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Iron bioavailability for piglets: 
The effects of iron status, vitamin C and cooked or uncooked meat 
A thesis presented in partia l fulfi lment of the req u i re ments for the 
Deg ree of Master of Science (Nutritiona l Science) 
at Massey Un iversity, Palmerston North, New Zealand 
Patricia J .  Clayton 2002 
Abstract 
Worldwide, iron deficiency affects more than 1 bi llion people. People with iron deficiency h ave 
symptoms of fatigue, intolera nce to the cold and poor behaviou r and psychomotor development 
problems. This is partly because the amount of iron p resent in food is not the amount that is 
available to the body. 
T h e  bioavailability of iron is a key component in understand ing the complexities of iron 
deficiency. 
Using an animal model involving 4-wee k old anaemic pig lets, th is study investigated several 
aspects of iron b ioavailabil ity. These we re :  
The re lationship between iron status and iron absorption, the d ifference i n  b ioavailab i lity o f  meat 
iron and non-haem iron and whether supple mentary vitamin C can aid in the absorption of 
d ie ta ry iron, and the effect of temperature and cooking of meat on iron b ioavailabil ity. 
D ietary i ron bioavailability was measu red both in iron deficient and non-iron deficient piglets, by 
measuring changes to the composition of the red cel l  mass, serum iron concentrations and the 
binding capacity of iron transport p rote ins over a period of 28-days. 
Experime n t  1 showed that meat iron was more bioavailable than the inorganic iron in a vegetable 
based d iet. Also, in the anaemic pig let, 500 ppm of vitamin C in the diet was able to enhance the 
availabil ity of the non-haem iron from a d iet consisting of food choices from a typical human d iet. 
Expe riment 2 showed that a d iet containing meat iron was able to return iron deficient pig lets to 
haematologic normality more read ily than a d ie t  consisting of milk p rote i n  and inorgan ic iro n .  
Also, cooking meat i n  a steam-heated circulating water bath was beneficial i n  increasing the 
d ig e stib i l ity of the d iet and also increasing the availabil ity of the meat iron . 
The findings of this study reflect the conclusions d rawn from similar human studies, thereby 
p roviding further evidence of the su itab il ity of the p ig let as a model for the human in future 
stu d ies of iron bioavailab il ity. 
2 
Acknowledgements 
Sincere appreciation is expressed to my supervisors, Dr. P . C .  H. Morel and Associate Professor R. 
W .  P u rchas for the ir gu idance and encouragement th roughout the study .  
I would a lso like to tha nk 
M rs. Rosa l i nd Power in ana lysing blood samples. 
Dr. Phi lip Pearce fo r laboratory ana lysis of serum and UIBC iron . 
Dr. David Simcock for laboratory ana lysis of the iron content of feed sa mples. 
Also Ms. Karin Weidg raaf, Ms. Jo Melai, M s. Laurence De Caster a nd M r .  Edwa rd James in help ing 
to ca re for the a n i ma ls used in th is study and a lso in the col lection of blood samples. 
The An ima l Eth ics Committee at Ma ssey Un iversity, Palme rston North, approved the experimental 
protocols of the studies described herein . 
Appl ication N u mbers: MU Eth ics ( 0 1/25) and ( 0 1/82) 
Th is study was Funded by Meat New Zea land 
3 
List of Tables 
Table 1: P roposed mecha n isms that could a id the absorption of 
non-haem iron by vita min C .  
Page 
23 
Table 2: A summary of several stud ies that evaluated the effectiveness 24 
of vita min C in increasing the bioava ilabi l ity a nd absorption of 
n on -haem iron from the d iet . 
Table 3: Proposed mecha n isms that could explain why h istidine can increase 27 
the bioava ilability of hae m iron 
Table 4: A su mma ry  of the effects of heat p rocessing on iron content of 28 
meat and meat prod ucts. 
Table 5 :  Accepted physiological va lues of red blood cell parameters i n  29 
the healthy p ig .  
Table 6: Accepted physiolog ical va lues of white cell parameters in the 30 
norma l healthy pig . 
Table 7 :  Cha racteristic of the pig that make it a n  appropriate model for 3 1  
stud ie s  of iron a va ilability. 
Table 8 :  Cha racteristics o f  the five treatment groups used i n  experiment 1. 36 
Table 9 :  Cha racteristics of the five treatment g roups u sed i n  expe riment 2 .  36 
Table 10: The composition of the experimental d iets % on an a s-fed basis, 
includ ing add itiona l mixing wate r .  3 7  
Table 11: Estimated analysis of the experimental d iets used in Expe riment 1 38 
Table 12: The composition of the experimental d iets % on an a s-fed ba sis, 38 
includ ing add itional mixing water .  
- -------- - - - --- - ----- -----
4 
Table 13: Estimated ana lysis of the experimental d iets used in Experiment 2 39 
Table 14: Red b lood ce l l  parameters, u n its and a bbreviations. 4 1  
Table 15:  Red cell cla ssification using a 3 x 3 matrix 
Table 16: Wh ite blood cel l  pa ra meters, u n its and abbreviations. 
42 
42 
Table 17: The sign ifica nce of d ietary treatment on feed intake and growth rate. 46 
Table 18: Least-sq uares mea ns of feed intake (gl week). 46 
Table 19: Least-squares mea ns of growth rate (gl day) 48 
Table 20: The sign ifica nce of d ietary treatment on blood parameters. 49 
Table 21 : Least-squares mea ns of red blood ce l ls count (x 1012 I L) . 49 
Table 22: Least-sq uares mea ns of blood haemog lobin concentration (g I L) 5 1  
Table 23 : Least-sq uares mea ns of he matocrit ( L  I L) 52 
Table 24: Least-sq uares mea ns of mean cell vo lume (f I L) 54 
Table 25 : Least-sq ua res mea ns of mean cell haemog lobin (x 1012 I L) 55 
Table 26: Least-squares mean s  of mean corpu sc le, haem concentration 56 
(MCHC g I L) 
Table 27 : Least-sq uares mea ns of corpuscular haemoglobin constant (gl L) 57 
Table 28: The sign ifica nce of d ietary treatment on red blood cell cha racteristic 58 
Table 29 : The Least-sq uares means of red blood cel l  cha racteristics 58 
5 
Table 30: The sign ificance of d ietary treatment on serum iron a nd 62 
iron b inding proteins 
Table 31 :  The least-sq ua re s  means o f  serum iron content (�tmol 1 L) 63 
Table 32: T h e  least-sq ua re s  means of unsaturated iron b ind ing capacity 64 
(�tmol I L) . 
Table 33: The least-squa res means o f  body HGB Fe content ( mg )  66 
for each of the treatment g roups. 
Table 34: The sign ifica nce of d ieta ry treatment on a d ifferentia l white 67 
blood cell counts. 
Table 35:  T h e  least-sq u a res means values for white blood cells 68 
( 1 09 cells I L). 
Table 36: The least-squa re s  means values for neutrophi l cel ls ( 109 cel ls I L). 69 
Table 37:  The least-squa res means values for lymphocyte cells ( 1 09 cel ls I L). 70 
Table 38: The least-sq ua re s  means values for monocyte cells 70 
( 1 09 cells I L ) .  
Table 39: The least-sq ua re s  means values for Eosinoph ils cells ( 1 09 cells 1 L). 7 1  
Table 40: The least-sq u a re s  means values for basophil cells ( 109 cells 1 L) . 7 1  
Table 41: The sign ifica nce of d ietary treatment on feed intake 72 
and g rowth rate 
Table 42: Least-sq uares mea ns of feed intake (glday) 73 
Table 43: Least-squares mea n s  of g rowth rate (glday) 74 
6 
Table 44: The sign ifica nce of d ieta ry treatment on complete blood counts 75 
Table 45: Least-squares mea ns of red blood ce lls count (x 10 12 1 L) . 76 
Table 46: Least-squares mea ns of blood hae mog lobin concentration (g 1 L) 77 
Table 47: Least-sq uares mea n s  of he matocrit ( L  1 L) 79 
Table 48: Least-sq uares mea ns of mean cell volume (f 1 L) 80 
Table 49: Least-sq uares mea ns of mean cell haemog lobin (x 10 12 I L) 8 1  
Table SO: Least square means o f  mean corpuscle, haem concentration 83 
(pg )  
Table 51 :  Least-sq uares means of corpuscular haemog lobin constant (g IL) 82 
Table 52: The sign ifica nce of d ieta ry treatment on red b lood cell cha racte ristic 83 
Table 53: The Least-sq uares means of red blood cell cha racteristics 83 
Table 54: The sign ifica nce of d ieta ry  treatment on serum iron. 88 
Table SS: The least-squares means of se ru m  i ron content (�tmol I L) 88 
Table 56: The least-squa res means of body HGB Fe content (mg )  
for each o f  the treatment groups. 90 
Table 57: The sign ifica nce of d ietary treatment on a d i fferentia l wh ite 9 1  
blood ce ll counts. 
Table 58: The least-sq uares means values for wh ite blood cells 92 
( 1 09 cells I L). 
Table 59: The least-sq ua res means values for neutrophil cells ( 109 cells 1 L. 93 
7 
Table 60: The least-sq uares means va lues for lymphocyte cells ( 1 09 cel ls / l). 93 
Table 61 :  The least-squares means va lues for monocyte ce lls 94 
( 1 09 cel ls / L ) .  
Table 62: T h e  least-sq ua res means va lues for Eosinophils cel ls ( 109 cel ls 1 L ) .  94 
Table 63: The least-sq ua res means va lues for basoph i l  cells ( 109 cells / L) . 95 
Table 64: A comparison of changes in blood parameters experiment 1 .  106 
Table 65: A comparison of changes i n  blood pa rameters expe riment 2 .  108 
Table 66: A comparison of changes in b lood pa ra meter in the human and 1 09 
p i g let mode l .  
8 
List of Figures Page 
Figure 1 :  Least-square s  mea ns of tota l feed inta ke (g) by week for 47 
each of the five treatment groups. 
Figure 2: Least-squares mea ns of g rowth rate (g 1 day) for 48 
each of the five treatment groups. 
Figure 3 :  Least-sq uares mea ns of red blood ce lls (RBC) x 1 0 . e 1 2  IL 50 
by week for each of the five d ieta ry treatment g roups. 
Figure 4 :  Least-squares means of haemog lobin (HGB) g I L by wee k 5 1  
for each of the five treatment groups. 
Figure 5 :  Least-sq uares mea n s  of he matocrit (HCT) L 1 L by week 53 
for each of the five treatment groups. 
Figure 6:  Least-sq uares means of mean ce ll volume (MCV) f L by 54 
wee k for each of the five treatment g roups. 
Figure 7 :  Least-sq uares means of mean ce ll haemog lobin (MCH) pg by 55 
wee k for each of the five treatment g roups. 
Figure 8: Least Square s  mean of red b lood ce l l  cha racteristic 6 1  
(volume and haemoglobin cone . )  a s  a percentage o f  th e  tota l 
for each of the five treatment groups at the beg inn ing of the tria l .  
Figure 9 :  Least Square s  mean of red b lood cel l  characteristic 62 
(volume and haemoglob i n  cone .) as a percentage of the tota l 
for each of the five treatment groups at the end of the tria l .  
Figure 10:  Least-sq uares mea n s  of se ru m  iron values (pmol IL) b y  week 63 
for each of the five treatment groups. 
9 
Figure 11 :  Least-squares mea n s  of unsaturated i ron bindi ng capacity 65 
( UIBC)�tmol I L by week for each of the five treatment g roups. 
Figure 12 :  Least-squares mea n s  of body hae mog lobin Fe content ( m g )  66 
by week for each of the treatment g roups. 
Figure  13 : Least-squares mean s  of white blood cel l  volume (e h )  g I L 
by week for each of the five treatment g roups. 
68 
Figure 14: Least-squares mea n s  of feed i ntake (g) by week for each of 73 
the five treatment g roups. 
Figure 15 :  Least-squa res mea n s  of growth rate (g I day) by week for 
each of the fi ve treatment g roups. 
Figure 16:  Least-squares mea n s  of red blood cells ( RBC) x 1 0 . e 1 2  IL 
by week for each of the five dietary treatment g roups. 
Figure  17 :  Least-squares mea n s  o f  blood haemog lobin concentration 
( H GB )  g I L by week for each of the five treatment g roups. 
Figure 18 :  Least-sq ua res mea n s  o f  he matocri t  ( H CT )  L 1 L 
by week for each of the five trea tment g roups. 
Figure 19 :  Least-squares mea n s  o f  mean ce ll volume (MCV) f L 
by week for each of the five trea tment g roups. 
Figure 20: Least-squares mea n s  of mean cell haemog lobi n (MCH ) pg 
by week for each of the five treatment groups. 
Figure 21: Least Sq uares mean of red b looo cel l  cha racteri stic 
(volume and haemog lobi n cone . )  a s  a percentage of th e  
tota l f o r  each o f  th e  five treatment g roups a t  the begi nning 
of the tria l .  
74 
76 
78 
79 
80 
82 
82 
10 
Figure 22: Least Sq uares mean of red b lood ce l l  characteristic 
(volume and haemog lob in cone .) as a percentage of the 
tota l for each of the five treatment g roups at the end of the trial .  
Figure 23: Least-squares mea n s  of seru m iron values (pmol /L) 
by week for each of the five treatment g ro ups 
Figure 24: Least- squa res mea n s  of body HGB Fe content ( mg )  by week 
fo r each of the treatment groups. 
Figure 25: Least square means of wh ite blood cell volume (e h )  g I L 
by week for each of the five treatment g roups. 
87 
89 
90 
92 
Figure 26 : Formation and destruction of red b lood ce lls and recycling of 1 22 
the haemog lobin components 
1 1  
Table of Contents 
Page 
Abstrac t. ___________________2 
Acknowledgements 3 
list of Tables 4 
list of Figures 10 
Table of Contents 13 
Chapter 1 
1 Introductio n. ________________ 17 
Chapter 2 
2 Review of literatur e.  _____________ 19 
2 . 1  The physiological role of iron 
2 . 2  Iron d igestion 
2 . 3  Iron a bsorption mechanisms 
2 . 4  Iron absorption i s  related to iron status 
2 . 5  Regulation of iron a b sorption 
2 . 6  Vita min C increa ses non-ha e m  iron a bsorption 
2 . 7  Effect o f  meat o n  iron b ioava ilabil ity. 
2. 8 The effect of heat p rocessing of meat on iron availa bi l ity 
2 . 9  Mea su res of iron status 
2 . 1 0  Using an a n ima l model to study iron deficiency i n  the h u ma n  
2 . 1 1  Sum ma ry 
Chapter 3 
3 Experiments 1 and 2 33 
3 . 1  Introd uction 
3 . 2  Material s  a n d  methods 
3 . 2 . 1  An i m a l s  
3 . 2 . 2  Housing 
3 . 3  Experime n ta l  design 
3.4 Diets a nd feed management 
3 . 4. 1 Diet 
12  
3. 4.2 Feed management 
3 . 5  Blood Samp les 
3.6 Da ta analysis 
Chapter 4 
4. Results experiment 1 45 
4. 1 .  Inta ke 
4.2. G rowth 
4.3. Haematology 
4. 3 . 1  Red blood cells 
4 . 3 . 2  Blood haemog lobi n concentration 
4.3.3 He matocrit 
4 . 3 .4 Mean ce l l  volume 
4 . 3 . 5  M e a n  cell haemoglobin 
4 . 3 . 6  Mean corpuscle hae m  concentration 
4 . 2 . 1  Corpuscula r  haemog lobin consta nt 
4.4 Red cell matrix 
4. 4 . 1  M 1  
4.4.2 M 2  
4.4.3 M 3  
4.4.4 M 4  
4.4.5 M S  
4.4.6 M6 
4.4.7 M 7  
4.4.8 MS 
4.4.9 M 9  
4 . 5  Iron 
4.5. 1 U n saturated iron binding capacity 
4.6 Iron retention 
4.7 White cells 
4. 7 . 1  Neutrophil 
4.7.2 Lymphocytes 
4.7.3 M onocyte 
4.7.4 Eosi nophi l 
4.7.5 Basophil 
13  
Chapter 5 
s. R esults ex periment 2 74 
5 . 1  Intake 
5 . 2  Growth ra te 
5 . 3  Haematology 
5 . 3 . 1  Red b lood cel l s  
5 . 3 . 2  Blood haemog lobin concentration 
5 . 3 . 3  Hematocrit 
5 . 3 . 4 Mean cell vo l u me 
5 . 3 . 5  Mean cell haemoglobin 
5 . 3 . 6 Mean corpuscle haem concentration 
5 . 3 . 7  Corpusc u la r  haemog lobin consta nt 
5 .4 Red cel l  matrix 
5.4. 1 M 1  
5.4.2 M2 
5.4.3 M 3  
5 .4.4 M4 
5 . 4 . 5  M S  
5 .4.6 M6 
5 . 4.7 M 7  
5 . 4 . 8  M8 
5.4.9 M9 
5 . 5  Iron 
5 . 6  Iron retention 
5 . 7  White ce l ls 
5 . 7 . 1  Neutroph il 
5 . 7 . 2  Lymphocytes 
5 . 7 . 3  Monocyte 
5 . 7 .4 Eosinophil 
5 .7 . 5  Basophil 
Chapter 6 
6. Discussion 101 
-�----�- - -
14 
Chapter 7 
7. The utilisation of finding s. __________ 115 
8. References _______________ 117 
9 .  Appendice s. _______________ 123 
1 5  
Chapter 1 
1. Int roduction 
Iron is an essentia l e lement, in the d iets of humans a nd a n imals. The m ineral is widely ava i la b le 
in a nu mber of foods, including : meat, l iver, shellfish, egg yolk, bea ns, leg u mes, d ried fru its, n uts 
a nd cereals, b u t  despite its ava ilabil ity, iron deficiency is p reva lent ( P ippard 1995 ) .  Worldwide the 
WHO (World Hea lth Organisation ) has estimated that more than 1 b il l ion people have sy m ptoms 
of fatigue, intolera nce to the cold a nd poor behaviour a nd psychomotor development problems 
as a re sult of iron deficiency. This is pa rtly beca use the a mount of i ron present in food is not the 
a mount that is ava ila b le to the body. To be a ssimilated, minerals, includ ing iron in itially have to 
be b ioava i la b le .  M atzke ( 1998) p roposed that b ioava ilabil ity of a n utrient can be d ivided into 
th ree constituent pa rts. These are :  
• The n u trient in the intestinal lu men must be ava ilable for absorption . 
• Absorption a nd retention of the n utrient in the body. 
• Utilization of the nutrient by the body. 
The concept of b ioava i labil ity of d ieta ry iron is the key to understa nd ing the complexities of iron 
deficiency. Reg re ttably bioava ilability is not an a b solute, a nd as a result it can be affected by the 
iron status of the ind ividual, and a lso the source of the iron. In add ition other d ieta ry nutrients 
can be i n h i b itory to iron's bioava ilabil ity. 
The a i m  of this study wa s to eva luate the model of the anaemic p ig let to assess the 
bioava ilabi l ity of iron from the h u man d iet. P rior resea rch has concentrated on the effects of 
indiv id u a l  n utrients in the d ie t  that may e ither e nhance or inhibit the absorption of iron . 
Additional ly, these studies have used a n i m a l  models that consumed d iets, conta ining ing red ients, 
which h u man subjects would not eat at a l l .  
Using 3-week old a naemic piglets, the gene ra l  objectives of Experiment 1 were: 
1 .  To determine the effect of iron status on iron a bsorption. 
2 .  To determine the bioava i labil ity o f  d ietary iron from meat and n on -meat sources. 
16 
3 .  To determine the effects o f  vita min C on the absorption o f  non -haem iron when fed a s  
pa rt o f  a complete diet. 
Spec ific objectives were : 
• To test the effectiveness of meat and non-meat sou rces of iron to bring about a return of 
iron deficient piglets to hae matolog ic normality. 
To compare iron bioa vai labil ity in non-iron deficient piglets ( 2 1  days old) and iron­
defic ient piglets receivi ng non-meat i ron daily in their diet. 
To compare iron bioa vai labi l ity using iron-deficient pig lets ( 2 1  days old) con suming non­
meat iron diets, with either 250 ppm or 500 ppm of supplementary vita min C, to 
determine if vitamin C ca n increa se the absorption of non-hae m i ron . 
Food processing affects the i nitia l phase of the bioava ilability model, by determi ning the a mount 
of the mineral that is available fo r absorption . The meat used in experiment 1 was in a raw, 
unp rocessed state. 
