  morpho l o g i c a l  
f e a t ur e s  o f  s e c r e t o ry c e l l s  w i thout a l l ud ing t o  the h i s to ­
c h em i c a l  d e f i n i t ion o f  t h e  s ec r e t o ry p r o duc t . Wi thin t h i s  
c l a s s i f i c a t i on s e rous c e l l s  h a v e  s ma l l  e l ec t ron den s e  homo ­
g en e ou s  g ranu l e s  whi l e  mucous g r anul e s  a r e  l ar g e  and c l o s e ly 
p a c k e d  w i th a homo g en e o u s  e l e c t r o n  lucent m a t r ix . S ec r e t o ry 
g ranu l e s  o f  s e romucous c e l l s  have cha r a c t e r i s t i c s  c ommon 
to b o th s e r o u s  and muc o u s  g ranu l e s . 
The fundamental qu e s t i on o f  whe t h e r  c e l l s  that s e c r e t e  
s i g n i f i c ant amount s o f  p ro t e i na c ious m at e r i a l  w i th wa t e r  
and e l e c t r o l y t e s , c o u l d  b e  c a l l e d  " s e r o u s "  r ema ins unansw e r ed� 
the t e rm " s e rous" deno t i ng a wa t e ry s e cr e t i o n  wi th ions but 
no t p ro t e i ns or g l ycopro t e i ns . P i nks t a f f  ( 1 98 0 )  r e f e r r e d t o  
a l l  three c e l l  typ e s ,  s e r ous , s e romuc o u s  and sp e c i a l  s e rous , 
a s  " s e rous " c e l l s  and r e p o r t e d  tha t a l l  " s e rous " c e l l s  
a r e  g enera l l y i nvo l v e d  i n  the synthe s i s o f  p ro t e ina c i ou s  
mat e r i a l , t o  " on e  deg r e e  o r  ano th e r " . Re cen t l y , t h e  t e rm 
" p r o t e o s e rous " h a s  b e en s ug g e s t e d  f o r  " s erou s "  c e l l s  
( B i r t l e s , 1 98 1 ) . Th i s  may b e  t h e  m o s t approp r i a t e  t e rm 
a f t e r  a c a r e fu l  app ra i s a l  o f  t h e  e x i s t i ng l i t e r a t ur e . I n  
the p r e s ent s tu d y , t h e  t e rm p ro t eo s e rous ha s b e en a dop t e d 
f o r  future d i s c u s s i o n . 
1 4  
I n  g e n e ra l , t h e  s e c r e t o ry c e l l s  o f  a n  endp i e c e  a r e  
e i th e r  p ro t e o s e rous o r  muc ous , but mixed endp i e c e s  w i th 
p r o t e o s e rous a n d  muco u s  c e l l s  a r e  p r e s ent in s om e  g l an d s . 
The demi l un e  c e l l s o f  s a l ivary g l ands l o c a t e d  d i s ta l  t o  an 
endp i e c e , p ro du c e  ma i n l y  a p ro t e o s e rous s e c r e t ion . C on t ­
r a c t i l e  myo ep i th e l i a l  c e l l s  wh i c h  l i e b e twe en the b a s a l  
l am i n a  and s ec r e to ry c e l l s  have a l s o  b e en r e p o r t ed 
( G a r r e t t  and Emm e l in , 1 979) . 
1 . 2 . 1  P r o t e o s e rous C e l l s  
P ro t e o s e r o u s  c e l l s  i nvo lved i n  s e c r e t ion o f  wat e r ,  
e l e c t ro l y t e s  a n d  pro t e i n a c ious mat e r i a l  a r e  o b s e rv e d  i n  
two b as i c  fo rms in s a l ivary g l ands . F i r s t ly , a pyram i da l ­
shap e d  c e l l  i n  a n  a c inar endp i e c e  a n d  s e condly a cubo i d a l  
c e l l  i n  a t ub u l a r  endp i e c e  ( Young and van L ennep , 1 97 8 ) . 
The l a t t e r  form i s  u s ua l l y  de s c r i p t ive o f  the bovine p a ro t i d  
and vent r a l b u c c a l  g l ands . 
A py r am i d a l  endp i e c e  c e l l ,  w i t h  a b ro a d  b as e  and a 
na r row ap ex i s  g en e r a l ly sp e c i a l i s e d  for synthe s i s  o f  b a s i c  
p r o t e i ns and s e c r e t ion o f  s ome wa t e r  and e l e c t r o ly t e s . The 
b r o a d  b a s a l  c y t o p l a s m  c on t a ins c o n s i derab l e  amount s o f  
rough endop l a s m i c  r e t i cu l um ( RE R )  and the narrow ap e x  hou s e s  
sma l l , dens e s e c r e t o ry g r anul e s . 
S e c r e t o ry c e l l s  wh i c h  a r e  p r e d om inantly s p e c i a l i s e d  f o r  
wa t e r  and e l e c t r o ly t e  t ranspo r t , howev e r , r eq u i r e  a g r e a t e r  
s ur fa c e  a r e a  t o  vo l ume r a t i o  (Youn g  and van L ennep , 1 9 7 8 ) . 
I n  the ruminan t th i s  cha r a c t e r i s t i c  i s  c l e a r l y  d emons t ra t e d  
1 5  
by the p r e s enc e o f  e x t ens i v e  b a s o l a t e r a l  info l d i ng s o f  t h e  
p l a sma memb r ane , an ap i c a l  a r e a  w i th s ev e r a l  s e c r e t o ry c an ­
a l i c u l i and we l l  d eve l op e d  m i c r o v i l l i .  The s e  c e l l  s u r f a c e 
mo d i f i c a t ions g en e r a l ly f a c i l i t a t e  w a t e r  equi l ib r a t i o n  
( van L ennep , Kenn e r s o n  and C omp t on , 1 9 7 7 ) . 
Th e p r e s en c e  o f  t i ght jun c t i o n s  ( z onu l a  o c c ludens ) 
b e tw e e n  the l a t e ra l int e r c e l l u l a r  cana l i cul i and the ap i c a l  
s e c r e t o ry cana l i cu l i (Young and van L e nn ep , 1 9 7 8 )  may 
addi t i o na l ly cont r ibute to the e s t ab l i s hment of an i o n i c  
g ra d i ent . Thus i t  i s  t emp t ing t o  p o s t u l a t e  that o v e ra l l , 
c ub o i da l  c e l l s  w i t h l a t e r a l  s ur f a c e  mo d i f i c a t i o ns a r e  mo r e  
a dv an t a g eous fo r w a t e r  and e l e c t r o l y t e  t rans p o r t  in a dd i t i on 
t o  synth e s i z ing and s e c r e t ing s om e  p r o t e i ns and g lyco ­
p r o t e i n s . 
Ab ove f ind i n g s a r e  p a r t i cu l a r l y  v a l i d  f o r  p a ro t i d  g l and 
p ro t e o s e r ous c e l l s  of sheep (van L enn ep e t  a l . ,  197 7 )  and 
c a t t l e  ( Shackl e f o r d  and Wi l b o rn , 1 9 6 9 )  a l though in the�·la t t e r  
sp e c i e s  ev i denc e h a s  accumul a t e d  i n  f avour o f  a s i gn i f i c a n ­
t l y h i gh p ro t e in a n d  g l ycop ro t e i n c o n t ent in p a ro t i d  s a l i v a  
( T ab l e  1 . 2 ) .  
Apa r t  from s t ru c tura l  mo d i f i c a t i o ns o f  the cytop l a sm i c  
memb r ane p ro t e o s e r o u s  c e l l s  o f  rum inants do no t p o s s e s s  
unu s u a l  featur e s . The p o s i t i on o f  t h e  sphe r i c a l  nuc l eu s , 
f o r  e xamp l e , i s  g o v e rned by the numb e r s  o f  s e cr e t o ry g ra n ­
u l e s  p r e s ent ; when t h e  c e l l  i s  r ep l e t e  w i t h  g ranul e s  t h e  
nuc l eu s  occup i e s  the b a s a l  thi r d  o f  t h e  c e l l , fo l low i ng 
the i r  d i s c harg e t h e  nuc l eu s  b e come s mo r e  c en t ra l ly p l a c e d  
(van L ennep e t  a l . ,  197 7 ) . I n  a c c o r d anc e w i th a l ow l ev e l  
o f  p r o t e i n s ec r e t ion g ene r a l ly l i t t l e  RER h a s  b e en d e s c r ib e d  
i n  p r o t eos e rous c e l l s  ( Shackl e f o r d  a n d  W i l b o rn ,  1 9 6 9 ) . RER 
app e a r s  as f l a t t en e d  c i s t e rnae s t a c k e d  on top of one another, 
p a ra l l e l  w i th t h e  b a s e  o f  the c e l l a n d  f i l l ing the p e r i ­
nuc l e a r  cytop l a s m .  The Bo l g i  c omp l ex i s  l oc a t e d  i n  t h e  
s up r anuc l ea r  p o s i t i on wh i ch i s  typ i c a l  o f  c e l l s eng a g e d  i n  
p ro t e i n synth e s i s . A sys t em o f  v e s i c l e s , l ame l l a e , v acuo l e s  
and s a c cul e s  a r e  p r e s ent i n  add i t i o n  t o  curved s t a c k s  o f  
1 6  
three t o  f i v e  smo o t h  memb r ane c i s te rna! e l emen t s  o f  Go l g i ­
ER- lys o s om e  ( G E RL )  s y s t em (Nov i ko f f , Nov i kof f ,  Qui n t ana and 
Hauw , 197 1 ) . Mi to chondr i a  are usua l l y  e l ong a t e d  a n d  p a r t i c ­
u l a r l y  abun dant i n  p r o t eo s e ro u s  ce l l s  o f  the s h e ep p a r o t i d  
(van L ennep e t  a l . ,  1 97 7 ) . The i r  d i s tr ibut i on ,  a l t hough 
r andom shows a p r e f e r ence f o r  the l a t er a l  cytop l a s m i c  mem­
b rane s  (Young and v an Lennep , 1 97 8 ) . 
The s e c r e t o ry g ranul e s  o f  p r o t e o s e rous c e l l s  c o n s i s t  
o f  a t r i l am i nar membr ane enc l o s ing a ma t r ix w i t h  o n e  o r  
mo re e l e c t r on den s e inc l u s i on s  (Young a n d  van L ennep , 1 97 8 ) ; 
the s i z e a n d  s hap e o f  the g ranu l e s  and the e l e c t r o n  m i c r o ­
s cop i c  app e a ranc e o f  the i r  m a t r i x  may v a ry depend i ng o n  the 
f ixa t i on p r o c edure.. I n  s h e ep two g r anu l e  typ e s  have b e en 
o b s e rv e d  b y  van L ennep e t  a l . ,  ( 1 97 7 ) . The majo r i ty o f  
g ranu l e s  c l a s s i f i e d  a s  type I w e r e  d e s c r ibed a s  sma l l  and 
e l e c t r o n  dense wh i l e  typ e I I  g ranu l e s  were l arg e r  w i th a 
l e s s  e l e c t ron den s e mat r ix and had a t endency t o  fu s e  w i th 
one ano the r ,  a f e a ture typ i c a l  o f  muc ous s e c r e t o r y  g r anul e s . 
Th i s  l a t t e r  cha r a c t e r i s t i c  wa s r e s p on s i b l e  f o r  the rum inant 
p ro t e o s e r o u s  c e l l s  b e ing p r ev i ou s l y  l ab e l l ed a s  " s p e c i a l  
s e rou s "  b y  Shac k l e fo rd and Wi l bo rn , ( 1 969) . 
1 .  2 .  2 Mucous C e l l s  
Tub u l a r  endp i e c e s  w i th cub o i d a l  s e cr e t o ry c e l l s  s p e c i a l ­
ised fo r s ynthe s i s  o f  muc ous g ranul e s  do no t d i s p l ay t h e  
comp l e x  l a t e ra l  s ur fa c e  mo d i f i c a t i ons o f  p ro t e o s er ous c e l l s  
(Young a n d  van Lennep , 1 97 8 ) . The ap i c a l  p l a sma memb rane 
i s  usua l l y  devo i d  of m i c r ov i l l i  and s e c r e t o ry c a na l i cu l i 
are r a r e l y  p r e s ent . B a s a l  and l a t e r a l  p l asma m emb rane s a r e  
smo o th o r  s l i g h t l y  p l i c a t e d  w i th s ome int e r d i g i t a t ion 
b e twe en a djac ent c e l l s . 
The nucl eus i s  d i s p l a c e d  t owa r d s  the b a s e  o f  the c e l l  
owing t o  the l a r g e  volume o c c up i e d  b y  the s e c r e t o ry g ranu l e s. 
The RE R c o ns i s t s  o f  c l o s e l y p ac ked c i s ternae in the b a s a l  
reg i o n but i t s  p r e c i s e  d i s t r i but i o n and concen t r a t ion va r i e s  
with the s e c r e t o ry cyc l e  ( P inks t a f f , 198 0 ) . G o l g i  comp l ex e s  
1 7  
a r e  l ar g e  w i th s ev e r a l  d i l a t ed c i s t e rnae on the i r  c onc ave 
s i de . M i t o c ho ndr i a  a r e  l o c a t e d  p r e dominant l y  i n  the b a s a l  
p a r t  o f  t h e  c e l l . 
The mucous s e cr e t o ry g ranul e s  a r e  l arg e r  than p r o t e o ­
s e ro u s  s e c r e t o ry g ranu l e s  and c o n s i s t  o f  an e l ec t ron luc ent 
ma t r i x .  The g ranu l e s  o f t en app e a r  fu s e d  wi th adja cent g r a n ­
u l e s  whi ch p r o duc e t h e  c h a r a c t e r i s t i c " fo amy" app e a r a n c e  
o f  t h e  cy top l a sm (Young a n d  v a n  L ennep , 1 97 8 ) . P ink s t a f f  
( 19 8 0 )  had e x t ens ive l y  r ev i ew e d  the f ine s t ruc ture o f  
muc o us g ranu l e s  wi th r e f e r en c e  t o  sp e c i e s  v a r i a t i o n  and 
f ix a t i on a r t i fa c t s . 
1 .  2 .  3 Dem i l une C e l l s  
I n  s a l i v a ry g l ands o f  mo s t  s p e c i e s  the mucous tubu l e s  
a r e  o ft en " c ap p e d" b y  c r e s c ent - s hap e d  s e c r e t o ry c e l l s  
r e f e r r e d  t o  a s  dem i l un e s .  The mo rp ho l o gy and s e c r e t ions 
o f  demi l un e s  vary among s p e c i e s  and in d i f fe rent g l ands 
o f  the one s p e c i e s  but g en e ra l l y  the s e c r e t o ry p ro du c t s  
a r e  p r o t e o s e rous . P ur e l y muc o u s  demi l un e s  a r e  r a r e  a n d  have 
b e en rep o rt e d  in only a few sp e c i e s  ( P inks t a f f , 1 98 0 ) . 
The a r r an g ement o f  a demi lune d i s t al to an endp i e c e  has 
r e s u l ted in much s p e cu l a t i o n  a s  t o  the mode of r e l e a s e o f  
t h e i r  s e c r e t o ry mat e r i a l  t o  the l umen o f  a t ubu l e . Shackl e ­
f o r d  and W i l bo rn , ( 197 0 )  have r ep o r t e d  t h e  p r e s en c e  o f  a 
s y s t em o f  i n t e rce l l u l a r  canal i cu l i in d em i l une s o f  bov ine 
m an d ibular g l ands . The s e  may i n t e rconn e c t  t o  commun i c a t e  
w i t h  a demi lune t h a t  h a s  d i r e c t  a c c e s s  t o  t h e  l umen o f  i t s  
a s s o c i a t e d  t ubul e ( P i n k s t a f f , 1 9 8 0 ) .  
Func t i o na l ly ,  p ro t e o s e rous s e c r e t i o n s  o f  the dem i l une 
c e l l s  may p r o v i de a " f l u shing" e f f e c t  fo r t h e  v i s c ous mat e r ­
i a l  s e c r e t e d  by the endp i e c e  muc ous c e l l s . 
1 .  2 .  4 Th e Myoepi the l i a l  C e l l s  
Myo ep i the l ia l  c e l l s  are f r equent ly found b e twe en the 
endp i e c e  s e c r e t o ry c e l l s  and the b as a l  l amin a . They a r e  
s l ende r ,  s p indl e-s hap e d  c e l l s w i t h  l ong b ranch i n g  cyto ­
p l a s m i c p ro c e s s e s  wh i ch en c i r c l e  c e l l s o f  the s e c r e t o ry 
endp i e c e s  and i nt e r ca l at e d duc t s . 
1 8  
Myo ep i the l i a l  c e l l s  have b e en a s s o c i at e d  w i t h a cont r a c ­
t i l e  r oie .  P r e s s u r e  e x e r t e d  dur ing c ont r ac t i on may e xp e l  
s e c re t e d  ma t e r i a l  from t h e  endp i e c e  l umens i n t o  the.int e r ­
c a l at e d  duc t s  and a l s o  p re vent b ac k f l ow o f  s al iva i n t o  the 
endp i e c e l ume n . P i nks t a f f  ( 198 0 )  has s ug g e s t e d  t h a t  myo ep i ­
the l i a l  c e l l s  may addi t i o na l ly s up p o rt the s e c re to ry end­
p i e c e  and int e r c a l a t e d  duc t s  dur i n g  s e c re t i o n . 
1 .  2 .  5 The Duc t a l  Sys t e m  
I n  cont r a s t t o  s e c r e t o ry endp i e c e s , c l as s i f i c a t i o n  o f  
the sa l ivary g l and duct s y s t em i s  l e s s  cont r o v e rs i a l . The 
b as i c  p a t t e rn o f  the d r a i n a g e  duc t s  w i thin a s a l iva ry g l and 
b e g i ns w i th an i nt e r ca l a t e d  duc t  wh i ch conne c t s  a s e c r e t o ry 
endp i e ce to an int r a l ob u l a r  duc t . The s e  emp t y  into a con­
f l uen c e  o f  d r a inage ve s s e l s  t e rm e d  int e r l obul a r  and int e r ­
l ob a r  duc t s , dep ending up o n  the i r  p o s i t ion . The l a t t e r  
ana s t o mo s e  t o  fo rm a main e x c r e t o ry duc t  o r  i n  s ome g l ands 
into mul t ip l e  e x c r e t o ry ducts . The ma in exc r e t o ry duc t  ( o r  
duc t s ) t e rmina t e s  at the o ra l  muco s a . 
1 .  2 .  6 I n t e r c a l at e d  Duc t s  
I n t e rc a l a t e d  duc t s  v a ry in l ength , di ame t e r  and c e l l  
he i gh t ; gene r a l ly the duc t  wa l l s  a r e  comp o s e d  o f  cub o i d a l  
ep i t h e l i a l  c e l l s  b u t  may r ange f r o m  squamous t o  l ow c o l umn a r  
( P inks ta f f ,  1 98 0 ) . I n  b o v ine p a r o t i d  g l ands the i n t e r c a l ­
a t e d  duc t ep i t h e l ium n e a r the s e c r e t o ry endp i e ce h a s  b e en 
de s c r i b e d  a s  l ow cub o i da l  chan g i n g  to l ow c o l umna r  ne a r  
the junct ion w i t h int r a l o b ul a r  duc t s  ( Shack l e fo rd and 
Wi l b o rn , 1 969) . 
I n  ca t t l e , in t e r c a l a t e d  duc t  c e l l s  have s ho rt ap i c a l  
m i c r o v i l l i  a n d  minima l  l a t e r a l  and p l asmal emma! mo d i f i c a t i ons 
19 
( Sha c k l e fo rd and W i l b o rn , 1 969) . The intracy t op l a s m i c  
comp o n e n t s  d o  n o t  p o s s e�s unu s ua l  f e a t ur e s . A l a r g e  sphe r ­
i c a l  c e n t r a l ly-p l a c e d  nuc l e us o c cup i e s  a l ar g e  a r e a  o f  the 
cy top l a sm wh i ch cont a i n s  a few mi t o chond r i a  in add i t io n  to 
a Go l g i  comp l e x  and s ca t t e r e d  RE R c i s te rna� . 
P r e v i ous l y , i nt e r c a l a t e d  duc t s  we re cons i d e r e d  a s  p a s ­
s iv e  c ondui t s  fo r t rans f e r  o f  s e c r e t o ry p ro duc t s  from end­
p i e c e s  into i n t r a l obul a r  duc t s ; howe ver a c c o r d ing t o  Y o ung 
and van Lennep ( 1 97 8 )  and P inks t a f f  ( 1 98 0 )  th i s  is an o v e r ­
s imp l i f i cat i on .  H i s t o chem i c a l  a n d  u l t r a s t ruct ura l s tud i e s  
have d emon s t r a t e d  a p o s s i b l e  s e c r e t o ry ro l e  fo r the s e  
duct s  i n  s ome s p e c i e s . S e c r e t o ry g ranul e s  have b e en r ep ­
o r t e d  i n  e p i t h e l ia l  c e l l s o f  i nt e r c a l a t e d  duc t s  o f  s h e ep 
p a ro t i d  ( van L ennep e t  a l . ,  197 7 )  and bo vine mandi bu l a r  
g l ands ( Shack l e fo rd and W i l b o rn ,  1 97 0 ; B l oom and C a r l s o o , 
197 4 ) . 
1 .  2 .  7 I n t ralobul a r  Duc t s  
I n t e rc a l a t e d  duc t s  a r e  cont inuous with i n t ra l obul a r  
duc t s  wh i ch o ften e xh i b i t  d i s t inct e o s inop hi l i c  b a s a l  
s t r i a t ions . T h i s  cytop l a s mi c  f e a t u r e  i s  r e s pons i b l e  fo r 
int r a l ob u l a r  duc t s  b e i ng f r equent l y  r e f e r r e d  t o  a s  " s t r i a t ed ' 
duc t s .  Howeve r, the t e rm int r a l o bu l a r  duct s e ems mo r e  app r o p ­
r i a t e  a s  b a s a l  s t r i a t i o n s  a r e  no t e v i dent in int r a l ob u l a r  
duc t  c e l l s  o f  a l l  s a l iva ry g l and s . I n  addi t i on t h e  " g ran­
u l a r "  duc t s  o f  rodent s i n t e rp o s e d  b e tween int e r ca l a t e d  and 
s t r i a t e d duct s a r e  a l s o  p a rt o f  t h e  int r a l o b u l ar duct sys t em. 
Ham and C o rmack ( 1 9 7 9 )  have d e s c r i b e d  t h e  p r in c i p a l  c e l l 
typ e l ining i n t r a l obul a r  duc t s  t o  b e  t a l l  c o l umna r  w i t h 
b a s a l  s t r i a t i ons ; howeve r ,  cub o i d a l  c e l l s  w i t h  p o o r l y  dev e l ­
op e d  b a s a l  s t r i a t i ons h a v e  b e en f o und i n  s ome sp e c i e s  
( Yo un g  and van L enn ep , 1 9 7 8 ) . I n  ruminant s the duc t  c e l l s 
a r e  further s p e c i a l i s e d  i n t o  th r e e  typ e s , " l i ght c e l l s " , 
" da rk c e l l s "  and b a s a l  c e l l s  ( Sh a c k l e fo r d and W i l b o rn , 196 9 ;  
19 7 0 ; B l oom and Car l s o o , 197 4 ) . B a s a l  cel l s  are ra r e ly s e en 
but when p r e s ent a r e  c l o s e  t o  the b a s a l  lamina and do not 
2 0  
e x t end t ow a rds the d u c t  l ume n . The i r  cy t op l as m  c o n t a ins 
s c a t t e re d  RE R ,  few m i t o chond r i a  and s ome int ra c e l lul a r  
f ib r i l s . The dark c e l l s  a r e  chara c t e r i sed b y  an e l e c t ro n  
dens e cytop l as m i c  ground s ub s tanc e , a n  i rr egu l a r  nuc l eus 
and c l os e ly p ac k e d  c y t op l a s m i c  o rgane l l e s . The mo re c ommo n  
l igh t ce l l  has a s ph e r i ca l , cent r a l ly-p l aced nuc l eus and 
e x t ens i v e  b as a l  memb r ane f o l dings ho u s ing ab undant m i to ­
chondr i a  o r i en t e d  p ar a l l e l  t o  the p l a s m a l emma! fo l di ngs . 
The b a s a l  s t r i at i o n s  are due t o  i n fo l d i ngs o f  the b a s a l  c e l l  
memb r an e . L a t e r a l  p l a s ma l e mma! f o l d i ng s  a re ob s e rve d t ow ­
a r ds t h e  b a s e  o f  t h e  ce l l . 
I n  gene r a l , s t r i at e d  duc t s  are we l l  deve l op e d  i n  p ro t e o ­
s e rous g l ands but ppo rly deve l op e d  o r  ab s ent i n  mucous 
g l ands and mino r s a l ivary g l ands , w i th the excep t i on of the 
ven t r a l  b u c c a l  gl and s  o f  ruminan t s . A l though the ab o ve 
p a t t e rn has b e en ob s e rve d i n  s al iva ry g l ands o f  s he e p  ( van 
L ennep e t  al . ,  1 9 7 7 ) , the p aro t i d  gl an d s  of ca t t l e  a re an 
e x c ep t i o n  in that the int r a l obul a r  duc t s  show a mark e d  l ac k  
o f  b a s a l  s t r i at i ons ( Shack l e fo rd and W i lb o rn , 1 9 6 9; P a l , 
Chandr a  and Bha r a dw a j , 1 9 7 2 ; P inks t af f ,  1 9 8 0 ; Vigno l i  and 
Nogue i r a , 1 9 8 1 ) . H oweve r s t r i at e d  duc t s  are w e l l  deve l o p e d  
i n  b ovine mandibul a r  and s ub l ingua l g l ands . 
I t  i s  gen e r a l l y  a c c ep t e d  that s t r i at e d  duc t s  o f  s h e ep 
enab l e  t h e  animal t o  s e cr e t e  a hyp o t on i c  s a l iva ( depending 
on i t s  s o dium s ta tu s )  by r e ab s o rp t i on o f  s o d i um i on s  ( Y o ung 
and van L enn ep , 1 9 7 9 ) . The abundant b as a l  m i t o chondr i a  
s ugges t a p o s s ib l e  act ive t rans p o r t  me chan i s m  fo r s t r i a t e d  
duc t s  wh i l e  the l ac k  o f  mi t o chondr i a  o r  b as a l  s t r i a t i o n s  
may i n d i c a t e  s e c r e t ion o f  an i s o t on i c  s al iva w i th l i t t l e  
r e ab s o rp t i on o f  i on s . 
Ap a r t  f rom b a s a l  p l a smal emma! i n f o l dings the s t r i a t e d  
duct c e l l s  s how ap i c al mo d i f i c at i on s  o f  two var i e t i e s ; m i c ro ­
vi l l i  and " ap i c a l  b l eb s " , ( P inks t af f ,  1 9 8 0 ) . "Ap i c a l  b l e b s "  
have b e en v a r i ous l y  des c r i b e d  a s  f i x a t i on a r t i facts o r  
s t age s o f  an ap o c r ine s e c r e t i on p ro c e s s ( s e e  Yo ung and 
van L ennep , 1 9 7 8 ) . The cy top l as m  o f  i nt ralobul a r  duct c e l l s  
2 1  
cont a ins a f ew s c a t t e re d  RE R c i s t e rn a e  and a G o l g i  comp l ex 
s i tuat e d  ap i c a l  t o  the nuc l e u s . 
I nt ra l o b ul a r  du c t s  a r e  a dd i t i onal l y  e ngag e d  i n  the syn­
thes i s  o f  s e c r e t o ry mat e r i al ( Young and van L ennep , 197 8 ; 
P i nks t af f , 198 0 ) . Enzyme s s uc h  as ka l l i k r i en , p e r ox i da s e ,  
l y s o zyme and the s e c re t o ry c omponent o f  I gA h ave b e en i de n ­
t i fi e d  i n  s t ri a t e d  duct c e l l s  (Young a n d  van L e nn ep , 1 97 8 ) . 
I n  s h e ep and c a t t l e , an a d d i t i on a l  intraep i th e l i a l  duc t  
c e l l  has b e en de s cr i b e d  i n  t h e  wal l s  o f  intra l o b ul a r  duc t s . 
I n  ca t t l e , the s e  c e l l s  h ave b e en c a l l e d  " int r a s t r i a t e d" duc t  
c e l l s  b y  S h a ck l e fo rd and K l ap p e r  ( 196 2 )  and B i r t l e s  ( 198+) 
wh i l e  i n  s h e ep , v an Lennep e t  a l . ( 1 9 7 7 )  des c r i b e d  them a s  
" g l obul e l euco cy t e s " . One o f  t h e  a ims o f  the p re s ent s tudy 
i s  to inve s t igate th e s e  " in t r a s t r i at e d" duct c e l l s  mo r e  
c l o s e ly .  
1 .  2 .  8 E x c r e t o ry Duc t s  
The e xc r e t o ry duc t s  a r e  u s ua l ly i nt e rpo s e d  b e twe en 
intralobu l ar duc t s  and the m a i n  e xc r e t o ry duct . Mo re t h an 
one typ e o f  e xc r e t o ry duc t  m a y  b e  p r e s ent ; an i n t e rl o b u l a r  
duc t  l o c a t e d  in t h e  int e r l o b a r  conn e ct ive t i s s ue s ep t a  b e t ­
ween l o b u l e s  and an int e rl o b a r  duc t i n  conne c t i v e  t i s s ue 
s ep t a  b e tw een l o b e s . 
Th e e p i the l i um o f  e xc r e t o ry duc t s  c an v a ry cons i de r ab l y  
r anging f r om s imp l e  co l umna r  t o  s t r a t i f i e d  cub o i da l  o r  
s t rat i f i e d  c o l umn a r .  Typ i c a l l y  t h e  m a i n  exc r e t o ry duc t i s  
l ine d b y  s t ra t i f i e d  co l umna r  o r  in s ome gl ands , w i th p s e udo ­
s t r at i fi e d  co lumn a r  ep i th e l i um w i th gob l e t c e l l s . 
H i s t o chemi c a l  s tudi e s  h av e  shown that exc r e t o ry duct s  
p o s s e s s  c ons i de ra b l e  s e cr e t o ry ac t iv i ty : Ka l l i k re ins and 
succina t e  dehydrogena s e  are among the many e n zyme s i s o l a t e d  
( s e e  P i nk s ta f f ,  1 9 8 0  fo r r ev i ew o f  e x c r e t o ry duc t  s e c r e t o ry 
p r o duct s ) . Youn g  and van L e nnep ( 19 79) have i n d i c a t e d  that 
excret o ry duct s  m ay b e  i nv o l v e d  i n  further mo d i f i c a t i o n  o f  
t h e  s al iva f rom the s e cre t o ry endp i e c e s  and s t r i a t e d  
duc t s . 
2 2  
The e xc r e t o ry duct l in i n g  chang e s  t o  s t r at i f i e d  squam­
ous ep i t he l i um a s h o r t  dis tance b e fo r e  i t  b e come s c ont inuous 
w i th the o r a l  c avi ty . 
2 3  
1 . 3  Bovine S a l i vary C o mpo s i t i o n  
The comp o s i t i on o f  b ovine s al i va c an b e  d i v i d e d  i n t o  
a n  inorgan i c  c omponent c omp o s e d  o f  e l e ct ro ly t e s  and wa t e r ,  
and an o r g an i c  comp on e n t  o f  muco s ub s t anc e s , p r o t e i n s  and 
ure a . 
On the b a s i s  o f  nume rous m i c ropunc ture s tud i e s  o n  t h e  
m ajo r s al ivary g l an d s  o f  va r i ou s  s p e c i e s , i t  i s  now g en e r ­
a l ly a ccep t e d  that s a l ivary g l an d  endp i e c es s e c re t e  an i s o­
t on i c  p r imary flui d h av i ng an e l e c t ro l y t e  c ompo s i t i o n  
s im i l a r  t o  p l a s ma ( Sc hneye r ,  Y o un g  and Schneye r ,  1 9 7 2 ) . 
The s e conda ry o r  f i n a l  s al iva tha t eme rg e s  into the o r a l  
c av i ty i s  p r i mary s a l i v a  wh i ch h a s unde rg one duc t a l  mo d i f ­
i ca t ion . 
Al though the c omp o s i t i o n  o f  e l e ct ro ly t e s  e l ab o ra t e d  b y  
an endp i e ce a r e  s e conda r i l y  mo d i fi e d b y  t h e  duc t  s y s t em , 
t h e r e  i s  no e v i dence t o  sug g e s t  that the muco s ub s t an c e s  
s e c re t e d  s imu l taneous l y  b y  t h e  e n dp i e c e  c e l l s  a l s o  und e r g o  
further mo d i f i ca t i on . I t  i s  g e n e r a l ly a s s umed that wat e r  
s e c r e t e d  a l o n g  wi th e l e ct r o l y t e s  d i s s o l v e s  and flu s h e s  out 
th e mo re v i s c ous muco s ub s t an c e s  ( Young and van Lennep , 1 9 7 9 ) .  
1 . 3 . 1  S e c r e t ion o f  W a t e r  and E l e ctro lyt e s  
As e a r ly a s  t h e  l as t  century , Ludw i g  ( 1 8 5 1 ) , o b s e rv e d  
t h a t  s a l i va c o u l d  b e  s e c re t e d  a t  p re s s ures in exce s s  o f  
b l o o d  p r e s s u r e . I nv e s t i g a t i o n s  b y  He i denhain ( 1 8 7 8 )  furt h e r 
e s tab l i s h e d  that s al i v a  was p r o du c e d  a s  a r e sul t o f  a s e c r e t­
o ry proce s s  rather t h an an ul t r a f i l t r a t e  o f  p l a sma , b y  
o b s erving chang e s  i n  int ens i ty a n d  durat ion o f  ne rvou s  s t i m ­
u l a t i on wh i c h  c aus e d  s a l ivary fl ow r a t e s  to vary w i d e ly wi th 
s ub s equent c h an g e s  i n  s al i va ry e l e ct ro lyte comp o s i t i o n .  
Thays en , Tho rn and S c hw art z ( 1 95 4 )  extended H e i denh a i n ' s  
( 1 8 7 8 )  find i n g s  and fo rmul a t e d  a two s ta g e  hyp o t he s i s  o f  
s a l ivary s e c�e t i on t o  account fo r the va r i a t i on i n  e l e c t r o ­
l y t e  comp o s i t ion wi t h  s al ivary f l ow rat e . I n  the f i r s t  
s ta g e  i t  wa s p o s tul a t e d  that a p re curs o r  ( p r imary )  s a l i va 
2 4  
was p ro du c e d i n  the e n dp i e c e r e g i on w i th an e l e ct ro ly t e  con ­
centrat ion s i mi l a r t o  p l asma . I n  the s e cond s t a g e , i t  was 
p ro p o s e d  t h a t  p ri ma ry f lu i ds d e l ive re d t o  the duc t  s y s t e m  
unde rwent changes i n  e l e c t ro ly t e  comp o s i t i on by a duc t a l  
me chan i s m  w i th a l im i t e d  max imum trans p o rt c ap a c i ty . 
1 .  3 .  2 P r i mary S al i va 
I t  h a s  b e en e s t ab l i shed t h a t  p rimary s al iva p r o du c e d by 
s e c re t o ry e n dp i e c e s  is due t o  an act i ve t rans p o r t  p ro ce s s  
i n i t i a t e d  b y  neural s t imul at i on . Th i s  s e c re t o ry p r o ce s s may 
invo l v e  the t rans fe r o f  s o di um and o th e r  ino r g an i c i ons de r ­
i ve d  from p l as ma a c ro s s  the d i s t a l s e gment o f  the s e c r e t o ry 
endp ie ce i n t o  i t s  l umen ( S chn e ye r  an d Emme l in ,  1 9 7 4 ) . W a t e r  
i s  t raris fe r r e d  p as s i ve ly s ince n o  e v i de n c e  e x i s ts tha t  
a c t ive t rans p o rt o f  w a t e r  o c cu r s  ( S chne y e r  e t  a l . ,  1 9 7 2 ) . 
As y e t , there i s  no w i de ly a c c e p t e d  theory o r  mo de l t o  
a c c ount fo r w a t e r  and e l e c t ro l y t e  t r ans p o rt i n  s a l i va ry 
g l an ds . E v i dence p e r t a in in g  t o  th i s  p ro ce s s  i n  s e c r e t o ry 
endp i e ce s  i s  us ual ly b y  ana l o gy w i th ava i l ab l e  da t a  on i o n i c 
t rans p o rt a c ro s s  in t e s t ina l e p i t he l i a  and prox ima l t ub u l e s  
o f  the k i dn e y . D e s p i t e  the p au c i ty o f  da t a  avai l ab l e  on 
e p i the l i a l  memb rane p o t en t i a l s  i n  s e c r e t o ry endp i e ce s , i t  
h a s  b e en p o s tul a t e d  th at endp i e c e e p i the l ium i s  o f  the 
" l e aky" typ e ( S chney e r  e t  a l . ,  1 97 2 ) , w i th p o t en t i a l i ty to 
t rans p o rt l arge vo l ume s of f l u i d  i s o t on i c a l ly . I t  i s  w i de l y  
h e l d  ( P e t e r s en ,  198 0 )  al though n o t  p ro ve d ,  tha t t r an sp o r t e d  
i ons and w a t e r  p a s s  v i a  p a race l l ul ar chann e l s  acro s s  int e r ­
c e l lul a r  jun c t i on a l  comp l e x e s  and that an act i ve t r ans c e lf 
l u l ar f l ux o f  at l e a s t  one i on i s  l ik e ly to o c cur . 
One mo de l fo r i on i c  t rans p o r t  p rop o s e d  by Fr i z z e l l , 
F i e l d  and S chul t z  ( 19 79) invo l ve d  a t i gh t ly c o up l e d  e l e c t r o ­
n e u t r a l  N a+C l - eo-t r an s p o rt p r o t e in l o c at e d  i n  t h e  b as al 
p l asma memb rane o f  the s e c re t o ry c e l l . Mo s t  se cr e t o ry ep i ­
the l i a ma i n t ain N a+ and K
+ 
i on s  at e l e c t rochemi c a l  p o t ent i a l s  
w i de l y d i s p l aced from equi l i b r i um .  I t  h a s  b e e n  p o s tul at e d  
that the d i f fus ion o f  Na+ into the cy to p l as m  concen t ra t e s  
C l  in t he cy t op l asm a g a i n s t an elec t r o chemi c a l  grad ient 
es t ab l i s hed by N a
+ , K+ - AT P a s e  l o cated in the b a s o l ateral 
p l asma memb rane . Under th i s  p ro p o s ed mo del Cl  wo u l d  then 
move p a s s i vely i n t o  the endp i ece l umen acro s s  the ap i c a l  
cel l memb rane b y  an unde f i ne d  mechan i s m .  Th i s  i on f l ow i s  
thought t o  in i t i a t e  a s ec ret i on p ro ces s (elec t ro s t a t i c a l ly 
and o smo t i ca l ly) in wh i ch mo s t  o f  t he secreted i ons and 
water enter the l umen by a p a racel l u l ar route , comp o sed o f  
intercel l u l a r  sp a ces and s ec re t o ry c ana l i cul i .  S uch a 
mechan i s m i s  known t o  be t hermo dynam i c al ly s o un d  ( Co o k  and 
Youn g , 1 9 8 1 )  b ut an exp lana t i on rema ins t o  be f o und to 
demons t ra te how jun c t i on a l  c omp lexes c an be a dequately per ­
meab le t o  i ons and wa ter and yet maint a in a h i gh reflex i o n  
coef f i c i ent t o  t he secret i on ion ( H i l l , 1 9 8 0 ) . 
D i amond and B o s sert ( 1 9 6 7 )  p ro p o sed a mo del wh i ch emp h­
a s i sed t he geome t r i ca l  fea t u res o f  ·e�i thel i a  s pec i a l ised 
2 5  
fo r i on i c  t r ans p o r t . The l a ter a l  i n tercel l u l a r  channe l s  
i den t i f ied un i vers a l l y  i n  e l ec t r o l y te t rans p o r t ing ep i thel i a  
may es t ab l i s h  s t anding o s mo t i c  g r a dien t s  tha t  c o u l d  f a c i l i t ­
a te the p r o duc t i on o f  an i s o to n i c  secret i on even though s e c ­
retory r a tes may be changed by u p  t o  th ree o r ders o f  mag ­
n i tude . 
Wel l deve l o ped l a tera l i nterce l l ul a r channe l s  and s e c ­
reto ry c ana l i cu l i have been des c r ibed i n  par6t i d  g l an d  
p r o teo serous cel l s  o f  the b o v i dae ( Y o ung and van Lennep , 
1 9 7 8 ) . 
The ino r g an i c  comp o s i t i o n  o f  the p r imary s ecret i on i n  
ruminant s a l ivary g l ands h a s  received l i t tle a t tent i on unt i l  
recen t l y  when Comp t on , Ne l s on ,  Wr i gh t  and Young ( 1 9 8 0 )  i nves ­
t i g a ted p a ro t i d  s a l iva o f  s heep in re l at i on t o  the s o d i um 
s tatus o f  the a n ima l . 
The p r imary s a l iva s o d i um c oncen t rat ion o f  s o d i um rep lete 
sheep was found to be i s o t o n i c  and i ndependent o f  s a l i va ry 
flow rate . However , in s o d ium dep l eted sheep p r imary s a l iva 
s o dium c oncen t r a t i on was very l ow and the p l a s ma - l i ke 
2 6  
o s mo l a l i ty o f  the p r imary f lu i d  was comp o s ed o f  an un i den­
t i f i e d  s o l u t e . A c c o r d ing to Compton et a l . ( 198 0 )  th i s  
s o lute may b e  a l ow mo l e cu l a r  w e i g�t o rg an i c  c omp o und wh i c h  
i s  r e ab s o r b e d  and u t i l i z e d  by the duc t c e l l s  s in c e  i t  was 
abs ent from final s a l i va .  Th i s  un i d en t i f i ed o r g an i c  com­
p ound wo u l d  a l s o  have to be non�i on i z e d  o r  p o o r l y  i on i z e d  a s  
t h e  s umme d  s o dium and p o t a s s i um concen t r a t ion i n  p r ima ry 
s a l i va c o r r e sponds c l o s e ly to t h e  s umme d chl o r i d e , pho s ph a t e  
and e s t ima t e d  b i carbonate conc e n t r a t i on s . 
I n  cont r a s t w i th the s o d i um conc e n t r a t ion o f  p r imary 
s a l iva , p o t as s ium concent rat i on d i d  no t d i f f e r  s i g n i fi c an t ly 
b e tw e en s o d i um rep l e t e  and s o d i um dep l e t e d s h e e p . On the 
b as i s  of ava i l ab l e  m i cropun c t u r e  dat a , Young and van L enne p  
( 1979) c on c l uded that p o t a s s i um i s  concentra t e d  i n  the cyto-
+ + p l asm o f  e n dp i e c e  c e l l s  by b a s a l ly l o c a t e d  N a  , K ATP a s e and 
that the i o n  e n t e r s  s al i va a c ro s s  the ap i c a l  memb r ane by a 
p a s s ive p ro c e s s .  
C omp t o n  e t  a l . ( 1 98 0 )  in t he i r i nv e s t i g a t i ons o f  the 
anion cont ent of p r imary s a l i v a  conc l u d e d  that the s o dium 
s t atus o f  t h e  anima l had l i t t l e  e f fe c t  o n  the conc ent r a t i o n s  
and excr e t o ry p a t t e rns o f  s a l ivary an i on s . O n  ave rage the 
c onc ent ra t i ons of b i c arbona t e ,  c h l o r i d e  and pho s p h a t e  i ons 
did not d i f f e r  s i g n i f i c ant ly b e twe en s o d ium rep l e t e  and 
s o d i um dep l e t e d  s h e e p . I n  a dd i t i on a l l  pho spha t e  i ons 
app e a r i n g  i n  final s a l iva were found t o  have b e en s e c r e t e d  
dur ing t h e  forma t i on o f  p r ima ry s a l iva , whi l e  b i c a rbona t e  
i ons app e a red to b e  s e conda r i ly s e c r e t e d  b y  t h e  duc t  s y s t em .  
Chl o r i d e  i ons ent e r ing p r imary s a l iva a c ro s s  t h e  s e c r e t o ry 
endp i e c e  e p i t h e l i um c ou l d b e  r e ab s o rb e d  dur ing s e c ondary mo d ­
i f i c a t i o n  o f  s a l iva b y  the duc t  sys t em . 
1 . 3 . 3  F i n a l  S a l i v a  
The s e cond s t e p  o f  s a l i va fo rmat i on o r i g i na t e s  di s t a l ly 
in the s t r i a t e d  and exc r e t o ry duc t s  o f  the s a l i v a ry g l ands . 
The duc t s  r e ab s o rb s o dium and ch l o r i de i ons and s e c r e t e  
p o tas s i um and b i carb ona t e  i on s . Re ab s o rpt i on b y  t h e  duc t 
2 7  
ep i the l i um o c curs a t  a fas t e r  r a t e  than s e c re t i on and duc t a l  
p e rmeab i l i ty to wa t e r  i s  l ow ,  henc e , f inal s a l i va i s  m a i n l y  
hyp o t o n i c  ( S chney e r  and S chne y e r ,  1 9 6 0 ) . Howev e r ,  t h e  
magni tude o f  the i on ic chang e s  in f i n a l  s a l iva o ft en d ep ends 
on th e s p e c i e s , the g l and and the nature and i n t e n s i ty o f  
the s t imul a t i o n  o f  s e cr e t o ry c e l l s  ( S chney e r  and Emme l in ,  
1 9 7 4 ) . F o r  e xamp l e ,  in rum in an t s  the f inal s a l i v a  p r o du c e d  
b y  the p a ro t i d  g l and m a y  b e  e i th e r  hyp o tonic o r  i s o t on i c  
dep ending o n  the s o dium s ta t u s  o f  the animal ( C omp ton e t  a l . ,  
1 9 8 0 )  whe r e as s a l iva fo rme d b y  the mandibul a r  and s ub l ingual 
g l ands has b e e n  s hown b y  Kay ( 1 9 6 0 )  t o  be hyp o t o n i c . 
Ul t r a s tructura l ly ,  s t r i a t e d  and e x c r e t o ry duc t  c e l l s  
show morpho l o g i c a l  f e a t ur e s  charac t e r i s t i c  o f  s e c r e t o ry and 
ab s o rp t ive ep i th e l i a ( Sc hney e r  et a l . , 1 9 7 2 ) . The p re s en c e  
o f  l umina l c l e f t s  b e tween adjacent c e l l s  and b a s a l  i n f o l d ­
ing s p ro v i de s  f l u i d  co l umns o r i en t e d  i n  l a t e r a l  and b a s a l  
as p e c t s  o f  c e l l s . T i gh t  jun c t i ons l o c a t ed a t  t h e  b a s a l  
ext r em e t i e s  o f  t h e  c l e f t s  p r event d i r e c t  c on t i nu i ty w i t h  
l a t e ra l  i n t e rc e l l u l a r  s p a c e s .  T h e  p r e s ence o f  lum i n a l  
c l e ft s  c r e a t e s  a doub l e  s t an d ing g r a d i ent in s e r i e s  wh i ch 
ensur e s  l um in a l  hyp o ton i c i ty . The f i r s t  gradi ent c o ul d b e  
fo rm e d  i n  the l a t e r a l  i nt e r c e l lu l a r  s p a c e  b e tween t i gh t  
j un c t i on s  and b a s a l  b o rd e r  o f  the ep i th e l i a l  l ay e r  by a c t i v e  
transp o r t  o f  s o d i um o u t  o f  t h e  c e l l  into th e i n t e rc e l l u l a r  
spa c e . The s e cond g r a d i en t  c ou l d  b e  c r eated i n  t h e  lum i n a l  
c l e ft f rom t h e  ap i c a l  c e l l  b o rd e r s  t o  t h e  t i g h t  j un c t i on 
and e s t ab l i sh e d  b y  p as s i ve s o dium t r ansport f r om l um i n a l  
f l u i d  i n t o  the c e l l .  S inc e the p e rme ab i l i ty o f  duc t e p i th ­
e l ia t o  w at e r  i s  g e n e r a l ly l ow ,  b o th s tand ing g ra d i e n t s  c o n ­
tr ibute t o  l um in a l  hyp o ton i c i ty .  O t h e r  s t anding g r a d i en t s  
fo rm e d  l uminal l y  b e tween m i c rovi l l i  and bas a l ly b e tween 
p l a s ma l emma! p l ic a t ions or i n fo l di ng s  also c o n t r ibute to 
the deve l opment o f  lum inal hyp o t on i c i ty .  
The f inal s a l iva o s mo l a l i ty o f  t h e  sheep p a ro t i d  i s  
ad d i t i ona l ly dep e n dent on t h e  s o d i um s t a tus o f  t h e  an ima l 
(Compton e t  a l . ,  1 9 8 0 ) . The duc t  s y s t em s  o f  the s h e e p  
paro t i d g l ands w e re un i que i n  p o s s e s s ing the c a p a c i ty 
t o  c e a s e  a l l n e t  s o dium and p o t a s s i um t r ans p o r t  a c t ivi ty i n  
s o dium r ep l e t e  an i ma l s .  The f i n a l  s a l i va p ro duc e d  was 
t h e r e f o r e  i s o to n i c  and fa i l e d  to e xh i b i t  flow d e p e ndency 
wh i l e  s o d i um dep l e t e d animal s  p ro du c e d  hypo ton i c  s al iva 
p a r t i cu l a r l y  at l ow f l ow r a t e s  w i th max imum duc t a l  s o d i um 
r e ab s o rp t i on .  
2 8  
Repo r t e d  l e ve l s  o f  p o t a s s i um i n  p a ro t i d s al i va o f  s o d i um 
dep l e t e d  anima l s  may b e  i nd i c at ive o f  a h i gh e r r a t e  o f  
duct a l  s e c r e t i o n  t han that wh i ch o ccurs dur ing t h e  f o rm at i o n  
o f  p r imary s al iva . I n  s o d i um r e p l e t e  an imal s ,  t h e  f i n a l  
s al ivary p o t a s s ium c oncent r a t i on was l ower than i n  p r im a ry 
s al i va i nd i c a t ing t h a t  p o t a s s i um w a s  mo s t ly r e ab s o rb e d  by 
the duc t e p i the l i um .  
The b i c arb on a t e  content i n  f i n a l  p aro t i d  s a l i v a  o f  the 
s heep was c ons i de r ab l y  g r e a t e r  than i n  p r im a ry s a l iva i nd i c ­
a t ing that duc t a l  s e c r e t i on o f  the i o n  has o c c u r r e d  e i the r 
d i r e c t l y  o r  ind i r e c t l y , (by ab s o rb ing hy dro g en i on s ) . Fur ­
thermo r e , s al ivary b i carb o na t e  conc e n t r a t ion w a s  n o t  d ep e n ­
d ent o n  t h e  s o d i um s t atus o f  t h e  anima l . 
Pho s p h a t e  and c h l o r i de l ev e l s  a l s o  app e a r e d  t o  b e  un i n ­
f l uenc e d  b y  t h e  an imal ' s  s o d i um s t atus . B o t h  p h o s p h a t e  and 
chl o r i de i ons in f in a l  s a l iva w e r e  r ep o r ted t o  b e  t o t a l ly 
d e r iv e d  f r om the p r imary f lu i d  w i th no duc t a l  s e c r e t i on but 
s ome chl o r i d e  app e a r e d  to be r e ab s o rb e d  dur ing s e c ondary 
modi f i c a t i on by t h e  duc t s y s t e m . 
I t  i s  unc e r t a i n , without furth e r  i nv e s t i g a t i o n , whe the r 
the f inding s  o f  C o mp t on e t  a l . ( 198 0 )  could e q ua l l y  app l y  
t o  b ov ine p a r o t i d  g l ands , a l t ho ugh e x i s t ing r ep o r t s  ind i c a t e  
t h a t  p a ro t i d  s e c r e t i ons o f  c a t t l e  a r e  l i kely t o  b e  pure l y  
i s o t on i c  ( P inks t a f f ,  198 0 ) . 
I n  a dd i t i on t o  C omp t on e t  a l . ( 19 8 0 ) , the i n o r g an i c  c om­
p o s i t i on o f  final s a l iva in r uminan t s  has b e en s tu d i e d  b y  
Phi l ip s o n  and Mang an ( 1 959) ; K a y  ( 1 96 0 ) ; B a i l e y a n d  B a l ch 
( 196 1 )  and revi ewe d  by Chur ch ( 19 7 6 )  and B a r t l ey ( 1 97 6 ) . 
Kay ( 196 0 )  inves t i gated  the e l e ctrolyte compos i t ion  of  
parot id ,  mandibular and subl ingual g land s ecretions  as  wel l  
a s  the "res i dual"  sal iva from the minor sal ivary g lands o f  
sheep and cat t l e . 
The paro t i d  g lands were found by Kay to cont inuous ly 
s ecrete  large quanti t i e s  of  wel l - buffered isotonic s al iva 
whi l e  the mandibular and sub l ingual g lands s ecreted  re lat ­
ive ly sma l l  quanti t i e s  o f  weakly buffe red hypotonic  s a l iva 
wi th a high mucous content . Kay ( 196 0 )  failed  to  ob s e rve 
the relationship between sodium s tatus and osmo lal i ty o f  
parot id  s al iva report ed  by Compton et  al . ( 198 0 ) , but 
reported a negat ive correlat ion b e tween Na+ and K+ and 
- - - + -HC03 and HP04 whi l e  the K and C l  l evels  were s imi lar 
to those  of  parotid  s al iva . Mandibular  s al ivary s ecret ion 
in part i cul ar , was fdund to be  influenced  by the rate of  
s e cret ion and the Na+ : K+ ratio  was  depre s s ed by  s odium 
dep l etion . 
Of the minor sa l ivary glands invest igated  by Kay ( 196 0 )  
the ventral buccal  was found t o  b e  ident ical t o  the parotid  
with respect  to  compo s i tion of  the s ecret ion , h i s to logy 
and rate of  s ecret ion per  gram of s ecretory t i s sue . The 
palatine , int ermediate  and dorsal  buccal  and pharyngeal  
g lands were p redominantly mucous s ecre t ing with a s imilar  
ionic  compos it ion to  parotid  sa l iva , although the Na+ 
2 9  
l evels  differed  with l i ttle  or  no  reduction in  concentrat ion 
with sodium deplet ion . The minor sal ivary glands were also  
found to produce i sotonic s ecret ions and the ir  comb ined 
vo lume of  s a l iva was approximately  equal to that  s ec reted  
by the paro t id g lands . 
Sal ivary nitrogen i s  an inorganic  e l ement o f  con s i derab l e  
metabol ic importance to  rumen microorganisms . The n i trogen 
content of  bovine s a l iva i s  mainly in the form o f  urea  and 
sa l ivary prote ins . Parotid  sa l iva contributes approximately  
7 5 - 8 5 %  of  the total  ni trogen as  urea  and the  prote in s ecre t ­
ed  by the mandibular  g land provides  the remaining 1 5 - 2 5 % , 
particularly during feeding when much higher concent rat ions  
of  nitrogen are ut i l i z ed (Phi l l ipson and Mangan , 1 9 5 9 ) . 
The s t imulus for the s ecret ion o f  sa l iva and contro l o f  
e l ectrolyte composit ion of  s a l iva i s  mainly provided by 
parasympathetic  and sympathe t i c  nerve s . The contro l of  
s a l ivary s ecretion w i l l  b e  d i s cuss ed  in  Section 1 . 4 .  
1 . 3 . 4  Organic Secretory Products  
Like inorganic  cons t i tuents ,  the  organic compos it ion o f  
s a l iva var i e s  cons i de rably b e tween spec i e s  and between d if ­
ferent g lands of  the s ame spec i e s . The nature o f  the s e cr ­
etory s t imulus i s  an  addit ional  factor  which may cause 
variat ions in  compos i t ion o f  s a l iva . 
General ly, the o rg anic content  o f  s a l iva i s  p rovided  by 
the mandibular , sub l ingual and minor s a l ivary g lands 
(Leeson, 1 9 6 7 ) , but the paro t id  g lands are  known to  synth­
es ise  and s ecrete  s ome prote inacious material , the  compos ­
i t ion of which varie s  depending on species  (Young and van 
Lennep , 1 9 7 8 ) . For example , s i gni ficant quant i t i e s  of  
organic material have been i s o l ated  from ruminant paro t id  
s a l iva (Tab l e  1 . 1 ) (Patterson , Brightl ing and Titchen , 
1 9 8 2 ) . 
3 0  
Organic  components o f  sa l ivary s ecretion · can b e  divided 
into two b road categorie s ,  dependent on whether the secre t ­
ory product i s  derive d  directly  from plasma o r  synthes i s ed  
and s ecre ted  by the g landular  epithel ium (Young and van 
Lennep , 1 9 7 9 ) . Extrins ic  prote ins are derive d from plasma 
and s ecret ed  without modificat i on via  the g landular  epith­
e l ium into  the  s al iva . Organic  compounds such as  a lbumin , 
o rosomuco i d ,  cae rulop lasmin , B - l ipoprot e in ,  t rans ferrin , 
B 2 -macroglobul in , urea and immunoglobul ins are s ome extr ­
ins ic prote ins ident ified i n  s a l iva . O f  the sa l ivary immun ­
oglobul ins , I g G  and I gM appear  to b e  extrins ic , whi le I gA 
i s  probab ly intrins ic , be ing synthes i s ed in g landular  
p lasma cel l s  and subs equent ly modified  by conj unct ion to  
s ecretory component synthe s i s e d  by  s t r iated duct c e l l s  
Tabl e  1 . 1 BOVINE SALIVARY PROTE INS I 
( Low Mo lecular  We ight Fract ion)  
Apparent Number of Bands 
Protein Parotid Mandibular Sublingual Int . Buccal PAS Molecular observed upon 
Band Gland Gland Gland Gland Reaction Weight ± S . D .  isoelectric focussing 
1 + l ( a) (b) + 1 ( c) - + ++ ? 2 
2 ? ? ? ? + ? 5 
3 + 3 ( a) + 3 (b )  - + ++ ? 6 
4 +++ - - traces - 140000 ± 8000 3 
5 ++ - - traces ++ 36000 ± 2000 4 
6 + - - traces + 36000 ± 2000 4 
7 + - - + - 53000 ± 2000 1 
8 - +++ - + - 50000 ± 2500 5 
9 - - +++ + - 43000 ± 2000 3 
10 ++++ - - + - 32000 ± 2000 5 
10s ++++ - - + - 32000 ± 2500 6 
10d ++++ - - + - 25000 ± 2000 3 
-
--------
( Source : Dr . W . T .  J ones , D . S . I . R . ,  Palmerston North , New Zealand . ) 
(Young and van Lennep , 1 9 7 8 ) . 
Mucous c e l l s  and to  a l e s s er extent proteoserous cel l s  
are  respons ib l e  for producing mos t  of  the intrins ic  pro t ­
e ins  o f  s a l iva , namely g lycoprot e ins or  mucosubs tances , 
po lypeptide s  and en zymes .  G lycoprote ins and po lypeptides  
produced by the s ecre t o ry endpieces  cons t i tute the  predom­
inant intr ins ic  organic  components ,  s ince , in ruminants ,  it 
has b een reported that the enzyme content of s a l iva is 
negl igib le  (Church , 1 9 7 6 ) . 
Sal ivary gland duct cel l s  have also  b een impl icated in 
the  product ion of intrins ic  proteins (Young and van Lennep , 
1 9 7 8 ) . Glycoproteins and pep t i des  o f  ductal  o rigin include 
the s ecretory component of I gA ,  enzymes such as ka l l ikrein 
and carbonic  anhydras e  and numerous growth factors manife s ­
t ing  various funct ions (Barka , 1 9 8 0 ) . A nerve growth 
factor , and an ep ithel ial  g rowth factor  known as " Parot in" 
have been i s o lated  from bovine parotid  g l and duct  cells  by 
Ho ffman , McAus lan , Rob e rtson and Burnett  ( 1 9 7 6 ) . 
3 1  
I n  the present s tudy an attempt has b een made t o  examine 
h i s tochemically the compos i t ion of the intr ins ic  p rote ins 
produced by  bovine s a l ivary g l ands . 
1 . 3 . 5  Synthesi s  and Secre t i on o f  Prote ins and Muco ­
s ub s tances 
The synthes i s  of exportab le  p roteins from s ecretory 
c e l l s  i s  primari ly contro l led  by genet ic  informat ion s tored  
in  the  DNA molecule s e gregat ed  in the  cell  nucleus (Ham and 
Cormack , 1 9 7 9 ) . One double - s t randed DNA molecule cons i s ts 
o f  four nuc l eotide bas e s , adenine , thyamine , guanine and 
cytos ine . The s equence  of  the bases  on the DNA s t rands 
de termines the o rder in which amino acids  required for 
protein synthesi s  appear  on a p ept ide chain or protein 
mo lecule . The genet i c  info rmation on DNA s trands are next 
t rans lated  with a comp lementary base  s equence into a s ing l e ­
s tranded me s s enger RNA (mRNA) mo lecule which migrates  from 
the nucleus to  the cytopl asm to ini t iate  pro t e in synthe s i s . 
The mRNA mo lecules  are in  turn attached to ribosomes pres ­
ent in  RER ;  mult iple  r ibo somes a l i gned on a mRNA mol ecule 
are  referred  to as polys omes . 
The amino acids required for protein synthes i s are 
extracted  from the extrace l lular  amino ac id  pool  into the 
c e l l  by act ive t ransport  (van Venrooi j , Kuij per - Lens tra 
and Krame r ,  1 9 7 3 ) . Aft e r  uptake , the amino ac ids are  
attached to t ransfer RNA (tRNA) mo l ecule s ,  and then trans ­
ferred  to ribosomes bound  to  mRNA . The recogni t ion of  mRNA 
s i t e s  with complementary bas es  on tRNA release s  amino ac i ds 
from tRNA mo l ecules  to  react  with a terminal amino acid  in 
an e l ongat ing po lypept ide chain found in polysome s . The 
s ecretory polypeptide mol ecule then enters  the lumen of the 
RER c is ternae through membrane pores  and is  s eques tr ated  
from the cytoplasm (Palade , 1 9 7 5 ) . 
3 2  
The s ecretory po lypeptide mo lecules  from RER a r e  t rans ­
ferred  by trans it ional vesicles  to the Gol g i  comp l ex ,  con ­
dens ing vacuole s  and finally to  secretory granules . The 
funct ion o f  the Golgi  complex i s  mainly to accept the trans ­
i t ional ves ic le s , modi fy the ir  contents  and d i stribute the 
s ecretory products via condens ing vacuoles  t owards the cell  
apex  in  preparat ion for  export extrace l lularly . 
Carbohydrate mo lecul e s  required  for incorporation into 
the po lypept ide backbone of  a g lycoprote in molecule  appear  
to  b e  synthes is ed in the Go lgi  complex (Palade , 1 9 7 5 ) . 
N - acetyl glucosruane and N- acetylgalactosamine are added to the 
po lypept i de chain with s ia l ic  acid  and fucos e  as  t erminal 
re s i dues ; the  membranes of the Gol g i  comp l ex contain the 
nece s s ary g lycosyltrans ferase  enzymes requi red for transfer 
of  res idues . Sulphat ion of  glycoprote ins i s  a lso  probably 
comp leted  in the Go l g i  complex .  
In  the next s tage o f  the synthet i c  proc e s s , the s ecre t ­
ory product s  from the Golgi  complex are transported  in Go lgi  
ve s i cles  to  condens ing vacuoles , where the s ecretory product 
3 3  
i s  concentrated  b y  a wi thdrawal o f  water  (Palade , 1 9 7 5 ) . 
Condens ing vacuol es deve lop firs t into immature and then 
mature s ecretory granule s  by a further proce s s  o f  conden ­
sat ion . In  a proteoserous c e l l  thi s results  in  the format ion 
of  discrete  homogeneous e l ectron dens e  granule s . In con­
tras t , s ince very l i t t l e  condensat ion occurs in mucous c e l l s ,  
the granules  of  these  cel l s  a r e  larger  and e l ectron lucent . 
The s ecretory granule s  thus formed are s tored  in the ap i cal  
cytoplasm  and e i ther d i scharged  into the  lumen or  if  no 
s t imulat ion occurs , degraded by lyso somal enzymes  (Young 
and van L ennep , 1 9 7 8 ) . 
Discharge o f  s erous and s eromucous granul e s  occurs by 
exocytos i s  (Palade , 1 9 7 5 )  where the membrane o f  the secret ­
ory granule fus e s  with the apical  p l a sma membrane bordering 
the c entral  lumen or an int� rce l lular  s ecretory canal iculus 
of an endpiece . The fus ion of  the two membranes i s  fo l lowed 
by perfo rat ion which allows the granule  contents to es cape 
into the lumen without dis rupt ion •o f  the integrity of the 
plasma membrane or loss  o f  cytoplasm . In  sal ivary glands 
cel l s  chain exocytos i s  is a common feature . This invo lves 
fus ion of membranes of a who le  row of granule s  each act ing 
as an extens ion o f  the lumen (Young and van Lennep , 1 9 7 8 ) . 
After  exocytos i s  surface membrane i s  recovered  and re turned 
to the Golgi  c i s t e rnae , p re sumab ly to be reut i l i zed  for 
newly synthe s i s ed  s ecretory products  (Palade , 1 9 7 5 ) . How­
eve r ,  the preci s e  pathways t aken by surface membrane to  
reach the  Gol g i  complex rema ins to b e  e s tab l i shed . The 
availab l e  evidence (Herzog , 1 9 8 1 )  indicates a l ikel ihood  
that both  a direct  route from surface membrane to Go lgi  
complex  and an indirect route invo lving lysosomes , may be  
present in  different cel l  types  for  cel l  membrane turnover . 
The mechani sm of  d i scharge  of  typical mucous granules  
appears to di ffer from that of  pro teos erous g ranules  
(Young and van Lennep , 1 9 7 8 ) . Although exi s t ing evidence i s  
far from conc lus ive , Kim , Nas j l e t i  and Han ( 1 9 7 2 )  have 
suggested  that ap ical mucous granules  upon s t imulation fus e 
to form a large  mucous droplet , whi ch when discharged 
through a wide gap in the ap ical plasma membrane l eaves an 
open space  wi thin the cel l . The mucous droplet  was found 
34 
to carry port ions o f  p lasma membrane with it , inc luding i t s  
own outer  membrane . Thi s  l o s s  o f  integrity o f  the c e l l  mem­
brane during exocytosi s  i s  in sharp contrast  to  proteo s erous 
c e l l s  which are b e l ieved to  recycle  the  membranes of  s ecre t ­
ory granules . Mucous cel l s  are thus p robably capab l e  o f  
regene rating a new c e l l  membrane with  the a i d  of  nume rous 
cytopl asmic ves i c l e s  around the intracel lular space . How­
eve r ,  Tandler  and Poul s en ( 1 9 76 )  have indicated  that mucous 
granul e s  are normal ly discharged by a process  of  exocytos i s  
s imi lar  t o  that ob s erved in proteos erous cel l s . I t  i s  
therefore unclear  whether the obs e rved difference re flects  
variat ions  in the  mechanism  of  exocyt o s i s  or whether earl i er  
ob servat ions were due t o  f ixat ion art i facts . 
The above de s cript ion of  synthes i s  and s ecret ion o f  
prote ins  and glycoproteins has mainly concentrated  on  pro t eo ­
s erous and mucous ce l l s . The synthetic  and s ecretory mech­
ani sms  for  other types  of  c e l l s  appear  to vary (Palade , 1 9 7 5 ) . 
In  p l a sma cel l s , for  example , the concentrat ion s tep has 
not b een ident i fi ed , intracellular  s to rage i s  reduced  in  
durat ion or el iminated and discharg e o f  s ecretory material  
seems to  occur continuou s ly . Addit ional ly the equival ent 
of  a s ecretory g ranule  has  not yet  b e en identified  ( s e e  
Palade , 1 9 7 5 ) . 
1 . 3 . 6  Compos i tion of Bovine Sal ivary Mucosub s tances  
Sa l ivary gland  mucosub s tances a re  biochemical ly and 
his tochemically s imilar  to tho se  derived from gas trointes ­
t inal , resp iratory and reproduct ive t ract ep i thel i a .  
I n  this  s ection ,  the b iochemi cal  aspects  of  mucus wi l l  
b e  reviewed with particular  reference  to bovine s al ivary 
g land  mucous s ecret ions . Histochemical  aspe cts  wi l l  b e  
examined in Chapter  2 .  
Ep ithe l ial mucous s ecret ions con s i s t  mainly of g lyco -
3 5  
pro teins , compounds with a polyp ep t ide backbone and a t tached  
carbohydrate re s i dues which may b e  e i ther ac idic o r  neutral  
( Shreeve , 1 9 7 4 ) . Of the amino a c ids ident ified in the pro t ­
ein  core , s e r ine and threonine repres ent more than S O %  of 
the total amino acid  compo s i t ion ,  other amino ac ids that 
occur in apprec iable  amounts  inc lude alanine , g lycine and 
prol ine (Herp , Wu and Maschera , 1 9 79 ) . Herp and his  a s soc ­
iates  have a l s o  reported  that the predominant carbohydrate  
group s that occur in g lycopro teins  are s ial ic ac id  and 
N- acetyl galac to samine , with fucos e , galactose  and mannose  
occas ional ly p re sent in  variab l e  p roport ions . The carbo­
hydrate s ide chains are  j o ined by  0 - glycosidi c bonds to  
hydroxyl res idues o f  threonine and  s erine , via  an  inte r ­
mediary compound , N - acetylgalactosamine , while  the i r  t er­
minal non- re ducing po s i t ions are  a lways occup ied by s ia l ic  
acid  and fucos e  res idues . S ial ic  ac id  in  part icular imparts  
cons iderab l e  v i s cos i ty to sal iva b ecause  the mutual repul ­
s ion of carboxyl group s s t iffens the random co i l  o f  the 
glycoprotein molecule ( Shreeve , 1 9 74 ) . Glycopro teins  are 
a l s o  thought to be  sulphated (Pamer ,  Jerzy Glas and Horowit z , 
1 9 6 8 ) ; such sulphated g lycoprote ins have a high val ine con­
tent in the p rotein core but the exact nature of  the sulph ­
a t e  res idue or  i t s  chemical comb inat ion with the p ro t e in 
core  is  poorly  unde rs tood .  
According t o  Shreeve ( 1 9 7 4 )  o ther properties  o f  g lyco ­
proteins are : -
( i )  frequent branching o f  s ide chains 
( i i )  there are few or  no repeat ing units  pres ent 
( i i i )  the monos accharide res idues number  about 2 5  o r  
l e s s . 
Apart from g lycoprote ins s ome  acid  mucopolysaccharide s  
may also  b e  p re s ent in ep ithe l ia l  mucosubstances . However ,  
acid  mucopolysaccharide s  are predominantly loca l i s ed  in 
connective t i s sue e l ements and cons i s t  mainly of  chondroi t in 
sulphates and hyaluronic  ac id  re s idues on a polypept i de 
backbone . I n  contras t to glycoprote ins , acid mucopolysac­
charides  contain a smal ler  polypeptide component and the 
3 6  
s ide chains are l inear  with l i ttle  o r  n o  branching (Shreeve , 
1 9 7 4 ) . However , i t  i s  not unl ikely that ep i thel ial  muco ­
s ub s tances  may cons i s t  of  a p rotein core  which could carry 
a mixture o f  carbohydrate res i dues of  g lycoproteins and 
ac id  mucopolys acchari des . Therefore , in general , use  o f  
t h e  term mucosubs tanc e when re ferring to  ep ithe l ial  s ecret ­
i ons  indicates  the pre sence o f  both glycoproteins  as  we l l  
a s  some acid  mucopolysaccharides . The preference for the 
t e rm "mucosubstance" ins tead of glycoproteins or  mucopoly­
s accharides  wi l l  be  d i scus sed  in Chapter  2 .  
I n  bovine sal ivary s ecret ions the mucosubs tances  and 
proteins ident i fied  have been clas s i fied  into three  g roups : 
( i )  bovine sal ivary mucoprote in (BSM) , ( i i )  o e sophageal  
mucoprotein  (OSM) , and ( i i i )  low mol ecular we ight  prote in 
( LMP ) (Jones ,  Gurns ey , B irt le s  and Re i d ,  1 9 7 7 ) . The h igh 
mol ecular  weight , BSM and OSM fract ions (Tab l e  1 . 2 ) are 
g lycoprot e ins with protein content s of 6 0 %  w/w and 3 0 %  w/w 
r e spectively , of which  69 % and 7 5 %  of the protein cons i s ts 
o f  val ine , threonine , s erine , alanine , g lycine and prol ine . 
OSM has an unusually  h igh val ine content ( 2 1 % ) . The maj or 
c arbohydrate  components  ident ified were s ial ic  ac id  and 
amino sugars . Herp e t  al . ( 1 9 7 9 )  further  ident i fied  e i ght 
di fferent derivatives  of  s ial ic  ac id in bovine sa l ivary 
mucoprotein with N - acetylneuraminic aci d ,  N - glycolylneur­
aminic acid  and N - acetyl , 9 - o- acetylneuramini c ac i d  as  the 
p redominant types .  OSM is s e creted by the sub l ingual , 
intermediate  and dors al buccals , palat ine and pharyngeal  
g l ands , whi le  BSM i s  s ecreted  by the mandibular  g lands . 
The l ow molecula r  we ight protein  fraction (Table  1 . 1 ) 
has  b een s eparated into twe lve component s  or  bands (Jones , 
B roadhurs t and Gurnsey , 1 9 8 2 ) . The twelve proteins  have 
b een numbered in order of the ir  e lectrophoretic  mob i l i ty .  
O f  the twelve proteins  the maj or i ty were pres ent in s ecre t ­
i ons produced by the parot i d ,  ventral and intermediate  
buccal g lands , with  b ands 4 and 1 0  predominant in  parotid  
and ventral  buccal s a l iva . Band 3 and band 8 have been 
i dent i fied  in mandibular sal iva whi le  band 9 was found to be 
Tab l e  1 .  2 BOVINE SAL IVARY PROTE INS I I  
(High Mol e cular  We ight Fraction)  
BSM Fract ion OSM Fract ion 
Amino Aci d  comp . % niole .  % comp . % mo le . % 
Glycine 1 8 . 2  2 3  6 . 7 8 
Val ine 8 . 4  1 0  2 1 . 0  2 6  
Serine 1 5 . 5 2 0  1 0 . 2 8 1 3  
Threonine 6 . 0  7 1 8 . 3 2 8  
Alanine 1 3 . 3 6 1 0 . 9  1 4  
P ro l ine 8 . 5 3 1 1  8 . 5 4 1 1  
Lys ine 1 . 6  2 1 .  2 3  1 
H i s t idine 0 . 8 5 1 0 . 8 6 1 
I s oleucine 2 . 9  4 1 .  3 5  2 
Tyros ine 1 . 0  1 1 . 1 3  2 
Phenylalan ine 1 . 3  2 1 . 1 3 2 
Arginine 4 . 5 6 4 . 5  6 
Aspart ic 4 . 5  6 3 . 6 8 4 
G lutamic 7 . 4  8 6 . 7 8 
Sec reted  by the Secreted by the 
mandibular g lands , subl ingua l and 
with 6 0 %  w/w mino r glands , with 
protein and 3 0 %  w/w protein 
2 0 %  w/w s ial ic  and 2 0 %  w/w s ialic  
ac i d .  aci d .  
(Source : Dr . W . T .  Jones , D . S . I . R . ,  Pa lme rs ton 
North , New Z ealand . ) 
pre s ent i n  subl ingual s al iva . Bands 2 and 7 were  further  
clas s if ied  as  extrins ic prote ins becaus e they cross  reacted  
with ant ibodies  ra i sed agains t bovine s erum . Bovine s a l iv­
ary bands 1 ,  2 , 3 ,  5 and 6 were  found  to  b�  g lycoprote ins 
whi le  the remainder were c la s s i fied  as  pept ides . 
3 7  
1 . 4  Contro l o f  Sa l i vary Secret ion 
Sal iva i s  usual ly secreted  in  re spons e to s t imul ation 
from autonomic innervation of  the g lands or by pharmacol ­
o gical  agents  mimicking the act ion o f  autonomic  neurotrans ­
mitters (Emmel in ,  1 9 6 7 ) . Sal ivary gland endpieces  and 
ducts  are innervated  by parasympathetic  and sympathetic  
s ecretomotor  fibres  but the parotid  g lands of  sheep  and 
cattle  exhibit  a spontaneous s e cret ion (Kay , 1 9 6 0 )  that 
p ers i s ts in  the ab s ence of neuronal and humoral s t imul i 
(Babkin , 1 9 5 0 ) . 
In  the p roduction and flow o f  s a l iva parasympathet ic  
3 8  
and sympathetic  nerves  general ly cooperate , a l though , par ­
t i cularly in  sympathet ic innervation , variations b e tween the 
di fferent s a l ivary g l ands are  cons iderable . The s ecretomo tor 
nerves in general  init iate the formation of  primary sal iva 
in endpieces  and e ffec t  the ab s o rpt ion and s ecret ion  o f  
certain ions  by the ducts . In  addition they s t imulate  exo ­
cytos is  o f  g ranules  containing macromol ecules such as  glyco ­
proteins from the endpieces  as  we l l  as  ka llikre in from the 
ducts (Emme l in ,  1 9 6 7 ) . 
The compos i t ion of  s a l iva i s  a lso  influenced by the 
nature of the s ecretory nerves . Parasympathet ically evoked 
s a l iva exhib i ts cons i derable  species  variati6n in the excre t -
+ + - -ory patterns of  the maj or  e l e c trolytes , Na , K , C l  , HC03 
and P04 -
- i ons (Young and van L ennep , 1 9 7 9 ) . The durat ion 
and intens i ty of  parasympathet i c  s t imulat ion a l so  a ffects  
the  inorganic  compos i t ion of  s a l iva (Schneyer et  a l . ,  1 9 7 2 ) . 
The inorgan ic  compos i t ion immed iately after s t imula t ion 
differs from that during s teady s tate  s ecretion owing to 
a l terations  in cel lular permeab i l i ty .  An increase  in  inten­
s ity of  the s t imulus changes  s a l ivary flow rate w i th a 
consequent change in e lectro lyte concentrat ions . The s teady 
state  s ecretory l eve l s  reached by mos t  ions are a l s o  highly 
variable  bo th among species  and individual g lands (Young and 
van Lennep , 1 9 7 9 ) . 
3 9  
Sympathet i ca l ly evoked sa l iva has  received compara t ively 
l i t t l e  at tent ion , however , a - sympathetic  receptors when 
s t imulated produce  a respons e s imi lar  to parasympathe t i c  
s a l iva whi le  t h e  S - sympathetic  response  e l ic it s  only a 
sparce  quanti ty of  vis cous sal iva r i ch in organic mater ial  
(Young and van  Lennep , 1 9 7 9 ) . 
The s ecret o ry and absorpt ive processes  o f  the duct s  have 
l ong  been suspected  to be control l e d  by autonomic nerves 
(Schneyer and Ernme l in ,  1 9 7 4 ) . I t  appears that sympathetic  
s t imulat ion usua l ly modi fies  parasympathetic  effects  in  
contro l l ing ductal  e lectrolyte tran sport activity wi th 
l i tt le  effect  on  ductal  water permeabi l i ty (Young and van 
Lennep , 1 9 7 9 ) . 
Myoepithe l ia l  cel l s  which s e rve  to accelerate the 
sa l ivary flow upon contract ion are innervated ma inly by 
sympathetic motor  fibres  that exer t  the ir  e ffect via a ­
receptors ( Garrett  and Emrnel in � 1 9 7 9 ) . However ,  both para­
sympathetic  and  sympathetic  motor nerves appear to  act  in  
cooperat ion to  produce maximal cont ract ion o f  the  myoep ith­
e l i a l  ce l l s  ( Ga rrett  and  Emrnel in ,  1 9 7 9 ) . 
In  contrast  to s ecretory endp i e ce , duct and myoep i thel ­
ial  c e l l s , the c lass ical  antagonism  between parasympathetic  
and  sympathetic  effects  i s  found in  the  innervat ion o f  the 
vas culature to  the s a l ivary glands . The blood  ves s e l s  of  
s a l ivary glands rece ive sympathe t i c  vasocons trictor  nerves 
( act ing on a - re ceptors )  and parasympathetic  vasodilator  
nerves (Emmel in ,  1 9 6 7 ) , both of  whi ch contribute to the  
adaptation o f  the  b lood  flow to  the requirements of  the  
g landular t i s s ue . Blood  ves s e l s  to  the  glands are  addi t ­
ionally control led  by vasodilator  p o lypept ides  such as  
physalaemin and  eledo i s in (Emrnel in , 1 9 7 1 )  and subs tance P 
from bovine hypothalamus (Leeman and Hammerschlag , 1 9 6 7 ) . 
The s e  substanc e s  induce secret ion which cannot  be  inhibited  
by specific parasympathetic  and sympathetic  blockers ( Emrnel i� 
1 9 7 1 ) . Recently , additional po lypept ide neurotransmi t ters  
have been shown to po s s e s s  vasodi la tory act ivity . Thes e  
4 0  
vas oactive inte s t inal  po lypept i de (VIP )  containing ne rve 
fibres  have been i dent i fied  in close  contact with s ecretory 
endp ieces , ducts  and b lood  ves s e l s  (Polak and B loom , 1 9 8 0 ) . 
As a resul t o f  sympathet ic s t imulat ion , s al ivary g land duct 
c e l l s  are reported to release  ka l l ikre in , which could fur ­
ther  modi fy s a l ivary flow rate by cons tr iction o f  arterio l e s, 
d i l a t ion of  venules , increas e in capi l lary permeab i l ity and 
re l ea s e  of prostaglandins and cyclic  GMP (0r s tavik and 
Gautvik ,  1 9 7 7 ) . Duct c e l l s  are addit ionally known to  be 
under  endocrine contro l , particularly from mine ralocort ico i d  
hormones  such as  a ldo s t e rone . I n  ruminant s a l ivary g lands 
aldo s t erone act ion on duct ce l l s  could increase  sodium 
reab s o rpt ion in sodium depleted  sheep (Blair - We s t , Coghlan , 
Denton , Nel s on ,  Wright and Yamauchi ,  1 9 6 9 ) . 
Neural control  o f  s a l ivary s e cret ion in ruminant species  
has b een inves t igated  w ith reference to  the sheep paro t id  
(Coat s , Denton , Goding and Wright , 1 9 5 6 ; Kay , 1 9 5 8 ; 
Pat terson and Titchen , 1 9 7 9 )  and mandibular (Ar iyakulkaln , 
1 9 8 1 )  g l ands . Prel iminary finding s of  parasympathet ic  and 
