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f� STUDY OF THE CVi.RIA.t"1 IUSPONSE OF NEV/ ZCA.Lh.ND ROMNEY E-dES 
SEQUENTII�LLY S UPEROVUL!'. TED VI  TH PREGNi..NT 
fii;�RE 18 SERDri  GONADOTROPHIN 
A thesis presented in partial fulfilment of the requirements for 
the degree of Master of ;�gricul tural Science 
in J:'.nimal Science 
at 
j·,;assey University 
If.,IN Ji.JviES CLP.RKE 
1973 
ABSTP..ACT 
A series of experiments investigated the nature and causes of 
ovarian refractoriness in ewes sequentially treated with Pregnant 
Mare's Serum Gonadotrophin (P.I'vl.S.G. ) . 70 ewes were subjected to 
the following treatments over 3 oestrous cycles of the 1972 
breeding season:- 1. Injected with P.M.S. G. at each of 3 cycles 
(cycles 112 and 3); 
2. 1\vo injections of P.M.S.G. (at cycles 1 and 3) 
separated by a normal oestrous cycle; 
3. Injected with P.M.S.G. at two successivG 
cycles (cycles 2 and 3); 
4. Injected at 1 cycle only (cycle 3). 
These treatments were replicated at 1000 i.u. and 1500 i.u. P.r�i.S.G. 
and 9 ewes acted as an uninjected control group. The ev!es were 
blood sampled and slaughtered at the end of these treatments and 
ovulation data were obtained by recovery of the reproductive 
tracts. The terminal ovulation rates showed that ewes were 
refractory to a second injection of P.M.S.G. and this condition 
persisted. The refractoriness was to some extent alleviated by the 
spacing of injections (Treatment 2 above). 
Biological Inhibition Tests (using mice) analysed the plasma 
of the above ewes for evidence of anti-gonadotrophins. Although 
such factors were not detected in the blood of these ewes, the 
test did reveal antibody production against P.M.S.G. in the plasma 
of a further group of ewes which hcd been chronically treated with 
the hormone for 6 weeks. It was concluded that ovarian refractori­
ness, which is rapidly attained in sequentially trented ewes, is 
iii 
not duo to the development of serological antibodies against the 
exogenous gonadotrophin, 
Another experiment 1 carried out early in the 1973 breeding 
season, investigated ovaric-,n follicle development in 30 ewes which 
V/Gre soquentially  treated with P.M.S.G, for up to 3 oestrous cycles. 
Ewes were laparotomiscd or kil led on Day 10 of the oestrous cycles 
fol lowing treatment and measurements on follicle development wore 
tr:,ken. !. group of control ev1es were observed nt a similar timo to 
the treated ewes. 
Counts on ovarian surface fol licles differed little between 
treated and control ev1cs, at each of  the observations. However, the 
ovaries of slaughtered ewes were sectioned to al low estimation of  
total ovarian fol liculnr populntions and to  make some assessment of  
foll icular atresia. Zwcs slaughtered after 1 injection of P,fi1.S. G, 
had lower numbers of normal nntral fol licles per ovary then did 
control ewes or ewes observed at similar times after 2 or 3 
injections. 
It wes suggested that exhaustion of ovarian follicular 
populations may precipitate a refractory condition but that this 
condition persists  because of an endogenous hormonal imbalance. 
�urther work should  be done to investigate this latter possibility. 
A CKNOViLEDGEME N 'rS 
The author is specially indebted to his supervisor Dr. M.F. 
McDonald for his invaluable guidance and assistance in experinental 
work and advice during the preparation of this manuscript. 
Special thanks are due to Professor R.E� Munford for his 
advice end aid in statistical analysis and computer operation; 
to Professors A.L. Rae and D.S. Flux for their helpful discussion 
and advice; to Mr. �. Feirhall for the care of the animals and to 
�·Jr. c. Muir for technical assistance. 
Ackno\'lledgewent is made to Mr. P.H. \'\hitehond and farm staff; 
�.;r. l\"1, Birtles and Dr. H .  Batten for assistance in histological 
preparations; staff of the Small h.nimal Production Unit; staff of 
the fiiasscy University Library; staff of the Longburn Freezing ":Iorks; 
r'irs. P. Collins for the typing of this manuscript; and all those who 
assisted in mouse dissection. 
Financial assistance from the New Zealand Wool Board, The George 
hlley Trust and The Robcrt Gibson Trust, is gratefully acknowledged. 
PHE F /  .. CE 
This investigation was conducted at the Sheep Production 
Centre, part of the :Uepe.rtment of Sheep Husbandry, Masscy University. 
The experimental work was carried out between i.pril of 1972 and May 
of 1973 and represents original research by the author under 
supervision of :Ur. M. F. McDonald, Reader, Sheep Husbandry Department, 
Massey University. 
Chap te r 
I 
T A B L E 0 F CONTENTS 
ABST�11' .. CT ii 
t.CICNOHL�DGEMENTS iv 
PREFi' .. CE V 
LIST CF T/.BLES 
LIST OF FIGtmES 
INTRODUCTION 1 
R:;;vw�·: CF LITZRI'.TURE 3 
Pregnant Mare's S erw:: Gonadotrophin <1 
Factors affecting response to P . M.S .G. 6 
1 .  Breed 6 
2. Livcweight nnu Nutrition 7 
3. S eason of Ldministration 8 
4.  l.ge of Ewe 9 
5. Day of Injection 9 
6. Nature of P.�. S . G. 9 
Mode of t.ction of P.fvi.S.G. 10 
Chemical Char�cteristics of P.M.S .G. 1 3  
Im unology o f  P.l\l.S. G. 13  
E¥Je Ovarian Veig.'lt Response of P.M.S.G. 16 
Assay of Gonadotrophins and r�nti-gonadot rophins 16 
aefractoriness o f  the Ovary wi th S equential 
P.M.S .G. Treatment 18 
Follicular Growth and Atresia in t h e  Ewe Ovary 23 
Purpose and scope o f  t h e  Investigation 26 
Chapter 
I I  
Ill 
27 
Sheep and their Management 27 
Mice and their Husbandry 29 
Experimental Design 29 
Experimental Proc�lure 30 
Experiment 1 30 
Zxperiment 2 33 
Experiment 3 35 
!;.nalysis of Dato. 38 
SYNCHRCNISf.TION i�ND OESTrtOUS :f:HENOMENA 40 
Factors affecting the Onset of Oestrus 40 
1. P.M.S.G. �dministration 40 
2. Injection of Progesterone 40 
Effect of Sequential P.M.S.G. Treatment on 
synchronisation of oestrous cycles subsequent 
to Progestagen treatment 41  
1 .  Variation in the Time of Onset of  Oestrus 4 1  
2. Manifest�tion of oestrus 42 
CH/.PT�R SUNiM!li.W 42 
IV OV&RI�N RESPONSE TC P.M.S.G. IN SEQUENTI�LLY 
TP.J!:1'. TED EYIES 43 
Transformation of Discrete Data 43 
Ewes Ovulating v 44 
Incidence of Cystic Ovaries 44 
Ovulation :late 44 
1. Corpora Lutea hll Ewes (Expt. 1)  44 
Chupte!: 
V 
2 .  Corpora Lutea ;�wes Ovuluting (Expt, 1) 45 
3. Corpora Lutea. • kll Ewes (Expt. 3) 45 
Corpora Lutea plus Follicles 46 
1. Corpora Lutor. plus Follicles greater than. 
3 mm, : /:...11 Ewes (Expt. 1 )  4G 
2.  Corpora Lutea plus Follicles greater than 
3 mm. : Ewes Ovulating (Expt. 1) 46 
3. CorporQ Lutea plus Follicles greater thon 
1 mm. : 1.11 J:wos (Expt. 3) 46 
Ovarian \'/eight 47 
1. Ovarian i'leight All Ewes (Expt. 1) 47 
2. Ovo.rio.n '.'.'oigl1.t Bwes Ovulating (�x�t. 1) 47 
3. DopenGonco o f  ova rian weight on ovaria n 
activity 47 
Difference in Ovulation riute betwe�• the Risht 
and Left Ovaries 
Ovulation �ate of Cont rol �wes (Expt. 3) 
Effect of Seas on 
CHAPTE� SUNJMi.;.!1Y 
DETECT! ON OF i:.NTI -GONf .OOTR.OPHI NS 
48 
49 
49 
50 
Immature l'Ylouse Uterine Weight Response t o  P.M.S.G. 50 
Test f or fo.nti�gonuclotr ophins in Plasma of Gwes 
of Experiment 1 � 
Test for Anti-gonndotrophins in Plasma of 6wes 
Ch r onically T reated >lith P�M,S!G, 51 
CJlAPTEf:t SUW".RY 52 
Chapter 
VI 0\'i�hii..N FOLLICUL'..R DEVELOPMENT OF Er!ES 
SEQUENTI!l.LLY �-t3i:..TI:l2.J!!.T".ti P.M.S.G. 
Surface Observations 
Total Ovc.rian i: .. ctivity 
Relationships bctwe0n Total Ovsrian Datn and 
Surface Dnta 
CHLPTEi-1 SUM.M/.;W 
53 
53 
55 
56 
57 
VII DISCUSSION 58 
Synchronisation and Oestrous Fh�nomena 
Effect of Sequential P.M.S.G. Treatment on 
Synchronisation 
D�welopmont of Ovnrinn P.ofractoriness in Ewce 
58 
61 
Sequentially Treated �ith P.M.S.G. 64 
1. Ovarian rtesponse and the Development of 
::-tofractoriness to P.M. S. G. 64 
2. Development of hnti-gonadotrophins 73 
3. Follicul�r Dynamics of the Ovary 78 
4. General Discussion on Uefractoriness to 
P.M.S.G. 
:REFERENCES 
h.PP:::i:NDICES 
84 
80 
Table 
1-1 
3-1 
3-3 
3-4 
3-5 
3-6 
3-7 
3-8 
L I S T 0 F Ti� BLES 
Literature reporting refractory conditions of the 
ova:;:-y after :;:;reGJlnnt IRare' s Serum Gonadotrophin 
treatment 
Effect of :i?.r!l.S.G. adr.iinistration on the time 
interval between progestogen sponge withdrawal and 
the onset of oestrus 
Effect of a progesterone injection on the time 
interval between progestaGen sponge withdrawal 
and the onset of oestrus 
Effect of �.M.S.G. administration on the occurrence 
of oestrus following progestagen treatment 
Effect of a progesterone injection on the 
occurrence of oestrus following progest�gen 
sponge treatment 
Treatment effects on the synchronisation of oestrus 
one oestrous cycle from progestagen treatment:­
Zxperiment 1 
Treatment effects on the synchronisation of oestrus 
two oestrous cycles from progostagcn treatment:­
Experiment 1 
Treatment effects on the manifestation of oostrus:­
Experiment 1 
Effect of sequential �.M.S.G. treatment on the 
manifestation of oestrus 
4-1 Effect of sequential P.M.s.G. treatment on the 
number of ewes ovulating 
4-2 
4-3 
4-4 
4-5 
4-6 
Effect of sequential P.M.S.G. treatment on the 
incidence of cystic ovaries 
Ovarian response all ewes 
Ovarian response ewes ovulating 
Comparison of means : ovariun response of all ewes 
(Experiment 1) 
Comparison of means : ovarian response of ewes 
ovulating (Experiment 1) 
19 
f 40 
f 40 
f 40 
f 40 
f 41 
f 41 
f 42 
f 42 
f 43 
f 44 
f 45 
f 45 
f 45 
f 45 
Table 
4-7 
4-8 
4-9 
Effect of sequential P,M.S.G. treatr.1ent compared 
with untreated eV!os : Corpora Lutea (transformed 
data). 
Effect of sequential F. M, S. G, treatment compared 
with untreated ewes : Corpora Lutea plus follicles 
greater than 1 mm, (transformed data) 
Relationship between ov2rinn weight and ovarian 
activity : regression analysis 
4- 10 Ovulation rct� of untreated owes Effect o f  season 
5-1 
6-1 
6-2 
6-3 
6-4 
6-5 
6-6 
6-7 
6-8 
Comparison of moans after analysis of covariance : 
mean uterine weights of mice given F,M.S.G. alone 
or with plasma of sheep treated with P.M.S.G. 
Ovarian surface activity observation one 
Ovarian surface o.ctivity observation two 
Ovarian surfc.ce activity observation threG 
Ovarian surface activity observation at slaughter 
Relationships between ovulation rate and ovarian 
activity at Day 10 of the previous oestrous cycle 
control ewes 
Relationships between ovulation rate and ovarian 
activity et Day 10 of thG previous oestrous 
cycle : treated ewes 
Estimates of total ovarian follicle population : 
numbers of antral follicles per ovary (transformed 
data) 
Estimation of the proportion of normal antral 
follicles per total antral follicles 
6-9 Estimation of the total normal antral follicle 
population : number of antral follicles per ovary 
(transformed data) 
f refers to following page 
f 45 
f 45 
f 48 
f 48 
f 51  
f 53 
f 53 
f 53 
f 54 
f 54 
f 54 
f 55 
f 55 
f 55 
1-1 
2- 1 
2-2 
L I S T 0 F F I G U R E S 
Dose response relationships for owes super­
ovulated with .?resnnnt I�bre' s Scrum Gonadotrophin 
Ewe livoweights at the beginning and end of 
Experiment one 
Zwe liveweight changes during Experiment three 
2-3 Experimental design : �xperiment one and 
Experiment two 
Design of Experiment two 
2-5 Calendar of Events for Experiment ono 
2-6 Design of ��xporimcnt thr.:::o 
2-7 In vivo measurement o:f ovarian follicles with 
��-
sterile slide c�llipers 
2-8 2 mm. ovarian slices semi-embedded in 70'/o alcohol 
3-1 Distribution of onset of oestrus following sponge 
withdrawal 
3-2 The effect of a progesterone injection on tlw onset 
of oestrus of owes given c,n intravaginal 
f 4 
f 27 
f 27 
f 30 
f 30 
f 32 
f 35 
't 36 
f 36 
f 40 
progestogen sponge treo.tmont f 40  
3-3 Effect of repeated gonadotrophic stimulation on 
the synchronis ation of oestrous cycles 
4-1 Effect of sequential F.M.S.G. treatment on the 
number of ewes ovulating 
4-2 
4-3 
4-4 
4-5 
Cystic condition of the ovary 
Effect of sequential I'.M.S.G. treatment on ewe 
ovulation rate : rnw data 
Effect of sequential i"'.M.S .G. treatment on ewe 
ovulation rate : transformed data 
Superovulated ov�ries taken from a ewe given one 
injection of P.M.s.G. (Trt.H41 Expt. 1) 
36 ovulations 
f 41 
f 43 
f 44 
f 45 
f 45  
f 45 
Figure 
4-6 
4-7 
4-8 
4-9 
Inactive ovaries of a evo in Treatment Hl (Expt.  1 )  
Ovaries observed a t  Day 10 of the oestrous cycle 
Ovarian slices showing luteinisation of follicles 
Effect of sequential ?.l"l.S,G. treatment on 
ovulation rate of ewes ovulo.ting : raw data 
4-10 Effect of sequential P.i'il.S.G. treatmont on 
f 45 
f 45 
f 45 
.'1 46 
ovulation rate of ewes o�tlnting : transformed data f 46 
4-11 Effect of sequential �.M.S.G. treatment on the number 
of corpora lu tea pluG follicles great0r than 3 nm. 
in all ewes : rou dato f 46 
4-12 �ffect of sequontiB.l i'.l':i.S.G. treatment on the 
number of corpora lu tea plus follicles greater 
than 3 mm. in all eYJes : trnnsformed data f 46 
4-13 Effect of sequent ial :?. Tvi. s. G. treatment on the 
number of corpora lutea plus follicles greater 
than 3 mm. in ewes ovulating : raw data f 46 
4-14 Effect of sequential P.�:l.S. G. treatment on the 
number of corpora lutea plus follicles grea ter 
4-15  
4-16  
than 3 rrun. in owes ovulating : transformed data f 46 
Effoct of sequential P.fvl.S.G. treatmen t on ovnrian 
-.;;eight : all e•.':es f 46 
Effect of sequential P.iVl.S.G. treatment on ovarian 
weight : owes ovulating f 46 
4-17 Comparison of e-.:10s soquontially tre:ated with �'.M. S. G. 
and c ontrol ev.'os : ovulation rate f 46 
4-10 Comparison of ewes sequontinlly treated with �.M.S.G. 
and control ewes : corpora lutea plus follicles 
greater tha n 1 mm.  f 46 
5-1 Immature mouse uterine weight response to  P.M.S.G. f 50 
5-2 Straigh t line and curvilinear equations for 
immature mouse uterine �eight response t o  P.M.S.G. f 50 
5-3 l'Yiean uterine weights of mice treated with P.M. s. G. 
and sheep plasma : raw data f 51 
Fig.!:!Fe 
5-4 
6- 1 
6-2 
6-3 
6-4 
6-5 
G-6 
6-7 
Menn uterine weights of mice treated with P.M.S. G. 
and sheep plasma : transformed data 
Observations on follicle numbers in ewes treated 
up to three times with P.M.S.G. 
Normal follicle sectioned through the ovum 
Cross section of u normal tertiary follicle 
Cross section through the luteinised wall of n 
cystic follicle 
Sarly atresin of a tertiary follicle 
�trosia of a tertiary follicle 
Late atresia of a tertiary follicle 
f refers to following pnge 
f 5 1  
f 53 
f 56 
f 56 
f 56 
f 56 
f 56 
f 56 
I N T R 0 D U C T I 0 N 
I N T R 0 D U C T I Q N 
11Tho t t · ou s and1ng deficiency of the Now Zealand 
nheep is low reproductive rate and yet it is 
in this aspect that a groat potential exists." 
(Coop, 1972 ) 
Pregnant Mare's Serum (P.M.S,) as a source of gonadotrophin is 
of potential value for increasing the reproductive rate of sheep. 
Superovulation with this hormone preparation makes rapid proliferation 
ol a small number of animals possible. The overall efficiency of this 
treatment may be facilitated by ovma transfer. 
Studies in New Zealand have shmm Pregnant Mare's Serum 
Gonadotrophin (P.M.S.G.) capable of increasing litter size of the 
Romney owe (Vfallace, 1954; Larsen, 1971) .  However, the largo litters 
produced after gonadotrophic stimulation are most susceptible to pro-
natal loss than those of normal size (Cumming, 1965)  and the ability 
of P.M.S.G. to effectively increase fecundity in the ewe is thus 
limited. To realise the full reproductive potential of the ewe, 
fertile ova from superovulated animals may be transferred to a number 
of recipient ewes for development. This would avoid the problem of 
uterine overcrowding. 
;;a th the introduction of "exotic" breeds of sheep into New 
Zealand, rapid multiplication of the small number imported would 
aid efficient dispersal of their influence throughout the country 
and hence, offer desirable characteristics to the national flock. 
2 
To obtain large numbers of offspring from the few parent individuals, 
" . " the exot1c e\"'IGs could be superovulated and mated to rams of the 
same breed. As this system would predispose to loss of whole litters 
in the pro-natal stages, it may be more desirahlo to transfer fertile 
ova frori1 the superovulated 
.,, cxotics" to ewes of o more cor.uuon bread. 
This type of operation has been suggested by Clarkc ( 1973 ) .  
If donor ewes wore able to be superovul�ted et successive 
oestrous cycles, the number of ova �vailr-ble for transfer could be 
incro::sed thus onhnncing the efficiency of multiplication procedures. 
l .. lthough so(luential superovulntions would be dosirable, such treat-
ments nro not feasible. �ith increasin� numbers of P.M. S . O • . treatlilents, 
owes bccolile progressively loss responsive to the injected horlilone. 
Tho r.oduced ovulation rutec obtained Yli th ::;equontinl gonadotrophic 
stimulations is referred to as "rofractorincss" in response to the 
exogenous hornone. The development of a refractory condition of the 
ovo.ry with administration of a protein horri1one (e. g. P.M.S.G.), may 
be duo to the production of sorologicnl [mtibodios ugc.inst tho hormone 
molecules. However, other explanations include loss of sensiti?ity 
oi' tho ovc.ries, oxho.ustion of nature follicles, or nn endogenous 
�ori!!oncl imb2..l::-.nce being augmented by largz n.-:Jounts of exogenous 
GOnc-.dC't rophin. 
If the factors rasponsible for refrnctoriness to P.M.S.G. were 
overcome, th0n sequentinl treatr.1ent of ewos could bocome feasiblu 
o..nd the yield of ova over a short period of· time would be potentic.lly 
l�rger. TI1is project was designed to elucidate causes of refractori-
ness in ewes sequontinl ly treated with P.M.S.G. nnd methods of ovor-
coming the condition were sought. 
C H A P T E R I 
R E V I � W 0 F L I T E 3 A T U R E 
CHAPTER I 
REVIEW OF LIT:3RATURE 
The indication, by Cole and ·i�•iillcr (1933), tho.t P.M.S • .  
augmented the rnt�nifcstation of oestruo in anoestrous ewes, created 
c. widGspread interest in the hormone preparation cs a source of 
gonadotrophin. Since this observation, P.M.S.G. has been utilised 
both for the induction of oestrus and ovulation in anoestrous 
animals end the superovulation of cyclic animals • . Extensive use of 
P.M.S. o.s a laboratory source of Follicle Stimulating Hormone (F.S.H.)­
likc activity ho.s o.lso contributed to knowledge on the preparation's 
physiological and biochemical properties. LiteraturG related to the 
use of P.M.S.G. in domestic o.nimcls is reviewed below. The present 
state of knowledge on the gonadotrophin's mode of action is o.lso 
considered. 
By virtue of their protein moeity, gonadotrophins become 
effective antigens when injected into heterologous species. P.Ivi.S.G. 
conforms to this pattern end the review presented in this chapter 
embodies a consideration of antibody formation o.gainst this molecule. 
h resum� of information available on Biological Inhibition Tests, 
used to detect anti-gonadotrophins, ia also pertinent to this study. 
Finally, the incomplete knowledge of sheep ovurian follicular 
dynamics will be reviewed. 
4 
Pregnant� Serum�Gonadotrophin 
The capacity of P.M.s.G. to exhibit F.S.H.-like activity in 
sheep has been reported in many studies. Efforts have been diroctod 
towards its use in:-
- Inducing out of season breeding, being used alone (Cole � �· 1 
1945; Robinson, 1950) or in conjoction with progostngens (Dutt, 1953 ; 
�obinson, 1954; McDonald, 1961; Gordon, 1958b; 1963b; 1972; Roborts 
and �dgar, 1966): 
- Augm�:::nting fecundity during the breeding season (Robinson, 
1951; :Jallace, 1954; Gordon, 1£l58a; 1963a; Gumming and McDonald, 19G7; 
Ne>rton � �·, 1971; Tempest and Bonz, 1970; Bindon _s! E:.!_. 1 1971; 
Hunt e!, p..l. 1 1971): 
Endocrinological studios, utilisin5 its F.S.H.-like properties 
(Short .£! !'8-.· 1 1963; McCrackon 2,! �· 1 1969; Hunt � al. 1 1971): 
-Production of ova for transplantation to foster 'recipients' 
followine; fertilization in the 'donor' (A.vcrill and Rowson, 1959: 
La:::-son, 1971) and for studies on the in vitro fertilization and 
culture of ova (Moor and Cragle, 1971). 
Notwithstanding the vo.lue of P.IVi.S. as a source of experimental 
gonndotrophin, the hormone also has practical value ns a supcrovu!L'.tory 
hormone (Gordon1 1963Q; 1972; Findlay and Vaughn, 1964; McDonald, 
1966; �obinson1 1967c). Gordon (1969) has pointed out the practical 
difficulties in the use of the hormone at the ferm level. 
Superovulatory responses to P.M.S.G. in sheep hnvc been recorded 
by a number of authors. Results of some of these studies are shown 
in Fig. 1-1 which plots ovulation response (number of Corpora Lutea) 
with increasing doses of P.M.S.G. 

<! w 1-::::> 
...J 
<! a: 0 a.. a: 0 
u 
u.. 0 
a: w al 
� ::::> z 
14 
13 
12  
11 
10 
9 
8 
7 
6 
5 
4 
FIGURE 1-1:-
DOSE RESPONSE RELATIONSHIPS FOR EWES SUPEROVULATED 
WITH PREGNANT MARE'S SERUM GONADOTROPHIN 
a 
_...oa 
-
..-
......--
....ob 
--
2 -
.-· 
--. --· ---
_.-·...--- . - - - � '1- - - - � -
- -
. - . -  
P.M.S.G. {L"-1 
I I I I I I I I 
200 400 600 800 1 000 1200 1400 1600 
5. 
Although a definite trend is seen with increasing dose rates 
of P.M.S.G., most workers (Robinson, 1951; Wallaca, 1954; Averill;. 
1953; Larsen, 1971) report marked variation of ovarian response in 
indivicual animals. This effect is greater at higher doses. 
Response becomes progressively less �ith increasing doses (Averill1 
1958) nnd falls off above some critical value (Robinson, 1951). The 
incidence of cystic and luteinised follicles increases with dose 
level (Robinson, 1951 and Larsen, 1971). 
Hammond .£.! !:!· ( 1942) and Moore and Shel ton ( 1964) obtained a 
clear relationship between dose level and number of antral follicles 
when injecting sheep with Horse Anterior Pituitary extracts (H.A.P. ). 
These �orkers could not achieve linearity of response with P.M.S.G. 
Yiallace (1954)1Holst (1969) and Bindon £!_ !:.!.· (1971) reported a 
relationship between P.M.S.G . dose level and the number of corpora 
lutea in sheep ovaries (see Fig. 1-1). Because of the large variance 
amongst animals on each dose level, Robinson (1951) was able to plot 
a linear curve within the limits of the standard deviations of the 
mean responses to increasing dose. Lamond (1964b) derived a linear 
increase in log-ovulation rate with log-dose of P.M.S.G.  The inclusion 
of follicle counts with numbers of corpora lutea as a measure of 
ovarian response, produced larger standard errors associated with the 
mean response than when ovulation rate was considered alone (Lnrsen, 
1971), Holst (1969) has shown significant dose effects of P.M.S.G . 
in inducing increases in the number of 'persistent' follicles and the 
number of corpora lutea plu.s 'persistent' follicles. 
6 
To counteract differences in response recorded by individual 
workers in different environments, Averill (1958) suggested the use 
of regression analysis, when drav.ring comparisons. The slope of the 
response lino would then offer a com<:�on denominator for all trials. 
Hecc:mtly, Hunt �!:l.· (1971) have noted a better ovarian 
response (number of corpora lutea) when sheep F.S.H. was used in 
preference to P.b.S.G. for superovulating sheep. 
1. Breed 
Factors _l'>.ffecting Response to 
Preg::1ant f{are' s Serum G�adotrophin 
Some of the discrepancies between results obtained by different 
authors (sec Fig. 1-1) in owe response to P.M.S.G. may be resolved 
when breed differences, or differences in inherent fecundity, are 
taken li<to nccount. Robinson (1951) used broods of a higher natural 
fecundity (natural ovulation rnte of 1.4-1.9) than did 'ilallace ( 1954) 
(1.13-1.17) and produced a steeper curve than the latter. 
McDonald and Ch'ang (1966) found differences in ovulation rates 
betv1een normal owes of the same strain when they were exposed to 
different environments. Using P.h.S.G., Braden .s,! !:.!· (1960) showed 
diffexential responses between ewes of the same breed which were 
obtained from different sources. '.lallnce ( 1954) has described a 
simile.r effect when treating Romnoy ev:es of different strains. 
The finding that breeds of an inherently higher fecundity will 
respond better to superovulation is borne out by the work of Bradford 
� 21· (1971) yet Bindon £! �· (1970) have found that ewes of a 
higher natural ovulation rate may need higher doses of P.M.S.G. to 
7 
effect maximal superovulation, than ewes of comparatively lower 
fecundity • .  
2. Liveweight end Nutrition 
Hnmmond (1952) postulGteG that high plane feeding of ewes 
produced more follicles with an antrum than did low plo.ne feeding. 
The influence of livewei�lt on ovarian activity has subsequently 
been verified (Coop, 1966; Allison, 1968). However, 'iiellace (1954) 
found increased nutrition, resulting in elevated liveweight 
(flushing), to hcve little effect on ewe ovulatory response to 
P.M.S.G. The increase in ovulation rate,. effected by the: hormone, 
was notoo on all planes of nutrition but was larger in ewes on 
a sub-maintenance diet than for flushed ewes (Allen and Lamcing, 
1961). In agreement with these latter authors, Lomond (1963) noted 
that administration of P. M. G. G. may compensate for nutrient 
deficiencies on low planes of nutrition, Lnmond also presented 
tentative evidence that, for ewes suffering from undernutrition, 
responses to gonadotrophin may be more closely aligned to bodyweight. 
Recent work (Newton £1 �· 1 1970; Bradford � �· , 1971) 
indicates little evidence of a relationship between liveweight of 
the ewe and the ovulatory response to P.M.S.G. Tait (1971) showed 
no evidence of an interaction between response to P.M.S.G. and 
nutritional levels when consic1ering number of lambs born per ewe. 
!l.llison ( 1973) and Eastvmod ( 1973) reported that ewes fed on a high 
plane of nutrition (higher liveweight) showed a better ovarian 
response to P.M.S.G. than those on a low plane of nutrition (lower 
liveweight) but the differences were statistically not significant 
in both casca. 
8 
The fasting of heifers after a low plane of nutrition will 
cnuse n decrease in ovulation response to P.M.S.G. but will not do 
so after n high plane of nutrition. Fasting also lends to a greater 
vorietion in the heifer ' s response to  the hormone (Lamond1 1970). 
Breed and inherent focundity thus seem more likely to  cause 
differences in response to P .M.S . G. than differences in liveweight . 
3. Sanson of hdministration 
3onsonol differences in the inciconce of multiple ovulat ions in 
owes have been notee by Radford (Hi59). l'ilcDonald nnd Ch'ang (1966) 
reported a similar variation during the breeding season of Romney 
ewes vhich showec negligible changes in liveweight over tho period of 
obscrvc.t ion. Those latter authors noted the natural ovulation rate  
of cvms t o  rise between the first ::�nd third oestrous cycles of the 
brao0ing season. 
3rsden et nl. (1960) dotectec bet ter superovulntory responses to  
P.�.'I.S.G. when trcntint;; ewes c�1rly in the bret."ding senoon in preforence 
to trcntment late in cnoestrous. Ho�cver ,  with non-sup0rovulatory 
dose:J, P.M. S . G. may induce similar ovcricn responses in anoestrous 
end cyclic e\"'cs ( Laraond, 1962) . './ith Suffolk ewes , A.verill (1958) 
noted a slightly (non-significant ) greater ovulatory response to  
P .h .S . G. t reatment in the breeding season 1 corapared with progesterone­
F-.M.S. G. treatment in the non-breeding season. A smell difference 
between ewes superovulcted in different b reeding seasons was also 
evident end may have been due to the time of the season in which 
the t reatment was imposed. 
Variations within a season in ewe sensitivity to  oestrogen 
have been recorded by  Raesido and McDonald ( 1959) 1 Renrdon nnd 
Robinson (1961) end Gibson and Robinson (1971).' Liveweight and 
9 
nutritional status affect this response �!though light and 
temperature play o dominant port (Gibson and Robinson , 197 1 ) .  
I':.obinson ( 1951) ,  i>.vcrill  ( 1958) c.nd Gordon ( 1963n) have all  
reported that there is no  differential response in ewes of 
different ages when the anim�ls  ere sup�rovuluted with P.M.S.G. 
5 .  £ay of Injection 
Ldministrntion of P.M.S.G. between days 12 and 15  of the 
oestrous cycle will  usunlly effect similar ovulation rntes in 
e\7es (Robinson, 1951; 1'iallnce, 1954; Bindon � !!!.·, 1960; 
Cwmaing, 1965; Bindon � �·, 197 1; Larson, 1971 ) .  
There is  an interaction between F.fu.S.G. nnd progestagens which 
affects the ovnrinn response \?hen the two hormones are used 
concurrently (Lamond , 1964a;b ) .  Type of progestogen pretreatment 
muy influence t ime after treetment at which P.M.S.G. should be 
injected, for optimal ovulation results  from the gonadotrophin, 
( Lomond 1964b; Roberts and Edgar , 1966 ) .  Hulet and Foote ( 1967 and 
1969 ) noted that P.�.s.G. treatment on the day of termination of 
progestogen synchronisation, yielded lower ovulation rates than if 
the same ewes were injected again during the fol lowing oestrous 
cycle. 
6. Nature of P.M.S.G. 
Whole serum appears to offer a better response in ewes (Gordon, 
1958a) and in cows (Rowson, 1951 and Brock and Rowson, 1952) than 
freeze-dried (whole serum) powders. The latter are in turn better 
than purified powders. This fact may account for some of the 
10 
diff0renccs in response that are sc8n in Fig.  1- 1 .  For example , 
�obinson ( 195 1 )  usud whole scrum anc.� obtained considerably larger 
responses than 'Jnl lc.cc ( 1954 ) who used a dried preparation . 
t!iodc of action of 
Frcl}nan t Marc ' s 3 e rum "g_onadot rophin 
Lamond ( 1964n)  stated that : 
" 
Knowledge nbout the way whereby gonadotrophins 
r :  
act on the ovary is meagre • 
c:':ormver , with recent cdvcmccs in assay t echniques it i s  now 
easier to monitor hormonal fluctuations � �  ( see Pant et a�. ,  
1972 ) .  S tudy of the cndocrinology of reproduction is also nided by 
the use cf ovnrinn transplnnts (McCracken _£! £.!.· 1 1969 ) which 
Gno.bL::  blood f lowinf, to  cnc1 from tho gl2.IlC: to be r.10ni tored in the 
conscious o.nimal . 
There are suggestion::; thr.: t t�w ef:ccct of  P . L S . G . is  mediated 
indirect ly , through the pi tui to.ry .  For example 1 H.cmnels e.nd 
C 'S teen ( 1967 ) exhibited a. significant c1ecrea.se in pituitary 
goncdotrophin ( L . H .  and F . S . H . ) of ro.ts t ro2.tGd with this hormone. 