The general objective of the second expe riment wa s to determine the effects of cooking 
temperature on i ron bioavaila bility in meat. 
Specific objectives were : 
To determine the effectiveness of iron in meat to retu rn anaem ic pig lets to haematologic 
norma lity. 
• To determine the effect of cooking meat on b ringing anae mic piglets back to 
hae matologic norma lity with meat that had been subject to either no heat treatment, 
heated to 60° C or 90° C. 
• To compare i ron bioavailability using 2 g roups of 5 piglets with either anaemia or positive 
i ron status receiving a non-haem dietary alternative . 
1 7  
Chapter 2 
2. Review of Literature. 
Kie s  a nd Me End ree ( 1982) commented that if a l l  nutrients found in food were digested, 
a b so rbed a nd made avai lable to the h u man or ani ma l ,  the science a nd practice of nutrition would 
be si mple. Unfortunately resea rch has shown dietary nu trients differ i n  their bioavai lability 
( Matzke 1998 ) .  
T h e  avai labi lity of nutrients i s  d etermined b y  a nu mber o f  factors, which i nclude the che mica l a n d  
physica l cha racteristics o f  t h e  foods containing th e  n utrients ( Lee 1984 a n d  Clydesdale 1 982) ,  
other constituents o f  the diet ( H a llberg et al., 1987) , t h e  digestive a nd absorptive processes for 
the specific nutrients ( Ferraris a nd Dia mond, 1989) and the physiologica l condition of the person 
or ani m a l  consu ming the food ( Ray a nd Enns, 2000 ) .  
This review o f  literature concentrates on these issues as they relate to dieta ry i ron . 
2.1 The physiological role of iron. 
Atomic numb<?r 
Fe 
Iron 
55.847 
2-8-14-2 
Electron 
Symbol 
Nam<> of<> l<>m<>nt 
Atomic weight 
Iron is an essentia l element that is req uired by h u mans 
and a ni ma ls. It i s  pri m a ri ly used i n  the production of 
hae mog lobi n and myog lobi n ,  the oxygen ca rrying 
proteins of the blood and muscles, respectively (Totara 
and Reyn olds-Gra bowski, 1996 ) .  Additiona l ly, elemental 
i ron can exist in two valance states and is therefore able 
to provide power, through oxidation and reduction 
reactions, to a numbe r of biochemica l pathways (Voet 
and Voet, 1995). These include those used in the man ufacture of enzymes needed for DNA 
synthesi s  and ATP (adenosi n e  t riphosphate) production. Regrettably the sa me redox properties of 
i ro n  can a l so  be detri menta l to biological systems generating oxidative radicals, which da mage 
biologica l components such as lipids, protein s  and DNA ( Roy and Enns. 2000). Therefore the 
a b sorption a nd metabolism of i ro n  need to be effectively regu lated . 
1 8  
2.2 Iron Digestion. 
Unable to synthesise elemental i ron, the body obtains the iron it requires from the d iet, where it 
is found in a large nu mber of foods including meat, liver, shel lfish , egg yolk, beans, legu mes, 
d ried fruits, nuts and cerea ls (Zijp et a l .  2 00 0 ) .  
There a re two forms o f  dietary iron a )  h a e m  i ron, which is found mai n ly in meat a nd b )  non­
haem i ron that makes up most of the iron in vegetables and cerea l grains ( Lomba rd et a l .  1997) . 
Haem i ron is contained within a porphyri n  complex associated with specific proteins. The acid 
conditions found in the stomach dena tu re the protein s, ena bl ing the porphyrin complex to be 
relea sed . The porphyrin complex re ma ins una ffected by further changes in pH a nd is di rectly 
assi mi lated into the enterocyte cel ls in the wa ll of the sma l l  intesti ne . 
Non-haem i ron a lso relies on mechan ica l  and enzymatic d igestio n ,  together with the series of pH 
changes a long the length of the digestive tract, to faci litate its release from the food we eat 
( Lombard et a l .  1 997). In add ition to freei ng i ron from other food components, Lombard et a l .  
( 1997) proposed d igestion also h a s  a further role, i n  the reduction o f  i ron from the Fe 3+  (ferric) 
state to the Fe 2+ ferrous form. The fe rrous form is more read ily a bsorbed across the sma l l 
intestine, despite the existence of specific transporters for both fe rric and ferrou s i ron (Conrad et 
al. 199 9 ) .  The acid conditions i n  the sto mach a re thought to in itiate the red uctive process. 
Intesti n a l  mucins a re then able to chelate the non-haem iron maintaining it in the more soluble 
ferrous form (Fe 2+ ) (Con rad et a l .  1994) . Inevitably move ment of the che la ted i ron from the acid 
environment of the stomach to the sma l l  i ntesti ne entails an increase in pH . The increased pH 
can ca u se  the precipitation of chelated iron causing the formation of an in so luble i ron residue 
that cannot be absorbed (Kies and McKendree 1982 ) .  The d ietary nutrients ca lci u m ,  phytate a nd 
polyphenols inhibit iron absorption by p recipitating chelated iron in a si milar way (Hal lberg et al., 
1987 ) .  
2.3 Iron ab sorption m echanisms. 
The e nterocyte cells differe ntiate between haem and non-haem i ron so that the two sou rces of 
dieta ry iron are absorbed using d ifferent pa th ways (Conrad et a l .  1999). Iron is moved from the 
lumen of the i n testine across the apical membrane and into the enterocyte usi n g  one of th ree 
pathways. Con rad et a l .  ( 1999) reported that non-haem i ron is absorbed using both the 
mobi lferri n -i ntegrin pathway and a diva lent cation transporter ( DCT- 1 ), whe rea s haem i ron is 
absorbed directly i nto the enterocytes by the haem i ron upta ke pathway. 
20 
DCT-1 is a p roton symporter that transports both non-haem ferrous iron a nd other divalent ions, 
from the i n testi n a l  l u men i n to the enterocyte. Body iron stores, dietary iron and posttra n slationa l 
modification reg u la te the expression of the DCT-1 p rotein (Conrad et a l .  1 999). 
The mobilferri n -i n teg ri n  pathway fa ci litates the absorption of dietary i norganic ferric i ron . 
The ferric iron is reduced to the ferrous form by a me mbrane-bound ferrireductase enzyme 
before entering the a bsorptive cell via !33 i n tegrin i n  combination with mobilferri n, both of which 
have been identified by Powell et a l .  ( 1 999) a s  tra ns-membrane p roteins. 
The haem iron uptake pathway facilitates the absorption of haem iron into the enterocyte. 
The iron is contained within a p orphyri n  complex, which enters the absorptive cell as an i ntact 
meta lloporphyri n . Once i n side the cell the porphyri n  comp lex is enzymatically degraded by haem 
oxygenase, releasing the iron ( Ra ffin et al . 1974) . 
The basolatera l  me mbrane then mediates the transfe r of the iron to the b lood p lasma and then 
to the rest of the body. As free i ron is toxic to biological systems, i ron is transported out of the 
enterocyte a nd into the body p lasma bound to the i ron transport p rotein tra nsferrin (Conrad e t  
a l .  1999 ) .  
2.4 Iron absorption is related to iron status. 
Iron absorption i s  complicated by regulation of iron uptake occurri ng at the two inte rfaces of the 
e n te rocyte between the apica l and basolatera l membrane ( Roy and Enns, 2000). The apical 
membra ne of the differentiated e nterocyte faces the i ntesti na l  l u me n  and i s  speci a li sed for 
transport of haem and fe rrou s iron i n to the cel l .  The basolateral m embrane then mediates the 
transfer of iron transported i n to the i ntesti n a l  epitheli a l  cells to the rest of the body (Totara and 
Reynolds-Grabowski ,  1 996) . 
Ferrari s  ( 1 989) reported that the i n testinal a b so rptive capacity for i ron is closely matched to 
dieta ry i n takes and body req ui rements, in order to p rovide just enough absorptive capacity, 
without wa sting biosynthetic energy on unneeded t ra n sporters. 
The body conserves much of its i ro n  by recycling it from wom out red b lood cells, but 
unfortunately i ron i s  a lso i rreversi b l y  lost from the body i n  the shedding of hair, epithe lia l  and 
mucosal cells a nd in sweat, u ri ne ,  faeces, bile a nd blood lost d u ri ng menstruation . Although these 
losses a re proporti onate to body stores of iron (Conrad and Umbreit, 2000), the lost iron needs 
to be replaced . Also changes in physiological state such as growth a nd preg nancy a lso req uire 
2 1  
i ncrea sed amounts of iron to be absorbed from the d iet for blood and tissue synthesis ( F u rugouri 
and Kawabata 1976) . 
Iron is essentia l and can only be obta ined from the d iet; therefore iron transportation proteins 
need to be continual ly expressed to operate at low d ietary iron leve ls. But as iron is toxic to 
bio log ica l systems, high levels of iron need to repress the transporter so as to protect the 
organism against the risk of intoxifica tion . 
Therefore when body iron levels fa ll, iron uptake from the sma l l intestine is increased until the 
iron re serves a re replete aga in ( Roy et al. 2 00 0 ) .  Cox and Peters ( 1980) has shown that d u ring 
the time the body ha s a negative iron balance iron absorption is up-reg ulated, the reby increasing 
the rate at which the ion transporte rs become fully sa tura ted with iron, while the rate of transfer 
in to the enterocyte cel l  remains unchanged ( Ferraris et a l .  1989 ) .  This is better exp la ined using 
the M ichae lis Menten expre ssion :  V0 = Vmax (s) I K m  + (s) .  
Vmax continues to increase until the transporters become saturated with iron and Vmax reaches 
a plateau, while km rema ins uncha nged . 
When h igh levels of d ietary iron a re continua l ly con su med, the iron transport protein tra nsfe rrin 
becomes saturated with iron; the iron is not absorbed a nd transported into the blood plasma but 
excreted a s  the transporters are repressed to prevent toxicity. Also the rate at which iron is 
transferred in to the ente rocyte cell rema ins unchanged, therefore any additiona l iron that may 
have been transported into the enterocyte cells before they were repressed is trapped, and is 
subseq uently lost from the body as these intestina l ce lls a re sloughed of a s  the tissue is tumed 
over (Roy and Enns 2000 ) .  
2.5  R egula tion of iron abso rption. 
The mecha nism by which iron homeostasis is ma intained within the body is poorly understood 
( Roy et a l .  2000) .  Like many of the body's reg u latory processes, it is hypothesised that reg u lation 
takes place on many d ifferent levels, so that when iron levels drop below a critical level iron 
upta ke is increased u ntil the reserves a re replen ished . Roy at al. (2000) proposed that a n  
imba lance between the rate o f  erythropoiesis o f  the ma rrow a nd its iron supply induced iron 
absorption . Furugouri et a l .  ( 1976) demonstrated that iron was actively absorbed in the p ig 
d u ring growth . The rap id  increase in body mass caused the Fe content in mucosal cells to drop 
thereby stimulating active absorption of iron. 
Iron is d istributed throughout the body; typica lly in humans 60-70% is found in haemog lobin, 20-
30 % in the iron storage proteins ferritin and hemosiderin and 3-5 % in myog lobin . Very sma l l 
amounts a re found in haem-conta ining enzymes and cytoch rome (0.2 %) and approxi mately 
22 
0 . 1% of the body's iron is found in transferrin, which transports iron in the plasma a nd extra 
va scular flu id (Worwood 1997 ) .  
2.6 Vitamin C increases non-haem iron absorption. 
Seve ra l studies have shown that ascorbic acid or vita min C enhances the a b sorption of d ietary 
non-haem iron . Clydesda le ( 1 982) proposed that vita m i n  C might a id in the a b sorption of non­
haem iron in several ways as outlined in Ta ble 1 .  
Table 1 :  P roposed mechanisms that could a id the absorption o f  non-haem iron by vita m i n  C. 
(Adapted from Clydesdale ( 1982) in Nutritional b ioava ilability of iron . Pg 5 5 ) .  
Characteristic 
1. p H  
2 .  Complexation 
and th e  
oxidation­
reduction 
potentia l  
Possible mech a n is m . 
The majority of d ietary non-haem iron is ferric iron ( Fe  3+) . P rior to 
a ssimilation the ferric iron is red uced by a series of pH changes a long 
the digestive tract to the more soluble ferrous form. 
The acidic p roperties of vita m in C could contribute to th is process both 
by increasing ionisation and by favouring the ferrous state, which has a 
g reater solubility at the pH of the intestine ( 10-1 M )  than d oe s  ferric iron 
( 10-ls M ) .  
Nojeim a nd Clydesd a le ( 198 1 )  found that low stomach p H  together with 
reduction potentia l d iffe rences between a scorbic acid and ferric iron 
facilitated the formation of a reversible a scorbate-i ron complex. This 
would p redominately leave more of the soluble ferrou s  iron ava i l a b le for 
a bsorption . 
U nfortunately the effectiveness of vita min C is l i m ited as the contin ued 
complex formations cause the pH to increase, changing the ion isation 
state of the i ron . The reversible complex then d isassociates leaving 
more of the ferric form . 
In addition to increasing non-haem iron b ioava ilabi l ity a nd absorption, vita min C is a lso req u i red 
in the tran sfer of iron fro m the storage proteins ferritin, h emosiderin a nd siderophylin to 
protoporphyrin for haemog lobin synthesis ( M onsen 1982) .  
Table 2: A summary of seve ra l  studies that eva luated the effectiveness of vita min C in i ncreasing 
the bioavai labil ity and absorption of non-haem i ron from the diet. 
Amount of vita m  i n  C Iron sou rce Effect on Iron Sgecies Reference 
added to diet bioava ila bilit¥ 
2 5 - 1 000 mg FeCi] 2 - 1 0  g reater H u ma n  Cook and M onsen 
absorption ( 1977) 
5 1 -247 mg/ d FeCi] No d iffe rence H u man Cook & Reddy 
(20 0 1 )  
25 mg 19, 22 and 20% G ui nea pig M ilne and Omaye 
1 00g/bodyweight i ncrease in rbc 1, hgb ( 1980) 
and het va lues 
respectively 
0 . 0 1 M  FeCI2 1 1 .5-19.6% i ncrease Rat Conrad and Schade 
Increase ( 1968) 
1 0 .4 g /Kg diet FeS04 Rat Wienk et a l .  ( 1997) 
330,660,990 (ppm) FeS04 3% increase Pig Yen and Pond 
( 198 1 )  
1 rbc : red b lood cell; hgb : haemog lobi n ;  het : haematocrit 
Ta b le 2 shows a su mmary of several stud ies using h u man and a n i m a l  subjects that have 
eva luated the effectiveness of vitamin C in increasi n g  bioavai lability and absorption of non -haem 
i ron from the diet, however it i s  difficu lt to compare the results of all these studies as they used 
different methodolog ie s. 
Yen et a l .  ( 1981), Milne et a l .  ( 1980) and Wienk et a l .  ( 1997) used i ron-deficient subjects to 
study the effectiveness of vita min C in i ncreasing bioava i lability and absorption of non-hae m iron . 
This was in contrast to Cook and Reddy (200 1) whose subjects were not deficient in i ro n .  
Additiona lly diet composition va ried between th e  studies. Cook e t  a l . ( 1 997) u sed a se m i  
synthetic diet contain ing c o m  oil, ova lbumin, a n d  dextri m a ltose . Whereas Yen e t  a l .  ( 1 98 1 ) ,  M i lne 
et a l .  ( 1 980) and Wienk et a l .  ( 1997) eva luated the effects of vitamin C using experi men ta l  diets 
formu lated specifically for the age and species of the su bject, and Cook and Reddy (200 1 )  u sed a 
complete 'western-type' diet in their evaluation. 
Also the stud ies used subjects both capable of vita min C synthesi s ;  (the piglet a nd rat studied by 
Yen et a l .  198 1 ,  and Wienk et a l .  1997, respectively) and those fo r which vita min C is an 
essential dietary nutrient. (The human and guinea pig studied by Cook et a l .  1997, Cook and 
Reddy 200 1 and Milne et a l .  1980, respective ly) . 
The leve ls and sou rces of vita min C u sed a lso va ried between the studies. The a ni m a l  stud ie s  
using synthetic L-ascorbic acid whereas the h u ma n  study o f  Cook and Reddy ( 200 1 )  calcu lated 
vitamin C content of the complete trial diet from dietary components. 
24 
The mea sure ments of the effectiveness of the vitamin C a lso vari ed .  The animal studies were 
repletion type studies that measu red the ra te at wh ic h  some blood pa ra meters i ncreased to 
norma l p hysi ologica l levels. Whereas the study by Cook and Reddy (200 1 )  used extri n sically radio 
iron-labe l led b read and hamburg e r  b u n s  to measure non-ha e m  iron absorption . 
Conseq uently the results of the studies a lso varied . Cook et a l .  ( 1977) gave 63 male subjects a 
semi synthetic meal containin g  corn oi l ,  ovalbumin, and dextrimaltose. Vita min C was added to 
the mea l s  i n  varying a mounts ranging fro m  2 5- 1000 mgs. As a resu lt of the i ncremental i ncreases 
in the vita min C content of the diet, a sim u lta neous i ncrease i n  the a b sorption of non-haem iron 
was a lso observed . Cook et a l  reported that the rate of absorption a ppeared to be logarith mica l ly 
related to the vitamin C conte n t  of the diet so that the larger a mounts of vita min C produced a 
prog ressive rise in the increase of iron absorption . 
Yen et a l .  ( 198 1 ) ,  and Wienk et a l .  ( 1 997) reported initi a l  increases i n  iron absorption during the 
initia l stages of their respective studies, but both failed to firmly esta b lish the existence of any 
linear relationship between the leve l of vita mi n C in the diet a nd the a bsorption of non-haem iron 
in both the pig let a nd rat; i n te resti n g ly both of these species a re able to synthesise enough 
vita mi n  C fro m g lucose to fulfi l  their req uirements. This cou ld suggest that i t  is solely the iron 
deficiency that i s  sti m u lating the active absorption of iro n from the small i ntesti n e  or 
a lternati ve ly, i f  vita min C is involved, its level in the p lasma rather than that of the 
ga stroi ntesti n a l  tract may be a n  i m porta n t  factor. 
However, M i l ne et a l .  ( 1980) was able to reprod uce the benefits of supplementary vita min C on 
the a b sorpti o n  of non-haem iron . Using 36 ma le guinea pigs, Milne et a l. ( 1980) eva luated the 
effect of vitamin C on copper a nd iron metabolism . The semi-puri fied diets fed to the anima l s  
contained 0 ,  0 . 5  or 2 5-mg/ 100g vita mi n  C .  Prior t o  beginning the study th e  guinea pig s received 
a daily sub-maintenance dose of vitamin C i nd ucing hypovitaminosi s .  Milne et a l .  ( 1980) then 
concluded that both plasma vita min C a nd i ron leve ls appea red to be related directly to vitamin C 
i ntake . P lasma iron was a lmost twice as high in the ani m a ls receiving 25 mg of vitamin C per 100 
g body weight than i n  animals receiving n o  supplemental vitamin C. 
I n  trying to reproduce desirable experi me ntal conditions to examine the effects of vitamin C on 
i ro n  metabolism Milne et a l .  ( 1980) may have i nadvertently created a situation where vita min C 
a p peared to be related to the absorpti o n  of non-haem iron when i n  fact the g uinea pig's body 
was replenishing the sma l l body store s  of vita min C .  
Cook a nd Reddy ( 2 00 1 )  compared the effect o f  vita min C on non-haem iron a bsorption from a 
complete diet rather than from a si ng le mea l and found that the faci litati n g  effect of vitamin C on 
i ro n  a bsorption from a complete mea l was far less pronounced than that from single meals. 
H owever in this study Cook a nd Reddy u sed both male and fe male subjects who had a positive 
iron status (serum ferritin concentrations that were g reater than 1 2  J.Jg I L ) .  The regu lative 
25  
mechanisms proposed by Roy et a l .  ( 2000) suggest that the radio-label led i ron would not be 
actively absorbed in these circu mstances a nd atte mpts to enhance i ron absorption i n  a subject 
with positive iron status would also be i neffective; as the toxicity of excess iron would in itiate the 
repression of the i ron transporters in the sma ll i ntesti ne until such time when body i ron levels 
began to decrease . A lso the role of Vita min C in enhancing the absorption of iron is i n  
mai ntain ing i t  i n  th e  Fe 2+  state rathe r than i ncreasing the rate a t  which the b od y  assi m i lates it 
and to do this the vita min C ha s to be present in the stomach at the sa me ti me as the non-haem 
iron (Cook et a l 1977), which cou ld n ot be gua ranteed when subjects were a l lowed to consume a 
self selected diet. 