sympathetic  e ffects  on bovine parotid  and mandibular  g lands 
have been reported by Phi l l ipson and Mangan , �9 5 9 ) . 
The innervation o f  p arasympathetic  secretomotor  nerves  
to  the sheep parotid  g l and , produces sal iva rich in  inorg ­
anic  ions , water and s ome  protein (Young and van L ennep , 
1 9 7 9 ) . The sympathet ic  re spons e o f  the sheep parot id  has 
been previous ly inves t igated  with reference to  an a - symp ­
athet ic motor  e ffec t  caus ing a mechanical  expuls ion of  
s a l iva from the paro t id  g land (Coats  et  a l . ,  1 9 5 6 ) . How­
ever , more recent ly an addit ional S - sympathetic  s ecretory 
component has  been repo rted  for the sheep parotid  by 
Patterson and Ti tchen ( 1 9 7 9 ) . Thi s  was shown to  cause  a 
net  increase in the amount o f  fluid  formed accompanied by 
a s i gni ficant e levat ion in the protein content of  paro t i d  
s a l iva . Pat terson et  a l . ( 1 9 8 2 )  extended the above findings 
and concluded  that pro t e in concentrat ions o f  paro t id  sa l iva 
were increased  mainly upon feeding and waned rap idly after  
feeding . However i t  was  not es tab l i shed whe ther para-
sympathetic  a s  we l l  a s  sympathe t i c  influences contribute 
to the increa s e  in pro t e in s ecre t ion  obs erve d on commence ­
ment o f  fee ding . Pat t erson e t  a l . ( 1 9 8 2 )  addi tiona l ly 
+ 
reported  on an increase  in K , C l  and Mg ions during  
sympathet ic innervation o f  the  paro t id  g lands , with an 
unexplained p o s i t ive correlat ion b e tween magnes ium and 
proteiri concentrat ions in paro t id  s a l iva . 
4 1  
Neural control o f  the sheep mandibular  g land has b een 
rec ently inves t igated by Ariyakulkaln ( 1 9 81 ) . The mandib ­
ular  g land mucous cel l s  were demons trated  to be  predominantly 
under  parasympathet ic  control  whi l e  the demi lunes we re under 
sympathetic  control . Furthermore , the mandibular s a l ivary 
protein secret ion appeared to  be p rimari ly mediated  via  a 
a -adrenergi c  mechanism  and the s ecretion of water , e l ectro ­
lytes  and mucus appeared  to be  under parasympathet ic  inne r ­
vat ion . 
In  addit ion to parasympathe t i c  and sympathet ic  e ffect s , 
i t  is  we ll e s tabli shed  that s al ivary s ecret ion in ruminants  
is  influenced  by re fl ex control  (Coats  et  al . ,  1 9 5 6 ; Kay , 
1 9 5 8 ; Ash and Kay , 1 9 5 9 ; Kay and Phi l l ipson , 1 9 5 9 ) . The 
receptors respons ib l e  fo r re flex control are found in the 
oral  cavity , o esophagus , rumen and ret iculum . The se  recep ­
tors  s t imulate  or inhibit  sal ivary s ecret ion during feeding 
and ruminat ion and are effect ive especially for fibrous 
d iets (Kay , 1 9 66 ) . The various example s  cited above s e rve 
to  i l lus trate  the pos s ib i l i t ie s  that may exis t  which could 
vary the flow rate  and compos it ion  of  s a l iva at  the  neuro ­
e ffector l eve l . 
1 . 5 Mucosal  Immunity 
Within the l as t  two decades  cons i derable a t t ent ion has 
been focus s ed  on immune mechanisms of mucosal s ur faces  
s ince the s e  are  int imate ly a s sociated  with potent ia l ly 
pathogen i c  organ i sms and cont inuous ly expos ed  to  ant igenic  
s t imula t ion from environmental  and dietary ant ig ens . The 
mucosa  o f  the oral  cavity in part icular is  as s o c iated  with 
such s t imul i .  
The s urfaces  o f  a l l  mucous membranes are p r imarily  pro ­
tected by a layer  of  mucus which i s  effective a ga inst  
attack from microorganisms . Mucous s ecretions may contain  
attachment s i t e s  for  pathogenic  bacteria  that phys ica l ly 
entrap and immobo l i s e  the o rganism .and prevent infection or  
penetrat ion of  underlying ep i the l ial  cells  (L indley , 1 9 8 0 ) . 
The protective role  o f  mucus is  addi tionally augmented  by 
the pres ence of ant ibacterial  prote ins such a s  lyso zyme and 
lactoferrin . Lys o zyme , for example , interact s  with s ia l i c  
ac id  res i dues o f  the mucus l ayer and agains t meuraminic 
ac id  containing bacterial  c e l l  wal l s  (Creeth , Bridge  and 
Horton , 1 9 7 9 ) . Lactoferrin complexes  iron (an e s s ential 
mineral  for microorgani sms ) present in s ecre t ions , thereby 
denying i t  to the organism ( Emery , 1 9 8 0 ) . Ruminant sa l iva 
is  part i cularly r ich in mucus (Jones et al . , 1 9 7 7 ) , and its  
protect ive functions may extend beyond the oral  cavity to  
the ruminant fores tomach whi ch lacks mucus producing ce l l s  
and immunoglobulin  synthe s i s ing p lasma cells  a s  ment ioned 
in Section 1 .  
4 2  
Humoral  immuni ty at mucosal surfaces is mediated  prim­
ari ly by immunog lobul ins such as s ecretory I gA ,  I g G , I gG 2 , 
I gM and I gE (Toma s i , 1 9 7 6 ) . The maj or  immunog lobul in in 
bovine s al iva is S i gA (Mach and Pahud , 1 9 7 1 )  which is com­
posed  of two monomers ( 7S )  j o ined by a polypep t i de (J chain) 
to produce dimeric  ( l l S )  I gA ;  the l atter  mo lecule  is  bound 
to a g lycoprote in called  s ecretory component ( SC ) , synth­
es ised  by epi the l ial  ce l l s  (Tomas i ,  1 9 7 6 ) . Pl asma c e l l s  of  
sal ivary gland s t roma produce  the  dimeric I gA and the  J 
chain , whi le  the endpiece  duct c e l l s  are thought to  produce 
the s ecretory component (Young and van Lennep , 1 9 7 8 )' .  I t  
i s  l ike ly that the b inding o f  the SC with dimeric  ( l lS )  I gA 
i s  e s s ential  for trans ce l l ul ar transport of  s i gA from sub ­
ep ithel ial  s i tes  t o  the duct lumen . 
4 3  
The presence o f  p lasma c e l l s  di fferentiate d  to produce 
s i gA from sal ivary glands , without evidence of any p recursor  
c e l l s  s ugge sts  that b la s t  c e l l s  des t ined for synthes i s  of  
dimeric  I gA are  primed e l s ewhere then migrate to  sal ivary 
gland t i s sues  and different iate  into plasma c e l l s  (Tomas i ,  
Larson , Challacombe and McNabb , 1 9 8 0 ) . The s i t e  o r  s i tes  
of  or ig ins of  b l a s t  cel l s  p rogrammed to  reach s al ivary g land 
t i s sue and the ant igen recognition s i tes  in s a l ivary g l ands 
are unknown . However ,  it appears  that a common mucosal  
assoc iated  lympho id  t i s sue  sys tem (MALT) whi ch inc ludes  the 
gut , lung , mammary g lands , sal ivary and lacrimal  g lands 
could be involved in the t ransport of  precur sor  ce l l s  of  · 
s i gA among each of  the s ecretory s ites  (Tomas i et  al . ,  1 9 8 0 ) .  
Thi s  may enhance  pro l i fe ra t ion and re tention of  precurso�  
cells  l ocal ly in s i tes  such as  the  sal ivary g l ands (Bienen­
s tock , Befus and McDermo t t , 1 9 8 1 ) . 
The effector  funct ions of  s i gA are vari ed  depending on 
the nature of the ant igen ( Dobbins , 1 9 8 2 ) . I n  general , the 
clos e a s sociat ion between s i gA and mucus (Clamp , 1 9 8 1 )  can 
interfe re with the adhes ion  of  organisms to ep i thel ial  
surface s  and p revent the i r  penetration into underlying 
t i s sue . 
I n  ruminants , a di s t inction has been made between s i gA 
l eve l s  i solated  from parot id and non - parotid  s a l iva (Watson 
and Lascel les , 1 9 7 1 ; 1 9 7 3 ) . I t  s eems that in  sheep the man ­
dibular  glands s ecrete  very high concentrations  of  s i gA 
compared  with the parot ids  (Cripps  and Lasce l l es , 1 9 7 6 ) . 
H i sto log ical s tudies have shown a relative abs ence of  p lasma 
cel l s  from parotid  t i s sue when compared with the mandibular . 
Watson  and Las celles  ( 1 9 7 3 )  have further indicated  that 
paro t id  sal iva owing to i t s  greater  total s a l ivary volume 
4 4  
c ould  probably act  as  a di luent of  I gA s ec reted  by  the 
mandibular and po s s ib ly  the sub l ingual g l ands . The contin­
uous flow of  large  quant it ie s  o f  parot id  sa l iva coul d ensure 
that  the g land rece ive s relat ively l i t t l e  local ant igenic  
s t imulat ion (Watson and Lascel l e s , 1 9 7 3 ) . 
In  the p res ent s tudy ,  a his tological  examination was 
conducted  t o  identify those  s a l ivary g lands o f  cat t l e  which 
represent the maj or  contributors  of  sa l ivary s i gA .  
In  addit ion t o  s i gA ,  I gG ,  I gG 2 , and I gM have been 
i dent i fied  in s igni fi cant concentrat ions in ruminant s a l iva 
(Tab l e  1 . 3 ) (Watson and Lascel l e s , 1 9 7 3 ) . The l evel s  o f  
I g G , I gG2 and I gM were 3 . 5  - 5 . 0  t imes  h i gher in non -parot id  
s a l iva . I gG was reported  as  b e ing s e lect ive ly t rans ferred 
across  the g landular ep ithe l ium with some local  �ynthe s i s  
whi l e  mos t  o f  the I gG 2 and I gM were derived from plasma with 
minimal local  synthes i s . The s ecre t ion o f  I gM appears  to  b e  
fac i l i tated  by comp l ex ing with J - chain conta ining po lymers 
and membrane bound s ecretory component as  a prerequi s i t e  for 
ep i the l ial  trans fer ;  the mechani sm i s  anal ogous to transport  
of  s i gA (Tomas i et  a l . , 1 9 8 0 ) . I gE ,  a l though de scribed in 
external s ecret ions (Tomas i ,  1 9 7 6 )  has no t been quantitat ­
ive ly a s sayed  in  ruminant sal iva (But l er , 1 9 8 1 ) . The abso l ­
ute  concentrat ion of  I gE in s a l iva i s  very smal l , however 
the rat io of sal ivary to  s erum I gE i s  high (Tomas i ,  1 9 7 6 ) . 
The rol e o f  I gE in immediate  hypersens i t ivity react ions i s  
wel l  documented (Strob e r ,  1 9 8 2 ) , thus i t  is  tempting to 
p o s tulate  that sa l ivary I gE may be  involved in poorly­
unders tood mucosal  a l l ergic  react ions . A po s s ible  transport  
mechani sm ( i f  any) for  s ecretory I gE at  mucosal  surfaces  
has  not been inve s t igated , a l though Mayrhofer , Ba z in and 
Gowans ( 1 9 7 6 )  have s ug ges ted  that in the rat , s ecret ion o f  
I g E  may invo lve intraepithel ial  mas t ce l l s  ( o r  " g lobule  
l eucocytes " )  which were  found to contain  intracytoplasmic 
I gE .  
Studie s on mucos a l  immuni ty thus far have largely 
focus sed on humoral  immune respons e s  in i t iated by B - lympho -
Table  1 . 3  
Species  
Cattle  
Sheep 
Goat  
IMMUNOGLOBUL IN  COMPOS I T I ON OF ADULT 
RUMINANT SAL IVA (mg/ 1 0 0  ml ) 
I gA 
5 6  
2 0  
2 0  
I gG 
? 
1 0  
1 0  
3 
? 
? 
I gG 2 I gM 
1 1 
? trace 
? t race 
from But ler  ( 1 9 8 1 ) . 
I gE 
? 
? 
? 
cyte  act ivated cel l - mediated  immuni ty a t  mucosal  s ites . 
Whi l s t  the factors  contro l l ing migrat ion and l ocal i s ­
a t ion o f  T -b las t c e l l s  in mucosal  t i s sue s are unknown , i t  
4 5  
has  been reported  by Guy - Grand , Gr i s ce l l i  and Vas s al i  ( 1 9 7 8 )  
that they are derive d from lymph nodes and subs equent ly 
migrate  to  the muco s a  where  mos t  can l a ter  be  identified  as 
intraepi the l ial  lymphocytes . Subpopulat ions of the se  c e l l s  
a r e  known to  contain cytoplasmic granu l e s  wi th s taining 
character i s tics  s im i l ar to mas t  ce l l s  ( Rudzik and Bienenstock , 
1 9 7 4 ;  Mayrhofer ,  1 9 8 0 ) . Exi s t ing evidence reviewed by 
Mowat and Fergus son ( 1 9 8 1 )  s ugges ts  that mucosal  cel l ­
mediated  immuni ty involve s primarily  T - suppressor  and 
T - cytotoxic  ce l l s  in the ep i thel ium .  The  suppressor  c e l l s  
may protect  the ep ithel ium from immune mediated damage 
caused by cont inuous ant igen- ant ibody and ant igen - cell  
interactions while  the  cytotoxic cel l s  c ould provide a 
f i rs t  l ine of  cel l -mediated de fence when a group of  ep i ­
the l ial  c e l l s  are de s troyed by a pathogen . The T - helper  
cells  located mainly at  subep ithel ial  s i tes  may be  respons ­
ible  for produc ing lymphokine s ,  after  interaction with 
antigens . 
Inve s t igat ions p ertaining to the ro l e  of  sub ep ithe l ial  
o r  mucos al mas t  cel l s  in  immonoregulat ion at  mucosal  surfarei 
are  only at a pre l iminary s tage , however exi s t ing evidence 
s uggests  that the se  c e l l s  are probably derived from a 
l ymphocyte  subpopulat ion and are funct ionally impl icated  in 
inflammatory respons e s  of  the gut (Cas tro , 1 9 8 2  ; Haig , 
Mckee , Jarrett , Woodbury and Mi ller , 1 9 8 2 ) . 
C e l l -mediated immunity in sal ivary g l and t i s sue seems 
to have been totally  unexplored .  However ,  the findings 
de scribed above regarding intestinal CM! may apply to o ther  
mucos al s i tes . In  ruminants , the lack  of  B or  T cells  or  
their  precursors  in the  fores tomachs , h i ghl ights  the  probab l e  
importance o f  the p ro tect ive funct ions o f  sal ivary gland 
lympho id cells  and s ecret ions in the s e  a reas . In  the pre s ent  
s tudy an  intraepithel ial cell  of  unknown ident ity was 
located in the duct s  of  the parotid  and ventral  buccal 
g lands . One pos s ib i l i ty is  that this  cel l  l ine may be  
invo lved in  poorly unders tood  mucosal  immune react ions . 
4 6  
1 . 6  Sal ivary Secretions  and Bloat  Suscept ibi l ity 
B loat i s  a disorder of  ruminants caused  by a failure 
4 7  
to  s ucce s s fu l ly e ructate  gases  p roduced  in  the ret iculorumen 
during fermentat ive d igest ion . The retention of gas together 
with the format ions of  a s t able  foam , mechanica l ly interferes  
wi th respi rat ion and can b e  fat al wi thin an hour of  commen ­
cement o f  feeding (Clarke and Rei d ,  1 9 7 4 ) . 
Evi dence to  date , reviewed by Cl arke and Rei d  ( 1 9 7 4 )  
suggests  that bloat  i s  l ike ly t o  b e  a comp l ex mul t ivariate  
d i sorder wi th interact i ons of  environmental , p l ant , animal  
and microbi a l  factors . However ,  recent inves t igat ions have 
focus sed  a ttention mainly on sa l iva and i ts as s oc iat ion 
w ith b loat ·  s usceptib i l i ty in cattle  (Re i d ,  Gurns ey , Waghorn 
and Jones , 1 9 7 5 ; Gurnsey , Jone s and Re i d ,  1 9 7 6 ; Jone s et  al . ,  
1 9 7 7 ) . 
Animal s  that are g ene t ically predi sposed  to  b loat  and 
manife s t  a high suscep t ib i l ity (H . S . ) produce s i gnificantly 
l e s s  s al iva than animal s of low bloat susceptibi l i ty (L . S . ) 
(Mc into sh ,  1 9 7 5 ) . Studies  on the weights  of  individual 
s a l ivary g l ands indicate  that L . S . animal s have a higher 
we i ght of  s ecretory t i s sue , thereby contr ibut ing to  an 
inc reased flow rate  ( Gurnsey , Re i d ,  Jones and B irtles , 1 9 7 7 ). 
Biochemical analys i s  of  sal iva s ecreted by H . S .  and L . S .  
cattle  have demons trated  that the l ow molecular weight 
p rotein ,  b and 4 ,  i so lated  from parot id s a l iva , has been 
co rre lated  with bloat  suscept ib i l ity (Mc intosh  and Cockrem , 
1 9 7 7 ) . Band 4 content in the reticulo rumen of  H . S .  an imal s  
was  found to  be  s igni ficantly greater  than in  L . S .  animal s .  
I t  is  pos s ib le  that s a l ivary proteins  and mucosubs tances  
could  act  as  surfactants  to promote  the  formation of  a s table 
foam that could reta in the gas e s  produced  during fermentation .  
I n  the pre s ent s tudy , parotid  g land t i s sue from H . S .  and L . S . 
cattle  were  examined immunohi s tochemical ly  to obs e rve the 
d i s tribut i on of secretory ce l l s  re sponsible  for produc ing 
band 4 .  
4 8  
B a r t l ey ( 1 9 7 6 )  has s ug g e s t e d  a p o s s ib l e  i nvo l vement o f  
s a l i v a ry immuno g l o bul ins t o  b lo a t , b u t  at p r e s ent any a s s o c ­
i a t ion b e tween i mmuni ty and b l o at i n  the a b s e n c e  o f  a 
p r e l iminary inve s t i g at ion w ou l d  r ema in hypothe t i ca l . 
F r om hi s t o l o g i ca l  s tu d i e s  o f  p a ro t i d and v e n t r a l  b u c c a l  
g l ands o f  c a t t l e , p re l im ina ry o b s e rv a t ions h a v e  reve a l ed an 
a s s o c i a t ion b e tween the o c cu r r e n c e  o f  int r a s t r i a t e d  duc t  
c e l l s  and b l o a t s us c ep t i b i l i ty (B i rt l e s , 1 9 8 1 ) . I n  L . S .  
an ima l s  the numb e r s  o f  i n t r a s t r i a t e d duc t  c e l l s  obs e rv e d  
w e r e  g re a t e r  t h a n  i n  H . S .  a n ima l s .  I n  the p r e s ent inve s ­
t i g at i on the a b o v e  f in d i ng s w e r e  e x t ende d t o  examine t h e  
int ra s t r i a t e d  duct ce l l s  i n  g r e a t e r  d e t a i l and p o s tu l a t e  
a p o s s ib l e  r e l a t i o nship o f  t h e s e  c e l l s  t o  b l o a t  s u s c ep t i b ­
i l i ty .  
C HAPTER 2 .  BOV I NE SAL I VARY G LAND 
H I S TOLOGY AND H I STOCHEMI S TRY 
2 . 1  I n t r o duc t i on 
I nve s t i g a t i ons by Shac k l e fo r d  and K l appe r ( 1 9 6 2 ) ; 
Quin t a re l l i  ( 1 9 6 3 a ) ; S ha c k l e fo r d  ( 1 9 6 3 ) ; Sha ck l e fo rd and 
Wi l b o rn ( 1 9 6 8 )  and B i r t l e s  ( 1 9 8 1 )  h ave demons t r a t e d  in 
cons i d e r ab l e  de t a i l  the h i s t o l o gy and h i s tochem i s t ry o f  
4 9  
the maj o r  and mino r s a l i v a ry g l ands o f  c a t t l e . The h i s to ­
l o g i c a l  f e a t u re s o f  b o v i n e  s al i v a ry g l and endp i e c e s  and 
duc t s  have b e en de s c r i b e d  in de t a i l  e l s ewh e r e  ( S e c t ion 1 . 2 ) . 
The p u rp o s e  o f  the p r e s ent s tudy ha s b e en t o  r e - examine 
and e x t end p r e v i ous f i n d i n g s  wh e re ne c e s s ary and d e t e rm i n e  
t h e  h i s tochem i c a l  cha r a c t e r i s t i c s  o f  b o v ine s a l i v a ry g l and 
s e cr e t o ry p r o duc ts w i th the a i d  of h i s t o l o g i c a l  s t a ins and 
en zyme b l o c k i ng metho ds . Howev e r , s ince the c l as s i f i c a t i on 
o f  ep i the l i a l  mu cosub s tanc e s  rema ins cont rove r s i a l  ( Re i d  and 
C l amp , 1 9 7 8 )  owing to c o n fus i on b e tween the u s a g e  o f  b i o ­
chem i c a l  and h i s to chem i c a l  t e rm i no l o gy , a s u i t ab l e  c l a s s i f ­
i c a t i on sys t em w i l l  b e  i n t r o du c e d  w i th cons i d e ra t i on t o  
b o t h  d i s c ip l i ne s .  
E p i t h e l i a l  s ec r e t o ry p ro duc t s , gene ra l ly r e fe r r e d  t o  a s  
muc u s , h a v e  b e en t r ad i t i o na l l y d e s igna t e d  as e i t h e r  g l y c o ­
p ro t e ins o r  a c i d  mucop o l ys a c cha r i de s . Howev e r , mucus o f  
ep i th e l i a l  o r i g in ,  cons i s t s p r e dom i nan t l y  o f  a c i d i c  and 
neu t r a l g l ycop ro t e ins w i t h an i n s i gn i f i c ant muc op o l ys a c ­
c h a r i de cont ent (Youn g  and v an L ennep , 1 9 7 8 ; Re i d  and C l amp , 
1 9 7 8 ) . 
The b i o ch emi s t ry o f  g lycop r o t e ins has b e e n  p r e v i o u s l y  
de s c r ib e d  in S e c t i on 1 . 3 . 6 .  E s s en t i a l ly the a c i d i c  n a t u r e  
o f  t h i s  comp ound i s  d u e  t o  i t s h i g h  s i a l i c a c i d  content , 
c a rb o xy l  g ro up s  o f  non - s i a l i c  a c i d  o r i g in and sulpha t e  
g r oup s . An a c i d  g l ycop r o t e in t h e r e f ore has a n  ex c e s s  o f  
n e g a t i ve cha r g e s  wh i l e  a neut r a l  g l ycop r o t e in has 
p r e do m inant ly p o s i t i v e  c h a r g e s  f r om f r e e  amino g roup s 
( S h r e e v e , 1 9 7 4 ) . 
I n  cont r a s t t o  g l yc op r o t e in s , a c i d  muc opo l y s accha r i d e s  
c o n s i s t  o f  a p o l ypep t i de c o r e  and cons i d e r ab l e  amoun t s  o f  
r ep e a t ing po l y s accha r i de uni t s  w i th l i t t l e  o r  no b ranch ing 
so 
o f  s i d e  chains , the mono s accha r i de c ontent b e ing cons i de ra b l y  
h i g h e r  ( S O  un i t s o r  mo r e  f o r  a g i ven mo l ecul e )  than t h a t  o f  
g ly c o p r o t e ins ( Re i d  a n d  C l amp , 1 9 7 8 ) . The mono s accha r i de s  
usua l ly i dent i f i e d  a r e  hya luron i c  a c i ds and d e r iv a t ive s o f  
chon d r o i t in s ul p hat e . The s e  two g r oup s a r e  ma i n l y  r e s p o n ­
s ib l e  f o r  t h e  h i ghly a c i d i c  natur e o f  mucopo l y s a c char i d e s . 
Unl i k e  g l ycop r o t e ins wh i c h  a re ma i n l y  found i n  b o dy f l u i d s  
and e p i the l i a l  s e c r e t i o n s , t h e  a c i d  mucopo lys a c c ha r i de s  a r e  
p r e do m inan t l y  d i s t r ibut e d  in b on e , c ar t i l ag e  and conn e c t ive 
t i s su e  g round s ub s t anc e s  ( Ham and C o rmack , 1 9 7 9 ) . . I n  
g en e ra l , mucu s , conn e c t i v e  t i s sue g r ound s ub s t anc e and 
s ke l e t a l  s t ructur e s  can b e  compo s e d  o f  a mixture o f  b o t h  
g l ycop r o t e ins and muc op o l y s a c cha r i d e s  w i th t h e  l a t t e r  m a i n l y  
found i n  conn e c t ive t i s s u e  e l emen t s , conj ug a t e d  t o  sma l l  
quan t i t i e s  o f  g lyc op r o t e i n s  (Ham and C o rmack , 1 9 7 9 )  wh e r e a s  
t h e  f o rmer a s  i dent i f i e d  i n  mucus , i s  l ik e l y  t o  cont a i n  v e ry 
sma l l  quant i t i e s  o f  muc o p o lys acch a r i d e s  such a s  uron i c  a c i d  
(Young and van L ennep , 1 9 7 8 ) . 
The d i f f e r ence o f  op i n i o n  b e tw e e n  b i ochem i c a l  and h i s to ­
chem i c a l  c l as s i fi c a t i on o f  ep i t h e l i a l  muco sub s t anc e s  p r im ­
a r i ly s t em f r om an inab i l i ty t o  d emons t r a t e  h i s t o chem i c a l l y 
that c arbohy d r a t e  and p r o t e i n  cons t i tuen t s  o f  mo l e cul e s  such 
a s  g lycop rot e i n s  and muc o p o l y s a c cha r i de s , b e l ong t o  the s ame 
or d i f fe r ent mo l e cu l e s . The h i s t o chemi c a l  f indings a r e  
g en e r a l ly a t t r i b ut e d  t o  r e a c t i v e  g roup s  p r e s ent i n  s i d e  
cha in s  o f  mono s accha r i d e  o r  o l i g o s accha r i de un i t s , f o r  
examp l e  glyc o l  g roup s , c a rb o xy l i c  and sulph a t e  g roup s . 
H i s t o c hemi s t s  c ommonly u s e a var i e ty o f  bas i c  dye s  t h a t  
r e a c t  with ac i d  ra d i c l e s  a n d  chem i c a l  react i on s  s p e c i f i c  fo r 
neu t r a l  g roup s to d i s t ingui s h  b e tween the two s i de chains 
a s  they oc cur in g lycop r o t e i ns and muc op o l ys accha r i de s . Add ­
i t i on a l  info rm a t i on c an b e  d e r i v e d  f rom enzyme hyd ro l ys i s  o r  
b l o c k i n g  metho d s  wh i ch r emov e sp e c i f i c  r e a c t ive g ro up s  
( C u l l i ng ,  1 9 7 4 ) . H i s t o c h em i c a l  r e s u l t s  a r e  thus c l a s s i f i e d  
ac c o r d ing to whe th e r  an ac i di c  o r  n e u t r a l  g r o up h a s  b e e n  
s ta i ne d .  
Howeve r ,  i t  i s  i napp r op r i a t e  f o r h i s t o ch emi s t s  t o  
c o n t inue t o  u s e the t e rms g lycop r o t e i ns and muc op o l y s a c ­
cha r i d e s  s omewh a t  a rb i t ra r i l y  whe n  de s c r ib ing the n a t u r e  
o f  a s i de g r o up , a s  th e s e  two c ompo unds a r e  b i o chemi c a l l y 
d i f f e r ent . A m o r e  app r op r i a t e  fo rm o f  t e rmino l o gy wo u l d  
b e  t o  adopt t h e  t e rms " ac i d g lycop r o t e in" and "neu t r a l  
g l y co p r o t e in" when de s c r i b ing muc o s ub s t anc e s  o f  a n  e p i th ­
e l i a l  o r i g in , a s  s u g g e s t e d  b y  Young and van Lennep ( 1 9 7 8 ) . 
I de a l l y , t h e  t e rms ac i d  and neu t r a l  muc o s ub s tanc e s  wou l d  b e  
even mo r e  app r o p r i a t e , s ince t h e s e  wou l d  incl ude any a c i d  
m�c op o lys a c c h a r i de s p r e s ent ( a l b e i t  i n  sma l l  amoun t s )  i n  
ep i th e l i a l  muc o u s  s e c re t i ons and s i d e  g roup s wh i ch c anno t  
b e  d e t e c t e d  b y  ex i s t in g  s t a in s  a n d  h i s t o c h emi c a l  m e th o d s . 
F r om s a l i vary g l ands th e r e fo r e , p r o t e o s e rous s e c r e t i on 
c o n s i s t s mo s t l y  of ino rganic i o n s , wa t e r  and s ome n e u t r a l  
g l ycopro t e i n s  w i th l i t t l e  e v i de nc e o f  a c i d  glycop r o t e ins , 
whe re a s  muc o u s  s e c r e t ions are c omp o s e d  p r e dom inan t l y  o f  
b o t h  a c i d i c  and neut r a l  glycop r o t e i ns ( o r  muc o s ub s t an c e s ) . 
5 1  
I t  i s  hop e d  that the above s ugg e s t i o n s  may a l l ev i a t e  
s ome o f  t h e  c on fu s i o n  as s o c i a t e d  w i t h  c l as s i f i c a t i on o f  
ep i th e l i a l  s e c r e t o ry produc t s  and c o n t r ibute t owar d s  a 
c l e a r e r  unde r s tanding o f  the d i s t inc t i o n  between b i o chemi c a l  
a n d  h i s t o c h e m i c a l  t e rmino l o gy . 
The h i s t o chemi c a l  p r o c e du r e s  u s e d  f o r  th i s  s tu dy have 
b e e n  s e l e c t e d  t o  d i s t ingu i sh b e tween neu t ral and a c i di c  
s i d e  g roup s o f  muc o s ub s t anc e s  and t o  i de n t i fy s ep a r a t e l y  
c a rboxy l  a n d  s ulpha t e  a c i d  r a d i c l e s . Two en zyme s , hya l u r ­
o n i da s e  and neuram i n i da s e  w e r e  addi t i o na l l y u s e d  t o  c on f i rm 
the p r e s en c e o r  ab s enc e o f  ac i d  muc opo l y s ac c har i de s  and 
s i a l i c a c i d s  r e s p e c t ively , in b ov ine s a l i va ry g l and t i s s u e . 
The p r e s e n t  s tudy w i l l  a l s o  r ep o r t  o n  pre l iminary 
f in d ings t o  h i s to l o g i c a l ly e s t ab l i sh the maj o r  s ou r c e  o f  
immuno g l obul i n s  i n  b o v in e  s a l iva , from an as s e s sment o f  
p l a sma c e l l  numb e r s  i n  the conne c t iv e  t i s s ue s t roma o f  b o th 
m a j o r  and m i n o r s a l i v a ry g l an d s . I t  has b een p r e v i ou s l y  
r e p o r t e d  b y  W a t s on and L a s c e l l e s  ( 1 9 7 3 )  that non p a ro t i d  
s a l iv a  o f  s he e p  c on t a i ns 3 . 5  - 5 . 0 t im e s  more immun o g l obul ­
i n s  than p a ro t i d s a l i v a  w i t h  t h e  mand ibul a r  g l and b e ing 
the maj o r  c on t r ibut o r  ( C r ipp s a n d  L a s c e l l e s , 1 9 7 6 ) . How ­
e ve r ,  in c a t t l e  the s al ivary g l an d  o r  g l ands r e spons i b l e  
f o r  s ec r e t i o n  o f  immun o g l obu l i n s  have s o  far not b e en i d e n ­
t i f i e d ,  a l though e s t i ma t e d  v a l u e s f o r  I gA ,  I g G , I g G 2 and 
! gM in bovine s a l iva h ave been r e p o r t e d  by Mach and P ahud 
( 1 9 7 1 ) . 
Ant ibo d i e s  p ro duc e d  a g a in s t  e a ch o f  the bovine s a l i v a ry 
p ro t e in b an d s  4 ,  8 ,  9 and 1 0  w e r e  r e a c t ed with app ro p r i a t e  
s a l ivary g l an d  t i s s u e  t o  ident i fy immuno h i s tochem i c a l ly 
t h e  d i s t ribut i on o f  c e l l s  sp e c i a l i s e d f o r  synth e s i s  o f  
b ands 4 ,  8 ,  9 and 1 0  r e sp e c t i v e l y . P a r o t i d  g l and t i s su e s 
o f  b o t h  l ow and h i g h  b l o a t s u s c ep t ib l e  c a t t l e  w e r e  e xam ine d 
f o r  d i s t r ibut i on o f  b and 4 ,  t h e  s a l i v a ry p ro t e i n p re do m i n ­
a n t l y  imp l i c a t e d w i t h  b l o a t  s us c ep t ib i l i ty i n  c a t t l e  
( S e c t ion 1 . 6 ) . 
A f ew p a r a f fin wax emb e d d e d  s e c t i ons f rom s h e e p  p a ro t i d  
t i s sue s w e r e  a l s o  s tudi e d  t o  o b s e rve any morpho l o g i c a l  
c hang e s  a t t r ibutab l e  t o  sp e c i e s d i f f e r en c e s  b e tween s h e ep 
and c a t t l e  p a ro t i d  g l ands . 
5 2  
2 . 2  Ma t e r i a l s  and Metho ds 
2 . 2 . 1 T i s s u e  s ampl i ng and f ix a t ion pro c e dure 
The h i s to l o g i c a l  s e c t i o ns us e d  fo r mos t of t h e  s t a in i ng 
and h i s t o chem i c a l  me tho ds we r e  p rep a re d  from p re v i ou s l y  
emb e dd e d  p a r a f f i n  wax b l o cks s upp l i e d  b y  Mr . M . J .  B i rt l e s , 
Depa r tment o f  P hy s i o l o gy and Ana t omy , Mas s ey Univ e rs i ty ,  
P a l me r s ton No r t h , an d cont a ined t i s su e  s p e c imens f i x e d  i n  
fo rmo l s al ine and Bouins f l u i d  ( Cu l l in g , 1 9 74 ) . Al l b l o c k s  
h a v e  b e en p re p a r e d  w i t h  t i s s u e  s amp l e s  r emove d from b o t h  
r i gh t  and l e ft , maj o r  and minor s al iva ry g l ands o f  c a t t l e  
( Bos taurus ) . Mo s t  t i s s ue s amp l e s  e x am i ne d w e r e  f rom a 
mat u r e  animal w i th a l ow b l o a t  s co re . 
F o r  the l at t e r  p a r t  o f  the s t udy fur ther s amp l e s  fo r 
h i s to l o g i c a l  e x amina t i o n  w e r e  g at h e r e d  f rom two anima l s  
w i th known b l o a t  s us c ep t i b i l ity ( on e  L . S .  and the o th e r  
H . S . ) . B o th an ima l s  w e r e  s tunn e d  w i th a c ap t i ve b o l t  a n d  
k i l l e d  b y  exs anguina t i o n . A t  d i s s e c t i on ,  t h e  he ad was 
s ep a r a t ed at t h e  l ev e l  of the fourth or f i fth c e rv i c a l  
ve r t eb r ae w i th the upp e r  o e s ophagus and ep i g l o t t i s  i nt a c t . 
The s al iva ry g l ands from b o t h  t h e  r i g h t  and l e f t  s i d e s  o f  
5 3  
t h e  h e a d  w e r e  d i s s e c t e d  i n  t h e  f o l low i ng o rd e r : - mand i b u l a r, 
p a ro t i d ,  s ub l ingual , d o r s a l  b u c c a l , i nt e rme d i a t e  b uc c a l  and 
vent r a l  buc c a l . The head wa s r e - o r i en t e d  fo r s amp l e s  t o  b e  
t a k en from t h e  p o s t e r i o r  tongu e , e p i g l o t t i s  and s o f t  p a l at e . 
S amp l e s  r emo v e d  f rom e a ch g l and w e r e  s ep a r at e ly p l a c e d  in 
t h e  f o l l ow in g  f ixat i v e s : - Bouins f l u i d ,  1 0 %  neut r a l  f o rmo l 
s a l in e  and Z en k e r - fo rmo l ( Cu l l in g , 1 9 7 4 ) . Further s amp l e s  
w e r e  p ro c e s s e d  fo r e l e c t ron m i c r o s c opy and u t i l i z e d  f o r  a 
s tu dy o f  int r a ep i the l i a l  g ranu l a t e d  duc t c e l l s  i n  t h e  p a r o tid 
and vent r a l  b u c c a l  g l ands ( s e e  C h ap t e r  3 ) . 
T i s s ue s  i n  f o rmo l s a l ine wer e l e f t  f o r  2 - 3  d ays wh i l e  
tho s e  in B o u i n s  f l u i d  w e r e  l e ft f o r 2 4  hours b e fo r e  b e i ng 
t ran s fe rr e d  i n t o  7 0 %  a l coho l . T i s su e s  i n  Z enker - fo rmo l 
w e r e  fixed f o r  1 2 - 1 5  hours and t h e n  w a s h e d  i n  runn ing t ap ­
wa t e r  fo r 6 - 8 hours . 
2 . 2 . 2  P a r a f f in wax p ro c e s s i ng and m i c ro tomy 
F o l l ow ing f i xat i on t h e  t i s s u e s  w e r e  l oade d into a 
Shandon �l l io t  aut omat i c  t i s s ue p r o c e s s o r  (Wat s on ,  V i c t o r  
L t d . )  fo r o v e rn i g h t  dehy dr a t i on , c l e a r ing an d para f f in wax 
imp r e gna t i on . The p r e l i m inary dehy d rat i on s t ep i n c l ud e d  
chan g e s  o f  an h o ur ' s dur a t ion e a c h  i n  inc r e a s ing conc e n ­
trat i o n s  o f  e t hy l  a l coho l ( 7 0 % , 9 5 % , 1 0 0 % , 1 0 0 % ,  1 0 0 % ) . 
5 4  
The c l e a r ing s t e p  ut i l i z e d ch l o ro fo rm ( 1  hou r )  and two 
chang e s  of xy l en e  ( 1  hour e ac h ) . I mp r egna t i on was c ar r i e d 
out a t  5 6 ° C  w i th two chang e s  o f  p a r a f fin wax ( 2  hour s e a ch) . 
T i s s ue b lo c k s  w e r e  then r emove d  from the p roc e s s o r  and 
emb e dde d i n  5 6 ° C  M . P .  p ar a f f in wax . S e c t ions w e r e  p re p a re d  
f rom wax b l o c k s  w i th s amp l e s  c u t  a t  5 - 6 � m  t h i ckne s s  us i ng 
a Re i chart s l i di n g  m i c ro tome ( S e l by - Wi l t on L td . ) .  I nd iv i d ­
ual s e c t ions w e r e  f l o a t e d  o n  warm wat e r  at about 4 6 - 4 8 ° C  
an d then at t a ch e d  onto s p a r s e l y  a l b umen i s e d  g l a s s  s l i de s  
( 3" x 1 " ) an d l e f t  at room t emp e ra t u r e  fo r about 3 0  minut e s  
b e f o r e  b e in g  a i r  d r i e d  a t  6 0 ° C  o v e rn i ght . 
2 . 2 . 3  S t a i n ing and h i s t o chemi c a l  methods 
B e fo r e  s t a i n ing the s e c t i o n s  w e r e  dewax e d  in two c h ang e s  
o f  xyl ene , r in s e d  i n  ab s o lut e a l co h o l  and 7 0 %  al coho l r e s ­
p e c t i v e l y  an d w a s h e d  i n  runn ing t ap wa t e r . 
The fo l l ow in g  s t a i n s  and h i s t o c hemi c a l  metho d s  w e r e  
app l i e d  t o  s e c t ions f rom a l l  t h e  maj o r  and minor s a l i va ry 
g l an d s : -
1 .  Haema t o xy l in an d e o s i n  ( H & E ) 
2 .  A l c ian b lue pH 1 . 0  an d pH 2 . 5 I H&E 
3 .  Al c i an b l ue pH 2 . 5  I pho s p h o tung s t i c  a c i d  haema -
t o xy l i n  (PTAH ) 
4 .  P e r i o d i c  a c i d  S ch i ff ( PA S )  
5 .  Al c i an b lue ( p H  2 . 5 ) and PAS 
6 .  Pheny l hy dr a z ine - p e r i o d i c  a c i d  Sch i f f  ( PAP S )  
7 .  Toludine b l ue 
8 .  A z ure A at pH 1 ,  2 ,  3 and 4 
9 .  A l c i an y e l l ow 
1 0 .  Hal e s  C o l l o i da l  I ron 
1 1 .  B IAL r e ag ent 
1 2 . D i a s t a s e  fo l l ow e d  by PAS 
1 3 . Ace ty l a t ion and PAS 
1 4 . Acety l a t ion , s ap on i f i c a t i o n  and PAS 
l S . Me t hy l a t ion and A l c i an b l u e  a t  pH 1 . 0  and 2 . S  
1 6 . Me thy l a t ion , s ap o n i f i c a t i on and A l c i an b l u e  a t  
p H  1 . 0 and 2 . S  
1 7 . Neuram i n i da s e  fo l l ow e d  by A l c i an b l u e  a t  pH 2 . S  
1 8 .  Hya l u ro n i da s e  f o l l ow e d  by A l c i an b l u e  a t  pH 1 . 0  
1 9 .  Methyl - G reen pyronin 
ss 
T h e  s t a in i ng p ro c e du re s  u s e d  a nd the r e a c t i o n  mechani sms 
wh e r e  app l i c ab l e  are l i s te d  i n  App e nd i c e s  I and I I .  
A ft e r  s t a in i n g , s e c t i o n s  w e r e  d e hydra t e d  in 7 0 %  a l cohol 
and ab s o lu t e  a l c o hol r e s p e c t i v e l y , c l e a r e d  i n  xyl ene and 
mo unt e d  in D . P . X . me dium ( B . D . H .  chemi c a l s ) . 
P r e p a r e d  s l i d e s  w e r e  e xam ined w i th an Olymp us mi c ro s c ope 
a t  four d i f fe r ent magn i f i c at i ons , name ly x4 0 ,  x l O O , x4 0 0  
and x l O O O . 
B o u ins f ix e d  t i s s ue s amp l e s o f  s he ep p a r o t i d  g l and we r e  
a l s o  c u t  and s t a ine d w i t h  H & E  and A l c i an blue a PTAH . 
2 . 2 . 4  I mmunoh i s t o ch e m i s t ry 
Ant ib o d i e s  t o  b ov ine s al ivary p ro t e in bands 4 ,  8 ,  9 
and 1 0  w e r e  e a ch s ep ar a t e ly r a i s e d  i n  rabb i t s  and p rep a r e d  
b y  D r . W . T .  Jone s , App l i e d  B i o chemi s t ry Divi s i o n ,  D . S . I . R . , 
P a lme r s ton N o r th . An ant ib o dy s pe c i f i c  t o  a b o v i n e  s al i v a ry 
p r o t e i n was then r e a c t e d  w i th the r e sp e ct ive t i s s ue ant i g en ; 
band 4 and 1 0  ( Pa ro t i d  g l and) , b an d  8 (Mand i b u l a r  g l and) 
and b and 9 ( Sub l ingua l  g l and) . 
The immuno h i s t o chemi c a l  me tho d u s e d  was the i nd i re c t  
5 6  
e n z yme l ab e l l e d ant i b o dy t e chn ique ( S t e rnb erge r ,  1 9 7 4 ) . 
Rabb i t  ant i b o v i ne s a l ivary p ro t e in was us e d  a s  p r im a ry ant i ­
s e rum . The s e conda r y  ant i s e rum was g o a t  ant i rabb i t  I g G 
conj ug a t e d  w i th ho r s e ra d i s h  p e r o x i d a s e .  S i t e s  o f  a n t i b o dy 
t o  ant i g en a t t a chme n t  w e r e  de t e c t ed by us i ng e i the r 3 . 3 
d i aminob en z i d i n e  ( DAB ) o r  3 - am i no - 9  e thy l carb a z o l e , b o t h  
c ompl exed w i th hy d ro g en p e rox i de .  
P r o c e d u r e  f o r  i n d i r e c t  en zyme l ab e l l e d an t i b o dy m e t h o d  
P a r a f f i n  w a x  s e c t i ons f r o m  p aro t i d ,  mandibul a r  and s ub ­
l in g�a l g l an d s , p re v i ous l y  f i x e d  in Bouins flu i d ,  w e r e  
p re p a r e d  a s  de s c r i b e d  in S e c t i on 2 . 2 . 2 .  
P a ro t i d  t i s s ue s amp l e s  from b o t h  L . S .  and H . S .  c a t t l e ,  
int e rme d i a t e  b uc c a l  g l and and t h e  s h e ep p aro t i d  w e r e  
addit ion a l l y  e xamine d f o r  t h e  d i s t r ibut i o n  o f  p ro t e i n 
b and 4 .  
1 .  Dewax s e c t i o n s  and b r i n g  t o  w a t e r . 
2 .  T r e a t  s e c t i o n s  w i th 2 %  bov ine s e rum a l b um e n  ( B SA )  
i n  pho s p h a t e  buffe r e d  s a l ine ( PB S )  f o r  5 m i nu t e s  
t o  reduce n on - s p e c i f i c  b i nding . Shake o f f  
e x c e s s  B SA . 
3 .  T r e a t w i th r abb i t  ant i b o v i ne s a l ivary p ro t e i n 
d i l ut e d  1 : 1 0 0 0  i n  2 %  B SA in P B S  for 3 0  m i nu t e s  
i n  mo i s t  chamb e r . 
4 .  W a s h  w i th P B S , minimum o f  th r e e  chang e s , 1 5  
m i nut e s  e ach with c o n s t an t  s t i r r ing . 
5 .  T r e a t  w i th 2 %  BSA i n  P B S  f o r  1 0  minut e s . 
6 .  Re a c t  w i t h  p e ro x i da s e - l ab e l l e d  goat ant i rabb i t  
immuno g l ob ul in ( d i l u t e d  1 : 6 0 )  for 3 0  minu t e s  
i n  mo i s t  chamb e r . 
7 .  W a s h  w i t h 2 %  BSA i n  P B S  ( op t i o n a l ) . 
8 .  W a s h  in P B S  thre e chang e s  5 m inu tes e a c h  w i th 
c o ns tant s t i r r ing . 
9 .  I nc ub a t e  w i th s o l u t i o n  o f  DAB ( 7 5  mg ) i n  0 . 0 0 1 %  
hydro g en p e r o x i d e  i n  0 . 1 5 M t r i s chl o r i de b u f f e r  
pH 7 . 6  fo r 1 0  - 3 0  m i nut e s . 
1 0 .  Wa s h  b r i e f ly in t r i s ch l o r i de b uf fer . 
1 1 . R i n s e in d i s t i l l e d  w at e r .  
1 2 . C o unt e r s t a in with 1 %  aqueous l i ght g r e e n  s o lu t i on 
f o r  about 5 - 1 0  s ec o n ds . 
1 3 .  D e hydra t e , c l ea r  and mount in D . P . X .  
Cont r o l s e c t i o n s  were s im i l a rl y  t r e a t ed e x c ep t t h a t  
p r e immune r abb i t  I gG wa s s ub s t i tut e d  f o r  the p r ima ry ant i ­
s e rum to d e t e c t  non - immuno l o g i c a l  b ind ing o f  y - g l o b u l ins . 
Endo g en e o u s  p e ro x i da s e  a c t i v i ty was n o t  s upp re s s e d  s in c e  
p revi ous f i nd ings h a v e  indi c a t e d  t h a t  s uch a c t i v i ty i s  
m inimal i n  bovine s a l ivary g l an d  t i s s u e  ( W . T .  Jone s , 
p e r s onal commun i c a t i o n ) . 
The d i s t r ibu t i on o f  I gA s yn t h e s i s i n g  pl asma c e l l s i n  
p a ro t i d ,  mand ibul a r  and s ub l i n g u a l  g l ands was e x am i n e d  b y  
s ub s t i tu t i n g  g o a t  ant ihuman I gA ( 1 : 1 0 )  as p r ima ry an t i s e rum 
in s t ep 2 and rabb i t  ant i g o a t  i mmuno g l ob u l in in s t ep 6 .  
2 . 2 . 5  Pho tomi c rography 
Pho t o m i c r o graphs o f  a r e a s  r e p r e s e nt a t ive o f  mo s t  o f  t h e  
s a l i v a ry g l ands e x amined f rom h i s to ch em i c al metho d s , w e r e  
t aken us i n g  a L e i c a  3 5  mm r e fl e x  c ame r a  moun t e d  on a L e i t z  
O rtho lux m i c ro s c op e  (Me d i c a l  S upp l i e s  (N . Z . ) L td . ) . 
E xp o s ur e  t ime s we r e  c a l cu l a t e d  w i th a m i c ro s i x  L e xpo s u r e  
m e t e r .  Ag fachrome S O L  and K o dach r ome S O  nega t i ve f i lms 
were us e d .  Co l o u r  p r int s us e d  in t h i s  thes i s  w e r e  p r o c e s ­
s ed by Ko dak (N . Z . ) L t d . f ro m  3 5  mm t ransparenc i e s . 
5 7  
2 . 3 Re s u l t s  
H i s t o l o g i c a l  f in d i n g s  f ro m  the maj o r  and mino r s a l i v a ry 
g l ands a r e  de s c r i b e d  b e l ow and the r e s u l t s  from h i s t o ­
chemi c a l  r e ac t i on s  have b e en s umma r i z e d  i n  Tab l e  2 . 1 .  
2 . 3 . 1  P a ro t i d  G l and 
5 8  
The b o v ine p a ro t i d  g l an d  was comp o s e d  o f  t ubul a r  s e c ­
r e t o ry en dp i e c e s  w i t h  c ub o i da l  c e l l s  s ur rounding a n a r r ow 
l umen . The s e c r e t o ry endp i e c e  c e l l s  s ho w e d  no e v i dence o f  
cy t op l as m i c  b a s op h i l i a  and h a d  a g ene r a l l y  va cuo l at e d app e a r­
anc e . Th e c ent ra l l y - p l a c e d  s p h e r i c a l  nuc l e i w e r e  l ar g e  and 
c o n t a in e d  one o r  two nuc l e o l i .  Wi th r e s p e c t t o  the hi s t o ­
ch emi c a l  r e ac t i ons u s e d ,  the cytop l a sm o f  s e c r e t o ry c e l l s  
s howed l i t t l e  e v i de n c e  o f  r e a c t ivi ty w i t h  ba s i c s t a i n s , 
a l though i n  one an i ma l  a c l us t e r  o f  c e l l s  pos i t ive t o  
Al c i an b l ue a t  pH 2 . 5  w a s  found ( F i gu r e  2 . 1 . 2 ) . Howev e r , 
t h e  o c c a s i ona l s e c re t o ry c e l l  o f  mo s t  an i mal s examined wa s 
d i s t inc t l y  PAS p o s i t i ve ( F i g ure 2 . 1 . 1 ) , d i s t r ib u t i o n  o f  
( d i a s t a s e - r e s i s t ant ) PAS p o s i t ive mat e r i a l  was s up ranuc l e a r  
and s omet imes c l o s e  t o  the ap i c a l  b o rde r . 
I n t e r c a l a t e d  duc t s  l e a ding from the s e cre t o ry endp i e c e s  
w e r e  l on g  and b r anch e d  and l ined b y  ep i th e l i a l  c e l l s  wh i c h  
w e r e  ini t i a l ly f l a t t en e d  b ut b e came cub o i da l  n e a r  t h e  i n t r a ­
l ob ul a r  duc t s . The cytop l a s m  was mod e r a t ely b a s op h i l i c  
( w i th H &E ) . The nuc l e i  o f  duc t c e l l s  c l o s e  t o  the endp i e c e s  
w e re f l a t  and dens e wh i l e  t h o s e  n e a r  t h e  int r a l o b u l a r  duc t s  
w e r e  ovo i d .  
The i n t r a l obul a r  duc t s  had a c onvo l ut e d  app e a r an c e  w i th 
mo de ra t e l y l a r g e  d i am e t e r  l umina . The e p ith e l ium was cub ­
o i dal o r  l ow c o l umn a r  w i th c ent ra l ly p l a c e d  nuc l e i . The 
c y t op l a s m  o f  s ome i n t r a l obul a r  duc t  c e l l s  wa s no t ab ly PAS 
p o s i t ive whi l e  the a p i c a l  c e l l  memb rane s o f  s ome w e r e  f r e ­
quen t ly mo d i f i e d  w i th cytop l asmic p ro t ru s ions o f  " ap i c a l  
b l eb s "  wh i ch cont a i n e d  PAS p o s i t i ve ma t e r i a l  ( F i gure 2 . 2 . 1 ) .  
The re w a s  l i t t l e  o r  no e v i d enc e fo r t h e  pre s en c e  o f  b a s a l  
Tab l e  2 . 1  H I STOCHEM I CAL RE SULTS O F  BOV I NE MAJOR AND M I NOR SAL I VARY GLANDS 
Salivary 
� M 0'1 Ill Ul � 
Glands u M M ::1 0 '0 u M M Ill Ill 0 +J � ;:1 � � M M Ill Ill u u u M M 
Q) 
111 ..0 Ill Ill Ul Ill Ill Ul u u Ul u u Ul Ul � ::1 � Ul Ill Ill Ul 
M M Ql ;:1 ;:1 Ql u u Ql ::1 ::1 Ql Q) Q) Q) 0 s 0 Q) Q) Q) Q) 'O M  ::1 ;:1 � 0'1 0'1 � ::1 ;:1 � ..0 ..0 § 
� � � ·.-i ·.-i � 0'1 0'1 � 
·.-i Ill ..0 Ul ..0 ::1 � Ul � ::1 ..0 Ul ..0 ::1 Ul ·.-i Ul ·.-i ::1 � Q) � ::1 � Ul � ::1 +J � ·.-i ::1 ·.-i M ·.-i ::1 ·.-i M ::1 M � ;:1 � M  +J ::1 +l M  Q) ::1 <ll M � g  :>. M  0 +J '0 0 '0 ·.-i M 0 M ·.-i • 0 • ·.-i Q) 0 Q) '.-i Ill 0 Ill ·.-i +J 0'1 +J ·.-i � ·.-i � � � u � s -§ g -§ ID +J u +J s o.. u o.. s M U M S Ul � Ul e m u � ID Ill Q) Ill ::1 Ill Q) � ::1 � Q) 
g. �  
O.. <ll Ill ::1 m <ll 0 0 0 Q) ..c: ::1 
Method A. :>  � s � '0 U) s U) 'O H S H 'O  :::> '0 A< e ll< 'O A. +l  ll< 'd  ll< s ll< 'd  
PAS ( +) +++ + +++ ( +) +++ ( +) +++ (+) +++ ( +) +++ + +++ + 
Diastase + PAS (+) +++ + +++ (+) +++ ( +) +++ (+) +++ (+) +++ + +++ + 
Methylation (M) + PAS ( +) ++ + ++ ( +) ++ ( +) ++ (+) ++ (+) ++ + ++ + 
Saponification (S )  + PAS ( +) +++ + +++ (+) +++ ( +) +++ (+) +++ (+) +++ + +++ + 
Acetylation (A) + PAS - - - - - - - - - - - - - - -
(A) + ( S )  + PAS ( +) ++ + +++ (+) +++ (+) +++ ( +) +++ ( +) +++ (+) +++ (+) 
PAPS ( +) ++ + ++ - ++ - ++ - ++ - ++ - ++ -
Alcian Blue (AB) pH 1 . 0  - (+) (+) ++ - ++ - ++ - ++ - ++ (+ ) ++ (+ ) 
pH 2 . 5  ( +) ++ + +++ ( +) +++ - +++ - +++ ( +) +++ + +++ + 
(M) + (AB) pH 1 . 0  - - - - - - - - - - - - - - -
pH 2 . 5  - - - - - - - - - - - - - - -
L_ 
+ 
11 
rt 
11 
Ill 
0 
m 
en 
+ 
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m 
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8 
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m 
+ + + 
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I 
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I 
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+ 
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+ 
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+ + 
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+ + 
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+ 
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+ + 
+ 
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VJ N 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
I I 
� ::r: � Ill 1-' 
1-' 1-' 0 
s:: m 1-'· 
p. en § 1-'· 
:::1 () 
m 0 >< 
1-' m 
tJj 1-' 1-' 
1-' 0 I:-' 
s:: 1-'· � m p. Ill 
1-' 
H 
11 
0 
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::r: 
1-' 
� I I + I 
� 
+ + + I + + + + + 
I + + + 
+ + + I + + + + + 
� 
I + + I 
� 
+ + + I + + + + + 
I I I I 
+ + + I + + + + + 
I I I I 
+ + + I + + + + + 
� 
� � 
I + + + 
� � 
� 
+ + + I + + + + + 
I + + + 
+ + + I + + + + 
I + + + 
::r: z � 
"< m :3: 
Ill s:: � 1-' 11 
s:: � + 11 :3: 0 1-'· � m 
:::1 :::1 (/) rt 
1-'· 1-'· � ::r 
p. p. 0 
Ill Ill + p. en en 
m <!> � G; + + , 
� 
� 
� 
G; 6; ' '0 '0 (/) 
� 
- ::r: ::r: Ill 
(j) l-'  '0 '0 '0 N 1-' 1-' 1-'· ::r: ::r: ::r: . . Ill < lJ1 0 :::1 Ill N 1-' N g. �  . . lJ1 0 lJ1 
Parotid and 
I I I I I Ventral buccal 
+ � Mandibular + + I I I mucous � 
� 
� 
Mandibular 
+ + + + I demilunes � � 
+ + + Sublingual + + + + I + mucous 
Sublingual 
I I I I I demilunes 
+ ·  + + Int . buccal + + + + I + mucous 
Int. buccal 
I I I I I demilunes 
+ + + Upper buccal + + + + I + mucous 
I I I I I Upper buccal demilunes 
+ + + Palatine + ·  + + + I + mucous 
� Palatine + I I I I demilunes � 
+ + + Posterior tongue + + + + I + mucous 
+ + I I I Posterior tongue 
� demilunes 
+ + + Pharyngeal + + + I + 
mucous 
+ + I I I Pharyngeal 
� demilunes 
5 9  
s t r i a t i ons i n  i n t r a l obu l a r  duc t s  ( F i gure 2 . 2 . 3 ) .  A f r equent 
o c c u r r e n c e , p a r t i cu l a r ly in t h e  duc t al ep i t h e l ium of l ow 
b l o a t  s us c ep t i b l e  c a t t l e , w a s  a g ranul a t e d  c e l l  l y ing 
adj a c e n t  t o  the b a s a l  l am i na ( F i gu r e  2 . 2 . 2 ) .  Th i s  c e l l  
has b e e n  the s ub j e c t  o f  a s ep a ra t e  inve s t i g a t i on ( s e e  
Chap t e r  3 ) . 
Wa l l s o f  t h e  l a rg e r  i n t e r l o bu l a r  and int e r l ob ar duc t s  
w e r e  l in e d  by s t r a t i f i e d  c ub o i da l  e p i the l ium and f requent ly 
s ur rounde d by i r r e gul a r l y  a r ranged f ib rous c onne c t ive 
t i s s ue c ont a i n ing ma s t  c e l l s , n e rv e s , b l o o d  v e s s e l s  and 
l ymph a t i c s . P l a sma c e l l s  w e r e  i n f r e quent ly o b s e r v e d  i n  a l l  
p a ro t i d  t i s s u e  s amp l e s . I n t e r l o bu l ar and i n t e r l o b a r  duc t s  
s howe d no e v i denc e o f  ap i c a l  cytop l a smic p r o t ru s i o ns o r  
b a s a l  s t r i a t i on s  but g r a nu l a t e d  c e l l s  i dent i c a l  t o  tho s e  
found i n  the e p i the l i um o f  i nt ra l o b u l a r  duc t s  w e r e  o c c a s ­
i o na l l y  p re s en t . The ma i n  e x c re t o ry duc t  wa s l in e d  by 
s t ra t i f i e d  c o l umnar ep i t h e l i um w i th gob l e t  c e l l s  ( F i g u r e s  
2 . 1 . 3  a n d  2 . 1 . 4 ) .  T h e  cy t op l a s m i c  contents o f  t h e  g o b l e t  
c e l l s  r e a c t e d  w i th PAS a n d  a l l  the c a t i on i c  dy e s  u s e d  ( e g . 
A l c i an b lue , pH 1 . 0  and 2 . 5 , Al c i an ye l l ow ,  T o lud ine b lu e , 
A z u r e  A and c o l l o idal i r o n ) . 
A b r i e f  i nv e s t i g a t i o n  o f  the s h e ep p a ro t i d  g l and d i d  
no t r e v e a l  h i s t o l o g i c a l  f e a t u r e s  d i s s imi l ar t o  the bovine 
p a r o t i d ,  e xc e p t  that the i n t r a l ob u l a r  d�c t ep i th e l i a l  
c e l l s  w e r e  t a l l c o l umna r  w i th a c i doph i l ic b a s a l  s t r i a t i ons 
( F i g u r e  2 . 2 . 4 ) .  The s e  duc t s  w e r e  f r equent l y  o b s e rv e d  i n  
c l u s t e r s , l o c a t e d  c e n t r a l ly w i th i n  a l obu l e . 
2 .  3 .  2 Mand ibu l a r  G l a n d  
The e l ong a t e d  tub u l a r  endp ie c e s  o f  t h i s  g l and w e r e  
comp o s ed o f  t w o  c e l l  typ e s ,  t h e  s e c r e t o ry c e l l s  wh i ch s u r ­
rounde d a na r r ow lumen a n d  a demi l un e  c e l l  wh i ch o c cup i e d  
a p o s i t i on a t  t h e  b l ind e n d  o f  an endp i e ce ( F i gu r e  2 . 4 ) .  
The s e c r e t o ry c e l l s  g ene r a l ly w e r e  cub o i da l  i n  app e a rance 
w i th a f l a t t e n e d  b a s a l  nuc l eus and a charac t e r i s t i c a l ly 
" fo amy" cytop l a s m ,  wh i l e  t h e  demi l un e  c e l l s  w e re l a r g e  and 
r o unded w i th a sphe r i c a l  b a s a l l y - p l a c e d  nuc l eus . 
The cytop l a s m i c  c o nt ent s o f  s e c r e t o ry (mucou s )  c e l l s  
r e a c t e d  p o s i t iv e l y  w i th PAS , PAP S ,  Al c i an b lu e  (AB ) pH 1 . 0  
( w e a k ) , pH 2 . 5 , Al c i an y e l low (AY) , T o l ud ine b lu e , A z u r e  
A pH 4 a n d  Ha l e s  co l l o idal i ron ( F i gu r e  2 . 4 ) .  M e t hy l a t i on 
a t  6 0 ° C fo r fou r hour s  e l imin a t e d  b a s op h i l i a a t  b o th pH 1 . 0  
a n d  pH 2 . 5 .  Sub s e qu e n t  s ap on i f i c a t i o n  f a i l e d  to r e s t o r e  AB 
s t a in i ng at e i th e r  pH 1 . 0  o r  pH 2 . 5 .  Ac e tyl a t i o n , w i th a 
m i xture o f  a c e t i c  anhy dr i de and pyr i d i n e  e l im ina t e d  PAS 
s t a i n ing wh i ch was s ub s equent ly re s t o r e d  by s ap o n i f i c a t i o n . 
B a s op h i l ia a t  pH 2 . 5  was mar k e dly r e du c e d  by n e u r am i n i d a s e 
a c t i v i ty ( F i gu r e  2 . 4 . 1 0 ) , whe r e a s  hya lu r o n i da s e  a c t i v i ty 
6 0  
w a s  n e g a t i v e  wi th n o  r e du c t i on in de g r e e  o f  b a s op h i l i a a t  
e i th e r  p H  1 . 0  o r  p H  2 . 5 .  I n  cont r a s t t o  the s e c r e t o ry end ­
p i e c e  c e l l s , the d em i l un e s  h a d  d i s t in c t  e o s i noph i l ic g r a n ­
u l e s  ( F i g ur e  2 . 4 . 6 ) w i th i n  a mo dera t e l y  o r  w e a k l y  b as op h i l i c  
cytop l a s m . The d em i l une c e l l s  were w e a k l y  po s i t i v e  t o  PAS 
( F i g u r e  2 . 4 . 5 ) ,  AB pH 1 . 0  and pH 2 . 5 , AY and co l l o i da l  i r on 
b ut r e ac t e d  s t rong ly w i th the B I AL r e a g en t  ( F i gu r e  2 . 4 . 7 ) . 
The g r anu l e s  r e a c t e d  s t rong ly w i th a c i d  s t ains s u c h  a s  
e o s in ,  p h l ox ine and p h o spho tung s t i c  a c i d  haematoxy l in 
( F i gure 2 . 4 . 6 ) .  A l t h o ugh s ap on i f i c a t i o n  a ft e r  m e thyl a t i on 
p ro duc e d  no evi denc e o f  AB p o s i t ive (pH 2 . 5 ) s t a i n ing i n  
mucous c e l l s , the d e m i lune s d i d  n o t  d i sp l ay th i s  f e a t u r e  a s  
t h e re was e v i dence o f  we ak AB po s i t i v e  ( p H  2 . 5 ) s t a i ning 
r e s t o re d  by s ap on i f i c a t i on . 
The s e c re t o ry e n dp i e c e s  t e rmina t e d  abrup t l y a t  int e r ­
c a l a t e d  duc t s  wh i ch w e r e  l ine d by s imp l e  cub o i da l  ep i th e l ium .  
T h e  nuc l e i  o f  i n t e r c a l a t e d  duc t  c e l l s  w e r e  h i ghly i r r e gu l a r  
i n  s hape and the cy t o p l asm o f t en i nvag i na ted the nuc l e i  t o  
a b out thre e - qua r t e r s  o f  the l at t e r s ' d i am et e r . The wa l l s  
o f  the i n t r a lobul a r  duc t s  we r e  l in e d  by t a l l  c o l umna r  c e l l s  
w i th w e l l - deve l op e d  b a s a l  s t r i a t ions ( F i gure 2 . 4 . 5 ) .  The 
c e l l  nuc l e i  were l ar g e  and sphe r i c a l  and were l o c a t e d  m i dway 
b e tween the b a s e  and ap e x  of the c e l l . Overa l l , the s t r i at ed 
duct c e l l s  did not a p p e a r  to cons t i tu t e  a n  ent i r e l y  homo g e n ­
e o u s  c e l l  p opu l a t i o n , s i nce v a r i a t ions w e re ob s e rv e d  i n  
6 1  
s t a in ing cha ra c t e r i s t i c s o f  t h e i r  nuc l e i  and cytop l a sm .  The 
c y t op l a sm and ap i ca l  b o r de r s  o f  s om e  s t r i a t e d  duc t  c e l l s  
w e r e  a l s o  i n t ens e l y  PAS p o s i t i v e . The i rr e gu l a r  f i b rous 
c onne c t ive t i s su e  s u r ro unding s t r i a t e d  duc t s and t h e  l a r g e r  
in t e r l obu l a r  duc t s  c o n t a i n e d  s ev e r a l  p l a sma c e l l s  r e a c t iv e  
t o  ant i I gA ant i b o dy ( F i gu r e  2 . 4 . 1 1 ) , m a s t c e l l s , b l o o d  
v e s s e l s , n e rves and l ymphat i c s . I n t e r l o bu l a r  duc t s  w e r e  
c o mp o s e d  o f  cubo i d a l  o r  l ow c o l umna r  ep i thel i um a n d  d i d  
n o t s how w e l l - de v e l o p e d  b a s al s t r i a � i o n s  a l though t h e r e  wa s 
s om e  e v i d e n c e  o f  PAS p o s i t ive ma t e r i a l  i n  the i r  ap i c a l  
b o r d er s . 
The ma in duc t  o f  t h e  mand i b u l a r  g l and was comp o s e d  o f  
s t ra t i f i e d  c o lumna r  e p i th e l i um w i thout g o b l e t  c e l l s  ( F i g u r e  
2 . 4 . 1 2 ) . 
2 . 3 . 3  Sub l ingu a l G l and 
The s e c re t o ry endp i e c e s  w e r e  l on g  and tubu l a r  w i th f l a t ­
t en e d  demilune c e l l s . The s e c r e t o ry c e l l s  were p o s i t i v e  t o  
PAS , AB p H  1 . 0  and 2 . 5 , AY , T o l ud in e  b l ue , A zure p H  4 ,  PAPS 
and Hal e s  co l l o i da l  i ron ( F i gu r e  2 . 5 ) .  Aft e r  methy l a t i on 
and s apon i f i c at i on A B  s t a i n in g  was r e s t o r e d  a t  pH 2 . 5 .  
N e u r amin i da s e  a c t i v i t y  d i d  n o t  e l imina t e  basophi l i a com­
p l e t e ly s ince cons i de rab l e  AB s t a ining a t  pH 2 . 5  was o b s e rved 
a f t e r  enzymic d i g e s t i on .  S i m i l a r l y  hya l u ron i da s e  t r e a tment 
d i d  not e l iminat e  b a s ophi l i a  at e i t h e r  pH 1 . 0 or 2 . 5  
( F i g ure 2 . 1 1 ) . 
The demi lune c e l l s  o f  t h e  s ub l ingual g l and d i f fe re d  
mo rpho l o g i c a l ly and h i s t o c h em i c al ly from tho s e  o f  the man ­
d i b ul a r  g l and . The demi l un e s  were s l e nd e r  in out l ine and 
n o t  e a s i ly i dent i f i e d .  They di d n o t  app e ar to r e a c t  w i th 
b a s i c s t a in s  but w e a k  PAS po s i t ive mat e r i a l  wa s i dent i f i e d  
i n  s ome d e m i l une s .  
I n t r a l o bul a r  duc t s  w e r e  w e l l deve l o p e d  and l in e d  by t a l l  
c o l umn ar c e l l s  w i t h b a s al s t r i a t i ons ( F i gure 2 . 5 . 5 ) .  The s e  
s t r i a ted duc t s  w e r e  mo rpho l o g i c al ly s im i l ar t o  tho s e  o f  the 
mand i bul a r  g l and , a l though PAS p o s i t i ve mat e r i a l  in duc t  
c e l l s  was no t a c ommo n f e ature o f  the subl ingua l g l and . 
The i nt e r l o b u l a r  and i n t e r l ob a r  duc t s  w e r e  comp o s e d  o f  
s t r a t i f i e d  c ub o i da l  ep i th e l i um w i thout g o b l e t  c e l l s . 
An unu s u a l  f e a t u r e  o f  mo s t  s ub l ingua l g l and t i s su e s  
examined was t h e  l ar g e  numb e r  o f  p l a sma c e l l s  d i s t r i bu t e d  
i n  c l ump s b e twe en t h e  s e c re t o ry endp i e c e s  and s t r i a t e d  
duc t s  ( F i gu r e s 2 . 5 . 7  and 2 . 5 . 8 ) .  P l a sma c e l l s  w e r e  n o t  
ob s e rv e d  i n  s uch h i gh p ro p o r t i on s  i n  any o f  t h e  o th e r  maj o r  
o r  m i n o r  s a l i va ry g l an d s . Ano th e r  unu s u a l  c h a r a c t e r i s t i c  
ob s e rv e d  i n  s ome t i s sue s amp l e s  e x amine d , w a s  a n  app a rent 
h i s t o l o g i c a l  d i f f e r enc e b e tween c e rt a in a r e a s  of the g l and 
( F i g u r e s  2 . 5 . 3  and 2 . 5 . 4 ) . Some a r e a s  of t h e  s ub l ingu a l  
g l an d  l ac k e d  p l a sma c e l l s  and s t r i a t e d  duc t s ; ins t ea d  the 
endp i e c e s  w e r e  c l o s e ly p a c k e d  and i nt r a l obul a r  du c t s  were 
comp o s e d of l ow cub o i da l  c e l l s  w i thout b a s al s t r i a t i on s . 
P l a sm a  c e l l s  w e r e  o c c a s i ona l ly s e e n  but neve r i n  g ro up s  o r  
c l ump s b e tw e e n  endp i e c e s  and duc t s . 
2 .  3 .  4 Buccal G l an d s  
The s e  g l ands a r e  d i s t r ib u t e d  in t h e  s ub e p i t h e l i a l  
t i s s ue s  o f  t h e  do r s a l , i n t e rme d i a t e  and vent r a l  a sp e c t s  
o f  t h e  bucc a l  c av i ty . The h i s t o l o gy and h i s t o chemi s t ry o f  
the vent r a l  b u c c a l  g l an d s  we re i de nt i c a l  t o  that o f  t h e  
p a ro t i d g l an d s  ( s e e  S e c t i on 2 . 3 . 1 ) . 
The s e c r e t o ry endp i e c e s  o f  the do rs a l  and i n t e rmed i a t e  
b uc c a l  g l an d s  w e r e  mai n l y  t ub ul a r  w ith fl a t t en e d  demi l une 
c e l l s towa r d  the i r  d i s t a l  ends . S e c r e t o ry c e l l s  r e a c t e d  
p o s i t ively t o  PAS , AB p H  1 . 0  and 2 . 5 ,  AY , To l ud in e  b lu e , 
A zu r e  A pH 4 ,  Hal e s  c o l l o i da l  i ron and PAP S  ( F i gu r e s  2 . 6  
and 2 . 7 ) .  N eu ramini da s e  and hya l u roni das e r e a c t i v i ty wa s 
s im i l a r  to t h a t  o f  the s ub l ingu a l  g l and . The demi l un e s ,  
wh i ch w e r e  d i ff i c u l t t o  i dent i fy, d i d  not app e a r  t o  r e a c t  
w i t h  PAS o r  b a s i c s t a in s . 
6 2  
The int r a l obul a r  duc t s  cons i s t e d o f  c ub o i da l  ep i t he l ium ;  
no b as a l  s t r i a t i ons w e � e  ob s e rved but the g ranu l at e d  int r a ­
ep i the l i a l  c e l l  typ e f ound i n  t h e  duc t a l  ep i the l i um o f  t h e  
p a r o t i d  g l an d  was i n f r e quent ly ob s e rv e d  in t h e  duc t s  o f  
b o t h  do rs a l  a n d  int e rm e d i a t e  g l ands ( F i g u r e  2 . 6 . 3 ) . An 
unu sual f e a t ur e  o f  b o t h  g l an d s  was the p r e s enc e o f  c e l l u l a r  
d e b r i s  m i x e d  w i th s e c r e t o ry mat e r i a l  i n  t h e  l umen c o nt ent s 
o f  s ome s e c re t o ry endp i e c e s  and intra l ob u l a r  duc t s  ( F igure 
2 . 7 . 4 ) .  I nt e r l o bul a r  and int e r l ob a r  du c t s  w e r e  l ine d by 
c o l umna r  ep i t h e l i um w i t h g o b l e t  c e l l s  wh i l e  t h e  ma i n  duc t  
w a s  l in e d  b y  s t ra t i f i e d  c o l umna r  ep i the l ium w i th gob l e t  
c e l l s . The cont ent s o f  g ob l e t  c e l l s  r e a c t ed w i th PAS and 
b a s i c  s t a i n s . The c o nn e c t i v e  t i s s ue s t r oma e nc i rc l ing the 
l a rg e  duc t s  and the p a re nchyma f r e quent l y  cont a in e d  ma s t  
c e l l s  and p l a sma c e l l s . 
6 3  
B o th t h e  dors a l  and int e rme d i a t e  buc c a l  g l ands f requently 
c o n s i s t ed o f  sma l l i s l e t s o f  s e c re t o ry c e l l s  s im i l a r  in 
mo rpho l o gy and s t a i n i n g  p ro p e r t i e s  to p a r o t i d  g l and s e c r e t ­
o ry ce l l s  ( F i gure 2 . 7 . 3 ) .  The s e  i s l e t s  cont a ined th e i r  own 
int r a l obul a r  and int e r l obul a r  duc t a l  sys t ems . 
2 . 3 . 5  P a l a t ine G l ands 
The g l an du l a r  t i s s u e  b e n e a t h  the s o ft p a l a t e  was com­
p o s e d  of l a r g e  e l ong at e d  tub u l a r  endp i e c e s  w i t h a s s o c i a t e d  
d e m i l une c e l l s . The s e c re t o ry c e l l s  w e r e  po s i t i ve t o  PAS , 
AB pH 1 . 0 and 2 . 5 ,  AY , T o l ud i n e  b lue , A zure A pH 4 ,  PAPS 
and Hal e s  c o l l o i da l  i ro n  ( F i gu r e  2 . 8 ) .  The dem i l une c e l l s  
w e r e  r e l a t i v e l y  mo r e  c onsp i cuous ( F i gu r e  2 . 8 . 4 ) than tho s e  
o f  the do r s a l  and int e rme d i a t e  buc c a l  g l ands and r e a c t e d  
we a kly w i th PAS . The i n t r a l obul a r  duc t s  w e r e  s im i l a r  t o  
t h e  do r s a l  and int e rm e d i a t e  buc c a l  g l ands . The g ranu l a t e d  
i n t raep i th e l i a l c e l l typ e w a s  infrequent l y  o b s e rve d i n  t h e  
duc t s o f  t h e  p a l a t ine g l ands ( F i gure 2 . 8 . 3 ) .  
The int e r l o b a r  and int e r l o b u l a r  duc t s  we r e  l in e d  by 
c o l umna r  ep i t h e l i um w i t h o cc a s i o n a l  s c a t t e r e d  g ob l e t  c e l l s . 
T h e  conne c t i v e  t i s s ue s t roma s ur rounding the s e c r e t o ry t i s ­
s ue s and t h e  duc t s  f r e quent ly cont a i n e d  mas t  c e l l s  and p l a sma 
6 4  
c e l l s . 
Addi t i onal f e a t u r e s in common w i th th e do r s a l  and in t e r ­
m e d i a te b uc c a l  g l ands were the f requent o c cur rence o f  sma l l  
i s l e t s  o f  s e c re t o ry c e l l s  i den t i c a l  i n  mo rpho l o gy and s t a i n ­
i n g  p rope r t i e s  to tho s e  o f  t h e  p a ro t i d  g l and ( F i g u r e  2 . 8 . 1 ) 
and the p r e s ence o f  a n  atyp i c a l  s e c r e t o ry mechan i s m  ( F i gu r e  
2 . 8 . 2 ) .  
2 . 3 . 6  Po s t e r i o r  Tongu e  
G l andu l a r  t i s s ue found b e n e a th the e p i th e l i um o f  t h e  
d o r s a l  s u r f a c e  o f  the tongue wa s comp o s e d  o f  l a r g e  d i ame t e r  
t ub u l a r endp i e c e s  w i t h  f l a t t e n e d  demi lune ce l l s  o n  the i r  
b l i n d  ends ( F i gure 2 . 9 ) .  The s e c re t o ry c e l l s  w e r e  p o s i t iv e  
t o  PAS , A B  pH 1 . 0  a n d  2 . 5 , AY , T o l ud in e  b lue , A z u r e  A pH 4 ,  
PAPS and H a l e s  co l l o i da l  i ron . The dem i l une s w e r e  PAS and 
AB p o s i t i ve at pH 1 . 0  and 2 . 5  ( F i gure s 2 . 9 . 3  and 2 . 9 . 4 ) .  
The i n t r a l o b ul a r  duc t s  cons i s t e d  o f  c ub o i d a l  ep i the l ium 
w i th no e v i dence o f  b as a l  s t r i a t i on s  o r  g ranul a t e d  int r a ­
ep i t he l i a l  c e l l s  ob s e rved i n  t h e  int r a l o b u l a r  duc t s  o f  t h e  
p a r o t i d g l an d ,  t h e  b u c c a l  g l an d s  a n d  the pal a t ine g l ands . 
The int r a l obul a r  d uc t s  we r e  l inked t o  int e r l o bu l ar 
duc t s  wh i ch commun i c a t e d wi th t h e  e xt e r i o r  s u r f a c e  o f  the 
t ongue v i a  l a r g e  s ub e p i t he l i a l  e xt ra l o b u l ar duc t s  l ined 
by s t rat i f i e d c ubo i d a l  ep i the l i um ( F i gu r e  2 . 9 . 1 ) .  
P l asma c e l l s  and mas t c e l l s  were commonly o b s e rve d in 
t h e  s ub ep i th e l i a l  c onne c t ive t i s su e s  and b e tween s ec r e t o ry 
endp i e c e s  and duc t s . 
2 .  3 .  7 Pharynge a l  G l ands 
G l ands l o c a t e d  w i thin t h e  s ub ep i t he l i al conn e c t ive 
t i s s ue o f  the l ingua l surfac e o f  the ep i g l o t t i s  w e re c omp ­
o s e d  o f  s ho rt tubu l a r  endp i e c e s  w i th l a r g e  r o unde d dem i l un e  
c e l l s on t h e i r  b l ind ends ( F i g ure 2 . 1 0 ) . T h e  s e c re t o ry ce l ls 
w e r e p o s i t i v e  t o  PAS , AB pH 1 . 0  and 2 . 5 ,  AY , PAP S , T o l ud ine 
b l ue , A z u r e  A pH 4 and Ha l e s  co l l o i da l  i ron .  The dem i l un e s  
c o n t a ined abundant e o s inop h i l i c cytop l as m i c  g r anul e s  that 
w e r e  B I AL p o s i t i ve , PAS p o s i t iv e  ( F i gu r e  2 . 1 0 . 2 )  and w e a k l y  
r e a c t i v e  w i th A B  pH 1 . 0  and 2 . 5  and T o ludine b lu e . 
6 5  
The i n t r a l obul a r  duc t s  w e r e  l ine d by co lumna r  ep i the l i a l  
c e l l s  wh i c h  we r e  f r e quent ly mo d i f i e d  a s  s e cre t o ry c e l l s , t h e  
c o n t e n t s  o f  wh i ch r e a c t e d  w i t h  PAS and b as i c s t a in s . Mo rph­
o l o g i c a l l y , s ome of the mo d i f i ed ep i th e l i a l  s e c r e t o ry c e l l s  
w e r e  typ i ca l  g ob l e t  c e l l s  ( F i gure 2 . 1 0 . 3 ) , wh i l e  o the r s  h a d  
t h e  appe a rance o f  s e c re t o ry endp i e c e s  wh i ch s e emed to c o m ­
mun i c a t e  d i rec t l y w i th int r a l o b u l a r  duc t s  ( F i gu r e s  2 . 1 0 . 3  
and 2 . 1 0 . 4 ) . The i n t ra l o b u l a r  duc t s  c o mmun i c a t e d  with t h e  
s ur fa c e  v i a  int e r l ob ul a r  duc t s  and s h o r t  subep i th e l i a l  
e xc r e t o ry duc t s . 
The co nne c t ive t i s sue s t roma s u r r ounding s e c r e t o ry end ­
p i e c e s  and duc t s  c o n t a in e d  p l a sma c e l l s  and ma s t  c e l l s ; 
p l a sma c e l l  numb e r s  w e r e  u s u a l ly h i gh e r  than i n  o th e r  mino r 
g l ands ( F i gure 2 . 1 0 . 2 ) . 
2 .  3 .  8 I mmuno h i s t o chem i s t ry 
Ant i b o d i e s  to l ow mo l e c u l a r  we i gh t  p ro t e i n b ands 4 and 
1 0  r e a c t e d  w i th p a r o t i d  and v ent r a l  b u c c a l  g l and t i s su e s . 
Wh i l s t  o n ly s ome s e c re t o ry c e l l s  r e a c t e d  with an t i  b and 4 
ant i b o dy , ant i b an d  1 0  ant i b o dy po s i t iv e  ce l l s  w e r e  w i de ­
s p read ( F i g ure 2 . 3 ) .  P aro t i d  t i s s ue s amp l e s  o f  b o t h  l ow 
( L . S . )  and high ( H . S . )  b l o a t  s u s c ep t i b l e  ca t t l e  e xamine d 
had equa l quant i t i e s  o f  s e c r e t o ry c e l l s  that r e a c t ed w i th 
ant i  b an d  4 ant i b o dy .  B an d  4 wa s a dd i t iona l ly i dent i f i e d  
i n  the dem i l une s o f  the int e rmedi a t e  b uccal g l and ( F i gu r e  
2 . 6 . 4 ) .  Ant i b o v i n e  b and 4 an t i b o dy fa i l e d t o  c r o s s  r e a c t  
w i th b and 4 o r  e q u ival ent s e c re t o ry p r o t e i n  p r e s ent i n  
s h e ep p aro t i d  t i s s ue s . 
B an d  8 was f o und t o  b e  l o cal i s e d i n  the demi lune s o f  
the mandibul a r  g l and ( F i g ur e  2 . 4 . 8 ) and band 9 in the 
dem i l une s o f  the s ub l ingua l g l and ( F i g u r e  2 . 5 . 6 ) .  
Ant i b o dy t o  I gA r e a c t e d  w i th s ub ep i t hel i a l  p l asma c e l l s  
o f  the mandib u l a r  and s ub l ingual g l a n d s  ( F i gu r e  2 . 4 . 1 1 ) . 
P o s i t i ve s t a i n i n g  in a l l t i s s u e s  wh i ch c o n t a i n e d  t h e  
ant i g en ( s al i v a ry p ro t e in )  w a s  r e c o gn i s ed b y  d ep o s i t s  o f  
dark b r own t o  b l a c k  r e a c t ion p ro duc t g en e ra l ly d i s t r i b u t e d  
throughout t h e  cy t o p l asm o f  s pe c i fi c  s e c re t o ry c e l l s  w i th 
v a ry ing deg r e e s  o f  i n t ens i ty .  
6 6  
F I GURE 2 . 1  
PAROT I D  AND VENTRAL BU CCAL GLANDS 
2 . 1 . 1  
2 . 1 . 2 
2 . 1 . 3 
2 .  1 .  4 
D i a s t a s e - r e s i s t ant PAS p o s i t i ve ma t e r i a l  d i s t r i b ­
ut ed i n  s up r anuc l e a r  s i t e s  o f  s e c r e t o ry c e l l s . 
( PAS / Tart r a z ine ) .  Magn i f i c at ion : x 6 5 0  
A r a r e  ob s e rva t i on o f  a g roup o f  s e c r e t o ry end ­
p i e c e  c e l l s  w h i ch have s t a i ned f o r  carboxy l a t e d  
a c i d  muco s ub s t a n c e s . 
(Al c i an Blue pH 2 .  5 / H  and E ). Ma g n i f i c at ion : x 1 0 0  
Ma i n  exc r e t o ry duct l i n e d  by s t r a t i f i e d c o l umna r  
ep i th e l ium w i th g ob l e t  c e l l s . 
( H a l e s  co l l o i da l  i ron/ N eu t r a l  R e d ) . 
Magn i f i c at i o n : x 1 0 0  
Ma in excreto ry du ct l i ned by s t r a t i f i e d c o l umn a r  
ep i th e l ium w i t h g o b l e t  c e l l s  cont a in ing s u l ph a t e d  
a c i d  muc o s ub s t anc e s . 
(Al c i an B l u e  pH 1 . 0 / P TAH ) . Magn i f i cat i on : x 2 6 0  