The direct response of the ovary to P . i': . S . G . is displo.yed by the 
administration of the hormone to hypophysectomised mice and rats  
(Lnmond and �r.uncns 1959 ; ?lux o.nd Li , 1965 ) ._ Bowcvcr , ovulation 
wil l  not occur in the cbsenco of the hypothalamus (Quinn and 
Zarrow , 1964 ; 1965 ) ,  suggesting that neuro-endocrine pathways arc 
able to mediate and enhance ovarian response to the exogenous 
gonadotrophin. S t imulated fol licles will become luteinised 
(C0le , 1969 ) , but it i s  thought that F . M. S . G. must  elicit a 
11 
release of pituitary L .H .  before ovulntion may occur (Quinn and 
Zarrm-1 , 1964 ; De La Lastre , 1972 ) ,  Lamond anc Ernnwns ( 1959 ) 
have shovm that responses of mice given P . M. S . G . are lEJssened by 
hypophysectomy and suggest thnt em1ogenous L . H ,  is the l imiting 
factor in mouse  response.  ;.dministrc-.t ion of H. C.  G. to such animals  
will  enhs.nce the  effoct of :;.:-- , l::, S , G. ( Lamond anc: BinC.on , 1966) . 
;.port f:rom the role  played by enclog(.mous L .::-I . 1 in ovulatory response 
to ::: , I:l , S .G. 1 the exogenous hormone may itsel f contribut e to  the 
luteinising of fol licles and ovulnt ion of the same ( ':.' ill iams 1 
19<15a ; b ) .  •·· preparation of P. Ni. S .G. used by Lamond ( 1960) we.s 
estimated to hcvo approximately one third the L . H .  potency of 
H . C . G . 1 ns n source of L . n .  and Schmidt-Emendoff � �· ( 1962 ) 
found c prepcrntion of the hor�mone to  have an ? . S . H . /L . H .  ratio of 
2 .  10-like . 
To wohat extent  the L . H .  mocity in P. M. S . G . preparations may 
contribute  to the L . H .  required to ovulate st imulated follicles in 
the eYJc is not known. Bindon � �· ( 197 1 )  nnd .r'..l lison ( 1973)  have 
suggest ed tho use  of hypophysectomised sheep to clarify this  point . 
Pigon .2.!_ .9.!_. ( 1960) discovered o decrease in pituitnry L . H .  
content  when ewes were given P . Jiti , S .  G .  The importance o f  the pre­
ovulntory surge in L . H . is YJel l  documented ( see Pant £! �· 1 1972 ) .  
Hence the reliance of superovulation on an adequnto  supply of 
oncogenous L. H .  is easy to imagine. In  fact , differences in L. H .  
contents of circulcting blood or  pituitaries of  ewes may be  primary 
cause of different responses among ewes given P .M . S , G . (Bindon 
.� �! .. 1 197 1 ) ,  
12 
In the owe , P .M . S . G .  wil l  cause follicle growth in the 
nnoostrous period (Robinson , 1954) nnd effect ive superovulation 
during the breeding season (Lnrsen , 1971 and many others ) .  Using 
ovnrian t ransplants ,  Baird e!, .sl• (1968) found P. M. S . G . to cause 
release of  oostradiol- 17-Bcta from the ovary with a delay of  2+ 
days frow the time of infusion . Th e delay before a rise in L .  ii . 1 
effected by the same hormone,  is 4 1-46 hours (Cumming � �1 . , 1971). 
Although L .H .  infus icns wil l  increase the steroid output of  the 
autotransplanted ovary (HcCro.cken .£! �· 1 1969), which could in 
turn elicit the release of endogenous L. H .  (Short , 1972) , the 
above suggests  thnt P . rfl. S . G .  may itself  stimulate the di rect 
release of endogenous L . H .  f rom the pituitary. This could occur 
before a rise in circulat ing oestrogens and hence be a di rect 
effect of the gonadotrophin on the pituitary. This  argumen-t is  
bused on  the fact that P .M . s . s . wil l  produce a riso in circulat ing 
level s of  L . H .  before a rise in oestrogens . 
�n ovulat ing dose of L . H .  extracts may ( Braden £! �· , 1960; 
Hunt � �· 1 1971) or may not (Robinson , 1951) augment a more 
efficient superovulatory response t o  P. M. S . G . in ewes . 
Greep (1973) in reviewing the state of knowledge about 
gonadotrophins indicates that the primary site of action of F . S . H .  
i s  on the granulsoa cells  of  growing follicles.  �ction o f  such 
hormones is  thought to be mediated by cyclic A . M. P .* ( Greep , 1973). 
* 3 r -5 '  - Adenosine  Monophosphate 
Chemical Cht.,racteristics of 
Pregnant Mare ' s S erum Gonadotrophin 
13 
I nformat ion on the chemi stry o f  I� . M. S , G . has b een reviewed by  
Geshwind ( 19G3 ) and ;1apkoff ( 1969 ) .  
P. M, S . G. is a molecule consisting of peptide chains , 
carbohydrate  and sialic acid (Gospodurowicz ; 1972 ) .  E s t imates of 
molecular weight have ranged from 20 1 000 (Papkoff , 1969 ) to 68 1000 
(Morris , 1964 ) .  Gospodai·owicz ( 1972 )  decided that , with u molecular 
weight of  53 1 000 1 being reduced and alkylo.t ed to 23 1 000 ,  P . M. S . G .  i s  
an oligomeric molecule composed of  two sub-units • 
.!m,_munology of 
�regpunt Mo.re ' s S erum Gonadot rophin 
Soon after the discovery of P .M .  s .  G,  1 by Col o  and Hurt ( 1930) , 
S elye £! �· ( 1934)  detected the format ion of antibodies being 
produced against the exogenou s gonadot rophin . lli th thi s  realisation 
o. large interest \"IUS taken in the fi eld of anti-hormones . Reviews on 
the early work have been prepared by Col l ip et !:.!· ( 1940) 1 Thompson 
( 194 1 ) ,  Leathem ( 1949 ) 1 nowla.nds o.nd Parkes ( 1965 ) and Wright ( 1965 ) .  
Thompson ( 194 1 )  stated that the early literature seemed: -
" 
Almos t  hopel essly confused by the number of hormones , the 
variance of their sources , the wide variety of test 
animal s experimented u�on and the number of di fferent 
approaches used in the study of the problem" . 
14 
Rowlnnds anG Parkos (1965) decided that : -
" 
nesponses of animals to prolong0e: injections of 
anterior pituitary extro.ct s of hctorozoic origin is 
that the hypertrophy produced init ic.lly by the target 
organ is  not mnintainod indefinitely. Ult imately a 
s -tato of physiological hypophysectomy is rznch0d , In 
these circumstanc�s tho ovaries have lost their capacity 
to  respond to injected gonadotrophin" . 
But refractoriness of the ovaries was not thought due to  
inscmsi t i  vi  ty  of the organ by  other v1orkers. Follicular exhaust ion 
of the ovary waD excluded as L possibi l i ty beceusc cnimals incc.pable  
of further response to one gonadotrophin were sensitive to 
propD. :retions from other species Claf c:z .9,! al .• · 1 1964) , anC: 
:cefre.ctoriness was not soon to develop \'!hen homolosous extre.cts 
v.rerc injected ( in fnct pro-gono.dot rophic responses were reported 
(rCatzman �t �· , 1947) � 
t l  • " Ini t inlly  antibodies to hormones  \'lore co.lled antl.-hormones 
yot it  ,_-,as not known whothe;r ant i-hormones and nntibodies t o  
hormones '.'lore the sr.me (Geshvlind , 1963) . In fact poor correlat ions 
were observcC: bctwcon anti-hormonal �;ctivi ty unci any chemical 
methods used to  quantitate the extent of the reaction e . g. Van Don 
Znde ( 19·11) .  Some authors supported the vievJ that the more pure a 
hormone preparat ion was, the loss likely  it  was to be antigenic  
(Rmll£.'..11ds and Parkes, 1965) .  
P.M . S . G� i s  regarded as a good e�t igen when injected into 
hetorozoic species (Rovlands and :?arkes, 1965). This may be 
15  
nttribut0G to its  high content of  peptide sub-units  and its  
long  half life in the blood stream O)P..rlow 1 196 1 )  • · For  the lat ter 
reason n single injection was fotind to be us effective as 
multiple inject ions in causing increased ovarian weight in 
immL,ture mice (Connoll 1  1965 ) .  The rate of loss from the blood 
stronm appears to be independent of dose Lamond 1 1959 ) .  
Preparations of P.  !•,1, s .  G. nro re(';nrC:od as being very impure 
( soo F lux and Li , 1935 ) .  Hence the ndministrntion of the hormone 
may enhance the formation of n number of antibodies not related to 
the gonadotrophin molecules  (Vnn 0en 3nde ,  194 1 , Flux and Li , 1965 ) .  
However , fewer antigen-antibody complexes nre formed with more pure 
px·opcrn t ions .  
Components of preparations other than the hormone frnct ion may 
also bo potent antigens (Vnn Den i.!:nde , 184 1 ) .  Th e  fact that 
impurities may be more strongly antigonic than the hormone itself 
has been displayed by Segal .£!. £.!.• ( 1960)  and Segal ..£.! �· ( 1962 ) • 
.t�ntibodies to  P .M. S . G . include a fract ion rencting with horse 
scrum proteins .  These are partly cbsorbed by  normal mare ' s serum 
without any loss of nnti-P. M . S . G .  potency (Flux and Li , 1965 ) .  
Injecting normal marc ' s serum into ewes for two months wil l  induce 
antibody production of a lower titre than a similar treatment with 
:? .M. S . G . 1 demonstrnting thc.t the hormone fraction has specific 
antibodies associated with it (Cole £1 �· , 1957 ) .  
!3wo Ovarian Weight Respon se to 
P��t Marc '�rum Gonadotrophin 
16  
Robinson ( 1950 ,  195 1 )  anc �llen and Lamming ( 196 1 )  measured 
ovarian weight after stimulating ewos with P .M. S . G ,  and found 
line2.r relationships betvJCen ovarian weight and clo se level of th e 
hormone, !. strni 1�;ht line is  also obteined by the regroosion of 
ovarie.n weight o n  the n umb e r  of c orpora lutea present (Robinsoi.1 1 
195 1 ) .  
Hutchinson and Rob<:':;.�t son ( 1966) , observing unsti m ula ted ewes , 
have fo und a significant relationship between total follicular 
volume and ovarian weight in ovaries devoid of corpora lutea . 
AssaL._of Gonas_otroEhins  and t .. n ti-gom:.do tro��ns 
The val ue of using inmm.ture mice to assay gonadotrophins has 
been indicated by Brown ( 1955 ) ,  Green ( 1955) states that the 
immat ure mouse ovary secretes hormone within 12 ho urs o f  an 
injection of  Equine F ituitary Gonadotrophin . The degree of 
respo nse m ay then be quantitated by measuring uterine or ovarian 
weights.  
Uterine and ovarian responses in the mo use have been recorded 
with H . C . G. ( Larnond and Binclon , 1966 ) 1 v1ith F . S , H �  (Brown 1 1955 , 
I garashi and McC ann, 1964 and Bell , 1969 ) and wi th P .M .S . G . ( Green, 
1955 ; 1956 ; S uhi ro .£! �· 1955 ; S a samo to, 1972 ; Chri sti a n sen and 
E le fthe ri ou, 1972 ) .  
· H ypoph y sectom i sed m i ce may be u sed fo r the speci fi c assa y of 
F .S . H .  1 m e a s uri n g i ts a ugme ntatio n o f  the re sponse by H .- C . G .  
(Lam ond and Emmens ,  1957 ; U te roi a nd Me yer , 1967 ; Lamo nd and 
B i nd on, 1966 ; Bind on and L amo nd 1  1966 ) . Lamo nd and B i nd on ( 1966 ) 
17 
state that the uterine weight is a more sensitive i ndicetor of 
F . s . H .  potency th un o varian weigh t ,  even though the o vary is the 
primary tarGet of the hormone. Intraperitoneal injection increRses 
the sensitivity of th e assay of P . i'll.S .G.  in im;naturc mice , ns 
compared v:i th subcutaneous injecU.on (Sasamoto , 1972 ) .  Lin and 
Bailey ( 1965 ) ,  Zarrow �.i E-l . ( 1972 ) and Bell ( 1969 ) h::>ve 
adequctely demonstrated thnt the: response v2.ries significantly 
between strain s of mice.  
Cole and Srway ( 194 1 )  developed c �8  hour assay test  for 
Equine Pituitary Gonadotrophin with result s expressed in 
International U nits .  Th e tes t �as based on  measurinG the o varian 
response in 25-clay-old rats .  Cole .£! !:.!.· ( 1957 ) modified this 
assoy to measure the inhibition of response to injected P.I'ii . S.G. 1 
by antiserum against  the hormone. nesults  2-re reported by indicating 
the % inhibition of the response. Tho same method vms used by 
Pigon .£! E:l:.• ( 1960) to noasuro anti-P . M . S . G .  titres in chronically 
treatec', ewes and by Jainudeen et al. ( 1966 )  for a similr�r stuc1y with 
cattle  sequentially tree tee: vlith th e h ormone. Nakahnri ,2i �· ( 1964 ) 
employed immature mice to detect nntibodies against  P �i'ii . S . G. which 
were produced in cattle. Joh nson ( 1962 ) used both rf'.ts  and mice for 
the detection of anti-gonadotrophins.  
Since Ouch terlony ( 1949 ) de veloped the  tech nique of  
investigLting antibody-antigen systems by  moans of  double diffusion 
th rough a n  agar gel , the  methcd h as been widely used ( see noes-
Midgely 1969 ) •  
18 
In  particular,  Segal et al . ( 1960 )  and Segal et al . ( 1962 ) have 
� - - -
used tho technique to diagnose t he i��unology of gonadotrophins.  
Flux end Li ( 1965 ) have investigated the cross reactions amongst 
gonadutrophins by this means and compered the results  with those 
from biological inhibition test s ,  performed in hypophysectomised 
rats.  Cross reactions and impurities in the hormone-ant isera 
complexes complicate analysis by this method al though these draw-
backs may be overcome by the use of electrophoresis , chromfltography 
and the removal of unwanted antibodies by absorption with specific 
antigens (Flux and Li 1 1965 ru1cl Rees-1\iidgely , 1969 ) .  The presence 
of non-precipitating antibodies must always be considered when 
interpreting results from this method (Rees-Midgely 1 1969 ) and there 
aro some indications that antiGen-ant ibody complexes involving 
P . M . S . G . may be of this kind (Johnson , 1962 ) .  
Refractoriness of the ovary in ewe� sequentially 
treated with Pre�ent Mare' s Serum Gonadotrophin 
2.efractoriness duo to "anti-substances" was noted by Parkes 
( 19<12 ) .  \'Then rabbits vmre stimulated with Horse Anterior ?ituitary 
Jxtracts (H .A. P. ) the initial hypertrophy of the ovaries was 
followc� by a decrease in the number of fol licles developing with 
subsequent trontment s .  hdams ( 1953)  noted a similar effect . 
A summary of the literature pertaining to the refractory 
condition is  given in Table 1- 1 .  
Species 
Bovine 
Ovine 
Murine 
Rodentia 
19 
T/o.BLIJ: 1-1  
Literature reportinJLrefractor�����t��ons of the ovarJ[ 
after _ Pregnant Mare
's �erum 2onado�rophin treatment 
Luthors 
Dzuik �t a�,· ( 1948 ) ; l:illett £! !..!. · ( 1953 ) ; 
Cola .£!_ :El· ( 1957 ) ;  Hafez .2.1 &· ( 1964 ) ;  
Nakahari � �· ( 1964 ) ;  Jainudeen � �· 
( 1966 ) ;  Laster £! �· ( 197 1 ) .  
Pigon � �1 . ( 1960 ) ; Bulet and Foote ( 1967 , 
1969 ) ;  Larsen ( 197 1 ) .  
Fowler c.nd 3dvmrds ( 1957 ) ;  Edwards and 
Fowler ( 1960) ; Lin and Bailey ( 1965 ) ; 
Land and l\1cLaren ( 1967 ) .  
Largomorpha 
Johnson ( 1962 ) ; Groenwald ( 1963 ) .  
Pe.rkes ( 1942 ) ;  , .dams ( 1953 ) . 
Fowler and Edwards ( 1957 ) produced a refractory condition in 
mico by injecting P . Jit!. S. G .  and H . C . G. Zdwnrds and Fowler ( 1960 )  
decided that, since four succesGive ovul�tions could result from 
four sorios of gonadotrophic stimulation , follicular exhaustion was 
not likely to be responsible for rofr�ctorincss .  Lin and Bailey 
( 1965 ) substantiated this claim by producing one to ten 
superovulations in mice on similar treatments .  
Land and l'ilcLaren ( 1967 ) have postulated anti-P.M.S.G. :  t o  be 
responsible for refractoriness developing in mice subjected to  
successive superovulations . 
20 
Dzuik £! �· ( 1948) detected no consistent reduction in 
superovulstion when cows were repeatedly stimulated with 
gonadotrophins, the interval bat·v.reon trentments varying from 49 to 
359 days. However Ylillctt et .'?1· ( 1953) obtained n refractoriness 
by treating cows with a variety of gonadotrophins . Hafez et nl . 
( 1964) and Lnster _£! .£.!.• ( 197 1)  realised lmwr ovarian responses to 
retrco.tment of cows with P, i'ii, S , G , Cole et al. ( 1957)  and Jainudeen 
£1 �· ( 1966) demonstrated anti-gonadotrophic activity in cows 
repeatedly stimulated with P •. i':i, S . G .  1 the former using precipitation 
tests and biological inhibition tests and the latter using 
biologiccl inhibition tests alone , 
Colc � �· ( 1957)  and Nakahari � �· ( 1964) decided that 
antibodies to P . �,1 . S . G .  VJOUld only be produced in cattle if the 
animals were treated with supra-physiological levels of the hormone. 
!.n animal does not normally develop antibodies to endogenous 
gonadotrophins .  However , in some cases , when a protein hormone of 
a different species is introduced , the antibody formed against it 
also rco.cts with the endogenous hormones.. Hence ,  rats immunized 
with ovine L. H • . developed an antibody which neutralised endogenous 
L. H .  (\!nko.baynshi and Tamaoki , 1966) . Lin and Bailey ( 1965 and 
Ylitschi and Johnson ( 1960) hnvu also hinted that repented injections 
of P. M. S . G . may inhibit endogenous hypophyseal control. 
Jainudcen .£! �· ( 1966) decided that normal follicle development 
continu�� under the influence of  endogenous hormones while nnti-
bodies to injected P. M. S , G . rendered exogenous influenc0 incapable 
of stimulating the ovaries of cows . 
21  
Greonwald ( 1963 ) showed thut anti-P .M. S . G. would not 
interfere with the endogenous control of cyclic behaviour in 
hampsters . To reinforce this argument , Land and McLaren ( 1967 ) 
found that even though the response of mice to  Human Chorionic 
Gonadotrophin (H . C . G. ) ceased after repeated injections , normal 
cycles resumed later , in spite  of continued treatment . These 
latter authors thus concluded that the effect was that of an 
immunity to the injected hormone , not a refractoriness of the 
ovary. However they did not seek 
being present . 
any evidence of ant ibodies 
Antisera to one particular hormone may also inhibit the 
action of other exogenous hormones ( see Lunfeld and Eshkol , 1969 ) .  
The basis of this  cross-reactivity may l ie in the similarit y in the 
antigenic  sites on the hormones (Geshwind , 1963 ) .  Similarities i n  
taxonomic relationship between onimals  may also enhance cross­
reactivity (Katzman � �. , 1947 and Geshwind , 1963 ) .  
P .M. S . G. is sufficiently similar to Equine L. H .  to result in 
confluent patterns in agar gel diffusion tests (Desjardins and 
Hafs , 1965 ) .  These authors also saw Ovine L. H .  to be neutralised 
( biologically) by antisera to Equine L . H .  They al so saw that 
ant isera to P .M. S . G. and Ovine L. H .  would not inhibit the action 
of H . C . G. in hypophysectomised rats .  Ely and Chen ( 1967 ) al so 
showed antiserum to Ovine L. H .  to inhibit P . M. S . G. action . 
In considering the above results i t  i s  important to note  that 
because an antibody is agglutinating , precipitating or neutralising , 
it does not necessarily follow that these processes parallel ono 
another (Geshwind , 1963) . 
22 
i'igon � �· ( 1960 )  subjected owos to chronic t reatment with 
P . H.S . G . o.nd developed a potent antioerull) . The treatment resulted 
in o. lowered pituitary content  of L . r! .  which may be one of the 
fnctors responsible for the failure of such owes to ovuli:'.te .  
Rulet una Foote ( 1967 ) demonstrated a f2.ll off in  ovulation 
rate uhen ewos v:oro superovulated nt six succcssivo oest rous 
cycles .  I nvestigating thc phenomcner. more fu lly , Hulet und Foote 
( 1069 ) showed the dovolopment of  u d0finite refractory condition 
yet did not sock evidcmco of ::nt ibody production. Information on 
the dissipation of tho concUtion with time was revealed . Larsen 
( 197 1 )  found u similar effect in N�v. Zealand Romncy and Border-
Leicester-Romnoy cross ewes when the emus wore treated over thr0e 
succossive cycles ; n high incidonce of cystic and luteinised 
follicles resulted from tho t reatments .  
Conflicting reports on the effect s o f  doso level of 
administered hormone on tho refractory condition meke conclusions 
difficult .  Po.rl-;:es ( 1942 ) showed thc.t o. geometric rise in doso.ge 
every few do.ys fo.ilcd to ov..:rcome t�1.:;  incrm>sing insensi ti vi ty  of 
the ovary. �·:illE:tt � �· ( HJ53)  and Jainudcen et al . ( 1966)  
decided that increc.ses in doc8 of P. hl. S . G . would partially overcome 
the conc.a tion . This  vms not verificcl vti th similar t reatment of 
sheep ( Larsen 187 1 ) .  �i8on et �· ( 1960 ) reported that 200 I . U .  
olicitcd 8. more rf'.pid manufucturo of  nntibo<.lius in sheep than 
500 I . U. when P .M .S, G. was given at chronic levels. Higher doses 
wore seen to be more effective for antibody production in cov1s 
(Colc � �· , 1957 )  and rats ( Johnsor. , 1952 ) .  
23 
Antibodies to P .M . S . G . have boon noted in the blood streams 
of tro�tcd animals some considerable t ime after sequential 
trcatraents  with the ::-wrmone (Naknhari � .£-..!.· 1 1964 ; Jninuduen et 
£�· 1 1966) and have b e-:m poD tulctec to bo pres(;nt up to a yvnr 
from treo.tment C!illott .£!. �!.· I 1853 ; aulot n.nd Foote , 1969) . 
�-1o·:tover :;dgon ?t .£!· ( 1v60) s tatcc: th.c effect to  be  dissipntod 
over throo weeks subsequent to the t reatment . 
i'. soconc1 series of go:10.cot rophic injections 1 some time after 
thu fi:.:·st 1 will  effect en upsurge in antibody ti  t ro (Cola � £:..!.· , 
1957 ; ?igon .£! g. 1 1960 ; rclf;lc:<hari et nl . , 196,1 ) . This is  typical 
of en ir:unune-liko responso (l'!ossal , 1971) . 
Follicul�r�j:;rowth _2:!-:._c _9tresia in tho owe C?_Vo.rx 
Grmrth of ovcrinn fol licles in the ewe hns been studied by 
Grcnt ( 1934 ) , Mc:i:enzie and Torrill  ( 1937) , Kammnlndo .£! �· ( 1952) , 
S o.ntnlucito .s! .'!!.· ( 1960) 1 Hobortson &J.<-' Hutchinson ( 1962) . 
nutcilinson and Robortson ( 1966) Slllenton c.nd Hobert son ( 1971) . 
Holst  et al .  ( 1972) �nd Brand o.nd De Jong ( 1973) . Folliculnr 
dyncmics has boon investigated in the mouse (Peters and Levy , 1966 ; 
Pctersen , 1970) 1 the pig Ci.obinson nnd Hnlbo.ndov , 195 1 ;  Po.rlow et �· , 
1964 ) and the cow (Rc-.jc.koski,  1960 ; 3rickson 1966 ; Choudnry .:::..!. !':.!..· 1 
1930) . Folliculogencsis in a variety of mammnl s  has been rovienoo. by  
r.1nuleon ( 1969) , follicula:r atresiu by  Ingram ( 196 1 ) ,  
The proovulntory surge in follicular growth has been recognised 
for u conaidorablc  t ime (Grant 1834) . M�Cenzie and Terrill ( 1937) 
stet ed that the number of smnll follicles ( 1-2 mm. ) in the ewe ovary 
24 
was quite variable  and some individual s hed consistently  more  than 
otl1ers . "Great varintion" in the number of ovarian foll icles W'as 
elso observed by Hrend and Do Jong ( 197 3 )  in 33 normal Toxcl ewes . 
The letter authors found a p:cir of  ovnrios to cont ain 32 . 2% (Moan= 
84 . 2  , S .  3 .  =38 ,  1 )  normal nnd 67. 8% ( II'loan= 177 . 1 , s .  E .  = 103. 4 )  atretic 
fol licles with diamet0rs greeter  then 2 ��. However , of the normal 
tGrtiary fol licles , 02 . 7% hnd n diamutor of less th[�n 1 mm. Choudary 
C\ .�. ( 1960 ) found n moan relat ive proport ion of 23. 9% normal and 
76. 3% atretic fol licles present per p�i r of ovaries in cyclic heifers 
of unspecified breed . 
:cmnmaladc et al . ( 1952 ) and S nntnucito � �· ( 1960 ) stated that 
fol licle growth t .3J.1dod to be continuous throughout the cycle •. 
Robertson and Hutchinson ( 1960 )  and Hutchinson 'llld Robertson ( 1966) 
uphold this concept and considered that fol licles destined to  ovulate 
at the ensuing oestrus bq;in to  enle:.:-gc 4-5 days after the onset of 
the previous oestrus and then pause until  a pro-ovulatory surge in 
grov1th. However Smeaton and Robort son ( 197 1 )  point out that these 
conclusions  were deduced from observat ions on the ovaries of different 
o.nir.mls killed nt various stcgos of the cycle .  This lc.tter  s tudy 
tracocl the pat tern of  folliculc.r �rowth by injecting Indian ink into 
the fol licles and thus monitor�� sub sequent development of follicles 
previously observed , This methoa relied on the assumption that the 
fol licles continued to �row normal ly eftor the administration of ink . 
The stuQy suggested that thoro may be  thrue or more waves of fol licle 
growth during one cycle . Follicles enl�rging at the beginning of the 
cycle mny become atretic nnd not ovulD.to.  This  concept of fol liculcr 
25 
grm1th is consistent  with thG fact thr;t one mny show three penks 
of oestrndiol-17-betn secretion from the ewe ovary on days 3-4 , dnys 
6-G nnd days 1 1- 15 o.ftor oestrus (f\lnt tnor and Braden, 1972 ) .  Holst  
<ot �· ( 1972)  show those poc,ks to bo  concomitant wi th an increase  in 
follicular volume of the ovary. Brand and Do Jong ( 1973 ) have 
recently found only two gronth waves of follicles in sheep ovcries . 
The first wns detected from days 1- 10 and the second from duy 6 of 
ono cycle to day 1 of the next .  
Peters and Levy ( 1966 ) nnd Petorson ( 1970 ) have traced 
fol liculnr growth in thv mouse ovrcry by usinG ruc'lio-&ctive 
thymidine. 
Brcnd and De Jong ( 1D73)  sugr:;ost that the growth pattern of 
onlcrging fol licles  is influenced by  the levels  of circulatinG 
progesterone . Tho end of the first  wave of growth ,  �otectec in 
their study , was at th0 time one noulc expect progesterone levels  
to  ho.ve rouchod maximum level s in  the  blood nnd enl<,rgel!lent of 
prcovulctory follicles on day 16 was nt n t ime of expected low 
levels of plasma progest erone . 
?here is  n sharp incronso in the size of the corpus luteum of 
the o·;,o between 36 hours after ovu lnt ion nnd 5 dcys nfter onset of 
oestrus (Hutchinson cilld Robortson , 1066 ) . Lutenl regres�ion begins 
nbout day 10 (Grnnt , 1934 ; Scilltnlucito £! �· , 1960 ; Hutchinson and 
J.obort son 1 1966 ) . :<.apid d ogcnera t ion occurs between this time and 
the onset of the next oestrus (Hut chinson and l'tobcrtson, 1966). 
26 
PURPOS� kHD ;3COPZ OF THE INVESTIGATION 
Tho product ion of  c.ntibodios to  P . k . S . G . hns boon dcmonstrntoc 
in sheep , The fnl l in ovulatory response  of  cv,ros trented 
scquential ly with this  hormone hc.s boon postulntud due to th(� 
dcvclop;uent of  such anti-P . M . S . G . factors . 
T:w first  part of this project considered the dovelo;.:;mont of  
e. rcfr�·.ctory condit ion in  rcS >)Onso of ovtcs t roo.t cd with P . fli . S , G . 
ov.:Jr throc oestrous cycles . :f:lcsmc from these owes , o.nd thr..t of 
cues chronically t ruo.tcd with the r:;ono.cotrophin , wore tust cd by 
2. BioloGical Inhibition Test , usinr; immature mice. The hypothesis 
thnt ovo.rio.n rofractorinc:;,ss  in P . n . s . G . -tn;atcd ewes we.s duo to  
anti-Gonadotrophic  o.gont s could thus be tested .  
On th.o: bc.sis of  those invostigctions , it  r.ms considered 
nccecs�ry to olucidnto other possible ccsual fnctors in the 
development of c. rcfrnctcry stnto of  the ovc.ries in saquenti�. lly 
t r<Jr:tod o':ms , Hence in n further experiment , the possibility  that 
the supply of normal mnturc ( cntrr:.l ) Graafian foll icles was depleted 
by repeated t rontmcnt , wns examined . 
C H � P T E R I I 
M h T � R I A L S  A N D  M S T H O D S  
Chapter I I  
M A T E R I A L S A N D  M E T H O D S  
Sheep and their Management 
Ev1es used in this study were ' cast for age' ( five or more 
years ) Romneys with no records of previous lnmbing performance. 
Sheep for Experiment 1 were purchased in March , 1972 from a mixed 
line of ewes offered for auction at a local sale.  The animal s for 
Experiments 2 (8 sheep ) and 3 (40 sheep ) were ' cast for age ' from 
various flocks on the Massey University farms.  The latter ewes 
were consequently derived from an environment similar to that in  
which the experiments were conducted . 
All  ewes were rotationally grazed on Ryegrass-llhite  C lover 
pastures with yard and surgical facilities nearby. Sheep of 
3xperiment 1 were offered pasture adequate to effect only a slight 
loss of body weight over the duration of the trial .  The district 
suffered drought conditions during January-February, 1973 and sheep 
of Experiment 3 were subjected to feed restrictions nt this time. 
The drought ended on March 5th , 1973 and good autumn pastures were 
o.vailable  for the experimental period. Liveweight changes of  ewes 
over the duration of Experiments 1 and 3 arc shown in Fig. 2- 1  and 
Fig . 2-2 respectively.  
Ewes were identified by serially  numbered ear tags and 
corresponding numbers branded on their shoulders. Vasectomised 
rams ( ' t easers ' ) ,  fitted with S ire S ine mating harness , were run 
F I G U R E  2-1 :- EWE L I V EW E I G H TS AT T H E  B E G I N N I N G A N D  E N D  O F  EXPE R I M E N T  1 
� ::r:: 
(!) 
w 
� 
w 
57 K .g 
56 
> 55 
-I 
w 
� 
w 
� ::r:: 
(!) 
w 
� 
54 
53 
7/6 14/7 
F I G U R E  2-2 :- EWE L I V E W E I G H T  C H A N G ES DU R I N G  EXP E R I M E N T  3 
51  K g .  
50 
DATE 
L E G E N D  w 49 > 
:::i 
w TR EATM E N T  SYMBOL 
� • w 
2 • 48 
3 0 
4 A 
47 
25/2 27/3 1 2/4 26/4 
DATE 
with the ewes during al l experimental treatments  except when the 
latter had recently been laparotomised or mated . When laparotomies 
were performed the operated ewes were run without a ram for 3 days 
to prevent ' rape ' services while they recovered. The ram to ewe 
rat io was never less than 1/40 and was usually greater. 
Observations to  record oestrus in ewes were made at os .oo-
09 .00 hr.  and 17 . 30-18.30 hr. Marked ewes were withdrawn from the 
flock to increase the efficiency of oestrus detection in the 
remaining ewes.  The sheep were yarded to inspect the rump regions 
for crayon markings that signified service by the tenser rams.  
Prior to laparotomy , ewes were yarded and starved overnight . 
This diminished the risk of loss associated with anaesthesia and 
surgery. f;.fter operation , ewes wore placed in pens to recover and 
then allowed to move back to pasture in their own time. 
Ewes of Experiment 1 were subjected to progestagen treatment 
in the middle  of the breeding season and had therefore experienced 
a number of oestrous cycles prior to the treatment . Experiment 2 
was carried out in the non-breeding season and ewes were not treated 
with progestagens.  Synchronisation of  oestrous cycles of  ewes in 
Experiment 3 was initiated before the 1973  breeding season began. 
In  1973 ewes on the same farm cs that used for these experiments ,  
were first seen to display oestrus  on 18 February. This was 2 days 
after progestagen sponges had been inserted intravaginally tnto shoep 
of Experiment 3.  
29 
Mice and their Husbandry 
Mice were employed in a test for anti-gonadotrophins 
(Experiment 2 ) ,  They were descznded from the inbred NOS strain 
derived from a stock of albino mice of unknown origin and have 
been previously described by MacKenzie ( 1972 ) .  This strain is  
known to  be characteristically  oestrogen sensitive Ci.non , 1965 ) .  
Only immature females , weighing between 7-10 grams , and being 
approximately 25 days of age , were used. 
The mice were collected over a period of 4 months (December , 
1972 to March , 1973 )  by weaning litters that had reached the 
desired age or \'/eight . Both these factors were taken into account 
in order to obtain mice of a uniform size i . e . stage of maturity. 
Mice from larger l it ters took longer to roach the desired weight 
than those of smaller litters . 
Plastic mouse boxes , with a flooring of saw dust , were used . 
Pcl lcted food and water were provided ad libitum.  The mice were 
kept in the same room and held at ambient temperatures , 
Experimental Design 
The invest�gation involved three experiments : -
Exper�ent 1 :  The development of an ovarian refractoriness in 
ewee subjected to various sequences of P .M. S . G. injections  over 
three oestrous ..cycles. 