Therefore the evidence from these studies suggests that i ron status a nd diet composition a lso 
contri b ute to the effectiveness with which vita min C increases the b ioava ilabi lity and a bsorption 
of non -haem i ron from the diet. 
2.7 Effect of meat on iron bioava ilabili ty 
In add ition to vita min C, the principal enhancer of non-haem i ron absorption in the diet is meat 
( Seth and Ma honey 2000). During digestion, stomach acids denature the meat p rotein s  prior to 
them being b roken d own i n to sma l l peptides and free a m ino acids (Tota ra 1 99 6 ) .  Kroe et a l .  
( 196 3 )  found that nine a mino acids fou nd i n  meat were able t o  increase the u ptake of ferrous 
i ron, including histidi ne, g luta mine, g lutamic acid and methion ine and just th ree were able to 
i ncrease the upta ke of ferric iron. These were cyste ine, histidine and lysine. Cystei ne li ke vita min 
C is a red ucing agent that assists in the conversion of ferric iron into its fe rrous form . U n li ke 
cystei ne, histid ine is not a red ucing agent (Stryer 1995); therefore the increased uptake of i ron 
must be because of a factor other than red uction . 
Kies a nd McKendree ( 1982) proposed several ways that histidine could contribute to the 
increased bioava i labi lity of haem iron . These are shown in Table 3 .  
2 6  
Table 3 :  P roposed mechanisms that could explain why histidine, cysteine and lysi ne can i ncrease 
the bioavaila bi lity of haem i ron (Adapted from Kies a n d  McKendree ( 1982) in Nutritional 
bioavailabi lity of iron Pg 185) . 
Cha racteristic 
1 .  B u ffering 
changes i n  p H  
2 .  Chelation 
3. Transportation 
P roposed mechanism of histidine 
Amino acids like i ro n  a re sensitive to changes i n  pH, which ca use 
changes to their ionisation state. Therefore the a mi no acids cou ld act 
a s  buffering agents i n  the i ntestine and delay the increase of the p H  
towards neutra lity where the oxidation of i ron forms i nsol u b le 
p recipitates. 
Alternatively amino acids can form iron a mine chelates that act to 
enhance iron absorption . 
Amino aci d s  can stimulate i ron transport system s  within the ani m a l  
thereby i ncreasing the a b sorption of i ron . 
2.8 The effect of heat processing of meat on iron bioavailability. 
The digestibi lity and n utritive va lue of protein is affected by heat processi ng (Jansuittivechakul et 
al. 1 98 5 ) .  This is because p rotein s  a re se n sitive to both p H  and heat, which cause them to 
denatu re (Stryer 1984 ) .  Meat contain s  both haem a nd non-haem i ron (Lombard et a l. 1997) 
Studies by carpenter a nd Clark ( 1995) and Kristensen a nd Purslow ( 200 1 )  have found that d u ring 
heat p rocessing the haem i ron content of meat and meat products decreases. The decrease in 
the haem iron content of the meat is accompanied by an i ncrease i n  the non-haem i ron content. 
Table 4 shows a su m m a ry of several studies that have evaluated the effects of heat processin g  
on the hae m  a nd non-haem i ro n  contents of meat a nd meat products. 
27 
Table 4: A summa ry  of the effects of heat processing on the haem and non-haem iron content 
of meat and meat products. 
Meat or meat P rocessing Effect on Iron Species Reference 
p rod uct bioavai labi lity 
Ground beef Raw, boiled No effect Rat Ja nsu ittivecha ku I 
autoclaved et a l . ( 1 98 5 ) .  
Ground Beef Baking, M icrowave Decrease In -vitro Sch ricker and M i l ler 
( 1 98 3 )  
Beef (baked) Baked Decrease Rat Rot ruck et a I. 
( 1 979) 
Pork Steam heated Decrease In-vitro Kristensen a nd 
water bath Purslow (200 1 )  
Rabbit and Direct heat Decrease In -vitro, Garcia et a l .  ( 1996) 
Beef � reci�itates H uman 
The simi l a rities in methodology of the three in-vitro studies enable comparison of results to be 
made . 
Kristensen a nd Pu rslow ( 2 0 0 1 )  used the longissimus m u scle s  taken from p ig  carca se s  24 h rs after 
sla ughter to examine the effects of heat processi ng on the haem to non-haem i ron ratio in meat. 
Meat wa s tri m med of visi ble fat and connective ti ssue and coarsely minced through a 3.7-x l .6 c m  
plate .  Heat treatment of the meat was pe rformed in thermostatica lly circu lating water baths for 2 
hrs followed by cooling on ice water. Kristensen a nd Purslow found that a s  temperature was 
incrementa lly increased above 55°C the haem iron content of the meat gradua l ly decrea sed . This 
was fo l lowed by an increase in the non-haem iron content of the meat, which increa sed when 
heat-processi ng temperatu re exceeded 80°C. As little or no loss in haem iron content occu rred in 
samples heat-treated to temperatures below 55°C it can be concluded that the myog lob in 
containing the iron was sti l l  intact. Also the non-haem iron content is sta b le until temperatures 
a re i n  excess of 80°C . 
This is consi stent with the findings of Sch ricker and Mil ler ( 1983 ) who used seve ra l  experiments 
to determine the effects of cooki ng a nd chemical treatment on haem and non-haem i ron i n  meat. 
Sma l l 5 g sa mples of ground beef were subjected to the effects of baking ( 1 76°C) for 20 -40 
minutes and m icrowave cooking for 0 .5 a nd 3 minutes (600 watts). Schricker and Mi ller showed 
the existence of a linear i ncrease i n  the non-haem i ro n  content of the meat and the ti me of 
exposu re to the heat treatment. The experi ment was later refined and repeated with much larger 
samples of g round beef (225-270 g ) .  Taking both surface and i nterior sa m p les of the cooked 
meat for i ron eva luation .  The results showed that the smaller sa mples subject to harsh cooking 
28 
treatment had the g reatest decrease in haem iron, and that the haem iron content of the meat 
surface deteriorated more than the interior. 
The method of Garcia et a l .  ( 1996) was a l ittle less orthodox. Sa mples of rabbit and beef meats 
were subject to d i rect heat treatment u nti l  the degradation of the meats metmyog lobin had 
caused the colour of the meat to change from red to b rown . The visual interpretation of the 
colour change may have introduced error into the experiment (especially as there a re d i fferences 
in the colour of meat from diffe rent muscle g roups, and a lso the surface colour of the meat 
changes when exposed to oxygen . )  rather than subjecting sa mples of meat to a specific 
temperature treatment for a g iven period of time . H owever, the decrease in the haem iron 
content of the meat as a result of heat processing was consistent with other studies. 
Unfortunately comparisons of in-vivo stud ies between d ifferent specie s  a re more d ifficult 
Jansuittivecha ku l  et a l .  ( 1985) a nd Rotruck and Luh rsen ( 1979) both u sed the rat to mode l  iron 
bioava i la b i l ity a nd absorption in repletion type studies. They fou nd that in anaemic rats 
consuming experimenta l d iets, that ava ila b i lity of inorganic iron (ferrous s ulphate) from the 
control g roups was h igher than e ither meat (cooked beef) or haemoglobin iron . The opposite 
occurs in the h u ma n .  This suggests that the rat may not be a good model in which to study iron 
bioava i la b i l ity a nd a b sorption . 
2.9 Measures of i ron status. 
As body iron is found in the b lood, iron status can be effectively determined by measures of the 
nu mber and percentage of red cells, blood haemoglobin concentration, the mean corp u scu la r 
volume, and mean corpuscular haemoglobin concentration . 
The accepted values for these haematologic parameters in the pig a re shown in Table 5 .  
Table 5 :  Accepted physiological values o f  red b lood cell parameters i n  the healthy p ig. ( Egeli et 
a l .  1998) . 
Blood Para meter '" �' "' ,,, � '  '' '''�''' ' 
Red b lood ce l l  n u mber per u n it volume of b lood . 
Blood haemoglob i n  concentration in whole blood . 
Haematocrit ( RBC vol ume/whole blood vol u me ) .  
Mean red b l o od  cel l  vol u me . 
Mean red blood cel l  haemoglob in .  
Mean corpuscula r haemoglobin concentration within the red 
J\<:c:�pt�cl �IJge . 
5-8 x 1012 cells I L 
1 10 - 1 70 g I L 
0 .37-0 .50 L I L (or 37-50 % )  
50-68 f L 
14.4-2 0 . 1 pg (weight per red 
blood cel l ) .  
300-340 g I L 
29 
Red blood cel ls a re manufactured in the bone ma rrow . They a re  d istinct from other cells in that 
they lack a n ucleus a nd other cel l u la r  organelles necessary to re produce and carry out extensive 
metabolic activities. They have a natural lifespan of approxi mately 120 days. The number of red 
cells in the c i rculatory syste m is homeostatica lly reg u lated, so that the number of cells e nte ring 
the circu latory system equals those removed and destroyed , a lthough an exception to th is occurs 
d u ri n g  the physiolog ica l states of growth and pregnancy when the nu mber of red cells entering 
the circu lation increa ses due to increases i n  blood volume. The n u mber of red cells or 
a lternative ly the percentage of red cells i n  circulation (haematocrit) can be used to identify i ron 
deficiency (Worwood 1997 ) .  
The pu rpose o f  the red b lood cel l  is t o  deliver oxygen t o  the body's cells a n d  remove carbon 
dioxide. They a re able to accomplish this, as they contai n the specia lised protei n haemoglob i n .  
"The haemog lobin molecu le conta ins four peptide cha ins that are held together by non-cova lent 
attraction . Each of the polypeptides contains a tig htly bound haem, a substituted porphyrin with 
a centra l i ron ato m .  The fou r  chains are packed together and the haem groups are located in 
crevices near the exterior of the spherica l molecu le, one in each of the four subunits. Al losteric 
i nteractions enab le  the haemog lobi n to co-ord inately transport oxygen, carbon d ioxide a nd W 
ions" ( Stryer 1984) . Measu ring blood haemoglobin concentrations can a lso identify iron defi c iency 
(Worwood 1997). Additiona lly measurements of b lood haemog lob in concentrations in repletion 
type stud ies can be u sed to identify the a mount a nd type of d ietary iron that can restore the 
body's iron ba lance most effectively. 
A differential wh ite blood cel l count is u sed to monitor the health status of subjects. A high wh ite 
blood cell count is usua lly i n d icative of infection (Tota ra 1 996) and can resu lt in a depression i n  
t h e  levels of seru m  iron ( Beisel e t  a l .  1974) . While specific increases in the type o f  white cel l  can 
be used to point to the i n fectious agent i nvolved . 
Table 6 shows the accepted ranges of the white blood cell para meters in the pig . 
Table 6: Accepted physiological values of white cell pa ra meters in the norma l healthy pig . (Ege li 
et a l .  1 998). 
Blood pa ra meter 
White cell 
Ce ll type 
Neutrophil cells 
Lymphocyte ce lls 
M onocyte cells 
Eosi nophil ce lls 
Basophil cells 
Accepted ra nge 
X 1 09 cells I L 
1 0-23 
2 .5 - 1 0  
7.0- 1 5 .5 
0 . 3 2 - 2 .0 
0 .08- 1 . 76 
0-0 . 3  
% O f  white ce l l  mass 
100 
26.6-56.7 
3 5 . 5-62 . 0  
1 . 6-8.8 
0 . 1 - 5 . 6  
0-2 .7 
30  
2.10 Using an animal model to study iron deficiency in the human 
N u trition studies, using human subjects a re d ifficult to carry out. Therefore a n imal stud ies a re 
often i n itia l ly used to eva luate the costs a nd benefits of dietary nutrients. Several a n ima l species 
have been considered as a m od e l  for nutritiona l studies in the h u ma n .  These include the monkey, 
gu inea p ig ( Na ra singa et a l .  1977), a nd rat (Wienk et a l .  1997) ) .  More recently the p ig has been 
shown to be a promising model d ue to the simila rities in digestive a nd a bsorptive processes 
( M oughan and Rowan 1989 ) .  U sing sca ling techn iques Moughan et a/ ( 1 992) estimated that the 
d ig estive tract of a 3-week old piglet could be used in comparative studies to represen t  a 3-
month old infant. 
The pig has severa l characteristics that made it the most appropriate a n ima l model i n  evaluating 
iron bioava ilability from a comp le te d iet. These a re outlined in Table 7 .  
Table 7 :  Characteristic o f  the pig that make i t  a n  appropriate model for studies o f  iron 
ava ilabil ity, for h u mans. 
Cha racteristic 
1 .  Omn ivore 
2 .  Eats most 
food s .  
3 .  Iron status 
4. Vita m i n  C 
synthesis 
Like the h u man the p ig is omn ivorous e na b l ing it to d igest food from 
both a n i m a l  a nd pla nt origin ( Enca rta E ncyclopedia 2000). 
As the p ig eats most foods that are consumed by humans, the 
experimental d iets can be formulated to contain foods typical of the 
h u ma n d iet ( M oughan a nd Rowan 1989 ) .  
After b i rth the p i g let qu ickly becomes iron deficient, a s  it is bom with 
l i m ited iron reserves a nd the m i lk produced by the sow contains very 
little iron (Engl ish et a l  1 984) . I ron deficiency is usua lly p revented in 
the fa rmed pig by the admin istration of iron by intra muscular injection 
soon afte r  birth . Therefore dela ying the admin istration of iron into the 
pig let p rovides a similar scenario to that of a child or a d u lt suffering 
from iron deficiency ( More! 2001 persona l communication) .  
The pig can synthesise enough vitamin C from g lucose to meet its own 
requ i re ments, from approximately 1 week of age (De Rodas et a l .  
1998 ) .  Lee ( 1984) reported several studies, which found that the 
reducing p roperty of organic acids could increase the a b sorption of 
n on-haem i ro n .  Therefore, vita min C can be added to the pig's d ie t  
a l lowing its role a s  a reducing agent in the d ig estive tract t o  be 
3 J 
explored , without it being utilised by the a ni m a l .  
Despite these beneficia l cha racteristics a nd apparent physiolog ica l simila rities, ca re i s  a lways 
needed when the results from an ani m a l  study a re extrapolated to the huma n .  
2.11 Summary 
The ph ysiologica l roles of i ron include, the prod uction of haemoglobin a nd myoglobin, and in the 
provision of power to a n u mber of bioche mica l pathways. 
When the body has a positive iron bala nce i ron is stored in sma l l metabolite pools ena bli ng iron 
to be continual ly available to be used i n  its physiologica l roles. 
Iron stores can be dep leted either d ue to i ncrea ses in requirements for growth a nd pregnancy or 
insufficiencies in the a mounts of i ron bei ng obtained from the diet, the i ron bala nce i n  the body 
movi ng from positive to negative . Restoring the ba la nce of i ron i n  the body is difficu lt despite the 
fact that the dep leted i ron stores a ssist in i ron being absorbed from the gastroi ntesti na l tract . 
This is becau se  the i ron content of food is not that ava ilable to the body through absorptio n .  The 
bioavailabi lity of d ietary i ron is not an a bsolute va lue; iron status, food processi n g  and other 
components of the diet can infl uence how much of the dietary i ron is ab sorbed . 
An im a l  models can be a valuable resource, aiding in the quantification of bioavai labi l ity. The most 
suitable a n imal models have digestive a nd metabolic processes that are compara b le  to those of 
the h u ma n .  In stud ies of iron bioavai labil ity the pig let may be more su itable th a n  the rat mode l, 
as both the pig let and the human a ssimilate the two sources of d ietary i ron in simila r ways. 
32 
Chapter 3 
3 .  The use of piglets to evaluate iron bioavailability 
3 .1  Introduction 
Worldwide iron deficiency a ffects more than one b i ll ion people. They have symptoms of fatigue, 
intolerance to the cold and poor behaviour and psychomotor development problems. Th is is 
because the a mount of iron p resent in food is not the a mount that is ava i lable to the body. 
There a re two forms of d ieta ry iron; haem iron ma inly found in meat and non-haem iron that 
m a kes up most of the iron found in cerea l a nd g rains. 
The ava ilabil ity of non-haem iron is particularly susceptible to pH changes that occur a long the 
d ig estive tract. Additionally th is type of iron can a lso form comp licated associations with other 
d ieta ry n utrients, wh ich can change the a mount of iron that is available in the small intestine for 
a b sorption . Haem iron is less a ffected by p H ;  thereby increasing its availabil ity. 
The predisposition of non-haem iron to changes in ion isation state identified several nutrients 
that a re able to either buffer changes in pH or ma in ta in non-haem iron in its Fe2+ state e nabling a 
g reater proportion of iron to be ava ilable for absorption . These a re some amino acid s derivatives 
found in meat and a lso org a n ic acids .  
T h e  body's capacity t o  absorb iron is tightly regu lated ; because excess iron i s  toxic to biolog ica l 
syste ms, therefore the absorptive capacity of the sma l l intestine is closely matched to d ietary 
intakes and body req u i re ments. So that in states of iron deficiency within the body, iron is 
actively abso rbed fro m the gastrointestina l tract. 
The objective of th is stud y  was to eva luate the effect of i ron sta tus on iron absorption, to 
quantify the biolog ica l effectiveness of the d iffe rent forms of iron in retu rning an iron defi c ient 
s ubject to haematologic norma l ity using a repletion study and add itiona l ly whether the strateg ic 
use of vita min C, an organ ic acid, is a b le to increase the bioava ilabil ity of non-haem iron i n  
anae mic 3-week o l d  pig lets. 
A second experiment eva luated the effect of cooking temperature on iron bioavailability from 
meat. 
The data obtained from both experiments is compa red with data from human stud ies with the 
a i m  of validating th e  pig let as a suitable a n i ma l  model for further bioava ilability stu d ies. 
33 
3.2 Materia ls and methods 
3.2. 1 Animals 
Experiment 1 
Five sows on a commercial pig fa rm were randomly selected from a group of twelve, which 
represented the nu mbe r of sows that a re fa rrowed each week from a 300-sow comme rc ia l  p ig 
u n it .  Each of the hybrid sows had been cross-mated to a sire l ine boar, producing slaughter 
genera tion progeny. 
After a gesta tion period of 1 1 5  days, each of the five sows was induced to fa rrow. The sows 
prod uced 19, 14, 1 3 ,  14 and 13 pig lets, respectively. Using only the male pig lets from each litter, 
39 pig lets were identified by the insertion of a nu mbe red ea r tag. Of these p ig lets, 14 were 
injected shortly after birth with 200 mg of i ron (as iron dextran), while the rema in ing 25 p ig lets 
received no iron injection . The j u stification in iden tifying more p ig lets than were necessary for 
this study was to account for any variation in wea n ing weig ht and a lso any pre-weaning mortality 
that may occur. 
The pig lets receiving no su pplementary creep feed d u ring the pre-wean i ng period . Cross 
foste ring was perm itted , occurring in two i n stances. 
The an ima ls were weaned at 2 1  days. At weaning, the 39 p iglets were we ig hed . Animals used in 
' 
the experiment we re chosen so that each treatment g roup would conta in 5 piglets ( n = 5), one 
from each of the l itte rs. (An analysis of varia nce was used to dete rmine that there was no 
sig n ificant d ifference between pig let we ights) . Each g roup would consist of one pig let that had 
been injected with iron and 4 others that had not received any iro n .  
The pig lets were transported t o  Ma ssey Un iversity's Intensive Animal Research facil ity, where 
they had a two-day period of acclimatisation, prior to the commencement of the study. Du ring 
this transition period pig lets were offered a liq u id sta rte r d iet consisting of skim m ilk powder 
(40%) and casein ( 10%) at a rate of 400 g per pig let per day. After this brief period of 
acclimatisation, d ieta ry treatments were randomly a ssigned to the g roups. 
Experiment 2 
Using an imals fro m the same herd as experiment 1 .  5 sows were randomly se lected and induced 
to fa rrow after a gestation pe riod of 1 1 5  days. 
Using both male and fe ma le pig lets, 8 an imals we re randomly selected from five l itters. Each of 
the 40 p ig lets was identified by the insertion of a n u m be red ea r tag . 
1 5  of the 40 pig lets (3 from each litter) were ra ndomly chosen and were injected shortly after 
b i rth with 200mg iron . The re ma i n ing 2 5  piglets (5 from each litter) received 60 mg iron as iron 
34 
dextra n .  As in experi ment 1 the j u stification in identifying more pig lets than were necessary for 
this study was to account for any variation in wea ning weight and a lso a ny pre-weaning morta lity 
that may occur. 