F I G U RE 2 . 2 
PARO T I D AN D VENT RA L BUC CAL G LAN D I NT RALOB ULAR DUCT S 
2 .  2 .  1 
2 .  2 .  2 
2 .  2 .  3 
2 .  2 .  4 
D i a s t a s e - r e s i s t ant PAS p o s i t ive int ra l o bul a r  
d u c t  c e l l s  w i t h " ap i c a l  b l eb s " . 
( PA S / T ar t r a z ine ) . Mag n i f i c a t i o n : x 6 5 0  
D i s t r i but i on o f  intraep i t h e l i a l  g ranu l a r  duc t 
c e l l s  wi t h in t h e  wal l o f  an i nt ra lobu l a r  duc t . 
G ra nul ar duc t c e l l s  var i e d  cons i d e r a b l y  in 
m o rp h o l o g y  but conta ined nu c l e i  s im i l a r  to 
tho s e  o f  p lasma c e l l s . 
( B i s ma r ck Brown/ H a ema t o x y l i n ) . 
Ma g n i f i c a t ion : x 6 5 0  
I nt r a l o bul a r  duc t c e l ls l i n e d  by h i g h  cubo i da l  
ep i th e l ium w i thout b a s a l  s t r i a t i o ns . 
( PTAH ) . Magn i f i ca t i o n : x 6 5 0  
Sh e ep p ar o t i d  g l an d  in t ra l ob u l a r  duc t s  l i ne d 
b y  t a l l  c o l umna r e p i t h e l i um w i th ac i dophi l i c  
b a s a l s t r i a t i o ns . 
( P TAH ) . Magni f i c a t ion : x 6 5 0  
2 
F I GURE  2 . 3 
IMMUNOH I STOCHEM I STRY : D I STRI BUT I ON OF PAROT I D  AND 
VENTRAL BUCCAL G LAND SAL I VARY PROTE I N  BANDS 4 AND 1 0  
2 .  3 .  1 
2 .  3 .  2 
2 . 3 . 3  
2 . 3 . 4 
Cont r o l  s e ct ion  w i thout  ant ibody to  
s a l ivary pro t e in . 
( L i g h t  Green counter s ta in ) . 
Ma gni f i ca t ion : x 1 0 0  
D i s t r ibut ion  o f  ant i band 4 p o s i t ive  p ro t eo s e rous  
,... 
c e l l s . 
( Da rk  b rown p r e c ip i t a t e  w i th  DAB sub s t rat e ) . 
Magn i f icat ion : x 6 0  
Wide spread  di s t r ibut ion  o f  ant i  band 1 0  p o s i t ive 
p ro t eo s e rous c e l l s .  
( B l ack  pre c ip i t a t e  w i th  carba zo l e  sub s trate ) . 
Magn i f i ca t ion : x 1 0 0 
D i s t ribut ion  o f  both  ant i  b and 4 ( brown precip ­
i ta t e ) and ant i band  1 0  (b l ack p r ec ip i t ate )  
p o s i t ive  proteo s erous  c e l l s .  
Magn i f i c a t i on : x 6 5 0  