Experiment 2 :  Biological I nhibition Test for anti-gonadotrophins .  
Experiment 3.; Ovarian foll icular development in ewes super­
ovulated up to three t imes with P .M. S . G. 
Flow diagrams outline the design of Experiments 1 and 2 in 
Fig. 2-3 and Fig. 2-4 . Design of Experiment 3 is set out in Fig, 2-6. 
) 
30 
Experimental Procedure 
2:xperir.�ent 1 
Eighty-two animals  were available for t reatment . Ini t ic.lly , 
the oestrous cycles of  all  ewes w0re synchronised by progestagcn 
administration . This technique c.ided the conduct of  the experiment 
as all  ewes could be treo.ted vlithin chronological l imits ,  thus 
climinnting a temporal bias fron the treo.tments .  Furthermore , 
synchronisation facilitated simplification of treatment , in thnt 
time of injection , blood sc.mpl ing and slaughter of experimental 
units  could be  co.rried out wi th efficient utilization of labour and 
resources . 
Synchronisation was effected by the insertion of  intravo.ginal 
pessaries ( polyurethane sponcos ) impregnated with 40 mg. 6�methyl 
17.,(_- acetoxyprogesterone ( i':: . l:. . • P .  ) .  * The hormone was diffused into 
sponges in 3 ml . of 95% ethanol .  
Sponges were insertec on 24-5-72 and withdra>m on 7-6-72.  
All  ev1es in  the experiment '.'Jere weighed ( straight off pasture ) 
on the completion of progestagcn treatment and ago.in just prior to 
slaughter (Fig. 2- 1 ) . 
The shcop were allocated to  treatment groups by consul t ing a 
table of  random numbers . P . M. S . G .** was injected subcutaneously at 
dose level s of either 1000 i . u. or 1500 i . u .  Ewes on each dose 
level were subjected to the foll owing sequences of injectiono 
( see Fig. 2-3 ) : -
* Upjohn Company 
** Paines and Byrne Ltd. 
FIGURE 2-3:-
EXPER IMENTAL DESIGN : EXPER IMENT ONE  AND EXPER IMENT TVVO 
••  
TREATME.�N�T�------�---r------.---. 
A. Experiment 1 
SYNCHRONISATION LEF:..._ __ 
CONTROL 
OESTRUS 
B. Experiment 2 
500 i .u 
PMS 
1000 i .u  
PMS 
W ITH 
FREUND'S 
ADJUVANT 
2 SHEEP 
2 SHEEP 
WITHOUT 
FREUND'S 
ADJUVANT  
2 SHEEP 
2 SHEEP 
I ]BLOOD SAMPLE J >f SLAUGHTER 
Each line represents a 1 ewe 
sample I treatment group 
(9 x 4 = 36 ewes) 
3 
I - , 
t=:· 
' Biological I nhibition Test for , 
.... , antigonadotrophins in serum 
,. 
• 
,. G = Gonadotrophin 
. .  Treatments 1-4 replicated at dose levels of 1 000 i .u. and 1500 i .u. P.M.S.G. 
F IGURE  2-4:-
DESIGN OF EXPER IMENT TWO 
R EPL ICATE * *  0'1 Mouse P.M .S.G. dose 0'1 
X 3 * 0 c;· 
} 
9 ewes ctl Expt 1 1 � 
0.25 I .U .  ::I 
s: 
3 (D' 
0 
( 9 9 ewes :- 0 
c: 2 
.... 
� "' 
"' 
ctl 
Q) 
� (/) Treatment 3 :E N � c: 
9 
.... 
ewes 
1 "0 ;::-.· ';T c: ctl 1D ';T 0 '§- :::!. Groups l 
"' 
c: ::::J .... 0 "' ... ctl ctl ctl "' ... 
9 ewes ...... .... 
:E 
2 c: � "' 
3 ctl ... 
c: ctl !0' ...... 
:r 3 ... 0 .... "' ...... 3 1 .0 I .U .  ... 0 ctl 3 Q) 0 Expt 2 ctl :r Q) "' 4 ewes 0 :r 1 :r ctl ( 4 ctl ctl 
:E "0 Treatment ctl � 4 ewes 
t 
0 2 0 Groups l 
ctl 3 :E c;· ctl ctl � 
* Each l ine represents one ewe I treatment 
** 2 repl icates (1 ewe I repl icate) for each ewe treatment group 
31 
Treatment 1 :  Injected at each of three oestrous cycles ( cycles 1 , 2  
and 3 ) .  
Treatment 2 :  Two injections ( at cycles 1 and 3)  separated by a 
normal oestrous cycle ,  
Treatment 3:  Injected at  t�o successive oestrous cycles 
( cycles 2 and 3 ) .  
Trectmcnt 4 :  Injected ot one oestrous cycle only  (cycle  3 ) .  
The experiment wns thus of 4 x 2 factorial design .  Nine uninjected ,  
' synchronised ' e�es constituted a reference group , enabl ing 
comparative responses of the treatment to  be guaged , Further 
reference to  the experimental groups vlill  be made using the 
following nbbreviations : -
L1 , L2 1 L3 , L4 refer to animals given 1000 i .u . P . M. S , G. in 
sequences outlineG in treatments  1 to 4 above. 
H 1 1  H2 1 H3 , H4 refer to animals r;iven 1500 i . u .  P. M. S . G. in 
sequences outlined in treatments 1 to 4 above. 
�ither 9 or 10 sheep were designated to each of  these groups 
at the beginning of the experiment but 1 death frora undetermined 
cause (Group H l )  and 2 accidental prognancies (Group H3) caused 
three sheep to be excluded from the results . One ovary of o ewe in 
Group L3 was lost as reproduct ive tracts  were recovered from animals 
being slaughtered at the local freezing works and this  sheep was 
excluded from analysis of ovarian response data. 
Sequences of injection of P . M. S . G. as described above were 
designed to quantitato the ovulation rate  of sequentially treated 
32 
ev.res (Treatments  1 nnd 3) compared with owes given 1 injection 
(Treetmont 4 )  or control ewes ( uninjected ) .  Treatment 2 was 
included to investigate the terminal ovarian response of ewes given 
staggered injections .  Tv1o dose levels  of P .M. S .  G. were given to see 
whether the development of a ref:.:-actory condition is dependent of 
the amount of  gonadotrophin administered. 
The first  injections were made on the day of sponge withdrawal 
and subsequent injections were made on Day 12 of the oestrous cycle 
(Dcy 0 taken as  the  day of onset of oestrus ) .  Because of vc.riation 
in oestrous cycle lengths of individual ov.ros , the initial synchroni­
sat ion became less defined nn the experiment proceeded . :.!:wos wore 
injected on Day 12 of their indivh1ual cycles regardless o f  this . 
Five days after the onset of the third oestrus  from sponge 
wi thdrm:1nl , the ewes were blood seJi1plod .  Blood srunples were teken 
fror,1 the jugular V6in with 10 ml . vacutainers * (heparin coated gloss 
tubes uhich enable  sampling direct from the vein , by means of a two 
\'my noodle ) .  The  blood V�as centrifuged nnd the col lected plosmo wca 
doep�frozen for nnnlyeis in Exportoant 2 .  
:.nimcls  were slnughtered 6 dQys cftcr the onset o f  oestrus 
fol lowing their final treatment wi th P . lYI. S . G . Those ewes not showinG 
oestrus were slaughter<:.'d on o date derived thus : oestrous cycles 
lengths of all  ewes on a similar t i�catment were averaged and the 
ev1os having silent oestrus wore slnughtored 6 days after this period 
of time hnd elapsed since their last  oestrus . 
*Biolob .  
F I GU R E  2-5 :-
CA L E N DA R  O F  E V E N TS F O R  EXPE R I M E N T  O N E 
W E I G H  • 
• 
P.M.S.G.  I NJECTION 
O ESTR US P E R I OD 
SLAU G H T E R  
SYNCH R O N ISATI O N  
DATE 
24/s 71>6 9J6 r2/6 21/6 23/6.2�/6 4l7 6l7 9l7 14b 1d/7 ·16/7 1B/7 20/7 2s/7 
33 
Reproductive tracts were recovered from the slaughtered 
ewos nnG date. on ovarian response (ovarian weight , number of 
ovulutions , numbor of follicles greater thon 3 mm. and incidence 
of cystic of inactive ovnries ) wore recorded . 
A cnlendar of events of this experiment is  given in Fig. 2-5 .  
Experiment 2 
Mouse tests ior anti-gonadotrophins in the plasma of ewes from 
Experiment 1 were cnrriod out between December 1972 and February 1973.' 
!.nti-gonc.dotrophins in the plasmn of animals chronically 
t rectce with P .M . S . G . were also detected by this method. Eight 
' cast for age ' ewes were injected subcutaneously with either 500 i . u. 
or 1000 i .u .  P .M . S . G. Half the ewes were given their first 
injection in Freund ' s  adjuvnnt ( e  water in oil emulsion of the 
hormone with heat killed mycobacteria E'.dded ; Rees-lvlidgely,  1969 ) .  
Sheep vJerc injected on Monday nnd Thursday of ench week , for 
6 weeks ( 13- 1 1-72 to 22- 12-72) . Plc.smn was collected on the Friday 
of the final week of trentment.  
Tho Biologicnl Inhibition Tests used were s imilar to  the rat 
tests  used by Cole £1 �· ( 1957 ) 1 Pigon � �· ( 1960) , Flux £1 �· 
( 1965 ) 1 and Jainudeen £1 �· ( 1966 ) to  detect anti-gonadotrophins . 
This test relies on the inhibition of immature mouse uterine weight 
response to injected gonadotrophins by the introduction of c�tibodies 
to  that gonadotrophin .  
Determination of a suitable dosage of P .M. S . G. for stimulation 
of  uterine weight in immature mice was carried out by means of a 
pilot test over a range of cose levels (0. 2 i . u . to 1 . 0  i .u .  P. M. S . G . ) .  
34 
Four sheep from each treatment group in �::xperiment 1 were 
randomly selected to donate plesma samples for the test s  (9 groups , 
36 ewes ) .  Plasma. of 2 sheep per group rms tested in mice injected 
with 1 i . u.  P .M .S .G .  and the plasma of  the other 2 sheep of that 
group w�s tested in mice injected with 0. 25 i . u .  P .M. S . G. The 
hormone was given to the �ice as a single subcutaneous dose of the 
gono.dotrophin dissolved in o. 2 ml . of 0. 15% saline . o.  25 ml . samples 
of plasma from eac.'1 ewe t ested were injected ( intra-pori toneally)  
into the test mice .  F ive plasma samples from each ewe were injected 
into mice given 1 i . u. and five into mice injected with 0. 25 i .u .  
P . Ivi . S . G. This sampling procedure is  outlined in Fig.  2-4 . 
Mice were allotted to treatment  groups by random numbers. They 
\7ere weighed and earmarked before being placed in boxes . ;::ach box 
housed only one treatment group of 5 mice . i�fter being injected with 
P.M. S . G. and plasma , the mice were left for 72 hours . After re­
weighing �hey were killed by cervical  dislocation and the uteri were 
removed. These organs were dissected free of fat and any adhering 
tissue  and then weighed on a torsion balance. Weighing of the animals 
before and after t reatment allowed the detection of any adverse effects 
of injection • ·8 ·  plasma samples may have become contaminated during 
storage. 
Samples of those sheep chronically treated ( i . e .  given twice 
weekly injections of P. M. S . G . ) were t ested only in mice injected 
with 1 i . u . of gonadotrophin.  Procedure was the same as above 
except that all sheep' s sampies were tested. S ince there were only 
two sheep on each treatment , a sample from each sheep permitted 2 
replicate tests per ewe treatment group , to be  carried out .  
35 
Experiment 3 
Forty sheep were treated at the beginning of the 1973  
breeding season � Intravaginal pessaries of similar preparat ion 
to those used in Experiment 1 ,  were inserted on 16-2-73 and 
withdrawn on 28-2�73.  Because e�es had not experienced recent 
overt oestrus prior to the progestagen treatment , a 10 mg� 
injection of progesterone dissolved in 1 ml , of peanut oil , was 
injected intramuscularly 2 days before sponge withdrawal . I t  was 
hoped such an injection �ould provide a more effective progesterone 
priming of the ewe ' s capability to display overt oestrus than has 
previously been noted in ewes treated early in the breeding season 
( see Cumming , 1965 and Larsen , 197 1 ) .  On withdrawal of the 
pessaries some of the ewes were suspected to have experienced 
silent oestrus.  Laparotomy of tuo such ewes confirmed that they 
had ovulated.  
P . M, S . G. we.s injected on Day 12  of the cycle following sponge 
\"'i thdrawal . Subsequent inject ions were made on either Day 12 or 13  
of  the cycle.  
The experimental design is shown in Fig.  2-6 .  Ewes were 
designated to 4 groups of 10 animal s by random numbers . Group 
numbers \"!ere later rendered unequal by 1 death and 1 suspectod 
pregnancy. Ewes were either uninjected ( control ) or injected 
once , twice or thrice (Fig. 2-6 )  over three oestrous cycles . 
Day 10 of the oestrous cycle was chosen as a reference point 
to monitor ovurian activity� Observation at this point ul lov1ed 
meusurement of ovulation rate at the previous oestrus as well as 
SYNCH RON ISATION 
1 6/2 28/2 1 5/3 
FI GURE  2-6:-
DESI GN OF  EXPER IM ENT THR E E  
28/3 30/3 1 3/4 14/4 2 1 /4 
to 
30/3 
to 
2/4 
to 
2 1 /4 
*G = Gonadotroph i n  
* *  Lap  = Laparotomy 
to 
28/4 
to 
8/5 
DATE 
36 
enabling assessment of follicles likely to be stimulated by a 
following injection of gonadotrophin. Treated ewes were either 
laparotomised or slaughtered on Day 10 ( 10 ewes being slaught ered 
nfter the first 2 superovulations and the third treatment group 
and control  beinG slaughtered after the final suporo�tlation ) .  
" " "* Laparotomy wRs performed under light anaesthesia ( Nembutal r 1 
on a lapa:rotomy cradle.  t. ventral incision , slightly off the  mid-
line , al lowed exposure of the reproductive tract . Counts of corpora 
lutea and fol licles wore mo.co.  Foll icle sizes wore established by 
the use of  sterile slide co.l lipers ( see Fig.  2-7 ) and v1ero always 
made by the same pm·son . The follicles wer>:: assigned to  three 
groups ; 1-3 mm. ( small ) ,  3-5 mm. (medium) greater than 5 mm . ( large ) . 
Ovnrios recovered from the slaughtcrec animals  wc:re immediately 
scored as for lo.parotomiscd animals and fixed in 107o buffered 
formalin . i .fter fixat ion ( this time was variable  depending on the 
size of the ovaries ) the ovaries were placed in 50% alcohol for 
24 hours and then stored in 70'7o alcohol . To assess the t otal 
ovarian folliculer populat ion all  ovaries were s ectioned into 2 mm . 
slice::;.  Thene ovarian sl ices were surrounded by a thin layer of 
pn.raffin -.:mx to contain the sl ices in correct sequence , Such "plates" 
of material were immersed in trays of 70% alcohol . Fig.  2-Q shows 
ovaries sot in wax before being put in alcohol . 
Counts of  all antral fol licles were made on all ovarian slices . 
Addition of  the number of  follicles in al l slices from one ovary 
gave an indication of the total follicle population in that ovary. 
These counts were made with the aid of a large magnifying glass . 
* Pentobarbitone Sodiwn (l�bbott Laboratories ) .  
Fig.  2-7 : - _!E. �  measurement of ovarian fol licles with 
�tcrile slide  callipers 
Fig.  2-0 : - 2 mm. ovarian aliccs semi-embedded in 70o/� alcohol 

37 
Ten randomly selected 2 nun . slices from ovaries of ewes in 
each of the 4 experimental groups ( right  or left ovaries ) were 
selected for histological analysis .- i .. fter  processing in graded 
alcohols and xylene , these samples were embedded in paraffin wax 
and sectioned at 6rL. The sections were stained with Haematoxylin 
and Eosin . Antral fol licles were now able  to  be assessed for eny 
signs of atresia or lutcinisnt ion and the percentage of the total 
population examined that were atret ic ,  was computed. Gross 
measurements  were corrected with respect to  this figure which was 
derived for each separate group. 
The assessment of fol licular population may be summarised thus : -
Number of 
surface follicles 
( 3  categories ) 
I 
All  ovaries 
sl iced into  2 mm. 
sections 
I 
Number of antral follicles/ 
ovary (cross sectional 
observation ) 
I 
Sample - 10 
J 
slices/group Correction factor 
htretic follicles/total 
�= proportion of 
� normal follicles .  
follicles/ 
2 mm. slice (histological 
m casu remen t s ) 
Average 
number of 
normal 
follicles/ 
ovary (all 
ovaries ) 
30 
This procedure was not designed to measure every follicle in 
every ovary. Hov;evor an indication of tho follicule.r population in 
ovaries of aninals  on each treatment i s  at tained and some idea of 
the degree of atresia  of these follicles derived . The limitations 
of this  method will  be discussed in Chapter Seven . 
Analysis of Date 
2 
Analyses of d iscrete date utilised Chi or the G stat istic 
(Sokal and Rolf 1969 ) � 
Ovarian response data from Experinent 1 were from groups of  
unequal size. Ho·;mvor , a two woy onalyDis of  vnrianco for unequal 
sub-clas ses wns avo.ilable  on a computer programm� , and the 
experiment was analysed as for a 2x2 factorial design .  The general 
linear equation for the model analysing ovarian response was : -
where 
u = the overall mean when equal frequencies exist in each 
subclass 
t .  = effect of t reo.tment ( i  = 1 , 2 , 3 ,  or 4 )  � 
d . = effect of dose ( j  = 1 or 2 )  
J 
( td ) ij = individual interaction ef fects expressed as 
deviations from mean u.  
eijk 
= error peculiar to each yijk 
*courtesy of Prof . R. E .  Munford . 
39 
Bartlctt ' s t est for homogeneity of variances was carried 
out on discrete ovarian response clc,tn as a t ost of normality of the 
data distributions (Snedecor and Cochran , 1967 ) .  On the basis of 
thos0 tests  all discrete data '>lere transformed toJ;;;:t before nny 
tests assuming normality werG cnrriod out .  
Comparisons o f  treatment moans \lore made following analyses 
v1hich yielded statistically signific'l.nt result s .  Comparisons were 
made using the S tudent-Newman-Keul s  tost (Sokal and Rolf 1969 ) .  
r!Iouse uterino weights (Exporir.wnt 2 )  were transformed to 
loRaritlL�s and analysed by covariancd. Significant treatment 
effects \"lore further analysed by the Student-Newmen-Keul s  test . 
C H A P T E R I ll 
S Y N C H � 0 N I S A T I 0 N A N D 
O E S T R O U S  P H E N O M E N A  
� 
40 
Chapter I I I  
!3 Y N C H i1 0 N I S I. T I C N A N D  0 E S T R 0 U S  
p H E N 0 iVl E N  A 
Experiments 1 and 3 yielded date on progestagen synchronisation 
of oestrous cycles and o0strous phenomena of subsequent cycles.  
fHfects of repeated P.  M. S • . G.  treatment of  ewes were apparent as well  
as information on the  efficiency of synchronisation by intravaginal 
progestogen administration. 
Factors Affecting the Onset of Oestrus 
following �rogestagen Sponge withdrawal 
J .  P � M. S . G . administration 
Immediately followinG the cessat ion of progestagen treatment 
of ewes in Experiment 1 ,  some sheep were given P. M. S .  G.  This  did 
not significantly affect the t ime interval from sponge withdrawal to 
the onset of oestru s  (Table 3- 1 ) .  Distributions of the onset of 
oestrus  in ewes either given , or not given P .M. S , G. are shown in 
Fig.  3-1 .  
The number of ewer> showing overt oestrus immediately following 
sponge wi thdrewal was not affected by the administration of P.M .S  . •  G.  
(Table 3-3 ) .  
2 .  I njection of Progesterone. 
Synchronisation of ev1cs given a progesterone injection 2 deys 
before sponge withdrawal (Experiment 3) is compared with that of 
ewes given only M .A .P .  (Tables 3-2 and 3-4 and F ig. 3-2 ) .  There is  
a t emporal b ias in thi s comparison as the  two different t reatments  
were carried out at  different times of  the  year and in  different 
TABLE 3-1 
EFFECT OF P .M . S . G. ADMINIS�\TION ON YrlE Tihlli INTERVAL BET\qEEN 
PROGESTAGEN SPONGE ·vVITI-IDRAil?.L :.1-m THE ONSET OF OESTRUS 
�tment Number of ewes in Oestrus Total 
Dal':s from S£on�o ;'[i thdra_!!al Number 
of Ewos 
Day 2 Day 3 Day 4 Day 5 
Progostagon 16(47 .0"/ol 9 ( 26. 5%)  0 (23.  5% ) 1 ( 3. 0%) 34 
and P. M. S . G . 
Progestagen 13( 37 . 2% )  15(42. 8% )  7 ( 20 .Cff:, ) 0 35 
Alone 
Total 29 24 15  1 69 
G = 3. 264 - Not S ignificant 
TABLE 3-2 
;:;:FFECT OF 1. PROGESTERONE INJECTION ON THE TIME INTERVAL BEYviEEN 
P:aOGEST/.GEi'l' SPONGE VilTHDR/.W�.L /�ND YnE ONSET OF OESTRUS 
Treatment 
M.A. P • .1:-.Iono 
(Expt 1 )  
M. A . P .  and 
Progesterone 
(Expt 4 )  
Total 
G = 7 .76 - P <.0 .05 
Number of Ewes in Oestrus 
Days from Sponge V/1 thdrawal 
Day 2 Day 3 Day 4 
13( 37 . 2% )  15(42 .5%)  7 (20 .0%)  
20( 66 .7% )  9 ( 30.0%)  1 (3 .  3"/o) 
33 24 8 
Total 
Number 
of Ewes 
35 
30 
65 
.11.. ( )  Percentage of Ewes coming �nto oestrus that were in oestrus 
on the day indicated.  
T!..J3LE 3-3 
�:?FECT OF P .M .S , G. Ji.Di\UNIBTRt.TION ON TI:IE OCCURitENCE 
OF OESTRUS FOLLOWING PROG�STi .. GEN TR.:.Y.T!YiENT 
Treatment Number of 
Ewes Showing 
Overt Oestrus 
Progcstagen 34 
and P. M. S . G. · 
Progcstagen 
!.lone 
Total 
35 
69 
Number of Total 
E:wos Showing Number 
S ilent Oestrus of Ewes 
3 37 
8 43 
1 1  80 
CHI2 = 1 . 068 - Not S ignificant 
T/iliLE 3-4 
% of Ewes 
Showing 
Overt Oestrus 
9 1 . 9  
8 1 . 4  
!:i:FF;:!;CT OF !. PROGESTERONE INJ"GCTION ON THE OCCUlUllll�CE 
OF OZSTRUS FOLLOWING PROGi!:ST,.GEfi SPONGE TREJ'..TIIiENT 
Treatment 
M.l  .... Pr  
Alone 
i�'l • l-.. • P • and 
Number of 
Ewes Showing 
Overt Oestrus 
35 
30 
Progesterone 
Total 65 
Number of Total 
Swes Showing Number 
S ilent Oestrus of Ewes 
8 43 
10 40 
18 83 
2 
C"".til = 0. 193 - Not Significant 
% of Ewes 
Showing 
Overt Oestrus 
8 1 . 4  
75 .0  
F I G U R E  3-1 :-
DIST R I BU T I ON O F  ONSET O F  O ESTR U S  F O L LOWI N G SPO N G E  W I T H D R AWAL EXP E R I M E N T  ON E 
20 
A. Progestagen alone 
1 0  
en 20 ::l B. Progestagen and P . M . S . G .  er: f-en UJ 
0 
z 
en 1 0  UJ 
$. UJ 
LL. 
0 
er: UJ a:l 
::::! ::l z 
30 
C. Al l  ewes 
20 
1 0  
2 3 4 5 
DAYS A F T E R SPONGE W I T H D R AWA L 
F IGURE  3-2 :-
THE E F FECT OF  A PROGESTERONE I NJECTION ON  THE ONSET OF  OESTRUS OF  EWES 
G IVEN AN INTRAVAG INAL PROGESTAGEN TREATMENT 
20 
Cl) 
=> 
ex: 1 0  
1-Cl) w 
0 
z 
Cl) w 
� w 
u. 
0 
ex: 20 
w 
cc 
� 
=> 
z 
1 0  
2 
2 
3 4 
3 4 
A. M .A.P. and Progesterone ( Expt. 3) 
5 
B. M .A.P. alone ( Expt. 1 )  
5 
DAYS AFTER  SPONGE WITHDRAWAL 
41 
breeding seasons. Seasonal variation in owe response to  
progesterone is known to  occur (Lamond 1964c ) .  For this reason , 
tho comparison i s  not strictly valid but is  drawn because of  
indications that supplementation of M . ll.. . P.  treatment \7ith 
progesterone may bo effective in producing a more definea 
synchronisation .  
Progesterone supploment�tion o f  M.A . P .  treatment caused owes 
to come into heat ovor a &�orter time than if M,h, P.  was given alone. 
The dist ributions of the onsot of oest rus were significantly 
(P < 0.05 )  differ-mt for those two groups (Table  3-2 and Fig. 3-2) . 
Of those ewes showing oestrus , CO.O%  wore in oestrus over a 2 day 
period with M.l'. • .P. alone compered wi th 96 . 7% when Ivl. t  •• P. was 
supplemented with n progesterone injection (Table  3-2 ) .  
Supplementation o f  progestogen sponge treatment cid not 
significantly reduce the proportion of ewes showing silent heats 
(To.ble 3-4 ) .  
Effect o f  Sequential F . M . S . G .  Treatment on Synchronisation 
of Oestrous Cr,cles Subsequent to Progestogen Treatment 
1 .  Variation in the Time of Onset of Oestrus 
Treatments in Experiment 1 had a significant effect on the time 
of onset of oestrus,  one oestrou s cycle after sponge withdra\"Jel 
(P<:0.005 ). Treatments 1 and 2 caused a greo.ter dispersion in the t ime 
period in which ewes came into oestrus than did Treatments 3 and 4 
(Table 3-5 end Fig. 3-3 ) .  This trond was al so apparent two cycles 
from sponge withdrawal (Table 3-6 and Fig.  3-4) ,  but was less 
marked (P <:0.01 ) .  
Ti' .. BLE 3-5 
TIU.::i.TI\1ENT EFFSCTS ON THE �N.flill21HGi�TION OF ogsTR.US 1 
.Qt!_� _ _22;;STi:WUS CYCLG F�10I'ii PROG3ST/.GEIJ TRCLTMBNT: - EXPERilvlENT 1 
Treatment Dose Days from the first  display of Oestrus Total 
Number 
of Ewes 
(F . M. G . G  .. ) 
0 1 2 3 4 - 10 
1 L 3 2 2 0 3 
H 2 2 1 0 4 
Total 5 4 3 0 7 
2 L 0 1 6 1 1 
H 1 1 3 2 2 
Total 1 2 9 3 3 
3 L 0 4 3 2 0 
H 1 3 3 0 0 
Total 1 7 6 2 0 
4 L 1 1 5 2 0 
H 0 1 6 2 0 
Total 1 2 1 1  4 0 
Total 15 29 9 10 
Test of�epenctense:  G Statistic D . F .  
Treatment x Dose x Distribution 31 
Treatment x Distribution 12 
Dose x Distribution 4 
Treatment x Dose x Distribution Interaction 12 
10 
9 
19 
9 
9 
9 
7 
16 
9 
9 
10 
7 1  
G Significance 
43 .238 p (.0 .05 
35 . 8 18 P <:o.oos 
0 .088 Not 
S ignificant 
C . 848 Not 
Significant 
TABLE 3-6 
TP.:Y.TMI.mT :!:FFECTS ON THE SYNCHRONlShTION OF OESTH.US , 
T\'10 0.2:STROUS CYCLES FROM PROGESTi�GEN 'ffi&.TMENT : - l!:XPERIMENT 1 
Treatment Dose Days from the first display of Oestrus Total 
(P . M. S . G. ) Nur.1ber 
of Ewes 
0 - 7 8 9 10 1 1  - 16 
1 L 3 3 0 0 2 8 
H 0 3 0 0 5 D 
Total 3 6 0 0 7 16  
2 L 0 2 2 2 2 8 
H 0 4 0 2 2 8 
Total 0 6 2 4 4 16 
3 L 0 4 2 0 1 7 
H 0 2 3 0 0 5 
Total 0 6 5 0 1 12  
4 L 0 2 4 2 1 9 
H 0 5 0 1 1 7 
Total 0 7 4 3 1 16 
Total 3 25 1 1  7 14 70 
Test of Inde;eendence :  G Statistic D. F .  G SiJ:£!ificance 
- -
Treatment x Dose x Distribution I ndependence 31  56 . 526 P <0.01  
Treatment x Distribution 12 35.608 P(O.Ol 
Dose x Distribution 4 0 . 322 Not 
Significant 
Treatment x Dose x Distribution x Interaction 12 6 . 346 Not 
S ignificant 
(/) => a: I-(/) w 
0 
z 
(/) w 
::-: w 
u. 0 
a: w Ill 
� => z 
F IGURE 3-3:-
E F F E C T  OF R E PEAT E D  G O N A DO T R O P H I C  ST I M U L AT I O N  O N  
SYN C H RO N I SAT ION O F  O E S T R OU S  C YC L E S  
A. One cycle after sponge withdrawal 
10  
0 
1 0  
0 
10  
0 
1 0  
2 3 4 5 6 7 
DAYS FROM F I RST EWE  IN OESTRUS 
10 
0 
1 0  
0 
1 0  
0 
1 0  
0 
10  
B. Two cycles after sponge withdrawal 
12  1 3  
DAYS FROM F I RST EWE  IN  OESTRUS  
T R EATM E N T  
CONTROL 
2 
3 
4 
8 9 1 0  
CONTROL 
2 
3 
4 
1 4  15 1 6  
42 
The effect of  doses of P . M. S . G .  on the temporal distribution 
of onset of oestrus  was not significant at either of the two periods.  
Table 3-7 gives the results  of sequontially treated , laparoto­
mised ewes (Experiment 3 ) .  Uninjected owes still  came into  heat 
over a 1 day period whereas treated ewes were dispersed over 0 days . 
Laparotomy did not appear to affect the time of onset of oestrus . 
2 .  Manifestation of  Oestrus 
All ewes in Experiment 1 aisplayed overt oestrus  at the second 
oestrous period from sponge withdrawal.  fo.t the final observation , 
more  intensive P . M. S . G. treatments (Treatments 1 and 3 )  caused a non-
significantly  greater number of m;;es to  experience silent oestrus 
(Table 3-0 ) .  
Data from Experiment 4 (Table 3-9 ) showed that increasing 
numbers of sequential P . IYi . S . G. treatments  enhanced the incidence of 
silent oestrus (i=- < 0 . 05 ) . This effect does not appeo..r to have been 
duo to the number of laparotomies as control owes which \'!ore 
laparotomisod three times ,  continued to exhibit overt oestrus . 
CI-I!.?TEi1 SUMJVJARY 
1 .  i"a.dministration o f  � .M . S . G.  immediately following sponge with­
drawal had little  effect on oestrous behaviour at the first  
subsequent oestrous period. 
2. �n intramuscular injection o f  10 mg. progesterone ,  2 days before 
sponge withdrawal , may cause more effective synchronisation but 
probably does not decrease L�e number of ewes showing silent oestrus . 
These results  are not conclusive for want of appropriate  control 
animals.  
3.  Oestrous cycles of owes given progestagen via  the intravaginal 
route may remain effectively synchronised for 3-4 cycles. This 
effect is disrUpted by the administration of P. M. S .  G. 
TABLE 3-7 
Tiillt.TI11ENT �FFECTS ON THE r/JANIFESTi.TION OF OESTRUS : - EXPERIMENT 1 
Treatment Dose 
(P .M . S . G. ) 
1 L 
H 
Total 
2 L 
H 
Total 
3 L 
H 
Total 
4 L 
H 
Total 
Total 
Number of 
Ewes Having 
Overt Oestrus  
8 
8 
16  
8 
8 
16 
7 
5 
12  
9 
7 
16  
60 
G = 1 1 . 1 10 - Not S ignificant 
Number of Total Number 
Ewes Having of Ewes 
S ilent Oestrus  
2 10 
1 9 
3 19 
1 9 
1 9 
2 18  
2 9 
2 7 
4 16 
0 9 
2 9 
2 18  
1 1  7 1  
Ti .. BLE 3-8 
EFFECT OF SEQUENTIAL P. M. S • G. TREA'I'MENT ON 
MJJHFJ:STJ..TION OF OESTRUS 
Trco.tment 
Injection 
at 1 cycle 
Injection 
at 2 cycles 
I njection 
at 3 cycles 
Uninjectcd 
Control 
Total 
Number of 
�wes Showine 
Overt Oestrus 
10 
8 
6 
9 
33 
G = 8. 128 P (. 0.05 
Number of 
Ewes Showing 
Silent  Oestrus 
0 
2 
3 
0 
5 
Total Number 
of Ewes 
10 
10 
9 
9 
38 
% Ewes 
Showing 
Overt 
Oestrus 
100.0  
oo.o  
66. 7 
100.0 
C H � ? T E R IV 
0 V A R I A N n E S P 0 N S E T 0 
_P_. �_la. s_.�G-• ._�I __ N_�3 Q U E N T I A L L Y 
T R E A T E D E W E S 
43 
Chapter IV 
O V A !l ! A N  a E S P O N S B T 0 P . rA. -S ._,Q. 
I N S E Q U E N T I A L L Y E '!l E S 
This chapter reports ovarian response data of ewes in 
2xperiment 1 .  Some ovulation data from Bxparimont 3 i s  also 
included . Cbservations of  overies from sheep in Experiment 1 were 
mede o.t the end of tho experiment and are thus ' terminal ' responses 
of ewes to the treatments imposed .  The ovaries of sheep in 
F.xperiment 3 were observed ut a series of laparotomies and 
post-mortem .  
Results arc presented as  transfor�ed data and raw data are 
Given in �ppendix 1 .  