The piglets were rea red on the sow and received no supplementary creep feed d u ri ng the pre­
weaning peri od .  Cross fostering was perm itted , occurri ng in two instances, and 2/5 litters were 
mu lti-suckled . The piglets were weaned at 2 1  days. 
At wea ni n g ,  the 40 piglets were weig hed . Animals u sed in the experiment were chosen so that 
each trea t ment g roup would contain 5 pig lets ( n = 5), one from each of the litters. (An ana lysis of 
va riance was u sed to determine that there was no sig nificant difference between pig let weights) . 
Each g roup would consist of one piglet that had been i njected with 200mg iron and 4 others that 
had received 6 0 mg iron . 
The pig lets were tran sported to Ma ssey U niversity's Intensive Animal Research facility, where 
they had a period ( 7  days) of accli matisation prior to the commence ment of the study. This 
differs from the initia l study where piglets had a 2-day acclimatisation peri od .  The acclimatisation 
period was extended so that feeding regi me wou ld be more established at the begi n n ing of the 
study, thereby reducing the i ncidence of feed refusa l  and low inta kes that occurred i n  the initia l 
experi ment. During this transition period pig lets were offered a l iq u id sta rter d iet consisti ng of 
skim milk powder a nd casein at a rate of 400 g per pig let per day . After the period of 
accli mati sa tion piglets were randomly a l located to treatment g roups using sex as a block. 
3.2.2 Housing 
Experiment 1 
Pig lets were individual ly housed, in metabolism crates. The crates were situated in a controlled 
environ ment of Ma ssey U niversity's Intensive An imal Research faci lity. 
The metabolism crates were man ufactu red from ga lvanised meta l, each crate measuring 1 .5 x 
O . S m .  The crate floor was made from p u nched meta l, a l lowing urine and faeces to fa l l  to the 
floor of the room, thereby preventing caprophagy a nd the ingestion of i ron excreted in the 
faeces. Recycled pla stic covered 1/3'd of the floor a rea and provided a comfortable laying a rea for 
each piglet. An i nfrared heat la mp provided additiona l heat so that the temperature was 
maintained wit h in the animals thermo-comfort zone at 29°± 1 .5° C .  
Each of t h e  meta bolism crates contained a removab le  400 m m ,  ad-li b  feeder (Sta ll ion pla stics 
New Zea land ) a nd water was available at a l l times through a bite nipple d ri n ker. 
35 
Experiment 2 
As with experiment 1, a n imals were ind ividually housed in metabolism c rates that were situated 
in an environmenta lly contro l led room in Ma ssey Un iversity's inten sive a n imal research fac i lity. 
The pen d imensions and layout of the metabolism crates were as in experiment 1 .  
3.3  Experimental design 
Experiment 1 
The experiment was a one-way desig n with repeat measures, which u sed 3 experimenta l  
treatments a nd 2 controls, a s  shown i n  Tab le  8 .  
Table 8: Characteristics o f  t h e  five treatment groups u sed i n  experiment 1 .  
Grou Fe at birth Diet � (ppm) N o  a n i ma ls - --
c 0 mg Control 0 5 
CV250 0 mg Control + Vit C 250 5 
CV500 0 mg Control + Vit C 500 5 
Meat 0 mg Meat 0 5 
C+ 60 mg Control 0 5 
Experiment 2 
The experiment was a one-way desig n with repeat measures, which u sed 3 experimenta l 
treatments a nd 2 controls, as shown in Tab le  9 .  
Table 9: Characteristics of t h e  five treatment groups u sed in experiment 2 .  
Grou Fe at birth D iet Meat cooking conditions No a n i mals 
c 60 mg Control 5 
M60 60 mg M eat rv600C for >60 min 5 
M90 60 mg M eat rv900C for >60 min 5 
M r  6 0  mg Meat Uncooked 5 
C+ 200 mg Control 5 
The control d iet wa s fed to both C a nd C+ groups who d iffe red only in iron statu s. The meat d iet 
wa s fed to th ree trea tment g roups a nd contained a meat fraction that was e ither raw or had 
been cooked to a bout 600 or 9oo C .  
36 
3.4 Diets and Feed Management 
3.4.1 Diet 
Experiment 1 
Each p ig let received one of four d iets, as shown in Table 10 . The control d ie t  was fed to both the 
C and C+ g roups who d iffered on ly in i ron status. The d iets used ma inly food choices from 
h u m a n  d ie ts, but were balanced for d igestible energy (DE) and Lysine enabling them to be fed to 
the p ig lets without any detrimental effects. 
The composition of the experimenta l  d iets a re shown in Tab le 10 
P ig lets were fed their respective d iets from 23 days of age (experimental day 0) for a 28-day 
experimental period . 
Table 10: The ingredient composition of the experimental d iets (%) for experiment 1 on an a s­
fed basis, i n c luding the add itiona l mixing water .  
Ingred ients % 
Peas 
Skim milk powder 
Soybean mea l 
Soybean o i l  
T h reon ine 
M e th io nine 
Vita m in s + m inera ls (Excl. Fe) 
Ce l lulose 
D icalcium p hosphate (DICP) 
Sod i u m  Chloride (NaCI) 
Meat 
Water 
Wheat starch 
Fe heptasulfate 
Group 
C , C+ 
1 0  
1 0  
4 
2 . 5  
0 . 1  
0 . 12 5  
0 . 1  
1 . 5 
1 
0 .075 
0 
63 
7 . 5  
0 
Cv250 
1 0  
1 0  
4 
2 . 5  
0 . 1  
0 . 1 2 5  
0 . 1  
1 . 5  
1 
0 . 075 
0 
63 
7 . 5  
0 
CvSOO 
1 0  
1 0  
4 
2 . 5  
0 . 1  
0 . 125 
0 . 1  
1 . 5 
1 
0 . 075 
0 
63 
7 . 5  
0 
Meat 
0 
5 
0 
5 
0 
0 .075 
0 . 1  
1 . 5 
1 .2 5  
0 . 1  
2 5  
50 1 
1 1 .96 
0 .0 12 
water wa s added to the meat d iet because the meat contained approximately 75% water. 
2Fe heptasulfate and Vit C were added to the meat d iet to rnach the level in the C and C + d iets. 
Table 1 1  shows the ca lcu lated and ana lysed nutrient composition of the experimental diets. 
3 7  
Table 11 :  Calcu lated and ana lysed nutrient composition the experimental d iets used in 
Experiment 1 on an as-fed basis. 
Content o n  an as fed basis) Control CV250 CV500 Meat 
Calculated 
Digestible Energy DE (MJ /Kg 5 . 9  5.9 5.9 5 .82 
Lysine (g/ kg ) 5 . 2 7  5 . 2 7  5.27 5 . 79 
ea (g/ kg ) 4. 14 4. 14 4 . 14 4 .04 
Tota l H20 mi/L reconstituted d iet 672 672 672 7 1 1  
Analytical1 
Tota l Fe (ppm) 43 .4 40 .6 45 .9 4 1 . 2 
% Haem 0.8 0 . 8  0 . 9  7 . 7  
% Non-haem 99 . 2  99.2 99 . 1  9 2 . 3  
1Dr Roger Pu rcha s persona l commun ication 
Experiment 2 
The ing redient compositions of the experimenta l  d iets for Experiment 2 a re shown in Ta ble 12 . 
Table 12: The ingredient compositions of the experimental d iets (%) for Experiment 2 on an as-
fed basis includ ing add itional mixing wate r. 
Ingred ients % Group 
C, C+ M60 M 90 M r  
Skim m i l k  powder 5 0 0 0 
Soybean oi l 5 5 5 5 
Th reon ine 0 . 05 0 0 0 
Meth ion ine 0 . 125 0 .0 5  0 .05 0 . 05 
Vita m i n s  + m i nera ls 0 .08 0 .08 0 .08 0 . 08 
Ce l l u lose 1 . 5 1 . 5 1 . 5 1 . 5  
Dica lcium phosphate (DICP) 1 . 5 1 . 5 1 .5 1 . 5  
Sod i u m  Ch loride (NaCI) 0 . 05 0 .0 5  0 . 0 5  0 . 0 5  
Meat 0 2 5  2 5  2 5  
Water 6 7 . 5  50* 50* 50* 
Wheat starch 1 1 . 72 1 1 .72 1 1 . 72 1 1 .72 
Fe heptasu lfate 0 . 0 0 3 5  0 . 00025 0 . 00025 0 .00025 
Ca se in 7 . 5  5 5 5 
* Less water was added to the meat d iet because the meat conta ined approxi mately 75% water. 
Ta ble 1 3  shows the calcu lated and ana lysed nutrient composition of the experimental d iets. 
38  
Table 13:  Calcu lated and analysed nutrient composition the expe rimenta l d iets used in 
Experiment 2 on a n  as-fed basis. 
Content (on an Cr C+ Mr M 60 M 90 
(3S fed basis} 
Calculated 
Digestible Energy DE (MJ / Kg )  6 . 0  5 . 9 1  5.91 5 . 9 1  
Lysin e  ( g /  kg ) 7.7 8.6 8 . 6  8 . 6  
ea (g 1 kg) 5.2 4 . 6  4 . 6  4 . 6  
Tota l H 2 0  m i / L  reconstituted d iet 7 1 3  7 16 7 1 6  7 1 6  
Analytical1 
Tota l Fe (ppm) 2 5 . 3  2 6 . 1  26.7 30 .5 
% Hae m 5 . 9  3 1 .3 27.6 2 1 .8 
94. 1  
Pig lets were introduced to their respective d iets o n  day 0 of a 28-day experimen ta l  period . 
The meat wa s prepa red from 1 2 0  kg frozen topside of beef. This was thawed overnight at room 
temperature/ p rior to being m inced through an 8 mm plate. The l i q u id exudate from the thawing 
meat was collected a nd added to the minced beef. 
After m incing1 the beef was d ivided into 3 x 40 kg batches and packaged into 1 kg polythene 
enclosed cyl i nd rica l packs ( d i a meter of 70 m m ) .  The first 40 x 1 kg of beef was frozen without 
cooking to -17 .so C .  The rem a ining 80 x 1 kg of beef were cooked in stea m-heated water baths/ 
40 kg at 62° C and 40 kg at 92° C, respective ly. The M60 rolls were in the water bath for 
approximately 2 . 2 5  hours and the interna l temperature of a sample rol l  was at > 57° C for m o re 
than 60 m i n ute s ( maximum temperature = 59.2° C. The M90 rolls were in the water bath for 
approximately 2 hours and the interna l temperature of a sample roll was > 84° C for a bout 60 
m i nutes ( ma x i m u m  temperature =88° C ) .  The packages were then rapidly cooled and froze n .  
T h e  individ ua l 1 kg packages o f  meat were then thawed 2 4  h rs at 4° C prior to them being 
incorporated i n to the meat d iets. 
3.4.2 Feed management 
Experiment 1 
Pig lets were fed twice da ily (at approximately 8 .00 am and 5 .00 p m ) .  The p rovision of two 
sma l ler meals rather than a single feed would ensure any food that was consumed wou ld be fully 
d igested and released into the sma l l  intestines in sma l l  quantities. These cond itions maxi m ise the 
absorption of n utrients. The a mount of feed offe red per pig let began at 200g I feed/day a nd 
incre a sed incre menta lly by 200 g every 4 days. This methodo logy was determined d uring a pre­
tria l review to be more appropriate th a n  calculating feed i n ta ke base upon l iveweight. The 
39 
j u stification for this wa s the a i m  of the study was to determine bioava ilability of dietary iron by 
presenting each piglet with a fixed amount of iron each rather than to ach ieve maximum rates of 
g rowth day. 
The l iq u id d iet was freshly prepared for each feed by: 
• Weighing the m ixing water 
We ighing the d ry  ing redients 
• Combining the dry ingred ients with m ixing water in a food processor and blend ing the 
water and dry ing red ients together fo r 15 second s  to produce a l iqu id diet. 
• In the ca se of the meat d iet, the m inced meat ( 100% visua l-lean bull beef) was added to 
the mixing water p rior to the add ition of d ry ing redie nts. 
A raspberry l iquid concentrate flavour ( manufactu red by Ha n sell New Zealand) wa s added at a 
rate of 1g / l OOg dry d iet to increase the palatabil ity of the d iet beca use of poor inta ke d u ring the 
acclimatisation period . 
Pig s had unl imited access to the a l located feed from one feed to the next. P rior to each feed ing 
the feeder was removed from the metabolism crate, the weight of feed refu sa ls was recorded, 
and any uneaten food was re moved and d isca rded .  The feeders were then washed and rep laced . 
Experiment 2 
As in experiment 1 ,  the p ig lets in experiment 2 rece ived a fixed a mount of food each day that 
increased incrementa lly every fourth day. The d iets were prepa red using the method deta i led i n  
experiment 1 b y  being combined with a n  equal volume of water to provide a l iquid d iet. The d iets 
used in experiment 2 d id not con ta in raspberry flavouring beca use pig lets had an extended 
pe riod of acclimatisation d u ring wh ich feed inta ke was good . 
3.5 Blood Samples 
Experiments 1 and 2 
A blood sa mple was taken on days 0, 6, 14, 2 1  and 27 from each of the p ig lets in experiments 1 
and days 0, 7, 14, 2 1 ,  a nd 28 in experiment 2 under ha lothane anaesthesia . 
Approximate ly 8 ml of b lood was d rawn from the jugu la r  ve in of each anima l  using either a 2 0 G :  
1 "  o r  1 .5" needle a nd collected i n  5 m l  a n d  3 ml vacuta i ner tubes (Manufactured b y  Becton 
Dickinson Vacuta iner systems, Europe ) .  The 5 ml tubes, which conta ined no added anticoag ulant 
were stored overn ight at 4° C .  The clots of blood were then unstuck from the sides of the 
vacuta iner and the vacuta iner centrifuged a t  4° C, at 3000 rpm (g value 3040) for 2 0  m i nutes 
40 
until the se ru m  had sepa rated from the other b lood p roducts. The serum was pou red into pre­
label led d up l icate vials and frozen at -20° C. 
The 3 ml vacutainer contained the anticoa g u la n t  EDTA ( 0 . 068 m l  of 7.5% (KJ ) E DTA solution 
( 5 . 1 m g ) )  that p revented c lotting of the b lood sample . These sa mp les were not stored b ut sent 
to the Institute of Veterinary, Animal and Biomedical Science (Massey Un iversity) immediately for 
analysis u s i ng a n  "Advia 120" e lectronic cel l  counting apparatus ( manufactured by the Bayer 
Corp . Tarrytown, N .  York ) .  
The "Adv ia  1 20 "  ana lysed the b lood sa mples measuring the parameters shown in Table 1 4 .  The 
accepted p h ysiological values of the red blood cell parameters in a normal hea lthy p ig a re shown 
in Tab le 5. 
Table 14: Red b lood cell para meters, u n its a nd a bbreviations. 
Blood pa ra meter Abbrev. U nit Mea su re ment 
Red blood ce lls RBC 1 012 cells/ L Direct 
Blood haemoglob in concentration H G B  g / L  Direct 
Haematocrit HCT L / L  Direct 
Mean Cell Vo l u me MCV fl 
Mean cell haemoglob in M C H  p g  Ca lculated as: HGB/RBC 
Mean Corpuscular, haem concentration MCHC g / L  calculated a s :  
HGB/RBC x MCV 
Corpuscula r haemoglobin consta n t  CHCM g / L  calculated as: 
The sa me apparatus classified red cel l  characte ristics by red cel l  vol u me (V) and haemog lobin 
concentration (HC) u sing a 3 x 3 matrix. 
Red ce lls are a rranged in categories that have cell d imensions between V<30 fl, H C < 280 g/ L 
a nd V > 8 0  fl, H C  > 41 0  g 1 L1 using the la bel ling M l  through to M9. The layout of the matrix is 
shown in Table 1 5 .  The a lign ment of the matrix g ri d l ines is dependent on species, sex a nd age. 
The a lig n ment of the matrix for the pig lets used i n  this study is shown adjacent to Tab le  15. Th is 
places red blood cells o f  optimum size in the MS category ( Egeli et al. 1998) .  
4 1  
Table 15:  Red cell classification using a 3 x 3 matrix 
RBC VI HC Grid l ines 
Red blood cell matrix 
Haemog lobin concentration 
HC < 2 80 HC= 280-410 HC > 4 1 0  
V>SO 
cel l  volume V=30-80 
V < 3 0 
M 7  MS M9 
M4 MS M6 
M 1  M2 M 3  
The electron ic ce ll counter was a lso a ble t o  provide a differentia l wh ite cell count, isolating the 
number of type of white cells in each of the blood sa mples as shown in Ta ble 1 6 .  The accepted 
physiological values of the white blood ce ll parameters in a norma l hea lthy pig a re shown in 
Table 6 .  
Table 16: Wh ite blood cell pa rameters, un its and a bbreviations. 
Blood a ra meter 
White blood cell 
Neutrophil cell 
Lymphocyte cell 
Monocyte cell 
Eosinophi l cell 
Basoph il cells 
Abbrevia tion 
WBC 
NEUT 
LYM PH 
MONO 
EOS 
BASO 
Serum iron and TIBC and UIBC 
U n it 
1 09 cells I L 
1 09 ce lls I L 
1 09 ce lls I L 
1 09 cells I L 
1 09 cells I L 
1 09 cells I L 
Tota l serum iron and unsaturated iron binding capacity were measu red using Roche Fe d iagnostic 
kits in a "Cobas Fa ra N" ana lyser by the Institute of Food N utrition and Human Health ( Massey 
U n iversity) .  Serum iron was determined at acid pH in the presence of a red ucing agent ferrozine. 
U nder these conditions serum proteins are precip itated and iron is released from transfe rrin . 
Spectrophotometric ana lysis ( 560 n m )  was then used to determine the seru m tota l iron 
concentratio n .  
Se ru m  unsaturated iron bind ing capacity (UIBC) .  At a l ka l i ne pH a sta ndard a mount o f  fe rrous iron 
is added to serum and is sequestered by tra nsferrin, fil l ing a l l  ava i la ble bind ing sites on the 
protein . The re maining unbound ferrous ions are then measured calorimetrica lly by use of 
fe rrozine . The d ifference between the amount of unbound iron and the tota l a mount of iron 
42 
added is the UIBC. Serum total iron b in d ing capacity (TIBC) is the sum of serum tota l iron and 
U I BC .  
3.6 Data analysis 
An a na lysis of varia nce (ANOVA) was u sed to compare the mean i n itia l weight of the five g roups 
of pig lets. 
Statistica l analysis was carried out using a general linear mode l ,  within the P roc GLM procedure 
of the SAS System, 8 . 1 .  (SAS institute Inc. Cary, NC, USA) 
In Experiment 1 physical production, red blood cell pa ra meters and an evaluation of the wh ite 
cell counts were carried out using the statistical model: 
y = �t  + g roup ; + pig 1 (g roup) + week k + week x g roup + err ;1k, 
W h e re :  
y = va riab le .  
�t = overa l l  mean . 
Group= fixed effect of ;th g roup (see Table 8) . 
Pig = random effect t pig in the ;th g rou p .  
Week= fixed effect o f  the kth wee k .  
Week x g roup = interaction between week a nd g rou p .  
E r r  = refers to the residual error. 
In Experiment 2 the statistica l model was expanded to account for sex d ifferences in the p ig lets: 
y = � + g rou p ; + sex i + sex x g roup + p ig k (sex x g roup) + week 1 + week x g roup + err iiki. 
Where: 
� = refers to the ove ra ll mea n .  
Group = fixed effect o f  ;th g roup see Table 8 .  
Sex = fixed effect gender o f  the piglet. 
Sex x g roup = interaction between se x  and g roup 
P ig = random effect kth p ig in the th g roup . 
Wee k= fixed effect of the 1th week. 
Week x g roup = interaction between week and group . 
E rr =  refers to the res idual error with i n  the model 
�--����-��··- ·��-- ··��-
43 
Compa risons between the treatments in both experiments were made u sing least sig nificant 
d ifferences ( LS D ) .  
T h e  retention o f  body haemoglobin iron was estimated by : 
Initia lly using the following equation to calculate body haemog lobin iron content a s :  
Body haemoglobin iron = Livewe ight x 0.07 x haemoglobin concentration x 0 . 00 3 3 5  
(g) (g) (g/L) 
Where :  
0.07 = weight of blood i n  a p ig let a s  a proportion of body weight (7% ) .  
0 . 00 3 3 5  = Fe we ig ht a s  a proportion o f  haemoglobin weig ht. 