F I GURE 2 . 4  
MAN D I BULAR G LAN D 
2 .  4 .  1 
2 .  4 .  2 
2 .  4 .  3 
2 .  4 .  4 
C h a r a c t e r i s t i c  app e a r ance o f  t ub ul a r  endp i e c e s  
w i th d e m i lun e s .  
( G o mo r i  Aldehyde Fuchs i n ) . 
Mag n i f i c a t ion : x 1 0 0  
D i s t r i but i on o f  c a rboxy l a t ed a c i d  mu c o s ub s tanc e s  
i n  muc o u s  c e l l s .  
( A l c i an Y e l l ow/H and E ) . 
Magn i f i c a t i on : x 2 6 0  
T o l ud i n e  b l ue m e t a chroma s i a  o f  mu c o u s  c e l l s . 
N o t e  ta l l  co l umna r s t r i a t e d  du c t  c e l l s . 
Magn i f i c a t i on : x 2 6 0  
A c i d  muc o s ubs tances o f  muc o u s  c e l l s  d emo ns t r a t e d  
w i th Hal e s  co l l o i da l  i r on . No t e  w e a k  co l l o i d a l  
i r o n  s t a i n ing in dem i l un e s . 
( H a l e s  c o l l o i da l  i ron/ N eu t r a l  Red ) . 
Magn i f i c a t ion : x 2 6 0  