Transformation o f  Discrete Data 
Bartlett ' s tests on ovulatory data (number of corpora lutea) 
revealed that variances of the 0 treatment groups were heterogeneous 
(Appendix 2 ) .  The most suitable transformation for data involving 
counts and zero values i sJx + 1 (Sokal and Rolf  1969 ) .  f-dter this 
transformation had been applied to the number of  corpora lutea per 
0\"le ,  Bartlet t ' s test showed that heterogeneity had been reduced but 
variances just failed to  attain homogeneity for this variable.  As  
this transformation was considered the most likely to produce 
homogeneity ,  no other transformations  were t ested .  I n  the light of 
. the marked reduction in heterogeneity attained , all discrete  data 
wore t ransformed to� for analysis of  variance. For ease of 
computation , the transformed data wore increased by a factor of 10 . 
F IGURE  4-1 :-
EFFECT OF  SEQUENTIAL P.M .S.G.  TREATMENT ON THE  NUMBER OF  EWES OVULATING 
(!) 
z 
..... 
< ...1 
:::> 
> 
0 
Cl) w 
3: w 
� 
1 00 
80 
60 
40 
20 
L 1 ' L 2 L3 L4 H 1  H2 H3 H4 cc 
TREATMENT GROUP 
TABLS 4- 1 
�FFE:CT OF SEQUENTIJ.L P. 1'1:., S .  G ,  TRE!. TMENT OF THE 
NUMBBn OF OVUL/;.TING 
A .  Experiment 1 
�==---·--=-=·· 
Treatment Dose Total N��ber of Ewes Number of  Ewes Ovulating 
r::::.=,::1:·:::-=::::r.==-·=31=>7'" ..�. 
1 
Total 
c._��-........,. 
2 
Total 
3 
Total 
4 
Total 
Grand Total 
L 
H 
L 
H 
L 
H 
L 
H 
10 
9 
19 
9 
9 
10 
8 
7 
15 
9 
9 
13 
70 
Test of Independence : G St3tistic 
Ewes Ovulating x Treatment x Dose Indep��dence 
Ewes Ovulating x Treatment 
Ewes Ovulat ing x Dose 
3wcs Ovulating x Treatment x Dose Interaction 
Ewe Troatmont 
1 ( 3  injections ) 
2 (2  injections ) 
3 ( 1  injection) 
G = 6.7 16 P <o.os 
Total Number of 3wes 
9 
20 
30 
7 
6 
13 
9 
!3 
17 
5 
2 
7 
8 
9 
17 
54 
Significance 
P < 0 . 05 
P<  0. 01  
N.S. 
N . S .  
Number of Ev10s 
Ovulat ing 
7 
18  
30 
44 
Ewes Ovulating 
The presence of a corpus luteum was taken as denoting that 
ovulation had occurred . Table 4-1a shows that t reatments of 
Experiment 1 significantly  (P ( o .o l )  affected the numbers of ewes 
ovulating. Fewer owes ovulated when given sequential treatments 
with P. lvi. S . G. (Treatments 1 and 3)  than \'lhcn given a single injection 
(Treatment 4 )  or stagg�red inject ions (Treatment 2 ) .  The effects of 
dose and dose x tre£J.tment interaction were not s ignificant. Those 
results  are displayed in Fis. 1-1 .  
I n  3xperiment 3 1  incrcLsinr; numbers o f  sequential treatments 
nith P.M . S . G . increased the pro·Jortion of e·:ves which were anovular 
(P  (0.05 ) .  The effect i s  pnrtly masked (Table  4-lb ) because f c;mer 
<mes wore observed us the number of injections increased.· Furthermore , 
the stress of laparotomy may have enhanced the nnovular condition in 
these owes .  
Incidence of C]Lstic Ovaries 
�reatment , dose and trcatmQ�t  x dose interaction had no effect 
on the proportion of ewes in !:i.:xporiment 1 which developed cystic  
ovaries (Table 4-2 ) .  Ewes having cystic ovaries were only seen in 
Treatments  1 and 3 ( sequent ial treatments with P . M. S . G. ) .  The 
cystic concli tion evident in a evlc on Treatment  H3 is seen in Fig .  4-2.  
Ovulation rate 
1 �  Corpora Lutea � Al l ewes (Experiment 1 )  
Ovulation rate  was determined by counting the number o f  corpora 
lutea per owe.. Treatments  of Experiment 1 significantly affected 
(P (0 •. 05 ), the number of corpora lutea per ewe thnt wore observed at 
Fig.  4-2 : - Cystic condition of the ovary ( .thi:;  ovc.ry 'bakon 
from n ene on Trt. H3 , Expt . 1 )  

TABLE 4-2 
EFFECT OF SEQUENTIAL P.M . S .G.  TREATiV:ENT ON THE 
.!l!£!DENCE OF CYSTIC OVl';.::UES 
Tr2ntmcnt Do so  Total Number of  E\:;os 
1 L 10 
H 9 
Total 19 
2 L 9 
H 9 
Totc.l 18 
3 L 8 
H 7 
Total 15  
4 L 9 
H 9 
Total 18  
Grand Total 70 
Test of Independence : G S tatistic 
Number of  3wes with 
Cystic Ovaries 
1 
0 
1 
1 
0 
1 
1 
2 
3 
0 
0 
0 
5 
Significance 
Cystic Condition x Treatment x Doso Independence N . S .  
Cystic Condition x Treatment N . S .  
Cystic Condition x Doso N . S .  
Cystic Condition x Treatment x Dose Interaction N . S .  
---��------------�· ----�·�----------------------------
45 
slaughter (Table  4-3 ) ,  Effects o f  dose level and dose x treatment 
interaction were not s ignificant , Raw clata is  plotted in Fig. 4-3 
and Fig .  4-4 present s the same data aft er transformation, 
Comparison of t reatment moans 1 by the S tudent-Newman-i(otlls  
tes t , is  outlined in ;.pponC.ix 3.  Treatment  1 caused significent ly 
lower terminal ovulation ro.tes than Treatments 2 ( P<: O, Ol ) and 
4 (P< 0, 01 ) ,  Treatment 4 proc.1uccd significant ly greater ter:;1inal 
ovulation rates than Treatment 3 (P< o . 05 )  (Table  4-5 ) .  Differences 
in all other t rontment  comparisons were not significant ,  
2 .  Corpora Lutea : Ev10s ovule.ting (Experiment 1 )  
Consideration o f  the ovulat ion rates of  those ewes ovulating 
(Table 4-4 ) revealed that treatment effects remained significant 
(P < 0 ,05 ) .  Treatments 2 1 3  nnd 4 caused significantly greater 
t erminal ovulation rates per ewes ovulatin[S than Treatment 1 .  
(P < 0 .05 1  P < 0 .0 1  and P <0,05 respectively ) (Table  4-6) . Date. from 
these ewes are presented in Fig.  4-9 and Fig .  4- 10. 
3 .  Corpora Lut ea : All  ewes (Experiment 3 )  
Table  4-7 and Fig.  4- 17 display data of  ewes given 1 1 2  or  3 
sequential injections of P .M. S , G . , compared with control animals.  
idter one st imulation ,  the  treated ewes had significantly  greater 
(P < 0.001 ) ovulat ion rat os th£m the control animals.  Ovulation 
rates of ewes t reated 2 or 3 times with P. M.S.G. did not differ 
from those of cont rol animals  observed at the same t ime. 
F I G U R E  4-3:-
E F F ECT O F  S E QU EN T I A L  P.M.S.G.  T R EATM E N T  ON EWE OVULATION R A T E  : R A W  DATA 
<( w f-. => 
..J 
<( ex: 
0 c.. ex: 
0 u 
LL 
0 
ex: w al 
� ::::> z 
1 0  
8 
6 
4 
2 
0 1 000 1 500 
F I G U R E  4-4 :-
L E G E N D  
TR EATM E NT SYM BO L  
1 • 
2 • 
3 0 
4 0 
CONT R O L  .. 
P.M.S.G.  ( i . u . )  
E F F ECT O F  SEQU E N T I A L  P.M.S.G.  T R EATM E N T  ON E W E  OVU LATI ON RATE : TRANS F O R M E D  DATA 
30 
0 
>< 
� 
<( w f-::::> 
..J 
<( 20 ex: 
0 c.. ex: 
0 u 
LL 
0 
ex: w al 
� ::::> z 
0 1 000 1 500 
P . M .S.G.  ( i .u . )  
TABLE 4-3 
OW.RIP..N RESPONSE : /.LL E\iES 
2stimated Moans and S t andard Errors 
Variable Means S tandard Errors 
DOSE TREATMENT 
Low High 1 2 3 4 
Corpora Lutea 21 . 56 2 1 . 07 14 . 61 26 . 43 13 .41  25. 81  
( transformed counts ) .:!:,1.  74 .:!:,1 .79 .!,2 . 40 �2 . 47 ±2 . 70 .:!;.2 . 47 
Corpora Lutea + 26 . 66 3 1 . 53 22 . 75 28 . 77 33. 26 3 1 . 58 
Follicles > 3mm. !1 . 65 .:t,1 . 70 _:!:2 . 28 .:!,:2. 33 .:!:,1 . 8 1  .t2 . 33 
( transformed counts ) 
Ovarian weight 7 . 35 9 . 63 5 . 87 6. 69 1 1 . 09 10. 31  
(Gms . ) :t,l . 17 ±1 . 20 !1 . 61 ±1 . 65 ;:2 . 56 !:,1 . 65 
Analysis  of  Variance 
Source of D. F .  Mean S quares 
Variat ion 
Corpora Corpora Lutea + Ovarian 
Lutea Fol licles > 3mm. weight 
Dose 1 4 . 14 410 . 58 ** 90 .04 
Treatment 3 603. 97 ** 376. 0 1  * 1 1 5 . 43 
Dose x t reatment 3 143. 88 1 1 1 . 50 55. 8 1  
Residual 62 109 . 43 90 . 36 49. 20 
• P <0.05 
•* P <0.01 
Tf.BLE 4-4 
OVARIAN RZSPONSE : EHES OVULii.TING 
Estimat ed Means e.nd S tandard Errors 
Variable  
Corpora Lutea 
( trensformed counts ) 
Corpora Lutea + 
follicles ) 3mm. 
( t ransformed counts )  
Ovarian weight 
(Gms, ) 
i�.nalysis  of Variance 
Source of 
Variat ion 
D. F .  
Dose 1 
Treatment 3 
Treatment x dose 3 
Residual 46 
* P (. 0,05 
•.• P (  0,01  
Neans 
DOSii: 
Low High 
24 . 18 25.70 
+1 . 93 +2 .08 
27 . 37 30, 85 
!_1 . 82 !,1 . 95 
7 , 37 11 . 68 
!o 28 .;t1 . 30 
Corpora 
Lutea 
4, 25 
359 ,,67 * 
54 . 21 
108,-54 
and S tandard Errors 
� 
TREA. TIVIENT 
1 2 3 4 
16. 89 27 . 45 28 , 98 26. 69 
:!;,.2 . 09 :t_2 . 52 ::!:,3. 93  ±_2, 52 
19 . 97 29 . 98 34 . 23 32 . 32 
;!:.2 . 72 !ja .  38 ;t3. 70 �. 38 
4 . 60 6. 97 13  .. 51  10. 73 
±.1 · 93 .±,1 . 60 !,2.:62 �1 . 60 
Mean Squares 
Corpora Lutea + Ovarian 
Foll icles 3mm. weight 
200. 43 200.96 * 
516. 53 ** 216 . 32 ** 
65 . 18 7 1 . 89 
96, 25 48 .. 04 
TI.BLE 4-5 
OVARI!.N fui.:SPONSE OF t.LL Eii'ES (EXPERIMEN!.Jl 
Compe.rison of Treatments Difference of means 
Corpora8 Corpora Lutea +8 Ovarian 
Lutea Follicles ) 3mm. Weight 
TRT 1 VS TRT 2 1 1 . 82 ** 6 .02 0. 02 
1 1  1 1 1  3 3 . 80 10. 51 * 5. 21  VS  
1 1  
1 
" 
1 1 . 02 ** 8 . 03 * 4. 43 vs 4 
" 2 " 3 8 .02 4 . 48 4 . 30 VS 
" 
2 " 4 0 . 62 2 . 01 3, 61 vs 
" 3 " 4 7 .  39 * 1 . 67 0.77 VS 
LOV/ dose vs High dose 0 ,40 4 . 06 * 2 . 2C 
TABLE 4-6 
COiVIPf.RISON OF MEJlNSA : OV!.RH.N il.ESPONSE OF EWES OVULf�TING 
(EXPERIMENT 1 )  
Comparison of Treatments Difference of  means 
Corpora8 Corpora Lutea + 8 Ovarian 
Lutoa Follicles > 3r.un. Weight 
TRT 1 VS Tt1T 2 10. 55 * 10 .00 2, 10 
" 
1 
" 
3 12 ,00 ** 14. 26 ** 0 .90 ** vs 
" 1 1 1  4 10. 55 * 12. 34 **  6. 12 * VS 
" 2 " 3 0.76  4 . 25 ** 6. 54 * VS  
" 
2 " 1 . 54 2, 34 3.76 vs 4 
" 
3 
1 1  
2 . 2 1  1 . 9 1 2 . 77 vs 4 
Low dose vs Hig.'l dose 1 ,  6 1  3, 46 4 . 31 . 
* P ( o.os A - Student-Ne\vman-Keuls Test 
** P < 0.01  B - Difference in Transformed Means 
TABLE 4:-7 
EFfi3CT OF S3C�WNTIAL P .M . S . G . 'ffii3:�NT1 COliiPARED iHTH 
UNTR�t.TED Ei:ms : CORPORli. LUTEL (TRJ'.NSFORMED DATA/ 
OBSEFI.Vi.TION MEAN NillVJBER OF COHPOR:. L!JTEA /;.Nfi.LYSIS OF V t.RIJ..NCE 
��� 
STD. ERROR (/X+l) 
P.  M. s .  G·. T.:t:.<;ATEDB CONTROLC MEAN SQUA.F.J�;s F 
(30)D 
**�:' 
1 25. 90 14 . 45 ( 10)  Between = 901 . 97 19. 83 
+ 1 .  20 :!;.2. 22 Hi thin = 49. 50 
2 16. 41  (20)  13. 59 ( 9 )  Between = 50�07  2 .;07 
:!;.1 .09 ,:!::1 . 63 Within = 24. 16 
3 13 . 57 ( 9 )  16 . 44 ( 9 )  Between = 37 . 13 1 . 93 
!, 1 . 46 !1 . 46 Vlith:i.n = 19 • 19 
TABLE 4-0 
EFF3CT OF SEQUENT!i�L P , l\1 , S .  G. 'ffi3/.T!Vlli:NT COMPi.RED WI'lli UNTI1Ei'l.TED �'lES 
COP..POB.io. LUTEi� PLUS FOLLICLES GI1El .. TEI1 THJ.N 1 mm. ( TRh.NSFORlV:iED D!.T/;. ) 
i':. 
OBSi3RVJ.TION MEAN NUMBER OF CORPOlli.. LUTEA+ ANALYSIS  OF VJ.RIANCE 
FOLLICLES 1 mm. + STD .  ERROR (JX+ l )  
P ,  M. S ,  G.  TREATE:D B CONTROLC MEAN SQUARES 
1 43 . 38 (30)D 39. 25 ( 10)  Between = 127 . 92 
!, 1 . 90 !3 . 29 V!ithin = lOO . 73 
2 35 . 17 ( 20)  32 .03 ( 9 )  Between = 60. 89 
_:t2.02 ;t3.02 \'li thin = 02. 26 
3 32 .03 (9 )  37 . 17 (9 ) Between = 1 18. 69 
:+_:3. 25 !;3. 25 Within = 95. 23 
A - Experiment 3 data 
B - Treated once , twice or three times respectively with P.M. S . G. 
before observations ono , two and three. 
C - The same animal s  laparotomised twice and then killed .  
D - ( )  Number of ewes/treatment group. 
••• P <.  o.cxn 
F 
1 . 17 
0.74 
1 . 24 
Fig.  4-5 : - Sul,)crovulatecl ovaries taken from a ewe given one 
injection of P . M. S . G. ( Trt . H4 , Expt . 1 ) .  36 ovulations . 
Fig.  4-6 : - Inactive ovaries of u ewe in Treatment El  (Expt . 1 ) .  
Corpora albicans denote response to previous 
gonadotrophic  stimulation. 

Fig.  4-7 Ovaries  observed at Day 10 of the oestrous cycle .  
Ewe 6 - control , e�e 2 2  - superovulated .  
F i g ;  4 - 8  Ovarian slices showing luteinisut ion o f  fol l icles .  
No. 12 - non-ovulatory . No ' s 18 and 23 contain 
corpora lutea. 

46 
Examples of superovulated ovaries from ewes in Experiment 1 
( taken 6 days after the onset of oestrus )  are shown in Fig.  4-5 and 
4-6. Ovaries of ewes in Experiment 3 ( 10 days after the onset of 
oestrus ) are seen in Fig. 4-7 . The cross sections seen in Fis .  4-8 
are through luteinised follicles,  At  Day 10 of the oestrous cycle ,  
luteinised follicles may resemble  corpora lutea but the two are 
cistinguished by the presence or absence of an ovulation point . 
Corpora L�tca�lus follicles 
1 .  Corpora Lutea + follicles greater than 3 mm. : All ewes 
(Experiment 1 )  
Both treatment and dose hcd significant effects (P< 0. 05 and 
P <0 .0 1  respectively) on this measurement (Table  4-3) . Interaction 
between the two main effect s was not significant . The number of 
corpora lutea + follicles ) 3 mm. of ewes on Treatment 1 was 
significantly  lower than owes on Tre:::.tr.wnts 3 (P < 0.05 )  and 4 
(P ( 0, 05) . The high dose of P . �r. . S . G .  ( 1500 i . u . ) caused a 
significantly greater response (P  ( 0.01 ) than did the low dose 
( 1000 i . u . ) 
These data are plotted in Fig.  4- 1 1  and 4- 12 .  
2 .  Corpora Lutea + follicles greater than 3 mm . Ewes ovulating 
(Experiment 1 )  
When data from ovulating ewes only  are analysed (Table  4-4 ) 1  
treatment and dose effects  remain significant (P < 0 .05 and P ( 0.01 1 
respectively ) .  Treatments 2 1 3  and 4 produce significantly greater 
response in this measurement than Treatment 1 (P < o.ol)  (Table 4-6 
and Figs . 4- 1. 3  and 4-14 )__.. 
3 .  Corpora Lutea + follicles gpeat,t:;r thnn 1 mm. : All ewes 
(Experiment 3 )  
Ewes treated once , twice o r  three times with P . M, S . G .  do no� 
F I G U R E  4-9 :-
E F F ECT OF S EQU E N T I A L  P . M .S.G.  TR E ATM ENT ON OVU LA T I O N  RATE 
OF EWES OV U LATI N G  : R AW DATA 
<( UJ 1-::::> ....I 
<( a: 
0 Q.. 
1 2  
1 0  
8 
a:: 6 
0 u 
LL 
0 
a:: UJ 4 CO 
� ::::> z 
2 
1 000 
FI G U R E  4-10 :-
L E G E N D  
TR EATME N T  SYMBO L 
1 • 
2 • 
3 0 
4 0 
CON T R O L  .& 
1 500 
P.M.S.G.  ( i . u . )  
E F F ECT O F  SEQU E N T I A L  P.M.S.G.  T R EATM ENT ON OVU LAT ION RATE O F  
EWES OVU LATI NG : T RANSFO R M E D  DATA 
30 
0 
..... 
X 
� 2 5 
<( UJ 1-::::> ....I 
<( 20 a:: 
0 Q.. a: 
0 u 
LL 
0 
a:: 15  UJ CO 
� ::::> z 
1 0  
1 500 
P.M . S . G .  ( i . u . )  
F I G U R E  4-1 1 :-
E F F ECT O F  SEQU E N T I A L  P . M .S.G.  T R EATM E N T  ON TH E N U M B E R  O F  C O R P O R A  L U T E A  
P L U S  F O L L I C L ES G R EAT E R  THAN 3 mm. I N  A L L  E WES : R AW DATA 
(/) 15  
::::> 
..J E a.. E 
L E G E N D  
<t M 
UJ z 1-
::::> <t 
..J J: 1 0  1-
TR EATM E N T  SYM BO L  
1 • 
<t a: a: 
0 UJ 1-a.. <t a: 
0 UJ a: (.) (.!) 
2 • 
3 0 
4 0 
u. (/) 5 0 UJ 
a: ..J 
UJ (.) 
CONTROL • 
al ..J 
� ..J 
::::> 0 
z u. 
0 1 00 0  1 500 P.M.S.G.  ( i . u . )  
F I GU R E  4-12 :-
E F F ECT OF SEQU E N T I A L P.M.S. G .  TR EATM E N T  O N  T H E  N U M B E R  O F  C O R P O R A  L U T E A  
P L U S  F O L L I C L ES G R E AT E R  THAN 3 mm.  I N  A L L  E W E S  : TRANSF O R M E D  DATA 
(/) UJ 
..J 
(.) 
0 .... 
X· 
40 
� 
� 30 
..J 
a.. 
<t E 
UJ E 
1- M ::::> 
..J � 20 
<t J: a: 1-
� a: 
a: UJ 
0 1-
(.) <t UJ u. cc 0 (.!) 
cc 
UJ 
al 
� 
::::> 
z 
1 0  
0 1 000 1500 P.M.S . G .  ( i .u . )  
en 
::> 
...1 
a.. 
<t w 
1-
::> 
...1 
<t 
a: 
0 a.. 
a: 
0 
u 
u. 
0 
a: 
w 
CO 
� 
::> 
2 
en 
w 
...1 
u 
...1 
...1 
0 u. 
en 
::> 
...1 a.. 
<t 
w 
1-
::> 
...1 
<t 
a: 
0 a.. 
a: 
0 
u 
u. 
0 
a: 
w 
CO 
� 
::> 
2 
F I G U R E  4-13 :-
E F F ECT O F  SEQU E N T I A L  P.M.S.G.  T R EATM ENT O N  T H E  N U M B E R  O F  C O R PO R A  L U T E A  
P L U S  F O L L I C L ES G R EAT E R  THAN 3 mm.  I N  A L L  E W ES OVU LAT I N G  : R A W  DATA 
1 5  
E 
E L E G E N D  
M 
2 
<t T R EATM E N T  S Y M B O L  
J: 
1-
a: 
1 • 
1 0  
w 
1-
2 • 
<t w 
a: 
3 0 .... 
(!) 
en 
4 0 
w 
...1 
u CONTR O L  
... 5 
...1 
...1 
0 u. 
0 1000 1 500 
P.M.S. G .  ( i . u . )  
F I G U R E  4-14:-
E F F ECT O F  SEQU E N T I A L  P.M .S. G .  T R EATM ENT O N  T H E  N U M B E R OF C O R P O R A  L U T E A  
P L U S  F O L L I C L ES G R E AT E R  THAN 3 mm. I N  EWES OVU LAT I N G  : TRANS FO R M E D  DATA 
40 
0 .... 
X 
� 30 
E 
E 
M 
2 20 <t 
J: 
l-
a: 
w 
1-
<t w 
a: 
(!) 
10  
0 1 000 1 500 
P.M .S . G .  ( i . u . ) : 
F I GU R E  4-15 :-
E F FECT OF SEQU E N T I A L  P.M.S.G.  TR EATM ENT ON EWE OVAR IAN WE I GHT : ALL EWES 
15 Grams 
L E G E N D  
TR EATM E N T  SYMBO L  
1-
:I: 10  (.!) 
1 • 
w 
::: 
2 • 
2 
: 
<t .. a: 
<t 5 > 0 
3 0 
4 0 
CONTRO L ... 
0 1000 1500 P.M.S.G. ( i .u . )  
FIGU R E  4-1 6 :-
E F FECT O F  SEQU ENTIAL P.M.S.G. TR EATM ENT ON EWE OVA R I A N  WEI GHT : EWES OVU LAT I NG 
1-:I: (.!) 
w 
::: 
2 
<t 
a: 
<t > 0 
15  Grams 
10  
5 
0 1000 1500 
P.M.S.G. ( i .u . )  
<( w 
1-
::> 
..J 
<( 
0: 
0 c.. 
0: 
0 
u 
u.. 
0 
0: 
w 
cc 
� 
::> 
z 
<( 
w 
1-
::> 
..J 
<( 
0: 
0 c.. 
0: 
0 
u 
u.. 
0 
0: 
w 
cc 
� 
::> 
z 
F IGURE  4-17 :-
COMPAR ISON OF  EWES SEOUENTIALLY TREATED WITH P.M.S.G.  AND 
CONTROL EWES : OVULATION RATE 
1 0  
8 
6 
4 
2 
1 2 
OBSERVATION 
F IGURE  4-18 :-
3 
L EG END  
CONTROL 
P .M .S.G.  
COMPAR ISON OF EWES SEOUENTIALLY TREATED WI TH P.M .S .G .  AND  CONTROL 
EWES : CORPORA LUTEA PLUS FOLL ICLES GREATER  THAN 1mm .  
E 
E 
..-
z 
<( 
::c 20 
1-
0: 
w 
1-
<( 
w 
0: 
(!) 1 0  
Cl) 
w 
..J 
u 
..J 
..J 
0 u.. 
+ 
1 3 2 
OBSERVATION 
47 
sign ificantly differ from control animal s  in the number o f  corpora 
lutea + follicles ) 1 mm. These data are presented in Table  4-8 and 
Fig . 4- 18.  
Ovarian \/0ight  
1 .  £varian weight /1.11 eves (Experiment 1 )  
Treatments  of  Experiment 1 exerted no significant effects  on 
ovarian weights  (Table 4-3) . Dose effects and dose x t rcatment o 
interaction were also not significant . 
2.. �an weight : Ewes ovulating (Experiment 1 )  
By neglect ing non-ovulat ing ewes , significant treatment 
(P <: 0.0 1 ) and dose (P < 0.05 )  effects were revealed in the analysis 
of ovarian weights .  �wus o f  Treatment 1 had lower ovarian weights  
than ewes of Treatments 3 ( P <O .O l ) and 4. (P <..0 . 05 ) .  Treatment 3 
produced greater ovarian weights  than Treatment 2 (P <:0. 05 )  and 
the high dose of P .M. S . G . increased ovarian weight more than the 
low dose.  Mean ovnrinn weights of  the treatment groups are shown in 
Fig.  4- 16 .  
3.  Dependence of Ovarian i'leight  on Ovarian Act ivity 
Regression analysis on al l ewes in Experiment 1 (Table 4-9a ) 
showed that ovarian weight was significantly affected by the number 
of corpora lutea (P < 0 • .001 )  and even more so by the number of corpora 
lutea + fol licles ) 3 mm. (P < 0.001 ) The regression analysis  
presented in this table is for an  overall  relat ionship.  The 
individual regression coefficients for each t reatment group are 
given in Table  4-9b . 
40 
There was no significant relationship between ovarian weight 
und the number of corpora lutea , or the number of corpora lutea + 
foll icles ) 3 mm. 1 in the control animals .  Large posi tivo 
regression relationships wore seen between ovarian weight and 
number of corpora lutea and between ovarian weight and number of  
corpora lutea + follicl0s ) 3 mm. , in OVJes treated once. Tnese 
counteracted the small ,  and negative regressions of other single  
eroup relationships to produce the  overall  regression relationship 
soon in Table  4-9n, 
Ewe ovarian weight wo.s not significant ly affected by the 
number of  corpora lutea present  when ewes wore given throo 
sequential  injections but if  fol licle  counts V/Cre also considered a 
significant positive rcgreGsion was obtained . 
Difference in Ovulation ftnto between the Hight and Left Ovaries 
Ewes of Experiment 1 ovulated more often on the loft ovaries 
( 185 corpora lutea)  than on the right ovaries  ( 163 C. L. ) 
2 
(Chi = 1 . 267 1 N . S , ) .  This trend was reversed when ewes of 
GXperimcnt 3 were considered . I n  these latter ewes the total 
number of corpora lutea observed on the right  ovaries was 147 , the 
. 2  
l eft  ovaries having 135 (Ch� = 0 .4290 ,N . S . ) .  As  neither of these 
trends were statistically significant , there is no evidence that 
the right  ovary is more active than the left . 
Tt..BLE 4-9 
RELATIONSHIP BET'/!E�N OV!.Rlt.N ·:!EIGHT 11.ND OVJUUAN i.CTIVI TY 
REGHESSI ON i�Ni:..LYSIS 
A. Ovcrull R�ossions 
!"!ELiiTI ONSHIP TESTED 
DEPENDENT 
Vf'.RI.'.BLS 
Ovarian ";it . 
1 1  
INDE�ENDENT D .F .  RZGR. 
V /;.RI/.BLE C02FF , 
Corpora Lutoa GD 0. 3952 
Corpora Lut ea + 69 
Follicles)3mm. 
0. 4793 
B . Groue �egressions 
RZLt.TI ONSHIPS TESTED 
DEPENDENT INDEPENDENT GROUP D .  F .  REGR. 
Vl.RI/..BLE VARii.BLE COEFF . 
OVli..RIJ".N VIT, Corpora 
" 
Lutea 
1 1  1 1  
1 1  " 
1 1  " 
" 1 1  
" " 
1 1  " 
" " 
" 
Corpora Lutea 
1 1  + Follicles ) 3mm 
" 1 1  
" 1 1  
" " 
" " 
" 1 1  
" " 
" 1 1  
L l  
L2 
L3 
L4 
Hl  
H2 
H3  
H4 
cc 
L1 
L2 
L3 
L4 
H1  
H2  
H3  
H4 
cc 
0 
7 
6 
7 
7 
7 
5 
7 
7 
0 
7 
6 
7 
7 
7 
5 
7 
7 
0.0401 
o. 2679 
0 . 2831 
o.7U12  
o.o23G 
0. 3082 
0. 12 13 
o. 6037 
-0.4403 
0 .6103 
0. 2556 
o. 3247 
0. 6539 
0 •. 159 1 
0. 3097 
0. 4090 
0. 7783 
-o. 1758 
l1EG1.1SSS ION AN/.LYSIS  
STD.  
ERROR rf• P 
0.0623 6 .  34 p (0.001  
0.0623 7 .  69 P <o.oo1  
REGR.ESaiON /.Nio.LYSIS  
STD. 
ERROR p 
0. 2541  0. 1598 N . S .  
0. 1091 2 .0456 N. S .  
0. 0672 4 .  2823 p (0.01  
0. 2558 
0. 2255 
0 .0509 
0. 4959 
0. 1055 
0. 3015 
0. 1765 
0, 0963 
0. 1085 
0. 1997 
0.0488 
0.0624 
0. 9584 
3 .0539 
0 .. 1055 
6 .. 0550 
0. 2448 
5. 7213  
1 . 4868 
3.4597 
2 . 6542 
3. 0055 
3. 2744 
3."2603  
4 .9631 
0. 4267 
P (  0 .01  
N. S .  
P ( 0.001 
N . S . 
P <. 0.01  
p (0.05 
P <_ o.os 
P < 0.05 
P < o.o5 
p (0.01  
N ..S .  
o. 0755 10. 3986 p ( o. 001  
0. 1 100 1 . 4898 N..S . 
A - T Test based on Null  Hypothesis ; Testing whether regression slope 
differs significantly from zero� . 
Ti".BLE 4� 10 
OVUL!. TI ON R-\ TE OF UNTRE/� TED Il:FFBCT OF SE/ .. SOH 
�be£ of Ovulations/Ewo 
B 
OBSERV/.TION 
�����--��-------� 
( 10 ) C 'IWO ( 9 )  ONE 
Number of :Swes hcving 0 C . L . 0 
1 1  1 1  1 1  1 1  1 1 1  9 
1 1  " " 1 1  2 
" 1 
" 1 1  " 1 1  3 " 0 
7Yiean3 and ;3 tandard Srrors 
���-�� 
Number of Corpora Lutea 
( TrPnsformod Counts ) 
SOURCE OF Vfi.D.l!-..TION 
Between Observation 
Srror 
Comparison of Means 
COMPf�iUSON OF 
OBS 3 VS OBS 
3 vs OBS 
2 vs OBS 
A Ewes of Experiment 3 
ONE ( 10) 
� 
14 . 45 
;!:0. 61 
D.  F .  
2 
25 
M.? .•ANS 
2 
1 
1 
'lliUEE ( 9 )  
2 0 
6 3 
1 4 
0 2 
lVISJ-'..N j; STD.  EaR OR 
OBSEr1VATION 
'J.'I.'/0 ( 9 )  THREE ( 9 )  
13. 57 16 . 85 
::,p. 64 �.64 
ME/.N SC�U/.RES F 
26. 06 
SIGNIFICANCE 
P< 0.001 
P(0.001 
N . S .  
B Observations 1 , 2 and 3 respectively related to the 2nd , 3rd and 
4tll oestrous cycles of the breeding season .  
C - ( ) Number /Group 
*** P <o.oot 
49 
Ovulat ion Rate of  Cont rol Ewes (Experiment 3 )  3ffect of 
Season 
Table  4-10 shows that the ovulat ion rat e  of unt reated ewes in 
Experiment 3 was significant ly affected by the stage of the 
breeding season . These ewes were observed to have significant ly 
more corpora lutea on thei r ovaries at the fourth oestrus of the 
breeding season than at the second . This  effect was partly due to 
the animals having more twin and t riple ovulations at the fourth 
oestrus than at the second and thi rd .  
CHf.PTER SUMMf>.RY 
1 .  S equent ial treatmunt with P .  �j;. s .  G.  significantly decreases th e 
proport ion of ewes ovulating and the ovulatory response to the  
hormone .  Ovulation rate per ewe is  reduced by  successive treatment 
\'Jhcther one considers all  ewes or only those ewes ovulating. 
2. The number of corpora luten + fol licles ) 3 mm . is decreased by 
sequential treatment but this effect is not apparent if  corpora lutea 
+ fol l icles .>  1 mm. are counted .  
3. Differences between ovarian weights of ewes treated up to three 
times with P . M. S . G. are on ly detect ed by eliminat ion of anovular 
ewes from the analys i s .  Ovarian weight is  highly correlated to  the 
numb er of corpora lut ea present vmen ewes have been superovulated 
once with P.M. S . G. This  relationship lessens with  sequent ial treat­
ment but if corpora lut ea + fol licles are counted high correlat ions 
with ovarian weight are maintained . The relationship between 
corpora lutea and ovarian weight of cont rol ewes is not significant . 