(value from Thoren-To l l ing, 1975) 
Then u sing reg ression ana lysis between cumula tive iron intake ( FEI) and whole body 
haemoglobin iron (HGB Fe) so that the equation for iron retention in each p ig let becomes: 
HGB Fe; = a + b x FEI; + e; 
Whe re 
HGB Fe = Body haemog lobin iron retention 
a= intercept (the expected va lue of the dependant variable when the x-va riable is zero). 
b= slope (the expected change in the dependant va riable g iven a unit change in the x-va riable . 
FEI= c u m u lative iron inta ke (feed intake x tota l iron content of the d iet). 
e = resid ua l error 
Then the ind ivid ua l intercept (a ) and the slope (b) were ana lysed with the following linear model :  
Y;i = 11 + g roup; + e;i 
Where :  
Y =  va ria ble 
�� = overa l l  mean 
Grou p = fixed effect of the ;th g roup . 
e = residual error 
Comparisons between the g roups were made u sing least sig n ificant d ifferences ( LSD). 
44 
I n itia l haematology results sh owed unequal CHCM a nd MCHC values ind icating that the setting s 
used by the "Advia 1 20 '  in cell counting, laser l ight scattering, and flow cytometry were 
inappropriate for the severity of iron deficiency. A con sultant from the mach ine's manufacturer 
( Bayer) accounted for these d iffe rences by ta king the data gene ra ted during the haematology 
ana lysis and adjusting it to account for low body Fe . The va lues reported for red b lood cells, 
haematocrit and M C HC in both experiments 1 a nd 2 were calculated using these adjusted 
settings. 
45 
Chapter 4 
4. Results (experiment 1) .  
Table 17 the shows the sig n ificance o f  the effects of g roup, pigs with in group, week a n d  week x 
g rou p on feed intake a nd g rowth rate . The statistica l model expla ined 94% ( r-squared = 0 .94) 
and 90% ( r-squa red = 0 .90) of the variation between the g roups for intake and g rowth ra te 
respectively over the 4-week study period . 
Table 17: Significance levels of the effects of g roup, pigs within g roup, week and week x g roup 
on feed intake and g rowth rate .  
Intake 
Growth rate 
Grou 
* * *  
* * *  
P ig i9.!:2_u� Wee k Week x g roup R2 
* * *  * * *  * * *  0 . 94 
* * *  * * *  * * *  0 . 90 
n s :  not sig nifican t  p > 0 .05; * p < 0 .05; ** p< 0 . 0 1 ;  * * *  p < 0 . 001 
4.1 Intake 
Statistica lly sig n ificant d iffe re nces ( P <  0 . 0 0 1 )  were observed between the g roups and there were 
a lso sign ifica nt d ifferences between weeks, and p ig s  within groups. There was a lso a sig n ificant 
interaction between week x g roup. The variable week x g roup was tested to identify cha nges that 
took place over the 4-week study period . The results presented in Table 18 and also in F ig u re 1 
show the least-squares means of the values for feed intake (g I pig let I day) by week, obtained 
from each of the trea tment g roups. 
Table 18: Lea st-squares means of feed intake (gl piglet I day) on an as fed basis. 
Group Week 1 Wee k 4 Mean value week 1 Residual Standard 
to week 4 Deviation {RSD} 
CV 250 206 7 70b 440b 13 1 . 1  
CV 500 1 58 7 16ab 407ab 1 3 1 . 1  
c 187 596a 367a 1 3 1 . 1  
C+ 324 1 3 18c 832c 13 1 . 1  
Meat 198 1 274c 7 1 8c 13 1 . 1  
a ,  b, and c means within a column, with a common superscript (or with no superscrir,t) are not significantly different from each ether (LSD; p<O.OS) 
46 
1 600 
> 1 400 "' "0 -
iii 1 200 E 
c 1 000 "' -
en 800 ...... 
G.l � 600 "' .... 
c ... 400 "0 G.l 200 G.l u.. 
0 
0 
� 
1 2 3 
Week of experiment 
4 5 
-+- CV250 
--cvsoo 
c 
C +  
� Meat 
Figure 1: Least-squa res means ( ±  S. E) of feed intake (g/ pig let/ day) by week for each of the 
five treatment g roups. 
In itia lly feed inta ke was not sign ificantly d ifferent a mong the g roups of p ig lets. As the study 
prog ressed, changes in intake between the g roups beca me more apparent, with the C+ g roup 
consuming significantly more food than the other g roups. 
Throughout the study the lowest intakes were recorded in groups consu ming the C, CV250 and 
CVSOO d iets. By week four of the study period feed inta kes in the meat and C+ g roups were 
sig nificantly h ig her than the C, CV250 or CVSOO. The feed intake of piglets in the C+ g roup was 
twice that of the C, CV250 and CVSOO g rou ps. 
4.2 Growth Rate 
Statistica lly sig n ificant d iffe rences (P<0 .00 1 )  were observed between the groups and there were 
a lso sig n ifica nt d ifferences between weeks, and p ig s  with in groups. There was a lso a sig n ificant 
interaction between week x g roup. The results presented in Table 19 and also in F ig u re 2 show 
the least- squa res mea n s  of the values for g rowth rate (g /pig let/ day) obta i ned from each of the 
treatment g rou ps. 
47 
Table 19: Least-squares mea n s  of g rowth ra te (g/pig let/ day) 
G roup Week 1 Wee k 4 Mean value week Residual Standard 
1 to wee k 4 Deviation RSD) 
CV 250 �l ab  172 . 5 3 60.4 a 60 .4 
CV 500 -67.4 3 193 . 5  a 52.8 a 60.4 
c -35 . 1  a b  174 . 2 3 6 1 . 7 a 60.4 
C+ 27.8 b 456 . 2  b 2 1 8 . 0  b 60.4 
Meat -28 .5 a b  426 . 6  b 190 . 2  b 60.4 
a ,  b, and c means within a column, with a common superscript (or  with no superscrif.t) are not significantly different from each ether (LSD; p<O.OS) 
480 
4 0 0  
- 3 2 0  >-� "' 
"' 'tJ ... - 2 4 0  .... 
.c � .... 3: Cl 1 6 0  0 ·a ... -� Cl ....... 80 
0 
-80 
1 2 3 
week  of experiment 
4 
--+-- C v 2 5 0  
--- C v S O O  
c 
C +  
--*- M ea t  
Figure 2: Least-squares mean s ( ±  S .  E) o f  g rowth rate (g/day) by week for each o f  the five 
treatment g roups. 
Leve ls of feed intake determ ined the ra tes of growth amongst p ig lets in all of the groups. The 
i n itia l poor feed intake resu lted in nega tive rates of g rowth in fou r  of the five treatment g roups. 
O n ly piglets in the C+ . group reported sma l l positive increases, wh ich were sign ificantly d iffe rent 
from other g roups. Th roughout the study the lowest g rowth rates were reported in pig lets in the 
C, CV250 and CV500 g roups. By wee k fou r  of the study there we re no sign ificant d iffe rences 
between the meat and C+ g roups. Both g roups had rates of g rowth g reater than the C, CV250 
and CV500 g roups. 
48  
4.3 Haematology 
Table 20 the shows the sig n ificance of the effects of g roup, pig within g roup, wee k and week x 
g rou p on a complete red blood cell count (CBC). The r-squa red values ind icate how much of the 
va riation between the treatments can be explained, by the statistical model shown in section 3 .6 
(data a na lysis) : Whe re the CBC cha ra cteristics and their abbreviations a re l isted in Table 14. 
Table 20: S ig n ificance levels of the effects of group, p igs with i n  g roup, week and week x g roup 
on b lood pa rameters. 
Group P ig (group) Week Week x g roup 
RBC ***  ***  ***  ***  0 . 89 
H G B  ***  ***  ***  *** 0 .96 
HCT ***  ***  ***  ***  0 . 95 
MCV ***  ***  ***  ***  0 . 98 
M C H  ***  ***  * **  ***  0 . 99 
MCHC ***  ***  ***  * 0 . 93 
***  ***  ***  
n s :  n o t  sig n ificant p > O . OS; * p < O .OS; ** p <  0 . 0 1 ;  ***  p < O . O O l  
4.3.1 Red Blood Cells 
Statistica lly sig n ificant d iffe re nces ( P <  0 .00 1 )  were observed between the groups and there were 
a lso sign ifica nt d ifferences between weeks, and p ig s  within g roups. There was a lso a sign ificant 
interaction between week x g roup for red b lood cells. The res ults presented in Table 2 1  and a lso 
in F ig u re 3 show the least-sq ua res mean s  of the values for red b lood cells (x 1 012 I L) obtained 
from each of the treatment g roups. 
Table 21: Least-sq uares means of red blood cells count (x 1 012 I L) 
Group Wee k O Week 4 Change from week Mean value 
0 to week 4 week 0 to week 
4 
Cv250 3 .5 3 a 4. 2 2  
Cv500 4 . 0 1  a 4.92 b 
c 3 .45 a 4.29 a 
C +  5 . 99 b 6. 1 1  c 
+ b 
Residua l Sta nda rd 
Deviation (RSD) 
0 .47 
0 .47 
0 .47 
0 .47 
a , b, and c means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; p< 0.05) 
49 
7 
6 . 5  
....... 
...I 6 
-..!!! 
-ai 5 . 5 V 
N 5 ... 0 � 
>< 4 . 5  
......... 
u 4 r:a 
0:: 
3 . 5 
3 
± 
0 1 2 3 
Week of exper iment 
4 
-cv2SO  
-cv soo  
c 
C+  
- Meat 
Figure 3:  Least-sq uares means ( ± S . E) of red blood cells ( RBC) x 10 12 /L by wee k for each of 
the five treatment groups. 
By desig n p ig lets in the C +  g roup began the study with red blood cell counts that were 
sig n ificantly d iffe rent from the other grou ps. This was because the pig lets in this g roup received 
a n  injection of supplementary iron shortly after birth, whereas iron was only ava ila ble to the C, 
CV250, CVSOO and meat g roups throug h their respective d iets. 
Red blood cell prod uction increased in l ine with d ietary iron intake throughout the d u ration of the 
study so that p ig lets with the h ighest inta ke s  had the greatest increases. Increa se s  in red blood 
cel l  counts were g reatest in iron deficient p ig lets, with p ig lets in the meat and CVSOO g roups 
having red b lood ce l l  counts that were sig nificantly d ifferent from th ose piglets consu ming e ither 
the C or CV250 d iets. Little change was observed in the C -J:: group a s  these pig lets had red b lood 
ce ll counts that were a l ready within the norma l physiolog ica l range as shown in Tab le 5. 
50 
4.3.2 Blood Haemoglobin Concentrations (HGB) 
Statistica lly sig n ificant d iffe rences ( P <  0.00 1) were observed between the g roups and there were 
a lso sign ifica nt d ifferences between weeks, and p ig s  within g roups. There was a lso a sig n ificant 
interaction between week x g roup. The results p resented in Tab le  22 and a lso in F ig u re 4 show 
the lea st-squares means of the values for haemoglobin (g 1 L) obta ined from each of the 
treatment g roups. 
Table 22: Least square means of blood haemog lobin concentration (g I L) . 
Group Week 0 Week 4 Change from Mean va lue Residual Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 42.0 a 46 .0 a + 4 b 44 .0 a b 6 . 12 
Cv500 46.6 a 57.0 b + 10.4 b 50 .2 b 6 . 12 
c 38.2 a 42 .61a + 4.41 b 41.2 a 6 . 12 
C+ 1 13.4 b 101 .4 c - 12 
a 107.7 d 6 . 12 
Meat 42 .2 a 64 .4 b + 22.2 c 53 .4 b c 6 . 12 
a , b, and c means within a column, with a common superscript (or with no superscri�) are not significantly different from each other (LSD; p<O.OS) 
1 20 T T .J.. -
c ..... 1 1 0 
:c - T T T 1 00 0 C\ i--i -+- Cv2 50 c, - 90 0 c --a- cvsoo 
E .2 80 cu ..., c 
nJ nJ 70 .c l:; C+ "'C c 60 0 cu � M eat 0 V - c 50 tO 8 40 -' 
30 
0 1 2 3 4 
Week of experiment 
Figure 3: Least-sq ua res mea n s ( ±  S. E) of blood hae moglobin concentration (HGB) g I L by 
week for each of the five treatment g roups. 
5 1  
By design, at the beg inning of the study the pig lets in the C +  group had blood haemog lobin 
concentrations that were sign ificantly d ifferent from a l l the other g roups. The C+ g roup reported 
a sma l l  decrease in b lood haemoglobin concentrations over the duration of the study, ind icative 
of decreasing iron reserves d u ring a period of increased req u i rements as the a n imal g rows. 
Ag a i n  in the groups conta i n ing iron deficient p ig lets, blood haemoglobin concentrations increased 
i n l ine with feed inta ke . The meat g roup, with the h ighest feed intake amongst the iron deficient 
g roups had the g reatest increases in blood haemog lobin concentration. Overa l l  there we re no 
sig n ificant d ifferences between haemoglobin concentrations in the C, CV250 a nd CVSOO, desp ite 
weekly d ifferences. Even though pig lets in the C, Cv250, CvSOO and meat groups saw increa se s  
in blood haemoglobin concentrations, at the e n d  o f  the study haemoglobin concentrations in 
these an ima ls we re still 50% lower than norma l physiolog ical va lues for blood hae mog lobin 
concentration as shown in Table 5. 
4.2.3 Hematocrit (HCT) 
Statistica lly sig n ificant d iffe rences (P< 0.00 1 )  were observed between the groups and there were 
a l so sign ifica nt d iffe rences between weeks, and pigs with in g roups. There was a lso a sig n ificant 
interaction between week x g roup. The results presented in Table 23 and also in F ig u re 5 show 
the least-squares means of the values for hematocrit (L I L) obtained from each of the treatment 
groups. 
Table 23 : Least-sq uares means of hematocrit ( L  I L) 
Group Wee k O Week 4 Change from Mea n va lue Residual Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 0 . 1 5 a 0 . 1 7 a + 0 . 02 b 0 . 16 a b 0 . 02 
Cv500 0 . 1 6 a 0 . 2 1  b + 0 . 0 5  c 0 . 18 b 0 . 02 
c 0 . 1 3 a 0 . 1 5 a + 0 . 02 b 0 . 1 5 a 0 .02 
C+ 0 .36 b 0 . 34 ( - 0 . 02 a 0 . 3 5  b d 0 . 02 
M eat 0 . 1 5 a 0 . 24 b + 0.09 d 0 . 1 9 b e  0 . 02 
a ,  b, c and d means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; 
p<O.OS) 
52 
0 . 4  
T T ;L. 
0 . 3 5  T .J.. 
- T ...I ..L 
......... 0 . 3  
� 0 . 2 5 -+- Cv250 
� - cv5 00 'i: 0 . 2  c u 0 
� C+ la 0 . 1 5  
E . . ---....- Meat G.J -
la 0 . 1 
J: 
0 . 0 5 
0 
0 1 2 3 4 
Week of experiment 
Figure 5: Least-squa res means ( ± S .  E) of hematocrit ( HCT) L I L by week for each of the five 
treatment g roups. 
Haematocrit is an alternative measurement of red blood cell nu mber. It measu res the percentage 
of red ce lls in whole blood . Therefore reflecting the changes obse rved in red b lood cell counts as 
we ll as their size. 
4.3.4 Mean Cell Volume {MCV) 
Statistica lly sig n ifican t  effects ( P <  0 . 00 1 )  were obse rved between the g roups and a lso there were 
sig n ificant d iffe re nces between weeks, and pigs with i n  g roups. There was also a significan t  
interaction between week x g roup. The results presented i n  Table 24 a nd a lso in Fig u re 6 show 
the least-squares mea n s  of the values for mean cel l  volume (f L) obtained from each of the 
treatment g roups. 
53 
Table 24: Least-squares mea n s  of mean cel l  volume (f L) 
Group 
Cv250 
Cv500 
c 
C+ 
Meat 
Week 0 
4 2 . 2  c 
39.9 b 
38.8 a b  
60 .3 d 
37.9 a 
Week 4 
3 5 .2 b 
34.6 b 
32 .0 a 
56.2 d 
39.9 c 
Change from Mean value week 
week 0 to week 0 to wee k 4 
4 
- 7 .0 a 37.5 b 
- 5.3 a 35 .7
a b 
- 6.8 a 34.4 a 
- 4 . 1  b 56.8 c 
+ 2 .0 c 37.5 b 
Residual Sta nda rd 
Deviation ( RSD) 
1 .46 
1 .46 
1 .46 
1 .46 
1 .46 
a , b, c and d means within a column, with a common superscript (or with no superscri�) are ncx significantly different from each other (LSD; 
p<O.OS) 
6 5  
� 
Q) 
E 5 5  .. � � .. -+- Cv2 5 0  � 
0 
> - --- cv5 0 0  
= ...I 4 5  c Q) 't-u - C +  
c 
ItS 3 5  --..- M eat Q) 
::t 
2 5  
0 1 2 3 4 
Week of experiment 
Figure 6: Least-sq uares mea n s  ( ± S. E) of mean cel l  volume (MCV) f L by week for each of the 
five treatment g roups. 
The mea n cell volume of red ce lls in the C+ group were sign ificantly d ifferent from a l l  other 
g roups throughout the study, a nd a lso maintained within a na rrow range, within norma l 
physiological ra nges. Other g roups in the study had variable MCV values, which in itia l ly declined 
prior to reach ing a platea u .  Subsequent increases in the MCV va lues we re then obse rved in the 
CV500 and the meat g roups. The low MCV values ind icate the red blood ce lls in circulation a re 
s ma l l .  This is because insufficient iron wa s available for their manufactu re . 
54 
4.3.5 Mean Cel l  Haemoglobin 
Statistica lly sig n ificant effects ( P <  0 .001) were obse rved between th e  g roups a nd a lso there were 
sig n ificant d iffe rences between the weeks, and pig within g roups. There was a lso a sig nificant 
interaction between week x g roup. The results presented in Table 25 and a l so  in F ig u re 7 show 
the lea st-squares means of the values for mean cell haemoglobin (p g) obta ined from each of the 
treatment g roups. 
Table 25 : Least-squares means of mean cell haemog lobin . ( p  g )  
Group 
Cv250 
Cv500 
c 
C+ 
M eat 
Wee k O 
1 1 .88 b 
1 1 .56 a b 
1 1 . 12 a 
18.98 c 
10.8 a 
Week 4 
9.60 b 
9.56 b 
8.87 a 
16.8 d 
10.8 c 
Change from Mean value from 
wee k 0 to week week 0 to week 
4 4 
- 2.28 a 10 .45 b 
- 2 .0 a 10.22 b 
- 2.25 
a 9.47 a 
- 2 . 18 a 17 .80 c 
o b 10.41 b 
Residual Standa rd 
Deviation ( RSD) 
0.39 
0 .39 
0.39 
0.39 
0.39 
a ,  b, and c means within a column, w�h a common superscript (or with no superscriJX:) are not significantly different from each cxher (LSD; p<O.OS) 
20 
c .,... 
.c ..... ..... 
.2 
en 
1 6  0 
e ...... 
cu en "' 
�. �. -+-Cv250 
-a-Cv500 
c 
.C Q.  _ ....... 
a; 12  V 
c 
"' 
- C+ 
.............__ ___....__ M ea t  ��.----- -
cu -- -- --
� 8 
- - -
0 1 2 3 4 
week of experiment 
Figure 7 :  Least-squares mea n s ( ±  S. E) of mean cel l  haemoglobin ( M C H )  p g by week for each 
of the five trea tment g roups. 
55  
As seen in measurements of mean cell vo lu me, mean ce l l  hae mog lob in concentrations in the C+ 
group were sig nificantly d iffe rent from the others. As the supplementary iron injected shortly 
after b irth would have provided adequate reserves of iron so that each red blood ce l l  
manufactu red contained a n  optima l concentration of haemoglobin . Iron deficient pig lets had n o  
iron reserves that could b e  used in the man ufactu re o f  red blood ce l l  s o  that t h e  red cells of 
these an i ma l s  we re poorly haemog lobin ised . As feed intake increa sed during the last weeks of 
the study the decline in MCH concentrations ceased . A small increase in the MCH concentration 
was observed in pig lets consuming the meat d iet, ind icating that meat iron was be ing more 
read ily a ssim ilated . 