F I G U RE 2 . 4  ( C ont i nu e d )  
2 .  4 .  5 
2 .  4 .  6 
2 .  4 .  7 
2 .  4 .  8 
D i s t r i but ion o f  PAS p o s i t i ve mu c o s ub s t anc e s  
i n  dem i l un e s  and a m i x t u r e  o f  a c i d i c  and 
neut r a l  muc o s ubs tan c e s  in mu c o u s  c e l l s . 
( PAS/Al c i an B lue pH 2 . 5 / H  and E ) . 
Mag n i f i c a t i on : x 6 5 0 
A l c i an B l ue p o s i t iv e  muc o u s  c e l l s  and 
d em i l un e s  w i th ac i do p h i l ( d a r k  b l ue ) 
g r anul e s . No t e  d i s t i n c t  a c i do p h i l i c  b a s a l  
s t r i a t i o ns o f  i nt r a l o bu l a r  duc t s  and s ub ­
e p i t h e l i a l  p l a s ma c e l l s  s c a t t e r e d  b e tw e e n  
e n dp i e c e s  and duct s .  
( P TAH/Al c i an B l ue pH 2 . 5 ) .  
M a gn i f i c a t i o n : x 6 5 0 
B IA L  p o s i t ive s i a l i c a c i d  r e s i du e s  i n  
d e m i lune c e l l s . 
M ag n i f i c a t i o n : x 6 5 0 
D em i lune c e l l s  p o s i t i ve f o r  s a l i v a ry 
p r o t e i n band 8 .  
( L i g ht G r e en count e r s ta i n ) . 
Ma g n i f i c at ion : x 2 6 0  

F I G U RE 2 . 4 ( C ont inu e d )  
2 .  4 .  9 Muc o u s  c e l l s  s t a i ned for c a rb o xy l a t e d  
a c i d  muc o s ub s t anc e s . No t e  a c i doph i l i c 
cont e n t  o f  demi l une s . 
( A l c i an B l ue pH 2 . 5 / H  and E ) . 
Magn i f i c at i o n : x 2 6 0  
2 . 4 . 1 0 N e u rami n i da s e  a c t i v i t y , s hown by l o s s  o f  A l c i an 
B l ue s t a i n i ng o f  mucous c e l l s ;  c a rboxy l a t e d  a c i d  
muc o s u b s t an c e s  c an the r e fo r e  b e  a t t r i b u t e d  to 
s i a l i c ac i d  re s i dues . No t e  weak A l c i a n  B l ue 
s t a i n i ng of ma s t  c e l l s  a t  pH 2 . 5 .  
( N euram i n i da s e /A l c i a n B l u e  pH 2 . 5 / H  and E ) . 
Magn i f i c a t i o n : x 2 6 0  
2 . 4 . 1 1 An immuno h i s t o chemi c a l  d emo n s t r a t i o n o f  p l a sma 
c e l l s  wh i c h  have r e a c t e d  w i th ant i I gA .  
( L i g ht G r e e n coun t e r s t a i n ) . 
Magn i f i c a t i o n : x 6 5 0 
2 . 4 . 1 2 Ma i n  exc r e t o ry duc t : s t r a t i f i ed co l umna r 
ep i the l ium w i tho ut g ob l e t c e l l s . 
( P TAH/ A l c i an B l u e  pH 2 . 5 ) .  
Magn i f ic a t i o n : x 6 5 0  

F I G U RE 2 . 5 
S UB L I N G UA L  G LAND 
2 .  5 . 1  
2 .  5 .  2 
2 .  5 .  3 
2 .  5 .  4 
S e c re t o ry e n dp i e c e c e l l s  comp o s e d  o f  mucous c e l l s  
c o n t a i n i ng a m i x t u r e  o f  ac i d i c  and n e u t r a l  
muc o s ub s tanc e s . No t e  p r e dom inant l y  p r o t e o ­
s e r o u s  d em i l une s . 
( PA S / Al c i an B l ue pH 2 . 5 ) .  
Mag n i f i c a t i o n : x 6 5 0  
D i s t r i but i o n  o f  s u lpha t e d  muc o s ub s t an c e s  i n  
muc o u s  c e l l s . 
( A l c i an B l u e  pH 1 . 0 / H  and E ) . 
Ma g n i f i c a t i o n : x 2 6 0  
The cha r a c t e r i s t i c  h i s t o l o g i c a l  ap p e a r a n c e  
o f  the s u b l i n g u a l g l ands , w i t h num e r o u s  p l a sma 
c e l l s  a n d  s t r i a t e d  duc t s . 
(A l c i an Y e l l ow/ H and E ) . 
Magn i f i ca t i on : x 2 6 0  
A r e a s  o f  sub l i ngua l g l a nd t i s s ue wh i c h d i ff e r e d 
f r om t ho s e  s h own i n  F i g . 2 . 5 . 3 .  N o t e  l ack o f  
p l a s ma c e l l s  a n d  s t r i a t e d  duc ts . 
( A l c i a n  Y e l l ow / H and E ) . 
Magn i f i c a t i o n : x 2 6 0  

F I G U R E  2 . 5 ( C o n t inue d )  
2 .  5 .  5 
2 .  5 .  6 
2 .  5 .  7 
2 .  5 .  8 
I n t r a l o b u l ar du c t s  w i t h  t a l l  c o l umn a r  c e l l s  
and d i s t inct a c i doph i l i c b a s a l  s t r i a t i o ns . 
( PTAH ) . Magn i f i c a t i o n : x 6 5 0  
D i s t r i b u t i on o f  p r o t e i n  b and 9 i n  d em i lune 
c e l l s . N o t e  nume rous s ub e p i th e l i a l p l a sma 
c e l l s . 
( L i g ht G r een c o unt e r s t a i n ) . 
Mag n i f i c a t i o n : x 6 5 0  
A l a r g e g ro up o f  p l a s ma c e l l s  d i s t r i b u t e d  
b e tw e en s e c r e t o ry endp i ec e s  and duc t s . 
( H and E ) . Magn i f i c a t i o n : x 2 6 0  
P l a s ma c e l l s s t a i ne d  w i t h M e thy l  G r e en - Pyro n i n . 
No t e  pyron inoph i l i a o f  p l a sma c e l l  c y t op l a s m i c  
RNA . 
Magn i f i c a t i o n : x 6 5 0  

F I G U RE 2 . 6 
I NT E RME D I AT E  BUCCAL G LAN D 
2 .  6 . 1  
2 .  6 .  2 
2 .  6 .  3 
2 .  6 .  4 
Muc o u s  c e l l s cont a in ing a m i x t u r e  o f  neut r a l  
an d ac i d i c  mu c o s ubs tance s .  No t e  i n t r a l o bul a r  
duc t s  l i n e d  b y  cubo i d a l  ep i t he l i um .  
( PA S /Al c i an B l u e / H an d E ) . 
Magni f i c a t i on : x 2 6 0  
Muc o us c e l l s c ont a in i ng PAS p o s i t i v e  rnuco s ub s t an c e s . 
( PAS/ Ta r t r a z i ne ) . 
M a gn i f i c a t ion : x 1 0 0  
An i n t ra l o bul a r  duct wi th t h r e e  int r a ep i the l i a l  
g ranu l a r  duc t c e l l s s im i l a r  t o  tho s e  o f  the 
p a r o t i d  a n d  vent r a l  buc c a l  g l an d s . 
( A l c i an B l ue pH 2 . 5 / H  and E ) . 
M a g n i f i c a t i o n : x 6 5 0  
P r o t e o s e r o u s d e rn i lunes c o n t a i n i ng s a l ivary 
p ro t e i n b and 4 .  
( L i g ht G r e en c o un t e r s t a i n ) . 
M a g n i f i c a t i on : x 2 6 0  

F I GURE 2 . 7  
DO RSAL ( UP P E R) B U C CAL G LAND 
2 .  7 . 1  
2 .  7 .  2 
2 .  7 .  3 
2 .  7 .  4 
To l ud i ne b l ue meta chroma s i a  o f  muc o u s  
c e l l s . Not e nume rous i n t e r l o bul a r  and 
i n t ra l ob u l a r  duc t s . 
Magni f i c at i on : x 2 6 0  
The d i s t r i but i o n  o f  s u l p ha t e  g r o up s  in 
s ome muc ous c e l l s . No t e  i n t en s e  A l c i an 
B l ue s t a in i ng o f  ma s t  c e l l s  a t  l ow pH . 
(Al c i an B l u e  pH 1 . 0 / H  and E ) . 
Mag n i f i cat i o n : x 2 6 0  
A g r o up o f  p r o t e o s e rous c e l l s  s c at t e r e d  
among mu cous endp i e c e s . N o t e  s ep a r a t e  
duc t a l  sys t em w i t hin p ro t e o s e ro u s  i s l e t . 
(A l c i an B l u e  pH 2 . 5 / H  and E ) . 
Mag n i f i c a t i on : x 6 5 0 
A p o s s i b l e  ho l o c r i n e  s e c r e t o ry m e c han i sm . 
No t e  c e l l u l a r ma t t e r  m i x e d  w i t h s e c r e t o ry 
p r o duc t s . 
(Al c i an B l u e  pH 2 . 5 / PTAH ) . 
Magn i f i c a t i o n : x 6 5 0 

F I G U RE 2 . 8  
PA LAT I NE G LANDS 
2 .  8 . 1  
2 .  8 .  2 
2 .  8 .  3 
2 .  8 .  4 
D i s t r i bu t io n o f  PAS po s i t i v e  mu co s ub s t a n c e s  
i n  mu cous c e l l s . No t e  n ume r o us p r o t e o s e ro u s  
c e l l s  s c a t t e r e d  among muc o u s  c e l l s . 
( PAS/ Tar t ra z i n e ) . Mag n i f i c a t i o n : x 2 6 0  
A p o s s ib l e  h o l o c r i ne s e c r e t o ry mechan i sm 
w i t h i n  s o me s e c r e t o ry endp i e c e s . N o t e  
c e l l u l a r  ma t t e r  m i x e d  w i t h mucus a n d  p r e s e n c e  
o f  s u lpha t e d  muc o s ub s t an c e s  i n  muc o u s  c e l l s . 
(A l c i an B l u e  pH 1 . 0 / H  and E ) . 
Ma g n i f i c a t i o n :  x 2 6 0  
An i n t ra e p i t h e l i a l  g ranu l ar duc t c e l l  l o c a t e d  
w i t hi n  th e wa l l  o f  a n  i n t r a l o bu l ar duc t . 
(Al c i an B l ue pH 2 . 5/ PTAH ) . 
Mag n i f i c a t i o n : x 6 5 0  
A typ i c a l  s e c r e t o ry e n dp i e c e  wi th c a rb o xy l a t ed 
a c i d  muc o s ub s tanc e s  i n  mucou s c e l l s  and 
p r e domin a n t l y  p ro t e o s e r o u s  d em i l un e s .  
( A l c i an B l ue pH 2 . 5/ PTAH ) . 
Mag n i f i c a t i o n : x 6 5 0  

F I G U RE 2 . 9 
P O S T E R I O R TONGUE G LAN DS 
2 .  9 .  1 
2 .  9 .  2 
2 .  9 .  3 
2 .  9 .  4 
S u b ep i th e l i a l  excret o ry duc t s  l o c a t e d  be t w e e n 
muc ous e ndp i e c e s  and d o r s a l  s u r f a c e  o f  t o ngue . 
( A l c i an Y e l l ow/ H and E ) . 
M a g n i f i c a t i o n : x 2 6 0  
S e c r e t o ry endp i e c e s  c o n t a i n i ng muc ous c e l l s  
s t a i ne d  w i t h  c o l l o i da l i r o n . N o t e  l a r g e  
i n t e r l o b u l a r  duct s .  
( H a l e s  c o l l o i da l  i ron ) . 
M a g n i f i c a t i o n : x 2 6 0 
D i s t r i b u t ion o f  Al c ian B l u e  p o s i t i ve sulpha t e  
g r o up s  i n  demi l une s . 
( A l c i an B l ue p H  1 . 0 / H an d E ) . 
Ma gn i f i c a t io n : x 6 5 0  
D i s t r i b u t i o n o f  PAS po s i t i ve muco s u b s t ance s in 
d e m i l une s . Mu cous ce l l s  hav e s t a i n e d  for b o t h 
a c i di c  and neu t r a l  muc o s ub s t anc e s . 
(A l c i an B l ue pH 2 . 5 / PAS ) . 
Magni f i c a t ion : x 6 5 0  

F I G U RE 2 . 1 0 
PHARY N G EA L  G LANDS 
2 . 1 0 . 1 D i s t r i but i o n  o f  g l andu l a r  t i s s ue in t h e 
e p i g l o t t i s .  No t e  e l a s t i c  c a r t i l ag e . 
(A l c i an B lue pH 2 . 5 / PAS ) . 
Magni f i c a t i o n : x 1 0 0  
2 . 1 0 . 2 Muc o u s  c e l l s  cont a i n i ng a m i x t u r e  o f  b o th 
n e u t r a l  and a c i d i c  muc o s ub s t an c e s . D em i l une s 
c o nt a in PAS p o s i t ive ne u t r a l  muco s ub s t anc e s . 
N o t e  num e rous p l a s ma c e l l s  b e tween s e c r e t o ry 
endp i e c e s .  
(Al c i an Blue pH 2 . 5 / PAS ) . 
Mag n i f i c a t i o n : x 6 5 0  
2 . 1 0 . 3  I n t r a l o b u l a r  duc t  ep i t hel i a  l in e d  w i t h  
gob l e t  c e l l s . 
(Ha l e s  co l l o i da l  i r on/Neut r a l  Re d ) . 
Mag ni f i c a t i on : x 6 5 0  
2 . 1 0 . 4 S e c r e t o ry endp i e c e s  wh i ch commun i c a t e di rect ly 
w i t h  i n t r a l o bul a r  duc t s . 
(A l c i an B l u e  p H  2 . 5/ PTAH ) . 
Magni f i c a t i o n : x 6 5 0  

F I G U RE 2 . 1 1  
HYALURON I DASE A CT I V I TY 
2 . 1 1 . 1  Demons t rat i o n  o f  s u lpha t e  g roups i n  pharyng ea l 
g l and m uco us c e l l s  an d mat r i x  o f  c a r t i l a g e , 
s hown b y  A l c i an B l ue s t a i n i ng at l ow pH . 
( Al c i an B l ue p H  1 . 0 / H  and E ) . 
Magni f i c a t i o n : x 2 6 0  
2 . 1 1 . 2  Hya l u ro n i da s e  a c t i v i ty h a s  no t i c e ab ly r e duced 
s t a i n i n g  i n t en s i ty of s u lphat e g ro u p s  f o und in 
t h e  c a r t i l ag e  ma t r i x .  S t a i n ing i n t en s i t y  o f  
s u lpha t e g r o up s  di s t r i but e d  in mu c o u s  ce l l s  
r ema i n s  un a l t e r e d .  S i mi l a r  r e s u l t s  were 
o b t a i n e d f rom muco u s  c e l l s  o f  the mandibu l a r , 
s ub l ing ual and the m i n o r  g l and s ( w i t h th e 
e xc ep t i on o f  t h e  v ent r a l  bucc a l ) ;  a r e s ul t 
whi ch s ugg e s t s that muco u s  c e l l s  p robably 
con t a i n a s u l p h a t e d  g l yc op r o t e i n ( or a 
s i a l o - s ulpha t e d  g l ycopro t e i n )  wh e r e a s  the 
c a r t i l a g e ma t r i x  i s  l i k e l y to con t a i n ac i d  
mucop o l y s a c c ha r i d e s s uch a s  chond ro i t in 
s u lpha t e s . 
( Hyalu r on i da s e / A l c i an B l ue pH 1 . 0 / H  and E ) . 
Magn i f i c a t ion : x 2 6 0  