4.  There is no evidence to  suggest that the right ovary i s  more 
active than the left . 
5. I n  untreated ewes , a significant increase in ovulation rate  may 
be seen at the beginning of the breeding season. 
C H � P T E n V 
u E T E C T I O N O F  � N T I - G O N A D O T R O P H I N S 
50 
Chapter V 
� E C T I 0 N 0 F A N T I - G 0 N A D 0 T R 0 P H I N S 
Result s  presented in this chapter involve t est s on plasma of 
sheep from Experiment 1 ,  v1hose ovarian data were r:;i ven in 
Chapt er IV.  
Innature Mouse U terine �'ieight Respon se to P. M . S . G . 
Fig.  5- 1 shows the P .  I·ll. S .  G .  dose-uterine weight  response 
curve for immatu:re mice injectec wi th 0 . 1 - 1 . 0  i . u .  P . M . S . G . 
Linear increases in uterine v1eight with log- increments in dose 
were seen between 0. 1 i . u . and 0 . 8 i . u . P . M. S . G. (Fig.  5-2 ) .  The 
l inear increase did not continue above this dosage and 1 i . u . was 
seen to be slight ly supra-optimal fo r s timulat ing increased 
ut erinG weight . 
Doses chosen for the ant i-gonadot�ophin tests  wore 0 . 25  i . u. 
and 1 . 0  i . u . P. M. S . G. The lm:wr dose wps selected to be  mid-way 
in the log phase of the cu rve in Fig.  5-2 (where linear in�reases in 
response VJere seen with log-increases in dose ) . I t  \'!US thus in the 
mos t  sensit ive region and able to  detect small chnnges in uterii.1c 
weight , which could be due to ant ibody inhibition of response .  The 
higher close ,  being supra-optimal , would only be coJnteractod by 
largG amount s  of  nnti-P . M . S . G. 
Test  for Anti-gonadotrophins in the Pla�a o f  �wos of Experiment 1 
Fig.  5-3 gives the raw means (unt ran sfo rrned ) for the ut erine 
weight s  of mice given P . rll . S . G .  alone and P .M . S . G . plus plasma f rom 
sheep of Experiment 1 and chronically treated ewes. Fig.  5-4 
presents  the same data after logarithmic t ransformation . �nalysi s  
was performed on the t rcns formed dnta , using mouse body weight at 
1-:::c (.!) 
w 
$. 
w 
2 
a: w 1-:J 
60 
50 
40 
30 
20 
10  
F I G U R E  5-1 :-
I M M AT U R E  MOUSE U T E R I N E  W E I G H T  R ESPONSE TO P.M.S.G.  
0. 1 o.2 
P.M.S. G .  ( Lu . )  
0.4 0.8 1 .0 
1· 70 
1· 60 
1 ·50 
1-J: 
(.!) 
w 
:: 1·40 w 2 
([: 
w 1-::::> 
,.... 
,.... 
1-30 
+ 
2$. 
Cl 
0 
...J 
1 ·20 
0.1  
F I GU R E  5-2: -
STRA IGHT  L I N E  AND CURV I L INEA R EQUATIONS 
FOR IMMATURE MOUSE UTER INE  
WE IGHT  RESPONSE TO P. M.S.G 
Curv i l i near Equat ion 
for 0 · 1 ,  0 · 2 , 0 ·4 , 0 · 8 ,  i . u. 
0.2 0.4 
Straight l i ne Equat ion 
for 0· 1 ,  0 · 2 ,  0 · 4 , 0·8 , i . u. 
0.8 
• 
P.M.S.G . ( i .u . )  
1 .0 
51 
slaughter as a covariete,  The use of this covariate reduced 
the error monn square of uterine weight by 9 .77% , compared with 
the error mean squcrc of nnalysis of variance. Adjusted mec.ns and 
sto.ndard errors of mean uterine weight for each treo.tment group in 
Fig.  5-3 and 5-4 are given in J•ppendix 4 ,  
Sub-cutaneous injection o f  P. M. S . G.  led t o  significc.nt 
(P (0,01 ) incrcnscs in mouse uterine weights  over control animals .  
1 .0  i . u .  go.ve significantly grenter increases than did 0. 25 i . u .  
( P  < 0 .05 ) .  Responses o f  the mice on the two dos e levels were not 
sie;nificantly altered by the injoction of 0, 25 ml , plnsma. from ewes 
of Experiment 1 .  These results  are sum�aris�i in Table 5- 1 .  
Tes..:L for J.nti•gonadotrophins in the plasma of Ewes Chronically 
Treo.ted with  P, M. S. G .  
Plasma from ewes chronically  treated with P . M. s . G. for 
6 weeks inhibited mouse uterine weight response to 1 .0  i. u.  ot the 
same hormone (Fig.  5-3 and 5-4 ) .  The inhibition wa• ' not 
statistically significant when the plasma of ewes given 1000 i . u. 
P . IY:. S . G. ( twice weekly ) , was given to the mice. However , the 
plasma of ewes given 500 i .u .  P .M , S , G. under the same regime did 
inhibit  the mouse response to 1 .0  i . u .  P.M . S , G, 
I f  ewes >Jere given their f irst injection of  P .M. S . G .  in 
Freund ' s  adjuvant , their plasma was able to produce s ignificant 
(P  (. 0 ,0 1 )  inhibition of mouse uterine weight response to the 
gonadotrophin. 
't"/hether or not Freund ' s  adjuvant  was given , the plasma of owes 
given 500 i . u .  P.M . S . G. caused greater inhibition (not significant ) 
of mouse  uterine weight response than did plasma of  ewes given 
1000 i 1 u . (Fig. 5-3 and 5-4 ) .  
MG 
50 
40 
..... 
I 
(.) 
w 30 
3 
UJ 
z 
c.:: 
UJ 
..... 
::J 20 
10 
F I G U R E  5 -3 
M E A N  UT E R I N E  WE I G H T S  OF M I C E  T R E AT E D  W I T H  P. M.S.G. A N D  S H E E P  P L A S M A :  R AW DATA 
NO PMS PMS L 1 
P M S  1 i . u  · 2 5  
I . U  
L 2 L 3  
1 · 0 i.u. PM S + p l a s m a  
L 4  H 1  H 2  H 3  H 4  C C  
0· 2 5 i.u.PM S + p l a  s m a  
� 1 - 0 i .u.PM S + p l a sma 
L 1 L 2  L 3 L 4  H 1  
r � ...... 
H 2  H 3  H 4  CC 500 1000 500 1000 
+ F  + F 
T R E ATME N T  GROUP 
180 
160 
0 0 
)( 
1- 140 J: 
() 
w 
3: 
UJ 
z 
0:: 120 UJ 1-::::> -
+ )( -
8 -' 
80 
tiJ 
N O .  
PMS 
F I G U R E  5 - 4 
M E A N  UTE R I NE W E I GHTS OF M ICE TREATED WITH P. M . S. G. AND S H E E P  PLA S MA : TRA N S FOR M E D  DATA 
PMS P M S  L 1  
1 i. u. · 2 5  
i .u .  
L 2  L 3  l 4  H 1  H 2  H 3  H 4  
0·25 i.u.PM S + pl a s m a  
-------- ----------
c c  l 1  l 2  
T R EAT M E N T  GROU P 
l 3  l 4  H I  H 2  H 3  H 4  c c  
1 ·0 i . u P M S + p l a s m a  
500 1000 500 1000 
+F + F 
TABLE 5-1 
1.. 
COMP,'JUSONS OF iVEt..NS f.FTZR. iUU;.LYSIS  OF COVf-.HIANCE : MEAN UTE� 
\"TEIGHT OF MICE GIVEN P . �i . S , G. /LLONE OR 7!ITH PL.C.SIVJi;. OF C:V/ES TR&'.TED -
VTI T:i P.M. S . G. 
C.OM:P/i..RISON 
MOUSE T�li!:i:.'IWIE.t�T GROUP VS 
P .M, S , G. ALONE 
(0. 25 i . u . ) 
P. M. S , G, ALONE 
( 1 . 0  i . u . ) 
P . M. S . G. ALONE 
P , M. S , G, ALONE 
VS 
VS 
VS 
vs 
vs 
MOUSE TR:_i:f. Tr1iENT GROUP 
Uhinject'ed P< 0,01  
L'nirijoc'ted F <. 0.01  
P . M . S . G, ( 1 . 0  i . u. ) + Plasma P ( 0.05 
of 3wcs given 500 i . u . 
P .M , S . G . alone 
P . M. S , G, ( 1 . 0  i . u. ) + Plasma P ( O .. O l  
of Ewes given 500 i .u .  
P . M. S , G. + Frounc r d  
P ,M. S .G. ( l . O . i . u. ) + Plasma P ( O. O l  
of  Ewes given 1000 i .  u .  
P . M. S , G . + Fround•s  
h - Only including comparisons of  means that showed significant 
differences . 
B - Level of significance of comparison between two groups . 
52 
CHAPTER SUMMi..HY 
1 .  Uterine growth of  immature mice was stimulated by dosages of 
0. 1 - 1 . 0  i .u .  P . M, S . G. Linear increases in this responoe were 
seen with log-increases in dose between 0. 1 - o. s i . u. P, M, S . G. 
2 .  The injection of plasme from sheep treated a t  up to three 
sequential cycles with P. M, s .  G •.  did not inhibit  the mouse uterine 
weight response to P. M. S . G, , suggesting that no anti-gonadotrophins 
had developed in these sheep, 
3, Plasma of chronically treated ewes inhibited response to 
P�M. S , G . , suggesting the presence of anti-gonadotrophins.  
C H � P T E R VI 
0 V h R I A N F 0 L L I C U L A a D E V E L 0 P M E N T 
O F  E � E S S E Q U E N T I A L L Y  
T R E A T E D U I T H P.M. S.G . 
53 
Chapter VI 
0 V A R I A N F 0 L L I C U L A H -"""D;...;;E;.....;V,....;:;;E;...;:;;L-.,;:;O.....;;..P�M;,;....;E;;;.• ..;;N;;..,;T;;..__..;O;;..,;;.,F 
E W E S  S S Q U E N T I A L L Y T H. E f-.. T E D VI I T H P .M. S .G .  
Experiment 3 was designed to  investigate ovarian follicular 
development of ewes treated v1ith P .M. S . G. up to  three successive 
t imes . Laparotomies nnd recovery of genital tracts after slaughter 
alloV�ed ovaries of such e"Wes t o  be examined at  successive cycles.  
Data on surface follicles were recorded at laparotomy and post­
mortem.  Cross sect ional scores of antral follicles and the 
calculation of the percentage of these that were normal ,  provided 
further information on the treated ewes when the ovcries were 
recovered at slaughter. Relationships between parameters at 
successive cycles ( repeated observations on the same animals)  were 
also investigated , as v1erc the relationships between cross­
sectional fol licular counts and surface ob servations . 
Surface Observetions  
Tables 6-1 ,  6-2 and 6-3 present analyses on transformed count s 
of visually appraised foll icular development ,  token fron owes 
treated once 1 twice and three times respectively.  f.s the controls 
varied in ovulation rato r:.nd fol licular clevelopmant from cycle to 
cycle ( see Table 4-10) 1 the treated ewes are compared to control 
ewes observed at the same time.  The tables only show the data for 
total ovo.rian follicular response. The right and left ovaries were 
found to bo sufficiently  s imilar to justify pooling the data from 
both. Data on the counts of corpora lutea and of corpora lutea + 
total follicles from these sheep are shown in the previous chapter 
( Table 4-7 and 4-8 ) .  
Tt.BLE 6-1 
OVARIAN Slr.:W/'.CE J�CTIVI TY : OBSERVi•TlON ONE 
Means and S tandard Errors 
TREA TiflENT N ME/ .N NUiVIBER OF FOLLICLES /SHEEP .:!:. STD .  ;�l\.I10R ( THl\.NSFORMED DA Ti'.. ) 
SMALL 
P . M. S . G. 30 22. 89 
.:!:,1.  78 
CONTitOL 10 19 . 08 
:t,3. 00 
J�alysis of Variance 
souncE oF 
Vf.FUATION 
D. F .  
MEDIUM LARGE 
22 . 53 10.; 80 
!,1 . 60 .:!:.1 .06 
27 . 63 19 . 47 
,!2. 78 !, 1 . 04 
SIVl! .. LL + MEDI UM 
31 . 32 
!1 . 87 
33. 32 
;!:3. 25 
'""" · "' s,..u · R"'s"· l'l.l.t!,.h�l'( '-J i J. � 
------------------·------ �------------------
BETilEEN 
GROUP 
ER..'10R 
1 
30 
SMALL MEDIUM 
108. 57 194 . 64 
95. 63 77. 29 
A - F rat ios all non-significant 
Li;.t1GE SMi'.LL + MEDIUM 
3. 44 38 . 62 
3L.l . 21 105 . 91 
Mi!:DI UM + L.t.RGE ALL FOLLICLES 
20.01  35 . 43 
:!1 . 00 .:!:. 1 . 07 
32 . 6'.1 37 . 70 
:!;3. 26 .±,3. 25 
l.VillDIU!'!l + Ll'o.RGE ALL FOLLICLES 
30. 27 38 . 62 
106 . 9 1  105 .91  
---
TI.BLE 6-2 
OV!:.RII..N SURF..:"..C::!: i'..CTIVlTY : OBSERv,:.TION TWO 
Means and Gtandard Errors 
T:illl. TMENT N MEAN NUMB!ZR OF FOLLICLES/SHi..::E:? !. STD Er:::-:OU ( TI1b .. l'JSFOHMSD Di-i.TA ) 
SN'Ji.LL IVTEDIU!Vi 
P .M. S . G. 20 25. 0 1  18 . 10 
±2. 02 !,1 . 2 1  
CONTROL 9 21 . 54 w. 56 
:t_3 .01  ;t, 1 . oo  
Analysis of Varian� 
SOURCE OF .  D. F .  V J..::nt-.. TI ON -
-
SMALL MEDIUM 
BET\"lED:N 1 74 . 83 0. 97 
GROUPS 
ERi.10il 27 3 1 . 63 29 •. 39 
-
A - F rntios all non-significant 
L/.RG::;; 
16. 56 
!;1 . 52 
17 . 20 
�2. 17 
Lt .. klG� 
2. '40 
46 •.  60 
Sl\1il.LL + !'/LSD! Ul'vi 
29 . 57 
:!,2. 1[l 
26 . 74 
!,3. 25 
A l\'�kN SO;U!i.RES 
M:3DIIDII + IJ�RGE f�LL FOLLICLES 
2 1 . 98 32 . 15 
!.1-. 24 ;t,2 . 1 1  
2<1 .03 30. 56 
:!:1. 05 !,3. 15 
--
SW.LL + MEDIU!'!. MEDIUM + Ll�:CiGE ,:..LL FOLLICLES 
48 . 62 26 . 15 15. 59 
G5 . 36 31 ..  02 89 . 49 
Tf.BLE 6-3 
OVARIIJN SURFACE �CTIVITY : OBSEaV�TION TI1REE 
Means and S tandard Errors 
TnEh.TMENT N 
P .M. S . G. 9 
CONTROL 9 
Sl\flf�LL 
2 1 . 54 
:t,2. 16 
2 1 . 46 
±.2 . 16 
Analysis of Variance 
SOimC3 OF 
Vi.RIATION 
BET'!lEEN 
GROUP 
ERHOR 
D. F .  
1 
16 
SMli.LL 
0.03 
42. 24 
r�iEAN NUIV1B:3R OF FOLLICL3S/EVJE !. STD EP.ROR 
MEDIUM 
10. 56 
!,3. 00  
23, 32 
::3.00 
MEDIUM 
101 . 80 
81 . 35 
Lll.RGE 
17 . 20 
.:!:,1 . 65 
17 . 69 
,!1 . 65 
Ii"RGE 
0.72 
24. 56 
Sl\U'.LL + MEDIUM 
30 . 50 
:!:,3. 50 
27 .06 
!t3� 58 
MEii..N sqUiLRES 
SM/I.LL + MEDIUM 
4 . 6 1  
1 15 .47 
MEDIUM + lJ'.fi.GE 
27 . 00 
.;!:3. 70 
20. 70 
!,3 . 70 
1\JEDI ffivl + Lll.RGE 
4 1 .03 
129 .03 
t .. LL FOLLICLES 
30. 56 
!,3. 5 1 
12. 35 
!,3. 51  
f...LL FOLLICLES 
53. 4 1  
1 1 1 . 22 
Cl) w ...J u 
...J ...J 
0 
u.. 
u.. 0 
a: w al 
::= :::::> 2 
20 
10 
10 
10 
0 
20 
10 
0 
F I G U R E  6-1 :-
0BSE RVATIONS ON F O L L I C L E  NUMBERS I N  EWES TR EATED 
UP TO TH R E E  TIMES WITH P.M .S.G. 
OBSE RVATI ON ONE OBSE RVATION TWO OBSE RVAT I O N  TH R E E  
1 -3 3-5 > 5 TOTAL 1 - 3  3-5 > 5 TOTAL 1 -3 3-5 > 5 TOTAL mm . 
FOL L I C L E  S I Z E  C ATEGORY 
54 
Trends in all parruneter�:; , at onch of three cycles are shown 
in Fig.  6-1 .  There was litt le  variation in the total number o f  
surface follicles present in the ovaries o f  ewes given either one , 
two or three sequentiel treatments ·. A tendency towards increasing 
numbers of smaller follicles , tmd decreasing numbers of larger 
fol licles , with increasing numbers of sequential treatments  is not 
marked . The number of follicles in each diff0rent size category 
for treated animal s did not diff er from those of the controls at any 
of the three observations . 
Table 6-4 shows the ovarian surface data of the three treated 
groups at slaughter.  These groups differed significently in  
ovulation rate (P < O � OOl ) .  Ewes given one injection had signifi­
cantly greater ovulation rates th�n ewes given two or three 
injections while the latter two groups did not differ. Swes of 
Treatment three ( 1 injection ) had significantly more (P< 0 .05 )  
follicles greater than 3 mm.  (med. + lgo. ) than ewes o f  treatment 
two ( 2  injections ) .. However ,  ewes of Treatment one ( 3  injections )  
did not differ from owes of  Treatment three in this  measurement .  
ffuen the total sum d corpore lutea and all foll icles i s  
considered , ewes given one injection showed a significantly larger 
score than the other two groups.  
The relationships between variates over two immediate  oestrous 
cycles are analysed for control animals in Table  6-5 and for ewes 
treated up to three t imes with P.M . S . G• in Table  6-6. These  
relationships were investigated by  regression analysis and the 
number of corpora lutea at any one observation are related to 
laparotomy observations at  Day 10 of the previous cycle.  In  the 
TABLE 6-4 
OVARIAN SURFACE t.CTIVITY : OBGEilVt.TION f.T SLi.UGHTER 
Moans end Standard Errors 
TRiY.TMENT N 
1 ( 3  Injections ) 9 
2 ( 2  Injections)  20 
3 ( 1  Injection ) 30 
/�alysis of Variance 
SOURCE OF 
V I•?. I A. TI ON D. F .  -
I'YIE/..NS .:!:. !3RROH3 Or TRt'..NSFCRrvi�D COUNTS 
CORPORh S!VU.LL FOLL . fl'iED .  FOLL . LGE . FOLL. SM.  + fv:C�D .  i\T:m. + LGE . 
LUTEA. FOLL . FOLL . 
16. 448 2 1 . 46 23. 32 17 . 69 30 , 08 27 . 00 
!.1. 73  _:t3. 50 ,;t3.03 !;2 . 30 .:!:,4 . 27 .;t2. 9 1  
15. 45A 26. 17 10·. 07 10 . 07 3 1 .00 23. 37A 
;t1 . 16 !;2 . 34 !;.2.03 _:!1 . 60 �2. 06 ,:!:1 . 95 
AB ·'· 24. 43 25 . 9'-:1 22. 33 22. 33 35•44 32.  394• 
tfJ. 95 .:!:1 . 9 1  .:t,1 . 66 ;t1.  30 ±.2·. 34 .!.h 59 
MEAN S(.')Ul•RES 
CORPOH.A SMJ' .. LL FOLL. MED. FOLL. LGE . FOLL . SM. +fli.3D. M2:D . +  LGE. 
LUT31l. FOLL. FOLL . 
BETWEEN GROUPS 
*** 239 . 45 65. 70 
1 10.4 1  
100. 72 
02 . 90 
56. 20 
5 1 . 20 
79 . 07 
164 . 69 
* 203. 60 
ERROR 27. 24 
A 1B  - Groups with the sruno subscripts arc significantly  diffor�nt mocns 
* P <o.o5 
** P <o.ot 
*** P < 0.001 
76. 24 
ALL 
FOLL. 
34 .0 1  
:!::,4 .03 
34 . 00 
!2 . 70 
4 1 . 24 
j:2 . 21 
1'..LL 
FOLL. 
171 . 57 
146. 59 
C . L.  + 
FOLLICLES 
37 . 1l· 
!.3. 137 
B 36. 30 
,t2 . 59 
47 , 19AB 
±.2. 12  
C. L. + 
FOLLICLES 
* 360 . 62 
135 . 16 
TABLE 6-5 
F..3Lf .. TIONSHIPS BETWE3l'� OVUL.".TION H.t•TE !.ND OVJ'.F:Ii' .. N ACTIVI TY 
Ci-! DAY 10 OF THE ?i1EVIOU3 OE3Tl.10US CYCLE : CONTROL B\IES . 
A B RELi:.TIONSHIP REGR. S1'D. D. F ,  T P 
COEFF . EP..R0�1 
L . C. L. 3 vs L . C. L . 2 -0. 1595 0, 6906 7 0. 2283 N, S .  
L.Sm.Fol l . 2 0 . 2309 0, 2479 7 0, 9314 N . S .  
L , l'iicd . Fol l .  2 -o. 6930 o. 3746 
L. Lge .Foll . 2 -0. 3523 0. 3470 
L . Tot ,Foll . 2 0.0020 0, 2299 
L .C . L . +Foll2 0.0030 0. 2290 
L . C .L . 2 vs L .C .L . 1 -0. 6667 0. 2 102 
L.Sm.Foll . 1 0. 2293 0. 1294 
L .Med .Foll . 1 0,0060 0 ,0803 
L .Lge.Foll . 1 0. 2525 0. 2496 
L , Tot .Foll . 1 0 .0431  0,0836 
L . C. L . +Foll 1 0. 0234 0,0062 
R . C . L. 3 vs R .C . L . 2 1 ,01 16 0, 4061 
n . sm.Foll . 2 0. 4932 0 , 2572 
R .Med ,Foll . 2 -0. 4126 0. 4235 
R.Lge. Foll . 2 0. 1705 0 , 4466 
R .Tot. Foll . 2 -0. 6536 0, 3029 
a .c . L . +Foll2 -0. 4774 0. 39 17 
ll . C . L . 2 vs R , C . L . 1 0. 5442 0, 1426 
R, Sm.Foll . 1 0,0366 0 .09 14 
R.Med .Foll . 1 0 ,0222 0 . 1010 
R . Lge .Foll . 1 0.2096 0 ,07 15 
R . Tot ,Foll . 1 0.077 1 0. 0744 
n .c . L . +Fol l . 1 o .0062 o.0596 
7 1 . 0499 
7 1 . 0129 
7 0, 0121 
7 0.0165 
7 3.0554 
7 1 . 7720 
7 0.0747 
7 1 . 0116 
7 0. 5155 
7 0. 27 14 
7 2. 00 10 
7 1 . 9 160 
7 
7 
7 
7 
0, 9742 
o. 3817 
2. 0570 
1 . 2187 
7 3. 0 160 
7 o. 3676 
7 o. 2198 
7 2 . 9314 
7 1.0362 
7 1 . 4463 
N . s .  
N . S .  
N . S .  
N . S .  
P<. 0.05 
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
N . S .  
P < o.o1  
N . s .  
N . S . ·  
p (0 ,05 
N . S .  
N . s .  
A - L .  = Left , R.  = Right , C .L .  = Corpora Lutea ,  Subscripts denote 
Observations 1 , 2 and 3 .  
B - T Test based on Null Hypothesis ;  Testing whether regression 
slope differs significantly from zero. 
Tf...BLE 6-6 
&:L!.TIONSHIPS BETIIEEN OVULATION lU.TE AND OVrJ.UAN li.CTIVITY 
ON DAY 10 OF THE PREVIOUS OSSTHOUS CYCLE 
HEL!. TI £NSHIP 
T.sSTED 
REGR. 
COEFF. 
1 . 1035 
L .Sm.Fol l . 2-0. 2296 
L .Mod .Foll2-a , 2032 
L . Lge .Foll2 0 . 4326 
L .Tot .Foll2-0. 1507 
L . C .L+Foll2-0. 1 174 
L . C . L . 2 vs L . C . L . 1 0. 24 15 
L . Sm.Foll . 1-0. 1352 
L .Mcd , Foll 1 0 , 5026 
L .Lgc.Foll 1 0 .49�1 
L . Tot .Foll 1 -0. 0675 
L . C . L+Fol1 1 -0, 0675 
0. 5016 
R . Sm.Foll2 -0 . 4275 
R .Mcd .Foll2 0 . 1569 
� . Lgc. Foll2 -0. 1760 
�. Tot .Foll2 -0. 1916 
:-;. .c .  L+Foll2 0. 1046 
n . c . L . 1 -0.0151 
H . Sm . Foll 1 -o . 3403 
it .Med .Foll 1 0 . 1030 
U. Lgc.Foll 1 -0. 4350 
� . Tot .Foll 1-o. 1413 
R. C. L+Foll 1 -o.0901 
STD,  D. F .  � P 
:.!:�E�OR 
o. 4545 7 2 .  4279 p < 0.05 
0. 19 19 
0. 6474 
0. 54 14 
0. 1047 
0. 2345 
0. 1965 
o. 31 13 
0 . 2 127 
0 .4413  
0 , 2672 
0. 2672 
0. 6230 
0. 2762 
0.4257 
0 .7330 
0. 25G5 
0. 2547 
0. 3620 
0 . 1740 
0 . 3325 
0. 3050 
0. 231 1  
0. 2090 
7 1 . 1960 N . S .  
7 0. 3138 N . S ,  
7 0. 7990 N . S . 
7 0. 7740 N . S .  
7 0. 5006 N . S .  
0 1 . 2290 N . S .  
0 0 . 4343 N . S .  
o 2 .  3629 P <o.o5 
0 1 , 1100 N . S .  
0 0. 2526 N . S .  
Q 0. 2526 N . S .  
7 0 , 2526 N . S .  
7 1 . 5447 N . S .  
7 0. 3685 N . S ,  
7 0. 2409 N . S .  
7 0. 7469 N . S .  
7 0. 4 106 N . S .  
0 0 .04 16 N . S  • . 
0 1 . 9523 N . S . 
0 0. �527 N . S  • . 
8 1 . 1432 N . S . 
s o.e1o9 N . s . 
0 0 .�693 N . S .  
A - L .  = Loft , R .  = aight , C . L . = Corporn Lutea.  Subscripts refer 
to Observations 1 , 2  and 3 .  
B - T Test based on Null  Hypothesis ; Testing whether regression 
slope differo significantly from zero. 
55 
control  animals 1 corpora lutea scan on the right ovaries of evJCs 
at the s econd observation v.rere significantly related (P < 0. 05 )  to 
the number of large foll icl es seen 6-8 days before oestru s  
( observation 1 ) . The cor�ora lutea seen at the s econd observation 
were related to the number of corpora lutea s een at the previous 
observat ion ( R . C . L. 2 vs R . C . L . 1 nnd L . C . L . 2 vs L . C . L . 1
)* (Tab l e  6-5 ) .  
The regression coefficionts  of these relationships ere opposi t e  in 
sign and hence no real trends aro apparent . 
Of the P. M . S . G. treat ce animals , very few significant 
relationships were found. Ovulat ion rate  of the left ovaries at 
the third oest rus was positively correl ated to the ovulat ion rate 
of the samo ovaries at the second oestrus and this regression was 
significant (P < 0.05 ) .  Ovulat ion rate o f  the left  ovaries at the 
second oestrus \Vas significant ly related to medium sized fol l icl es 
( 3-5  mm. ) i .. t Day 10 of th e previous cyc le (F <.. 0 .05 ) .  ;".gain , the 
significant relct ionships  obtainec ho.ve little  real implicat ion 
bec�use there ere no consi stent t rends .  
Total Ovarian hctivi ty 
The number of  ant ral fol l icles in serial 2 mm. slices gave an 
est imation o f  the total ovarian activity and the number of  foll icles 
that were potentially  abl e to be  stimul�t ed by gonadotrophin . The 
number of nn t ral fol licles per ovary wus estimated by counting the 
number per 2 m�. section anc summing them for each ovary. Table 6-7 
•· Regression of corpora lutea on ri ght/left ovaries at the second 
ovulation , on corpora lutea o f  the same ovari es at ovulation one .  
TABLE 6-7 
ESTIMATI:S OF TOTl:.L OV!�IUAN FOLLICLE :20PyL:�TION : NUMmm OF !�NTil.AL 
FOLLICL.il:S/OVAHY (TPJ'..NSFO�WJED DATA ) .  
Means + S tandnrd Errors 
TilE!. TiYIENT 
G�10UP 
NUMBER/GROUP MEJ'.N NUI'till�r. OF f...NTRhL FOLLICLES 
PJ:;:J. OV :.CI.Y .:!: S •. E .  ( ../X+i ) 
1 
( 3  I njections ) 
2 
( 2  Injections ) 
3 
( 1  Injection )  
4 
( Uninjccted ) 
4 1 . 50 + 3.·53 
10 47 . 1D + 3 •.  35 
10 40 •. 97 + 3. 35 
9 36. 57 .:!:. 3 •. 53 
Analxsi� of Variance 
SOU:::C3 OF Vi.r.Iii.TION 
BET\,.,SEH G:-tOUPS 
D. F .  
3 
34 
lVIEAU S'iUARES 
10 1 . 22 
1 12 . 52 
TABLE 6-0 
F p 
1 . 61 N . S .  
ESTII'II!•TION OF 'ISE PnOPORTION OF NORJ',!AL f�NT�1i.L FOLLICLES/TO'I'AL 
TUEi\. TiviENT GilOUP 
1 ( 3  Injections)  
2 (2  Injections ) 
3 ( 1  Injection) 
4 (Uninjected ) 
ANTRAL FOLLICLES 
NUMBE� OF SAlVlPLES 
OBSEl1VIm 
10 
10 
10 
10 
% NOil.MAL LNTRAL FOLLICLES/TOTAL 
ANTPJi.L FOLLICLES (CCR.HGCTION 
Fi'.CTOR) 
31 . 2% 
3 1 . 2% 
13 . f1%  
33.0% 
TABLE 6-9 
ESTHIILTI:S OF THE TOTJ..L NOUMi�L f.NTH!l ..L FOLLI CLE POPULJ�TION : 
NUMBE:1 OF NORMAL f.N1YU .. L FCLLICLi3S/OV/.l1Y ('I'RANSFOHV!ED DATA ) .  
Means + Standard Errors 
Ti:\l!:A TI!'£NT GROUP NUMBEP./GROUP M13h.i'l' NUMBER OF NOPJilAL ANTPJ'.L _ 
FOLLICLES P:<:U OVJ.RY ±., S . E . ( JX+ 1 )  
1 ( 3  Injections)  9 24, 03 + 1 . 63 
2 ( 2  Injections ) 10 27 . 7 1  + 1 . 55 
3 ( 1  Injection ) 10 17 . 87 + 1 . 55 
4 (Uninjocted ) 9 22 . 59 + 1 . 63 
� alysis of Variance 
DEG��S OF FREBD�1 l\2hN SQU/.:KES F p 
BET,'/EEN GaOUPS 
Comparison of Means 
TiGl'.TMENT VS 
1 VS 
1 vs 
1 VS 
2 vs 
2 vs 
3 VS 
3 
34 
TIGJ'. TMENT 
2 
3 
4 
3 
4 
4 
17 1 . 41  
24 . 10 
S IGNIFIC.l\.NCE 
N . S .  
P <: o.oo1  
N .S . 
P<0.001  
N . S .  
P (0,01  
7 , 1 P<.O ,CXJl 
56 
presents  the datn obtained from these counts .  There was no 
si gnificant difference between treatment groups in the number  of  
antral follicles per ovary. 
Histological examination of 10 of the 2 mm. slices per 
treatment group anablod estimation of tho number of normal nntrul 
follicles per total antral follicl es .  An  averaGe fiGure was 
obtained for each group and these are given in Table  6-8. The 
figures ( correction factors ) presented in thi s table were used to 
correct the dat� of Table 6-7 (mean number of antral fol l icles per 
ovary ) . 
Photomicrographs showing various stages of  foll icular atresia 
are soGn in Fig. 6-2 to 6-7 . 
The £roportion of normal antral follicles/ total antral  
fol licles of ovaries recovered from ewes on  Treatment  3 ( 1  injection ) 
rtas lower than that of the other three groups (Table 6-0 ) .  Th0 
other three groups wore comparab le in this measure . 
Analysis  of follicle populntions corrected for the degree of 
atresia i . e. normal follicles per ovary , is given in Table  G-9 and 
the applicat ion of the correction factor reveals  that  owes of 
Treatment  3 had a significantly lower (P< 0 .001 ) number of normal 
fol licles per ovary than the other groups .  
Rolationshi£s Between Total Ovarian Data and Surface Data • 
Relationships between estimated total ovarian activity and 
ovarian surface observations wore tested by regression analysi s. 
The estimated total number of antral follicles per ovary and the 
estimated total number of normal antral follicles per ovary were 
related to the number of surface follicles per ovary and the 
Fig. 6-2 : - Normal fol l icle sectioned throu�h the ovum : 
a ovum ; b - cumulus cells ; c - granulosa cel l s ;  
d - theca intozna ; o - theca externa (x  163 ) .  
Fig. 6-3:- Cross  section of a normal tertiary fol l icle : 
a - granulosa ; b - theca interne. ; c - theca 
externa {x 65 ) .  

Fis.  6-4 : - Cross section of the luteinised wall  of n cystic  
follicle :  a - theca interna ; b - lutein cells ;  
c - thecn externa ( x  1GB) . 
Fig, 6-5 : - Enrly atresia of a t ertiary follicle :  a - granulosa 
cells  moving into the antrum (x  168 ) .  