4.3.6 Mean  corpuscle, haem concentration (MCHC) 
Statistica lly sig nificant d iffe rences (P< 0 . 00 1 )  were obse rved between the g roups and there were 
also sign ifica nt d ifferences between the variables wee k, and p igs with in groups. There was a lso a 
sign ificant inte raction between week x g roup for MCHC. The results presented in Table 26 show 
the least-squares means of the values for MCHC (g I L) obtained from each of the treatment 
groups. 
Table 26: Least-squares means of mean corpuscle, haem concentration (MCHC g 1 L) 
Group Week 0 Week 4 Change from 
week 0 to week 4 
Cv250 282 a 273 a - 9 
Cv500 290 a 276 a - 14 
c 286 a 277 a - 9 
C+ 3 14 b 299 b - 1 5  
Meat 285 a 2 7 1 a - 14 
Mean va lue from 
week 0 to week 
4 
279 a 
286 b 
283 a b 
3 13 ( 
278 a 
Residua l Sta ndard 
Deviation ( RSD) 
5 . 57 
5 . 57 
5 . 57 
5 . 57 
5 . 57 
a ,  b, and c means within a cdumn, with a common superscript (or with no superscri>() are not significantly different from each ether (LSD; p<O.OS) 
MCHC va lues in pig lets in the C+ g roup were sign ifica ntly different from a l l  other g roups 
th roughout the study. There were no sign ifica nt d ifferences between the other g roups rega rd less 
of d iet and iron statu s. 
56 
4.3.7 Corpuscular haemoglobin constant (CHCM) 
Statistica lly sig n ificant d iffe rences (P< 0 . 0 0 1 )  were observed between the g roups and there were 
a lso sign ifica nt d ifferences between a lso between weeks, and pigs within g roups. There was a lso 
a sig n ificant interaction between week x grou p .  The resu lts presented in Table 2 7  show the least 
square mean of the values for corpuscu la r haemoglobin constan t  obta ined from each of th e  
treatment groups. 
Table 27: Least squ a re means of corpuscu la r haemog lobin consta nt (g 1 L) 
Group Wee k O Week 4 Change from Mean va lue Residua l Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 276 . 6 a 2 7 2 . 6  a - 4 .0 275 .3 a 5 . 2 5  
Cv500 284 . 4 a 278.4 a - 6.0 282 .8 a 5 . 2 5  
c 281 . 0  a 2 77 . 5  a - 3 .5 279 . 5  a 5 . 2 5  
C +  3 1 3 . 4  b 299.2 b - 1 4 . 2  3 1 2 . 1  b 5 . 2 5  
Meat a 273 . 2  a - 6 .2 a 
a ,  b, and c means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; p<O.OS) 
C H CM values in pig lets in the C+ g roup were sign ifica ntly d ifferent throughout the study. There 
was no sig nifican t  d iffe rence between the other g roups regardless of d iet and iron status. 
4.4 Matrix analysis 
Ta ble 28 shows the s ig n ificance of the effects of g rou p, p igs with in groups, weeks a nd week x 
g roup on the outcome of red blood cell cha racteristics, when they were ana lysed and placed in 3 
x 3 matrix ( M 1  to M 9 ,  see Ta ble 1 5 )  describing the haemoglobin concentration ( HC) and cel l  
volume (V) . The r-squa red va lues ind icate how much o f  the va riation between the g roups can be 
explained by the statistical model shown in section 3 . 6  (data ana lysis) : 
57  
Table 28: Significance levels of the effects of group, pigs with i n  group, week and week x g roup 
on red blood cel l  cha racte ristics. 
Grou Pig (g ro.!:P Week Week x g rou p R2 
M 1  * *  * * *  * * *  * * *  0 . 73 
M 2  * * *  * * *  * * *  * * *  o .ss 
M3 * * *  * * *  * * *  * * *  0 . 92 
M4 * * *  * * *  * * *  * * *  0 .93 
M S  * * *  * * *  ns O .S6 
M6 * * *  * * *  * * *  * * *  O .SS 
MS * * * *  * * *  ns O .S6 
M 9  * * *  * * *  * * *  * * *  0 . 9 1  
n s :  not sig n ificant p > O . OS; * p < O .OS; * *  p <  0 . 0 1 ;  * * *  p < 0 .00 1 
The re su lts presented in Ta ble 29 show the least-sq ua res mea n s  of the va lues for red blood cells 
cha racterised as M 1 -M9 obta ined from each of the treatment g roups 
Table 29: The least-sq uares means of red blood cell cha racteristic M 1 -M9. 
C E LL WEEK 
M 1  0 
4 
M 2  0 
4 
M 3  0 
4 
M 4  0 
4 
M S  0 
4 
M 6  0 
4 
M 7  0 
4 
M S  0 
4 
M 9  0 
4 
CV2SO 
2 1 .4ab 
s.oa 
1 3039 b 
1 7474 b 
4740 b 
9346 c 
49.6 b 
4 .6 b 
1 1 7S6 a 
7720 a 
1S9S a b  
S436 b 
2 1 6.0 b 
2 7 1 .0 b 
4S.2 a 
206.4 c 
G ROUP 
cvsoo c C+ M EAT 
1 2 . 0a 30.Sab 64.4b 9 . 0a 
S.6a 6 . 1a 2 0 2 . Sc 1 0 3 .6b 
10S76 b 1 02 3 1  b 43 2 7a 1 1 2 S 1  b 
202S1 c 13S99 b 6929 a 27S01 d 
6261 b 6S7 1  b 9 a 7747 ( 
SS90 c 1 1 1 0S c s a 42 13 b 
2 9 . 6 b 3 l .S b 1 S47.0 a 26.4 b 
2 . 6  b l .S b 377.0 a 1 0 .4 b 
143S6 a 1 0604 a 43736 b 1 1 619 a 
1 0 1 99 a 6 1 S6 a 4 1 3 1 6 b 14704 a 
3 4 1 S  b 3409 b 2 1 S a 372 1 b 
9 179 c SS92 c 1 92 a S076 b 
No cells were identified in th is category 
3 14 .S b 
3 2 S . 2  b e 
7 2 . 2  b 
2 1 9 .4 c 
290.4 b 
2 7 1 . 2  b 
S7 .2 b 
290 . 5  d 
79 .0 a 2S l .S b 
3S.6 a 204.6 b 
3 . 4 a 90.S b 
2 .4 a 92.S b 
RSD 
3S .9 
3 S .9 
3370 
3370 
1S 1S 
1 S 1S 
147.3 
147.3 
7340 
7340 
146S 
146S 
S 2 . 5  
S 2 .5 
36.7 
36.7 
a , b, and c means within a column, with a common superscript (or  with no superscri!X) are not significantly different from each other (LSD; p<O.OS) 
4.4.1 Ml.  
Statistica lly sig nificant effects ( P <  0 .0 0 1 )  were obse rved between the groups a nd th e re were a lso 
sig n ificant d iffe rences between weeks, and pigs with in g roups. There was a l so  a sign ifica n t  
58  
interaction between week x g roup . The results presented in Tab le 29 show the least-squares 
means of the va lues for red b lood cells characterised as M 1 obta ined from each of the treatmen t  
g roups. 
4.4.2 M2. 
Statistica lly sig nificant effects ( P <  0 .0 0 1 )  were obse rved between the g roups and there were a lso 
sig n ificant d ifferences between weeks, a nd pigs with i n  groups. There was a lso a sign ificant 
interaction between week x g roup for M 2 .  The results presented in Table 29 show the least­
squ a re s  mea n s  of the values for red blood cells cha ra cterised as M2 obtained from each of the 
treatment g roups. 
4.4.3 M3.  
Statistica lly sig n ificant effects ( P <  0 .0 0 1 )  were observed between th e  g roups and there were a lso 
sig n ificant d iffe rences between weeks, a nd pigs wit h i n  groups. There was also a sign ificant 
interaction between week x g roup. The results presented in Table 29 show the least-squares 
mean s  of the va lues for red b lood cells characterised as M3 obta ined from each of the treatment 
g roups. 
4.4.4 M4. 
Statistica lly sig n ificant effects (P< 0 .0 0 1 )  were observed between the groups and there were a lso 
sig n ificant d iffe rences between weeks, a n d  pigs with i n  groups. There was a l so  a sign ificant 
interaction between week x g roup. The results presen ted in Table 29 the least-sq uares means of 
the va lues for red blood cells characterised as M4 obtained from each of the treatment g roups. 
4.4.5 MS.  
Statistica lly sign ificant effects ( P <  0 .0 0 1 )  were observed between the g roups a n d  there were a lso 
sign ificant d i ffe rences between weeks and pigs with in g roups. Week x g roup was not a significant 
interaction . The results p rese nted in Table 29 show the least-sq uares means of the values for red 
blood cells cha racterised as MS obta ined from each of the treatment g roups. 
59  
4.4.6 M6. 
Statistica lly sig n ificant effects (P< 0.001) were observed between the g roups and there were also 
sig n ificant diffe rences between weeks, and pigs within g roups. There was also a sig n ificant 
interaction between week x g roup. The results presented in Table 29 show the lea st-squares 
means of the va lues for red blood cells characterised as M6 obta ined from each of the treatment 
g roups. 
4.4.7 M7 
There were no cells identified in th is category 
4.4.8 MS. 
Statistica lly sig n ificant effects (P< 0 .0 0 1 )  were observed between the g roups and there were a lso 
s ig n ificant d ifferences between week and p igs within groups. There wa s not a sign ifica nt 
interaction between week x g roup. The results presented in Table 29 show the least-squares 
means of the va lues for red blood cells characterised as MS obta ined from each of the treatment 
g roups. 
4.4.9 M9. 
Statistica lly sig n ificant effects (P< 0 .0 0 1 )  were observed between the g roups and a lso between 
the variables week, and pigs within g roups. There wa s a lso a sign ifica nt interaction between 
week x g roup. The results presented in Tab le  29 show the least-squares means of the va lues for 
red blood cells characterised as M9 obtained from each of the treatment g roups. 
Red ce lls were classified by volume a nd haemoglobin concentration u s ing a 3 x 3 matrix. 
Throughout the study, the classification of red blood cells reflected the iron status of the p ig lets. 
P ig lets in the C +  g roup maintained approximately 90% of red blood cells in the optimum mS 
positio n .  Whereas the iron deficient p ig lets had many red cel l  in the lower matrices categories, 
ind icating that insufficient iron had resu lted in the manufacture of red b lood cells that were smal l 
and had lower concentrations of haemog lobin . The red blood ce lls have a l ife span of 
a p proximately 1 2 0  days therefore d u ring th is study, which lasted just 28 days only sma l l  changes 
in red cell cla ssification were evident. By wee k 4 of the study pig lets consu ming the meat d iet 
60 
increased the nu mber of red blood ce lls classified as m S .  Where a s  the nu mber of red cel l s  in this 
category decreased for C, CV250, a nd CVSOO groups. 
Fig u res 8 a nd 9 show the red blood cell characteristics at the beg inning and aga in at the 
completion of the expe ri ment. 
a.. 100% � � D m9 E 
= 
c 80% D m8 
nJ D m6 � 60% 0 • ms � 
� 
0 D m4 
� 40% 0 D m3 nJ 
Ill 20% • m2 nJ 
u 
1:0 D ml 
0:: 0% 
CV250 CVSOO c C+ meat 
d iet 
Figure 8: Least-sq uares means ( ±  5. E) of red blood ce l l  characteristic (volume and 
haemoglobin concentration ) a s  a percentage of the tota l for each of the five treatment g roups at 
the beg inn ing of the tria l .  
6 1  
100% 
L. Qj 80% .c 
E 
::I 
c 60% ru mS  
.... 0 
.... 
� 40% 0 
� 0 
ru 20% 
Ill ru 
u 0% c:Q 
0:: 
CV250 CVSOO c C+ meat 
diet 
Figure 9: Least-squares means ( ± S. E) of red b lood cel l  characteristic (volume a nd 
haemog lob in concentration ) as a percentage of the tota l for each of the five treatment g roups at 
the end of the trial .  
4.5 Serum Iron Concentration. 
Table 30 shows the sign ificance of the effects of group, p igs with in g roups, week and wee k x 
g roup on serum iron and iron binding p rote ins. The r-squared values ind icate how much of the 
variation between the treatments can be expla ined by the statistical model shown in section 3 . 6  
(data ana lysis) : 
Table 30:  Significance level of the effects of g roup, pigs with in group, week a nd week x 
group on serum iron and iron binding proteins 
Iron 
UIBC 
HGB Fe 
G rou 
* * *  
* * *  
* * *  * * *  
Week 
n s  
* * *  
* * *  
Week x 
ns 
ns 
* * *  
0 . 70 
0 . 77 
0 . 9 1  
ns : not sig n ificant p > O . O S ;  * p < O .OS; * *  p <  0 . 0 1 ;  * * *  p < 0 .001 
62 
Statistically sig n ificant diffe rences ( P <  0 .00 1 )  were observed between the groups. There were no 
sig n ificant d ifferences between weeks, pig with in groups and week x grou p .  The results 
presented in Tab le  31 and a lso in F ig u re 10 show the least-squa res means of th e  va lues for tota l 
seru m iron obtained from each of the treatment g roups. 
Table 31:  The least-squares means of serum iron concentration ( � mol 1 L) 
Group Week 0 Week 4 Change from Mean va lue Residual Sta ndard 
week 0 to week 4 week 0 to week Devia tion ( RSD) 
4 
Cv250 1 . 2a 3 .0 a + l . S a 1 .9 a 6.63 
CvSOO 1 . 1  a 2 . 3  a + 1 .2 a 1 . 3 a 6.63 
c 1 . 1  a l . S a + 0 . 4 a 1 . 3 a 6.63 
C+ 1 0 . 2  b 2 2 .2 b + 12 b 1 9 . 0  b 6 .63 
Meat o .s a 6.8 a + 6 a 2 .7 a 6 .63 
a ,  b,  a n d  c means within a column, with a com mon superscript (or with n o  superscript) are not sign ificantly different from each other (LSD; p<O.OS) 
-
..J 30 
- T � 25 1 1 T :::J ........ -+-Cv250 
...., 20 c: -cv5oo cu 
...., 15 c c: --, 
0 u C+ c: 10 0 --.-- Meat loo. 
E 5 :::J 
loo. 
cu 0 V) 
0 1 2 3 4 
Week of experiment 
Figure 10: Least-sq ua re s  mea ns ( ±  S. E) of se ru m  iron values (�tmol I L) for each of the 
treatment g roups 
63 
By desig n ,  Serum iron concentrations were h ighest in the C+ g rou p .  This reflected the d ifference 
in the ad m in istration of supplementa ry  iron (as shown in Table 8). Initially there we re no 
sig n ificant diffe rences between the serum iron concentrations of the other four g roups. The 
serum iron concentration increased across a l l  g roups throughout the duration of the study. Of the 
iron deficient p ig lets, the meat g roup showed the l a rgest increa se s, wh ich may be a result of 
d iffe rences in the b ioava ilabi l ity of the diffe rent forms of iron found between the d iets. 
4.5.1 Unsaturated iron bind ing capacity {UIBC} 
Statistica lly sign ificant effects ( P <  0 .0 0 1 )  were observed between the groups and there were a lso 
sig n ificant diffe rences between weeks. The va riables pigs with in g roups and the interaction 
between week x group were not sign ifica nt. The results prese nted in Table 32 and a lso in Fig u re 
10 show the least-sq ua res mea ns of the va lues for unsatura ted iron b inding capacity obtained 
from each of the treatment g roups. 
Table 32: The least-sq ua res means of unsaturated iron b ind ing capacity (�tmol 1 L) . 
Group Wee k O Week 4 
Cv250 159.4 b 1 30 . 6  b 
Cv500 163 .8 b 1 3 2 .8 b 
c 158 . 8  b 1 37.8 b 
C+ 79 .0 a 69.6 a 
Meat 168 . 0  b 142.8 b 
Change from 
week 0 to week 4 
-- --
- 28.8 
- 3 1 . 0  
- 2 1 . 0  
- 9.4 
- 25.2 
Mean va lue 
week 0 to week 
4 
1 28 b 
1 2 5  b 
129 b 
6 1 a 
139 b 
Resid ual Sta nd a rd 
Deviation ( RSD) 
2 3 . 4  
2 3 . 4  
2 3 . 4  
2 3 . 4  
2 3 . 4  
a ,  b ,  and c means within a column, with a common superscript (or with no superscript) are nol: significantly different from each ol:her (LSD; 
p<O.OS) 
64 
2 0 0  
1 8 0  
- 1 6 0  ..... 
- 1 4 0  
0 1 20 
E 1 00 :::J ....... 
8 0  u 
a:l 60 1-4 
� 40 
2 0  
0 
0 
T T 
l--l 
1 2 3 
Week of experiment 
4 
-+- Cv250 
- cvsoo 
c 
C+ 
_._ Meat 
Figure 1 1 :  Least- squares means ( ±  S .  E) of unsaturated iron bind ing capacity (11mol I L) for 
each of the treatment g roups. 
U nsaturated iron b inding capacities reflected the d ifference in iron status between the groups. 
The iron deficient p ig lets had h igher U I BC levels than the non-iron deficient pig lets i n d icating that 
there we re ma ny unfilled bind ing sites on the iron b inding prote ins ( ma in ly transfe rrin )  in the 
blood of these a n i m a ls. 
4.6 Iron Retention 
The iron retention efficiency was ca lcu lated as the slope of the relationship between iron intake 
and body H B G  Fe (see page 44). The results presented in Ta ble 33 and a l so  in F ig u re 12 show 
the least-squa res means of the values for body H G B  Fe obta ined from each of the treatment 
g roups. Statistica lly sig n ificant effects ( P <  0.001) were observed between the g roups and there 
we re a lso sig n ificant diffe re nces between weeks a nd between pigs with in g roups. There was a lso 
a sig n ificant interaction between week x g rou p .  
65 
Table 33: The least-squares mea n s  of body haemoglobin iron content ( mg) by wee k for each of 
the treatment g roups. 
Group Week O Week 4 
Cv250 5 2 . 8 a 78.4 a 
Cv500 5 7 . 8 a 9 1 . 1  a 
c 4 9 . 3 a 7 6 . 3  a 
C+ 146 . 3 b 2 78 . 0 c 
M eat 5 3 . 4 a 1 9 1 . 9 b 
Change from 
week 0 to week 4 
+ 2 5 . 6 a 
+ 34 . 0 a 
+ 2 6. 9 a 
+ 1 3 1 . 7 b 
+ 1 3 8 . 5  b 
Mean value 
wee k 0 to week 
4 
6 0 . 3 3  a b 
64.64 a b 
56 . 8 1  a 
1 8 8 . 2 3  c 
87. 3 5  b 
Residual Standard 
Deviation (RSD) 
2 3 . 7  
2 3 . 7  
2 3 . 7  
2 3 . 7  
2 3 . 7  
a ,  b, and c means within a column, with a common superscri pt (or with n o  superscri>A:} are not significantly d ifferent from each other (LSD; p<O.OS} 
350 
- 300 T en .J.. E 250 -+- Cv250 -
Ql T - cvsoo � 200 
a::! T 1 --. c � 150 T.--T- .J.. :::z::: .I.. .I.. C+ > 100 "C ------ Meat 0 
a:l 50 
0 
0 1 2 3 4 
week of experiment 
Figure 12:  Least-squa res mea n s ( ±  S. E) of body Haemoglobin iron ( mg) for each of the 
treatment groups. 
The regression a na lysis statistics of body HGB Fe x FEI are shown below. 
Intercept (a) 
Slope (b) 
c CV250 
50.6a 
0 .0 3 9a 
CV500 Meat C+ SE 
4.90 
0 . 0 1 9  
a ,  b ,  and c means within a column, with a common superscript (or with n o  superscri>A:} are not significantly different from ea ch  other (LSD; p<O.OS} 
66 
Body haemoglob in iron accum u la ted more rapidly in iron deficient p ig lets consuming the meat 
d iet than iron deficient pig lets consuming d iets conta i n ing non-meat iron. This ind icates the 
d i ffe rences in bioavai labi l ity between meat and non -meat sources of iron and a lso their a b ility to 
return iron deficient p ig lets back to haematologic normality more rapidly than non -meat sources 
of iron . Vita min C was not sign ificant in returning iron deficient subject back to haemato log ic 
norma l ity. 
4. 7 White blood cells (WBC) 
Wh ite b lood cells combat pathogens and other fore ign substances that enter the body. 
Table 34 shows the sig n ificance of g rou p, pigs within groups, weeks a nd week x g roup on 
d iffe rential wh ite b lood cel l  eva luations. The r-squared values ind icate how much of the variation 
between the treatments can be expla ined by the statistical model shown in section 3.6 (data 
analysis) . 