2 . 4  D i s c u s s i o n  
T h e  maj o r  and mino r s a l ivary g l and e n dp i e c e s  o f  c a t t l e  
c o n s i s t e d  o f  tubul a r  endp i e c e s  c o mp o s e d  o f  s e c re t o ry c e l l s  
s p e c i a l i s ed fo r s ynthe s i s and s e c r e t i on o f  muc o s ub s t anc e s ; 
t h e  comp o s i t i o n  o f  the s e  wa s demon s t r a t e d  by a range o f  
h i s t o c h em i c a l  m e t h o d s . 
I n  g en e r a l , the h i s t o c hem i c a l  f i n d i n g s  suc c e s s ful l y  
e s t ab l i s h e d  t h e  c l as s i f i c a t i o n  o f  s e c r e t o ry c e l l s  i n t o  two 
d i v i s i on s : p ro t e o s e r ous and muc o u s . The s e c r e t o ry c e l l s  
that c o n t a i n e d  only s c a t t e re d  PAS p o s i t i v e  mat e r i a l , s yn ­
t h e s i s ed s ome n e u t r a l  g l ycop r o t e i n s  ( s e e  App end i c e s  I and 
I I )  and we r e  c l a s s i fi e d  a s  p ro t e o s e ro u s  c e l l s . The s e  c e l l s  
a r e , howeve r ,  p r imar i l y s p e c i a l i s ed f o r  the s e c r e t i o n  o f  
w a t e r  and e l e c t ro l yt e s . The s e c re t o ry c e l l s ,  wh i c h  c o n ­
t a in e d  muc o s ub s t anc e s  tha t r e ac t e d  s t rong ly w i th PAS and 
b a s i c  s t a in s  s uch a s  Al c i an b l ue , Al c ian ye l l ow ,  T o l ud ine 
b l ue , A z u r e  A and c o l l o i d a l  i ro n  c on s i s t e d  of a mixture o f  
b o th n e u t r a l  and ac i di c  g l y c o p r o t e i n s  ( s e e App e n d i c e s  I 
and I I ) , s uch c e l l s  w e r e  c l a s s i f i e d  a s  muc o us c e l l s . The s e  
c e l l s  may a dd i t i o na l ly have a s e c o n d a ry r o l e  i n  the s e c r e t ­
i o n  o f  s ome wa t e r  and e l e c t ro ly t e s . 
6 7  
Th e p a ro t i d  g l and s e c r e t o ry p o r t i o n  wa s comp o s e d  p re dom­
inan t l y  o f  p r o t e o s e rous c e l l s  wh i ch o cc a s i o na l l y cont a i ned 
d i a s t a s e  r e s i s t an t  PAS p o s i t ive ma t e r i a l  ( F igure 2 . 1 . 1 ) . 
Th i s  s ug g e s t s that in c a t t l e  t he p a ro t i d  g l and c o u l d  b e  
r e s p on s i b l e  f o r  the synth e s i s  and s e c r e t i on o f  s ome n e u t r a l  
g l ycop r o t e ins in ad d i t i on t o  s e c r e t ing l a rge quant i t i e s  o f  
wa t e r  and e l e c t ro l yt e s . S e ve r a l  g l y c op r o t e i n c omp one n t s  
from b o v i n e  s a l i va have r e c en t l y  b e e n  i s o l a t e d  by Jone s e t  
a l . , ( 1 9 8 2 ) ; g lycop r o t e i n b ands 1 ,  3 ,  5 and 6 i n  p a r t i c u l ar , 
a r e  s e c r e t e d  by the p a ro t i d  g l an d s  ( T ab l e  1 . 1 ) . I n  a d d i t i on, 
t h e r e  w a s  s om e  e v i dence t o  in d i c a t e  that the p a ro t i d g l ands 
may a l s o  b e  s e c re t i ng s ma l l  quant i t i e s  of s i a l i c  a c i d  
( F i g u r e  2 . 1 . 2 ) s inc e i n  a few t i s s u e  s amp l e s  examine d , 
i s o l a t e d  s e c r e t o ry c e l l s  had r e a c t e d  w i th AB (pH 2 . 5 ) ,  AY 
and c o l l o i da l  i r on , al l b a s i c  s t a i ns s p e c i f i c  f o r  c a r b o xyl 
r a d i c l e s . AB (pH 2 . 5 ) and T o l ud ine b l ue p o s i t i v e  ma t e r i a l  
was a l s o  o c c a s i on a l l y i den t i f i e d  i n  t h e  lume ns o f  i n t ra ­
l obul a r  and int e r l o b u l a r  duc t s , fu r th e r  d emon s t ra t i ng t h a t  
ac i d i c  g l y c o p ro t e i n s  s uch a s  s i a l i c  ac i d  may b e  s e c r e t e d  b y  
t h e  b o v i n e  p a ro t i d g l ands , a l b e i t  in sma l l  quant i t i e s . 
6 8  
H i s to l o g i ca l  o b s e rvat i on s  o f  p a ro t i d g l and duc t s  r e v ­
e a l e d  a n  unu sual a b s ence o f  b a s a l  s t r i a t i ons i n  t h e  i n t r a ­
l ob u l a r  duc t s  ( F i gu r e  2 . 2 . 3 ) where c on s i de rab l e  wa t e r  and 
e l e c t ro l y t e  t r ansp o r t  ac t iv i ty is exp e c t e d  to o c c u r  ( Young 
and van L ennep , 1 9 7 9 ) . B a s a l  s t r i a t i o n s , wh i c h  h o u s e  num ­
e rous m i t o chond r i a  ( van L ennep e t  a l . , 1 9 7 7 )  a r e  u s ua l ly 
foun d  in duc t s  s p e c i a l i s e d f o r i o n i c  t r anspo r t . The above 
f ind i ng p e r t a i n i n g  t o  b o v i ne p aro t i d int ralobu l a r  duc t s  c o n ­
f l i c t ed w i t h  ob s e rv a t ions ma d e  o n  s h e ep paro t i d g l and t i s ­
s u e s  ( F i gu r e  2 . 2 . 4 ) whe r e  " s t r i a t e d" ( in t ral obul a r )  duc t s  
we r e  abun d a n t . I n  g ene r a l , s tr i a t e d  duc t s  o f  m o s t  mamma l i an 
s p e c i e s  a r e  p rominent in the p a ro t i d  g l and , nex t the man­
d ibul a r  and l a s t l y  the s ub l i n g ua l  ( L e e s on ,  1 9 6 7 ) . Howeve r ,  
the b o v i n e  p a ro t i d g l and c l e a r l y  depar t s  from t h i s g en e r a l  
ru l e , b e c au s e  o f  r e l a t i v e l y  p o o r ly deve l op e d  i nt r a l ob u l a r  
duc t s . 
A l though b a s a l  s t r i a t i o n s  we re a b s ent from p a r o t i d  g l and 
int r a l o b u l a r  duc t s ,  there wa s s ome e v i dence to s ug g e s t t h a t  
these may p o s s ib l y  b e  invo l v e d  i n  synthe s i s  o f  p r o t e in -
a c i o us ma t e r i a l , s in c e  d i a s t a s e - re s i s t an t  PAS p o s i t ive g r an ­
ul e s  w e r e  s ome t im e s ob s e rved i n  the cy t o p l a s m  o f  s ome duc t  
c e l l s  ( F i g ure 2 . 2 . 1 ) .  A t  p r e s ent a s a t i s fa c t o ry e xp l an a t i o n  
cann o t  b e  o f fe r e d  f o r  the imp l i ca t i on o f  th i s  PAS s t a in i n g , 
ap a r t  f r o m  a t t r i but i ng i t  t o  a neut r a l  g lycop r o t e i n p r e s en t  
i n  s ome duc t  c e l l s .  Ano t h e r  feature o f  int ra l obu l a r duc t  
ce l l s  wa s the p re s en c e  o f  PAS p o s i t ive ap i c a l  c y t op l a s m i c  
p r o t ru s i o n s  o r  " ap i c a l  b l e b s " ; al though such " ap i c a l  b l eb s " 
hav e  b e e n  p r e v i o u s l y  a t t r i but e d  t o  s t a g e s  o f  an ap o c r i ne 
s e c r e t o ry mechan i s m ,  the i r  r e a l  s i gn i f i c anc e is s t i l l  d e b a t e d  
b y  mos t au tho r s  ( s e e  Young a n d  van L ennep , 1 9 7 8 ) . 
Go b l e t  ce l l s , wh i ch s t a i n e d  i n t en s e ly fo r b o t h  neut r a l  
and ac i d i c  muc o s ub s t anc e s , w e r e  p re s ent . in t h e  m a in e x c r e t ­
o ry duc t o f  t h e  b o v ine p a ro t i d g l an d s  ( F igure s 2 . 1 . 3  and 
2 . 1 . 4 ) .  Ove ra l l , s ome mucu s  c ou l d  t h e r e fo re s t i l l  b e  s e c ­
re t e d b y  the p a r o t i d  g l ands , i n  a dd i t i on t o  i t s  v o l uminous 
wa t e ry s e c r e t i on . 
6 9  
I n  c o n t r a s t t o  the p a ro t i d g l and , the man d i b u l a r  g l an d  
s e c r e t o ry c e l l s  c o nt a i n e d  mucus , comp o s e d o f  a c i d i c  and 
neu t ra l  g lycop ro t e i ns . The PAS me t ho d ,  t o g e t h e r  w i t h  ac e t ­
yl a t i on and s ub s e quent s ap on � f i c a t i on ( s ee App e n d i x  I )  
e s t ab l i s he d  the p r e s ence o f  g lyco l g ro up s  in muc o s ub s tanc e s . 
B i o ch em i c a l ly , t h e s e  b e l on g  t o  g a l a c t o s e , manno s e ,  a c e t y l ­
g l uc o s am i n e , a c e t y l g a l ac t o s amine , fuc o s e  and s i a l i c  a c i ds , 
( Cu l l i n g , 1 9 7 4 ) . The a c i d i c  s id e  g roups o f  muc o s ub s t an c e s ,  
inve s t i g a t e d  by us e o f  b a s i c  s t a i n s  and b l o c k i n g  methods 
( s e e App endix I )  were i den t i f i e d  a s  c a rboxy l g r o up s  and 
s u l p h a t e  g roup s wh i ch , in e p i the l i a l  s e cret i on s , b e l ong t o  
de r i va t i ve s  o f  s ia l i c a c i ds and s u l p h a t e d  g ly c o p ro t e ins 
( Re i d  and C l amp , 1 9 7 8 ) . 
Th e en zyme hya l uro n i da s e  u t i l i z e d to de t e rm ine t h e  
o r i g in o f  s u l ph a t e  g roup s i n  muc ous s e c r e t i on s , d i d  no t 
r e duce o r  e l im i n a t e  AB s t a in ing a t  p H  1 . 0  ( F i g u r e  2 . 1 1 ) . 
Th i s  ru l e s out t h e  p o s s ib i l i ty that s i gni f i c an t  quant i t i e s  
o f  ac i d  muc op o ly s a c cha r i de s  s uch a s  c hondro i t in sulpha t e s  
and hay l uron i c  a c i d s , cou l d  b e  p r e s e n t  i n  b o v i n e  s a l iva ry 
g l and muc o s ub s t anc es . AB (pH 1 . 0 ) s t a in ing o b s e rved a f t e r  
hya l u ro n i da s e  t re a tment c ou l d  the r e f o r e  be a t t r i b u t e d  t o  
s u lp h a t e d  g lycop r o t e in s . T h e  bovine mandibu l a r  g l an d , how ­
e ve r , d i d  no t app e a r  t o  b e  a maj o r  s ource o f  s ulpha t e d  
g lycop r o t e i ns owing t o  ve ry weak A B  (pH 1 . 0 ) s t a i n ing 
o b s e rv e d  in o n l y  a few muc o u s  c e l l s . 
The s i a l i c  a c i d  cont e n t  o f  t h e  mandibul a r  g l and wa s 
h i s t o c hemi c a l l y  analy s e d  b y  neuram i n i da s e  d i g e s t io n  ( F i gu r e s  
2 . 4 . 9  a n d  2 . 4 . 1 0 ) . The u s e  o f  t h i s enzyme a s  we l l  a s  me t hyl­
a t i o n  ( s e e  App e nd ix I )  s uc c e s s fu l l y  ab o l i s h e d  b a s op h i l i a  a t  
p H  2 . 5 .  I n  p a rt i c u l a r  me t hyl a t ion , fo l l owe d by s ap o n i f i c ­
a t i on , fa i l e d  t o  res t o re A B  s t a in i ng a t  pH 2 . 5 ; th i s  
su g g e s t s  that s i a l i c a c i d  r e s i du e s  p re s ent w e r e  remo v e d  by 
neu r am i n i da s e a s  wel l as a c i d  hydro ly s i s . A l t hough the 
p r e s e n c e of s i a l i c  a c i d  r e s idu e s  were e s t ab l i s he d  by neur­
ami n i da s e  ac t i v i ty , t he B I AL r e a g ent ( s p e c i f i c  f o r  s i a l i c  
ac i d ) f a i l e d  t o  r e a c t  wi t h  s i a l i c  a c i d  re s i du e s o f  muc o u s  
c e l l s  ( F i gure 2 . 4 . 7 ) .  Th i s  anoma l o us r e s ul t  m a y  h av e  b e en 
c au s e d  b y  the hyd ro l y t i c  a c t i on o f  concentra t e d  HC l a c i d  
he a t e d  t o  7 0 ° C  f o r  the B I AL reac t i on and the p r e s en c e  o f  
he a t  l ab i l e  s i a l i c  a c i d  r e s i due s . 
The met achroma t i c  purp l e  r e a c t i o n  from T o ludine b l ue 
7 0  
and A z u r e  A me t h o d s , was furt h e r  p ro o f  that mandibu l a r  g l and 
muc o u s  c e l l s  c o n t a in e d  c o n s i d e r ab l e  amount s  o f  a c i d i c  muc o ­
s ub s t anc e s . The b l ue o r t ho chroma t i c  c o l our o f  To l ud in e  b l ue 
is g e n e ra l ly i nd i c a t ive o f  non - a c i di c  ma t e r i a l  wh i l e  a c i d i ty 
as a r e s ul t o f  c a rb o xy l  a n d  sulpha t e  group s  s hows a purp l e  
o r  purp l e - re d  r e a c t i o n  ( Cu l l ing , 1 9 7 4 ) . S i a l i c  a c i d s  i n  
p a r t i cu l a r  imp a r t a weak p u rp l e  co l o ur ( F igure 2 . 4 . 3 ) wh i l e  
a c i d  muc op o l y s a c c ha r i de s  s uch a s  c hondro i t i n  sulph a t e s  and 
hy a l u r o n i c  ac i ds p ro duce a n  i n t e n s e purp l e - r e d  c o l our . 
A z u r e  A me t achroma s i a  a t  pH 4 . 0 fur ther e s t ab l i s he d  that 
muc o u s  c e l l s  are l i ke ly to con t a i n  p re dominan t l y  c a rb o xy l a t ed 
muc o s ub s t anc e s , s i nc e purp l e  me t a chroma s i a  wa s no t o b s e rved 
a t  l ow pH . On t h e  o th e r  hand , s t rong ly ac i d i c  mucop o l y s ac ­
ch a r i de s found i n  the e l a s t ic c a r t i l a g e  o f  t h e  ep i g l o t t i s  
fo r e xamp l e , we re s t rong l y  met achroma t i c  at l ow p H  (pH 1 . 0 ,  
2 . 0  and 3 . 0 ) .  
I n  marked c o n t r a s t  t o  endp i e c e  mucous c e l l s , t h e  d em i ­
lun e s  o f  the b o v i ne mand i b u l a r  g l an d  con t a i n e d  s t ro n g l y  
ac i dop h i l i c  g ra nu l e s  ( F i gu r e  2 . 4 . 6 ) .  Th i s  a c i dop h i l i a  
cou l d  b e  a t t r i bu t e d  t o  the p r e s en c e  o f  h i s t o c h em i c a l ly 
de t e c t a b l e  b a s i c  p ro t e in s . Ant i b o dy to bov i ne s a l ivary 
p ro t e in band 8 ,  for examp l e ,  r e a c t e d  int ens e l y w i t h  the con­
t e n t s  o f  the demi lune c e l l s  ( F i gure 2 . 4 . 8 ) .  B and 8 i s  
t h e re f o r e  l i ke ly to b e  d e r ived f ro m  mand ibu l a r  g l and demi ­
lun e s  and may a l s o  b e  re s p ons i b l e  for some o r  a l l o f  the 
a c i do p h i l i a . 
The demi l un e s  we r e  a l s o  � e a k l y  p o s i t ive t o  PAS and AB 
a t  pH 1 . 0  and 2 . 5 ;  the d em i lune s e c re t i ons c o u l d  t he re fo r e  
c o n t a i n  s ome s i a l i c a c i d  a n d  s u l p h a t e d  g l ycop ro t e i ns . 
S i a l i c  ac i d ,  i n  p a r t i cu l a r , wa s p o s i t i ve l y  i de n t i f i e d  by 
7 1  
t h e  B I AL r e ag en t  ( F i g u r e  2 . 4 . 7 ) ; t h i s  s ug g e s t s  that unl i k e  
ih  muc ous c e l l s , carboxy l  g ro up s  o f  s i a l i c  a c i d  de r i va t ives 
in d e m i lun e s  are p rob ab l y  ma s ke d  by b a s i c  p ro t e i n s  and 
rema i n  mo s t l y  un r e a c t ive t o  b a s i c  s t a in s  s uch as Al c i an b l u e  
( F i gu r e  2 . 4 . 9 ) . Me t hy l a t i o n ,  f o l l owed b y  s ap o n i f i c a t i o n  
r e s t o r e d  s ome AB s ta i n i n g  ( p H  2 . 5 ) in dem i lune s  but f a i l ed 
t o  r e s t o r e  AB s t a ining o f  muc ous c e l l s . Unl i k e  the muc o u s  
c e l l s , the demi l une s a r e  t hus l i k e ly t o  p o s s e s s  mo s t ly 
b ound s ia l i c  a c i d  de r i va t i v e s  no t s us c ep t ib l e  t o  hydro lys i s . 
N e u r am in i da s e , fo l l ow e d  b y  AB s t a i n ing (pH 2 . 5 ) fa i l e d  t o  
r e s t o r e  b a s op h i l i c  s t a in i n g  o f  demi lune s ,  a r e su l t which 
p a ra l l e l e d  n e u ramin i da s e  r e a c t i v i t y  o f  mucous c e l l s . Th i s  
s ug g e s t s  that s i a l i c  ac i d  o f  dem i lun e s , a l though no t h e a t  
l ab i l e  i s  l ik e l y  t o  b e  s u s c ep t i b l e  to neuram i n i da s e a c t i v i ty . 
The i n t r a l o bul a r  duc t s  o f  t h e  bovine man d i bu l a r  g l a nd , 
in c on t ras t t o  tho s e  o f  t h e  p a ro t i d  g l and , we re we l l  deve l ­
o p e d  w i th d i s t inct ac i dop h i l i c b a s a l  s t r iat i o n s  ( F igure 
2 . 4 . 6 ) .  By i mp l i c a t i o n  t h e  mand i b u l a r  g l and s t r i a t e d  duc t  
c e l l s  cou l d  b e  o f  c o n s i de rab l e  imp o r t an c e  fo r wat e r  and 
e l e c t ro ly t e  t ransp o r t  ( Y o ung and van L ennep , 1 9 7 8 ) . 
The muc o u s  ce l l s  o f  t he subl ingual g l an d s  we r e  PAS and 
AB p o s i t ive a t  pH 1 . 0  a n d  2 . 5  ( F i gure 2 . 5 ) . The s e c r e t o ry 
p ro du c t s  a r e  t hus p ro b a b l y  c omp o s e d  o f  neut r a l  g lycop r o t e i ns , 
s i a l i c a c i ds and s u l p ha t e d  g l ycop r o t e ins . The s i a l ic a c i d  
s y n t h e s i s e d  b y  s ub l ingu a l  g l and mucous c e l l s  d i d  n o t  app e a r  
t o  b e  neuram i n i da s e  l ab i l e ,  a r e s u l t  b y  comp a r i s on w i t h  
t h a t  o f  man d i b u l a r g l and mu cous c e l l s , s ugg e s t s that s i a l i c 
a c i d  i s  p r o b a b l y  found i n  mo re t han one fo rm in bov ine 
s a l iv a . Me t hy l a t i on , f o r  e xamp l e , fa i l ed t o  remove mo s t  
o f  t h e  s ia l i c  a c i d  r e s i du e s ; cons e quen t l y  A B  s t a i n ing (pH 
2 . 5 ) was r e s t o r e d  a f t e r  s apon i f i c a t i o n , a p ro b ab l e  ind i c ­
a t i on that s ub l ing ua l g l and s i a l i c  ac i d  re s i due s w e r e  n o t  
r e mo ve d  b y  hy d ro l y t i c  a c t i on . I n  cont r a s t  t h e  s i a l i c  a c i d  
c o n t e n t  o f  mand ibul a r  g l an d  muc ous c e l l s  was b o t h n e u r ­
ami n i da s e  and h e a t  l ab i l e  and a f t e r  methy l a t i on AB s t a in ing 
(pH 2 . 5 ) was n o t  re s to r e d  by s ap on i f i c a t i o n . 
Hy a l uron i d a s e  a c t i v i t y  was n e g a t ive in s ub l ingua l g l and 
muc o u s  c e l l s ; th i s  s u g g e s t s  that AB s t a in i ng at pH 1 . 0 
was p ro b a b l y  du e to the p re s ence o f  s u lphated g lycop r o t e in s  
( F i gu r e s 2 . 5 . 2  and 2 . 1 1 ) . 
7 2  
T h e  demi l une s o f  the s ub l ingua l g l and we r e  w e a k l y  p o s ­
i t i v e  t o  PAS and reac t e d  i mmuno h i s t o chem i c a l l y  w i t h ant i b o dy 
to p ro t e i n  b an d  9 ( F i gu r e  2 . 5 . 6 ) ;  the r e f o re , t h e  s e c r e t o ry 
p r o du c t s  a r e  mo s t ly p ro t e o s e rous . 
T h e  int r a l obul a r  " s t r i at e d" duc t s  o f  the s ub l i ngua l 
g l an d  by comp a r i son w i th the man d i b u l a r  g l and , w e r e  e qu a l l y  
we l l  deve l op e d  ( F i gure 2 . 5 . 5 ) and l i k e l y  t o  b e  invo l v e d  i n  
w a t e r  and e l e c t ro ly t e  t r a n sp o r t  t o  the s ame e x t en t  a s  i n  
t h e  mandibul a r  g l ands . Thus in c a t t l e  we l l  deve l op e d  
s t r i a t e d  duc t s  w e re found only i n  the mand ibu l ar and s ub ­
l in g u a l  g l an d s  wh i l e  t ho s e  o f  the p a ro t i d we r e  p o o r ly 
dev e l op e d . 
The muc o u s  c e l l s  o f  t h e  mino r g l ands , in g ene r a l , c o n ­
t a i ne d h i s t o c h emi c a l ly s im i l a r  muc o s ub s t anc e s  to tho s e  o f  
s ub l i n gual g l ands , b u t  p o s i t i ve s t a ining for s ul p h a t e  g ro up s  
w a s  c ons i s t e n t l y  g re a t e r  than f o r  b o t h  s ub l i ngua l and man ­
d i b u l a r g l an d s . 
O f  the b u c a a l  g l ands , the v e n t r a l  buccal was s t ructur ­
a l l y and h i s t o chem i c a l l y  i dent i c a l  t o  the p a ro t i d . I t  
app e a r s l i k e l y  that ve n t r a l  bucc a l  g l ands may a c t  t o  s up ­
p l e m e n t  the p a ro t i d  s a l i v a ry s e c r e t i on . The d o r s a l  and 
i n t e rmed i a t e  buccal and the p al a t ine g l ands were p re domin ­
a nt ly s p e c i a l i s e d f o r  t h e  synthe s i s  and s e c r e t i o n  o f  a c i d i c  
a n d  n e ut r a l  muc osub s tanc e s  ( F i g u r e s  2 . 6 , 2 . 7 and 2 . 8 ) .  
a l t h ough a m in o r  p ro t e o s e rous c omp onent was frequent l y  
o b s e rved . L ob ul e s  o f  p ro t eo s e rous c e l l s we r e  o f t en s c a t ­
t e r e d  ami ds t the mucous endp i e c e s ; the i r  s e c r e t i o n s  c o u l d  
7 3  
c o n t r ibute s i g n i f i c ant ly to t h e  v o l ume o f  s a l iva s e c r e t e d  
b y  t h e  mino r g l and s . T h e  do r s a l , int e rmed i a t e  buc c a l  and 
p a l a t ine g l an d s  were a l s o  unu s u a l  in p o s s e s s i n g  what app e a red 
t o  b e  a h o l o c r i ne s e c r e t o ry mechani sm s in c e  e p i th e l i a l  c e l l s  
an d n e c ro t i c nuc l e i  w e re o f t en found i n  duct lumen s mixed 
w i t h  muc o sub s t anc e s  from the endp i ec e  c e l l s  ( F i gu r e s 2 . 7 . 4  
and 2 . 8 . 2 ) .  The func t i o n a l  s i g n i f i c ance o f  s uc h  a s e cre t o ry 
me c h an i s m p r e s ent only i n  s ome s al iva ry g l ands i s  d i f f i cu l t 
t o  a s c e r t a in w i thout p re hap s a p r e l imina ry inve s t i g a t ion 
u t i l i s ing s e r i a l  s e c t i o n s  of t i s s ue s  f rom the r e sp e c t i ve 
g l an d s . 
The p o s t e r i o r  t ongue had h i s t o chemi c a l l y s imi l a r  muc o ­
s ub s t an c e s  t o  tho s e  o f  t h e  s ub l ingua l , int e rme d i a t e  and 
do r s a l  b uc c a l  and p a l a t i ne g l ands . Ne i th e r  l o bu l e s  o f  
p ro t e o s e ro u s  c e l l s  no r e v i de n c e  o f  an a t yp i c a l  s e c r e t o ry 
me c h a n i sm was p re s ent ( F i g ure 2 . 9 ) .  The dem i l une s o f  t h e  
p o s t e r i o r  tongue g l ands o f t en c o n t a i n e d  a c i d i c  a n d  neut r a l  
muc o s ub s t an c e s  ( F i g ur e s  2 . 9 . 3 and 2 . 9 . 4 ) in cont ra s t  t o  
demi l un e s  o f  do rs a l , i n t e rmedi a t e  buc c a l  and p a l a t ine g l ands 
wh i c h  app e a r e d  to e l ab o r a t e  a p ro t e o s e rous s e c r e t i on . 
The pharyng e a l  g ro up o f  g l an d s  h i s t o l o g i c a l l y  d i f fe re d  
f r o m  the o th e r  min o r  g l ands . They app e a r e d  t o  p o s s e s s  
s h o r t e r  t ub u l a r  endp i e c e s  and l a r g e  round demi l une c e l l s  
wh i c h  cont a ine d nume r o u s  ac i dop h i l ic g ranul e s  ( F i gure 2 . 1 0 ) , 
i n  a dd i t ion t o  PAS , AB ( p H  2 . 5 ) and B IAL po s i t iv e  mat e r i a l . 
Th e demi l un e  s e c r e t i ons a r e  t hu s  l i ke ly t o  con t a in s ome 
s i a l i c ac i d  and neut r a l  muc o s ub s t ance s .  The comp o s i t i on o f  
muc o us c e l l  s e c r e t ions w e r e  howeve r h i s t o ch em i c a l ly s im i l a r  
t o  t ho s e  o f  t h e  o t he r m i n o r  g l ands . 
The int r a l ob u l a r  duc t ep i th e l ium o f  the pha ryn g e a l  
g l ands f r e quen t ly cont a ined gob l e t  c e l l s  an d s e c r e t o ry e nd ­
p i e c e s  wh i c h  s e emed t o  c ommun i c a t e  di r e c t ly w i th t h e  duc t  
e p i t he l i a  ( F i g ure s 2 . 1 0 . 3  and 2 . 1 0 . 4 ) . S imi l a r  mo d i f i c ­
a t i ons o f  int r a l o bu l a r  duc t ep i t h e l i a  w e re n o t  o b s e rved in 
the o t h e r  s a l ivary g l an d s  examin e d .  Func t i on a l ly , howeve r ,  
a duct ep i t h e l ium w i t h  s e c r e t o ry c e l l s  c oul d p ro v i de an 
a d d i t i o n a l  s upp ly o f  muc o s ub s t an c e s  to s uppl eme nt tho s e  
s e c re t e d  b y  muc ous and demi l un e  c e l l s . 
An i nv e s t i g a t i o n  o f  p l a sma c e l l  numb e r s  i n  b o t h  maj o r  
a n d  mino r s a l ivary g l and s o f  c a t t l e  r e ve a l ed t h a t  mo s t  w e r e  
d i s tribut e d  in t h e  c onne c t ive t i s s ue s t r oma a n d  sub ep i th ­
e l i a l s i t e s  o f  the s ub l ingual g l ands ( F i gure 2 . 5 . 7 ) and 
s e c onda r i l y , in s imi l a r  l o c a t i o n s  o f  t h e  pharyng e a l  g l ands 
( F i gu r e  2 . 1 0 . 2 ) . The s ub l ingua l g l ands i n  p a r t i c u l a r  
app e a r e d  t o  b e  t h e  maj o r  s o u r c e  o f  immuno g l obul i n s  found 
7 4  
i n  bovine s a l i va . S o me p l a sm a  c e l l s  w e r e  a l s o  f r equen t l y  
o b s e rve d i n  t h e  conn e c t ive t i s s ue s t roma o f  t h e  mandibul a r  
g l ands u s u a l l y  b e tw e e n  s e c re t o ry endp i e c e s  and s t r i a t e d  
d u c t s , wh i l e  o n l y  a few s uch c e l l s  w e r e  p re s en t  i n  s ub ep i th ­
e l i a l  s i t e s  o f  int e rm e d i a t e  and d o r s a l  b ucca l ,  p a l a t ine and 
p o s t e r i o r  t o ngue g l an ds . On t h e  o th e r  hand , the p a ro t i d  
a n d  vent r a l  bucc a l  g l ands con t a in e d  n e g l i g i b l e  numb e r s o f  
p l a s ma c e l l s  and a r e  t h e r e fo r e  un l i ke l y  t o  b e  maj o r  c o n ­
t r ibut o r s  o f  irnmuno g l obul ins t o  bovine s a liva . 
S e c r e t o ry I gA ,  t h e  maj o r  immunog l o b u l in f o und i n  b o v i n e  
s a l iva (Wa t s on a n d  L a s c e l l e s , 1 9 7 3 ) , i s  p ro b a b l y  s ynthe s i s e d  
a n d  s e c r e t e d  by the maj o r i ty o f  p l a sma c e l l s  i n  b o v i n e  
s a l iva ry g l ands . The p r e s enc e o f  s i gA i n  p l a sma c e l l s  was 
i mmuno h i s t o chem i c a l ly demons t ra t e d  ( F i gure 2 . 4 . 1 1 ) . I mmuno ­
h i s to chem i c a l  s t ud i e s  a l s o  demons t ra t e d  t h e  � i s t r i but i o n  o f  
b ovine s a l i vary p ro t e i n b ands 4 ,  8 ,  1 0  and 9 i n  s a l ivary 
g l and t i s su e s  ( F i g ur e s  2 . 3 , 2 . 4 . 8 , 2 . 5 . 6  and 2 . 6 . 4 ) .  Mo s t  
p ro t e o s e r o u s  c e l l s  o f  the p a ro t i d and ventral bucc a l  g l ands 
r e a c t e d  w it h  ant i b an d  4 and an t i  band 1 0  ant i b o dy . P ro t ­
e o s e r ous c e l l s  p o s i t i ve for ant i b and 1 0  ant i b o dy were 
g ene r a l l y  mo r e  w i de sp r e ad than tho s e  p o s i t i ve fo r ant i b and 
4 ant i b o dy ( F i gu r e  2 . 3 ) .  B i o ch emi c a l  s t udi e s  have a l s o  
e s t ab l i sh e d  a g re a t e r  p ropo r t i on o f  p ro t e i n b and 1 0  i n  
p aro t i d s a l i va than b and 4 ( W . T .  Jone s , p e r s on a l  c ommun ­
i c at i on ) . B and 4 f rom p a ro t i d  s a l iva h a s  b e e n  c o r re l at e d  
w i th b l o a t  s u s c ep t i b i l i ty i n  c a t t l e  ( Mc i nt o s h  and Cockrem , 
1 9 7 4 )  but b i o chemi c a l  t e s t s  have s hown that p a ro t i d  g l ands 
o f  b o th L . S .  and H . L .  an ima l s  cont a in e d  app ro x imat e l y  equa l 
7 5  
quan t i t i e s  o f  p ro t e in b and 4 ( W . T .  Jone s ,  p e rs onal c ommun ­
i c a t i on) . I mmuno h i s t o c hemi c a l  i nve s t i g a t i ons o f  p a ro t i d  
t i s s ue s amp l e s  a l s o  r e v e a l e d  t h a t  b o t h  s t r a ins h a d  e qua l 
p r op o r t i o n s  o f  ant i  b an d  4 p o s i t ive p ro t e o s erous c e l l s . 
Howeve r ,  only H . S .  an i m a l s  a r e  known t o  s e c re t e  b and 4 in 
s i g n i f i c an t  quan t i t i e s , a r e s u l t  whi ch s ug g e s t s  that p o o r ly 
und e r s t o o d  c e l lul a r  r e g u l a t o ry mechani sms in p a ro t i d  p ro t e o ­
s e r o us c e l l s  o f  L . S . and H . S .  anima l s  may b e  re spons i b l e  
fo r the s yn t he s i s  and s e c r e t i o n  o f  b and 4 .  
Band 4 wa s addi t i o na l ly f o un d  in t h e  d emi l un e s o f  t h e  
i n t e rme d i a t e  b uc c a l  g l ands ( F i gu r e  2 . 6 . 4 ) ; th i s  l a t t e r  
g r o up o f  g l an ds was a l s o  unu s u a l  i n  s e c r e t ing t r ac e s  o f  a l l  
t h e  s a l i va r y  p ro t e i n s  i s o l a t e d  f r om b o v i n e  s a l iva ( T ab l e  
1 . 1 ) .  Emb ryo l o g i c a l l y , t h e  i n t e rme d i a t e  b uc c a l  g l ands may 
h a v e  r e t a i n e d  t h e  g e n e t i c ma t e r i a l  requ i r e d  f o r  t h e  synt h ­
e s i s  o f  a l l t h e  s a l i va ry p ro t e i n s  throughout t h e  animal ' s  
l i f e t ime . 
CHAPT E R  3 .  PAROT I D  G LAND I NT RAE P I THELIAL 
DUCT CELLS 
3 . 1  I n t r o du c t i on 
7 6  
P r e v i o u s  h i s t o l o g i c al s t ud i e s  o f  ruminant p a ro t i d  g l an d s  
h a v e  a lways revea l e d t h e  p re s en c e  o f  a g ranu l a r  c e l l typ e , 
p r e s umab l y  non - ep i the l i a l  in o r i g i n  and o f  doub t f u l  i den ­
t i ty ,  d i s t r i but e d  w i t h in the e p i t h e l ium o f  int r a l ob u l a r  
duc t s . S h ack l e f o r d  and K l ap p e r  ( 1 9 6 2 )  a n d  B i r t l e s  ( 1 9 8 1 )  
d e s i g na t e d  th e s e  c e l l s  in the bovine p a r o t i d  g l and a s  
" i n t ra s t r i a t e d  duc t  c e l l s " wh i c h  c l o s e l y re s emb l e d b o t h  
m a s t  c e l l s  and p l a s m a  c e l l s . Van L ennep e t  a l . ( 1 9 7 7 ) , 
f r o m  a s tu dy o f  the s heep p a ro t i d g l and , obs e rv e d  two g r a n ­
u l a r  c e l l  typ e s  l o c a t e d  w i t h i n  the ep i th e l ium o f  s t r i a t e d  
duc t s . T h e  mo s t  c ommon typ e w a s  a n  int r a ep i the l i a l  mas t 
c e l l , found a l s o  in t he conn e c t i ve t i s s ue s t r oma s u rrounding 
the s t r i a t e d duc t s . The o t h e r  typ e , r e f e rre d t o  a s  a 
" g l obul e l eucocyt e" , was charac t e r i s e d b y  the p o s s e s s i on 
o f  l a r g e  i n t racytop l a smic e o s i nop h i l i c g r anul e s . R e c e n t l y , 
V i gno l i  and Nogu e i r a  ( 1 9 8 1 ) , f rom a h i s to l og i c a l  and h i s t o ­
c h em i c a l  s tudy o f  t h e  p a ro t i d  g l and o f  a bov i d a e  s p e c i e s , 
Bos  indi c us , r ep o r t e d  the p r e s ence o f  " in t r a ep i t he l i a l  ma s t ­
o cy t e s "  a b undan t l y  d i s t r ibut e d  wi thin t h e  int r a l ob u l a r  duc t  
e p i the l i um .  P a l  e t  a l . ( 1 9 7 2 ) , who exam i ne d t h e  p a r o t i d  
g l ands o f  the I nd i an buf f a l o  (Buba Z us b uba Zi s )  have a l s o  
r e p o r t e d  a n  " int r a e p i the l i a l  cy s t" w i th s t a i n i ng chara c t e r ­
i s t i c s  s im i l a r  t o  t h a t  o f  a ma s t  c e l l , f requent l y  p re s ent 
w i th in t h e  ep i th e l i um of the e x c r e t o ry duc t . I t  i s  unc l e a r  
wh e th e r  t h e  r e f e re n c e  was t o  a n  unknown c e l l typ e . 
The p a rot i d  and the h i s t o l o g i c a l l y  i dent i c a l  ve n t r a l  
b uc c a l  g l and e x am i n e d  i n  t h e  p r e s ent s tu dy , c ont a ine d s ev ­
e ra l  i n t r a ep i th e l i a l  g r anul a r  c e l l s  w i t h in t h e  wa l l s  o f  
i nt r a l ob u l a r  du c t s  and i n f r equent l y  i n  the wa l l s  o f  l a rg e r  
i n t e r l ob u l a r  duc t s . S imi l ar c e l l s  we r e  a l s o  r a re ly ob s e r ­
v e d  w i t h i n  int r a l ob u l ar duc t ep i t h e l i a o f  the do r s a l  and 
i n t e rme d i a t e  b u c c a l  g l ands and p a l a t ine g l ands . 
l nt ra e p i t he l i a l g r anu l a r  duc t  c e l l s , i n  g en e r a l , 
r e s emb l e d  ma s t  c e l l s  i n  mo rpho l o gy and s t a in i ng cha r a c t e r ­
i s t i c s , b u t  p o s s e s s e d nuc l e a r  c o n f i gur a t i o ns s im i l a r  t o  
tho s e  o f  p l a sma c e l l s . 
F o r  fu rthe r d i s cu s s i o n , t h e  t e rm int r a ep i t h e l i a l  g ran ­
u l a r  ( l E G )  duc t  c e l l  h a s  b e en adop t e d  i n s t ead o f  int ra ­
s t r i a t e d  duc t c e l l  b e c au s e  b a s a l  s t r i a t i o ns a r e  n o t  a 
f e at u re o f  int ra l o bu l a r  duc t s  o f  the bovine p aro t i d  g l and 
( S e c t ion 2 . 3 . 1 ) and l E G duc t  c e l l s  were a l so o c c a s i ona l l y 
p r e s ent w i thin the i n t e r l o b u l a r  duc t  ep i t hel i um .  
7 7  
The p re c i s e  n a t u r e  o f  the l E G duc t  c e l l s  we r e  examine d 
by h i s to l o g i c a l , h i s t o chemi c a l  and ul t ra s truc tur a l  methods . 
S i nc e  l EG duc t  c e l l s  o f  the p a ro t i d  g l and have a l s o  b e en 
i mp l i c a t e d  with b l o a t  s u s c ep t i b i l i ty i n  c a t t l e  ( S e c t i o n  1 . 6 ) 
t h e  p r e s ent s tudy h a s  ext ende d thi s f in d i ng t o  c omment on 
the po s s ib l e  func t i o n  of the s e  c e l l s  i n  r e l a t i o n  t o  b l o a t . 
As f a r  as i t  i s  known , the re have b e en no rep o r t s  y e t  
o f  de t a i l ed inve s t i g a t i ons p e r t a in i ng t o  unknown o r  p o o r l y  
d e f in e d  c e l l  typ e s  l o c a t e d  i n  s a l i v a ry g l and du c t s .  
3 . 2  Ma t e r i a l s  and M e thods 
7 8  
H i s t o l o g i c a l  s e c t i o ns o f  p a ro t i d  and vent r a l  b uc c a l  
g l an d  t i s s u e s  p r ep a r e d  e a r l i e r  f o r  h i s t o ch emi c a l  e xamina t i on 
( S e c t i on 2 . 2 ) we r e  a dd i t i ona l l y us e d  fo r a l i gh t  mi c r o s cop i c  
e va l ua t ion o f  I E G  duc t c e l l s . The cytop l a smi c contents o f  
th e s e  c e l l s  w e r e  a l s o  c l o s e ly inve s t i g a t e d  with the a i d  o f  
t h e  fo l l ow i n g  a dd i t i on a l  metho ds : -
1 .  P h l o x ine - Ta r t ra z ine 
2 .  B i s ma r c k  b rown 
3 .  N e u t r a l  r e d  
4 .  F a s t r e d  2 B  
5 .  F l u o re s c e in conj ug a t e d  ant i  I gA 
6 .  S i l v e r  imp re gnat ion 
( s e e App e n d i x  I I  fo r s t a in i ng p ro c e dure s ) . 
H i s t o l o g i c a l  s e c t i ons from the s h e e p  p a ro t i d  g l and and 
ab oma s um ,  p r ev i ous l y  f i x e d  i n  B o uins f l u i d  and s t a i ned w i t h  
A B  at pH 1 . 0  and 2 . 5  and PTAH , were a l s o  examine d fo r t h e  
p r e s ence o f  i dent i c a l  o r  c l o s e l y r e l a t e d  c e l l typ e s  to the 
l E G  duc t  c e l l s  o f  c a t t l e . 
From the d i s s e c t i on o f  two anima l s  ( S ect i on 2 . 2 ) ,  s am ­
p l e s  o f  p a r o t i d  t i s s ue w e r e  s ep a ra t e l y taken fo r a n  e l e c t ron 
m i c ro s cop i c  e xamina t ion of the l E G  duc t  c e l l s . 
P r epa r a t i on o f  t i s s ue s  for e l e c t ron m i c ro s copy 
T i s s ue s  w e r e  i n i t i a l ly p l ac e d  in p e t r i  ' d i s h e s  wh i ch 
conta i n e d  a sma l l  quan t i ty o f  mo d i f i e d  Karno vsky ' s  f i xa t ive . 
T i s sue b l o ck s  � - 2  mm 2 w e r e  cut w i th a s c alp e l  an d a l l owe d t o  
f i x  fo r a t  l e a s t two hour s . 
Modi f i e d  Karno v s ky ' s F i xa t i ve 
P r e p a re 2 0  ml s o f  1 0 %  s o lution o f  p a ra fo rma l dehyde by 
d i s s o l v ing 2 g rams of p a r a fo rma l dehy de powde r in 2 0  ml s o f  
d i s t i l l e d wa t e r  and he a t ing to 6 0 ° C  in a fume cup b o a r d . Add 
a few drops o f  lN NaOH unt i l  the s o l u t i on c l e a r s . Al l ow t o  
c o o l  b e fo r e  us e .  P r e p a re f i x a t ive w i th : 
0 . 2M S o dium c a c o dy l a t e  b u f f e r  5 0  ml s 
1 0 %  p ar a fo rma l de hyde in w a t e r  2 0  ml s 
2 5 %  g l utara l dehy d e  in wa t e r  1 0  m l s  
a dd di s t i l l e d  wat e r  t o  1 0 0  ml s .  
A .  F ix e d  t i s s u e s  w e r e  wa s he d  in t h r e e  chang e s  o f  0 . 2M 
S o d i um c a c o dy l a t e  buffe r ( p H  7 . 2 ) fo r app ro x ima t e l y  
2 hours . 
B .  P o s t f ixat i o n  was c a r r i e d  out in 1 %  O s o4 f o r  1 hour w i t h  
s u f f i c i ent O s o 4 t o  c o v e r  t h e  t i s s u e s . Samp l e s were 
t hen washed f o r  30 minut e s  wi th s o d i um c a c o dy l a t e  
b u ff e r . 
C .  F i x e d  t i s s ue s  we re dehy dr a t e d  i n  a g rade d a l c o ho l 
s e r i e s  a t  room t emp e ra t ur e . 2 5 % , 5 0 % , 7 0 % , 9 5 %  and 
2 x 1 0 0 %  conc e n t r a t i on s  w e re us e d , 2 0  minu t e s  each on 
s t i r rer wi th t o p s  on s amp l e  b o t t l e .  Some t i s s ue 
s amp l e s  were l e ft o v e rn i gh t  in 1 %  urany l a c e t a t e  in 
7 0 %  a l coho l at 4 ° C t o  i n t ens i fy s t a in ing . 
D .  The t i s s ues w e r e  i n f i l t r a t e d  w i th 2 x 2 0  minu t e  chang e s  
o f  p ropyl ene o x i de us ing a me chan i c a l  s t i r rer in a 
fume cup b o ar d .  
E .  Emb e dding med i um was p repared u s i n g  a m i xture o f  2 5 %  
r e s in and 7 5 %  p ropy l en e  o x i de . T h e  emb e dd ing medium 
( Durcupan ACM F l uka )  c o n s i s t e d  o f  an epoxy r e s in ,  
ha r dene r ,  a c c e l e r a t o r  and p l as t i c i z e r .  Fo r 1 0  m l s  o f  
r e s i n  the above components we r e  m i xed i n  the f o l l ow i n g  
p roport i on s : -
Epoxy re s in (A) 5 . 4 7 g rams 
harde n e r  ( B )  4 . 7 9 g r ams 
acc e l e r a t o r  ( C )  0 . 2  ml 
p l a s t i c i z e r  ( D )  0 . 1  ml 
Component s A and B w e re we i g h e d  and i n cub a t e d  at 6 0 ° C  
for about 1 0  minu t e s  t o  ensure a g o o d  mix . Componen t s  
7 9  
C and D were n e x t  added c a r e ful ly from a 1 m l  s y r i ng e . 
The comp onent s w e r e  mixed t ho ro ugh l y  unt i l  the s o l u t i o n  
c l e a re d .  
F .  P ropy l ene o x i de was remo v e d  a f t e r  i n f i l tra t i on and t h e  
e mb e ad ing m e d i um o f  2 5 %  re s in a n d  7 5 %  p ro py l ene o x i de 
w a s  a dd e d  and l e f t overn i ght on a s t i rrer . The fo l l ­
owing mo rning c o n t e n t s  w e r e  rep l a c e d  by fre s h  1 0 0 %  
r e s in ,  l e ft f o r  6 - 8  hours , then emb e dded in f re s h  
1 0 0 %  r e s in i n  g e l a t ine c ap sul e s  and hardene d for 4 8 -
7 2  hours a t  6 0 ° C . 
G .  App r o x ima t e l y  2 �m t h i c k  s e c t i ons w e re c u t  w i t h  a g l a s s  
kn i f e  o n  an LKB ul t r a t ome , s t a in e d  w i th To l ud in e  b lu e  
and e x amine d wi t h  a l i gh t  mi c r o s c o p e  t o  no t e  t i s s ue 
c on t en t s . Thin s e c t i ons ( 6 0 0  �) o f  int r a l obu l ar duc t  
ep i th e l i um we re cut and f l o a t e d  o n t o  copp e r  g r i ds . 
H .  S e c t i ons were s t a i n e d  w i th urany l a c e t a t e  ( 5  minu t e s ) , 
was h e d  w i th 5 0 %  a l c o h o l  and d i s t i l l e d wa t e r . Gr i ds 
w e r e  dr i e d  o n  f i l t e r  p ap e r  and s t a i ne d in l e a d  a c e t a t e  
( 5  minut e s ) , f o l l owed b y  rins ing i n  d i s t i l l e d wa t e r . 
I .  S e c t i ons we r e  e x am in e d  and pho t o g r aphed u s ing a P h i l ip s  
EM Z O O  e l e c t r o n  micro s co p e . 
8 0  
3 . 3  Re s u l t s  
3 .  3 . 1  H i s t o l ogy 
I E G  duct c e l l s  w e r e  f r e quent ly l o c a t e d  wi t h i n  the wa l l s  
o f  i n t r a l obul a r  duc t s  o f  the p a ro t i d  and vent r a l  b uc c a l  
g l ands , adj acent t o  th e b a s ement l am i n a  o f  the duc t  ep i th ­
e l ium . Usual l y , two to s even l E G  duc t c e l l s  we re o b s e rv e d  
in mo s t  intral o b u l a r  duc t c r o s s  s e c t i o n s  ( F i gure 3 . 1 ) . 
Very r a re ly , s ome c e l l s  app e a r e d  t o  have b e en s h e d  into 
the duc t l umen ( F i gure 3 . 1 . 6 ) and o th e r s  were found a t  s ub ­
ep i t h e l i a l  s i t e s  c l o s e  t o  a duc t  ( F i gu r e  3 . 1 . 5 ) .  The ran g e  
o f  s h ap e s  exh ib i t e d  va r i e d  w i de ly , mo s t  had i r r e gu l a r  out ­
l in e s  wh i l e s ome w e r e round o r  oval s hap e d  w i t h  e c c ent r i c ­
a l ly - l o c a t e d  nuc l e i . 
The cytop l a s m  o f  l E G  duc t c e l l s w a s  o ft en vacuo l a t e d  i n  
app e a r ance a n d  rema ine d r e f r a c t o ry t o  mo s t  h i s t o l o g i c a l  
s t a i n s  b ut con t a i n e d  num e r o u s  r o und g r anul e s  o r  g l ob ul e s  
tha t s howe d c on s i d e rab l e  var i a t i on i n  s i z e and quant i ty p e r  
c e l l  ( F i gure 3 . 3 ) . Nuc l e i  o f  l E G  duc t  c e l l s  were charac ­
t e r i s t i c a l ly roun d o r  oval in s hap e and o ft e n  o c cup i e d  a n  
e c c en t r i c  p o s i t i o n . The d i s tr ibut i on o f  chroma t i n aroun d  
the p e r iphe ry o f  the nuc l eu s  app e a r e d  s imi l a r  t o  the " c a r t ­
whe e l " p a t t e rn o b s e rve d i n  nuc l e i  o f  p l a sma c e l l s . S i gn s 
o f  c e l l  divi s i on o r  e v i de n c e  o f  pykno s i s  in nuc l e i  we r e  n o t  
ob s e rve d .  
Some d i f f i cu l ty was encoun t e r e d  i n  an a t t emp t t o  mak e  a 
di s t inct ion b e tw e en ma ture and immat u r e  I E G  duc t  c e l l s , 
a l though a common o b s e rva t i on was that the maj o r i ty o f  
c e l l s  s e eme d t o  deve l op wi t h in t h e  duct ep i th e l ium . The 
granular c o n t e n t s were p robab ly r e l e a s e d  a t  a part i c u l a r  
s t a g e  o f  t h e  c e l l  cyc l e  o r  p o s s i b l y  o n  r e c e ival o f  a s t im­
u l u s  to " d e g ranu l ate" . The cytop l a s m  o f  s ome c e l l s  was 
comp l e t ely devo i d  of g r anu l e s  ( F i g u r e  3 . 3 . 6 ) wh i l e  o t he r s  
w e r e  r ep l e t e  w i th g ranul e s  o f  var i o u s  s i z e s  ( F i gure 3 . 3 ) .  
A p o t ent i a l  p r e cur s o r  c e l l  was n o t  i den t i f i e d  in t h e  
8 1  
v i c in i ty o f  int r a l o b u l a r  duc t s  no r was the r e  any ev i de n c e  
t o  s ug g e s t  t h a t  l E G  duc t c e l l s  h a d  p r e v i ous ly m i g ra t e d  f ro m  
the s ub ep i th e l i a l  c onne c t ive t i s s u e  v i a  t h e  b a s ement l am i n a  
into t h e  duc t  e p i th e l ium . 
8 2  
l E G duc t  c e l l s  no t e d  i n  the e p i t h e l ium o f  intr a l obul a r  
duc t s  o f  the thr e e  mino r g l ands , do r s a l , i n t e rme d i a t e  b uc c a l  
and p a l a t ine g l an d s  were s im i l ar t o  t ho s e  o f  t h e  p a ro t i d  
g l an d s  ( F i gure s 2 . 6 . 3  and 2 . 8 . 3 ) . 
Mature s ub e p i t he l i a l  o r  muc o s a l  ma s t  c e l l s  o c c a s i on a l l y  
ob s e rv e d  c l o s e  t o  t h e  i n t r a l obul a r  duc t s  o r  b e tween s e c r e t ­
o ry t ubul e s  o f  t h e  p a r o t i d  and ven t r a l  buc c a l  g l ands we r e  
p o lymo rph i c  in a p p e arance ( F i gu r e  3 . 2 ) .  Some muc o s a l  ma s t  
c e l l s  were r e l a t i ve l y  f l a t t ene d o r  e l ong a t e d  and cons i s t e d  
o f  s p h e r i c a l  nuc l e i with s c a t t e r e d  c hromat in , whi l e  o t h e r s  
we r e  rounde d o r  ova l - s h ap e d  w i t h  nuc l e i  s im i l a r  t o  t ho s e  o f  
p l as ma c e l l s  ( F i gure 3 . 2 . 4 ) .  Muc o s a l  ma s t  c e l l s  o f  a l l  
o th e r g l ands we r e  s imi l a r t o  tho s e  o f  p a ro t i d  and v en t r a l  
b uc c a l  g l ands b u t  had a t endency t o  b e  d i s t r ibut e d  n e a r  
p l a s ma c e l l s ; i n  such in s t anc e s  t h e  c l o s e  r e s emb l an c e  b e t ­
we e n  nuc l e i  o f  b o th muc o s a l  mas t  c e l l s  and p l a sma c e l l s  was 
s t r i k in g l y  e v i d e n t . Ma s t  c e l l s  ob s e rved in l o o s e  a r e o l a r  
conn e c t ive t i s s ue o f  a l l  g l ands we r e  e l ong a t e d  with l o n g  
tap e r ing ends . The i r  nuc l e i  were o f t en s im i l a r t o  tho s e  o f  
mu c o s a l  ma s t  c e l l s b ut a t  t ime s we r e  den s e l y  s t a in i n g  w i th ­
o u t  p e r ip h e r a l  chroma t in . 
3 . 3 . 2  H i s t o chemi s t ry 
H i s to chem i c a l  r e ac t i ons o f  l E G  duc t  c e l l s  have b e e n  
s ep ar a t e ly s umma r i z e d  in Tab l e  3 . 1  t o g e the r with r e a c t i o n s  
o b s e rved fo r m a s t  c e l l s . 
The cytop l a sm o f  l E G  duc t c e l l s ,  a l tho ugh re frac t o ry t o  
mo s t  hi s t o l o g i c a l  s t a in s , w a s  markedly p o s i t ive t o  AB a t  
b o th p H  1 . 0  and 2 . 5  ( F i gures 3 . 1 . 1  and 3 . 1 . 2 ) .  T h e  c o n t en t s  
o f  the g r anu l e s  showed mode r a t e  r e a c t i v i ty t o  PAS ( F i gure 
3 . 1 . 3 ) ,  Phl o x i n e  ( F i gure 3 . 3 . 2 ) ,  E o s in , P TAH , F a s t Re d 2 B  
T ab l e  3 . 1 H I STOCHEMI CAL RE SULTS OF 
l EG DUCT CELLS AND MAS T  CELLS 
Histochemical I IEG duct cells Method Result Comments Mast cells 
H & E + weak eosinophilia + 
Alcian Blue pH 1 . 0  ++++ strong cytoplasmic ++++ 
basophilia 
Alcian Blue pH 2 . 5  +++ cytoplasmic basophilia +++ 
PAS ++ moderate reactivity -
Alcian Blue/PAS +++ cytoplasmic basophilia ++ 
Alcian Yellow ++ moderate basophilia + 
Neutral Red +++ yellow-red granules +++ 
(metachromatic?)  
Bismarck Brown +++ basophilic  granules +++ 
Colloidal Iron +++ cytoplasm - blue I + 
granules - dark brown 
Phloxine ++ acidophilic granules -
Phloxine/AB +++ cytoplasm - basophilic +++ 
granules - acidophilic 
Toludine Blue ++++ metachromatic granules ++++ 
(purple) 
Azure A pH 1-3 - -
Azure A pH 4 ++++ metachromatic granules ++++ 
(purple)  
Fast Red 2B ++ orange-brown granules + 
Methyl Green- ++ cytoplasm - pink/purple + 
Pyronin granules - orange/brown 
PTAH/AB +++ cytoplasm - blue -
granules - dark purple 
S ilver ++ cytoplasm - negative ++ 
Impregnation globules - +ve · (brown) 
Anti-IgA - -
Formalin-induced +++ yellow-green +++ 
autofluorescence fluorescence 
( F i gu r e  3 . 3 . 8 ) and s i l v e r  imp r e gnat i on ( F i gure 3 . 3 . 5 ) . I n  
add i t i o n  they w e r e  s t ro n g l y  me t a chroma t i c  t o  To l u d ine b l ue 
( F i gu r e  3 . 3 . 1 ) and A z ure A at pH 4 .  When b o th Al c i an b l ue 
( a  b a s i c  s t a i n )  and Phl o x ine ( an a c i d  s t a i n )  w e r e  us e d  t o g ­
e t he r ,  s ome I E G  duct c e l l s  cont a i n e d  only p h l o x in e  p o s i t i ve 
g ran u l e s  wh i l e  o thers had an Al c i an b l ue p o s i t ive cy t op l asm 
wh i ch conta i n e d Phl oxine p o s i t ive g ranul es ( F i gure 3 . 3 . 7 ) .  
I E G  du c t  c e l l s d i d  n o t  app e a r  t o  r e a c t  w i t h  f l uo r e s c e in 
conj u g a t e d  an t i  I gA an t i b o dy but i n s t e a d  s howe d  fo rma l in ­
indu c e d  aut o f l uo re s cence ( s e e  Appe n d i x  I I ) . 
S t aining r e a c t ions o f  s ub e p i th e l i a l  o r  muc o s a l  mas t 
c e l l s  o f  p a ro t i d  and ven t r a l  b uc c a l  g l ands c l o s e ly p a r a l ­
l e l e d  tho s e  o f  I E G  duc t  c e l l s , a l though mas t c e l l s  w e r e  
PAS n e gat ive . Muco s a l  mas t  c e l l s  o f  a l l o th e r  g l ands w e r e  
h i s t o chemi c a l l y  s imi l a r  t o  I E G  duc t  c e l l s  o f  t h e  p a ro t i d  
and vent r a l  b u c c a l  g l ands but w e r e  a l s o  PAS n e g a t i ve . 
8 3  
G ranu l a r  " in t ras t r i a t e d" duc t  c e l l s  o f  t h e  s h e e p  p a ro t i d  
g l an d  cont a i n e d la rge e o s inop h i l i c  g l o bul e s  and an AB 
p o s i t i ve ( p H  1 . 0  and pH 2 . 5 ) cy t o p l a sm ( F igure 3 . 1 . 8 ) .  
S i m i l ar c e l l s  e xamine d i n  t h e  s h e e p  abomasum a l s o  h a d  l a r g e  
e o s i nop h i l i c  g ranu l e s  b u t  the i r  cy top l as ms were r e fr a c t o ry 
t o  Al c i an b l ue s t a i n in g . A comp a r i s on b e tw e en s h e ep and 
c a t t l e p a ro t i d  g l and i n t raep i t h e l i a l  duc t  c e l l s  r e v e a l e d 
cons i de rab l e  va r i a t i o n  in g ranul e s i z e s  in c a t t l e , wh i l e  
t ho s e  o f  s h e e p  were r e l a t i ve ly un i fo rm .  E o s inoph i l i c  gran ­
u l e s  o r  g l o b u l e s  were a l s o  mo r e  obvi ous in s heep than in 
c a t t l e . 
3 . 3 . 3  U l t ras t ru c t ure 
Ul t r a s t r uctural ly , I E G  duc t  c e l l s  s howe d  cons i de rab l e  
mo rpho l o g i c a l  va r i a t i on an d we r e  l o c a t e d  b e tween adj acent 
e p i t he l i a l  duc t c e l l s ; int e rc e l l u l a r  at tachment s s uc h  a s  
d e s mos ome s w e re no t p r e s en t  ( F i gure 3 . 4 ) . The r e  was how­
e v e r s o me e v i dence o f  cy t o p l a s mi c f i n ge r - l i ke p ro c e s s e s 
e x t ending f r o m  surfac e s  o f  I E G  c e l l s  into in t e rc e l l u l a r  
F I GURE 3 . 1 
MORPHOL O GY AND D I S T R I BUT I ON OF I NTRAE P I T HE L IAL GRANULAR 
(l EG )  DUCT CELLS O F  THE PARO T I D AND VEN T RAL BUCCAL GLAND S  
3 . 1 . 1  
3 . 1 .  2 
3 . 1 .  3 
3 . 1 . 4 
Charact e r i s t i c appe a rance o f  l E G  duct c e l l s  
l o c a t e d  a dj a c ent t o  the b a s a l  l amina o f  
intra l o b u l a r  duc t s . 
(Al c i an B l u e  pH 2 . 5 / H  and E ) . 
Magni f i c a t i on : x 6 5 0  
l E G duct c e l l  w i th s t rong ly s ul p ha t e d  cytop l a s m i c  
c ont ent s a n d  smal l a c i dop h i l i c g ranul es . N o t e  
r a r e  b inuc l e a t e d  l EG duct c e l l  and chromat in 
pat t e rn o f  nucl e i . 
(Al c i an B lue pH 1 . 0 / H  and E ) . 
Magni f i c a t ion : x 1 6 5 0  
Mo derat e PAS r e a c t i v i ty o f  l E G du c t  ce l l s . 
( PAS / T a r t ra z ine ) . Magn i f i c a t i o n : x 6 5 0  
Neut r a l  r e d  p o s i t i ve (Me t a c h roma t i c ? )  int r a cy t o ­
p l a s m i c  g r anul e s  o f  va r i o u s  s i z e s  d i s t r i but e d  
w i t h i n  l EG duc t  c e l l s . One c e l l  (bot t om r i g h t ) 
i s  wi thout i n t r a cy top l a s m i c  g ranu l e s  but s hows 
an i n t a c t  nuc l eus . 
(Neut r a l  Red) . Magn i f i c a t i o n : x 1 6 5 0  

F I G URE 3 . 1  ( C on t i nue d )  
3 . 1 . 5 
3 . 1 .  6 
3 . 1 . 7 
3 . 1 . 8 
A r a r e  s ub e p i t he l i a l  I E G  duc t c e l l . No t e  
h e t e rog e n e o u s  app e a ran c e  o f  g ra nul e s  and 
e c c e nt r i c nuc l eu s . 
(Al c i an B lue p H  1 . 0 / Ta r t r a z i ne ) . 
Magn i f i c a t i on : x 1 6 5 0 
A g r oup - o f I EG duct c e l l s  l y i ng w i th i n t he 
l umen o f  a duc t . 
(A l c i an B l u e  pH 1 . 0 / H  and E ) . 
Magn i f i c a t i on : x 6 5 0  
A s em i - t h i n  ( 2 �m) s e c t i o n  o f  p a ro t i d  t i s s u e  
f i xed i n  K a rno v s ky ' s f i xa t i v e . No t e  i r r e g u l a r  
app e a ran c e  o f  I EG duct c e l l s  and t h e i r  nu c l e i . 
( Ep o n  e m b e d d e d/ T o l udine B l ue ) . 
Magn i f i c a t i on : x l 6 5 0  
S h e e p  p a ro t i d  g l and " i nt r a s t r i a t e d "  duc t  c e l l  
w i th l a r g e ac i d o ph i l i c  g l o bu l e s  and b a s o p h i l i c 
( p H  1 . 0 ) cy t o p l a s m .  S i m i l a r  c e l l s  i n  the s h eep 
a b oma sum c o n t a i ned l a r g e  a c i dop h i l i c g l o b u l e s  
b u t  d i d  no t s how c y t op l a s m i c  b a s o p h i l i a .  N o t e  
p e r iphe r a l  d i s t r i but i o n  o f  chroma t i n w i t h i n  the 
nu c l eus wh i c h i s  s im i l a r  to tha t o f  a p l a s ma c e l l . 
(A l c i an Blue pH 1 . 0 / H  and E ) . 
Magni f i c at i on : x 1 6 5 0 
7 
F I G U RE 3 . 2  
MO RPHO LOGY AN D D I STRI BUT I ON OF MAS T  C E L L S  I N  BOV I NE 
SAL I VARY G LANDS 
3 . 2 . 1  
3 .  2 .  2 
3 .  2 .  3 
3 . 2 . 4  
S ub ep i the l i a l  ( muc o s a l )  ma s t  c e l l s  o f  the 
p a ro t i d  an d ven t r a l  buc c a l  g l ands , l o c at e d  
a dj a c ent t o  an in t r a l o b u l a r  duc t . Such c e l l s  
w e r e p o l ymo rph i c  i n  app e a ra n c e  and c o n t a ine d 
s ma l l  b a s oph i l i c  g r anul e s . 
( Ha l e s  co l l o i d a l  i r on/ N e u t r a l  Re d ) . 
M a g n i f i c a t i on : x 6 5 0  
P a ro t i d  g l and muco s a l  ma s t  c e l l  ( a  s im i l a r  c e l l  
t o  tho s e  i n  F i g .  3 . 2 . 1 ) .  
( B i smarck B rown/Haema toxy l i n ) . 
Magn i f i c a t i o n : x 1 6 5 0  
Muco s a l  ma s t  c e l l  o f  t h e  mand i b u l a r  g l and .  
Such c e l l s  we re d i s t r i b ut e d  c l o s e t o  p l a sma 
c e l l s  and c o n t a ined a nuc l eu s  w i t h p e r iphe r a l ly 
d i s t r ibut ed chroma t i n . 
( T o l u d i n e  B l u e ) . Mag n i f i c a t i o n : x 1 6 5 0 
A few p l a sma c e l l s  and a ma s t  c e l l , s c a t t e r e d 
wi thin conn e c t i v e  t i s s ue s o f  t h e  p h a ryng e a l  
g l ands . No t e  mo rp ho l o g i c a l  s im i l a r i t i e s  b e tween 
the two c e l l typ e s , i n  p a r t i c u l ar , the chroma t i n  
d i s tr ibut i o n  p a t t e r n  o f  nu c l e i . 
(Al c i an B l u e  pH 1 . 0 / H  and E ) . Mag n i f i c a t ion : x 1 6 5 0  
3 
F I G U RE 3 .  3 
H I S TO L O G I CAL AND H I STOCHEMI CAL F EATURE S OF C YTO P LASM I C  
G RANULES I N  l EG DUCT C E L L S  
3 .  3 .  1 
3 .  3 .  2 
3 . 3 . 3  
3 .  3 .  4 
l EG duct c e l l s  w i t h int r a cy t o p l a sm i c  
me t a chromat i c  g r anu l e s  o f  v a r i o u s  s i z e s : 
c e l l  cy t o p l asms are r e f r a c t o ry t o  s t a i n i ng 
an d the nuc l e i  a r e  not vi s i b l e .  
( T o l ud i n e  B l ue ) . Magni f i c a t i o n : x 1 6 5 0  
Ac i dophi l i c n a t ure o f  l EG duct c e l l  g r anul e s , 
d emons t r a t e d  by s t a i n i ng w i th P h l o x i n e  ( an 
a c i d  dy e ) . No t e  e c c en t r i c  nuc l eu s  w i t h  
p e r i p h e r a l ly d i s tr ibuted c h r omat i n . 
( P h l o x i n e / T a r t r a z ine ) . Magn i f i c a t i o n : x 6 5 0  
T h e  h e t e ro g en e o us na t u r e  o f  l EG duct c e l l  
g r anul e s . 
(Al c i an B l u e  pH 1 . 0 / T a r t r a z i ne ) . 
Magni f i c a t i o n : x 1 6 5 0  
A s im i l a r  c e l l  to tho s e  s hown i n  F i g s  3 . 3 . 1  
and 3 . 3 . 3 , s t a i ned w i t h Neut r a l  R e d . The 
y e l l ow c o l o u r  found in s ome g r anu l e s  s ug g e s t s 
that N e u t r a l  Red may have me t ac hroma t i c  
p r op e r t i e s . 
Magni f i c a t i o n : x 1 6 5 0  

F I G U RE 3 . 3  ( C ont i nu e d )  
3 .  3 .  5 
3 .  3 .  6 
3 .  3 .  7 
3 . 3 . 8  
Two l E G  duc t c e l l s  demo ns t r a t e d by s i lv e r  
imp r e g na t i o n . Argyrophilic s t a i n i ng o f  g ranul e s  
s ug g e s t s  the l i k e ly p r e s e n c e  o f  a b i o g en i c 
am i n e . No t e  p a r t i a l ly " de g ranul a t e d" c e l l . 
( S i l v e r  imp r e gnat i o n/ L i g h t  G r e en ) . 
Mag n i f i c a t i on : x l 6 5 0  
A " de g r anu l a t ed" l EG duc t  c e l l . No t e  app e a rance 
o f  nuc l eu s  w i th " c a r twh e e l "  d i s t r i b u t ion 
p a t t e rn of chroma t in . 
( S i l v e r  imp r e g n a t ion/ L i g ht G r e en ) . 
Magn i f i c a t i on : x / 6 5 0  
Two h i s t o ch em i c a l l y d i f fe rent l E G du c t  c e l l s . 
The c e l l  o n  the l ef t  c o n t a i n s  a c i dop h i l i c 
ma t e r i a l  whi l e  the ce l l  o n  t h e  r i g h t  contains 
a m i x t u r e  of b a s oph i l i c  and a c i do p h i l i c mate r i a l . 
( A l c i an B l u e  p H  1 . 0 / Ph l ox i ne / Ta r t ra z ine ) . 
Ma gn i f i c a t ion : x 6 5 0  
l EG duc t  c e l l s  wi th F a s t R e d  2 B  p o s i t iv e  intra ­
cytop l a sm i c  g r anul e s , s ugg e s t i ng that a b i o g en i c  
am i n e  ( e g . 5 - HT )  i s  p r o b a b l y  s t o r e d  and/ or 
synthe s i s e d by the g r anu l e s . 
( F a s t Red Z B / Ha emat o xy l i n ) . 
Magn i f i c a t i o n : x 1 6 5 0  

F I GU RE 3 . 4  
U LTRA S T RU CTURAL F EATURE S OF l EG DUCT C E L L S  
3 .  4 .  1 
3 .  4 .  2 
Typ i c a l  e l ec t ron mi c ro s c op i c  app e a r an c e  o f  an 
l E G duct cel l l o c at e d  b e tw e e n  int r a l o bu l a r  duc t 
ep i t h e l i a l  c e l l s . F o ur b a s i c  g r anu l e  typ e s a r e  
c ommonly found ( s e e t ex t ) . No t e  v a c u o l a t e d  
app e a r an c e  o f  c y t op l a sm and p ro t e i n a c i ous 
c rys t a l l i ne ma t e r i a l  ( a rrows ) l o c a t e d  in s ome 
g ranu l e s . The nuc l eus i s  no t v i s i b l e .  
A d e sm.osome ( D )  i s  p r e s ent b e t ween t w o  int r a ­
l o bul a r  duc t c e l l s  but in t e r c e l l u l a r  a t t achments 
are no t p r e s en t  b e tween l EG duc t c e l l s  and 
ep i t h e l i a l  c e l l s . 
Mag n i f i c a t ion : x 1 0 5 0 0  
An l EG duc t c e l l  s im i l a r  t o  tha t s hown 1n 
F i g . 3 . 4 . 1 . N o t e  vari a t i o n in g r a nu l e  
s t ructu r e  and t h e  p r e s enc e o f  c ry s t a l l i ne 
ma t e r i a l  in s ome g r anu l e s . The nuc l eus i s  
n o t  v i s i b l e . 
Mag n i f i c a t i o n : x 6 3 0 0  

F I GU RE 3 . 4  ( C ont i nu e d )  
3 . 4 . 3  
3 . 4 . 4  
l E G  duct c e l l  w i th mo s t l y typ e I V  g r anu l e s  
and cytop l a s m i c  " f ing e r" l i k e  p ro c e s s  ( P )  
o n  c e l l  s ur fa c e . 
Magn i f i c a t i o n : x 1 0 5 0 0  
Two g r anul e s  ( typ e s  I I  and I V )  a r e  s hown at 
a h i g h e r  mag n i f i c a t ion . The typ e IV g ranu l e 
c o nt a ins s om e  c ry s t a l l ine ma t e r i a l . 
Magn i f i c a t i o n : x 4 7 4 0 0  