-
-- ---- -- -
Fig.  6-6 : - fl.trcsic. of a terti::.ry foll icle ;  disintegration of the 
granulosa (x 65 ) .  
Fig.  6-7 : - Late  atresia  of a tertiary follicle ;  granulosa 
cel ls dispersed throughout the antrum (x 65 ) .  

57 
number of  corpora lutea per ovary. None of these regression 
relationships were found to be significo.n � .  The regression 
coefficients  ere given in hpp�<dix 5 .  
CH.h?TEH SUrtlM!.RY 
1 .  [;.lthough there i s  no evidence of a reduction in total 
ovarian population with increasing numbers of sequent ial treatments  
v1ith P. M . S . G . 1 there is  a trend towards increasing numbers of  small  
fol l icles and decreasing numbers of large follicles.  None of  these 
trends  were shown to  be st�tistically  significant . 
2 .  The number of follicles greater than 3 mm. was seen to  b e  l ess  
in ewes treated twice with P .M . S . G. thnn in  ewes treated once 
( observation 10 days af ter the onset of oestrus fol lowinG 
inject ion ) .  
3 . Relationships bctv1een ovule.t ion rate  and ovarian activity 
( surface ) observed on Day 10 of the previous cycle are poor. 
However , relationships between estimated total ovarian activity 
and observed surface act ivity are not significant , indicating that  
surface observat ions do not represent total ovarian activity.  
4 .  The number o f  total normal antral follicles per ovary was seen 
to be  significantly  lower 10 days after one injection with P.M. S . G .  
than at a similar time after a second and third injection. 
C H A P T E R VI I 
D I S C U S S I 0 N 
50 
Chapt er VI I 
D I S C U S S I 0 N 
Synchronisation and Oestrous Phenomena 
Oestrous cycles were synchronised with progestogen sponges 
to assist  t he conduct of the experiments ·. 
i.fter 14 days int rnva(jinal admini st ration of  i'!l. l  •• :;:>� to 
cyclic ewes (Expt . 1 ) ,  injection· of P . M. S . G . was observed not to 
affect the t ime after sponge wi thdrawal in whi ch evtes came into 
oestrus. Neither did this  inject ion of P � M� S . G . reduce th e nwnber 
of ewes showing silent oestrus.  
Tiobinson and Smith ( 1967 ) have shown the administrat ion of  
P . Ivl. S . G. after sponge withtlravml to increase the number of  
anoest rous c�es brought into oest rus with  progesta�on sponge 
t reo.tment . Furthermore 1 ewes wore brought into heat earlier  \?hen 
P .M . S . G. was given.  �ttempting to induce early breeding , Gordon 
( 1D7 1a)  showed that more ewes ¥/ere mated after progestagen t reat­
ment when P . f,J . S . G . was given.  With ev:es given progesterone 
treatment early in the season , Rob inson ( 1955 ) found that P � M. S . G . 
\"/OUld give an earlier and a more pr :;dictable onset of oestru s ,  but 
later stated (nobinson , 1961 ) that thi s  effect was more marked in 
younger ewes.  Ford ( 1966)  derived no advantage in giving 
gonadotrophin to cycl ic ewes treated with  progestagens .  The 
indications are therefore , thc.t P. M. S . G.- may effect a (Sreater 
oestrous response in ewes treated with progestaGens in the 
anoestrous s eason or early in  the breeding season .  The effect seems 
less marked when mature mvcs nre t reated , late in the b reeding 
59 
season. The synchronisation obtained in ewes of  �xperiment 1 
substantiates t�.is  thought . 
Injection of 10 mg.  progest erone in oil , on Day 12 of a 
11.1 day intravaginal fv'i.I� . P .  treatment , may provide a more 
effective synchronisntion of e£'.rly-brcd ewes than M .A. P. 
treatment alone.  
Robinson at E-l.· ( 1960 ) showed a linoar decline ,  with t ime of  
insert ion , in  the amount of residual progestagon in  an inserted 
intravaginal sponge�  Proccstagon t runtment early in tlw season is  
thought to prime oestrogen rece�tors (Raeside and McDonelc ,  1959 ; 
Gibson and Robinson , 1'07 1 ) .  I t  was therefore thouGht tlmt injection 
of progesterone in the let ter s tnges of an intravaginal trcetment 
may supplement the depleted suppl y  in the sponge and give o. more 
effective priming of ocstro�cn roco)tors . 
Bind on and nobert s ( 196<1 )  decreased the vc.riabil i ty in t ime 
of onset of o�st rus fol lowinc; synchronisation by the combined u se 
of short- and long-acting  p::toGestagens.  
Gordon ( 197 1b ) suggest ed that minor modificat ions in 
progestagon treatments  may improve the oestrous response of early 
bred ewes and that thi s  vms probably related to the amount of 
progestagen absorbed .  However , Gordon ( 197 1d ) found that the 
addition of 400 mg . of progesterone , to an intravaginal sponge 
already impregnated with progestagcn ,  did not improve the oest rous 
response in such ewes and only gave marginal superiority in cyclic 
ewes . Robinson £! �· ( 1960 ) and aobinson ( 197 1 )  indicated that 
this effect may be  achieved by modifications  in preparation of 
progestagen spongos . 
60 
Although synchronisat ion in Experiment 3 was better than 
thut achieved in Exporimon t 1 1  comparison between tho two t rials 
is  l imited . Lamond and Bindon ( 1962 ) 1 Lamond ( 196,! c )  and 
Robinson ( 197 1 )  have demonstrated seasonal variation in owe 
response to progostagens and for this reason , comperisons in 
Tables 3-2 and 3-4 arc not s trictly  valid . No control e-,,cs were 
e.vailable  to guagc the effoctiveness of a progcste:;:-ono injection 
Eer � but the given comparisons were carried out in order to 
obtain some indication of whether such a practice hold3 c.ny 
future promise .  
Ewes came into oestrus over a more compact period ,-;hen owes 
wore given an inject ion of prof1ostcrone and M.  t • •  P .  rather than M. A.  P.  
alone. A Greater numbe:::- of  eues tJCro in oest rus on Day 2 
following withdrawal of  spon;;cs.  The progesterone injection 
ap:�ears to have produced a better synchronisation .  The rcsul -::s of 
?.obinson et al . ( 1960 ) indicate that there is  considerable between­
owe variation in the amount o:i proge:stagen absorbed from progcstagon 
improgn£,tod sponges . Administrntion of progesterone on Day 12 of 
t reatment may overcome this variation and ensure that circulating 
levels of <..dministerod pror;estagcns , at the termination of 
t reatment 1 are more uniform betvJCcn. animals .  
The number  of encs showing overt oestrus was not improved by  
the administration of  progesterone ,  compared with progcstaGen 
t reatment late in tho breeding season . I t  appears ns  though the 
injection did not aid the priming of oestrogen receptors . 
Cumming ( 1065 ) shovtcd that 32-30% of Romney ewes experienced silent 
oestrus when given a constant daily dose of progesterone during 
61  
February. Larsen ( 197 1 )  found this  figure t o  be 19% with 
intravaginal M. A. P .  treatment of similar ewes during March. I t  i s  
l��own that ewes often experience cyclic activity and silent oestru s  
a t  t he  end o f  t h e  anoestrous season (Mcl{onzie and Terrill ,  1937 and 
nob ins on , 1950 ) .  Ewes not experiencing oestrus  aft er M • .t:. . P . /  
progesterone t reatment probc:.bly  experiencec silent  oestrus  
( confirmed by  laparotomy in  t\•Jo ewes ) . Maybe those ewes coming 
into  oest rus hnc previously  expcrioncec: s ilent oestrus ancl those 
shoning silent oestrus post-treatment had not . This animal 
variation in the t ime of nc.turrcl silent oestrus may be responsible  
for the inability  of hormonal  treatment to c.ffect the inciclcnco of  
s i lent oestrus at this t ime of  the year. There is scope for further 
s tudy in this respect . 
Thorough investisation of  the effect of  c progesterone 
injection , as given in this s tudy , may be fruitful . Hith the use  
of  appropri2.te control animal s ,  the true value of the pract ice could 
be revealed. 
Effect of  S equential  i' . iVi. S . G. Treatment on Synchronisation 
Synchronisation in ewes given M . il. . P .  only  ( control ewes : 
:�xpt . 1 )  remained effect ive over three oest rous cycles . The 
distribution of the number of ewes in oestrus on any particular 
clay , fol lowing M. A. P . synchronisat ion is  t ypically  skewed ( see 
Clarke � �. , 1966 ) in these ewes.  However the distribution of  
the day of  onset of oestrus becomes more dispersed in nature dur ing 
the subsequent two oestrous periods .  Ewes coming into oestrus over 
three days at oestrous p�riods 1 and 2 were distributed over 4 days 
62 
at  their third oestrus  (FiG. 3-3 ) .  Edey and Thwaites ( 1966 ) have 
reported that once oestrus  is synchronised in ewes then the degree 
of dispersion at the next cycle i s  small .  This has been 
substnntiated by !lobinson .£! !:!• ( 1967 ) who maintained that the 
second oestrus after synchronisat ion would be convenient for 
artificial inseminat ion ns the depressed fertil ity seen in ewes 
inseminated immediately fol lowins synchronisation is not evident 
one cycle later. Hancock and Hovell ( 1962 ) and Larsen ( i97 1 )  were 
able to use the second synchronised cycle as a defined period over 
which ovum transfer could b e  carried out .  
The results  of  Experiment 1 show that oestrus of  ewes given 
progestagen wil l  remain effectively synchronised for three oestrous 
cycl es. Laparotomy at each of the three cycles ( on Day 10 of the 
cycle )  does not alter this synchronisation markedly) . 
Synchronisation iG  not wel l preserved in owes treated with 
gonadotrophin (Table 3-9 ) .  The administration of P .M. s , G. affects 
the synchronisation to different degrees , depending on the cycle( s )  
at which i t  i s  given .  ;::wes superovulated immediately after 
synchronisation ( Trts .  1 + 2 - Expt . 1 ) show a greater dispersion 
in their oestrous periods at the second cycle than ewes not treated 
at this  point .  Ewes treated with P ,M. S . G. over three cycles come 
into heat over a period of 16  days  after the third injection and 
thus synchronisation was completely destroyed by this t ime. The 
staggerinc of  inj0ctions  with P.M. S . G  •. (Trt . 2 - Expt . 1 )  also 
caused considerable d ispersion in the onset of oestrus when 
compared with ewes given two or one injections (Trts .-3 and 4 -
Expt . 1 ) .  
63 
The influence of  sequential  P , M. S . G. injections on 
synchronisation does not facilitate ovum transfer work. The 
dispersing influence of  the gonadotrophin , as compared with ewes 
given progestagen alone ,  would moan thct superovulated donors and 
synchroni sed recipients would become progressively asynchronous 
with successive treatment .  
Variation in  superovulatory response may be responsible  for 
the dispersion of once synchronised oestrous periods , in animal s 
trcated with gonadotrophin. :::!:xtremo vnriation in response to  
P, M, S , G, is  wel l documented ( e. g .  Uobinson 195 1 )  and was also 
evident in this study ( see ;.ppendix 1 ) .  Excessive populations  of  
corpora lutea and lnrge fol licl es on the ovaries of  stimulated 
animals may cause  the varintion in t ime taken to display oestrus  
at n lo.ter cycle .  
Increasing numbers of  silent heats  in  ewes treated over 
three cycles would have rendered any statistical unalysis of  
cycle  length to br.:l suspect ( in Expt . 3 6 ewes came in  to oestrus  
after three injections ) .  For this  reason , oestrous cycle lengths 
were thought not to be a valid indicator of  any real trend e, g .  
effect of  sequential superovulation as discussed in  the above para­
graphs .  Unless some account of  silent  oestrus was taken , 
conclusions based on oestrous cycle length could be misleading, 
Resul ts of  3xperiment 1 showed the treatments to  have no 
effect on the number of  ewes showing overt oestrus (Table  3-7 ) .  
However , in Experiment  3 increasing numbers o f  sequential treatments 
with P. M. S , G, reduced the number o f  ewes displaying overt oestrus  
( ccmpared with control ewes ) .  The inconsistency between these tvto 
64 
experiments may be seasonal . Ewes could  be  expected to show silent 
heat more readily during the earlier part of the brooding season . 
Nevertheless , o.ll the cont rol eHes of Experiment 3 exhibited oestrus , 
��d the txend seen in the 3equentio.lly t reated ewes of this 
experiment is  pTobably real.  
�evelopmont of Ovarian �efractorinoss in 
Ewes Sequentially Treated wi th P .M.S . G . 
S tudies on ovarian refractorincss to P .M . S . G. in sequentially 
treated ewes involved investigat ion into more than one aspect of 
the condition. The result s obtained wil l be discussed in the 
following order : - 1 .  Ovarian response and the development of 
refractoriness .  2 .  Development of anti-gonadotrophins 
3. Folliculo.r dynamics of the ovary 
4 .  General discussion on refrectoriness to P . rfl , S . G .  
The first three sections will discuss the results  of Experiments  1 
to 3 and the fourth sect ion will  integrate all information on 
ovarian refractoriness.  
1 .  Ovarian response and the development of refractoriness t o  P .M. S . G . 
Ovarian refractoriness to P. M. S . G .  has been demonstrated in 
the ewes of both Experiment s 1 e�d 3.  
In Experiment 1 ,  ewes given three sequential injections 
(Trt . 1 )  had significantly lower terminal ovulation rctcs (numbers 
of corpora lutea )  than ewes given a single injection (Trt . 4 )  or 
two spaced injections  (Trt . 2 ) .  Ewes given two sequential 
injections (Trt . 3 )  also had significantly lower ovulation rates 
than those given one injection but the difference between 
treatments 2 and 3 failed to  attain s ignificance (0.05 (P {0. 10 ) . 
65 
When a period of  one oestrous cycle separated two injections then 
the evJC ' s terminal response to the hormone did not di ffer from 
that of ewes given one injection. 
\'!hen trea tcd ev:es were cor.lpared with control ewes (Expt . 3 )  , 
the first st imulation with P . M. s . G .  was able  to  superovulate 
nnimals  but the ovulation rotes of  ewes treated a second and third 
did not d iffer from those of cont rol animals  observed et the same 
time. 
The results  of this investigation indicate that refructoriness 
to P . M. S . G .  superovulation in ewes is rapidly at tained. The mean 
response of the ovaries to  a second injection during the cycle 
inllilediately fol lowinG the first injection will be considerably 
lower than the nonnal supcrovulatory response.  This very rapid 
development of refractoriness YIUS not previously noted by Hulet and 
Foote ( 1967 1 1969 ) but was by Lursen ( 197 1 ) .  In  one experiment , 
the former authors t reatec e'.'ICS <1 times with 1000 i . u .  P . M . S . G . , 
the interval between the second and third injections va�ying 
between 33 and 340 days and the intervals between the first and 
second and third and fourth injections being 16  days. I n  all cases 
refractorinoss was attained between the thifd and fourth injections 
but became less marked as the period between second and third 
injections  increased . They therefore suggested that there was a 
gradual dissipation of  the refrnctoriness with t ime. 'l'he results  
of �xperiment 1 indicate that the refractoriness may be dissipated 
more rapidly  than this after a single injection , A space of ono 
oestrous cycle between injections is sufficient to  enable  ewes to  
respond to  a second injection to  the same extent as  ewes super­
ovulated once. 
66 
In  a second GXilGriment 1 Hulet and Foote  ( 1969 ) treated ewes 
up to 6 times with P , l'li� S . G . nncl showed that ewes became 
progressively more refr::,ctory VJi th increasing numbers of t1·entmonts .  
I� the present investigation ,  there was litt le  difference in the 
number of corpora lutea of cues treated t·wice and ewes t reated 
three t imes . This further reinforces the idea that enimnl s 
rapidly lose sensitivity to tho exogenous hormone and responses to 
successive treatments vill  be lon compared to the initial treatr.1ent �  
Furthermore� i t  o.ppears c. s  thouc;h the condit ion persists . 
Hulet and Foote ( 1967 ond 1969 ) showed that the first 
injection , given on >Ji thdrm1ql of progosterone synchronisation 1 
nlwnys producad lower suporovulatory responses than P • .f!l. S . G. 
inject ion of the s:1llle mvoo o.t the  no:xt cycle,  ;'. similar si  tuo. tion 
could have existed in enos of Treatments 1 nnd 2 of  Experiment 1 in 
this study.  The effect has  b0en attributed to  an interaction 
between progeste1·one and P. rr: • .s . G . Olulet and Footc , 1969 ) but it 
must be noted that these authors used progesterone and the present 
study  employed progostagcn spon�e tre2.tmcnts .  Mooro and Holst  
( 1967 ) have shown that there i s .no significant difference in 
ovulation rates bet>.ceen ewes given either intrvaginal or intra­
muscular treatments of progesterone or synthetic progestngen 1 when 
anoestrous e\"ICS were given P .M. S . G. after treatment . · l�verill  ( 1958 ) 
reported that the ovulation rates of ewes given P. M. S . G. treatment s 
duting the breeding season ware not s ignificantly diff0rent to those 
of ewes given progesterone-P. M. S . G. t reatment during the o.noestrous 
season . Many authors huve shovm good responses to P . M. S . G. • 
immediately after progestcgen sponge treatment (e . g.  Holst , 1969 ) 
67 
and it is therefore di:.i'ficul t to decide whether ewes of 
Treatments  1 and 2 (Expt , 1 )  v;ould have had lowered superovulatory 
responses to P ,M, 8 . G , because of the prior administration of 
progestagen . The interaction reported by Hulet and Foote may not 
be so ext reme if proGestcgcn is used in preference to progesterone, 
If it is accepted that ev1os experienced some degree of superovulation , 
when injected with ? . r�1 . S . G .  immediately following synchronisation , 
then the lack of  apparent  refrc;ctorincss in ewes of Treatmen t 2 
(Expt . 1 )  is  a real effect . The possibility  of an int 0raction 
between P .M . S . G . and the proGostagen was avuided in :Sxperiment 3 
by commencing inject ions ono cycle  after synchronisation, In  this  
:.!:xperimcnt  it  was confirmed that refractoriness  wns established 
after only  one injection of the gonadotrophin .  
Jninudeen e t  al . ( 19G6 ) gave cows a total of 4 injecticns of 
P ,M, S . G. at interval s of 5-7 months , 31-34 days nnd 10-2 1 days 
( fi rst  injection = 2000 i . u . and subsequent injections were 3000 i . u . ) 
The second injection produced a superovulatory response equivalent 
to the fi rs t ,  in spite  of the increased dosage. The final two 
injection s fai led to produce ovulat ion rates greater than in 
untreated ewes . !.gain , a refractory condition was evident  at the 
second sequent ial treatment.  'ilillet et al . ( 1950 ) used more 
intensive gonadotrophin treatment s than arc normally used to 
superovulate domestic  animal s .  They also showed that animal s wil l  
very rapidly develop a refractoriness in  response to  exogenous 
gonadotrophins and this  condition persists  for a long period of 
time , Hafez � �· ( 1964 ) and Laster � �· ( 197 1 )  have sho� 
that refractoriness in cows i s  evident at a second sequential 
60 
superovulat ion . Larsen ( 197 1 )  obtained a similar effect in ewes 
regardless of increasing dose levels of P . M. s . G . �11 the 
observations of the above workers substantiate the rcsul ts of 
this investigation , in that  refractoriness i s  established after 
only one inject ion.  But there are  varying  opinions as to the 
period of time required to dissipate the condition .  The long 
period stated by Hulet and Footc ( 1969 ) may have been bece.use 
initial t reatment was in fact 2 sequential injections.  
The response of ewes t reat3d wi th a single injection of 
P .M. S . G. (Trt . 4 ;  Expt . 1 ;  Fig. 4-3 ) is comparable  to  results  
plotted in Fig . 1- 1 . The owes VJOro of comparc..ble  natural fecundity ,  
and of the  s£1Jlle breed , as  those used by V/al lace ( 1954 ) .  The dose 
response curve of ewes in this study is slightly steeper than that 
of \!allace' s ewes , but may be regarded as  similar . The responses 
to 1000 i . u.  and 1500 i . u .  P. M.S . G . are greater than those obtained 
by Tervit  ( 1967 ) and Larsen ( 197 1 )  but are simil ar to  these of 
Cumming ( 1965 ) .  
Differences in superovulatory response to  a single injection 
of P . M. S . G . between treatments  in thi s  study and treatment s imposed 
by other workers in the same environment may b e  due to  age factors 
( e . g .  Larsen 1 197 1 )  and seasonal di fferences in response ( e . g. 
Cumming , 1965 ) .  The lat ter of these two factors is the more l ikely ,  
as  there is  little  evidence of differential responses in  ewes of 
different ages (Robinson , 195 1 and J.veril l 1 1950 ) .  
The range in ovulatory response o f  ewes given 1500 i .·u. was 
greater than in ewes j':Sl,ven 1000 i .u .  P.M. S . G. (h.ppendix 1 ) .  
Robinson ( 195 1 )  Wallace ( 1954 ) t..veril l  ( 1958)  and Larsen ( 1971 )  
69 
have all  not ed thut there i s  un increase in individual ewe 
variation with increasing dose of P . M. S . G. 
Doso level and treatment x dose  interactions  did not have 
si[¥1ificant effects  on the nurnbers of corpora luteu observed in 
ewos cf Ex�orimcnt 1 .  Fig. 4-4 shows that , o. l  though these effects 
were not stqtist ico.lly  s ignific<'mt 1 some discussion on these 
trends may be justified providing the l imitations of significance 
ure appreciated .  r!i th  sequenti£', 1  t reatments ( Trts .  1 ond 3 ;  Expt . 1 ) ,  
lower doses ( 1999 i . t.� . ) produced hie;her terminal ovulc:.tion rates 
tho.n higher doses ( 1500 i .u . ) ,  Tho reverse trend wus seen in l oss 
int ensively treated evrcs ( Treatment s 2 o.nd <1; Expt . 1 ) ,  Ui th 
sequential treatments , high doses of gonadotrophin may eru1unce 
refractoriness by producin3 greater ovo.riun responses at the first 
injection .  This suggestion mey in part explain why ewes r;iven 
spaced injections  (Trt . 2 )  have a dose response curve of s imilar 
slope to that of ewes given a single injection . TI1e spece of one 
oest rous cycle possibly allmw the hypert rophied ovaries to regain 
normal size and function before restimulation. 
Although there was no incidence of  cystic  ovaries in 
Treo.tmcnts 2 and 4 (:3xpt . 1 )  whi le thoro was in sequential ly 
treated ewes ,  statist ico.l significance was not  at tained, Larsen 
( 197 1 )  hus indicated that successive stimulations are likely to 
cause cystic and luteinised follicl es to be more prevalent. 
However , Robinson ( 195 1 )  and ·.!al lace ( 1954 )  report that simil::lr 
effects are obtainabl e with administration of n single large dose 
of P .M. S . G. The two may be  similar in that they are both over­
stimulating the ovnri es. The development of cystic ovaries is  
70 
thus unl ikely to be a primary cnusal factor of refractoriness but 
may enhance i ts effect . 
Hulet and Foote  ( 1969 ) consider that refractoriness  
( lowering of  ovulatory response to  P .  M .S . G . ) is  in pE'.rt due to  
ewes becoming anovular. They vmro able  to  partially  negate  the 
eff ect of  rcfractoriness  by considering ovulation rates of only 
cv1es ovulating . A. Dilililc.r affect was apparent in the present  
stucly whon numbers of corpora lutea of  ovulating c>tes only were 
analysed .  Ovulating owcn siven three scqu,:mt ial injections  had 
significantly  lower terminal ovulation rates than all  other ewes 
in Experiment 1 .  This resul t implies that the failure to ovulate  
may be the  first  demonstrable  cause of  apparent refractoriness  as  
the el iminat ion of non-ovulating ewes rendered the terminal 
ovulation rat es of mtes on treatment 3 ( two sequential injections )  
s imilar to  those of  m·tes t3i ven one injection. This effect was 
reproduced to a lesser extent in Experiment 3 where 2/20 and 2/9 
owes failed to  ovulate Vli th £'. second injection and third injection 
respect ively.  !l.veril l ( 1950 )  has given some evidence o f  o. similar  
anovule>.r condit ion vthere m1es being treated with P.M. S • .  G.  for a 
second and third t ime within one breeding season , failed to ovulate.  
P.M.S. G .  is primarily  fol licle  stimulatory in action (Lamond , 
1960) and may stimulate the �rowth of many fol licles that do not 
ovulate (Lamond 1 1964 ) .  Scanlon � �· ( 1960 ) noted that only  63% 
of mature follicles ovulate when cows were stimulated with the 
hormone. I t  is therefore likely that counts  of corpora lutea + 
large follicles would provide a more accurate idea of total 
ovarian response to gonadotrophic stimulation than corpora lutea 
71  
alone. Lamond ( 1064 ) and Larsen ( 197 1 )  have found larger 
standard errors associated with means of corpora lutea + 
follicles than with means of corpora lutea only. In Experiment 1 
this effect was not evident when corpora lutea + follicles 
greater than 3 mm. were considered . The mean for these measure­
ments was si gnificantly less for ewes on Treatment 1 than for al l 
other groups of  ewes. t�dding these fol licle counts to corpora 
lutea causes Treatment 3 ( two sequential injections ) to differ from 
Treatment 1 and therefore the difference between these two groups 
is in the number of l arge fol licles . I t  is  interesting to  note 
that in Experiment  3 there was no difference between control ewes 
fu1d ewes given one ,  two or three sequential treatments  with P .M. S . G .  
when the number of corpora lutea + foll icles greater than 1 1nm. 
were counted.  Smaller  follicles were taken into account in this 
experiment whereas measurement s  in Experiment one neglected any 
follicles l ess than 3 mm. in size. I t  appears as thoush the 
population of ' larger'  follicles is more sensitive than thei r 
' smaller'  counterparts to gonadotrophic stimulation .  
Over all  treatment s of  Experiment 1 ,  the higher dose of  P.M. S . G .  
( 1500 i . u . ) st imulated a greater number o f  corpora lutea + 
follicl es greater than 3 mm. than did the low ( 1000 i . u . ) dose. 
This  result confonns to that of Holst  ( 1969 ) which shows that 
increasing doses wil l increase the number of large follicles as 
well as the count of cori_::.oru lutea + large fol licles .  Lamond 
( 1964 ) and Larsen ( 1971 ) have also reported increases in 
follicle growth with increasing level s of administered P.M. S .G .  
72 
Regression a�ulyses given in Table 4-9 show that ovarian 
weight is  dependent on the number of corpora lutea per ovary when 
eYJes arc treated once with  P .M . S . G . This relationship does not 
hold for uninjected ewes ant'\ does not always npply whon Gwes are 
given successive injections.  aobinson ( 1951 )  has shown tha 
ovarian weights of  trc2.ted cues to  be  significantly correlated 
Y/i th the number of corporn lutea present in ewes superovulated with 
?. M . S . G. and tllen anc Lamming ( 1961 ) have derived relationships for 
untreated , flushed ewes.  
Regrcssicn relationships for ovarian weight on corpora lutcn 
plus large follicles are better than if only corpora lutea arc 
considered. This  is  consi stent  with the ecrlier suggestion that 
this composite figure is a better indication of total ovarian 
- . " " act1vity than 1s  corpora lutea alone • 
S lopes of curves in Figs . �- 15 and 4-16 are , in majority ,  
similar to those of Figs . 4-4 and 4-5 showing that ovarian ,·might is  
related to the amount of luteai tissue.  TI1c high figure for group 
H3 in Fig � 4- 1 5  is a consequence of the number of owes on thi s  
treatment that had cystic ovaries ( see Table 4-2 ) .  This  effect 
is el iminated in Fig. 4- 16 because such ewes fail ed to ovulate.  
No evidence of a difference in activity between the l eft  and 
right ovaries was revealed in these studies . Mcl(enzie and Terrill  
( 1937 ) and Wal lace ( 1954 ) reported that the right ovaries of ewes 
were significantly  more active than the left but Hutchinson and 
�obertson ( 1966 ) were unable to  verify this�  
73 
In  Sxperiment 3 1  untreated owes wore observed et each of 
three successive oestrous cycles ( 2nd tc' L1th oestrous cycles of 
the breeding season) (Table '.1- 1 1 ) .  The ovulation rate  was 
signif icant ly greater 2.t the third observed oestrus  than at the 
first end second.  That ovulation rate increases from the 
beginning of the season , has been noted by �vorill ( 196� ) and 
McDonald and Ch ' ang ( 19G6 ) , using  �omn�y ewes . Observations  on 
control owes of the present study conform to the  pnttern suggested 
by those authors . 
Detection of , .n t i-rzonadotrophin..,!! 
Lin cmd Bailey ( 1965 ) have shown strain differences in mouse 
response to P.M. S . G . Bell  ( 1969 ) has also shown that some s trains 
of  mice may be relatively inse!lsi  tivo to gonadotrophic stimuletion 
whereas others are very sensitive.  The mice used in this  study wore 
known to be oestrogen sensitive (!.non 1965 ) end for this rec..son , 
only  low level s of ? . M. S . G . were rcquirod to elicit a uterine 
weight response in inunaturc fer.mles.  Suehiro ( 1055 ) and Lamond and 
Bindon ( 1966 ) showed responae curves of mice to plateau at  60 i . u .  
<md 10 i . u. respectively. The i,)lateau obtained in the mice of the 
present study was between o. n i .u .  and 1 . 0  i . u . and the sensitivity 
of  these mice i s  thus exemplified.  Supra-optimal doses frequently  
yield lower ovarian and uterli1e weight responses than sub-optimal 
doses (Suehiro � �. , 1955 ; Lamond and Bindon , 1966 ) nnd this was 
also noted in the mice of Experiment 3 (Fig .  5- 1 ) .  
Responses of mice given 1 . 0  i .u .  gave more uniform (between 
group ) response than mice given 0. 25 i . u.  P .M . S . G.  1 when injected 
with plc..sma (Figs. 5-3 and 5-� ) . The mice given the lower dose 
74. 
were in a far more sensitive dose range than those given the high 
dose and the  greater variat ion of  the former may be  a consequence 
of this .  
S tandard errors associc.ted with moan uterine weights of  mouse 
treatment groups W(:)re comparable  whether mice were given 1 . 0  i .  u .  or 
0. 25 i . u. P .M . S . G . (Appendix <.l ) .  Incref\sod dosage , producing 
greater increases in uterine weight , often increase the standard 
error associated with  the mean response (Lamond and Bindon , 1966 
and de la Lastra , 1972 ) .  ;�dministration of an ant iserum may 
(Sasamoto � �. , 1D72 ) 1  or may not (F lux end Li 1 1965 ) decrease 
s tandard errors of mean response  to P . M. S . G. at a particular dose 
level . This  is  maybe due to the inhibition in response ,  which gives 
lower mean values , and lo�or standard errors could be associated 
with those. No reduction in the standard errors of the mean uterine 
weight response to P .M. S . G ,  by the administration of plasma was 
seen in thi s study.  Thi s  is  explainable  in the mice not showing 
inhibition of response as the mean response is  not lowered . However , 
i t  may have been reasonable to  expect a decrease in standard errors 
associated with  the uterine weights of mice receiving plasma from 
chronically treated ewes but this was not evident .  
Failure t o  obtain any reduction in immature mouse uterine 
weight response to P .M . S . G. , by inject ing plasma of ewes from 
Experiment 1 ,  implies that no anti-P . M. S . G. factors were present 
in the blood of these ewes . The credence of concluding that the 
observed ovarian refractoriness was not due to antibody production , 
depends on the sensitivity  and validity of this test.  Detection of 
ent i-gonadotrophins in the plasma of chronically treated ewes 
75 
confirms that the test used was able to p ick up antibody 
act ivity and to indi cate  the relat ive potencies of the some . 
Similar tes ts have been used by Cole et al . ( 1957 ) 1 Pigon et al . 
� � � .--
( 1960 ) 1 Johnson et al . ( 1962 ) 1 Naknhari et al . ( 1964 ) and 
� -=- �-=---==- -
Jainudeen £! �· ( 1966 ) to successfully demonstrate mltibodios to 
P. M. S . G. Cola � �· ( 1957 ) have sho>m that anti-gonadotrophins in 
catt le behaved in a mar�er similar to  seroloGical antibodies,  
For the present stuGy 1 a biological method of anti-gonadotrophin 
determination wo.s selected in preference to a chemical method . 
Biological inhibition would indicate � � neutralisat ion of 
hormone action .  I t  is  this  consequence of  antibody react ion that 
is being investigated when one consicors refractoriness . Geshwind 
( 1963 ) points  out that the various methods used to  detect and 
quant itate antibody production may not necessarily parallel one 
another. Cole � �· ( 1957 ) exempl ified this when obtaining better 
indi cations  of anti-gonadotrophic activity when using biological 
neutrc.lisation tests tha..""l with precipitation tests .  
Rees-Midgely ( 1969 ) expresses some scepticism about the 
inferences drawn from tests which rely on neutralisation of 
biological activity. He states that neutralisation of a hormone i s  
a good indication o f  the presence o f  ant ibodies , but failure t o  
detect ant ibodies by this method does not automatically mean that 
they do not exist . l�ntibodies may be produced in too low a titre 
to be  detected by inhibition tests  and he suggests  that they 
should be combined with immunochemical procedures.  However ,  
Johnson ( 1962 ) presents  evidence t o  indicate that an antibody t o  a 
hormone may be present yet not produce procipitins in immuno-
chemical analysis and that the P .M.  S . G. -anti-P. M.S . G. complex 
76 
may be  of this type.  
Ouchter�ony doubl�diffusion t ests  (Ouchterlony,  1949 ) were 
at tempted on samples of plasma obtained from sheep in this study •. 
Failure to observe any precipitation between antigen and antibody 
impl ied no evidence of anti-gonadotrophins whereas inhibition was 
obtained when similar samples were injected into mice. The 
Ouchterlony tests were nbo.ndonod becausa it  was thought the antigen 
(P . IVl . S . G , ) was of too low a potency for this method. 