Table 34: The significance of d ietary treatment on a d ifferentia l wh ite blood cel l  count. 
Group Pig ( g roup) Week Week x group 
Wh ite blood cells ns * * *  * * *  ns 0 .68 
Neutrop h i l  cells ns * * *  ***  n s  0 .64 
Lymphocyte cells n s  * * *  * * *  n s  0 .73 
M o nocyte cells n s  ***  **  n s  0 . 5 7  
Eosinophil cells * *  * *  * *  ns 0 . 6 1  
cel l s  n s  * * *  * * *  ns 
ns: not s ig nificant p > 0 . 0 5 ;  * p < 0 .0 5 ;  ** p <  0 . 0 1 ;  * * *  p < 0 .0 0 1  
Statistica lly sig n ificant d iffe rences ( P <  0 . 0 0 1 )  were observed between p igs within groups a nd 
weeks. There were no sign ificant d ifferences between the g roups or no sign ificant i n te ractions 
between week x g roup .  The results presented in Table 35 a nd a lso in Fig u re 13 show the least­
squa re s  means of the values for white blood cells obtained from each of the treatment groups. 
67  
Table 35: The lea st-squares means va lues for wh ite blood cells ( 109 cells I L) . 
Group Week 0 Week 4 Change from Mean value Residual Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 8 . 4  1 7 . 8  a + 9.4 a 1 3 . 3  4 . 98 
Cv500 1 2 .4 1 7 . 7  a + 5.3 a 1 2 . 5 4 . 98 
c 1 1 . 7 1 5 . 1  a + 3.4 a 1 2 . 6 4 . 98 
C+ 1 2 .0 1 7 . 7 a + 5 . 7 a 16 . 8 4 . 98 
Meat 1 0 . 7  2 2 . 7  b + 1 2 . 0  b 1 7 . 6  4 . 98 ---
a , b, and c means within a column, with a common superscript (or with no superscript) are nct significantly different from each cther (LSD; 
p<O.OS) 
25 
23 
11) - 21  
Qj ....1 19 -+- Cv250 V -
'C 11) 17 --- cv500 
0 Q) 15 ,2 V c 
.c .... 13 .... C+ � 0  ·- '1""1 1 1  ---*-- Meat .c >< 9 3: ...... 
7 
5 
0 1 2 3 4 
Week of experiment 
Figure 13: Least-squares means ( ±  S. E )  of wh ite blood cell volume x 109 I L by week for each 
of the five treatment g roups 
A piglet weaned at 21 days is particularly vulnerable to infection, as the levels of passive 
\ i m m u n ity a re declining and the a n i mal's own immune system is not yet mature. ( Eng l ish et a l  
V 
1984 ) .  Wh ite blood ce lls combat pa thogens a nd other foreign substances that enter the bod y. 
Wh ite blood ce ll values for weaner piglets are normally between 10-23 x 1 09 cells I L. 
Initially a ll an ima ls had white blood cell counts that were within a narrow range of each other, 
regardless of treatment g roup. The stress a ssocia ted with iron deficiency, wean ing , and 
transportation, i n d ivid ual h ousing a nd d ietary changes pred isposed the a n imals to infection . Day 
6+ saw an increase in obse rved instance of scouring, vomiting and sneezing in a l l  g roups. This 
68 
corresponded to an e levation in wh ite b lood cel l  counts, n eutroph il, lymphocyte, monocyte a nd 
basophil ce lls in ind ividua l pigs a nd at specific periods du ring the study. 
Group was significa nt in the increasing number of eosinoph i l  cells. These types of ce lls a re  
produced a nd increased i n  number b y  the body t o  combat a llergens (Tota ra 1996) . This suggests 
that the a llergen may have been a basal feed ingredient of the meat d iet. 
4.7.1 Neutrophil cells 
Statistica lly sig n ifican t  d ifferences (P< 0.001) were observed between individ u a l  p ig s  within d iets 
a nd wee ks. D ie tary treatments a nd week x d iet were not sig n ifica nt. The results presented in 
Table 36 show the least-squares mean s  of the values for neutrophil cel ls obtained from each of 
the treatment g roups. 
Table 36: The lea st-squares means va lues for neutrophil cel l s  109 cells I L. 
Group Wee k O Wee k 4 Change from Mean va lue Residual Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 2.06 5 .56 a + 3 .5 a 3 .66 3 .24 
CvSOO 2 . 18 4 .88 a + 2 .7 a 2 .55 3 .24 
c 2 . 14  4 .30 a + 2 . 16 a 2 .80 3 .24 
C+ 4 . 18 4.22 a + 0 .04 a 4.66 3 .24 
Meat 2 .78 8 .66 b + 5 .88 b 6 .40 3 .24 
a ,  b, and c means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; p<O.OS) 
4.7.2 lymphocytes. 
Statistically sig n ifican t  d ifferences ( P <  0.001) were observed between pigs within diets a nd 
weeks. There were no significant d ifferences i n  d ietary treatment or sig n ificant i nteraction 
between week x d iet . The results prese n ted in Table 37 show the least-squares m ea n s  of the 
values for lymphocyte cells obtained fro m each of the treatmen t  g roups. 
69 
Table 37:  The least-sq ua res means values for lymphocyte cells ( 109 cells I L) . 
Group Week 0 Week 4 Change from Mean va lue Resid ual Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 5 .64 a 1 0 .42 + 4 . 78 b 8 .45 2 .0 
Cv500 8 .88 b 1 0 .98 + 2 . 1  a 8.78 2 .0 
c 8 .46 b 9 .49 + 1 .03 a 8.73 2 .0 
C+ 6 .48 a 1 1 . 54 + 5 .06 b 1 0 . 3 5 2 .0 
Meat 6.64 a 1 1 . 5 2  + 4 .88 b 9 .48 2 .0 
a ,  b, and c means within  a column, with a common superscript (or with no superscript) are ncx significantly different from each cxher (LSD; 
p<O.OS) 
4.7.3 Monocytes. 
Statistically sig nificant diffe rences ( P <  0 . 00 1 )  were obse rved between p igs with in d iets a nd 
weeks. There were no statistica l ly sig n ifica nt d iffe rences betwee n d ietary treatments a nd the 
week x d iet interactions we re n ot sign ificant. The results presented in Table 38 show the least­
squares means of the values for monocyte cells obta i ned from each of the treatment g rou ps. 
Table 38: The least-sq ua res means values for monocyte cells ( 1 09 ce lls I L) . 
Group Week O Week 4 Change from Mean va lue Resid u a l  Sta ndard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 0 .28 a 0 . 74 a + 0 .46 0 . 5  0 . 43 
Cv500 0 .82 a l . lO a + 0 . 28 0 . 7  0.43 
c 0 .64 a 0 . 60 a -0.04 0 . 5  0.43 
C+ 0 .60 a l . O a + 0 .40 0 . 9  0 . 43 
Meat 0 .90 b 1 .34 b + 0 .44 0 . 9  0 . 43 
a ,  b, and c means within a column, with a common superscript (or with no superscrirx) are not significantly different from each cxher (LSD; p<O.OS) 
4. 7.4 Eosinophils 
Statistically sig n ificant d iffe rences ( P <  0 . 0 0 1 )  were observed between the d ietary treatments, 
and there were a lso statistica lly sig n ificant d iffe rences between pigs within diets a nd weeks. 
There was no significant interaction between week x d iet. The results presented in Table 39 show 
the lea st-squa res mea n s  of the va lues for eosin oph i l  cells obtained from each of the trea tment 
g rou ps. 
70 
t 
Table 39: The lea st-squa res mean s  values for Eosinophils cells ( 109 cel ls I L) . 
Group Week O Week 4 Change from Mean value Residual Stand a rd 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
Cv250 0 .0 0 3 0 . 1 2  a + 0 . 12 0 .0 5  0 . 1 3 
CvSOO 0 .04a 0 . 16 a + 0 . 1 2  b 0 .0 6 a 0 . 1 3  
c 0 .0 2  a 0 .04 a + 0 . 0 2  a 0 .0 2  a 0 . 13 
C +  0 . 34 b 0 . 2 2  b - 0 . 1 2  a 0 .26 b 0 . 13 
b + 0 .3 0 ( a b  0 . 1 3 
a , b, and c means within a column, w�h a common superscript (or with no superscript) are not significantly different from each other (LSD; p< 0.05) 
4.7.5 Basophil cells. 
Statistica lly sig n ifican t  d iffe rences ( P <  0 .00 1 )  were observed between p ig s  within d iets and week. 
There were no significant d ifferences between d iet, and the week x d iet interaction was not 
sig n ificant. The resu lts presented in Table 40 show the least-sq uares means of the values for 
basop h i l  cel ls obtained from each of the treatment g roups. 
Table 40: The lea st-squares mean s  values for ba sophi l  cel ls ( 109 cells I L). 
Group Week 0 Week 4 Change from Mean va lue Residual Standa rd 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
Cv250 0 . 0 4  0 . 1 6  a + 0 . 1 2  a 0 . 09 0 . 09 
Cv500 0 . 1 2  0.24 a + 0 . 1 2  a 0 . 1 1  0 . 09 
c 0 .0 6  0 . 2 3 a + 0 . 1 7 a 0 . 1 2  0 . 09 
C +  0 . 0 8  0 . 24 a + 0 . 16 a 0 . 1 8  0 .09 
b + b 
a ,  b, and c means within a column, w�h a common superscript (or with no superscript) are not significantly different from each other (LSD; p<O.OS) 
7 1  
Chapter 5 
5. Results (experiment 2) . 
5 .1 Intake 
Table 41 shows the sig nificance of the effects of g roup, p igs with in g roups, weeks and week x 
g roup on feed intake a nd g rowth rate . The effects of sex, sex x group, a nd pig (sex x g rou p) 
we re a lso tested in experiment 2 .  
Sex, a s  the g roups contained both male and fema le p ig lets, sex x g roup ;  t o  test for the existence 
of an interaction as a result of either sex and grouping and pig (sex x g roup ); where p ig s  with in 
g roups were te sted for any interaction which may exist as a result of sex diffe rences. 
The r-sq uared va lues ind icate how much of the variation between the g roups ca n be expla ined , 
by the statistical model. 
Table 41: Sign ificance leves of d ietary trea tment on feed intake a nd g rowth rate 
Group Sex Sex x group Pig (sex x Week Week x R2 
grou ) _ gr� 
Feed intake n s  N s  n s  ***  * * *  ns 0 . 97 
Growth rate n s  Ns ns ** * * *  ns 0 . 90 
n s :  not sig n ificant p > O.OS; * p < O .O S ;  * *  p <  0 .0 1 ;  ***  p < 0 . 0 0 1  
Statistica lly sig n ificant effects ( P <  0 .0 0 1 )  were observed between p i g s  with in groups and a lso 
weeks. There we re no sign ifica nt d ifferences between g roups and between sexes. There were 
a lso no interactions between wee k x grou p and between sex x group. The results presented in 
Table 42 a nd a lso in Fig u re 14 show the lea st-squares means of the values for feed intake 
(g/day) obta ined from each of the treatment g roups. 
72 
Table 42: Least-sq uares mean s  of feed intake (g/day) 
Group Wee k 1 Week 4 
c 466 a 1067 
C+ 655b 1 143 
M 60 579 b 1 1 70 
M 90 455 a 1 1 7 1  
M r  593b 1 1 67 
Change fro m 
week 1 to week 4 
+ 60 1 b 
+ 488 a 
+ 59 1 a 
+ 7 1 6 ( 
+ 574 a 
Mean va lue 
week 1 to week 
4 
82 3 . 7 a 
950.2 b 
939.3 b 
9 1 1 .4 a b  
94 5 . 1 b 
Residual Standard 
Deviation (RSD) 
90 . 1  
90 . 1  
90 . 1  
90. 1 
90 . 1  
a ,  b ,  and c means within a col u m n ,  with a common su perscript (or with n o  su perscript) are not significantly different from each other 
(LSD; p<O.OS) 
-
1 300 -,--------------. 
1 200 -t-----------'1"-----1 
� 1 1 00 
� � 1 000 +---------,#-------.lb--'"'---------1 la 1: ;a 900 +-- ------/:' ---/-'---------1 
...,. E 
"C ·- 800 -t-------,# --v----------1 Q) c 
Q) la 7 0 0 --l---......'-/ LL. ........ 
C\ 600 
....... 
5 0 0  +- ·'------------1 
4 0 0  +---=--.-------,-----,-----1 
1 2 3 4 
Week of experiment 
-+-C 
-c+ 
M60 
M90 
--..- M r  
Figure 14 :  The least-squa re s  means o f  feed intake (g/day) by wee k for each of the five 
treatment g rou ps. 
The initia l d iffe rences between feed intakes in the p ig lets occurred as d iet was changed over 
from the sta rte r d iet, fed d u ring the accl imatisation period, to the experimental diets conta in ing 
raw or cooked meat or the inorganic sou rce of non-haem iron . Piglets in the C + ,  M60 and M r  
g roups had sig n ificantly bette r intakes du ring the transitio n .  B y  wee k fou r o f  the study there were 
no sign ificant d ifferences between the g roups. The significance of the changes in feed 
consumption from wee k 1 to wee k 4 was due partly to the initia l d iffe rences. 
73 
5.2 Growth rate 
Statistica lly sign ificant effects ( P <  0 .001) were obse rved between pigs within g roups a nd a lso 
between weeks. There were no sign ificant d iffe re nces between groups and between sexes. There 
we re a lso no sig n ifica n t  interactions between wee k x g roup and between sex x g roup. The results 
presented in Tab le  43 and a lso in Figure 15 show the least-sq uares means of the va lues for 
g rowth rate (g/pig let/day) obtained from each of the treatment g roups. 
Table 43: Lea st-squares mea n s  of growth rate (g/pig letjday) 
Group Week 1 Week 4 Change from Mean va lue Residual Standard 
week 1 to week 4 week 1 to Deviation (RSD) 
week 4 --- -
c 57 a 356 a b + 299 b e 250 .4 58.6 
C+ 163 b e 407 b e + 244 a b 3 1 1 .8 58 .6 
M60 134 b 332 a + 198 a 255.9 58.6 
M90 34 a 367 a e + 334 e 241 .4 58.6 
Mr 80 a b 332 a + 253 a b 248.7 58.6 
a , b ,  and e means w i th i n  a colu m n ,  with a common su perscript ( o r  with no su perscript) a r e  n o t  significantly different from each other 
(LSD; p< O. OS) 
500 -,--------------, 
450 +--------------1 > 400 -j----------r----==--��--1 
! � 350 -j-------��---=�'""""'l�--1 
E � 300 +-------*'"-----��E-----=-----1 
£ .!! 250 +------,-,L--.�-----------l 
� -� 200 -+----=--r--77'"-=----------l 
� ; 150 
- 100 +---=Jti<--------------l 
so -J---"1� ----------1 
I 0 -�-��,---,---,-------1 
1 2 3 4 
Week of experiment 
-+-C I 
--- c+ 
M60 
M90 
� Mr  
Figure 15:  The lea st-squa re s  means of g rowth rate (g/pig let/day) by week for each of th e  five 
treatment g roups. 
74 
_) ·-
Feed intakes shaped g rowth rates throughout the study. Sma l l  weekly d ifferences occurred 
between the g roups, but the mea n rates of g rowth were not significantly d iffe re n t  between 
g roups. 
5.3 Haematology 
Table 44 shows the sig n ificance of the effects of group, sex, sex x g roup, pigs withi n  g roups, 
weeks, a nd week x g roup on a complete red blood cell coun t  (CBC) . 
The r-squared values ind icate how much of the va riation between the g roups can be expla ined by 
the statistica l model, shown in section 3 . 6  (data a na lysis) : Where the CBC cha ra cteristics and 
their abbreviations listed in a re those l isted in Table 1 4 .  
Table 44: S ign ificance levels of d ieta ry treatment o n  complete blood count. 
Group Sex Sex x g roup P ig (sex x g roup) Week Week x group 
RBC * * *  n s  n s  * * * *  * * *  0 .86 
HGB * *  ns ns * * *  * * *  * * *  0 .90 
H CT * n s  n s  * * *  * * *  * * *  0 .87 
MCV * * *  n s  ns * * *  * * *  * * *  0 .97 
M C H  * * *  n s  ns * * *  * * *  * * *  0 .97 
MCHC * n s  n s  * * *  * * *  n s  0 .85 
CHCM * ns ns * * *  * * *  * * *  0 .86 
n s :  not sig n ifican t  p > 0 .0 5 ;  * p < 0 .05; * *  p <  0 . 0 1 ;  * * *  p < 0 . 0 0 1  
5.3.1 Red blood cells 
Statistica lly s ig n ificant effects ( P <  0 . 0 0 1 )  were obse rved between the g roups and there were a lso 
sig n ificant d i ffe rences between p igs wit h i n  g roups, and between weeks. There was a lso a 
sig n ificant in teraction between week x g ro u p .  Sex, and the inte raction between Sex x group was 
not sign ificant i n  the number of red b lood cel ls. The results p resented in Table 45 and a lso in 
Fig u re 16 show the least-sq u a res means of the values for red blood cells (x 1 0 . e 9/ L) obta ined 
from each of the treatment g roups. 
75 
Table 45: Least-sq uares means of red blood cells (x 10 .e9/L) 
Group Week 0 Week 4 Change fro m Mean value Residua l Stand a rd 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 5.0 6 . 4 a + 1 . 4 a  5 .S a 0 . 56 
C+ 5.4 6 . 7 a + 1 . 3 a 6 . 3  b 0 . 56 
M60 5. 2 8 . 7 c + 3 . 5 ( 7 . 1  c d  0 . 56 
M 90 4.8 1  8 . 2  c + 3 . 4 ( 6 . 7  b e  0 . 56 
M r  4.90 7 . 4 b + 2 . 5  b 6 . 4  b e  0 . 56 
a ,  b, and c means within a column, with a common superscript (or with no superscri!X) are not significantly different from each other (LSD; p<O.OS) 
10 
- 9 U) ...I 
G) ........ 8 V U) 
"C G) 0 V 7 O N  
- � 
.C O 6 "C 'I""' 
QJ >< Cl:: ._.. 5 
4 
T 
:!: 
0 1 2 3 
Week of experiment 
4 
-+-C 
-c+ 
m60 
M90 
� Mr  
Figure 16: The least-sq ua res mea ns of red b lood ce lls ( x 1 012/L) by week for each of the five 
treatment groups 
Blood sa mpl ing revealed the in itial nu mber of red blood ce l ls was h ig hest in the C+ g roup. Th is 
reflected the d ifferences in level of iron administered shortly after birth (200 mg versus 60 mg ) 
but they were not sig n ificantly diffe rent from the other g roups. 
As the study progressed, the number of red b lood cel ls increased across a ll g roups ind icating 
active erythropoiesis was taking place, as d ietary iron was a ssimi lated . The va riation in the rates 
of erythropoiesis ca n in essence be attributed to d ifferences in iron status and i ron b ioava ilabil ity. 
Not on ly between haem a nd non-haem iron as shown in the i n itia l study, but a lso the effect of 
processing on haem iron . So that at the end of the study the m60 and m90 g roups had 
sig n ificantly higher red blood cell counts than the mr g rou p .  Wh ich in tum was sign ificant from 
76 
eithe r the C or C+ group, but a l l  g roups had red blood cel l  counts that were within n orma l 
physiologically accepted levels ( a s  shown in Table 5). 
5.3.2 Blood haemoglobin concentrations (HGB) 
Statistica lly sig n ifican t  effects ( P <  0 .001) were observed between th e  d ietary groups and there 
we re a lso s ig nificant d iffe re nces between p ig s  within d iets, and between weeks There was a lso a 
s ig n ificant inte raction between week x g roup .  Sex a nd the interaction between sex x g roup was 
n ot sign ifican t  in blood hae m og lobin concentration .  The results presented in Table 46 and also in 
F ig u re 17 show the least sq uare mean of the values for blood haemoglobin concentration (g / L) 
obtained from each of the treatment g roups. 
Table 46: Least- square s  means of haemoglobin (g I L) 
Group Week O Week 4 Change from Mean va lue Residual Standa rd 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 60.5 a b 59 . 1  a - 1 .4 b 56.4 a 6 .81 
C +  97 .1( 79.3 b - 17 .8 a 90.6 ( 6.81 
M60 59 .7 a b 89.3 c +29.6
d 73.0 b 6 .81 
M90 57.9 3 87.5 b c +29.6 d 71.9 b 6 .8 1  
b 85. 1 b e + 18.6 
c 76.9 b 6 .81 
a , b, c and d means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; 
p<O.OS) 
77  
1 1 0 
100 ....... 