8 4  
s p a c e s  ( F i g ure 3 . 4 . 3 ) . 
The i n t racytop l as m i c  g ranul e s  w e r e  surrounde d by smo o t h­
s u r f ac e d  un i t  memb ra n e s  and meas ur e d  from 0 . 6  t o  5 . 0  �m i n  
d i ame t e r . D e s p i t e  cons i de rab l e  v a r i a t i on in the i r  mo rpho l o g� 
f o ur b as i c  g ranu l e typ e s  w e r e  r e c o gn i z e d : -
Typ e I :  sma l l  round homo g en e o u s ly e l e c t ron dens e 
g r anul e s ; 
Typ e I I : l a r g e  found o r  o v a l  homo g eneous ly e l e c t ron 
dens e g ranul e s ; 
Typ e I I I : l a r g e  round o r  o v a l  g r anul e s  w i th an e l e c t ron 
dens e out e r  c o r e  s ur r o unding a l e s s  e l e c t ron 
dens e inn e r  c o r e ; 
Typ e I V :  l ar g e  round o r  ova l e l e c t ron dens e g r anu l e s  
wh i c h  had a re t i c u l a r  app e a ran c e  p ro duc e d  
b y  p o c k e t s  o f  un e v e n l y  s c a t t e r e d  e l e c t ro n  
l uc e n t  mate r i a l .  
Trans i t i ona l g r anu l e  typ e s  w e r e  a l s o  p r e s ent with 
shrunk en ma t r i c e s  and g l obul a r  c rys t a l l ine inc lus i ons . 
The cytop l as m  o f  an ent i re I E G  duct c e l l  usua l ly h a d  a 
vacuo l at e d  app e a ran ce and o r g ane l l e s  s uch a s  RE R and Go l g i  
comp l e x  w e r e  n o t  v i s ib l e  b e c aus e o f  the g r anul ar conten t s . 
The nu c l e us , wh en o b s e r ve d , h a d  chroma t i n  p e r iphe ra l ly d i s ­
t r i b ut e d  a s  py r ami d a l - shap e d  p r o j e c t i ons ext ending t owa rds 
the c en t r e  o f  the nuc l eus . 
3 . 4 D i s cus s i on 
The p a r o t i d  a n d  vent r a l  b uc c a l  g l and I E G  duc t  c e l l s  
w e r e  f requent ly f o und in the b a s a l  a s p e c t  o f  the int ra l ob ­
u l a r  duc t  ep i t h e l i um .  The i r  g e n e r a l  o r i en t a t ion w i t h  r e s ­
p e c t  t o  the duc t  e p i th e l ium d i d  no t s ug g e s t  that th e s e  
we r e  m i g r a t o ry c e l l s , a l though a f ew we re occas i ona l l y 
ob s e rv e d  in t h e  duct lumen and a t  s ub ep i th e l i a l  s i t e s . 
The p re s e n t  inve s t i g a t ion has s uc c e s s fu l ly e s t ab l i s h e d  
t h e  mo rp ho l o gy and h i s t o chem i s t ry o f  I EG duc t  c e l l s  l o c a t e d  
w i th in the i n t r a l o bu l a r  duc t  ep i th e l ium o f  the p a ro t i d  and 
v e n t r a l  b uc c a l  gl ands . 
8 5  
I n  g en e r a l  I E G  duc t  c e l l s  w e r e  s imi l a r  t o  s ub ep i th e l i a l  
o r  muc o s a l  ma s t  c e l l s  b ut had nuc l e i  s im i l a r  t o  tho s e  o f  
p l a s ma c e l l s . Sub e p i th e l i a l  ma s t  c e l l s  were p o lymo rp h i c  i n  
ap p e arance w i th nume r o u s  un i fo rm l y  sma l l  b a s ophi l i c g r a n ­
ul e s . T h e  chroma t in d i s t r ibut i on p a t t e rn o f  the i r  nuc l e i  
v a r i e d  cons i de r a b l y  and a t  t im e s  s howe d  the " c a r twhe e l "  d i s ­
t r i b u t ion p a t t e rn o f  p l a s ma c e l l  nucl e i . The r e  was no 
e v i dence t o  s ug g e s t  tha t such ma s t  ce l l s m i g r a t e d  into the 
duc t  ep i the l i um to b e come I E G  duc t  c e l l s . M i g r a t o ry ep i th ­
e l i a l  ma s t  c e l l s  have howeve r r e c e nt ly b e en d e s c r i b e d  i n  
g a l l b l adder e p i th e l i um o f  sheep and c a t t l e  (Mo r a l e s , P e r ey ra , 
To l e do and Man t e s , 1 9 8 0 )  wh i ch w e r e  hi s t o l o g i c a l ly and u l t r a ­
s t ruc tura l l y s im i l a r  t o  s ub ep i th e l i a l  mas t  c e l l s  ( C . R . Mo r ­
a l e s , p e r s o na l commun i c a t ion ) . A comp a r i s on b e tween s ub ­
ep i th e l i a l  mas t c e l l s  and I E G  duc t  c e l l s  o f  the p a r o t i d  and 
ven t r a l  b u c c a l  g l ands r e v e al e d  tha t a l though b o th w e r e  p o l y ­
mo r ph i c  i n  app e a ranc e , I E G  duc t  c e l l s  s howed a va r i e ty o f  
g r anu l e  s i z e s  a n d  the i r  nuc l e i  i n  p a r t i cul a r , o f t en c l o s e ly 
r e s emb l ed tho s e  o f  p l a sma c e l l s , a feature only o c c a s i o na l l y 
ob s e rv e d  in s ub ep i the l i a l  ma s t  c e l l s . 
On the o th e r  hand a s p e c i f i c  a s s oc i a t i on , i f  any , b e t ­
we e n  I E G duc t  c e l l s  and p l asma c e l l s  i s  ob s cur e . F o r  exam­
p l e , there is n o  evidence y e t  t o  s ugge s t  tha t p l a sma c e l l s  
have b e en ob s e rv e d  a t  i n t r a ep i th e l i a l  s i t e s . I n  p a r t i cu l a r  
p l a s ma c e l l s  s p e c i a l i s e d f o r  synth e s i s  and s e c r e t i on o f  
immun o g l ob u l i n s  a t  muc o s a l  s u r fa c e s  have been mo s t ly ob s e r ­
ve d a t  sub ep i t h e l i a l  s i t e s  ( Toma s i  e t  a l . ,  1 9 8 0 ) . I n  b o v i ne 
s a l i v a ry g l ands numerous p l a sma c e l l s we r e  p r e s ent s ub ep i t h ­
e l i a l ly in t h e  s ub l ingua l and man d i bu l a r  g l ands b ut s e l dom 
in p a r o t i d  or v e nt r a l  b uc c a l  g l ands ( S e c t i on 2 . 4 ) . I t  i s  
the re fo r e  unl i ke l y tha t I E G  duc t c e l l s  a r e  typ i c a l  p l a sma 
c e l l s . Fur th e rmo r e , the maj o r i ty o f  h i s tochem i c a l  f in d in g s  
w i t h  t h e  exc ep t i on o f  the PAS r e ac t i on , were not -chara c t e r ­
i s t i c  o f  p l asma c e l l s . Mo rpho l o g i c a l l y ,  howeve r ,  I E G  duc t  
8 6  
c e l l s  may a t  t im e s  app e a r  s imi l ar t o  p l a sma c e l l s  w i th 
Rus s e l l  b o d i e s . Such c e l l s  a r e  c o mmon in p a tho l o g i c a l  c o n ­
d i t i on s  ( A z a r , 1 9 7 9 )  and have p r o b ab ly r e a ch e d  the f i na l  
s t a g e s  o f  the i r  s e c r e t o ry cyc l e  ( Wa l t e r  and I s ra e l , 1 9 7 9 ) . 
Al t h ough exp e r i m ent a l  evi den c e  i s  l acking , Ru s s e l l  b o d i e s  
a r e  l ik e l y  t o  c o n t a in i mmuno g l o bu l i n s  comp l e x e d  w i th b a s i c  
p ro t e ins . Th i s  could a c c o unt fo r t h e  i n t ens e PAS and a c i do ­
ph i l i c s t a in in g  usua l l y  repo r t e d  i n  Rus s e l l  b o dy c e l l s  
(Wh i t e , 1 9 5 4 ) . On the o th e r  hand , g r anu l e s  o f  I E G  duc t  
c e l l s  were f r e q uently b a s ophi l i c w i th o n l y  mode r a t e  PAS 
r e a c t iv i ty and a c i dophi l i a .  
U l t r a s t ru c t ur a l  e v i dence ( F i gu r e  3 . 4 ) showed that 
g r an u l e s  of I E G  duc t c e l l s  we r e  n o t typ i c a l  of e i th e r  ma s t  
c e l l s  (Az a r , 1 9 7 9 )  o r  Rus s e l l  b o d i e s  o f  p l asma c e l l s  ( A z a r , 
1 9 7 9 ) . Ma s t  c e l l  g r anu l e s  a r e  un i fo rm l y  sma l l  roun d  and 
e l e c t ron den s e wh i l e  Rus s e l l  b o d i e s  of p l asma c e l l s  a r e  
un i fo rmly l a r g e  w i th ma t r i c e s  o f  mo d e r a t e  e l e c t ron d en s i t y 
inve s t ed by a d o ub l e  memb rane o f  RE R .  I E G  duc t  c e l l s  w e r e  
no t un i fo rm i n  app e aranc e w i t h  h e t e r o g enous matr i c e s  s u r ­
rounded by un i t  memb rane s , an d t h e i r  nuc l e i  when ob s e rv e d  
we r e  i n t a c t  and sug g e s t ive o f  fun c t iona l o r  l iving c e l l s . 
H i s t o chemi c a l  s t ud i e s  demon s t r a t e d  the s t r ong cy t o p l as m ic 
b a s op h i l i a  o f  I E G duc t  c e l l s  wh i l e  a mi xture o f  b o th a c i d o ­
ph i l i c and b a s o p h i l i c  ma t e r i a l  w a s  iden t i f i e d  in the g r an ­
ul e s  ( F i gure 3 . 3 . 7 ) .  The cy top l a sm i c  content s a r e  p ro b ab l y  
c o mp o s e d o f  s u l phated a n d  carboxy l a t e d  p ro duc t s  wh i ch w e r e  
demon s t r a t e d  b y  s t a in i n g  w i t h  A l c i an b lue a t  p H  1 . 0  and 2 . 5  
r e s p e c t i vely . H i s t o chemi c a l  me tho d s  s uc h  a s  Fa s t  Re d 2 B  
( F i gure 3 . 3 . 8 ) ,  s i lve r imp r e gn a t i o n  ( F i gure 3 . 3 . 5 ) and form­
a l i n - induc e d  a u t o fluo r e s cence i n d i c a t e d  that the g r anu l e s  
a r e  l i kely t o  cont a i n  a b i o g e n i c  amine . Pre s enc e o f  n e ut r a l  
a n d  a c i d i c  s i d e  g roup s  i n  g ranu l e s  were ind i c a t e d  b y  the PAS 
r e a c t i on and T o l udine b l ue me tachroma s i a ,  r e s p e c t i ve l y .  
C ry s t a l l ine i n c lus ions i dent i fi e d  w i th i n  s ome g ranu l e s  
( F i g u res 3 . 4 . 1  and 3 . 4 . 2 ) cou l d  p r o b ab ly b e  a t t r ibut e d  t o  
t h e  p r e s ence o f  a g l obular p r o t e i n (A . S .  Cr a i g , p e r s on a l  
c ommun i c a t i on ) . The r e  was , howe v e r ,  i n s u f f i c i ent e v i denc e 
8 7  
to c on c l ude tha t the g ranu l e s  may b e  e l ab o r a t ing a s e c r e t o ry 
p r o duc t . The p o s s ib i l i ty a l s o  e x i s t s that th e g ranul e s  may 
be i nvo l v e d  in e i t h e r  t ranspo r t  o r  s t o r a g e  of s e c r e t o ry 
ma t e r i a l , o r  b o th . 
The r a r e  o b s e rva t i o n  o f  I E G  duc t  c e l l s  in do r s a l  and 
in t e rmed i a t e  b uc c a l  and p a l at i n e  g l ands is di f f i c u l t t o  
e xp l a in ,  exc e p t to s p e c u l a t e  tha t the i r  appe a r an c e  m a y  in 
s om e  way b e  a s s o c i a t e d  w i th the p re s en c e  of p r o t e o s e rous 
i s l e t s  in a l l  three g l ands ( S e c t i on 3 . 3 ) i dent i c a l  t o  tho s e  
o f  t h e  p a r o t i d and v en t r a l  b uc c a l . 
The p r e f e r e n t i a l  l o c a l i s a t i on o f  I E G  duc t c e l l s  i n  i nt r a ­
l ob u l ar duc t s  o f  the p ar o t i d  and v ent r a l  b uc c a l  g l an d s  
s t r o n g ly s ug g e s t s  that i n  c a t t l e , t h e  I E G  duc t  c e l l s  a r e  
p r i m a r i ly a s s o c i a t e d  o n l y  w i t h  a func t i on sp e c i f i c  t o  the s e  
two g l ands . Th i s  hyp o the s i s c an b e  further e x t ended t o  
s t a t e  tha t I E G  duc t  c e l l s  a r e  p r ob ab l y  an independent c e l l  
l in e , p o o r l y  unde r s t o o d  func t i o n a l ly and spe c i f i c  o n l y  t o  
c e r t a in t i s s ue · s i t e s  and o r g an s . For e xamp l e , the p a r o t i d  
and v ent r a l  b u c c a l  g l ands o f  l ow b l o a t  s u s c ep t i b l e  c a t t l e  
c o n t a in a h i gh e r  p r o p o r t i on o f  I E G  duc t  c e l l s  than h i g h 
b l o a t  s u s c ep t i b l e  c a t t l e  ( B i r t l e s , 1 9 8 1 ) . I t  i s  t emp t ing 
t o  s ug g e s t  that I E G duc t  c e l l s  i n  conj unc t i on w i t h a fun ­
c t i on s p e c i f i c  to the paro t i d  g l an d , a f fo r d cons i de r ab l e  
p ro t e c t i o n  t o  c a t t l e  a g a in s t  b l o a t . T h i s  pro t e c t ive ro l e  
may invo lve t h e  muc o s a l  immune s y s t em o r  an unknown s e c r e t ­
o ry p r o duc t e l ab o r a t e d  b y  the I E G duc t  c e l l s . 
Al though t h e  e x a c t  fun c t i o n  o f  I E G  duct c e l l s  r em a i n s  
unknown , a l i k e l y  o r i g in o r  i den t i ty cou l d  b e  e l uc i da t e d  
f r o m  an a s s e s sment o f  two p o s s ib l e  the o r i e s  b a s e d  on a l l  
t h e  ava i l ab l e  eviden c e  p e rt a in i n g  t o  the i r  morpho l o gy and 
h i s t o chemi s t r y . 
( 1 )  P a r o t i d  and vent r a l  buc c a l  g l an d  I EG duc t c e l l s  
a r e  ident i c a l  to s ub ep i t he l i a l  or muc o s a l  mas t 
c e l l s  p r e s ent in a l l  ma j o r and mino r s a l iva ry 
g l ands but l o c a t e d  i n t r a ep i the l i a l l y only in 
p a ro t i d  and vent ra l b uc c a l  g l and int r a l o b u l a r  
duc t s . The char a c t e r i s t i c morpho l o g i c a l  app e a r ­
anc e  o f  the i r  g r anu l e s  c o u l d thus b e  a t t r i bu t e d  
8 8  
t o  anatom i c a l  r e a s ons s i n c e  they a r e  l o c a t e d  i n t r �  
e p i th e l i a l l y  rathe r t h a n  i n  conne c t i ve t i s su e . 
( 2 )  Th e l E G  duc t  c e l l s  a r e  an indep endent c e l l  l in e  
c l o s e ly r e l a t e d  t o  b o t h  muc o s a l  mas t c e l l s  and 
p l a s ma c e l l s ; a l l  t h r e e  c e l l  typ es p r o b ab l y  s h a r e  
a c o mmon s t em c e l l but l E G duc t ce l l s  o r  t h e i r  
p r e cu r s o r s  a r e  sp e c i f i c  t o  int r a ep i th e l i a l  s i t e s  
a n d  i n  c a t t l e , func t i on a l ly a s s o c i a t e d  o n l y  wi th 
t h e  p a ro t i d  and vent r a l  b uc c a l  g l ands . 
Drawb a c k s  o f  the f i r s t  t h e o r y  a r e  tha t , a l though muc o s a l  
ma s t  c e l l s  s ha r e d  nume rous f e a tu r e s  i n  common w i t h  l E G duc t 
c e l l s , s uch a s  a nuc l eus s im i l a r  t o  that o f  a p l a s m a  c e l l  
and int e n s e b a s ophi l i c s t a in i n g , u l t r a s t ructur a l l y  t h e  two 
c e l l  typ e s  d i d  n o t  app e a r  to b e  i dent i c a l . Fur t h e rmo re , a s  
p r e v i ous l y  r e p o r t e d  b y  Mo ra l e s  e t  a l . ( 1 9 8 0 ) , i n t r a ep i the l ­
i a l  ma s t  c e l l s  o f  r uminants d i d  n o t  d i f f e r  from s ub e p i th e l ­
i a l  mas t c e l l s  and d e g ranu l a t in g  muc o s a l mas t c e l l s  o b s e rv e d  
s u b ep i the l i a l ly i n  t h e  p re s ent s tu dy cons i s t en t l y  c o n t a i n e d  
un i f o rmly s ma l l  round g ranu l e s , d i s s im i l a r  to tho s e  found 
i n  l E G  duc t  c e l l s  wh ich showe d a v a r i e ty of g ranu l e  s i z e s . 
I n  a ddi t i on i t  has b e en rep o r t e d  b y  C o l l an ( 1 9 7 2 )  t h a t  
i mm i grant c e l l s  common l y  ob s e rv e d  intraep i the l i a l ly s uch 
as lympho cy t e s  and e o s inophi l s , do no t d i f fe r  in mo rpho l o gy 
t o  s imi l a r  c e l l s  l o c a t e d  s ub ep i th e l i a l ly . Ano t h e r  p o s s i b ­
i l i ty ,  t h a t  l E G du c t  c e l l s may b e  d e r ived from muc o s a l  mas t  
c e l l s  wh i c h  have p r e v i o u s l y  m i g r a t e d  into the du c t  e p i the l ­
i um , i s  un l i k e ly s in c e  muc o s a l  m a s t c e l l s  o f  a l l  g l ands 
app e ared t o  be non - mi g r a t o ry whe r e a s  lEG duc t  c e l l s  s e emed 
t o  d i ff e ren t i a t e  only w i thin t h e  e p i the l i um of p a ro t i d  and 
v e n t r a l  b uc c a l  g l and i nt r a l ob u l a r  duc t s , adj acent to the 
b a s al l am i n a . 
The s e cond theo ry app e a r s  mo r e  p l aus ib l e  b e c au s e  o f  the 
charact e r i s t i c  fe a t ur e s  un i que t o  the l E G duc t  c e l l  s uch 
8 9  
as v a r i a t i on i n  g r anu l e  s i z e and s t ructure , an d the p r e s enc e 
o f  a c omb ina t i on o f  f e a t ur e s  c ommon t o  b o t h muc o s a l  ma s t  
c e l l s  and p l a s ma c e l l s , f o r  examp l e  sma l l  e l e c t r o n  den s e  
g r anul e s , b a s o ph i l i a a t  l o w  pH , m e t a chroma s i a , p r e s en c e  o f  
a b i o g en i c  ami n e , PAS r e a c t iv i ty and nuc l ear c h roma t i n  
p a t t e rn . 
I t  i s  we l l  e s tab l i s h e d  that p l a s ma c e l l s a re d e r i v e d  
f r o m  l ymphocy t e s  (Ham a n d  C o rmack ,  1 9 7 9 ) . E xp e r iment a l  
e v i d e n c e  t o  d a t e , in s upp o r t  o f  a lympho cyte o r i g in fo r 
muc o s a l  ma s t  c e l l s  i n  r a t  and man , h ave b e en r ev i ewed by 
Mi l l e r  ( 1 9 8 0 )  and B i enen s t o c k , B e fus , P e ar c e , D enbu r g  and 
G o o da c r e  ( unp ub l i she d ) . I t  i s  probab l e  that a l ymp ho cy t e  
s ub p o p u l a t i o n  s p e c i f i c  t o  int r a ep i th e l i a l  s i t e s  c ou l d  b e  
imp l i c a t e d  i n  the d e r iva t i on o f  I E G duc t c e l l s  o f  p a ro t i d  
and v e n t r a l  b u c c a l  g l an d s  o f  c a t t l e . Th i s  lymphocyte s ub ­
p o pu l a t ion may o r  may n o t  b e  c l o s e l y  r e l a t e d  t o  the p re c u r ­
s o r  c e l l s  o f  t h e  muc o s a l  ma s t  c e l l .  I t  i s  a l s o  t emp t ing t o  
s p e c u l a t e  on t h e  s i gn i fi c anc e o f  muc o s a l  mas t c e l l s  obs e rv e d  
in t h e  p r e s ent s tudy that s e em e d  t o  di fferent i a t e  in t h e  
v i c i n i ty o f  p l asma c e l l s  ( F i gu r e s  3 . 2 . 3  a n d  3 . 2 . 4 ) ; th i s  
c o u l d  s ug g e s t  a lympho cy t e  o r i g i n f o r  the muc o s a l  ma s t  
c e l l s  o f  c a t t l e  a s  w e l l .  
A p r e l imi n a ry h i s t o l o g i c a l  a n d  h i s t o chem i c a l  examin a t i o n  
o f  s h e ep p a ro t i d  g l and s t r i a t e d  duc t s  e s t ab l i s h e d  a n  app arent 
s p e c i e s  d i ffe r e n c e  b e tw e e n  i n t r a e p i the l i a l  duc t  c e l l s  o f  
s h e ep and c a t t l e . I n  s h e e p , typ i c a l  mas t c e l l s  w i th un i f ­
o rm l y  sma l l  c y t o p l a s m i c  g ranu l e s  and den s e l y - s ta ining 
nuc l e i  were o c c a s iona l l y o b s e rved intra e p i th e l i a l ly . I n  
a d d i t i on a g r anu l a r  c e l l  typ e wa s a l s o no t i c e d ,  e s s en t i a l l y 
s im i l a r  to I E G  duc t c e l l  o f  c a t t l e ,  w i t h  a nu c l eus s im i l ar 
t o  t h a t  o f  a p l a sma c e l l ; howeve r the g ranu l e s  w e r e  un i fo r ­
m l y  l arge , r o un d  and c o n s i de ra b l y  more e o s inophi l ic and 
we r e  s·urrounde d by Al c i an b l ue p o s i t ive b a s op h i l i c  ma t e r i a l  
( p H  1 . 0 and 2 . 5 ) ,  ( F i g u r e  3 . 1 . 8 ) . Van L ennep e t  a l . ( 1 9 7 7 )  
wh o e x amined th i s  c e l l  u l t ra s t ru c t ura l ly du r i ng an inve s ­
t i g a t i on o f  t h e  she ep p a ro t i d  g l and , rep o r t e d  the granu l e  
ma t r i c e s  t o  b e  homo g en e o u s . Th i s  f i nding con t r a di c t s  
ob s e rv a t i on s  made in the p r e s ent s tu dy wh e re t h e  g r anul e s  
o f  I E G  duc t c e l l s  i n  c a t t l e  app e a r e d  h e t e rog enous . The 
g r anu l a r  c e l l s  o b s e rve d in the ep i th e l i um o f  t h e  sheep 
aboma s um we r e  mo rpho l o g i c a l l y s im i l a r  t o  tho s e  of the s heep 
p a ro t i d  g l and s t r i a t e d  duc t s  but h i s t o chemic a l l y there was 
l i t t l e  evi denc e o f  cytop l a sm i c  b as op h i l i a ,  a l t hough the 
g r a nu l e s w e r e  d i s t in c t ly a c i dophi l i c .  Such c e l l s  in the 
sheep aboma s um h ave b e en v a r i o us l y  d e s c r i b ed as e i t h e r  
g l o b u l e  l eu c o c y t e s  (Kent , 1 9 5 2 ; Mur ray , Mi l l e r  and J a r re t t , 
1 9 6 8 )  o r  p l a s ma c e l l s  w i t h  Ru s s e l l  b o d i e s  ( Do b s on , 1 9 6 6 ) . 
T h e s e  p r e l iminary f i n d i n g s  s e em e d  t o  indfc a t e  that 
va r i a t i on of the ep i the l i a l  g r anu l a r  c e l l  typ e may b e  
p r e s e n t , dep end ing o n  s p e c i e s  and anatomi c a l  l o ca t ion . 
9 0  
CHAPTER 4 .  GENERAL D I SCUSS I ON 
9 1  
Ove r two decades a g o , K ay ( 1 9 6 0 )  i nve s t i g a t e d  rum i nant 
s a l i vary s e c r e t i ons i n  cons i de r ab l e  de t a i l  and r ep o rt e d 
b r i e f ly on t h e  h i s t o l o gy o f  t h e  maj o r  and mino r s a l i vary 
g l ands o f  s h e e p  and c a t t l e . The p a ro t i d  g l ands o f  b o th 
s p e c i e s  w e r e  c omp o s e d  o f  PAS n e g a t i ve cubo i da l - s h ap e d  s e c ­
r e t o ry c e l l s  ma inly s p e c i a l i s e d f o r  s e c re t ion o f  wa t e r  and 
e l e c t r o l y t e s , but s ub s e quent h i s t o l o g i c a l  and h i s t o c h emi c a l  
i nv e s t i g a t i o n s  b y  P a l  e t  a l . ( 1 9 7 2 ) , B i r t l e s  ( 1 9 8 1 )  and 
Vi g no l i  and N o g ue i r a  ( 1 9 8 1 )  have e s t ab l i s hed that in bovine 
p a r o t i d  g l ands in p a r t i cu l a r , t h e  s e c r e t o ry c e l l s  o c c a s i o n ­
a l l y  cons i s t e d  o f  d i a s t a s e  r e s i s tant PAS p o s i t ive ma t e r i a l . 
I n  t h e  p r e s en t  s tudy t h e s e  f ind i n g s  w e r e  con f i rmed ( F i g u r e  
2 . 1 ) a n d  i n  a dd i t i on s ome s ec r e t o ry c e l l s  w e r e  found to 
c o n s i s t  o f  w e a k  A l c i an b lu e  (AB ) (pH 2 . 5 ) and c o l l o i da l  i ron 
p o s i t ive ma t e r i a l , a f e ature a l s o  demons t ra t e d  by Pal e t  a l . 
( 1 9 7 2 )  and V i g no l i  and Nogue i ra ( 1 9 8 1 ) . Immunoh i s to chem i c a l  
i n v e s t i g at i on s  wi th ant i b o d i e s  t o  bovine s a l iva ry p r o t e i n  
b an d s  4 and 1 0  i s o l a t e d  b y  J o n e s  e t  a l . ( 1 9 8 2 ) , c o n f i rme d 
t h a t mo s t  b o v ine p a r o t i d  g l and s e c r e t o ry c e l l s  addi t i ona l ly 
c o n t a in e d  p r o t e i nac i o u s  ma t e r i a l  whi c h  was n o t  de t e c t e d by 
h i s t o l o g i c a l  s t a ins ( F i gu r e  2 . 3 ) .  Th i s  fur the r  s t rengthens 
the va l i d i ty o f  naming s e r ous c e l l s  a s  p ro t e o s e rous c e l l s  
( B i r t l e s , 1 9 8 1 ) . 
From an a s s e s sment o f  e xi s t ing h i s t o chemi c a l  and b i o che� 
i c a l  dat a ,  it may be c on c l ude d that s e c r e t i on s  of the b o v i n e  
p a r o t i d  g l ands a r e  p r e dominant l y  comp o s e d  o f  wa t e r , e l e c t ro ­
l y t e s  and p r o t e in s  w i t h  sma l l  amo un t s  o f  neut r a l  and ac i di c  
g l y c op ro t e i n s . The PAS p o s i t iv e  mat e r i a l  c an b e  a t t r ibut e d  
t o  p ro t e i n b and 5 ,  a g l ycop r o t e in wh i ch c on t a i n s  nume rous 
s i a l ic ac i d  s ubun i t s  ( W . T .  J o ne s , p e r s on a l  c ommun i c a t i on ) . 
T h e  Al c i an b lue and c o l l o i da l  i ron p o s i t i ve ma t e r i a l  r a r e l y  
f o und in s ome t i s s ue s e c t i ons e xamine d ,  may b e  d u e  t o  sma l l  
quant i t i e s  o f  h i s t o ch em i c a l ly de t e c t ab l e  s i a l i c  ac i d  r e s i d ­
u e s .  Ac c o r d i ng t o  V i g no l i  and Nogui e ra ( 1 9 8 1 ) , t h e  o cc u r ­
r e n c e  o f  PAS and A B  p o s i t iv e  mat e r i a l  in b o v i n e  p a r o t i d  
g l a n d s , can b e  a s s o c i a t e d  wi th the a g e  o f  t h e  anima l ; 
c a l ve s a g e d  b e tw e en 5 a n d  1 3  months had cons i d e r a b l y  h i gh e r  
l e v e l s  o f  g l y c o p ro t e i n s  t h a n  mo r e  mature anima l s . Th i s  
p h e n o menon wa s no t exp l o re d  i n  t h e  p re s en t  s tudy . S a l ivary 
p ro t e in b and 4 s e c r e t e d  b y  t h e  p ar o t i d  g l ands has b e e n  
co r r e l a t e d  w i th b l o a t  s us c ep t ib i l i ty in c a t t le (Mc i nt o s h  
9 2  
and C o c k ram , 1 9 7 7 )  and w a s , i n  t h e  p re s en t  inve s t i ga t i o n , 
e q u a l ly d i s t r ibuted in p ar o t i d  t i s s ue s amp l e s  o f  b o th L . S .  
and H . S .  anima l s . Thi s unu s u a l  f i nding i s  l ik e l y  t o  i nvo lve 
p o o r l y  unde r s to o d  c e l l u l a r  r e g u l a t o ry me chan i sms a s s o c i a t e d  
w i t h  p ro t e in s e c ret i on . Re cent l y , P a t t e r s on e t  a l . ( 1 9 8 2 )  
have r ep o r t e d  that p ro t e i n s e c r e t i on from t h e  s h e ep p a ro t i d  
g l an d s  invo l v e d  a S - a d r e n e r g i c  ( sympathe t i c )  comp onent 
p r om i n e n t  up o n  commenc ement of f e e d ing . I f  a s im i l a r  s e c ­
r e t o ry comp o n e n t  ex i s t s  i n  c a t t l e , a v a l u ab l e c o n t r i but i on 
t o  p r e s ent unde r s t anding co u l d  b e  made by examining the 
e ff e c t s o f  s - s ympa the t i c  s t imul a t i o n  on p ro t e i n  s e c r e t i on 
f ro m  L . S .  and H . S .  an i ma l s , e sp e c i a l ly in r e l a t i o n  t o  s e c ­
re t i o n  o f  p r o t e in b and 4 .  Al though the s ymp a th e t i c  e ff e c t s  
on r uminant p a ro t i d  s a l i va ry s e c r e t i on h a s  b e en c a r e fu l l y  
exp l o r e d ,  i t  i s  l ik e l y  t h a t  owing to s yne r g i s t i c  i n f l uenc e s , 
( B a b k in , 1 9 5 0 )  b o th symp athe t i c  and p ara s ymp a the t i c  i nn e r ­
va t i o n  may b e  imp l i c a t e d  i n  p ro t e in s e c r e t i o n  from p a r o t i d  
g l an d s . P a t t e rs o n  e t  a l . ( 1 9 8 2 )  a dd i t i o na l l y  c o r r e l a t e d  
p r o t e in s e cr e t ion w i t h  magne s ium i o n  c o n c en t r a t i o n  o f  p a r ­
o t i d  s a l iva . A p o s s ib l e  exp l ana t i o n  f o r  th i s  a s s o c i a t i on 
c o u l d  b e  the influence o f  e i th e r  a n  i n t r a c e l lu l a r  o r  a 
c e l l ul a r  memb rane p r o t e in t ran s p o r t  mechan i sm wh i ch ut i l i s e s 
ma g n e s ium i ons . 
A h i s to l o g i c a l  survey o f  t h e  p a ro t i d g l and duc t a l  sys t em 
s ho w e d  that b a s a l  s t r i a t i ons wh i ch hou s e  m i t o c hond r i a  
( P i n k s t af f , 1 9 8 0 )  w e r e  n o t  a f e a ture o f  the bovine p a ro t i d 
g l an d  i n t r a l ob u l a r  duc t s  ( F i g u r e  2 . 2 . 3 ) .  A s im i l a r  conc lus ­
i o n  was r e a ch e d  by Shac k l e fo rd and Wi l b o rn ( 1 9 6 9 ) , P a l  e t  a l . 
( 1 9 7 2 ) , P in k s t af f  ( 1 9 8 0 )  and V i gno l i  and Nogue i ra ( 1 9 8 1 ) . 
I n  t h e  p r e s ent s t udy , h oweve r ,  numerous b a s a l  s t r i a t ions 
w e r e  i dent i f i e d  i n  s h e e p  p aro t i d  g l and s t r i a t e d  duc t s  ( F i g ­
u r e  2 . 2 . 4 ) ,  a finding u l t ra s tructura l ly c on f i rmed by 
van L enne p  e t  a l . ( 1 9 7 7 ) . Kay ( 1 9 6 0 )  wa s no t awa r e  o f  the 
s i g n i f i c a n c e  o f  b a s a l  s t r i a t i o n s  in paro t i d  g l and duc t s  o f  
e i t h e r  sheep o r  c a t t l e  and conc l uded that the rum inant 
p a r o t i d g l an d s  p ro duc e d  a n  i s o t o n i c  s e cr e t io n  and we r e  
un l i k e  the g l ands o f  m o s t mammal i an sp e c i e s  wh i c h  p r o duced 
9 3  
a hyp o t on i c  s e c r e t i o n . Howeve r ,  Compton e t  a l . ( 1 9 8 0 )  rep ­
o r t e d  from m i c r opuncture i nve s t i g a t ions that the s he e p  
p a r o t i d  g l an d  duc t a l  s y s t em was capab l e  o f  wat e r  a n d  e l e c t iU ­
ly t e  t r an s p o r t  and s e c r e t ion o f  hyp o t o n i c  s a l i va . The 
l a c k  of b a s a l  s t r i at i o n s  in i n t r a l ob u l a r  duc t s  of b o v i n e  
s p e c i e s  s ug g e s t s that t h e s e  duc t s , unl i k e  tho s e  o f  the 
s h e e p  p a ro t i d  g l ands , a r e  no t a c t ively eng a g e d  i n  wat e r  and 
e l e c t r o l y t e  t ransp o r t  and thu s  s e c re t e  an i s o to n i c  s a l iva . 
The func t i o n a l  s i g n i f i ca n c e  o f  an i s o t o n i c  s e c r e t i o n  c an 
b e  a t t r i b ut e d  t o  a con s i de rab l e  s aving o f  o s mo t i c  ene rgy 
made from r e du c e d  duc t a l  t rans p o r t  of i o n s , thereby enab l ing 
the animal to achi eve a higher r a t e  of s e c re t i on . Ac c o r d ing 
to S hack l e fo r d , ( 1 9 6 9 )  i on i c  t r anspor t a t i on in the b o v ine 
p a r o t i d  g l an d s  i s  p r ima r i ly condu c t e d  by l a t e r a l  s u r f a c e  
mo d i f i c a t i o n s  o f  the p ro t e o s e r o u s  s e c r e t o ry c e l l s  wh i l e  
i n t r a l o b u l a r  duc t s  may h ave a s e c ondary ro l e . 
Ano ther s i gni f i c ant f e a t u r e  o f  bovine p a ro t i d  g l and 
i n t r a l obul a r  duc t s  wa s the p r e s en c e  of PAS p o s i t i ve m a t e r i a l  
a n d  ap i c a l  b l eb s  in s om e  duc t  c e l l s  (F i gure 2 . 2 . 1 ) . A l t hough 
s om e  o f  the PAS p o s i t iv e  ma t e r i a l was d i a s t a s e l a b i l e , the r e  
w a s  a s ig n i f i c ant amo un t o f  d i a s t a s e  re s i s t ant ma t e r i al 
wh i ch c o u l d  b e  a t t r i b u t e d  t o  n e u t r a l  g l ycopro t e i n s . A 
s i m i l ar c on c l u s i on was r e ache d b y  Shack l e fo rd ( 1 9 6 3 ) , P a l  
e t  a l . ( 1 9 7 2 ) , B i r t l e s  ( 1 9 8 1 ) a n d  Vi gno l i  and Nogue i r a  
( 1 9 8 1 ) . T h e  PAS p o s i t ive mat e r i a l  may b e  r e l e a s ed t o  the 
duc t l umen v i a  an ap o cr ine s e c r e t o ry me chan i s m  wh i ch coul d 
a c c o unt fo r the p r e s en c e  o f  ap i c a l  b l eb s . 
The ex c r e t o ry duc t  o f  the b o v ine p a ro t i d  g l and was 
c h a rac t e r i s e d  by nume rous gob l e t  c e l l s  (F i g u r e s 2 . 1 . 3  and 
2 . 1 . 4 ) wh i c h  c o n t a ine d ac i d i c  and neut r a l  mu c o s ub s t an c e s ;  
t h e s e  finding s p a ra l l e l e d ob s e rvat ions by Shack l e fo r d  and 
K l appe r  ( 1 9 6 2 ) , P a l  e t  al . ( 1 9 7 2 ) , B i r t l e s ( 1 9 8 1 )  and 
V i g no l i  and Nogue i ra ( 1 9 8 1 ) . The a c i d i c  muc o s ub s t an c e s  o f  
t h e  g ob l e t  c e l l s  w e r e  f ound t o  cont a i n  b o th c a rb o xy l i c  
a n d  sulpha t e  g roup s . The t o t a l  s e c r e t i o n  e l ab o r a t e d  by 
9 4  
t h e  bovine p a ro t i d  g l ands c o u l d the r e fo r e c o ns i s t  o f  wat e r , 
e l e c t r o ly t e s , p r o t e i n s , neut r a l  muc o s ub s t an c e s  and a c i d i c  
muc o s ub s t an c e s  comp o s e d  o f  s i a l i c a c i ds a n d  s u l p ha t e d  g ly c o ­
p r o t e in s , t h e  l a t t e r  s e c r e t e d  by gob l e t c e l l s .  
I t  i s  doub t ful whe t h e r  the p a ro t i d  g l ands o f  c a t t l e  c o n ­
t r i b u t e  s i g n i f i c ant l y  t o  t h e  ma intenanc e o f  t h e  muc o s a l 
i mmune s y s t em s in c e  p l a sma c e l l s  were infr equen t l y  ob s e rv e d  
i n  p a r o t i d  t i s s ue s . O n e  e xp l an a t i on c ou l d  b e  the h i g h  
con t inuous s a l iva ry f l ow r a t e  p ro v i d ing enhan c e d  p ro t e c t i o n  
a g a i n s t ant i g en i c  s t im u l a t i o n  ( Wa t s o n  a n d  L a s c e l l e s , 1 9 7 3 ) . 
Ano the r p o s s ib i l i ty i s  that l E G  duc t c e l l s  r a t h e r  than 
p l a sma c e l l s  may p r ima r i ly m e d i a t e  mu c o s a l  immun i ty , a l t h ­
o u gh t h e  e x a c t  mechani s ms invo l v e d  a r e  a t  p re s en t  unc l e a r . 
Th i s  hyp o t he s i s i s  s up p o r t e d  by the o b s e rv a t i o n that o f  a l l  
t h r e e  maj o r  s al ivary g l ands examine d ,  l EG duc t  c e l l s  were 
l o c a t e d  only in the du c t a l  ep i t h e l ium of the p ar o t i d  g l ands . 
B o t h  the mandibu l a r  a n d  s ub l i ng ua l g l ands con t a i n e d  s ub s t an ­
t i a l  numb e r s  o f  p l a sm a  c e l l s , b u t  no t l E G  duc t  c e l l s . 
Bovine mandibul a r  g l ands , un l i k e  the p a ro t i d  g l ands , 
w e r e  p r e dom inant l y  s p e c i a l i s e d  fo r the s e c r e t i o n  o f  muc o ­
s ub s t ance s .  Long tub u l ar endp i e c e s  o f  the mand ibul a r  g l ands 
w e r e  comp o s e d  o f  muc o u s  c e l l s  and dem i l un e  c e l l s , the i r  
h i s t o l o g i c a l  and u l t r a s t ructur a l  f e a t u r e s have b e en de s c r i b ed  
b y  Shackl e fo r d  and K l a pp e r  ( 1 9 6 2 ) , Shack l e f o r d  and W i l b o rn 
( 1 9 7 0 ) , B l o om and C ar l s o o  ( 1 9 7 4 )  and B i r t l e s  ( 1 9 8 1 ) . 
Muc ous c e l l s  o f  t h e  b ovine mandi b u l a r  g l and w e r e  found 
t o  contain neut r a l  a n d  c a rb o xy l a t e d  a c i d  muc o s ub s t anc e s , a 
c o n c lus ion a l s o  r e ach e d  by Shac k l e f o r d  and Kl app e r  ( 1 9 6 2 )  
and B i r t l e s  ( 1 9 8 1 ) . The comp o s i t i on o f  muc o s ub s t anc e s  was 
p r i ma r i l y  d emons t ra t e d  w i t h  PAS , AB and To l ud i n e  b lue s t a i n ­
i n g  t e chn i ques , a l though Quint a r e l l i  ( 1 9 6 3 a ) , from h i s t o ­
c h em i c a l  inve s t i g a t i on s  o f  mamma l i an s a l i v a ry g l ands , found 
t h e  bovine mandibul a r  g l and muc o s ub s t anc e s  to be T o ludine 
9 5  
b l u e  n e g a t i v e  and non - m e t achroma t i c , a r e s u l t d i sput e d  i n  
t h e  p re s en t  s tudy ( F i gu r e  2 . 4 . 3 ) .  Al though the t h e o r y  o f  
m e t achroma t i c  r e ac t i on s  i s  s t i l l  in s ome dou b t  ( Cu l l ing , 
1 9 7 4 ) , i t  i s  l i k e l y  tha t a l l  a c i di c  g roup s i nduc e e i t h e r  
purp l e  o r  p u rp l e - r e d  m e t achroma s ia depend ing o n  t h e i r  mo l ­
e c u l a r  w e i g h t  and p o l ym e r i c  n a t u r e . F o r  examp l e  s t r o n g l y  
a c i d i c  g ro up s found i n  c onne c t ive t i s s ue muc o p o l y s a c c ha r i de s  
r e v e a l  a d e e p  purp l e  t o  red m e t a chroma t i c  c o l o u r  n o t  evi de n t  
i n  e p i t he l i a l  muc ous s e c r e t i o n s . I n  the p r e s en t  s tudy , 
b ov i n e  s a l i v a ry g l and muc o s ub s t anc e s  e xh i b i t e d  purp l e  me t a ­
ch r omas i a ,  d emons t r a t i n g  the p r e s en c e  o f  mo d e ra t e ly s t rong 
a c i d  ra d i c l e s such a s  t ho s e  of c a rboxyl g roup s . The purp l e  
me t a chroma t i c  co l ou r  o f  the man d i bul a r  g l an d  muc o u s  c e l l s  
i s  m a i n l y  du e to the c a rboxyl g roup s o f  s i a l i c  a c i d  r e s i d ­
ue s , a r e su l t  con f i rmed b y  neuramini das e d i g e s t i o n  fo l l owed 
b y  AB s t a in in g  ( F i gu r e s 2 . 4 4 9  a nd 2 . 4 . 1 0 ) , and a l s o  f rom 
h i s t o chemi c a l  (Quinta r e l l i  et a l . , 1 9 6 0 )  and b i o chem i c a l  
( J o n e s  e t  a l . ,  1 9 7 7 )  s t ud i e s . T h e  maj o r s i a l i c  a c i d  r e s i d ­
u e s  i n  b ov i n e  man d i bu l a r  g l an d  s e c re t i ons have b e e n  i dent ­
i f i e d  a s  N - a c e ty lneuram i n i c  a c i d  ( 3 7 % ) , N - a c e t y l - 9 - 0 -
a c e t y lneuram i n i c  ac i d  ( 3 3 % )  and N - g lyco l y l n e u r am i n i c  a c i d  
( 1 7 % ) , ( H e rp e t  a l . ,  1 9 7 9 ) . 
Wh i l s t  t h e  s i a l i c  a c i d  d e r i va t ive s o f  the b o v i n e  s a l iv ­
a ry g l an d  s e c r e t i on s  have b e en w e l l  cha r a c t e r i s e d , t h e  
p r e s enc e o f  s ulphate g r o up s  in t h e  mand ibul a r g l an d  o r  i n  
b o v ine s a l i va ry muc o s ub s tanc e s , i n  g e ne ra l , h a v e  r e c e ived 
l i t t l e a t t en t i on . I n  the p r e s ent inve s t i g a t i o n  a few mucous 
c e l l s  of t h e  mandibul a r  g l ands conta ine d v e ry weak AB s t a i n ­
i n t  at pH 1 . 0 .  Th i s  c ou l d  b e  a t t r ibu t e d  t o  t h e  p r e s en c e  o f  
sma l l  amoun t s  o f  sulpha t e d  g lycop r o t e ins o r  s ia l o - s u lphat e d  
g l y copro t e i n s  ( Re i d  a n d  C l amp , 1 9 7 8 ) , ra the r than t o  de r i v ­
a t i v e s  o f  chondr o i t in s u lphat e s , s inc e A B  s t a in i n g  a t  l ow 
p H  w a s  r e t a i n e d  a f t e r  hyaluron i da s e  d i g e s t i on . AB s t a i n i n g  
a t  p H  1 .  0 a s c r ib ed t o  " s ul p homu c i n s "  has b e e n  demon s t r a t e d  
b y  P a l  a n d  Chandra ( 1 9 7 9 )  in t h e  mand ibul a r  g l and o f  the 
wa t e r  buffa l o , a c l o s e l y  r e l a t e d  bovi dae sp e c i e s . The i r  
h i s tochem i c a l  finding s app e a r e d  s im i l a r  t o  tho s e  ob s e rv e d  
in c a t t l e  i n  t h e  p r e s e n t  s t udy , excep t t h a t  t h e  autho rs have 
b ro a d l y  a t t r ib ut e d  AB s t a i n i ng a t  pH 2 . 5 to " s i a l omuc ins 
a n d  hya luron i c  ac i d" , n e i th e r  of wh i c h  were conf i rm e d  by 
e n z ymic d i g e s t ion . 
9 6  
I n  cont r a s t  t o  muc ou s  c e l l s ,  the h i s t o chem i s t ry o f  the 
b o v i n e  man d i b ul a r  g l an d  dem i l une s has r e c e iv e d  l i t t l e  
a t t ent i on . The demi l un e s w e r e  l a rg e  con s p i cuous c e l l s  
w e a k l y  PAS and AB (pH 2 . 5 )  p o s i t ive d emon s t ra t ing the 
p r e s ence of neut r a l  a n d  c a rb o xy l a te d  a c i di c  muc o s ub s t anc e s . 
S im i l a r  r e s u l t s  w e r e  a l s o repo r t e d  by Sha c k l e fo r d  and 
K l apper ( 1 9 6 2 )  and B i rt l e s  ( 1 9 8 1 ) . The p rominent a c idop h i l  
g r anul e s  o f  the d em i l un e s  ( F i gu r e  2 . 4 . 6 ) c o u l d  c o nt a in s ome 
e n do g eneous p e ro x i da s e  ( B l o om and C a r l s o o , 1 9 7 4 )  and bovine 
s a l i va ry p r o t e in b an d  8 wh i c h  w a s  l o c a t e d  immuno h i s tochem i c ­
a l ly i n  the demi l une s ( F i gu r e  2 . 4 . 8 ) .  
An unusual p rop e r ty o f  the demi l une a c i doph i l  g ranu l e s , 
n o t previ o u s ly inve s t i g a t e d , c o u l d  b e  i n  the i r  ab i l i ty t o  
" m a s k" r e s i du e s  o f  any s i a l i c  a c i d  p r e s ent i n  t h e  demi lun e s , 
s o  that s uc h  r e s i du e s  wou l d  r ema i n  inacc e s s ib l e  to h i s t o ­
l o g i c a l  s t a in s . Quint a r e l l i  ( 1 9 6 3b )  i nv e s t i g a t e d  t h i s  mas ­
k i n g  e ff e c t  o f  s i a l i c  a c i d  r e s i du e s  by b a s i c  p ro t e in s  and 
r e c ommended the u s e  o f  a p r o t e o l y t i c  e n z yme to unma s k  t h e  
r e s i due s . A c c o r d i n g  t o  Quint a r e l l i , s i a l i c ac i d  may b e  
p r e s ent i n  two d i f f e r e n t  fo rms , a b ound fo rm de t e c t e d  a f t e r  
p ro t e o lyt i c  a c t i on , and a n  unbound f o rm e a s i l y  d e t e c t e d  and 
s u s c ep t ib l e  t o  m i l d  a c i d  hydro l y s i s . I n  the p re s ent inve s ­
t i g a t i on a p ro t e o l y t i c  enzyme w a s  no t us e d  but the B I AL 
r e a c t i on (Cul l ing , 1 9 7 4 )  s p e c i f i c  f o r  s i a l i c  a c i d ,  y i e l de d  
a p o s i t ive r e sul t o n l y  i n  t h e  demi lune c e l l s . I t  i s  po s s ibl e 
t h a t  the m i l d  hy dro l y t i c  a c t i o n  o f  h e a t ing HC l ac i d  t o  7 0 ° C 
d u r ing the B I AL t e s t  w o u l d  have r emo v e d  s i a l i c  a c i d  r e s i due s 
f ro m  the muc o us c e l l s  wh i l e  unma s k ing tho s e  o f  the demi l une s .  
T h e  re d - br own r e ac t i o n  p r o duc t , sp e c i f i c  for s i a l i c  ac i d , 
w a s  the r e f o r e  l o c a t e d  only in t h e  dem i l u n e s  ( F i g ure 2 . 4 . 7 ) .  
T h i s  " unma s king" a c t i o n  o f  t h e  B I AL r e a c t i o n  i s  howeve r not 
c l e a r ,  a l though it i s  p o s s i b l e  that the B IAL t e s t  wou l d  
h av e  p r odu c e d  d i f f e r e n t  r e s u l t s  i f ,  a s  r e c ommended by 
C u l l ing ( 1 9 7 4 ) , f o rma l in vap ou r - f i x e d  s e c t i ons w e r e  u s e d  fo r 
t h e  t e s t  in s t e a d  o f  fo rma l i n - f i x e d  p a r a f f in s e c t i on s , u s e d  
i n  t h e  p re s e n t  inve s t i g a t i on . The l a t t e r  f ix a t ive w a s  a l s o  
foun d t o  enhance A B  s t a i n ing o f  mandi b u l a r  g l and demi l une s , 
( s e e  B i r t l e s , 1 9 8 1 ) . The r e a s ons fo r t h i s f i x a t ive - depen ­
d e n t  s t a i n i n g  e f f e c t  o f  mand i b u l a r  g l and dem i l une s we re 
no t inve s t i g a t e d , a l tho ugh it i s  l i ke ly tha t f o rma l i n  may 
s ub s t ant i a l l y  reduce t h e  ma s ki n g  e ff e c t  o f  b a s i c  p r o t e i n s . 
9 7  
The c o n t en t s  o f  s om e  dem i l une s we r e  a l s o  w e a k l y  r e ac t iv e  
t o  A B  a t  p H  1 . 0 , i nd i c a t ive o f  n e g l i g i b l e  amo un t s  o f  s u l p h ­
a t e d  o r  s i a l o '"" s ulpha t ed g lycopro t e ins . A s im i l a r  r e s u l t  was 
r ep o r t e d  b y  Pal and Chandra ( 1 9 7 9 )  i n  t h e  mand ibu l a r g l ands 
o f  the wa t e r  b u f fa l o , but it is unc l e a r  whe t h e r  t h e  r e f e r ­
e n c e was s p e c i f ic a l l y  t o  a demi l une c e l l  o r  an a l t e rnat ive 
c e l l  typ e  the autho r s  have d e s c r i b e d  a s  a " s e romu c o u s  end ­
p i e c e  c e l l " wh i c h  was a l s o  r e f e r r e d  t o  a s  a " s e r o u s  dem i ­
l un e " .  
The i n t r a l o b u l a r  duc t s  o f  the mand ibul a r  g l an d s  w e r e  
c omp o s e d  o f  t a l l  c o l umna r  c e l l s  w i th d i s t in c t  b a s a l  s t r i a t ­
i on s  ( F i g ur e s  2 . 4 . 3 and 2 . 4 . 6 ) .  The s e  s t r i a t e d  ( i nt r a l o b ­
u l a r ) duc t s  wou l d  b e  capab l e  o f  p ro duc ing a hyp o t o n i c  s a l iva 
( K ay , 1 9 6 0 )  by conduc t ing duc t a l  wat e r  and e l e c t ro l y t e  t r an s ­
p o r t  o f  i o n s  ( Young and van Lenne p , 1 9 7 9 ) . On the o t h e r  
h a n d  t h e  p a r o t i d  g l an d  i n tr a l ob u l a r  duc t s  l ac k  b a s a l  
s t r i a t i on s  ( F i gu r e  2 . 2 )  and p r o du c e  a n  i s o t on i c  s a l iva ( Kay , 
1 9 6 0 ) . Howev e r, two f e a t u r e s  i de n t i f i e d  i n  s om e  p a ro t i d g l and 
i n t r a l o bu l a r  duc t c e l l s , the p r e s en c e  of PAS p o s i t i ve mat e r ­
i a l  and ap i c a l  b l eb s , ( F i g u r e  2 . 2 . 1 ) w e r e  a l s o  no t i c e d  i n  
t h e  mand ibu l ar g l and s t r i a t e d  duc t s . The ma in e x c r e t o ry 
duc t  o f  the mand ibul a r  g l and , unl i k e  that o f  the p a ro t i d , 
d i d  n o t  c on t a i n  g ob l e t  c e l l s  ( F i g ure 2 . 4 . 1 2 ) , a f in d ing a l s o  
r ep o r t e d  b y  B i rt l e s  ( 1 9 8 1 ) . Howeve� gob l e t  c e l l s  have b e e n  
i d en t i fi ed i n  t h e  ma i n  e xc r e to ry duc t  o f  the s h e e p  mand i b ­
u l a r  g l an d  (Ar iyaku l k a l n , 1 9 8 1 ) . 
The mo rpho l o gy o f  t h e  bovine s ub l ingual g l and s e c r e t o ry 
endp i e c e s  d i f f e r e d  from tho s e  o f  the mand ibu l a r  g l and . The 
endp i e c e s , a l though tubul ar , c o nt a ine d f l a t t ene d ,  inconsp i c -
9 8  
u o us demi l une c e l l s  which c on t r a s t e d  s h a rp l y  i n  mo rpho l o gy 
w i th tho s e  of the mandibu l a r  g l ands . T h e  s e c re t o ry p r o du c t s  
o f  t h e  muc ous c e l l s  w e r e mo s t ly neu t r a l  and a c i d i c  muc o ­
s ub s t an c e s ;  th e s e  f i nd ing s p a r a l l � l e d o b s erva t i o n s  by 
S hack l e fo r d  and Kl app e r  ( 1 9 6 2 )  and B i r t l e s ( 1 9 8 1 ) . The 
a c i d i c  muc o s ub s t anc e s  were AB ( p H  2 . 5 ) ,  c o l l o i da l  i ron and 
T o l udine b l ue p o s i t i ve , but n e ur amini das e  d i g e s t i o n  fa i l e d  
t o  e l imina t e  AB s t a i n ing a t  p H  2 . 5 ; t h i s  demon s t ra t e s  that 
mo s t  s i a l i c  ac i d  r e s i du e s  of s ub l ingua l g l and muc ous c e l l s  
w e r e n o t  neuram in i da s e l ab i l e  and c ou l d b e  s t ruc tur a l ly 
d i fferent f rom tho s e  of the man dibul ar g l ands . B i o chem i c a l  
s tud i e s  have addi t i o n a l l y  e s t ab l i sh e d  a d i ff e r e n c e  in com­
p o s i t i on b e tween t h e  mucous f r a c t i o n s  o f  the two g l ands 
( T ab l e  1 . 2 ) .  
H i s t o ch emi c a l  demon s t ra t i on o f  s u l p h a t e  g roup s  in b o v i n e  
s ub l ingua l g l ands e s t ab l i s h e d  that muc o u s  c e l l s  we re r e a c ­
t ive t o  AB at pH 1 . 0 ( F i gure 2 . 5 . 2 ) ind i c a t ing the p re s en c e  
o f  a s u l p h a t e d  o r s i a l o- s ulpha t e d  g l ycop r o t e i n . I n  cont ra s t  
t o  the mand i bul ar g l an d , p r op o r t i o n a t e l y  mo r e  s ub l i ngual 
g l and muc o u s  c e l l s  w e r e  AB p o s i t ive a t  l ow p H . 
Mo s t  d e m i l un e s  o f  the s ub l i ngua l g l ands r e a c t e d  immuno ­
h i s t o chem i c a l l y w i t h  ant i b o v i n e  s a l ivary p ro t e in b and 9 
a n t i b o dy ( F i g ure 2 . 5 . 6 ) and a few s t a i n e d  we akly w i th PAS . 
The s e c r e t o ry p ro duc t s  o f  the demi l une s unl i k e  tho s e  o f  the 
mandib u l a r  g l and dem i l une s were ma i n ly p ro t e o s e rous with 
l i t t l e  e v i denc e o f  e i the r a c i d i c  or n e u t r a l  muc o s ub s t an c e s .  
S t r i a t e d  duc t s  o f  the b o v i n e  s ub l ingual g l ands were 
i dent i c a l  t o  tho s e  o f  the mand i b u l a r  g l ands , w i th t a l l  
c o l umna r  c e l l s  and d i s t in c t  b a s a l  s t r i a t i ons ( F i gure 2 . 5 . 5 ) .  
I n  c ont ra s t  to mo s t  s p e c i e s , i n c luding s heep , the p a ro t i d  
g l ands c o n s i s t  o f  we l l  deve l o p e d  s t r i a t e d  duc t s  wh i l e tho s e  
o f  the man dibul a r  and sub l ing u a l  g l ands are p o o r l y  deve l op e d  
( L e e s on , 1 9 6 7 ) . The p re s en t  i nv e s t i g a t i o n  has h i s t o l o g i c ­
a l l y e s t ab l i shed that in c a t t l e ,  s t r i a t e d  duc t s  are d i s t r i b ­
u t e d  o n l y  i n  the mand ibul ar and sub l ingua l g l ands , f i nd ing s 
wh i ch have n o t  b e e n  p r eviou s l y  r epo r t e d . Phy s i o l o g i c a l ly , 
w e l l  deve l op e d  s t r i a t e d  du c t s  a r e  u s u a l l y  a s s o c i a t e d  w i th 
a hypo t o n i c  f i n a l  s a l i va ( Young and van L enn e p , 1 9 7 9 ) . 
K a y  ( 1 9 6 0 )  d i d  n o t  r ep o rt the p r e s ence o f  s t r i a t e d  duc t s  i n  
e i th e r  man d i b u l a r  o r  s ub l ingua l g l ands b u t  f o und th e i r  
s e c r e t i on s  t o  b e  hyp o t o n i c . 
9 9  
An unu s u a l  f e a t u r e  o f  the bovine sub l ing u a l  g l ands was 
t h e  app ar e n t  hi s t o l o g i ca l  va r i a t ion from one re g i o n  to 
anothe r ( F i gu r e s  2 . 5 . 3  and 2 . 5 . 4 ) .  Som e  areas o f  s ub l ingua l 
g l and t i s s ue s amp l e s  e xamined were comp o s e d  o f  d e n s e l y  
p a cked s e c r e t o ry endp i e c e s  and p o o r ly d e v e l op e d  i n t r a l obul a r  
duc t s  w i thout b a s al s t r i a t i on s . P l a sma c e l l s , a c ommon 
f ea t u r e  o f  bovine s ub l ingual g l ands , w e r e  no t e v i de nt i n  
s uch r e g i on s . Howeve r, mo s t  a r e a s  o f  t h e  s ub l i n g u a l  g l ands 
c ons i s t e d  of s e c r e t o ry endp i e c e s  d i s t r i b u t e d  amon g  i rr e g ­
u l a r l y - a r r a n g e d  conn e c t i ve t i s s ue bund l e s  wh i ch c o n t a i n e d  
s ev e r a l  p l a s ma c e l l s  and we l l  deve l op e d  s t r i a t e d  duc t s . 
Shackl e fo r d  ( 1 9 6 3 )  r emarked b r i e fly on t h e  h i s t o l o g i c a l  
va r i a t i o n  o f  bovine s ub l ingual g l ands w i t h  r e f e re n c e  t o  " a  
p ronounc e d  d i fference i n  the r e l a t iv e  numb e r s  o f  muc ous and 
non - mucous e l emen t s  f rom one a r e a  t o  ano th e r " , b ut the r e  
wa s n o  men t i o n  o f  p l a sma c e l l s  o r  s t r i a t e d  duc t s . The 
s i g n i f ic a n c e  of t h i s h i s t o l og i c a l  var i a t i on i s  unknown ; 
t i s s ue s o f  o th e r  bov ine s a l iva ry g l ands e xami n e d i n  the 
p r e s ent s tudy did not exhib i t  r e g ional va r i a t i on . 
Hi s to l o g i c a l  and h i s tochem i c a l  s t ud i e s  o f  t h e  m ino r 
s a l ivary g l ands o f  c a t t l e  have b e en we l l  docume n t e d  by 
B i r t l e s  ( 1 9 8 1 ) . O f  the buc c a l  g l ands , the vent r a l  buccal 
wa s h i s t o l o g i c a l ly i d e n t i c a l  t o  the p a ro t i d .  Ruminant ve n ­
t r a l  buc c a l  g l ands inve s t i g a t e d  t o  dat e , have b e e n  r ep o r t e d  
a s  g ene r a l ly s im i l a r  t o  the i r  p a ro t i d  g l ands ( Young and van 
L e nnep , 1 9 7 8 )  and c o u l d  func t i o n a l l y s up p l ement the p a ro t i d  
s a l iva ry s e c re t i o n . 
The mucous c e l l s  o f  the i nt e rme d i a t e  buc cal g l ands con ­
t a ined a c i d i c  and n e u t ral mu c o s ub s t an c e s  wh i ch w e r e  h i s t o ­
c h emi ca l l y  s imi l ar t o  sub l ing u a l  g l and muco s ub s t anc e s , wh i l e  
t h e  demi l une s w e r e  p ro t e o s e rous and immunoh i s tochemi ca l l y 
1 0 0  
found t o  contain s al iva ry p r o t e in b and 4 ( F i gu r e  2 . 6 ) .  B i o ­
c h e m i c a l  inv e s t i g a t i o n s  have s hown the i n t e rme d i a t e  buc c a l  
g l ands t o  b e  unu s u a l  i n  po s s e s s ing t ra c e s  o f  a l l  b ovine l ow 
mo l e c u l a r  we ight s a l i va ry p r o t e ins ( Tab l e  1 . 1 ) . The s i g ­
n i f i cance o f  th i s  f i nd ing , r e l a t ive t o  a l l o t he r b ovine 
s a l ivary g l ands and p a rt i c u l a r ly in r e l at ion t o  futu r e  
inve s t i g a t ions i s  no t c l e a r , a l though i t  app e a r s  that g e n ­
e t i c mat e r i a l  requ i r e d  fo r s yn t he s i s  o f  b ovine s a l i va ry 
p r o t e in s  h a s  b e e n  r e t a in e d  by only the i n t e rme d i a t e  b uc c a l  
g l ands , i n  a dd i t i o n  t o  a n  ab i l i ty t o  d i fferen t i a t e  l E G  duc t  
c e l l s obs e rved i n  t h e  par o t i d  g l ands . 
The d o r s a l  b uc c a l  and p a l a t ine g l an d  mucous c e l l s  c on ­
t a ine d h i s t o chemi c a l l y  s i mi l a r  s e c r e t o ry pro duc t s  t o  tho s e  
o f  the i n t e rme d i a t e  b uc c a l  and s ub l i n gu a l  g l ands : the 
d e m i l un e s  were u su a l l y  r e f r a c t o ry t o  a l l  s t a in s  u s e d  a l t h ­
o u gh immunoh i s t o chemi c al s t u d i e s  we re no t conduc t e d  t o  
i nve s t i g a t e  the p o s s ib l e  d i s t r i but i on o f  s a l i v a ry p ro t e i n s . 
A r e a s  o f  p r o t e o s e r o u s  l ob u l e s  w e r e  f r equen t ly o b s e rv e d 
i n  do r s al and int e rm e d i a t e  b u c c a l  g l ands and the p a l a t ine 
g l ands . I t  i s  the r e f o re l i k e l y  that th e s e  thr e e  mino r 
g l a nd s  a l ong w i t h  t h e  vent r a l  buc c a l , c o n t r i b u t e  s i gn i f ­
i c an t ly t o  the vo l um e  o f  t h e  t o t a l  s e c r e t ion from the b o v i n e  
s a l ivary g l ands . Kay ( 1 9 6 0 )  p r e v i o u s l y  c onc l ud e d  t h a t  t h e  
t o t a l  vo l ume o f  s al i v a  s e c r e t e d  by the m i no r  g l ands , app r o x ­
i ma t e l y  equa l l e d the vo l ume s e c r e t e d  b y  t h e  p a r o t i d  g l ands . 
B i r t l e s  ( 1 9 8 1 )  p r e s en t e d  e v i de n c e  i n  favour o f  a p o s ­
s ib l e  ho l o c r ine s e c re t o ry me chani sm f o und in do r s a l  and 
i n t e rme d i at e  buc c a l  g l ands and p a l a t ine g l ands , f indings 
wh i ch we r e  confi rmed in t h e  p r e s ent s tu dy ( F i gure s 2 . 7 . 4  
a n d  2 . 8 . 4 ) .  The s ig n i f i c an c e  o f  thi s s e c re t o ry mechan i sm 
o b s e rved only in s om e  bovine s a l ivary g l ands is s p e cul a t ive . 
A t yp i c a l  s e c re t o ry me chan i sms h ave n o t  b e en r ep o rt e d  from 
inve s t i g a t i ons on s al ivary g l ands of o th e r  mamma l i an s p e c i e �  
Muc o u s  c e l l s  o f  s al ivary g l and t i s s u e s  i n  the p o s t e r i o r  
t o ngue w e r e  h i s tochem i c a l l y  s im i l a r  t o  thos e o f  the 
1 0 1  
s ub l ingu a l  g l ands , d o r s a l  and int e rmed i a t e  bucc a l  and 
p a l a t ine g l ands . The dem i l un e s , howeve r ,  o ft e n  cont a in e d  
c a rb o xy l a t e d  a n d  s ul ph a t e d  muco s ub s t anc e s  and PAS p o s i t iv e  
n e ut r a l  muco s ub s tan c e s  ( F i g u r e  2 . 9 ) ,  f e a t ur e s  no t o b s e rv e d  
i n  d em i l un e s  o f  e i t h e r  t h e  s ub l ingua l g l a nds o r  t h e  t h r e e  
p r e v i ous l y  des c r ib e d  m ino r g l ands . Add i t i on a l l y p r o t e o s e r ­
o u s  l obul e s  were no t p re s ent . 
The p h a ryng e a l  g l ands u s u a l ly d i sp l ay e d  mo rpho l o g i c a l  
f e a t u r e s  n o t  evi dent i n  the o th e r  mino r g lands . Th e s e c r e t ­
o ry endp i e c e s  w e r e  s ho rt and t ub ul a r  w i th co n s p i cuous demi ­
l un e s  tha t  cont a ine d a c i dophi l i c  g ranul e s , n e u t r a l  and c a r ­
b o xy l a t e d  muco s ub s t a n c e s . I n  g en e r a l  t h e  hi s t o c h em i c a l  
c o mp o s i t i on o f  t h e  demi l unes w e r e  s im i l a r  t o  tho s e  o f  the 
mandibul a r  g l ands b ut the pha ryng e a l  g l an d  d em i l un e s  s t a in e d  
mo r e  i nt en s e ly fo r muco s ub s t a n c e s . The s e cr e t o ry p ro du c t s  
o f  t h e  muc o us c e l l s w e r e  s im i l a r  to tho s e  from muc o us c e l l s  
o f  p o s t e r i o r  t ongue , p a l a t in e , do r s a l  and int e rme d i a t e  
b uc c a l  a n d  s ub l ingua l g l ands . 
The i n t r a l obul a r  duc ts o f  the pharyn g e a l  g l ands we r e  
u s ua l ly l ine d w i th g o b l e t  c e l l s  and t h e  s e cr e t o ry endp i e c e s  
o f t en app e a r e d  t o  dra in d i r e c t l y  into i n t ra l obul a r  duc t s  
( F i g ure s 2 . 1 0 and 2 . 1 0 . 4 ) ; t h i s  dr a i n a g e  p a t t e rn h a s  b e en 
r ep o r t e d  fo r s a l ivary g l ands o f  o t h e r  s p e c i e s  (Young and 
van Lenn e p , 1 9 7 8 ) . Howeve � b o th the a b o v e  f e a t u r e s  and 
p a r t icul a r ly gob l e t  c e l l s , w e r e  no t ob s e rve d in t h e  i nt r a ­
l ob u l a r duc t s  o f  o t h e r  b ovine maj o r  and m ino r s a l ivary 
g l ands . 
The p h a ryn g e a l  g l ands s h a r e d  in common w i th b o th s ub ­
l in g ua l  and the man d i bu l a r  g l ands , the p re s en c e  o f  nume rous 
s ub e p i the l i a l  p l a s ma c e l l s , d i s t r ibut e d  b e twe e n e n dp i ec e s  
and c l o s e t o  i n t r a l o b u l ar duc t s  ( F i gure 2 . 1 0 . 2 ) . Th i s  s i g ­
n i f i c ant p re l iminary find ing e s tab l i s h e d  that humo r a l  muc o s al 
i mmun i t y me d i a t e d  v i a  the bovine s a l iva ry g l ands i s  ma i n l y  
p ro v i d e d  b y  immuno g l obul ins synthe s i s ed and s e c re t e d  b y  . 
p l a sma c e l l s o f  the s e  thre e g l ands . The sub l ingual g l ands 
i n  p a rt i c u l a r  con t a i n e d  the maj o r i ty o f  p l asma c e l l s , mos t 
1 0 2  
o f  wh i c h  reac t e d  i mmunoh i s tochem i c a l l y with ant i b o dy t o  
s e c re t o ry I gA ,  t h e  m a j o r  immuno g l o b u l i n  o f  ruminant s a l i v a  
( Mach a n d  Pahud , 1 9 7 1 ) . O n  the o the r h and t h e r e  was l i t t l e  
e v i denc e t o  s ug g e s t  tha t p a ro t i d  and ventral b uc c a l  g l an d s  
c on t r i b u t e  s i gn i f i c an t l y  t o  humo r a l  i mmun i ty s in c e  p l a s m a  
c e l l s  w e r e  r a r e l y  f o und i n  the s e  g l an d s . Neve rthe l e s s , 
i mmuno l o g i c a l  s tud i e s  by Wa t s on and L a s c e l l e s  ( 1 9 7 1 )  p r e v ­
i o us ly e s t ab l i s he d t h a t  in ruminan t s , i mmuno g l obul ins s u c h  
a s  s i gA were l ow i n  p a r o t i d  s a l iva comp a red w i t h  l eve l s  i n  
n on - p a r o t i d  s a l i va . I t  i s , howev e r , a l t e rna t i v e l y  p rop o s e d  
h e r e , a l though n o t  p rove d ,  that p a ro t i d  and ven t r a l  buc c a l  
g l ands may cont r i b u t e  p r e dominan t l y  t o  c e l l u l a r  mu co s a l  
i mmune r e a c t i on s , m e d i a t e d  v i a  I E G  duc t  c e l l s  wh i ch we r e  
s p e c i f i c  t o  th e s e  two g l an d s  ( F i gu r e  3 . 1 ) .  
I mmuno l o g i s t s  who have r e c en t l y  fo c u s s e d a t t en t i o n  o n  
t h e  dive r s e  muc o s a l  c e l l  typ e s  a r e  g en e ra l ly o f  the op i n i o n  
that , i n  a ddi t i o n  t o  T - c e l l  s ubpopul a t i ons , muc o s a l  ma s t  
c e l l s , g l obu l e  l eu c o cy t e s  and int r a ep i t h e l i a l  lymp ho cy t e s  
a r e  a l s o  invo l v e d  i n  p o o r l y unde r s t o o d  muco s a l  c e l l - me d i a t e d  
i mmune r e a c t i ons ( B i enens t o c k  and B e fu s , 1 9 8 0 ; Toma s i e t  a l . ,  
1 9 8 0 ) . Toma s i  e t  a l . ( 1 9 8 0 )  s t a t e d  t h a t " th e r e  may b e  s ev ­
e r a l  s ub c l as s e s  o f  T - lympho cy t e s , s ome g ranu l a t e d  wi th ma s t  
c e l l - l i ke func t i ona l p ro p e r t i e s , o t h e r s  mo re typ i c a l ly 
l ympho i d  w i th h e l p e r and s upp re s s o r  funct ions . I nt raep i th ­
e l i a l  l ympho cy t e s  i n  p a r t i cu l a r  c o n t a i n  cytop l a smi c granu l e s  
and a r e  mo rp ho l o g i c a l ly s im i l ar t o  ma s t  c e l l s , w i th r ep o r t e d  
a l t e r a t ions i n  the i r  prop o r t ions dur i n g  gas tro int e s t i n a l  
d i s e as e s . "  Add i t i on a l ly ,  B i enens t o c k  ( 1 9 7 9 )  s t a t e d  that 
i n t raep i th e l i a l  l ymp hocy t e s  "may r e p r e s ent a c l o s e d  c e l l 
p opul a t ion , what ev e r th e i r  de r iva t i o n  t h ey have a r e s emb l an ce 
t o  b o th lympho cy t e s  and ma s t  c e l l s  and may a c t  a s  s p e c i f i c  
p as s i ve ly - s en s i t i s e d  s ent ina l c e l l s  a t  the muco s a l  s u r f ac e . "  
S ome i n t raep i the l i a l  granu l a r  c e l l typ e s  with mas t - l i k e  
s t a i n i n g  cha r ac t e r i s t i c s  a r e  known t o  p o s s e s s  n a t u r a l  k i l l e r  
a c t i v i ty ( J .  B i en e n s tock , p e r s on a l  c ommun i cat i o n ) . The 
r e l a t i o n s h ip of int r a ep i t he l i a l  lymph o c y t e s , i f  any , w i t h 
muc o s a l  mas t  c e l l s  and g l ob u l e l e uc o cy t e s , has not b e e n  
e s tab l i s he d .  
1 0 3  
I n  the p r e s en t  s t udy , the l E G  duc t  c e l l s  o f  p a ro t i d  and 
v e n t r a l  b uc c a l  g l an d s  exam i n e d  had features c ommon t o  b o t h  
l ymp ho cy t e s  and muc o s a l  mas t  c e l l s . I t  s houl d ,  howeve r ,  b e  
s t r e s s e d that the n a ture o f  the r e l a t i on s h ip s , i f  any , o f  
l E G  duc t  c e l l s  w i th i n t r a ep i t he l i a l  l ymp hocy t e s , muc o s a l  
ma s t  c e l l s  and g l o b u l e  l e uc o cy t e s , a r e  a t  b e s t  hyp o th e t i c a l, 
a l though f rom an o v e r a l l  a s s e s sment o f  the above da t a , a 
p o s s ib l e  r o l e  for l E G  duc t  c e l l s  i n  c e l l - me d i a t e d  immuni ty 
c anno t b e  i gnor e d ; a dd i t i ona l ly the e s t ab l i s h e d  a s s o c i a t i o n  
b e tw e en l E G duc t  c e l l s  a n d  b l o a t  s us c ep t ib i l i ty ( B i rt l e s , 
1 9 8 1 )  s u g g e s t s  tha t the app e arance o f  t h e s e  c e l l s  c ou l d  b e  
a s s o c i a t e d  w i th a p r o t e c t iv e  r o l e . 
I n  c a t t l e , th e r e fore , i t  s e ems t h a t  humo r a l  i mmun i ty t o  
the ent i re o r a l  c av i ty ,  o e s ophagus and t h e  r e t i c u l o rumen , 
r e g i ons whi ch l a ck l ympho i d  t i s sue , i s  p ro v i d e d  by immune ­
g l ob ul i n s  s e c r e t e d  f r om the s ub l ingua l , mandibul a r  and 
p h a ryng e a l  g l ands , whe re a s  c e l l u l a r  immun i ty t o  the s ame 
r e g i o n s  i s  p r obab l y  me d i a t e d  v i a  the l E G  duc t  c e l l s  o f  t h e  
p a ro t i d  a n d  vent r a l  b uc c a l  g l ands . 
To p o s t u l a t e  t h e  i dent i ty o f  l EG duc t  c e l l s , a c r i t i c a l  
e v a l ua t i on was ma de o f  r ep o r t s  de s c r i b i ng s uch c e l l  typ e s  
i n  ep i th e l i a l  l o c a t i ons . The c e l l  typ e wh i ch app e ar e d  ve ry 
s imi l a r  to the l EG duc t  c e l l  in mo rpho l o gy was the g l o b u l e  
l eucocy t e , a p o o r l y  de f i n e d  c e l l  typ e , f i r s t  de s c r ib e d  b y  
We i l l  ( 1 9 2 0 ) * ,  b u t  a s  y e t  i t  h a s  n o t  b e e n  p r e c i s e l y  i den t i f ­
i e d  ( s e e G re g o ry , 1 9 7 9 ) . I t  i s , how e v e r ,  e s t ab l i sh e d  tha t 
t h e  g l o b u l e  l euc o cy t e  i s  no t w i de l y d i s t r ib ut e d ,  but r e s t ­
r i c t e d  mainly to e p i the l i a i n  r e g i o n s  s uch a s  the r e s p i ra t o ry 
t r a c t  ( Kent , 1 9 6 6 ; Vandenb e rghe and B a e r t , 1 9 8 1 ) , g l andu l a r  
s t omach (Whur , 1 9 6 6 ) , sma l l  inte s t ine ( Kent , 1 9 6 6 ; Whur and 
J o hn s ton e , 1 9 6 7 ; Mu r ray e t  a l . ,  1 9 6 8 ; T akeuch i , J a rvi s and 
Sp r in z , 1 9 6 9 ) , co l on (Takeuchi et a l . ,  1 9 6 9 ) , b i l e  duc t 
( Rahko , 1 9 7 2 ) , ut e r ine ep i th e l ium ( K e l l as , 1 9 6 1 )  and u r i n a r y  
t r a c t  ( C an t in a n d  Ve i l l eux , 1 9 7 2 ) . 
* s ee G rego ry ( 1 9 79 ) . 
1 0 4 
A " typ i ca l "  g l ob u l e  l euc o cy t e  de s c r i b e d  i n  t h e  ab ove 
r ep o r t s  u s ua l ly c o n s i s t e d  of a n  e c c e n t r i ca l ly - p l ac e d  nuc l eu s  
w i th p e r i p h e r a l ly - di s t r ibut e d  chroma t i n  and a cy top l a sm 
cont a in i n g  sma l l  m e t a ch romat i c  ac i doph i l i c  g ranu l e s  or l a r g e 
a c i dophi l i c g l ob ul e s . A c r i t i c a l  ana l y s i s  o f  mo s t  r e p o r t s , 
howev e r ,  s howed t h a t  there a re sp e c i e s  a s  we l l  a s  h i s t o ­
chemi c a l  d i f fe r en c e s  de s c r i b e d  b y  mo s t  autho r s , a finding 
o r i g in a l l y  made by W e i l l  ( 1 9 2 0 )  who "not only empha s i s e d 