Chronic treatment s of sheep wi th P .M . S . G. (Expt . 3) produced 
results  similar to those of Pigon � �· ( 1960) ,  I t  appeared as 
though sheep �)roduccc:. antibodies to P ,M. S . G. more readily with 
a lower dose of the hormone . In  the present  s tudy sheep injected 
twice weekly with 500 i . u . P ,M , S . G. had greater antibody titres  
(not significnnt )  than those given 1000 i , u . , at the end of  6 weeks 
of t reatment • This is cont rary to results  obtained in c�ttle  
(Cole � �. , 1957 ) where lower doses are seen to  produce lower 
t itres of antibody,  
The P .M, S , G.  preparation used in this  study was of low potency 
(epprox, 40 i .  u. /mg. ) which is  similar to that used by Pigon � !:!.· 
( 1960) ( 37 . 6  i . u . /mg. ) and much lower than other workers have u sed 
to develop antisera ( e. g. Flux and Li , 1065 used a preparation with 
a potency of  2500 i . u . /mg. ) .  
Before detection of anti-P.M , S . G. in sheep , Pigon � �· 
injected approx. 16 mg. ( at a dose of  200 i . u. ) ,  approx. 80.4 mg� 
( at a dose of 500 1 . u . ) and approx. 267 mg, ( at a dose  of 1000 i . u. ) •. 
The amounts  of hormone injected into sheep receiving three 
injections of 1500 i , u . (e. g . Trt . H l ) � this study,  would be 
77 
approx. 30 mg. Of chronically treated sheep 1 those injected with 
500 i .u .  received a total of approx . 150 mg.  and those dosed at 
1000 i . u. 1 approx . 300 mg. 
If the study of Pigon £! �· ( 1960) presents a typical picture 
of  anti-P . M. S . G .  production is  sheep , if the sheep in that study 
were of the same weight as those used in the present study and i f  
the amount of nntigen introduced has an effect on the rate of 
antibody production ,  then certain deductions may be made.  I t  
appears as though sheep on all t reatments in  Experiment 1 did not 
receive sufficient antigen to produce an immune reaction , whereas 
chronical ly treated ewes were supplied with udequatc amounts.  Both 
Cole £! �· ( 1957 ) and Naknhari � �· ( 1964 ) have stated that 
treatment with gonadotrophins must be excessive and unphysiological  
to  obtain immune responses. One l imitation of this argument is 
that the amount of hormone int roduced �ay not be as important as 
other factors in enhancing antibody formation ( see Cole � �. , 1957 ) .  
Time factors are also important in the development of antisera.  
Jainudeen .£! �· ( 1966) show that if cows are dosed once with P . M. S . G . 
and then left  for 180 clays,  a second injection will produce rapid 
formation of  antibodies . This typical  effect is  also displayed in  
cows by the  study of Cole � �· ( 1957 ) and in rabbits  (Leathem1 
1947 ) .  I t  i s  likely that i f  sheep in this s tudy were left  for a 
period of months and then given an injection , or a second series of 
injections , appreciable  antibodies ti tres would have been detected . 
I n  contrast to studies where nnti-gonadotrophins are obtained by  
rotreatment of animal s some time after the first  injection( s ) 1  
some workers have shown that short term t reatments  will do l i ttle 
78 
to produce anti-P .M. S . G. factors . Cole et al . ( 1957 ) showed that 
1500 i . u . given every week , or 3000 i . u .  given every third week , 
results  in very l ittle formation of ant ibodies in cattle  ( treatment 
for about three months ) .  Polgc and Rowson ( 1973 )  reported that 
gonadotrophic  stimulation of pigs at each of three sequential 
oestrous cycles did not produce a refractory condition in these 
animals  and no evidence of anti-gonadotrophins was obtained . 
In  conclusion , it appears that the injection of sheep over 
a short space of time (2 oestrous cycle lengths sepurating three 
injections : 34 days ) is  unlikely to produce serological ant i­
bodies against ? . M. S . G . The present study ratifies this statement . 
Fol licular Dynamics of the Ovary 
Discussion in this  section relates primarily to results  
obtained in Experiment 3 and deals with  observat ions on foll icular 
dynamics in the ovaries of ewes sequent ially treated with ? . IYi. S . G. 
The choice of  Day 10 as a reference point to monitor  follicle 
development could have an important bearins on any conclusions 
drawn f rom observat ions  that VJDre made.  If the pattern of growth 
suggested by Smeaton and Robertson ( 197 1 )  is accepteG , then the 
time of observation would hr.ve coincided with the waning of the 
second wave of follicular growth and th e beginning of the third 
( sec Chapter 1 ) .  I f  Brand and de Jong ( 1973 )  have revealed a more 
likely pattern of fol licular growth , then the observations would  
have been made after the first wave and mid-way during the �ave 
that terminated in ovulation . In  this latter case observations 
would hnve given some indication of  fol l icles that would  respond 
to gonadotrophic stimulation 2 days after observation (Day 12  of  
79 
the oestrous cycle}. Brand and do Jong kil led 39 owes at 
different s·tages of the ocstrouc cycle and studied microscopic 
changes in follicles , account ing for follicular atresia . Smeaton 
and Robertson injected Indian ink into  fol licles  and follormd 
development in a much smo.ller number of 0\1€S . The work of Brand 
�nd de Jong i s  probably more informat ive than that of Jmeaton and 
�obert son because of the more sophisticated techniques �nd the 
le.rger numbors of  ewes urn�)loyod by the former authors � 
I t  has been shown that there was very litt le  difference in 
the total number of  corpora lutea plus follicles greater than 1 mm. 
when ewes treated vlith ? � r.J. S � G. ( once , twico or three t imes ) arc 
compared with control animals  (Table 4-0 ) .  I n  ewes superovulated 
once ,  about half of this composite  f iGure i s  accounted for by the 
count of  corpora lutea. In  the o.ppropriate cont rol G.nimal s ,  the 
figure is mainly r�erived from a count of follicles.  /.pparently , 
the injection of :-'. 1\I. S . G • was able  to  ovulate follicles that were 
analogous to  those observec: on the surface of  ovaries in the control 
animals . Hovmver , the injection must  ho.ve st imulated cxt:ra 
follicle growth as well  since the total number of  fol licles seen 
in treated ewes was not si�nificant ly d ifferent from the number 
in cont rol owes (To.ble 6- 1 ) .  
In animal� stimulated a second and third t ime , ovulation rates 
were not significantly greater than thoso of control enimal s ,  
indicating that a refractory condition had been attained.· Neither 
did the count of corpora lutea. plus foll icles greater than 1 nun. 
J 
J 
I 
00 
differ between control and tro�tod animal s at these two 
observations .  The first injection seems to st imulate ovulating 
and non-ovulating fol licles.  The latter are enlarged by the 
gonadotrophic stimulation and then become atretic. At the second 
and third injections neither ovulating foll icles nor non-ovulating 
fol licles were stimulated by the gonadotrophin . The fol licle 
stimulatory action of ? . M. S . G. seems to have become ineffective 
as well  as the ovulatory capacity.  
;";.! though P. M. S . G. is often thought of an an F . S . H . -like 
hormone , i t  may also display properties similar to L. H .  Lamond 
( 1959 ) and Schmidt-Emendorf � �· ( 1962 ) have suggested that 
approximately one-third of  the endocrinological act ion of P .M . S . G .  
may be  L. H . -like. It is not known to  what extent the L. H .  f raction 
of a !J. M. S .  G. preparation may contribut e to  the superovulatory 
response of ewes.  However, thoro is evidence to suggest that the 
exogenous gonadotrophin may exert an effect on the pituit�ry gland 
and thus the ovulatory capacity may not be by  d irect influence on 
the ovary. This mediation of response via the pituitary wil l  be 
discussed in more detail later.  
Inference from the data presented in Tables 6-1 to 6-4 is 
difficult .  Error mean squares (EMS ) for comparisons involving 
surface follicle  counts are large and thus ,  large standard errors 
are associated with the treatment group means.  This indicates 
extreme between-animal-within group variation and has been 
encountered by other authors (e . g. Brand and de Jong,  1973 )  when 
measuring similar variates � These lnrge s tandard errors make 
comparisons between treatment groups unlikely to be of statistical  
131  
significo.nce. The l ikelihood of uppo.rent real differences between 
groups is further Gecreased by the small numbers of animal s per 
�roup tho.t were on oomo of the treatment s.  For these statistical 
reasons 1 any real trends in Fig.  S- 1 are of little significance. 
From Fig. 6- 1 there is  some sum;estion that treatec: ewes 
consistently  show fol liclGs  in the 1 ln.r�e size ' category to bo a 
lesser proportion of the tot al foll icular population tho.n they are 
in the control ewes . This  trend i s  not statistically  significant.  
�ven if  the number of larse fol l icles per ovo.ry is  slight ly 
diminished in treated eves t21i s i s  of lit tle import to tho 
development of a refro.ctory condition . There are still  follicles 
present on the ovaries that would be  capable of responding to 
GOnadot rophic  stimulation . Smecton and Robertson ( 1971 )  show that 
follicles of approx . 2 ffim, in diameter may grow rapidly over the 
last 36-46 hours before ovulation .  Gonadotrophins should therefore 
be able  to enhance the growth of such foll icles .  J.efrc;ctorineso 
seems to be the inability  of the exogenous gonadotrophin to 
stimulate such fol licles .  
/;.11  relationships between ovarian surface data nnd estimated 
total ovarian activity were found to be statistically not­
significant . I t  the�ofore appeo.rs as though inference from results 
on surface follicular data are not representative of total ovarian 
activity.  Land ( 1973)  hao recently  presented evidence which 
suggests  that the number  of  fol licles destined to ovulate f rom 
ewe ovaries is  not determined until  within 3 days before 
ovulation . Maybe the observations of the present study were 
made too early to give informative indications of the imminent 
132 
effect of a gonadotrophic stimulation on the follicles observed 
at Day 10. This suggestion is supported by the fact thet 
ovulation rates were very rarely and inconsistent ly related to  
the observations made on Day 10 of the  cycle (Tables 6-5 and 6-6 ) .  
Cross-sectional and histological observations  may be more 
informat ive than the surface follicular data.  
Elimination of the proportion of  antral fol licles that were 
atretic  accounted for a very important unknown f rom the estimations 
of total ovarian activity.  The proportion of atretic foll icles 
that were included in  surface counts was not known and fo1· this  
reason , estimated total number of normal fol licles per ovary is 
probably a more accurate assessment of ovarion folliculcr status. 
The percentage of fol licles in the ovaries of control owes that 
YJere classed as being atretic compared agreeably wi th observations 
of  Brand and de Jong ( 1973) . This is  encouraging as the assessment 
of atresia may often suffer from the subjective nature of descrimin­
ations between normal nnd atretic fol licles.  The early stages of  
atresia are di fficult  to discern ( Ingram 1962 ) .  Brand and do  Jong 
were able to use more elaborate methods to differontinte bet\"Jeen 
normal and atretic fol licles as they sectioned each follicle 
serially.  This  was not done in  the present study  where s��ples 
were drawn from many ovaries and the state of a single follicle  
was assessed from only  one  microscopic  s ection per ovary , although 
criteria of fol licle  atresia wore basically  s imilar to those used 
by  Brand and de Jong.  It  is  debatable whether serial section of a 
small  number of ovaries i s  more informative than limited sectioning 
of a larger number. 
03 
The proportion of  antral follicles that were atretic was 
larcer in animals given one injection than in animals on other 
treatments  of Experiment  3. The number of normal fol licles  of 
owes in this group was thus lowered to become significent ly less  
than for e\"ies on the other three t reatments .  /.1 though ewes treated 
once With :r: .M . S . G . had significantly  greater numbers of follicles > 
3 mm. (Table G-4 ) when comp2.red with  ewe;s t reated two or  three 
times , the majority of thcne fol lic les were probably atretic .  
The low number of  no:..-mel antral fol licles observed in the 
ovaries of ewes t reated once with P • .  M. S . G . may be of pertinence to 
the development of a refractory condition.  These ewes had similar 
numbers of antral foll icles to emas treated two or three t imes 
(Table  6-7 )  but a larger proportion were atretic .  The atretic 
fol l icles were likely  to have been stimulated by the previous 
injection and have porsistoc1 to the point of observo.tion.  Their 
persistence may impair the growth of smaller , ' heo.lthicr' foll icles 
which would be capabl e of respondinrs to a further injection of 
gonadotrophin . Greenwald ( 1063)  has surmised that the persistence 
of such follicles might increase steroid production which would in 
turn decrease the release of endogenous gonadot rophin. I f  P .M . S . G. 
acts synergistically with cndogenouE gonadotrophin ( sec Chapter 1 )  
then i t ' s action could b e  impaired by this sequence of events .  
Furthermore , . the ovary at this  point is support ing corpora lutea 
and persistent foll icles . ·  S timulation with P .M . S . G . 2 days after 
observation may not be effective because normal follicles have 
little space in which to grow. Hulet and Foote  ( 1969 ) have 
indicated that sequential :?. M. S . G . treatment  may have a luteo­
trophic  effect and if  this caused persistence of corpora lutea ,  
the ovary may be  less  sensitive to gonadot rophins on two counts ;  
hormonal status of the onimal ( high circulat ing levels of 
progesterone) and persistence of  corpora lutea which may absorb 
the blood supply of L.H .  ( or Il , M. S .G . ) nnd inhibit fol liculnr 
growth. 
Suggestions in the last paragraph are unsubstantiated by any 
convincing experimental evidence but should be considered by any 
�orkers who further investigate the refractoriness  of ewes t o  
P. M. S . G . The state of the ovaries in ewes over the oestrous 
cycle subsequant to P .M. S . G .  injection has not been studied in 
detail .  Such a study could utilise laparoscopic techniques and 
would be valuable  in trying to assess the effects  of gonadotrophic  
stimulation. The relationships between endocrine status of ewes 
nnd concomitant changes in follicle growth have not been studied 
and much informat ion on these related events is required before 
a true understanding of the pattern of fol licular growth in ewes 
is obtained. 
General Discussion on ilefractoriness to P.M. "S ; G. 
I t  has b een shofm that refractoriness is  rapidly attained 
and much lower ovulation rotes wil l  be  achieved by n second 
injection of P , M. S , G. , given one cycle after the first .  Anti­
gonadotrophic activity is  unlikely to account for this rapid 
decline in response , probably because of a lack of t ime for the 
establiehment of on immune react ion and also because of the 
insufficient quantity of antigen injected . 
The mos t  prominent result obtained in a study of ovarian 
follicular development was that n significant ly lower number of 
85 
normal follicles per ovary arc observed 10 days after supcrovulatory 
response to one injection of P. M. S . G. compared to the number in 
animals on al l other treatments.  Lower ovulation rates in  response 
to  o second injection of P . &·I. S . G. arc likely to be due to  this 
lowered number of normal antral folliclos that are potentially able 
to respond to gonadotrophin. However , an apparent resto�.tion of 
the follicular population causcG the est imated number of normal 
follicles in ovaries of e·aos stimulated a second and third t ime 
not to  differ from similar counts in the ovaries of control 
animal s .  The lack of  normal , potentially ovulnting1 antral 
follicles is therefore unlikely to be  a persistent causal factor 
of refractoriness . 
Observation at Day 10 of the �oconu cycle ( following a second 
injection ) su(';gests t hat ovarian fol licular populations have been 
restored and yet the third st iwulation does not produce effective 
superovulation.  I t  is  at thi s  point that a third factor i s  
suggested a s  likely to cause refrnctoriness .  A review o f  literature 
on the mode of E'.ction of P . r:I . S . G. is outlined in Chapter Ono and 
i t  is  apparent that P . M . S . G . may have a direct effect on the 
pituitary by eliciting the release of endogenous gonadotrophin. 
This evidence has mainly boon gained from polytocous animals ( e . g. 
mice ) but there is now some suggestion of similar effects in 
sheep (monotocous ) .  Pi�on et al.  ( 1960 ) have shown that chronic 
� -
t roatmet1t of  ewes with P .M .  s .  G.  will markedly lower the pituitary 
L. Ii.  content of such animals.  The increasing prevalence of  owes  
becoming anovular , and the increasing incidence of  cystic and 
persistent luteinised follicles ,  in ewes treated sequentially 
06 
wit� P.M, S , G . further suggests thnt the L .H .  supplies of  these 
animals  are becoming e:r.:haustcc . The L . H . -like moeity of P. M. S . G, 
does not seeQ able  to ex�rt an ovulatory effect in sequential ly  
treated ewes and it  may bo that this fraction of the  exogo�ous 
hormone nets  synergisticnl ly with endogenous gonadotrophin , 
which v;ould be released on ndr:linistrution of P, i',1. S . G. 
Polge and nowson ( 1973 )  have recently reported that pigs w:·_ll  
not become refractory to gonadotrophic stiQulntion if  P .M . S . G o  io  
used in conjunction w). th H . C . G. 1 over three oestrous cycles .  
�!though there may be important differences between polytocous and 
monotocous ani�al s 1 thi s observation of Polgo and Rmvson ( 1973 ) 
may have important implications  with respect -::o the development of 
ovcrian refractoriness in ewes. Varied resul ts h3ve been obtnincd 
vfi th the use of H. C. G. in ewes but Hunt et al . ( 197 1 )  have shmvn 
,_,� c:=o<L"D 
good responsEJS with purified F . S . H . and L. H .  extracts .  U; is 
suggested that those results  should be examined in greater detail 
in an effort to see whether such hormonal t echniques would help ·�o 
overcome refractoriness in ewes . 
I f  refrnctoriness observed in ewes t reated with P .M . S . G. ovo::-
three oestrous cycles is related to the depletion of endogenous 
L .H . , n hypothesi s of the chain of events which causes tho 
condition could be proposed. The condition may be precipitated 
by an initial exhaustion of normal ant ral  foll icles which would  
reduce response to a second injection.  However,  due to sequential 
t reatment , L .H .  reserves (pituitary ) may al so become depleted so 
that when follicular populations are restored ( i . e. at the second 
observation in Experiment 3)  the ovaries are unable to respond to  
fJ7 
further stimulation because of  a lack of L . H .  to enhance the 
effect of the exogenous hormone. 
Tho staggering of  suporovulatory doses was seen to partial ly  
ovorcomu refractoriness .  This  may be explnined with reference to  
the  proposal in the preceoding paragraph. Wi th n speco of one 
oestrous cycle between injections , follicular populntions he.ve e. 
sufficient period of  t ime  to bo restored and atretic or cyatic  
follicles would be given t ime to  reGress .  Furthermore , pituitery 
reserves of L . H .  could  bo  replenished without any oxogonous 
int erference. A second injection of gonadotrophin i s  thus able  to 
rostimulnte the ovnrios to auperovulatory responses . 
Further work on the development of a refractory condition in 
ewes troo.tod with F .M . S . G. ovor a short period i s  prob2.bly 
justified .  I f  the condition were overcome,  the short term yields 
of largo numbers of ova could be  possibl e from n small number of 
owes . I t  is unlikely thnt ant i-gonadotrophic factors are 
responsible for the condition and limited information would be 
obtained from studies on follicular status of such ewes until 
further work is  done on animals in the normal state.  Investigation 
of endogenous L . H .  depletion seems a promising l ine of study. I t  
should be establ ished whether such a depletion does occur with 
short term treatment . I t  may then be  feasible to  rectify the 
condition by supplementation with rel iable  L . H .  extracts .  
R � F � H E N C E S 
88 
REFERENCES 
Adams , C . E .  ( 1953) Inhibition of  superovulation in the rabbit.  
Nature (Lond. ) ,  �: 82 � 
Lllen ,  D .M .  and Lamming,  G. E .  ( 1961 )  Nutrition and reproduction in 
the ewe. J .  Agric. Sci . , 56 : 69 
Allison , A . J .  ( 1968 ) The influence of  livoweight on ovulation rate 
in the ewe. Proc . N . Z .  Soc. Anirn.  Prod.  1 � : 1 15 .  
Allison , A . J .  ( 1973)  � ·  �· 
tlnon ( 1965 ) Mouse N�wslettcr 1965 ,  No . 3 , p. 52 
Animals Centre (M . R.C . ) .  
Laboratory 
Averil l ,  R .W. L. ( 1958 ) The production of living sheep eggs.  
J .  l\.gric. Sci .  1 §.2 : 17 . 
t.verill 1 R .W. L. ( 1964 ) Ovulatory J"..ctivity in mature ROOUloy ev1es in 
Now Zealand . N . Z .  J. Agric. Re�. , l :  514 .  
Averill R .W . L . and Rowson 1 L. E . I  • •  ( 1959 )  Attempts  at storage of  
sheep ova ot  low temperatures . J .  Agric . Sci.  1 � : 392 . 
Baird , D. T. , Goding , J . R. , Ichikawa , Y.  ond McCracken , J .h .  ( 1968 ) 
The secretion of steroids from the autotransplanted 
ovary in the owe , spontaneously , and in response to 
sys.tomic gonadotrophin. J.  Endocr. , � : 283. 
Bell ,  E . T. ( 1969 ) The assay of Foll icle Stimulating Hormone by the 
ovarian augmentation test in nine mouse colonies . 
J.  Reprod. Fert . , l£ :  546. 
Bindon , B. M. , Ch ' ang , T. S .  and Newton-Turner , H. ( 197 1 )  Ovarian 
response to Gonadotrophin by Merino owes selected for 
fecundity.  Aust.  J .  Agric. Res. , � : 809. 
69 
B indon , B .M .  and Robort s 1  E . M. ( 1964 ) Control of ovarian 
act ivity in owes with progestegons . J .  Reprod. Fort . , 
7 : 337 . 
Bindon , B. M. and Lamond , D. n. ( 1966 ) Diurnal component in the 
response by the mouse to gonadotrophin. J� Roprod. 
Fort�, 11_ : 249 . 
Bro.dcn 1 r • •  �·r. H . , Lamond , D. R .  and Radfo rd , H .M .  ( 1960 ) The cont rol of  
the time of  ovulation in  sheep. hust .  J .  �gric . _ non . , 
1 1  : 30:::> . 
Bradford , G. E . , Quirko 1 J . F .  and Hart ,  R.  ( 197 1 )  The natural and 
lnduced ovulation rat e of Finnish Landraco a::1d other 
breeds of shoep , £!.�!_!!1.:_!2:o.ch_ ,  J1 627 . 
Brand 1 A. and de Jong , W ,H . R .  ( 1973 )  Qual itative and quantitative 
micromorphological investigat ions of  the tertiary 
fol licle population during the oest rous cycle in the 
sheep . J .  Reg�d . Fort� ; � :  431 .  
Brock , H .  and Rowson , L. E �n .  ( 1952 ) The production o f  viable  bovine 
ova. J ._..!i�� S ci . , � : 479 . 
Brown , P. S .  ( 1955 ) The assay of gonadotrophin from urine of non­
pregnant human subjects .  J. Endocr. , 13 : 59 . 
Christiansen , C . M . and Eleftheriou , B . E .  ( 1972 )  Dose dependence of 
superovulation in mice to two inject ions of P .M. s �G .  
J .  ReE£od. F�. , 29 : 287 . 
Chouda.ry , J . B . 1 G ior,  H . T .. and Marion , G; B.  ( 1968 ) Cyclic changea 
in the bovine vesicular foll icles . J. Anim. Sci . , � 
460. 
90 
Clarko , J . N .  ( 1973)  Sheep immigrants arrive at S omes I sl �nd . 
Moat and Wool (N �.�.:) F ebrualf:l ,  p .  3 .  
Clarko1  J . N . 1 �tobert s ,  E . M. 1 Carter , A . H . and Kirton , I. . H. ( 1966 ) 
Hormonal synchronisation of  oestru s  in �1omney owes 
during the b reeding season . Proc. N . Z .  S oc. Anim. i'rod. , 
26 : 107 . 
Col e ,  H . H .  ( 1969 )  Physiological c..'1o.racterisation of gonndot rophins . 
I n  "aeproduct ion in Domestic f�nimal s
" ( cd .  Col e ,  H .H. 
and Cupps , P . T. ) p. 17 , Academic Press , New York and 
London . 
Col e ,  H . H . and Erway , J .  ( 194 1 )  48 hour test  for equine 
gonadot rophin with results  in I nt ernational Units.  
Endocrinology, � : 514 . 
Col e ,  Ii . H .  and Mil ler , a . F .  ( 1933 ) ,".rtificial induction of 
ovul�tion and o0s trus in the ewe during anoestrum. 
J .. m.  J .  f'hysiol .  1 � : 165.  
Cole , H . H. and Hart , G. H. ( 1930 )  The potency of t he blood serum of 
Mo.res in progressive s to.ges of pregnancy in effect ing 
the sexual maturity of the immature rat .  Am . J .  Physiol . 1 
� :  57 . 
Col e ,  H .  H .  1 Hrnnburger,  C .  nnd Nieman-Sorensen , A .  ( 1957 ) S tudies on 
anti-gonadot rophins with emphasi s on their fo rmation in 
catt le. �eta 3ndocrinol. , 26 : 286 . 
Col lip, J . B . 1 S elye , H .  and Thompson 1 D . L .  ( 1940) The Antiho:rmones . 
Biol.  nev . , 1 5  : 1 .  
91 
Coop , I . � . ( 1966 ) Effect of flushing on reproductive performance 
of ewes , J .  Agric. Sci. , §1 305. 
Coop , I , E ,  ( 1972 ) Protein production from sheep , goats end deer. 
Proc, N . Z .  Soc .  Anim. Prod. , B : 138 ,  
Connell 1  G ,M .  ( 1965 )  Pregn2>nt Mare' s Serum Gonadotrophin potency : 
effect of single nnd multiple inj�ct ions. Nature (Land. ) , 
207 : 412.  
Cumming , I .A .  ( 1965 ) A study of ovulation and early prenatal 
mortality in the New Zee.land Romney ewe. M.  Agri£. 
Sci . Thesis ,  Massey University. 
Cumming 1 I . L. , Brorm , J. M. , Blockey , M .A .  de B . , and Goding, J. R .  
( 197 1 )  Regulation of  the oestrous cycle in the ewe·. 
J .  Reprod. Fert . , 24 : 148 . 
Cumming , I . A .  and McDonald , M. F .  ( 1967 ) The production of ova by 
New Zealand Romney ewes following hormonal stimulation. 
N . Z. Jl . Agric. Res. 11£ : 226 . 
Dc ' la Lastra , M. , Forcell edo, M. L .  and S errano , C .  ( 1972 )  
Influence of the hypophysis on Pregnant Mare' s Serum 
Gonadotrophin induced ovulation in immature rats.  
J .  Reprod. Fert. , � :  23. 
Desjardins , c.  and Hafs , H .D .  ( 1965 )  Immunochemical and electro­
phoretic properties of Equine Luteinising Hormone. 
J. Anim. Sci .  1 � : 347 . 
Dutt ,  R. H.  ( 1953 ) Induct ion of oestrus and ovulation in anestral 
ewes by use of progesterone and Pregnant Mare S erum. 
J. Anim. Sci .· 1 E : 515. 
92 
Dzuik , P . J .  1 Donker , J . D. 1 !'lichol s ,  J . R .  and Petersen , �i; . E .  ( 1D58 ) 
Problems associatcG with thu t ransfer of ova in cattle.  
Tech . Bull . Min�ic. �xp. Sta. , No. 222. 
Gastwood , K . C .  ( 1973)  Pers . Comm. 
Bdey , T . N .  and Thwaites , C . J. ( 1966 )  Syn chroni sation of ewes at the 
second oestrus  after progostngon t reatment .  Proc. Aust . 
Soc. !Ulim. Prod . , � : 38. 
�dwards , R .G .  and Fowler , D. G .  ( 1960 )  Superovulation troetment of 
adult mice:  their subsequent fert ility and response to 
further t reatmen t .  J. Endocr. ,  � :  147 . 
Ely,  C . h .  and Chm1 ,  B .  ( 1966 )  Immunological stud ies of the 
Luteinising Hormone. 1 .  I dentification of the antigen 
components  of sheep Luteinising Hormone and the 
development of e serologica l ly specific ant i serum. 
J. Endocr. ,  79 362 . 
Ford , H. E.  ( 1966 ) Obs ervat ions on the use of progcstegen 
impregnat ed t ampons in a herd of Dorset Hill sheep. 
Vet . Rec. , ZQ : 461 .  
Falkenburg , J . l.. . ,  Hulet , C . V .  nnd Kal tenbnch 1 C . G. ( 197 1 )  C:ffects 
of hormone combinations on est rus , ovulat ion and 
fertility in ewes ,  J .  fUlim. Sci . , � :  1206 . 
Findlay , A . L . P. .  and Vaughn , S . M. ( 1964 ) The pract icabil ity of  
methods of controlling the oest rou s cycle and breeding 
s eason in the sheep : a review. Vet . Rec . , � : BOO. 
93 
Flux , D. s .  and Li , C . H .  ( 1965 ) Immunological croos reactions among 
gonadotrophins .  �eta Endocrinol . 1 i£ : 6 1 .  
:�owlor , a. E .  and Edwards , R. G. ( 1957 ) Induction o f  superovulation 
and pregnancy in mice by gonadotrophins.  J.  Endocr. , 
� : 37·1 . 
Gosh\7ind I .  I .  ( 1963 )  The c,'lcmistry and immunology of  aonado­
trophins .  In  "Gonadotrophins their chemical and 
biological properties and secretory control , " ( ed .  H . H .  
Cole ) , p , l : Freo@an , San Francisco. 
Gibson , W. R.  and 2obinson , T. J. ( 197 1 )  The seasonal nature of 
r0productive phenomena in sheep . 1 .  Variation in 
sensitivity to oestrogen . J .  �1eprod , Fort . , � : 9,  
Gordon , I .  ( 1950a ) The us� of serum gonadotrophin ( P .M . S . )  in  the 
control of fertility in sheep. 1 .  The hormonal 
augmentation of  fert ility in the ewe during the 
breeding season. J .  il.gric. S ci .  1 � : 123.  
Gordon , I .  ( 195Gb ) The use of scrum gonadotrophin ( P .fi'I, S . ) in  the  
control of  fertility in  the sheep. 2 .  Studies in the 
extra-seasonal production of lambs . J • .':.gric . Sci , 1 .2£ 
152.  
Gordon , I .  ( 1963a) I nduction of pregnancy in the anoestrous ewe by 
hormonal therapy. 1. Progesterone - Pregnant Mere' s 
Serum therapy during the breeding season . J.  f,gric. Sci . 1 
60 : 3 1 .  
Gordon , I .  ( 1963b ) I nduction o f  pregnancy in the anoestrous ewe 
by hormonal therapy. 2, Progesterone - Pregnant Mare ' s 
Serum applications during anoestrous. J.  l:..gric. Sc1. 1 
60 : 43. 
94 
Gordon , I .  ( 1969 )  Controlled reproduction in sheep and cattle.  
I r. Dept . t.gric. J . , §§. :  184 • 
Gordon , I .  ( 197 1a)  Induction of early breeding in sheep by 
standard and modified progestagen-PMS treatment s .  
J .  Agric.  Sci.  1 � : 337 . 
Gordon , I .  ( 197 1b ) SC90C0-progestorone applications in  the 
induction of early breeding in sheep. J .  �gric .  Sci . , 
� : 3'13.  
Gordon , I .  ( 197 1c)  Intravaginal SC98DO-progesterone treatments  in 
the synchronisat ion of cyclic sheep. J. Agric. Sci . , 
1.2 : 361 .  
Gordon , I .  ( 1972 )  Physiology and animal product ion - the years 
ahead . Proc. :'lorld Confr. <'•nim . Prod . ( 1973 ) , 5 : 34 . 
Gospodarowicz , D .  ( 1972 )  Purification and physiochemical 
properties of the �'rcgmmt Mare' s S erum Gonadotrophin 
( P .M . S . G . ) .  Zndocrinology,  £1 :  101 .  
Grant , n.  ( 1934 )  Studies on the physiology o f  reproduction i n  the 
ewe. Trans.  n. Soc. (Edin . ) lviii ,  ?t . 1 ,  No. l .  
Green 1 J . A .  ( 1955 ) Hormone secretion by the immature mouse  ovary 
after gonadotrophic s t imulation . Endocrinology, � : 62 1 .  
Green , J .A .  ( 1956)  Functional responses of the immature mouse ovary 
to different levels  of Equine Pituitary Gonadotrophin. 
Anat ! Rec . 1 E§ : 195.  
Greenwald , G . S .  ( 1963 )  Effect of ant i-PMS serum on superovulation 
in the Hampster.  Endocrinology, ll : 436. 
Greep , R. O.  ( 1973 )  A vista of research on the mammalian gonado­
t rophins.  Biol . �eprod. ,  � :  1 .  
95 
Hafez , E , S , E ,  1 Rajokoslci , 2 . 1 Anderson , P ,,B  • .  1 Frost , O. L.  and 
Smith , G. ( 1964 ) Problems of gonadotrophin induced 
multiple pregnancies in beef cattle.  Am. J. Vet •. Res . 1 
� : 1074 . 
i{ancock , J . L. and Hovel l 1 G. J , R. ( 1961 )  Transfer of sheep ova.  
J .  Reprod . Fert . , � : 295 , 
Hammond , J .  ( 1952 ) Proc,  6th Int . Congr. Anim . Husb ,  p. 38.  
Cited i .. llcn and Lnmming ( 196 1 ) ,  
Hammond 1 J .  (Jnr. ) ,  Hnmmond 1 J .  and Parkas , A . S .  ( 1942) Hormonal 
augmentation of fert il ity in 4heep . 1 .  Induction of 
ovulat ion and superovulation and heat in sheep . 
J .  /�gric. Sci . , � : 300. 
Holst , P, J ,  ( 1969 ) The ovarian response to  Pregnant Mare ' s Serum 
(P . M. S , ) administ ration aft er intravaginal progestagen 
treatment of cyclic c.nd anoestrous ewes . i:.ust.  J .  i .  gric.  
�. � :  1 143.  
Holst , P . J. 1 Mattner , A .V!, H, and Braden 1 P .E .  ( 1972 ) t.ssociation 
between ovarian fol licular developmen t and oestradiol-
17-beta secretion 3-4 days after oest rus in the owe. 
J .  Endocr. , � :  47 1 .  
Hul et 1 c . v. and Foote ,  w. c. ( 1967 ) Physiological factors affect ing 
frequency and rate of lambing . J. Anim. Sci . , � :  553, 
Hulet , c .v .  and Foote , \'! , C ,  ( 1969 ) Ovulatory response of the ewe to 
repeated injections of  P, M. S .  J .  Anim. Sci . , � : 457 .  
Hunt , W. L. 1 Addleman, D .  and Borgart , R .  ( 197 1 )  Induction of mult i­
ovulation in the ewe fol lowing synchroni sation of 
. estrus,  J,  Anim. Sci .  1 � : 49 1 .  