t h e  i n t e r s p e c i f i c  d i f fe re nc e s  i n  the g l o bul e s  but in t h e  
d e g r e e  o f  b a s op h i l i a o f  the cytop l asm . " * Fo r examp l e , i n  
the r a t  t h e  g l o b u l e l euc o cy t e s  w e r e  v e ry s im i l ar t o  the 
muc o s a l  mas t  c e l l  ( K ent , 1 9 6 6 ; Mur ray e t .  al . ,  1 9 6 8 ; C an t in 
and Ve i l l eux , 1 9 7 2 ; C o l l an ,  1 9 7 2 ; May r h o f e r  e t  a l . ,  1 9 7 6 ; 
H am and C o rmac k , 1 9 7 9 ; Vanden b e rghe and B aert , 1 9 8 1 )  b u t  
the i r  g ra nu l e  s t ru c t u r e  d i f f e r e d  f r o m  muc o s a l  mas t c e l l s  
and w e r e  more ac i do p h i l i c ; f e a t ur e s  wh i c h  temp t e d  Mu r r ay e t  
a l . ( 1 9 6 8 )  to p o s tu l a t e  t h a t  g l obul e l eucocy t e s  a r e  d e r i v e d  
f r om muc o s a l ma s t  c e l l s . However t h i s  v i ew h a s  no t r e c e i v e d  
w i d e s p r e a d  supp o r t  ( C o l l an ,  1 9 7 2 ; C an t i n  and Ve i l l eux , 1 9 7 2 ; 
Ru i t enb e r g  and E l g e r s ma , 1 9 7 9 ; Van denb e rghe and B a e r t , 1 9 8 1 )  
s in c e  t r an s i t i o n a l  c e l l  typ e s  and mas t c e l l s  wh i ch m i g ra t e d  
into the e p i t he l ium t o  b e c ome g l obul e l e ucocy t e s  w e r e  no t 
i de n t i f i e d .  I n  t h e  r a t , t h e  c u r r ent t r e nd the r e f o r e  i s  t o  
emp h a s i s e  the s im i l a r i t i e s  b e tw e e n  t h e  muco s a l  mas t  c e l l and 
the g l obu l e  l eu c o cy t e  (Ham and Co rma c k , 1 9 7 9 )  s uch a s : 
mo rpho l o gy and T o l ud ine b l u e  met achroma s i a  ( Co l l an ,  1 9 7 2 ) , 
AB s t a i n i ng at l ow p H  and p r e s ence o f  b as ic p ro t e in s  ( C ant i n  
and Ve i l l eux , 1 9 7 2 ) , cytop l a sm i c  I g E (May rho f e r  e t  a l . ,  1 9 7 6) 
and a p o s s ib l e  lymp h o cy t e  o r i g in ( Mi l l e r , 1 9 8 0 ) . 
I n  ruminan t s , a n d  p a r t i c u l a r l y  i n  t h e  sheep ab oma s um ,  
the g l obul e l euco cy t e  has b e e n  charac t e r i s e d  by l ar g e  a c i do ­
ph i l i c g l ob ul e s , a c y top l a s m  u s ua l l y  r e f racto ry to s t a i n ing 
and a nuc l eus s im i l a r  to tha t of a p l a sma c e l l  ( Kent , 1 9 5 2 ; 
Dob s on , 1 9 6 6 ; Mu r r ay e t  a l . ,  1 9 6 8 ; Rh a k o , 1 9 7 2 ) . But even 
though h i s tochem i c a l  s imi l a r i t i e s  s uch as AB s t a in i ng at 
l ow p H  and To lud i n e  b lue met ac hroma s i a  h ave b e en rep o r t e d  
* s ee G rego ry ( 1 9 79 ) . 
1 0 5  
(Murray e t  a l . , 1 9 6 8 ) , t h e  ma s t  c e l l  a s s o c i a t i on in rum i n ­
ant s  w a s  l e s s  c l e a r  s ince t h e r e  was no co rr e l a t i on b e twe e n  
a pp e a ran c e  o f  mas t c e l l numb e r s and g l o b u l e  l e u c o cy t e s  ( K ent,  
1 9 5 2 ; L awren c e , 1 9 7 2 ;  G r e g g , D i ne e n , Ro thwe l l  and K e l ly , 
1 9 7 8 ) . Dob s on ( 1 9 6 6 )  was o f  the op i n i o n  that i n  s h e e p, g l ob ­
u l e l eu c o cy t e s  w e r e  p l asma c e l l s  w i t h  Ru s s e l l  b o di e s ; how � 
e v e r  K en t  ( 1 9 5 2 )  p revious l y  e s t ab l i s h e d  that t h e  two c e l l  
typ e s  i n  sheep w e r e , a t  t ime s , mo rp ho l o g i c a l l y  s im i l a r  b u t  
h i s t o chemi c a l l y d i ffe rent , for e xamp l e ,  g l obu l e  l e ucocy t e s  
w e re s t rongly r e a c t i ve t o  PTAH (pho s p ho tung s t i c ac i d  haem ­
a t o xy l i n )  and me t a chroma t i c  w i t h  T o l u d i ne b l ue wh i l e  Ru s s e l l  
b o d i e s o f  p l asma c e l l s  w e re n e g a t i v e  t o  b o t h  s t ains . Th e s e  
two c e l l  typ e s  w e r e  a l s o  s tudi e d  b y  Murray e t  a l . ( 1 9 6 8 )  who 
demon s t r a t e d  ul t r a s t ructura l  d i f f e r e n c e s  b e tw e e n  g l obu l e 
l eu c o c y t e s  and Rus s e l l  b o d i e s  o f  p l a s ma c e l l s  and emphas i s ed 
t h e  u l t r a s t ruc t u r a l  and h i s t o chem i c a l  s im i l a r i t i e s b e twe e n  
g l ob u l e  l e uc o cy t e s  and muc o s a l  mas t c e l l s . Rhako ( 1 9 7 2 ) , 
f rom a n  inve s t i g a t i on o f  g l obu l e  l eu c o cy t e s  i n  the g a l l  
b l adde r ep i the l i um o f  sheep , c a t t l e  a n d  g o a t s  conc lu de d t h a t  
the g l ob u l e  l euc o cy t e  in a l l  thre e s p e c i e s  exhib i t e d  mo rp h ­
o l o g i c a l  as we l l  a s  h i s to chemi c a l  d i f fe renc e s ; f o r  e xamp l e  
s ulpha t e  g roup s w e re demon s t r a t e d  o n l y  in c a t t l e . I n  s he ep 
u t e r i n e  e p i th e l i um ,  an i n t r a ep i th e l i a l  g r anu l a r  c e l l  typ e 
t ent a t ive ly i de n t i fi e d  by K e l l a s ( 1 9 6 1 )  a s  a g l obul e l eu c o ­
c y t e , d i ffe r e d  i n  morpho l o gy and h i s t o chemi s t ry with g l ob u l e  
l euco c y t e s  o f  t h e  aboma s um .  
G l o b u l e  l e u c o c y t e s  have a l s o  b e en s tud i e d  i n  the c a t , 
( T ak e u ch i e t  a l . ,  1 9 6 9 ) , fowl ( Tone r ,  1 9 6 5 )  and m i c e  
( Ru i t e nb e rg and E l ge r sma , 1 9 7 9 ) . T a k euchi e t  a l . ( 1 9 6 9 )  i n  
p a r t i cu l a r  r ep o r t e d  h i s tochemi c a l  d i f fe renc e s  b e tween g l o b ­
u l e  l eu c o cy t e s  o f  the sma l l  and l ar g e  int e s t ine . 
Two c e l l  typ e s ,  the ma s t  c e l l  and the l ymp ho cy t e , have 
b e en c ommonly p ro p o s e d  a s  p re cu r s o r s  of g l o b u l e  l e ucocyt e s . 
Mu r r a y  e t  a l . ( 1 9 6 8 )  w e re o f  the op i n i on that g l obul e l eu c o ­
cy t e s  i n  rum inan t s  are de r i ved f rom muc o s a l  ma s t  c e l l s  
whe r e a s  Kent ( 1 9 5 2 )  has p rop o s e d  t h e  lympho cy t e  a s  the 
l i k e l y  p re curs o r  o f  the g l ob u l e  l eu c o cy t e  in s he ep . The 
1 0 6 
o r i g i n  o f  the g l ob u l e  l eu c o cy t e  in the c a t  i s  unknown , 
( F inn and S chwa r t z ,  1 9 7 2 )  however i n  fowl ( K i t a g awa , O g a t a , 
S u g i mur a , H a s h imo t o  and Kudo , 1 9 7 9 )  and in m i c e  ( Ru i t enb e rg 
and E l g e r sma , 1 9 7 9 )  thymus - dependent l ymp hocy t e s  h ave b e en 
demon s t ra t e d  a s  l ik e ly p re cur s o r s . Rud z i k and B i enens t o c k  
( 1 9 7 4 )  a s s o c i a t e d  t h e  g l ob u l e l e ucocy t e  w i th a g r anul a r  
lymp h o c y t e  s p e c i f i c a l ly found i n  t h e  g u t  muco s a l  e p i t he l i a 
but r a r e  in lymp ho i d  t i s s u e  and p e r i ph e ra l  lymp h n o de s . 
The func t i on o f  the g l obu l e  l e uco c y t �  is equa l ly o b s c u r e. 
I t  i s  howeve r e s t a b l i s he d  tha t in mo s t  s p e c i e s  g l obul e 
l e uco cy t e s  a r e  commonly p r e s en t  dur i n g  p a ras i t i c  i n fe c t i on s  
( s e e  G r e go ry ,  1 9 7 9 ) , b ut t h e  nature o f  t h e  r e l a t i on s h i p  i s  
unknown . Previ ous l y  Do b s on ( 1 9 6 6 )  a n d  Whur and Johns t o n e  
( 1 9 6 7 )  s ug ge s t e d  t h a t  t h e  g l ob u l e  l eu c o cy t e  i n  t h e  s h e ep 
abomas um and r a t  i l e um re s p e c t i v e ly , contained immuno g l o b ­
ul i n s , b ut the i r  f i n di n g s  w e r e  l a t e r  s hown to b e  i n  e r r o r  
(Whu r a n d  Whi t e , 1 9 7 0 ; Rud z i k and B i en e n s t o c k , 1 9 7 4 ) . 
Mu rray e t  al . ( 1 9 6 8 )  p ropo s e d tha t the t rans fo rma t i on o f  
muco s a l  mas t c e l l s  t o  g l o b u l e  l euco cy t e s  may b e  a s s o c i a t e d  
w i th t h e  s e c r e t i o n  o f  immuno g l obul in f rom p l a s ma c e l l s . 
Mayrho f e r e t  a l . ( 1 9 7 6 )  demon s t r a t e d  cytop l a s m i c  I gE in 
g l obu l e  l euco cyt e s  i n  the r a t  i l e um and were of the o p i n i o n  
tha t g l ob u l e  l eu c o c y t e s  may b e  invo l v e d  in t ran s p o rt a t i on 
o f  s e c r e t o ry I gE during p a ra s i t i c  i n f e c t i ons . Re c ent ly , 
H . R . P .  Mi l le r  ( p e r s o n a l  c ommun i c a t i o n )  has i s.o l a t e d  l a r g e  
amoun t s  o f  dopam i n e  and s ome chymo t ryp s in and t ryp s in from 
g l ob u l e  l e uc o cy t e s  b ut the i r  s i gn i f i cance was no t e xamin e d . 
B i en e n s t o c k  and B e fus ( 1 9 8 0 )  have s u g g e s ted that g l obul e 
l e uc o cy t e s  o f  the g a s t ro in t e s t inal t r a c t  may b e  imp l i c a t e d  
i n  c e l l - me di a t e d  i mmun e  r e a c t ions a t  muc o s a l  surfac es . I f  
thi s w e r e  s o , an inve s t i g a t i o n  o f  the i r  r o l e  i n  s al ivary 
g l an ds has y e t  t o  r e ach the p r e l imi n a ry s t age . Ove ra l l  
there i s , howeve r ,  a con s e n s us o f  op i n i on among autho r s  t h a t  
t h e  g l ob u l e  l euco c y t e  i n  a s s o c i a t i on w i th the muc o s a l  ma s t  
c e l l i s  in s ome way imp l i c a t e d  in i mmuno l o g i c a l  r e a c t i o n s  a t  
e p i th e l i a l  s u r fa c e s  ( s e e  G r e g o ry ,  1 9 7 9 and Cas t ro , 1 9 8 2 ) . 
T h e  I E G  duc t  c e l l s  o f  p a r o t i d  and ven t r a l  buc c a l  g l an d s  
1 0 7  
o f  c a t t l e  w e r e  f o und t o  b e  mo rpho l o g i c a l ly and h i s t o chemi c ­
a l ly s imi l a r  t o  g l o b u l e  l euco cyt e s  o f  mo s t  s p e c i e s , e s p e c ­
i a l ly the app e a rance o f  t h e  nuc l e us , va r i a t i on i n  g ranu l e  
s t ru c ture and h i s to chemi c a l  cha rac t e r i s t i c s , s uch a s  ac i do ­
p h i l i c ,  me tachroma t i c  g ranu l e s  and cytop l a s m i c  b a s o p h i l i a 
( F i g u r e  3 . 1  and 3 . 3 ) .  De s p i t e  s imi l a r i t i e s, I E G  duc t  c e l l s  
d i d  no t app e a r  t o  b e  i de n t i c al t o  g l o b ul e l e uc o cy t e s  r ep o r ted 
in ruminant s by Kent ( 1 9 5 2 ) , Ke l l a s  ( 1 9 6 1 ) , Mur ray et a l . 
( 1 9 6 8 )  and Rhako ( 1 9 7 2 ) . I n  p a r t i cu l a r  ul t ra s t ructural 
var i a t i ons in g ranul e mo rpho l o gy we r e  no t e d  b e tween g l o b u l e 
l eu c o cy t e s  o f  t h e  bovine aboma s um ( Murray e t  a l . ,  1 9 6 8 )  and 
I E G  duc t  c e l l s . G l obul e s  o f  the a b o mas a l  g l ob u l a r  l eu c o ­
c y t e s  were ac i do p h i l i c and s ome t im e s  r imme d b y  Al c i an b l ue 
s t a i n in g  ma t e r i a l  g i ving r i s e  t o  a " hal o - e f f e c t " . Th i s  
s ug g e s t s  that mo rpho l o g i c a l  and h i s t o chemi c a l  va r i a t i on s  o f  
t h e  g l obul e l eu c o cy t e  r ep o r t e d  i n  o the r s p e c i e s  ( K e l l as , 
1 9 6 1 ; Takeuchi e t  a l . ,  1 9 6 9 ; Rhako , 1 9 7 2 )  may a l s o  b e  p r e s ent 
in c a t t l e . Mo r e o ve r, the p r e s ent s t u dy has b e e n  the f i r s t 
t o  i nve s t i g a t e  a p o s s ib l e  g l obul e l e u cocyte i n  s a l ivary 
g l ands , and the i r  func t i o n , l ik e  tha t o f  g l o b u l e  l eucocy t e s  
i n  o th e r  s p e c i e s  and o r gans rema ins undet ermin e d . P r e v i o uslx 
van L ennep et a l . ( 1 9 7 7 )  r epo r t e d  p r e s ump t i ve g l obul e l e uco ­
cyt e s  i n  s t r i a t e d  duc t s  o f  the s h e e p  paro t i d  g l ands ; the s e  
c e l l s  were, howe v e r, no t de s c r i b e d  i n  d e t a i l  b ut cou l d  we l l  b e  
i dent i c al t o  tho s e  ob s e rv e d  in the p r es ent s tudy ( F i gu r e  
3 . 1 . 8 ) .  
A comp a r i s on b e twe en i n t racy t o p l asmi c contents o f  g l ob ­
u l e  l eucocyt e s  and I EG du c t  c e l l s  r e ve a l e d  tha t b o th p r o b ab ly 
c o n t a i n  a b i o g en i c  amine . H . R . P .  M i l l e r  ( p e r s onal c o mmun i c ­
a t i o n )  has i s o l a t e d  dopamine from g l o b u l e  l eucocyt e s  wh i l e  
5 - hy d ro xyt ryp t amine o r  a r e l a t e d  b i o g e n i c  amine was i dent i f ­
i e d  i n  I E G duc t  c e l l s  ( F i gure 3 . 3 . 8 ) . The p re s en t  inve s t i g ­
a t i on a l s o  e s t ab l i s h e d  t h a t  s i gA i s  unl i k e l y t o  b e  c o n c e n t ­
ra t e d  in the g ranule c o n t e n t s , but the p r e s en c e  o f  o th e r  
i mmun o g l obul ins , e s p e c i a l l y I gE cann o t  b e  ru l e d  o u t  b e c a u s e  
o f  i t s  as s o c i a t i o n , in the rat , w i t h g l o bul e l eucocy t e s  and 
p a r a s i t i c  infe c t i ons (May rho fe r et a l . ,  1 9 7 6 ) . I t  i s  no t 
c l e a r  whe th e r  s im i l a r  c o n d i t ions a r e  p r e s ent i n  c a t t l e ,  
1 0 8  
a l though g l obul e l euco cy t e s  have b e e n  co rre l a t e d  w i t h p a r a ­
s i t i c  i n fe c t i o n s  i n  t h i s  s p e c i e s  ( L awr enc e ,  1 9 7 7 ) . 
I f  I E G  duc t  c e l l s  a r e  g l obu l e  l e u c o cy t e s  the i r  a s s o c i a t ­
i o n  w i th sus c ep t i b i l i ty t o  b l o a t  s t i l l r ema ins unr e s o l ve d . 
Al tho ugh i t  i s  t emp t ing t o  p o s t ul a t e  tha t s i n c e  I E G  duc t  
c e l l s  a r e  mo r e  n um e rous i n  L . S .  anima l s , they may r e duc e 
b l o a t  s u s c ep t i b i l i ty b y  e l ab o ra t ing a s e c r e t o ry p ro du c t  o r  
by me d i a t ing c e l l ul a r  immun e reac t i on s . The e f f e c t s  o f  s uch 
a s e c r e t o ry p r o du c t o r  immun e r e a c t i o n s  cont ro l l e d from 
ruminant paro t i d  g l ands are l ik e l y  t o  extend into the 
r e t i cu l o rumen a n d  o ma s um ,  areas wh i ch s how a pauc i ty o f  
i n t r a e p i t he l i a l  c e l l s . I n  c on t r a s t , t h e  p r e s ence o f  I E G  
duc t  c e l l s  o r  s im i l ar i n t r a ep i the l i a l  c e l l p opul a t i on s  h a s  
n o t  b e en r ep o r t e d  i n  s a l i v a ry g l ands o f  mono ga s t r i c  s p e c i e s  
wh i ch , no t une xp e c t e d l y , have an a b un danc e o f  i n t r a ep i t h e l i al 
c e l l s , s uch as g l ob u l e  l eucocyte s ,  w i t h in the g a s t r i c  muc o s a, 
p a rt i c u l a r l y  dur i n g  infl amma tory d i s o r d e r s  and i n fe c t i on s  
o f  t h e  G I  tra c t .  
The p r e s en t  s t udy fai l e d  to e s t ab l i s h  that I EG duc t  
c e l l s  ( o r  p r e s ump t i ve g l o b u l e l eu c o cy t e s )  a s  they app e a r  
i n  b o v i n e  s a l i v a ry g l ands a r e  d e r i v e d  from muco s a l  ma s t  
c e l l s  (Mur ray e t  a l . ,  1 9 6 8 )  a l though c l o s e  s im i l a r i t i e s  
b e tween the two c e l l  typ e s  w e re no t e d . Rathe r ,  the w r i t e r 
favours a g ranu l a r  lympho cy t e  s p e c i f i c  to ep i t he l i a l  s i t e s  
a s  a l i k e ly p r e c u r s o r  c e l l  wh i ch may b e  r e l a t e d  to the 
muco s a l  ma s t  c e l l  or i t s  s t em c e l l . 
1 0 9  
CHAPT E R  S .  CONC LUS I ON 
B o vine s a l ivary s e c r e t i ons a r e  e l ab o r a t e d  by three 
ma j o r  s a l i v a ry g l ands , the p a ro t i d ,  mandib u l a r  and s ub l in ­
g u a l  and nume rous mi n o r  s a l iva ry g l ands emb e dded among s ub ­
e p i t h e l i a l  conne c t ive t i s s ue s and mus c l e s . The s a l ivary 
g l ands c ons i s t e d  o f  t ub u l a r  s e c r e t o ry endp i e c e s  comp o s e d  o f  
p r o t e o s e rous , muc o us a n d  demi l un e  c e l l s , s p e c i a l i s e d for 
s y n t he s i s and s e c r e t i o n  of w a t e r , e l e c t ro ly t e s , p r o t e i ns 
and muc o s ub s t anc e s . The duc t a l  s y s t em , wh i l e  funct i on ing 
a s  condu i t s  fo r t rans p o r t  o f  s e c r e t o ry p r o du c t s  from end­
p i e c e s , a dd i t i ona l ly p o s s e s s e d ep i the l i a  we l l  deve l op e d  fo r 
t rans p o r t  o f  wa t e r  and e l e c t r o l y t e s  and s ynthes i s  o f  g l y c o ­
p r o t e ins . 
The h i s t o chem i c a l  c omp o s i t i o n  o f  s al iva ry g l ands 
e xamine d a r e  s umma r i z e d  in T ab l e  5 . 1 .  
The p a ro t i d  g l ands w e r e  comp o s e d  o f  p ro t e o s e rous c e l l s  
s p e c i a l i s e d  fo r s e c re t i on o f  wa t e r  and e l e c t r o l y t e s  w i th 
s ma l l  amoun t s  o f  PAS p o s i t ive n e u t r a l  muc o s ub s tan c e s  and 
s a l i va ry p r o t e i n b ands 1 ,  3 ,  4 ,  5 ,  6 ,  7 ,  1 0 , 1 0 s and l O d .  
O f  thes e ,  b an ds 4 and 1 0  w e r e  t ra c e d  immuno h i s to chemi ca l ly 
t o  indi v i dua l p ro t e o s e rous c e l l s  o f  the g l and . The bovine 
p a ro t i d  g l ands w e r e  unu s u a l  i n  n o t  p o s s e s s ing " s t r i a t e d" 
i n t r a l ob ul a r  duc t s ; a l though th i s  may b e  s i gn i f i c ant in the 
s e c r e t ion of an i s o t on i c  s al i va , int ra l o b u l a r  duc t s  of t h e  
s he ep p a ro t i d  g l ands w e re no t i c e ab ly " s t r i at e d" . Th i s  
s in gul a r  d i ffe rence may h i ghl i gh t  the dan g e r s o f  b ro a d l y  
a p p ly ing t h e  " ruminant mode l "  a rb i t r a r i ly w i thout cons i d e r ­
a t i on t o  s p e c i e s  va r i a t i on . The int r a l ob u l ar duct c e l l s  o f  
t h e  bovine p a ro t i d  g l ands o cc a s i ona l l y  cons i s t e d  o f  PAS 
p o s i t i ve ma t e r i a l  and " ap i c a l  b l eb s " . Ano ther unu s u a l  fea t ­
u r e  o f  the p a ro t i d  g l an d  i n t r a l obul a r  duc t s , was the p r e s ence 
o f  a g ranul a t e d  int r a ep i t h e l i a l  c e l l  l o c a t e d  a dj a c ent t o  t h e  
b a s a l  l amina o f  the duc t  wa l l . Such c e l l s we r e  p o l ymo rp h i c  
i n  app e a rance with nuc l e i  s im i l a r  t o  tho s e  o f  p l a sma c e l l s  
and b a s ophi l i c  cy t op l as ms wh i ch cont a i n e d  a c i doph i l  
Tab l e  5 . 1 
Proteoserous 
cells . 
Mucous cells 
Demilunes 
Mucous cells 
Dernilunes 
Mucous cells 
Dernilunes 
Mucous cells 
Dernilunes 
H I STOCHEM I CA L  C LASS I F I CAT I ON 
OF BOVINE S AL IVARY G LANDS 
PAROTID AND VENTRAL BUCCAL GLANDS 
Immunohistochemically detectable intrinsic proteins 
eg.  band 4 and band 10 , and PAS positive band 5 .  
MANDIBULAR GLANDS 
Neutral glycoproteins and carboxylated acid rnuco­
substances . 
Neutral glycoproteins , carboxylated acid rnucosub­
stances and acidophilic basic  proteins , including 
protein band 8 .  
SUBLINGUAL GLANDS 
Neutral glycoproteins , carboxylated and sulphated 
acid rnucosubstances . 
Proteoserous , including protein band 9 .  Negligible 
amounts of acid rnucosubstances and PAS positive 
glycoproteins . 
INTERMEDIATE BUCCAL GLANDS 
Neutral glycoproteins , carboxylated and sulphated 
acid rnucosubstances . 
Proteoserous , including protein band 4 .  Negligible 
amounts of acid rnucosubstances and PAS positive 
glycoproteins . 
UPPER BUCCAL GLANDS 
Neutral glycoproteins , carboxylated and sulphated 
acid rnucosubstances . 
Proteoserous , negli'gible amounts of acid mucosub­
stances and PAS positive glycoproteins . 
Tab l e  5 . 1 ( cont . )  
Mucous cells  
Demilunes 
Mucous cells  
Demilunes 
Mucous cells 
Demilunes 
PALATINE GLANDS 
Neutral glycoproteins , carboxylated and sulphated 
acid mucosubstances . 
Proteoserous , negligible amounts of acid muco­
substances and PAS positive glycoproteins . 
POSTERIOR TONGUE GLANDS 
Neutral glycoproteins , carboxylated and sulphated 
acid mucosubstances . 
Neutral glycoproteins , carboxylated and sulphated 
acid mucosubstances . 
PHARYNGEAL GLANDS 
Neutral glycoproteins , carboxylated and sulphated 
acid mucosubstances . 
Neutral glycoproteins , carboxylated and sulphated 
acid mucosubstances and acidophilic basic proteins . 
1 1 0  
m e t a chroma t i c  g ranu l e s  o f  va r i o u s  s i z e s . I t  was p rop o s e d  
tha t g ranu l a te d  i n t r a ep i the l i a l  c e l l s  a r e  d e r i v e d  from a 
g ranu l a r  l ympho cy t e  s ubpopu l a t i on and a r e  p ro b ab l y  s im i l a r  
t o  the p o o r l y  de f i n e d  g l obul e l eucocy t e  c e l l  typ e d e s c r i b e d  
b y  mo s t  wo r k e rs , ( s e e G r e g o ry , 1 9 7 9 ) . The e xc r e t o ry duc t s  
o f  t h e  p a r o t i d  g l ands conta i n e d  numerous gob l e t  c e l l s wh i ch 
c o u l d  s up p l ement t h e  p a ro t i d  s e c r e t i on w i th a c i d i c muc o ­
s ub s t an c e s . 
Tub u l a r  endp i e c e s o f  the mandibul a r  g l ands w e r e  comp o s e d  
o f  muc o us c e l l s  a n d  p rominent demi l une c e l l s . The mucous 
c e l l s  cont a in e d  n e u t r a l  and a c i d i c  muc o s ubs tanc e s , the 
l a t t e r  w e r e  mo s t ly neu ramin i da s e l ab i l e  carboxyl g ro up s  o f  
s ia l i c  a c i d .  The demi lune s c o n t a i n e d  a c i dop h i l g ra nu l e s  
the cont e n t s  o f  wh i ch inc l u d e d  p r o t e in b and 8 ;  i n  a d d i t i on 
th e demi l un e s  app e a re d  to s yn t h e s i s e  neutral and a c i di c  
muc o s ub s tan c e s  mo s t  o f  wh i ch w e r e  " ma s k e d" b y  t h e  a c i doph i l  
g r anul e s . The in t r a l ob u l a r  du c t s o f  the man d ib u l a r  g l ands , 
un l i k e  t ho s e  o f  the p a ro t i d  g l and , w e r e  comp o s e d  o f  t a l l  
c o l umna r  c e l l s w i th b a s a l  s t r i a t i on s . Gob l e t  c e l l s , a 
f e a ture o f  the p a ro t i d  g l and e x c r e t o ry duc ts , w e r e  no t 
found in t h e  mandibul a r  g l an d . 
The muc o us c e l l s  o f  the s ub l ingual g l ands we r e  p o s i t iv e  
for neut r a l  a n d  a c i d i c  muc o s ub s t an c e s . The a c i d  g ly c op r o t �  
e i ns i n  p a r t i cul a r  we r e  no t n e u r ami n i da s e  l ab i l e  and app e a red 
to d i f f e r  h i s t o chem i c a l l y t o  t h o s e o f  the man d i b u l a r  g l and . 
A h i s t o c h em i c a l  eva l u a t i on fo r the p r e s ence o f  s u l p h a t e  
group s  i n c l u de d  hya luron i da s e d i g e s t i on fol l ow e d  b y  Al c i an 
b l ue s t a in in g  a t  l ow pH . S ub s e quent p o s i t iv e  s t a i n ing showe d 
that conn e c t ive t i s s u e  mucop o l y s ac cha r i de s  a r e  un l i k e ly t o  
b e  found i n  ep i t he l i a l  muc ous s e c r e t ions . A l c i an b l ue s t a i n ­
i n g  w a s  th e r e fo r e  a t t r ibut e d  t o  s ulpha t e d  g l y c op r o t e ins o r  
s i a l o - s ul p h a t e d  g ly c o p ro t e in s . Such r e s i du e s  w e r e  ma i n l y  
p r e s en t  i n  the s ub l ingua l g l an d s  and a l l  min o r  g l ands , 
excep t the vent r a l  bucc a l ; t h e  s u lpha t e  con t e n t  o f  the man ­
dibular wa s found t o  b e  ne g l i g i b l e . The s e c r e t i o n s  o f  the 
s ub l ingua l  g l and demi lun e s  w e r e  p r e dom inant l y  p r o e t o s e rous 
and c ont a in e d  p ro t e in band 9 .  The r e  was very l i t t l e  e v i dence 
o f  neut r a l  o r  ac i d i c  g ly co p ro t e i ns . The sub l ingual g l and 
" s t r i a t e d" int r a l o b u l a r  duc t s  by comp a r i s on w i th tho s e  o f  
the mand i bul a r  g l an d , w e r e  e qu a l l y we l l  deve lop e d . 
1 1 1  
The mino r g l an d s , w i t h t h e  e x c ep t i o n  o f  the vent r a l  
buccal , wh i ch w a s  i d ent i c a l  t o  t h e  p a ro t i d ,  w e r e  sp e c i a l i s e d  
fo r the s e c r e t i o n  o f  ac i d i c  and neu t r a l  muc o s ub s t anc e s . I n  
g ene ra l , the h i s t o c hemi c a l  f e a tu r e s  o f  the mino r g l and 
mucous c e l l s  were s imi l a r t o  tho s e  of the sub l i ngual g l and . 
The pharyn g e a l  g l ands howe v e r  con t a i n e d  large d em i l un e  
c e l l s  wh i ch were s im i l a r  i n  mo rpho l o gy and h i s t o chem i s t ry t o  
tho s e  o f  the man d i b u l a r  g l an d , a l though the demi l un e s  o f  
the fo rme r  we r e  no t i c eab l y  mo r e  a c i d i c .  The d em i l un e s  o f  
the p o s t e r i o r  t o n g u e  g l ands o c c a s i on a l l y  cont a in e d  s u lpha t e d  
muc o s ub s tance s ;  a f e ature a!so o b s e rve d i n  dem i l une s o f  the 
pharyn g e a l  g l an d s . Dem i l un e s  of t h e  i n t e rme d i a t e  b uc c a l  
g l ands w e r e  foun d  t o  cont a in p ro t e in b and 4 ;  i t  i s  no t : un ­
l i k e l y  that demi l un e s  o f  o the r mino r g l ands may s imi l a r l y  
con t a in p ro t e ina c i o us mat e r i a l  wh i ch i s  no t d e t e c t e d  b y  
h i s to l o g i c a l  s t a in s , s uch a s  PAS and A l c i an b l u e . I t  wo u l d  
b e  s a fe t o  a s s ume t h a t  s e c r e t i on s  from the s e  c e l l s  a r e  
ma inly p r o t e o s e ro us rather than pure l y  s e rous . 
Ab undant g ob l e t  c e l l s  i n  the i n t ra l o bul a r  duc t s  w e r e  
no t i c e d  o n l y  in t h e  pharyng e a l  g r oup o f  g l ands . Th e r e  w a s  
a l s o  a n  indi c a t i on t h a t  in th i s  g r o up , t h e  s e c re t o ry end ­
p i e c e s  c ommun i c a t e d  d i r e c t l y  wi th t h e  int ra l o b u l ar duc t s . 
Two unusual f e a ture s o b s e rved i n  t h r e e  o th e r  mino r 
g l ands , the int e rm e d i a t e  b uc c a , do r s a l  bucc a l  and p a l a t i n e , 
were ( 1 )  a p o s s ib l e  a typ i c a l  s e c r e t o ry me chan i s m  wh i c h  
invo l v e d  c e l l u l a r  d eb r i s  m i x e d  w i th muc ous s e c r e t i on s , and 
( 2 )  s c a t t e r e d  i s l e t s  of p r o t e o s e rous l obul e s  i dent i c a l  t o  
tho s e  o f  the p a ro t i d  and v e n t r a l  b uc c a l  g l ands . 
The humo r a l  muc o s a l  immune s y s t e m  o f  the b o v i n e  s a l i v a ry 
g l ands i s  l ik e l y  t o  b e  ma i n t a ined by immuno g l obul ins , s uch 
a s  s i gA ,  synthe s i s e d and s e c r e t e d  f r om p l asma c e l l s  d i s t r i b ­
uted mo s t ly in t h r e e  s a l ivary g l ands , the s ub l ingua l , 
1 1 2  
man d i bul a r  a n d  pharyn g e a l  g l ands . I t  was p ropo s e d t h a t  the 
p a r o t i d  and v e n t r a l  b u c c a l g l ands may be s p e c i a l i s e d f o r  
ma i n t enan c e  o f  c e l l - me d i a t e d  immun i ty a t  a muc o s a l  l e v e l , 
me d i a t e d  p ro b a b l y  by g r anul a t e d  i n t ra ep i the l i a l  duc t  c e l l s , 
wh i ch were t e n t a t i vely i dent i fi e d  a s  g l o b u l e  l eu c o c y t e s .  
H i s t o l o g i c a l  s tud i e s  p e r t a i n i n g  t o  b l o a t s u s c ep t i b i l i ty 
in c a t t l e , demons t r a t e d  that p ro t e in b and 4 o f  p a ro t i d  
s a l iva , wh i ch h a s  b e en c o r re l a t e d  w i th s u s cept i b i l i ty t o  
b l o a t , was e qu a l ly d i s t r i buted i n  p a ro t i d  t i s s u e  s amp l e s  o f  
b o th L . S .  and H . S .  animal s .  Exp l ana t i ons p ropo s e d f o r  th i s  
anoma l o us r e s u l t  were : a po s s ib l e  d i ffe rence in p a r a s ym ­
p a t he t i c  a n d  s ympathe t i c inn e rva t i on p a t t e rns i n  L . S .  and 
H . S .  anima l s  i nvo lving p a ro t i d  p r o t e in s e c re t i on ,  and the 
p re s ence o f  a p o o r ly unde r s t o o d  i n t r ac e l l u l a r  r e g u l a t o ry 
mechan i s m  fo r s e c r e t i o n  o f  p ro t e in b and 4 .  
The que s t i ons that a r o s e  du r i n g  the p r es ent s tudy , 
wh i ch co u l d  p ro v i de gu i danc e fo r fur t h e r  inve s t i g a ti ons a r e : -
( 1 )  The p hys i o l o g i c a l  s i gn i f i c ance f o r  the l a c k  o f  
b a s a l  s t r i a t i ons in t h e  i n t r a l o b u l a r  duc t s  o f  
the p ar o t i d  g l ands . 
( 2 )  To i dent i fy the p r e curs o r  c e l l  o f  the i n t r a e p i th ­
e l i a l  granu l a r  duc t c e l l , p r e f e r ablf from ep o n ­
emb e d d e d  s em i - th in s e r i a l  s e c t i o n s  a n d  t o  conduc t 
a c omp a r a t ive , ul t ra s t ru c tur a l  and cy t o chemi c a l  
s tudy o f  b o t h  the i n t r a e p i the l i a l  g r anu l a r  duct 
c e l l  and the muco s a l  mas t c e l l .  
( 3 )  A de t a i l e d  e xamina t i on o f  the app a rent a t yp i c a l  
s e c r e t o ry me chan i sm ob s e rved i n  thr e e  m i no r g l ands 
and i t s  p o s s ib l e  s i gn i f i c anc e . 
( 4 )  An u l t ra s t ructural inv e s t i g a t i on o f  p a ro t i d  p r o t e o ­
s e ro u s  c e l l s  in b o th L . S .  and H . S .  anima l s  t o  
e xam ine i n  d e t a i l  the i n t rac e l l u l a r  d i s t r i bu t i on 
p a t t e rn of b and 4 .  
1 1 3  
( 5 )  The s t imulus fo r the app e a r ance o f  i n t r a ep i t he l i a l  
g ranul a r  duc t  c e l l s  i n  p a ro t i d  g l ands o f  L . S .  
an ima l s  - a cha l l enging p r o b l em which c anno t b e  
comp l e t e ly r e s o l ve d  from h i s t o l o g i c a l  s tu d i e s  
a l one . 
S in c e  s e c r e t o ry t i s s ue s  a re n o t  found in the ruminant 
fo r e s tomachs , it  i s  the w r i t e r ' s  o p i n i on that the b i o chem­
i c a l  componen t s  o f  bovine s a l iva s uc h  a s  l ow mo l e cul a r  
we i gh t  s a l ivary p ro t e in s , h i g h  mo l e c u l a r  weight muc o s ub ­
s t anc e s , wa t e r  a n d  e l e c t r o ly t e s , c o u l d  e ach have a s p e c i f i c  
func t i on w i th i n  t h e  r e t i cu l orumen and oma s um .  Th e h i s t o ­
l o g i c a l  s t ruc t u r e  and h i s t o chemi c a l  comp o s i t i o n  o f  the 
s a l i vary g l ands s how t i s s u e s  s p e c i a l i s e d  for e i th e r  s y n t h ­
e s i s  o r  s e c r e t i o n  o r  b o th . 
I nve s t i g a t i o n s  into the func t i o n  o f  l ow mo l e cul a r  
we i gh t  p ro t e i n f r a c t i ons a r e  y e t  a t  a p r e l iminary s t a g e  
a l though band 4 h a s  b e e n  imp l i c a t e d  w i t h b l o a t  s u s c ep t i b ­
i l i ty .  H i gh mo l e cu l a r  w e i ght muc o s u b s t anc es , s uch a s  
a c i d i c  and neut r a l  g lycop r o t e ins c o u l d  p rovide a p ro t e c t iv e  
l ay e r  o f  mucus t o  t h e  rum i nant fo r e s tomachs to p r event 
ab r a s i o n  and de s s i c at i o n  o f  the l i n i ng ep i the l i a and r e s i s t  
b a c t e r i a l a dhe r e n c e  i n  a dd i t i o n  t o  s upply ing a va l uab l e  
s o u r c e  o f  n i t r o g en fo r ma intenan c e  and p ro l i fe ra t i o n  o f  
rum i n a l  m i c ro o r g an i sms . Add i t i o na l l y ,  s in c e  lympho i d  
t i s s ue s  a r e  s c a r c e  i n  t h e  muc o s a  o f  rumi nant fo r e s tomachs , 
immuno g l obul i n s  s uch a s  s i gA ,  synthe s i s e d  and s e c r e t e d  b y  
p l a s ma c e l l s  o f  s a l ivary g l ands c an enhanc e immuno p ro t e c t i o n  
t o  t h e  o r a l c a v i ty , o e s ophagus , r e t i cu l o rumen and oma s um ,  
a g a ins t harmfu l ant i g en i c  ma t t e r .  
APPEND I X  I 
The pr i n c ipl e s  o f  h i s t o chem i c a l  methods us e d  
fo r the d e t e c t i o n  o f  muc o s ub s t anc e s . 
1 1 4  
Ep i the l i a l  muc o s ub s t a n c e s  cons i s t  p r e domi n an t l y  o f  g lyc� 
p r o t e ins wh i ch a r e  p ep t i d e  ch a i n s  b e a r i ng num e rous cova l ent ly 
l in k e d  o l i g o s a c char i de un i t s . The s e  c a rbohy d r at e  un i t s  a r e  
s ix - me mb e r e d  py r ano s e  r in g s  wh i ch c o nt a in h i s t o chemi c a l ly 
de t e c t ab l e  r e s i dues s uch a s  1 , 2  g l yc o l  g ro up s , 1 , 2  amino 
hy d r o xyl g roup s , c a rboxyl a c i d  g ro up s  and s u l p hat e e s t e r s . 
Ep i th e l i a l  s e c r e t o ry g ly c o p ro t e i n s  that con t a in th e s e  
r e s i du e s  a r e  m a i nly neut r a l  s u g a r s , s i a l i c  a c i ds and s i a l o ­
s u l p h at e d  g ly c o p ro t i en s  a n d  s ulph a t e d  g l y c op r o t e in s . 
T o  e s tab l i s h  the i den t i ty o f  the ab ove g roup s  h i s t o l o g ­
i c a l  s t a ins c an b e  us e d ,  a s  we l l  as h i s t o chem i c a l  t e chn i ques 
s u ch as a c e ty l a t ion , s ap o n i f i ca t i o n ,  m e thyl a t i on and en zymic 
d i g e s t i on .  
A .  D emon s t r a t i on o f  n e u t r a l  s i de groups 
The p e r i o di c  a c i d - s ch i ff ( PA S )  method ut i l i s e s  p e r i o d i c  
a c i d  t o  o x i d i s e  hydroxy l  g roup s o f  adj a c ent c a rbon a toms t o  
a l d e hyde s ; th e s e a r e  t h e n  rend e r e d  v i s ib l e  b y  r e a c t i on w i th 
S c h i f fs r e a g ent , w i th wh i ch the a l dehy d e s  fo rm a s t ab l e  
p r o duct , p ink o r  dark r e d  i n  co l ou r . A c e tyl a t i o n , by us e 
o f  a c e t i c  anhydride in d ry py r i d i n e , b l o cks hydroxy l g roup s  
b y  c onve r t ing them t o  a c e ty l  e s t e r s thus p r event ing r e a c ­
t iv i ty with the S ch i f f s  r e a gent . A PAS p o s i t iv e  s ub s t an c e  
a ft e r  a c e ty l at i on wou l d  b e  PAS n e g a t ive , t h u s  indi cat ing 
that the o r i g inal r e a c t i on was due t o  a 1 , 2  g lyco l g ro up . 
A d i s t in c t ion b e tw e e n  PAS r e a c t iv i ty due t o  n e u t r a l  
s i d e  g roup s  o f  g ly c o g e n  mo l e cul e s  a n d  neu t r a l  g ly c op r o t e ins 
can be made us ing d i a s t a s e  d i g e s t i o n  wh ich s p e c i f i c al ly 
removes g ly c o g en . PAS r e act ivi ty a f t e r  d i a s t a s e  d i g e s t i o n  
c o u l d  the r e fo r e  be a t t r i b u t e d  t o  neut r a l  g ly cop r o t e ins 
o th e r  than g ly co g en . 
B .  Demon s t r a t ion o f  a c i di c  s i d e  groups 
1 1 5  
With c o n t r a s t to n e u t r a l  s i de g roup s  t h e r e  a r e  a mul t i ­
p l i c i ty o f  me thods ava i l ab l e  fo r the demo n s t r a t i on o f  ac i d  
g ro up s , the maj o r i ty b e ing cat i on i c  dy e s  s p e c i f i c  fo r c a r ­
b o xy l  and s u lpha t e  g ro up s . F o r  examp l e ,  A l c ian b l ue , 
A l c i an y e l l ow ,  c o l l o i da l  i ron , p e r i o d i c  a c i d - pheny l hy dr a z ine 
S ch i f f ( PAP S ) , T o l u d i n e  b l ue and Azure A .  In ad d i t i o n  t h e  
u s e  o f  a b a s i c  s t a i n  s uch a s  A l c i an b lue t o g e t h e r  w i th PAS , 
can b e  u s e d  t o  d i s t i n g u i s h  b e tw e en neut r a l  and a c i d i c  
g r oups a t  t h e  s ame s i t e . 
The mo s t  c ommo n l y  u s e d  c a t i on i c  dye i s  Al c i an b lu e  wh i ch 
c a n  be u s e d  at e i th e r  pH 1 . 0  o r  2 . 5  to i de nt i £y s u lphat e d  
and carb o xy l g roup s r e s p e c t i ve l y . A t  l ow p H  s u l p ha t e  
g r o up s  a r e  s uf f i c i en t l y  i on i z e d  t o  r e a c t  w i th the dy e wh e r e ­
a s  a t  pH 2 . 5 the r e a c t i v e  g roup s ioni z e d  a r e  mo s t l y  c a rboxyl 
g r o ups . 
Methy l a t i o n  p e r f o rm e d  a t  6 0 ° C  fo r 4 hours by u s e o f  1 %  
H C l  in me thy l al coho l e l im in a t e s  b a s ophi l i a du e t o  a c i d i c  
g ro ups : ca rboxyl g ro up s  a r e  c o nv e r t e d  t o  methyl e s t e r s 
wh i l e  s u l p h a t e  g roup s a r e  r emov e d  from g ly c op r o t e i n mo l e c ­
u l e s . Methy l a t i o n  c o u l d  a l s o  r emove c a rb o xyl g ro up s  o f  
s om e  s i a l i c  a c i d  r e s i du e s  w i th o u t  conve r t ing them t o  me t hy l  
e s t e r s . 
The e f f e c t s  o f  m e t hy l at i o n  can b e  r ev e r s e d  by s apon i f ­
i ca t i on w i t h  0 . 5 %  s o l u t ion o f  KOH i n  7 0 %  e thano l .  Th i s  
p r o c e dur e  r e s to r e s  b as oph i l i a  . o f  ca rboxy l g ro up s  e s t e r i f i e d  
b y  me thy l a t i on b u t  do e s  no t r e s t o re b a s oph i l i a  du e t o  s u l ­
p ha t e  g roup s o r  c a rb oxyl g roup s wh i c h  have b e en r emoved 
dur ing m e t hy l at i on . S aponi f i c a t i o n  c ou l d  al s o  b e  u s e d  t o  
r e s t o r e  PAS r e a c t ivi ty b l o c k e d  b y  a c e ty l a t ion . 
Carboxy l g ro up s  o f  s i a l i c a c i d  o r i g in can b e  s p e c i f i c ­
a l l y  i dent i f i e d  by r emoval o f  s i a l i c ac i d  un i t s  b y  u s e  o f  
t h e  en zyme s i a l i da s e  ( o r  neuram i n i da s e )  and s t a in ing s ub ­
s eq uent ly w i th Al c i an b l ue a t  pH 2 . 5 .  Any b a s ophi l i a 
• 
1 1 6  
a f t e r  neuram i n i da s e  d i g e s t i on i s  p r o b ab ly due t o  s i a l i c 
a c i d  r e s i du e s  not l ab i l e  t o  n e u r aminidas e and add i t i ona l ly 
t o  sma l l  amoun t s  o f  u r o n i c  a c i d ,  a l t hough the l a t t e r  has 
no t b e e n  i d ent i f i e d  in b o v i n e  s a l iva ry g l a nd muc o s ub s t anc e s . 
The na ture o f  the s u l p ha t e  g roup s c an be inv e s t i g a t e d  
by u s e  o f  t h e  enzyme t e s t i cu l a r  hya l u ro n i d as e  wh i c h  e x t ­
r a c t s  muc o p o l ysacchari de r e s i du e s  us ua l l y  found i n  con ­
n e c t iv e  t i s s u e s . Sub s equent A l c i an b lue s t a i n ing a t  l ow 
p H  cou l d  t h e r e fo re b e  a t t r ibut e d  to s u l p h a t e  g r oup s a t t a ched 
t o  g l ycop r o t e in mo l e c u l e s , such a s  s ul p ha t e d g l y c op r o t e ins 
or s i a l o - s u lpha t e d  g ly copro t e i n s  rather than chondro i t in 
s u l p hat e s . 
The a c i d i c  nature o f  the muc o s ub s t an c e s  c an b e  further 
e s t ab l i sh e d  by use of p e r i o d i c  a c i d - pheny l hydr a z i n e  Schi f f  
r e a c t i o n  wh i ch us e s  p h eny l hy dr a z ine t o  b l o ck p e r i o da t e ­
i nduc e d  a l d ehyde g r o up s from n e u t ra l  g l y c opro t e in s  l e aving 
a l d e hy de g roups o f  s i a l o - g ly c o p r o t e ins or s i a l o - s u l p ha t e d  
g l ycopro t e i ns to r e a c t  wi th t h e  S ch i f fs r e ag ent . 
The p h enomenon o f  m e t a chroma s i a  i n  th e p r e s en c e  o f  
t h i a z in dy e s  s uch a s  T o l udine b l ue and Az u r e  A i s  due t o  
t h e  ab i l i ty o f  t h e  dy e t o  imp a r t  a c o l ou r  tha t  d i f f e r s  f rom 
t h e  o r i g i n a l  co l our o f  the dy e .  Th i s  change in c o l our , due 
t o  a s h i ft to sho r t e r  wave l en g t h s  ( a hyps ochrom i c  s h i ft ) 
i n  the ab s o rp t i on s p e c t rum o f  the dy e , i s  cha ra c t e r i s t i c ­
a l ly indu c e d  b y  h i g h  mo l e cul a r  we ight t i s s ue s ub s t r a t e s  
wh i ch have an ion i c  r a d i c l e s  c l o s e  t o g e the r .  H i gh mo l e c ­
u l a r w e i g h t  connect i v e  t i s s ue mucop o lys a c char i de s  s uch a s  
chondro i t in sulpha t e s  and hya l ur o n i c  a c i ds imp a r t  a n  int ­
e n s e  v i o l e t - red me t a c hroma t i c  c o l our w i th th i a z i n e  dy e s  
and ep i t h e l i a l  muc o s ub s tan c e s o f  l ow e r  mo l e cu l a r  w e i ght , 
such a s  s i a l i c  a c i d s , imp a r t  a weake r purp l e  me t achroma t i c  
c o l o ur . 
APPEND I X  I I  
S t a i n ing and H i s t o chemi ca l T e c hnique s 
( 1 )  Haematoxyl in and Eo s i n ( H & E )  
Me t ho d : -
1 .  Dewax and b r ing s e c t ions t o  w at e r .  
2 .  St ain in M ay e r s  H a ema lum fo r 1 0  minu t e s . 
3 .  Rins e in t apwa t e r .  
4 .  Blue in S c o t t s  t apwa t e r  fo r 2 minut e s . 
5 .  Rins e in t apwa t e r .  
6 .  S t a i n  in 1 %  aqueous Eo s in Y fo r 2 m i nu t e s . 
7 .  Rins e r ap i dly in t apwat e r .  
8 .  Dehy d ra t e , c l e a r  i n  xy l ene and mo unt in D . P . X . 
( 2 )  A l c i an B l u e  (AB ) pH 1 . 0 and pH 2 . 5  
Me t h o d : -
1 .  Dewax and b r ing s e c t i ons t o  w a t e r . 
2 .  S t a i n  in 1 %  Al c i an b lue 8 GX in 3 %  a c e t i c  a c i d  
(pH 2 . 5 ) o r  1 %  Al c i an b l ue 8 GX i n  O . lN HC l 
(pH 1 . 0 ) for 1 0  m i nut e s . 
3 .  Rins e in t apwat e r .  
1 1 6  
4 .  Co unt e r s t a in w i th H & E , dehydrate c l e ar and mount. 
Re s ul t s : -
C a rb o xy l at e d mu co s ub s t anc e s  s t a i n  b l ue a t  p H  2 . 5 , 
S ulphat e d  muc o s ub s t an c e s s t a in b l ue a t  p H  1 . 0 . 
( 3 )  P e r i o d i c  Ac i d  S ch i ff ( PA S )  
Me t ho d : -
1 .  Dewax and b r ing s e c t i ons t o  wate r .  
2 .  Ox i d i s e in 1 %  p e r i o d i c  ac i d  for 8 m i nut e s . 
3 .  Wash in runn ing tap wat e r  fo r 5 minute s .  
4 .  Wash in t h r e e  chan g e s  o f  d i s t i l l ed wat e r . 
5 .  Re a c t  w i th S c h i f f s  r e a g ent f o r  1 0  m inut e s . 
6 .  Wash in runn ing t apwat e r  fo r 1 0  m i nut e s . 
7 .  S t a in in May e r s  H aema lum fo r 5 minu t e s . 
8 .  Rins e i n  t apwat e r .  
9 .  B l u e  i n  S c o t t s  t apwa t e r  fo r 2 minut e s . 
1 0 .  Rins e in t apwat e r .  
1 1 7  
1 1 .  Rin s e  in 7 0 %  and 9 5 %  a l c o h o l . 
1 2 .  Count e r s t a i n  i n  s a turat e d  s o lut i o n  o f  Tar t r a z ine 
in c e l lus o l ve ( e thy l en e  g l yco l mono e thyl e th e r )  
fo r 2 minu t e s .  
1 3 .  D i f f e r ent i a t e  and dehydr a t e  in 2 chan g e s  o f  
ab s o l u t e  al coho l . 
1 4 . C l e a r  in xy l en e  and mount in D . P . X . 
Re s ul t s : -
Neut r a l  g l ycopro t e i n s  s t a i n  p ink to dark red . 
P r ep a r a t i on o f  S chi ffs Re a g ent : -
To 4 0 0  ml o f  d i s t i l l ed wa t e r  warme d to 3 7 ° C add 2 g r ams 
of p ar a fuch s i n  and ag i t a t e  un t i l  d i s s o lv e d . Add 7 . 6  g rams 
of s o d i um met ab i s ulphi t e  and 2 ml o f  concent r a t e d  Hydro ­
ch l o r i c  ac i d .  Shake we l l  fo r 1 0  m i nut e s . S t o r e  i n  dark 
cupb o a rd at room temp e ra tu r e  overni ght unt i l  s o l u t i o n  
b e come s s t raw- c o loured . A dd 2 g r ams o f  a c t i va t e d  charco a l . 
Shake w e l l  and f i l t e r  b e fo r e us e .  S to r e  i n  r e f r i g e ra t o r .  
( 4 )  A l c ian b l ue and P e r i o d i c  a c i d  S ch i ff 
Method : -
1 .  F o l l ow AB me t h o d  S tep s 1 - 3 .  
2 .  S t a in with PAS me thod S t e p s  1 - 1 4 . 
Re s u l t s : -
Neut r a l  g lycop r o t e i n s  s t a i n  r e d . 
Ac i d  g lycopro t e i n s  s t a i n  b l u e . 
Mixed g ro up s  s t a i n  purp l e . 
( 5 ) Phenylhydr a z ine - P e r i o d i c  Ac i d  S ch i f f  (PAP S )  
Metho d : -
1 .  Dewax and b r i n g  s e ct i o n s  t o  wate r .  
2 .  Oxi d i s e  in 1 %  p e r i o d i c  a c i d fo r 8 minut e s . 
3 .  W a s h  in runn i n g  t apwa t e r  fo r 5 m i nut e s . 
4 .  B l o c k  p e r i o d a t e - i ndu c e d  a l dehyde s w i t h 0 . 5 % 
aqueous pheny l hydra z i n e  hy droch l o r i de i n  
c o p l in j a r fo r 3 0  minut e s . 
5 .  W a s h  in runn i n g  t apwa t e r . 
6 .  S t a in w i th Schi ffs r e agent fo r 1 0  m i nu t e s . 
7 .  W a s h  i n  runn ing t apwa t e r  fo r 1 0  minut e s . 
8 .  C o unt e r s t a in w i th t a r t r a z ine . 
9 .  Dehydr a t e , c l e a r  and mount . 
Re s u l t s : -
S i a l o  g l y c o p r o t e i ns and s i a l o - sulphat e d  g l y c o ­
p ro t e in s  s t a in red . 
( 6 )  Ac e tyl a t i on and PAS 
Me tho d : -
1 .  Dewax and b ring t o  w a t e r .  
2 .  T r e at w i th mixture o f  a c e t i c  anhyd r i de ( 1 3  ml ) 
and p y r i d i n e  ( 2 0  ml ) fo r 1 hour . 
3 .  W a s h  i n  wa t e r .  
4 .  S t a i n  w i th PAS t e chn i que S t e p s  2 - 1 4 . 
Re s u l t s : -
Ac e ty l at i on b l o c ks hy d r o xy l  g r o up s , wh i ch a r e  
then PAS n e g at i ve . 
( 7 )  Ac e tyl a t i o n , S apon i f i c a t i o n  and PAS 
Me tho d : -
1 .  Dewax and b r ing to w a t e r .  
2 .  Tre at w i th mixture o f  a c e t i c  anhy d r i d e  ( 1 3  ml ) 
and py r i d ine ( 2 0  ml ) fo r 1 hou r .  
3 .  Wash i n  wat e r . 
1 1 8  
4 .  T r e at w i th O . lN KOH for 4 5  minut e s  i n  c op l i n j a r .  
5 .  W a s h  i n  w at e r .  
6 .  S t a in w i th PAS t e chn ique S t ep t s  2 - 1 4 .  
Re s ul t s : -
S a p on i f i ca t i on re s t o r e s  PAS s t a ining o f  hy d roxy l 
g ro up s  b lo c k e d  by a c e ty l a t i on . 
( 8 ) D i a s t a s e  and PAS 
Me tho d : -
1 .  Dewax and b r ing t o  wat e r .  
2 .  Di g e s t  w i th d i a s t as e ( 1 : 1 0 0 0  in d i s t i l l e d wat e r )  
o r  t r e a t  w i t h s al i v a  fo r 3 0  minut e s  a t  3 7 ° C .  
3 .  Wash i n  w a t e r . 
4 .  S ta in w i th PAS techn i que S t ep s  2 - 1 4 . 
Re s ul t s : -
Di a s tas e removes g ly c o g en ,  PAS reac t i v i ty i s  
th e r e f o r e  s p e c i f i c  fo r n e u t ra l g l ycop ro t e in s . 
( 9 )  Me thyl at i on and Al c i an b l ue a t  pH 1 . 0  and pH 2 . 5 
Me tho d : -
1 .  Dewax and b r i ng s e c t i ons t o  7 0 %  a l c o ho l . 
2 .  P l ac e  i n  p rehe a t e d  1 %  HC l i n  me thy l a l c oho l 
for 4 h o u r s  at 6 0 ° C . 
3 .  Rins e i n  al coho l . 
4 .  S t a i n  w i th Alc i an b lue me thod Step s 2 - 4 .  
Re s ul t s : -
Ac i d  muco s u b s tan c e s  a r e  AB n e g at ive at b o th pH 1 . 0  
and pH 2 . 5 ,  a s  a c t ive m e thy l at i on e l im i n a t e s  
b a s ophi l i a . 
( 1 0 )  Me thyl a t i on , S apon i f i c a t i on and AB at pH 1 . 0 and 
pH 2 . 5 
Me tho d : -
1 .  Dewax and b r i n g  s e c t i o n s  t o  7 0 %  a l coho l . 
2 .  P l a ce i n  p r e h e a t e d  1 %  HCl in me thy l a l coho l 
for 4 hours at 6 0 ° C .  
3 .  Rins e  i n  a l co ho l . 
4 .  S ap o n i fy in 0 . 5 % KOH i n  7 0 %  a l coho l f o r  3 0  
minut e s  a t  Ro om T emp e ra t ur e . 
5 .  Wash i n  wat e r . 
6 .  S t a in w i th Al c i an b lue metho d St e p s  2 - 4 .  
Re s ul ts : -
N e g at ive AB s t a i n i n g  a t  p H  1 . 0  a s  sulpha t e  g roup s  
a r e  hy dr o ly s e d .  S ap on i f i c a t i o n  res to r e s  methy l at e d  
c a rboxy l  g ro ups t o  s t a in a t  AB p H  2 . 5 .  
1 1 9  
( 1 1 )  N e u r am i n i d a s e  and Al c i an b l ue at pH 2 . 5  
Me t h o d : -
1 .  Dewax and b r ing t o  wat e r .  
2 .  I ncub a t e  s e ct ions i n  s o lut i o n o f  n e u r am i n i da s e  
( C l . P e r fr ingens ) ( P . L . B i o chemi cal s I n c . ) ;  
p r e p a r e d  by : 
O . l M a c e t a t e  b u ff e r  pH 5 . 5 1 . 0  ml 
Cal c i um chl o r i de 1 0  mg 
Neuram i n i d a s e 1 0  mg 
I n cub a t e  o v e rn i ght at 3 7 ° C w i t h  c ont ro l 
s e c t ions i n  b u f f e r  a l one . 
3 .  Wa sh i n  t ap wat e r .  
4 .  S t a i n  w i t h  AB m e t h o d  a t  p H  2 . 5 , S t ep s 2 - 4 .  
Re s u l t s : -
N e ur ami n i da s e  e l im i na t e s  AB s t a i n ing a t t r i b ut e d  t o  
c a rb oxy l g roup s o f  neu rami n i da s e  l ab i l e  s i a l i c 
a c i ds . 
( 1 2 )  Hya l u ron i da s e and Al c i an b l ue a t  pH 1 . 0  
Me t ho d : -
1 .  Dewax and bring t o  wat er . 
2 .  I ncub a t e  s e c t i on s  in s o l ut i o n  of hy a l uron i das e 
( B ovine t e s t e s ) ( 1 5 , 0 0 0  un i t s )  ( P . L .  B i ochem­
i c a l s  I nc . ) ; p r e p a r e d  by : 
hy a l u r o n i d a s e  5 mg 
Pho s ph a t e  buffe r pH 6 . 7 5 ml 
I ncub a t e  o v e rn i ght a t  3 7 ° C w i t h  cont ro l 
s e ct ion o f  e l a s t i c  c a r t i l a g e  ( ep i g l o t t i s ) 
i n  buffer a l on e . 
3 .  Wash i n  t ap wat e r . 
4 .  S t a i n  w i th AB me t h o d  at p H  1 . 0  S t ep s  2 - 4 .  
Re s ul t s : -
Hyaluro n i da s e  remo v e s  chondro i t in sulphat e and 
hy a l u ro n i c  ac i d ;  s u b s equent AB s t a ining at pH 1 . 0 
i s  due t o  s ulphat e d  g l ycop r o t e i n s . 
1 2 0  
( 1 3 ) Al c i an Y e l low pH 2 . 5 
Me tho d : -
! .  Dewax and b r i n g  to wat e r .  
2 .  S t a i n  in 0 . 5 %  A l c i an Y e l low GX in 3 %  a c e t i c  
a c i d  (pH 2 . 5 ) for 1 0  m inut e s . 
3 .  W a s h  i n  t ap wa t e r . 
4 .  C o un t e rs t a i n  w i th H & E . 
5 .  Dehydrat e ,  c l e a r  and mount . 
Re s u l t s : -
Carboxy l a t e d  muc o s ub s t an c e s  s t a i n  ye l l ow .  
( 1 4 )  To ludine B l ue 
Me thod : -
! .  Dewax and b r ing t o  wat e r .  
2 .  S t a i n  in 1 %  aq . To l u d i n e  b l ue for 1 minu t e . 
3 .  Rins e in t ap wat e r .  
4 .  Dehy drate r ap i dly , c l e a r  and mount . 
Re s u l t s : -
Ac i d  muc o s ub s t anc e s  s t a i n  p u rp l e . 
( 1 5 )  Azure A pH 1 ,  2 ,  3 and 4 
Me tho d : -
! .  Dewax and b r ing t o  w at e r .  
2 .  S t a i n  at requi r e d  pH s o lut i o n  for 5 minut e s ; 
Prep a r e  Azure A 1 : 5 0 0 0  at pH 1 ,  2 ,  3 and 4 
w i th p h o s phat e - c i t ra t e  buffe r .  
3 .  R i n s e in t ap wat e r . 
4 .  Dehydra t e , c l e ar and mo unt . 
Re s ul t s : -
S t ong l y  a c i d i c  muc o s ub s t an c e s  s t ain at pH 2 and 
ab ove wh i l e  s i a lo - g lycop r o t e ins s t ain at p H  4 .  
( 1 6 ) H a l e s  C o l l o i da l  I ron 
Me tho d : -
! .  Dewax and b r ing t o  w a t e r .  
2 .  F l o o d  with s o lut i on o f  d i a lys ed i ron 1 vo l ume 
1 2 1  
and 2 M  a c e t i c  a c i d  1 vo l ume (mixe d j us t  b e fo r e  
us e )  fo r 1 0  m i nut e s . 
3 .  Was h  w i t h  d i s t i l l e d  wat e r . 
4 .  F l o o d  w i t h  a c i d - f e r r o c y an i de s o lution o f  
2 %  p o t a s s ium fe rrocyan i de 1 vo l ume and 
2 %  H C l  1 vo l ume for 1 0  m i nut e s . 
5 .  W a s h  w i t h d i s t i l l e d  wat e r . 
6 .  C o un t e r s t a in i n  May e r s  H a emalum fo r 5 m inut e s . 
7 .  Rins e in t ap wa t e r . 
8 .  Dehy d r a t e , c l e a r  and moun t . 
Res ul t s : -
Ac i d  muc o s ub s t an c e s  s t a i n  b r i ght b l ue . 
P r e p a r a t i on o f  d i a ly s e d  i ro n  s t o ck solut i o n : ­
d i s s o l ve 7 5  g r ams o f  F e C 1 3 in 2 5 0  ml d i s t i l l ed 
wat e r . 
Add 1 0 0 ml o f  g l ycero l , w i th cons tant s t i r r in g  
a d d  5 5  m l  o f  2 5 %  ammon i a  s o l u t ion . D i a l y s e  
r e s u l t i ng s o lut ion ag a i n s t  d i s t i l l e d  wa t e r  
fo r 3 days . 
( 1 7 )  B IAL r e a c t i o n  fo r s i al i c  a c i d  
Me tho d : -
1 .  Dewax an d b r ing to wa t e r .  
2 .  Sp r ay s e c t i on s  with B IAL re agent by u s e  o f  
S h andon sp ray gun . 
3 .  P l a c e  s e c t i o n s , f a c e  down , on a g l a s s  frame 
in a p r e h e a t e d  con t a i ne d ,  wh i ch h a s  on the 
b o t t om a thin l ay e r  of conc ent rat e d  HC l a t  
7 0 ° C ,  fo r 5 - 1 0  m inu t e s . 
4 .  D ry s e c t i ons in a i r . 
5 .  C l e ar in xy l ene and m o unt . 
Re s u l t s : -
Are a s  o f  h i gh concent ra t i o ns o f  s i al i c  a c i ds 
s t a in r e dd i s h  b rown .  
1 2 2  
P r e p a r a t i o n  o f  B IAL r e a g ent : -
o r c i no l  
c on e . HCl 
O . l M Cuso4 
2 0 0  mg 
8 0  ml 
0 . 2 5  ml 
D i s s o l ve the o rc ino l in HC l ,  a d d  Cuso4 s o l u t i o n  
and make up t o  1 0 0  ml w i th d i s t i l l e d  wat e r . Al l ow 
t o  s t and fo r 4 hour s . 
1 2 3  
( 1 8 ) B i s m a r c k  B rown 
( 1 9 )  
Me t ho d : -
1 .  Dewax and b r ing t o  wat e r . 
2 .  S t a in i n  B i sma r c k  B rown s o l ut i on fo r 3 0  minut e s . 
3 .  Rins e i n  7 0 %  a l c o ho l . 
4 .  Coun t e r s t a in in C a r az zi' s h a ematoxy l i n  f o r  1 - 2 
minut e s . 
5 .  Wash in runn ing t ap wat e r  f o r  5 minu t e s . 
6 .  Dehydr a t e , c l e a r  and moun t . 
Res u l t s : -
Su lphat e d  muc o s ub s t an c e s  and mas t c e l l  g r anul e s  
s t a in y e l l ow - brown . 
P r e p a r a t i o n  o f  B i s mar c k  B rown s o l ut i on : -
B i smarck B rown 0 . 5 
Ab s o lut e  a l co h o l  8 0  
1 %  HCl 1 0  
N e u t r a l Re d 
Me t h o d : -
1 .  Dewax and b r ing t o  wat e r .  
g 
ml 
ml 
2 .  S t a in in 1 %  aque o us s o l u t i o n  of Neut r a l  r e d  
f o r  5 minut e s . 
3 .  Dehyd r a t e ,  c l e a r  and moun t . 
Re s u l t s : -
N e u t r a l  r e d ,  a c a t i o n i c  dy e ,  s t ains ma s t  c e l l  
g ranul e s  dark re d .  
( 2 0 )  P h l o x ine - T a r t r a z ine 
Me thod : -
! .  Dewax and b r ing t o  water . 
2 .  S t a i n  i n  C a r a z z i ' s  ha emat o xy l in fo r 2 minu t e s . 
1 2 4  
3 .  S t a i n  w i th 0 . 5 %  pholxine i n  0 . 5 %  c a l c ium ch l o r i de 
fo r 3 0  m inut e s . 
4 .  Rin s e  i n  wa t e r  and 7 0 %  a l c o ho l . 
5 .  Count e r s t a i n  in T a r t r a z ine in c e l l o s o lve fo r 
2 minut e s . 
6 .  R i n s e  i n  ab s o l u t e  a l c o ho l . 
7 .  Dehy dr a t e , c l e a r  and moun t . 
Re s u l t s : -
Ph l o x ine , a n  ac i d  dy e , s t a in s  b a s i c  
p ro t e ins p ink t o  r e d . 
( 2 1 )  F a s t Re d 2 B  
Method : -
! .  Dewax and b r i n g  t o  wat e r . 
2 .  S t a i n  i n  0 . 1 % F a s t  Red 2 B  i n  O . lM v e r o n a l  
ac e t a t e  buf fe r  a t  pH 9 . 2 f o r  2 minut e s . 
3 .  Wash i n  runn ing t ap wate r .  
4 .  Coun t e r s t a in i n  C a raz zi's h a emat oxy l in f o r  
2 m i nu t e s . 
5 .  Rins e in t ap w a t e r . 
6 .  Dehydr a t e , c l e a r  and mount . 
R e s u l t s : -
B i o g en i c  amine s s t a i n  dark o range to r e d . 
( 2 2 )  Me thyl G r e en - Pyro n i n  
Method : -
! .  Dewax and b r ing to wat e r . 
2 .  F lo o d  with s t a in ing s o l u t i on o f : 
5 %  aqueous pyron in Y 1 7 . 5  m l  
2 %  aque o u s  methy l  g r e e n  1 0  m l  
di s t i l l e d  wa t e r  2 5 0  m l  
a n d  l e ave fo r 1 5  minut e s . 
3 .  Rins e rap i d l y  i n  d i s t i l l e d wat er and b l o t  
dry w i t h  f i l t e r  p ap e r . 
4 .  F l o o d  w i th a c e t o n e , l e ave fo r a few s e conds . 
5 .  F l o o d  w i th equal p ar t s  o f  a c e t one and xy l ene . 
6 .  C l e a r  i n  f r e s h  xy l ene and mount in D . P . X . 
Re s u l t s : -
S t a i ns RNA b r i ght p i nk - r e d ,  p a r t i cu l a r l y  u s e fu l  
f o r  i dent i f i c a t i o n  o f  p l a sma c e l l s . 
( 2 3 ) F l u o r e s cent ant ibody me tho d 
Me tho d : -
! .  Dewax and b r ing t o  wat e r . 
2 .  F l ood w i th 2 %  b o v i ne s e rum a lbumen ( B SA) 
for 5 m i nut e s . 
3 .  T r e a t  w i th fluo r e s c in e  c onj u g a t e d  ant i b o dy 
( g o at ant i - human I gA) (Mi l e s - Yeda L t d . , 
I nd i an a ) , 
di l ut e d  1 / 1 0  w i t h pho spha t e  buffe r e d  s a l ine 
( P B S )_ p H  7 .  1 .  
P l ac e  s e ct i o ns i n  p e t r i - d i s h e s  with mo i s t  
f i l t e r  p ap e r  o n  b o ttom o f  d i s h . I ncubat e f o r  
1 hour . 
Cont r o l s e c t i ons are incub a t e d  w i th PBS only . 
4 .  Rin s e  in PBS . 
1 2 5  
5 .  Wash i n  g l as s  j ar w i th us e o f  a ma gne t i c  s t i r r e r , 
in 3 chan g e s  o f  PBS , 1 5  m i nu t e s  e ach wash . 
6 .  Rins e i n  d i s t i l l e d  wat e r . 
7 .  Mount in g lyc e r o l  j e l ly and examine w i th 
fluo r e s cent m i c ro s c op e . 
Re sul t s : -
A p o s i t i v e  r e a c t i on ,  for e x amp l e  p l asma c e l l s  
synthe s i s i ng I gA ,  i s  demon s t r a t e d  by app l e - g re en 
o r  y e l l ow f l uo r e s c en c e ; howeve r  i f  cont r o l  s e c t i on s  
show s im i l a r  f l uo re s cent c o l ou r s , th i s  indi c a t e s  
forma l i n - i nduc e d  a ut o fl uo r e s c ence p ro duc e d  b y  a 
b i o g e n i c  amine such as 5 - Hy d r o xy t ryp t amine . 
( 2 4 )  S i l v e r  i mpregn a t i on method 
Me thod : -
1 .  B r i ng s e c t i ons t o  w a t e r . 
2 .  S t ain in f r e shly p r e p a r e d  0 . 0 5 %  aqueous s i l v e r  
n i t ra t e  f o r  3 hours a t  6 0 ° C .  
3 .  W i thout r i n s ing r e du c e  in a f r e s hly p r ep a r e d  
s o l ut i on o f  1 %  hy droqu inone a n d  sulph i t e  for 
1 minut e a t  4 5 ° C .  
4 .  R ins e in d i s t i l l e d  wat e r .  
1 2 6 
5 .  F ix i n  5 %  s o d ium t h i o sulphat e for 2 1 / 2 m i nut e s . 
6 .  R i n s e  in d i s t i l l e d  wat e r .  
7 .  D e hy dra t e ,  c l e a r  and mo unt . 
Re s u l t s : -
Argy r op h i l i c s ub s t ance s s uch a s  ma s t  c e l l  g ranu l e s  
s t a i n  dark b rown t o  b l a c k .  
P r e p a ra t i o n  o f  s i l v e r  s o l u t i on : 
M i x  S O  mg s i lver n i t ra t e , 1 0  ml o f  pH 5 . 6 
ac e t i c  a c i d - s o dium a c e t at e _ buffer in 9 0  ml 
o f  d i s t i l l ed wate r .  
( 2 5 ) Mal l o ry ' s Pho sphot ungs t i c  Ac i d  H a ematoxyl i n  ( PTAH ) 
Me t h o d : -
1 .  Dewax and b r ing t o  wat e r . 
2 .  T r e at w i th Lugo r s  i o d i n e  and s od i um th i o s u l p h a t e, 
2 minu t e s  each . 
3 .  P l a c e  i n  0 . 2 5 %  aqu e ous p o t a s s ium p e rmanganat e 
fo r 5 m i nut e s . 
4 .  R i n s e  i n  d i s t i l l e d wat e r . 
5 .  P l a c e  i n  5 %  oxal i c  ac i d  fo r 1 0  minu t e s . 
6 .  Rin s e  in di s t i l l e d  wat e r .  
7 .  Wash i n  t ap wat e r  f o r  5 m i nut es , r i n s e  wi th 
di s t i l l e d wat e r . 
8 .  S t a i n  i n  PTAH 1 2 - 2 4  hour s . 
9 .  Dehydr a t e  rap i dly , c l e a r  and mount . 
Re s u l t s : -
E o s inop hi l i c sub s t an c e s  s t a in dark purp l e  o r  b l ac k , 
nuc l e i  and c r o s s  s t r i a t i ons o f  mus c l e  f i b r e s  s t a in 
b l ue , c o l l a g en and g r o un d  s ub s t ance o f  bone , 
ye l l ow to b r i ck red . 
P r ep a rat ion o f  PTAH s o l u t i on : 
H aema t e i n  
Phospho t ung s t i c  a c i d  
D i s t i l l e d wa t e r  
1 g 
2 0  g 
1 0 0 0  ml 
D i s s o l ve haema t e i n and p h o s pho tung s t i c  a c i d  s ep ­
a r a t e l y  in d i s t i l l e d  w a t e r , u s ing g ent l e  h e at . 
1 2 7  
Whe n  co o l ,  co mb ine s o l u t ions and make up t o  1 l i t r e . 
Rip e n  s o l u t i o n  imme d i a t e l y  by adding 0 . 1 7 7  g r ams 
o f  p o t a s s i um p e rmangana t e  o r  expo s e  to l i g h t  and 
warmth f o r  S - 6  weeks . 
B I BL I OGRAPHY 
Ar iya kul ka ln , P .  ( 1 9 8 1 )  A s t udy o f  morpho logi c a l  and 
phys i o l og i c a l  changes i n  t h e  mand ibular gl an d  o f  
the s h e ep a s s o c i a t e d  w i t h  e a t i ng and d i r e c t  s t im ­
u l a t i o n .  Ph . D . thes i s , Ma s s ey Un ive r s i ty ,  
P a lme r s t on No r th , New Z e a l and . 
1 2 8  
Ash , R . W . & Kay , R . N . B .  ( 1 9 5 9 )  S t imul a t ion and inhib i t i on 
o f  r e t i culum cont rac t i on s , ruminat ion and p a r o t i d  
s e c r e t ion f rom the fo r e s t omach o f  cons c i o u s  s h e e p . 
Journ a l  o f  Phys i o l o gy 1 4 9 , 4 3 - 5 7 . 
Az a r ,  H . A . ( 1 9 7 9 )  The hema top o i e t i c  sys t em .  I n : D i ag ­
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& J o ne s , R . T .  J o hn Wi l ey & Sons . 
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G l an d s . "  New Y o r k : Ho e b e r .  
B a i l ey ,  C . B .  ( 1 9 6 1 )  S a l iva s e c r e t ion and i t s  r e l a t i on t o  
f e e d i ng in c a t t l e . 4 .  The r e l a t io n s h ip b e t we e n  
t h e  concentr a t i ons o f  s o d i um ,  p o tas s ium , c h l o r i d e  
and i n o r g an i c  pho sphat e i n  m i x e d  sal iva a n d  rumen 
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i t s  r e l a t i o n  t o  f e e d ing in cat t l e . 2 .  The com­
p o s i t i on and r a t e  of s e c r e t ion of mixed s a l iva i n  
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!i, 3 8 3 - 4 0 2 . 
B arka , T .  ( 1 9 8 0 )  B i o l o g i c al l y  act ive po lyp ep t i d e s  in s ub ­
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c t i o n . I n : Buf f e r s  i n  Ruminant Phys i o l ogy and 
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1 2 9  
Chur ch Dwi g ht Co . I n c . , New Yo rk . 
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r e spons e .  I n : I mmun o l o gy o f  the G a s t ro i nt e s t i n a l  
Tra c t . e d . Asqui th , P .  Chu rchi l l , London . 
B i en e n s tock , J .  & Be fus , A . D .  ( 1 9 8 0 ) . Muco s a l  immuno l o gy .  
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Muco s a l  I mmun i ty . I n : The Muco s al I mmun e Sys t em .  
Curr ent Top i c s  in V e t e r i n a ry Me d i c i ne and An imal 
$c i ence V o l . 1 2 . Mart inus N i j ho f f (pub l i s he r s ) 
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New Z e a l and . 
B l a i r - Wes t ,  J . R . , Coghl an , J . P . , Denton , D . A . , Ne l s on , J . , · 
Wr i gh t , R . D .  & Yamauchi , A .  ( 1 9 6 9 )  , I on i c  h i s t o ­
l o g i c a l  and vas c u l ar fa c t o r s  in the r e a c t i o n  o f  the 
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1 � 7 . P l enum P r e s s , New Y o rk . 
1 3 0  
C ant i n , M .  & V e i l l eux , R .  ( 1 9 7 2 )  G l obul e l euko cy t e s  and 
mas t  c e l l s of the urinary t ra c t  in magne s i um d e f i c ­
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No . 5 ,  G 3 2 1 - G 3 2 9 .  
Chu r c h , D . C .  ( 1 9 7 6 )  
o f  Rumi nant s " . 
" D ige s t i ve P hys i o l o gy and Nu t r i t i on 
Vo l .  1 ,  2nd Ed . Church (pub l i s he r s ) 
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Th e Muc o s a l  I mmune Sys t em .  Cur r ent top i c s  i n  
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C a t t l e . A Rev i ew .  Journa l o f  D a i ry S c i ence � '  
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Journa l o f  Phy s i o l ogy 1 3 1 , 1 3 - 3 1 . 
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A m i c r opunc ture inve s t i g a t i on o f  e l e c t ro l y t e  t r an s ­
p o r t  i n  the p a r o t i d  g l an d s  o f  s o d ium - rep l e t e  and 
• 
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1 3 2  
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