96 
Hutchinson 1 J . S . M. and ilobertson 1 H . A .  ( 1966 ) The growth of the 
follicle and corpus luteum in the ovary of the sheep . 
Res . Vet .  S ci.  1 1 17 . 
I garo.shi , M.  and McCann 1 S . M. ( 1947 ) 1. new sensitive bioo.ssay for 
follicle stimulatin8 hormone. Endocrinology1 Zi : 440. 
I ngram , D. L. ( 196 1 )  Atresia. In  "The Ovary" ( ed .  s.  Zuckermann) 1 
p , 247 : Academic Press , New York and London . 
Jainudeen 1 M . R. 1 Hafez 1 E . S . F . , Gollnick , P . D. and Moustafa , L .� .  
( 1966 ) il.nti-gono.dotrophins in  the serum of cows 
following repeatoo therapeutic  P . l'vl . S .  injections . 
Am. J. Vet . Res..:. ,  31, : 669 . 
Johnson , n. c.  ( 1962 ) Immune responses with placental gonedotrophins.  
Gen. Compnr. �ndocr�� � Suppl . 1 1  p. 22 .  
Kammalade , l"l. G. 1 i.'lelch 1 J .A. , No.lbandov , A . V. and Norton , H. Yl . 
( 1952 ) Pituitary activity  of sheep in relation to the 
breeding season . J. Anim. S ci .  1 Q : 646 .  
Ketzman 1 P .A . , Wade,  N .  J .  and Doisey 1 E . ;  • •  ( 1947 ) Gonadotrophic 
modifying action of sera of animals treated with 
hypophysoal extracts•  Endocrinology1 i1 : 27 . 
Lomond , D. l1 .  ( 1960 )  Induction of ovulation in mice with placental 
gonadotrophins .  J. Sndocr . , � :  277.  
Lwnond1 D. R. ( 1962 ) The effect of season on hormonally induced 
ovulation in Merino ewes. J .  Reprod . Fert . 1 1 : 1 1 1 .  
Lamond ,  D. n.  ( 1963 ) The effect o f  nutritive status on oestrus , 
ovulation and Graaffien follicles of  ewes . Aust . J .  
Agric. Res . , 14 403. 
97 
Lamond 1 D. R .  ( 1964a) Synchronise.t ion of ovarian cycles ln sheep 
and catt le.  i' .. nim. Breed . J�bstr. , 32 : 269 . 
Lomond, D. R. ( 1964b ) �uentitotive studies of the interaction 
between i='regnant Mare G erum and progesterone on 
ovarian function on the ewe. J.  Heprod . Fort . 1 7 : 17 1 .  
Lamond 1 D. R. ( 1964c ) Seasonal chonees in the occurrence of oestrus 
following progesterone supporession of ovarian function 
in the Merino ewe. J.  Reprod . Fert . 1 8 : 101 .  
Lomond , D .R .  ( 1970) The effect of P.M. S . G.  on ovarian funct ion of 
beef heifers as influenced by progestagens , plane  of 
nutrition nnd fasting. ;"..us t .  J. l'..gric . !1es . 1 ll 153.  
Lcmond , D. n.  and B indon , B .M. ( 1962 ) Oestrus , ovulation and 
fertility  following suppress ion of ovarian cycles in 
Merino ewes by progesterone. J. ::1epro� Fert .  4 : 57 . 
Lamond , D . R.  and Bindon , B. M. ( 1966 )  The biological assay of 
foll icle stimulating hormone in hypophysectomised 
immature mice, J .  Endocr. 1 � : 365 . 
Lamond , D. R.  and Emmens , C . W. ( 1959 ) The effect of hypophysectomy 
on mouse uterine weight response to gonacotrophins.  
J .  Endocr . , � :  25 1 .  
Land , R. B.  ( 1973 )  Ovulation rate of  Finn-Dorset sheep following 
uni lateral ovariectomy of chloroproma.zine treatment at 
different stages of the oestrous cycle.  J.  Reprod. 
Fert . , � : 99.  
90 
Land , R. B.  and McLarcn , I� . ( 19G7 ) The response of  female mice to 
repeated injections of Human· ·  Chorionic Gonadotrophin. 
J .  Reprod . F0rt . , j1 :  321 .  
Larsen , W.A .  ( 197 1 )  /� study of oomc factors affecting reproductive 
pcrformanco in Ne�-, Zealand Romncy and Border-Leiccster x 
Romney two-tooth 0\"IIJS , M. t.gric. Sci .  Thesis I r,lasscy 
University.  
Lestcr , D . B .  1 Turnman , E . J . , Stcphens 1  M.  and Renbarger , �. ;:;; . ( 197 1 )  
Ovulation rates of beef cows and heifers trcc.ted \"Jith 
equine gonadot rophin (PNE ) and chorionic gonadotrophin 
(HCG ) . J .  f..nim. Sci . , � 433 .  
Lc<.them,  J .H .  ( 1949 ) The antihormone problem in endocrine therapy • 
.lli:.£:_Progr. Horm . Hes . 1 .1, : 1 15 .  
Lin ,  T, P. and Bailey ,  D . Wo ( 1965 ) Difference between two inbred 
strains of mice in ovulatory response to repeated 
administration of eonadotrophins.  J .  Reprod. Fert � , 1Q 
253.  
Lunfeld 1 B.  and Eshkol , A; ( 1969 ) Immunology of follicle stimu�ating 
hormone and luteinising hormone, Vit . Horm. , � :  131 . 
Mouleon 1 P.  ( 1969 ) Oogensis and folliculogencsis .  In  "::1.eproduction 
in Domestic J'.nimel s" ( ed. H .H .  Colc and P. T. Cupps ) 
P• 187 : /.endemic Pres s ,  Now York and London • .  
McCracken , J .A .  1 Uno , A.  1 Goding , J . n. 1 lchikawa , I and Ba.ird ,  D. T. 
( 1969 )  The � Tivo effects of sheep pituitary 
gona.dotrophins on the secretion of s-teroids by the 
autotra.nsplanted ovary of the ewe. J . · Endocr. , � 425. 
99 
McDonald 1 M. F. ( 1061 ) S tudies of the response of the anoestrous owe 
treated with prog0steronc and Pregnant Mare Serun. 
�ric. Sc.b_, _!l� : 3G7 . 
McDonald 1 M . F .  ( 1966 ) Developments in the controlled breeding of 
sheep and their implications for the New Zealaad sheep 
McDonald 1 M. F .  and Ch ' ang ,  T. S .  ( 1965 ) Variation in ovarian 
activity of Ro��ey Marsh ewes .  Proc . N . Z .  Soc. �nim. 
Prod , 1 2f2 : DO.  
McKenzie ,  H.  ( 1972 ) i\-lammogonesis in the ovariectomised mouse.  
���i .  !���� Massey University.  
McKenzie ·, F . F .  e.nd Terrill , C . E . ( 1937 ) Estrus ,  ovulatio::l end 
Moore , N . W. and Sholton , J . N .  ( 1964 ) Response of the owe to  a horse 
anterior pi tuitary extract . J,  il<::.E_rod .  ��t· 1 Z. 79 . 
Moor , R . M. and Cragle ,  n. G.  ( 197 1 )  The sheep egg : Enzymet ic removal 
of the zona pel lucida and cul ture of eggs r.�� v�}�S· 
Morris ,  C , J. o , n, ( 19G4 ) On the mol ecu lar weight of Pregnant Marc 
Serum Gonadotrophin. Acta Endo�rinol�, �l!_J?Pl . 90 : 163 . 
Nakahari , T. , Yamauchi 1  M , , Kataska , A.  and Kaneda 1 Y .  ( 1964 ) 
S tudies on the antihormone against gonadotrophins in 
cattle.  Proc. 5th Int , Congr. Anim. Reprod , 1  Vol . 3 '  p . 330 . 
Newton , J . E . , Bctts , J. E .  and Largo, U .V .  ( 1970) Increasing l itter 
size in three breeds  of  sheep by superovulation . 
�ric. �ci. 1 1.2 : 355.-
li BRARY 
'MASSEY UN IVERSITY 
100 
Nossal , G. J. V.  ( 1969 ) f.ntibodies nnd immunity. Penguin books . 
Ouchterlony , 0 .  ( 1949 ) � vitro method for testing the toxin 
producing capacity of Diptheria bacteria. i.cta Path. 
Microb ial . (S can�. )  � : 186. 
Parkes 1 h . S .  ( 1942 ) Induction of superovulation and super-
fecundation in rabbits .  J. Endocr. , 3 : 260 .  
Pant , H . c . , Hopkinson , c . , and  Fitzpatr1ck 1 P. J .  ( 1972 ) Plasma 
oest radiol , progesterone and luteinising hormone 
concentrations durin g  the ovine oes t rous cycle.  
J .  Reprod . Fert . , � :  501 .  
Papko f f ,  H .  ( 1969 ) Chanistry o f  tho gonadotrophins . I n  Reproduction 
in Domestic ;',.nimal s" ( ed .  H . H .  Col e  and P. T. Cupps ) ,  
p . 67 : Academic �ress 1 New York and London. 
' ' - .. . " Parlow , A. F .  ( 196 1 )  In  Human Pitu1 tary Gonadot rophins 
( ed .  h .  Albcrt ) ,  p. 204 : Thomas , S pringfield , 
I l linois , Cited Col a ( 1969 ) .  
Parlow, A. F . , hndorson , L . L .  and Melanpy , R.M .  ( 1964 ) Pitui tary 
fol l icle stimulat ing hormone concent ration s in 
relat ion to the reproductive stages of  the pig.  
Endocrinology1 12 : 365 .  
Pedersen , T.  ( 1970 ) Determination of  fol licle growth rate in the 
ovary of the immature mouse. J .  Reprod . Fert . , � :  0 1 .  
Peters , H .  and Levy , E .  ( 1966 )  Cel l dynamics of the ovarian cycle.  
J.  Reprod . Fort . , 11 :  227 . 
Polge , c .  and r.owson , L. E .A. ( 1073 )  Recent progress in t echniques 
for increasing reproductive potential in farm animals.  
Proc� '1lorld Confr . Anim . Prod . ( 197 3 )  1 � : 1 .  
101 
Quinn1 D. L.  and Zarrow, M .X. ( 1964n) Inhibition of PMS induced 
ovulation in tho inuanturo rat. Endocrinology, Z! : 309 . 
Quinn , D. L.  nnd Znrrow , M. J[ .  ( 1964b) lnhibition of the release of 
ovulatinG hormone in immature rats with hypothalamic 
lesions . Endocrinology, 77 : 225• 
Radford , H . M. ( 1959) Variation in the incidence of twin 
ovulations in Merino ewes on a constant plane of 
nutrition. i;.ust. J .  /.gri·c . nes. , .!2 : 337 .  
nneside ,  J . F .  and McDonald, M.F .  ( 1959) Seasonal changes in the 
oestrus response by the ewe to progesterone nnd 
oestrogen. Nnturo (Lond . ) ,  lQi :  458 .  
najakoski 1 E .  ( 1960) The ovarian folliculnr system in sexually 
mnture heifers with special reference to seasonal, 
cyclical and left-right variations . Acta Endocrinol . ,  
Suppl .  52.  
;:,eardon , T. F .  nnd Robinson , T. J. ( 196 1) Seasonal variction in  the 
reactivity to oestrogen in the ovariectomised ewe. 
£::.ust . J .  iq:;ric. Res . , � :  320. 
Rees-Midgely,  ,".. , ( 1969) Immunological characterisation of the 
gonadot rophins . "- . . " I n  x !eproduct1on in DomestJ.c Animals 
( eel.  Cole, H . H. o.nd Cupps , P. T. ) , p·. 47, Academic Press , 
New York and London. 
Rennels ,  E. G.  nnd O ' Steen , rr. K.  ( 1967) Alterations in the LH and FSH 
content and weight of the anterior pituitary of 
immature female rc.ts trented with P.-M. S .  Endocrinology, 
80 : 82. 
102 
nobertson , G. E.  a nd Nalbondov , A .V .  ( 1951 )  Gonadotrophic  potency 
of  the pig pituitary duri ng the estrous cycle. 
J .  Anim. S ci. , lQ :  469 . 
aobertson , H. A .  and Hutchinson , J. S. M. ( 1962 ) The levels of  FSH 
and LH in the pituita ry of the ewe in  relation to 
follicular growth nnd ovulation .  J. Endocr. ,. 24 : 143.  
;.-tobinson , T. J ,  ( 1950)  The control of  fertility in  sheep. 1 .- Hormonal 
therapy in the induction of pregnancy in the anoestrous 
ewe. J. Agric. S c i.�' i2 : 27 5.  
Robinson , T. J .  ( 1951 ) The control o f  fertility i n  sheep. The 
augmentation of fertility by gonadotrophin t reatment of 
the ewe i n  the normal b reeding season. J, �gric .  S ci. , 
4 1  : 6 .  
Robinson , T. J .  ( 1954 ) The production o f  coincident oestrus a nd 
ovulation in  the a noestrous ewe with  progesterone and 
Pregna nt Mare Serum. J .  Endocr. , 12 : 1 17 .  
Robinson , T. J .  ( 1955 ) Endocrine relationships a nd the indu ction o f  
oest rus a nd ovulation in  the nnoestrous ewe. 
J. t.gric. S ci. , � : 37 . 
Robinson , T . J. ( 196 1 )  The time o f  o vulation end effi ciency of  
fertil ization following progesterone and Pregnant 
Mare Serum t reatme nt i n  the cyclic ewe. J .  fl..&ric. Sci. , 
.21 129 . 
Robinson , T. J .  ( 1967 ) Conclusions. In " The control of  the o varian 
cycle in sheep". (ed. T.H. Robinson ) 1  p. 237 : Sydney 
University Press. 
103 
Robinson 1 T . J.  ( 197 1 )  The seasonal nature of reproductive phenomena 
in the sheep. 2 .  Variation in fertility  following 
synchronisntion of oestrus .  J·. Reprocr. Fort . 1 � : 19 . 
�obinson , T . J .  1 Snlamon , 8 . 1 Moore , N .W .  nncl 3mith , J . F .  ( 1967 )  
The evaluntion of SC9GOO-impregnatcd intravaginal 
sponges for the synchronisation of  oestrus for lnrge 
scnlo nrtificial insemination of Merino ewes in summer 
and autumn. In  "The control of the ovarian cycle in 
sheep" 1 ( ed .  T .J .  Robinson ) : Oydnoy Univorsi ty  Press . 
Hobinson , T. J .  and Smith , J . F .  ( 1967 ) The evaluat ion of  SC9000-
impregnated int ravasinal sponges used wi th or without 
PMS for the advancement of the breeding season of  
Brit i sh broccJ owes . I n  "The control of  the ovarian 
cycle in sheep , ( ed .  T. J .  Robinson ) : Sydney University 
Press . 
Robinson , T. J . , Quinlivan , T. D .  and Baxt0r , c .  ( 1960 ) The relation­
ship between dose of progcstagen and method of  
preparation of int ravaginal sponges on their eff ective­
ness for the control of ovulation in sheep. J .  Reprod . 
!.!zt:., 17 : 47 1 .  
�1owlands 1  I . W .  and Parkas , A.s .  ( 1965 )  Hypophysectomy and tho 
gonadotrophins .  In  "Marshalls  Physiology o f  Reproduction" , 
Vol . 3 ,  ( ed .  ! • •  s .  Pnrkos )  p . 26 : Longmans , London . 
i1owson , L .E . A.  ( 1951 )  Methods of  inducing multiple ovulation in 
cat tle.  J .  Endocr.  7 : 260. 
104 
Sa.ntelucito , J .A . , Clegg , M. T.  and Cola , H . H . ( 1960 ) Pituitary 
gonadotrophins in tho owe at different stages of  the 
estrous cycle.  Enc1ocrinology, §_Q 273 ,  
Sasamoto , . S .  ( 1972 )  Blocc level s of PMS after a single injection 
into mice, �ndocrinol . Japan . ,  1£ 303. 
Scanlon , P . , S roonan , J .  end Gordon , I .  ( 1968 ) Hormonal induction of 
superovulation in catt le .  J .  Anim. S ci .  1 7q : 179.  
Schmidt-Elmondorff ,  H . , Lornino , J ,h ,  and Bell ,  E . T. ( 1962) S tudies 
on the biological activity of various gonadotrophin 
preparations.  J. Endocr. , � : 349 .  
Sogal , S . J. , Niu,  L .  and Hakim , s .  ( 1960) Immunochemical cnalysi s of 
the sheep pituitary fol licle stimulating hormone.  � 
Endocrinol . , Suppl . 52 : 493. 
S egal , S . J . , Lc.urenc0 1  K . A . , Perlbechs , M.  and Hakim , S .  ( 1962 ) 
Immunological ene.lysi s of sheep pitui tary 3onndotrophins.  
�' CompGr.  Enclocrinol . ,  Suppl .  1 : 12 
Selye ,  H . , Collip , J . B .  and Thompson , D . L.  ( 1934 ) Loss of sensitivity 
to  the anterior pituitary like hormone on the ovary of 
the hypophysectomised rat .  Proc.  Soc . Exp. Biol�,  N . Y .  1 
.ll : 566. 
Short 1 R. V. ( 1972 )  Role  of hormones in sex cycles .  In ' \:tuproduction 
in  mammals,  part 3 Hormones in  reproduction" , ( ed .  C . R .  
Austin and a. v .  Short ) p . 42 ; Cambridge University Press .  
Short , R.V. , McDonold 1  M.F .  and  Rowson , L .. E , ;'.. . ( 1963 ) Steroids  in 
ovarian venous blood of ewes before and after gonado­
trophic stimulation.  J .  Endocr. , 26 : 155.  
105 
Smoaton , T . C .  and �obertson , H. &. ( 197 1 )  Studies on the srowth and 
atresia of Grauffinn fol licles in the ovary of the 
sheep . -[:_ neprod . Fert . 1 31 : 243.  
Sncdecor , G . W.  nnd Cochran , -:;·r . G.  ( 1967 )  " " J tatistical Methods 
I owa S tate University r>ress .  
Soknl R. :\1.. and :ilolf ::? . J. ( 1Q60 ) "Biomctry" Freeman , San Francisco . 
Suhiro 1 M . , Kidokoro , M .  and 3c.to , T .  ( 1955 ) S tud ies on the Bioassay 
of Pregnant Mare S erum Gonadotrophic hormone usinc 
immature female mice . �ndocrinol . Japan . 1 � : 175 .  
Tait 1 R .M .  ( 197 1 )  Influence of nutritional status on hormone-
induced superovulation in normally cyclic  ewes . 
Can . J .  Anim. Sci . , §1. :  77 1 .  
Tempest , 'f[ . �ii . and Boaz , T. G.  ( 1070 ) The impact o f  hormone treatment 
on the reprocuctive and lamb rearing performance of 
Scottish :ialf-broc ewes.  Anim.  Prod . 1 _8. : 350 . 
Tervit 1 H . n .  ( 1967 ) S tudies on the � � cleavage and the in vitro 
culture of New Zealand Rowney sheep . M .  Agric .  S ci .  
Thesis : Massey Universi ty. 
Thompson I K. \1.  ( 194 1 )  J',.ntihormones . Physiol . neview. 2 1  : 500 . 
Uteroi , N. K. and Meyer,  R . K .  ( 1967 ) Uterine weight of the immature 
rat as a measur.� of augmentation of pituitary 
gonadotrophins by HCG.  Ferti l . S teril . ,  10 : 420 .  
van den Ende ,  M.  ( 194 1 )  �recipitins in  the serum of  rabbits  
immunised against serum gonadotrophin . J .  Endocr . , 2 
·'103.  
Vlal lace , L. n. ( 1954 ) Studies on the augmentation of  fertility in 
Homney ewes with Pregnant Mare Serum. J .  Agric. Sci . , 
45 : 60. 
106 
Vlakabayashi ,  K .  Tamaoki ,  B. I .  ( 1966 ) Influence of immunisation with 
luteinising hormone upon the anterior pituitary-gonad 
system of rats nnc� rabbits  with specific reference t o  
histological chc.ngcs and bio-synthesi s of luteinising 
hormone nnd stGroids .  Endocrinology ,  1£. : 47 1 .  
Ylillctt , E. L. , Buckner , P . J .  and McShan , Vl. H .  ( 1953 ) aefractoriness 
of cows rcpentecUy superovulated with gonndotrophins . 
J .  Dairy s e:�;. I 1Q : 1083.  
V!ill iams , P. c .  ( 1945a ) S tudies on the biological action of scrum 
gonadotrophin. 1 .  Decline in response after hypophysec­
tomy. J .  Endocrin . ,  � :  127 .  
�'lilliams 1 P . C .  ( 1915b ) S tudies on thG biological action of scrum 
gonadotrophin 2 . Ovarian response  after hypophysectomy 
and oostroger>. t rcntmen t .  J .  Endocrin'. 1 j_ : 1 3 1. .  
'\'li t ischi 1 E .  and Johnson 1 D .  c .  ( 1960 )  Effect o f  repeated inject ions 
of PMS in the V!cnver Finch . Anat. nee . I m : 401 .  
'Vlright 1 P .. H.  ( 1965)  Hormone antibodies in endocrinology. Vit .  Horm. 1 
� :  61 .  
Zarrowl M. X .  I Christenson l c . M. and Eleftheriou , B . E . ( 197 1 )  
S train differences in the ovulatory rGsponse of 
i�ature mice to PMS and to pheromal facilitat ion of 
PMS induced ovulation. Biol . Reprod . 1 ! : 52. 
• 
A P � E N D I C E S 
APPENDIX I 
Rl\.\7 DATA MEANS t"..ND R!.NGES OF OV!o.RIAN RESPONSE DATA (EXPERIMENT 1 )  
GROUP 
Ll  
L2 
L3 
L4 
H1  
H2 
H3 
H4 
cc 
£QEPORA LUTE!:. : CORPORA LUTEA + FOLLICLES 
IVIEI\.N NUMBER/ c:;·.m (AND Rfi.NGE ) ) 3 mm. : i'IJE;•N NUMBER/ EWE 
(AND PJ·l.NGE ) 
1 . 8  (0- 10 )  
6 . 1 ( 1- 1 2 )  
5 . 6  (0-20 ) 
4 . 9  (0-18)  
0. 9 (0-3 )  
7 .4  (0-10)  
2 .0  (0-12 )  
9 . 6 ( 1 . 36 )  
1 . 1  ( 1-2 ) 
3 . 7  ( 1- 1 1 )  
7 . 8  (2-12 )  
9 .  6 (2-20)  
9 .6  (2-29 ) 
2 . 7  ( 1- 18)  
9 .4  (Q- 18) 
16 .0  (9-20 ) 
13 .0  (5-40 ) 
2 . 6  ( 1-4 ) 
a.  Sew data 
A GrOU!J D. F .  
(n- 1 ) 
L 1  9 
L2 8 
L3 7 
L4 8 
H 1 8 
H2 0 
H3 6 
H4 8 
b .  Transformed 
APPENDIX I I  
t::..I:OI::::>i�--�-==--=-
Variance s2 
9 . 070 
15 . 027 
ti l .  307 
29 . 1 12  
O . BG 1  
37 . 528 
2 1 . 143 
127 . 50::> 
D . F .  = 7 
The variance 
data B 
Coded s2 
90. ?0 
150. 27 
513 . 87 
29 1 . 12 
8 . 3 1  
375 . 28 
2 1 1 . 43 
1275. 50 
L c 2 og coded o 
1 . 95751 
2 . 17696 
2. 7 1079 
2 . 46404 
0 . 93505 
2 . 57430 
2 . 32531 
3. 10551 
Chi2 = 39 . 7535'\'.* 
is  significantly heterogeneous 
��--=-.,.,.,�-==-=-:r� 
Group D .I? .  Variance s 2 Coded 8 2 Lo[S coded 82 
(n- 1 ) 
w� 
L1  9 0 . 4862 48. 62 1 . 6860 
L2 8 0 . 7219  72. 19 1 . 8585 
L3 7 2 . 07'16 207 . 76 2 . 3 176 
L4 8 0 . 9165 9t.l . 65 1 .  7961  
H1 B 0. 1083 10. 33 1 . 0346 
H2 8 0. 9507 95 . 07 1 . 9780 
H3 6 1 . 1077 1 10 . 77 2 . 0322 
H4 8 2 . 2244 222 . 44 2 . 347 1 
� 
2 17 . a2 10**  (o.o5 <. P <  o.ol ) D.  F .  = 7 Chi == 
Variance approaches homogeneity.  
h Group abbreviations arc described in Chapter I I .  
B Data transformed to .lx+ 1 .  
* p <. 0.05 
** P -(_ 0.01  
APPENDIX I I I  
STUDENT-NEWMAN-KEULS TEST TO COMPARE ��S OVUL!o.TION H.l�TE I 
EXPERIMENT 1 
1 .  Differences between moans when these means are ranked from 
smallest to l argest 
Rank 1 
Mean 14 . 612 
No. /group 19 
Group 1 
2 3 4 
18. 416 25. 807 26. 436 
15 18  18 
3 4 2 
Rank Mean No. /group Group 
1 14 . 6 12 19 
2 
3 
4 
18. 416 
25. 807 
26 . 436 
15  
18  
18 
1 
3 
4 
2 
3 .084A 
1 1 .  195 
1 1 . 824 
7 .  39 1 
8 . 020 0. 629 
M. S .within= 109. 4343  M. S .within= 10. 461 
Degrees of freedom = 62 Use D. F .  = 60 
2.  B� consultin� a table �ivin� the crit ical values of the 
S tudentized ran�e the fol lowin� table ma� be drawn up 
ICB= 2 3 4 
c 2 . 363 2 . 959 3. 312 Q 10(k , 6d )  
::: 
JM. S . within Q.  toCk 6o> =24 . 7 19 30. 954 34 . 647 
Q0.05(k , 60 )  = 2 . 829 3 . 399 3 .737 
JM. S • within Q o.o5(k 1 60 )  =29 . 594 35 . 557 39 .093 
Q0.01 (k 1 60 )  = 3 . 762 4 . 282 "1 • 595 
/M. S . within Q :c39 . 354 44. 794 48.068 o .010c 1 60 )  
3 .  For a difference between means to b e  significant at theot level 
of significance it must be equal to or greater than: -
A 
B 
c 
D 
D 
LSR = 'it_(k , oofM.S . with-· �n2 irJTn:;n2 
Difference between means 
k = 1+ difference in ranks of two means . 
Q = Value from studentized range for k items when the 0 . 10(k 1 60)  error variance has 60 degrees of  freedom. 0.01 
refers to n test being carried out at the 10% level of s ignificance. 
n1 and n2 
are the srunple sizes of the two means . 
4 .  Using the Q values from tho above table the following ranges 
may bo computed : -
a .  10% level of significance 
LSR1----)2 
r.sn .. 2 -74 
r.sn2 �3 
LSR3-74 
b .  
= 
34 . 647 X 6.0828 
26, 155 
8 ,058 ( sign )B 
30. 954 X 6,0828 
26. 155 
= 7 . 199 ( sign ) 
= 24. 7 19 X 5. 831 
23. 875  
c 
= 6 .037 (N . S . ) 
= 30, 954 X 5. 745 
23, 2379 
= 7 . 652 ( sign )  
= 24 . 7 193 X 5. 745 
23 , 2379 
= 6 . 1 1 1  ( sign)  
= 24 . 7 19 X 1 
4 . 243  
= 5. 826 (N .  S . )  
5% level of siGnificance ( testing only those ranges 
which were 
B 
LSRl � 4 
= 9.092 ( sign)  significant at  P(  0. 10 )  
LSl\ --73 
= 8 , 269 ( sign )  
LSR2-:)4 
= 8 .790 (N.  S .  ) 
LSR2�3 
= 7 , 316 ( sign )  
LSR1_,4 = Least significant range for comparing means with 
ranks of 1 end 4 (� this is not comparing treatments 1 and 4 . 
Indicates that these two means are significantly different at 
this level of significance , i . e .  the difference between the 
means is greater than the LSR. 
C Indicates that these two means are not significantly di fferent 
at this level of significance , i . e. the difference between the 
moons i s  less than the LSR. 
c .  1% level o f  significance (only test ing those 
ranges which were si gnificant et P <  0, 05 ) 
LSi11 � 4 
= 1 1 . 176 ( sign )  
LSR1 � 3 
= 10. 043 ( sign )  
LSR2 �3 
= 9 . 728 (N. S . ) 
5.  A table indicatins the differen ce between means may now be 
constructed : -
Ranks of the mean group difference significance of 
pairs pairs of means mean difference 
1 and 4 1 and 2 1 1 . 824 P < O.Ol  
i and 3 1 and 4 1 1 . 195 P <o.o1  
1 and 2 1 and 3 3 � 804 P > O. lO 
2 and 4 2 and 3 8 . 020 p <0. 10 
2 and 3 2 and 4 7 . 39 1  p < o.os 
3 and 4 4 and 2 0. 629 p )0. 10 
If comparison of means \1as not significant at the 5% level then 
means were said not to be significantly different.  However those 
which fel l into the P < 0. 10 category were reported as N. S.  
(P < 0, 10) 1 and those of P <..0, 10 were reported ns N. S .  
APPENDIX IV  
HEAl� UTGRINE YilliGHTS i .. HD  STil.NI1".RD ERRORS FOR MOUSE G:1.0UPS OF 
BIOLOGICAL INHIBITI On TJST (EXPBRIMENT 2 )  
MOUSE GROUP 
Uninjocted 
P. M. s. G. Alone 1 . 0. i . u. 
P. M. S .  G. r";.lono 0. 25 i . u . 
1 . 0  i . u. P. M. S. G. + plasma 
1 1  1 1  1 1  
1 1  1 1  1 1  
1 1  1 1  1 1  
" 1 1  " 
1 1  " " 
1 1  " 1 1  
1 1  1 1  " 
1 1  1 1  1 1  
1 1  1 1  1 1  
11 11 " 
" " " 
1 1  1 1  1 1  
SH!::D:P GROUP 
L1 
L2 
L3 
lA 
H1 
H� 
H3 
H4 
cc 
500 i , u .  P. M. S . G. 
500 i . u . P. M. S . G. 
+ adjuvant 
1000 1 .  u. P. M. S . G . 
1000 i � u .  P. M. S . G. 
+ a'djuvant 
0. 25 i . u . P. M. s . G.  + plasma Ll  
L2 
L3 
L4 
H 1  
H2 
H3 
H4 
cc 
1 1  " 11 
1 1  1 1  1 1  
" 11 1 1  
" " 11 
" " 1 1  
" I f  1 1  
" 1 1  " 
1 1  " " 
MEAN (:t;_ STD.  EHI10R) * 
86. 5 1  (.:!:, 5. 07 )  
163 . 60 (t 4 . 99 )  
137 . 60 <.:t. s .oo)  
162. 98 <.:t. 5 . 09 )  
159 . 73 <:t. 4 . 99 )  
167. 05 (:t_ 4 e 98 )  
165 . 55 (;t 4 . 91 ) 
159 . 87 <.:t. 5 .02 )  
159. 93 <.:t 4 . 99 )  
159 .95 <.:t. 5.02 )  
160. 46 <.::. 5.  00)  
161 , 39 <.:t L..l . 99 )  
137 . 60 (.:!:, 4 i 99 )  
106. 35 <.:t 4 . 98 )  
152 . 87 <.:t 4 . 99 )  
1 5 1  ... 53  <.:t 5 .·02 )  
150.93 <.:t. 4 .-98) 
143. 55 <:t. 4 .. 99 ) 
137 . 86 <.:t 5 . 00 )  
1 3 1  •. 72 <.:t 5 . 07 )  
127 .09 (:!:, 4 . 99 )  
137 . 50 <.:t. s.oo )  
150. 14 <.t 4.07 )  
* Transformed data transformation = log (X + 1 ... 1 )  x 100. 
APPENDIX V 
ill:LATIONSHIP BETWEEN ESTH/li.TGD TOTAL OVI.RIAN FOLLICUU.il DEVELOPMENT 
AND OVAr:.IAN SUI!Fi'.CE OBS£RVATIONS (EXPEI\IMENT 3 )  
RELATIONSHIP TESTED REGRESSION ST/i.NDARD 
CUOSS SECTIONAL vs SUH.FI.CE COEFFICIENT EiffiOR t 
..,...__,. 
COUNTS OBSElWATION 
LEFT OVi..RY 
ANTR./ • .L FOLLICLES vs c .L .  0. 17 0. 4 1  0. 4 1  N . S .  
VS Sm. Fol . 0, 26 0. 25 1 . 04 N . S .  
VS  Mod. Fol . 0.06 0. 21 0. 29 N. S .  
VS Lgc. Fol . 0, 31  0.41  o. 76 N . S .  
VS All Fol . 0, 13  0. 16 0 . 81  N. S .  
vs C. L .  + Fol . 0. 18  0. 10 1 . 00  N . s .  
NOI1.MAL ANTRAL VS C . L. -0. 12 0.09 1 , 33 N . S .  
FOLLICLES Sm. Fol . 0.09 0,05 1 . 80 N . S .  VS 
VS Med . Fol . -0, 04 0,06 0 , 66 N . S .  
vs Lge. Fol . 0. 04 o, 1 1  o, 36 N .S . 
VS All  Fol . 0. 04 0,05 o. oo N . S .  
vs C , L .  + Fol . 0,02 o.o5 0.40 N. S .  
niGHT OVI.RY 
ANTRAL FOLLICLES vs C .  L. 0,.48 Oa60 0.70 N . S .  
VS Sm, Fol . 0. 19 0 .43 0.44 N . S .  
vs Med. Fol . 0.07 0. 34 o. 2 1  N.S  .• 
VS Lge.  Fol . 0. 68 0 .69 0. 98 N. S .  
vs All  Fol .  0. 1 3  0.27 0. 48 N . S .  
VS c .L . · + Fol . 0. 22 0. 30 0. 73 N . s .  
NORMAL ANTRAL VS c . �. -o. 27 0, 67 0.40 N . S .  
FOLLICLES Sm. Fol . 1 . 1 1  0. 32 3. 47 N. S .  VS  
vs Med. Fol . 0. 2 1  0 .47 0 .45 N. S .  
vs Lge. Fol . -0. 32 6. 02 0. 39 N . s .  
VS All Fol . 0 .72  . 6. 33 2 . 18 N . S .  
vs C . L. + Fql . 0. 66 6. 34 1 . 94 N . S .