C ....l 
:c - 90 
.2 cn  Cl - 80 0 c 
E .2 cu .... 70 RI RI .c l:;  "0 c 60 
0 cu 
0 V - c 50 a::l 0 V 
40 
0 
-+-C 
'------"""-.... ---1 - c + 
m 60 
M90 
-=--,---- ----------.-------; ---....- M r  
1 2 3 4 
Week of experiment 
Figure 17: The least-squa res mea ns of blood haemoglobin concentration (g / L) by week for 
each of the five treatment grou ps. 
Ha ving receiving 200 mg iron shortly afte r birth, pig lets in the C+ g roup began the study with 
blood haemog lobin concentrations that were sig n ificant from the other g roups. In sp ite of that, 
p ig lets in this g roup saw an overa l l  decrease in blood hae mog lobin concentration . Th is was in 
contra st to p iglets in the othe r g roups who increased blood haemog lobin concentration. With the 
exception wa s the C g rou p whose haemoglobin concentration ove ra ll, remained uncha nge d .  
Desp ite these increases blood haemoglobin concentrations rema ined below optimal leve ls. 
5.3 .3 Hematocrit 
Statistically sig n ificant effects ( P <  0 .00 1 )  were observed between the g roups and there were a lso 
sig n ificant d iffe rences between pigs within g roups, and between weeks. There wa s also a 
sig n ificant inte raction between week x g roup .  Sex, and the interaction between sex x g roup were 
not sig n ificant in the level of haematocrit. The results presented in Table 47 a nd a lso in Fig u re 1 8  
show the lea st-squares mea n s  o f  th e  values for hematocrit ( L  I L )  obtained from each o f  the 
treatment g roups. 
78 
Table 47: Least-sq uares mea n s  of hematocrit (L I L) 
Group 
c 
C+ 
M 60 
M 90 
M r  
Week 0 
0 . 2 1 a b  
0 . 3 1  c 
0 . 2 1  a b  
0 . 20 a 
0.23 b 
Week 4 
0 . 20 a 
0 . 26 b 
0 . 30 c 
0 . 29 c 
0 . 29 c 
Change from Mean va lue 
week 0 to week 4 week 0 to week 
4 
-0.0 1 b 0 . 19 a 
-0.05 a 0.29 c 
+0.09 d 0.25 b 
+0.09 d 0.25 b 
+0.06 c 0.26 b 
Residual Standard 
Deviation (RSD) 
0. 02 
0.02 
0.02 
0.02 
0.02 
a ,  b, and c means within a column, with a common superscript (or with no superscript) are not significantly d ifferent from each other ( LSD; p<O.OS) 
0 . 3 5  
0 . 3 3  
......... 0 . 3 1 ...1 
....... 0 . 2 9  ...1 
......... 0 . 2 7  ...., 'i: 0 . 2 5  V 
0 0 . 2 3  ...., n:s 
0 . 2 1 E 
Q.l 0 . 1 9  n:s 
J: 0 . 1 7  
0 . 1 5  
0 1 2 3 
Week of Experiment 
4 
-+-C 
-- c+ 
m60 
M90 
---..- Mr 
Figure 18 :  The least-squa re s  means of hematocrit ( L  /L) by week for each of the five treatment 
g rou ps. 
As hae matocrit is an a lternative measurement to the nu mber of red blood cel ls it is not surprising 
that similar patte rn s  in the number of red b lood cell per volume of b lood were evident. At the 
beg inn ing of the study, hae matocrit levels in the C+ g roup were sign ificantly d ifferent from the C, 
m60, m90, a nd mr g roups, but the haematocrit level decreased as the study p rog ressed . In 
contrast haematocrit leve ls in the m60, m90 and mr g roups increased ind icating active 
eryth ropoiesis was taking p lace and a s  a resu lt the proportion of red b lood cel l  in the blood was 
a lso in creasing , a s  d ietary i ron was a ssimi la te d .  The variation in the proportion of rates of 
erythropoiesis was in essence being attributed to d ifferences in iron status a nd iron 
b ioava i la b i l ity . So again th i s  is a lso the reason for d ifferences in the level of haematocrit. 
79 
5.3.4 Mean cell volume 
Statistica lly sig nificant effects (P< 0.001 ) were obse rved between the groups and there were a lso 
sign ificant d ifferences between pigs with in groups, and between weeks. There wa s a lso a 
sig nificant inte raction between week x grou p. Sex a nd the interaction between sex a nd g roup 
were not sign ifica nt in the mean ce ll vo lume. The results presented in Table 48 and a lso in Figure 
19 show th e  least sq ua res mea ns of the values for mean ce l l  volume (f L) obta ined from each of 
the treatment grou ps. 
Table 48 : Least-sq uares means of mean cell volume (f L) 
Group Week O Week 4 Change from Mean va lue Resid u a l  Standard 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 43 .1  b 3 1 .8 a - 1 1 .3 b 35 .5 a 1 .36 
C+ 58 0 d 39.6 d -18 .4 a 47.7 d 1 .36 
M60 40.7 a 34.4 b -6 .3  c 35.6 a 1 .36 
M 90 42.6 a 36.3 c -6 .3 c 37.5 b 1 .36 
M r  46.0 c 39 . 1  d -6 .9 c 41 . 1  c 1 .36 
a ,  b, and c means within a column, with a common superscript (or with no superscript) are net significantly different from each ether (LSD; 
p<O.OS) 
6S 
- 60 
...I lt- ss - -+- C Cl.l 
E so - c +  
= 
0 4S m 60 > M90 40 
Qj ----*- M r  u 3S  --c .. .. la 30 Cl.l 
� 2S 
0 1 2 3 4 
Week of experiment 
80 
Figure 19 : The least squa re means of mean cell volume (f L) by week for each of the five 
treatment groups 
The data shows that C +  g roup had mean cell volume that was sign ificantly d ifferent from the 
other treatment g roups throughout the study. This despite a reduction in the volume of the red 
blood cells of 1 8 . 4  ft. The other treatment g roups a lso had decrea sing mean cel l  volume values 
for the first two-weeks of the study until cell vol u mes reached a p latea u .  Despite i m p rovements 
in iron status the p ig lets in the m60, m90 ,  mr a nd c g roups are still iron deficient. The mean cell 
volume values obtained suggests that the red cel l  m a ss conta i n s  large n umbers of i m mature 
reticulytes that a re sma l ler than mature e ryth rocytes. 
5.3.5 Mean cell haemoglobin 
Statistica lly sig n ifican t  effects ( P <  0 . 0 0 1 )  were obse rved between the groups and there were a lso 
sig nificant d ifferences between pigs within g roups, and between weeks. There was a lso a 
sign ificant interaction between week x g rou p .  Sex a nd the interaction between sex a nd g roup 
were not significant in th e  mean cell haemoglobin concentration . The resu lts presen ted in Table 
49 a nd a lso in F ig u re 2 0  show the lea st-squares mean s  of the values for mean cel l  haemoglob in 
(p g )  obtained from each of the treatment g roups. 
Table 49: Least-squares means of mean cel l  haemog lobin (p g) 
Group Wee k O Week 4 Change from Mean va lue Resid u a l  Standard 
week 0 to week 4 week 0 to week Devia tion (RSD) 
4 
c 12 a g a -3 b 1 0 a 0 .4 1  
C+ 18 b 1 2  b -6 a 1 5 c 0 .4 1  
M 6 0  1 2  a lO a -2 b 1 0 a 0 . 4 1  
M 9 0  1 2  a 1 1  a b -1 b e 1 1  a b  0 . 4 1  
M r  a 1 1  a b -2 b 1 2  a b 0 . 4 1  
a , b, and c means within a column, with a common supersctipt (or with no superscript) are not significantly different from each other (LSD; p< 0.05) 
8 1  
19 .5  
.E 17. 5 
.c 
0 
C'l 1 5 . 5  
0 
� -13 .5  
ta C'l 
::J: C... - -u. 5  
Q) 
u 
c 
ta 
Q) 
� 
9 . 5  
7 . 5  
0 1 2 3 
Week of experiment 
4 
-+- C 
--c+ 
m60 
M90 
-liE- Mr 
Figure 20 : The least-squa re s  mea ns of mean ce l l  haemoglobin (p g) by week for each of the 
five treatment groups 
The data shows that in itia lly the C+ g roup had mean cell haemoglobin concentration that was 
sig n ificantly d iffe rent from the other treatment groups. H oweve r Subseq uent mea su re ments both 
in the C+ group and the other trea tment g roups saw a systematic decrease in mean cel l  
haemoglobin concentration . Again this is because p ig lets are sti l l  iron deficient a nd the red b lood 
cells manufactured a re sma ll ,  and therefore contain smaller amounts of hae mog lob i n .  Decreased 
mean ce l l  haemog lobin concentration in the C+ g roups is a result of the turnover of existing red 
cel ls, which are rep laced by sma l ler immatu re red b lood cel ls. As iron reserves d ecl ine as the 
pig lets g row. 
5.3.6 Mean corpuscular haem concentration 
Statistica lly significan t  effects ( P <  0.001) were obse rved between the groups and there were a lso 
sig n ificant d iffe rences between pigs within g roups, and between weeks. There was no sign ificant 
interaction between week x d iet and between sex x g roup. The effects of se x  were a lso not 
sig n ificant. The resu lts presented in Table 50 show the least-sq ua res mea ns of the values for 
mchc obtained from each of the treatment g roups. 
82 
Table 50: Lea st-squares mean s  of mchc (g/L) 
Group Week 0 Week 4 Change from Mean va l ue Residual Standard 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 282 a 286 a +4 b 288 a 5.82 
C+ 309b 299 b -lO a  307 b 5.82 
M 60 285 a 297 b + 12 ( 291 a 5.82 
M 90 284 a 293 a +9 ( 290 a 5 .82 
M r  291 b 289 a -2 b 291 a 5.82 
a , b, and c means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; p< 0.05) 
5.3.7 Corpuscular haemoglobin constant 
Sta t istica lly sig n ificant effects ( P <  0 .0 0 1 )  were obse rved between th e  groups and there were a lso 
sig n ifica n t  d iffe rences between pigs with in g roups, a n d  between weeks. There was a lso a 
sig n ifican t  inte raction between week x d iet. Sex a nd the interaction between sex x g roup were 
not sign ificant. The results presented in Table 51  show the least-squa res mea ns of the values for 
CHCM obta ined from each of the treatment g roups. 
Table 51 :  Lea st-sq uares means of C H CM (g /L) 
Group Week O 
c 278 a 
C+ 308 c 
M60 281 a 
M 90 280 a 
M r  b 
Week 4 
285 a 
296 b 
298 b e 
294 b 
290 a 
Change from 
week 0 to week 4 
+7 b 
-12 a 
+ 1 7 (  
+ 14 (  
+2 b 
Mean value 
week 0 to week 
4 
285 a 
304 b 
290 a b 
289 a b 
290 a b 
Residu a l  Standard 
Deviation (RSD) 
5.69 
5.69 
5.69 
5.69 
5.69 
a , b, and c means within a column, with a common superscript (or with no superscript) are not significantly different from each either (LSD; p<O.OS) 
5.4 Red cell matrix 
Table 52 shows the significance of d ieta ry treatments, sex, sex x d iet, p ig (sex x d iet) weeks a nd 
week x d iet, on red b lood cell matrix evaluations. Where the n u mber of red blood cells a re 
categorised by cell haemoglobin concentration and volume and correspond to m 1- m9 groups of 
values with in a 3 x 3 matrix. 
82 
The r-squared values ind icate how much of the va riation between the treatments can be 
explained, by the expanded sta tistica l model shown in section 3 .6 (data ana lysis) :  
Table 52:  The sign ificance o f  d ietary treatment o n  com plete blood count. 
Diet Sex Sex x d iet Pig (sex x d iet) Week Week x d iet R2 
M l  * *  n s  n s  ***  * * *  * * *  0 .90 
M 2  **  n s  n s  ***  * * *  * * *  O .S9 
M 3  * * *  n s  n s  * * *  * * *  * * *  0 .9 1  
M 4  ns n s  n s  ***  ns * * *  0 . 77 
M S  * *  n s  n s  ***  ***  * * *  0 .92 
M6 * * n s  ***  ***  ***  O .SO 
M 7  n s  n s  n s  n s  ns ns 0 . 3 7  
M S  n s  n s  n s  ***  ***  n s  0 .6S 
ns: not sign ificant p > O . OS; * p < O .OS; ** p< 0 . 0 1 ;  * * *  p < O .OO l 
The resu lts p re sented in Ta ble 53 show the least-sq ua res mea n s  of the va lues for red blood ce lls 
characterised a s  M l -M 9 obta ined from each of the treatment groups 
Table 53 : The least-sq uares means of red blood cell characteristic M l -M9. 
CELL WEEK c 
M l  0 5330 b 
4 13 190 b 
M 2  0 2543 a b 
4 12 146 b 
M 3  0 43 a b 
4 247 ( 
M 4  0 1 7 7 5 6 ( 
4 1 1 3 3 1  
M S  0 1 5 9 1 6  a 
4 10570 a 
M 6  0 60 a b 
4 1 6 3  b 
M 7  0 5 1  a 
4 - 1  
M S  0 1 S6 a 
4 - 16 
M 9  0 
4 
C+ 
2 5a 
42SS a 
244 a 
7 1 50 a 
4 a 
7S a 
5 1 7 4  a 
1 142S 
374SO c 
290 2 1 b 
4 3 a 
1 2 6  b 
199b 
6 
1 14 2  b 
7 5  
GROUP 
M60 
S 2 2 3 c 
4 1 2 2  a 
32SO b 
179 1 2  c 
S2 c 
2 1 1  c 
1 4 10 2  b e 
13491 
16224 a 
27703 b 
97b 
6 1 a 
12 a 
7 
164 a 
-6 
M90 
5659 b 
3004 a 
2 52S a b 
1 3453 b 
49 b 
132 b 
1 5 2 1 6  b e 
15S03 
164 1 S  a 
2S95 1 b 
so a b 
40 a 
39 a 
5 
2 1 S  a 
- 1  
M R  
3 S 3 7 b 
2 9 2 2 a 
1 9 3 5 a b 
7672 a 
3 4 a b 
79 a 
1 1 26S b 
1 5 S 14 
2 2 341 b 
3 1 6S4 b 
7 4 a b 
40 a 
2 1 a 
1 5  
7 7 5 b 
- 1 1  
N o  cells were identified i n  th is category 
RSD 
1 75 1  
1 7 5 1 
2 3S 6  
2 3S6 
3 1  
3 1  
3S44 
3S44 
36S7 
36S7 
37 
37 
S 2  
S2 
3 1 5  
3 1 5  
a ,  b, and c means within a column, with a common superscript (or with no superscrirt) are not significantly different from each cther (LSD; p<O.OS) 
83 
5.4.1 Ml  
Statistica lly sig n ificant effects ( P <  0 .0 0 1 )  were observed between th e  g roups and there were a lso 
s ig n ificant d iffe rences between p igs within g roups, and between weeks. The re was a lso a 
sig n ificant inte raction between week x d iet. Sex, a nd the inte raction between sex x d iet were not 
s ig n ificant. The results presented in Table 53 show the least-squa re s  mean s  of the values for m 1  
obta ined from each of th e  treatment g roups. 
5 .4.2 M2 
Statistica lly sig n ifican t  effects ( P <  0 .0 0 1 )  were observed between the groups a nd there were a lso 
s ig n ificant d iffe rences between pigs (sex x g roup), and between weeks. There was a lso a 
sig n ificant inte raction between week x g roup .  Sex, and the i nteraction between sex x group were 
not significant. The results p resented in Table 53 show the least squa res means of the values for 
m2 obtained from each of the treatment grou ps .  
5.4.3 M3 
Statistica lly sig n ificant effects ( P <  0 .0 0 1 )  were observed between th e  g roups a nd there were a lso 
s ig n ificant d ifferences in pig (sex x g roup), and between weeks. There wa s a lso a sign ificant 
i nteraction between week x g roup . Sex, and the i nteraction between sex x g roup were not 
s ig n ificant. The resu lts presented in Table 53 show the least sq ua re s  means of the values for m3 
obtained from each of th e  treatment g roups. 
5.4.4 M4 
Statistica lly sig n ificant effects ( P <  0 .0 0 1 )  were obse rved between pigs ( sex x g roup) a nd there 
was a lso a sign ifica nt interaction between week x g roup . Sex, g roup a nd the i n te raction between 
sex x g ro u p  were not significant. The results presented in Tab le 53 show the lea st  squares means 
of the values for m4 obtained from each of th e  treatment groups. 
5.4.5 MS 
Statistica lly s ig n ificant effects (P< 0 .0 0 1 )  were obse rved between th e  groups and there were a lso 
sig n ificant d iffe re nces between pigs (sex x g roup), and between weeks. There was a lso a 
sig n ificant inte raction between week x g rou p .  Sex, and the inte raction between sex x g roup were 
84 
not significant. The results presented in Table 53 show the least squares mea ns of the values for 
m5 obta ined from each of the treatment g roups. 
5.4.6 M6 
Statistica lly sig nificant effects (P< 0 .0 0 1 )  were observed between the g roups and there were a lso 
sig nificant d ifferences between sex, p ig s  (sex x group), and between weeks. There was a lso a 
sig n ificant interaction between week x g roup .  Sex, and the interaction between sex x g rou p were 
not significant. The resu lts presented in Table 53 show the least squares means of the values for 
m6 obta ined from each of the treatment groups. 
5.4.7 M7 
There were no statistica l ly sign ificant intera ctions for m7. The resu lts presented in Table 53 show 
the least sq uares mea n s  of the values for m6 obtained from each of the treatment g roups. 
5.4.8 MS 
Statistica lly sig n ificant effects (P< 0 .00 1 )  were observed between pigs (sex x g roup) a nd 
between weeks. Group a nd sex, and the interactions between sex x g roup a nd week x g roup 
were not sign ificant. The results presented in Tab le 53 show the least squares means of the 
values for m8 obtained from each of the treatment g roups. 
5.4.9 M9 
There were no cells identified in this category m9 . 
The 3 x 3 matrix described the changes in hae mog lob in concentra tion and cel l  vol u me ove r the 
experimental period . In itially the C+ g roup had a g reater proportion of red cells p laced in the 
"id ea l" m5 category. This reflected, that having received 200 mg of supplementary iron shortly 
after b i rth that the piglets were not iron deficient and that the bone ma rrow had sufficient iron to 
manufactu re red ce lls. 
85 
The positive iron status would have inhibited iron absorption from the smal l  intestine, where a s  
the nega tive iron status i n  the other treatment g roups wou ld have enha nced the absorption of 
d ie ta ry  iron . Therefore the d imensions of the red cells changed over the experimental period . 
C+ a nd C g roups saw an increase numbe r of red cells in the lower matrix categories. The iron 
reserves in C+ g ro u p  would have become depleted as a change in physiological state increased 
the demand for i ro n .  The negative iron status in the C group wa s compounded by the 
consumption of d ietary non-haem iron, that has a lower bioavailabil ity than haem iron that was 
consu med by a n imals in the M60, m90 and m r  g roups. Therefore insufficient quantities of iron 
are reaching the bone marrow; as a result red cel ls with smaller a mounts of haemoglobin a re 
being released in to the circulatory system .  Like the C g roup i n it ia l ly the M60, m90 a nd m r  
g roups have large n u mber o f  cells in lower matrix categories. B u t  a s  these a n imals were 
cons u m i ng d iets conta ining iron that was more bioavailable, the measurements ta ken at the end 
of the study show some movement. The M60, m90 and mr g roups have increased red cel l  
n u mbe rs in the idea l categories a nd reduced th e m  i n  others. 
The short dura tion of this study compared with the life span of a red blood cel l  mea ns that 
a lthough changes are beg i n n ing to emerge it is not possible to see the whole picture a s  only a 
s m a l l  p roportion ( 2 3 % )  of the i n itia l red ce lls have been turned over. 
Fig u re s  2 1  and 22 show the red blood cell counts classified using the 3 x 3 matrix, as a 
pe rcentage of the tota l nu mber of red cells. 
86 
100% 
n:s D m8 � 80% 0 D m7 � 
'+- D m6 0 60% 
� • ms 0 
n:s 40% D m4 11) D m3 n:s 
u 20% m2 cc D ml � 
0% 
c C+  m65 m90 mr  
diet 
Figure 21 : The least squares means of red blood ce l l  cha racteristic a s  a percentage of tota l at 
the beg inn ing of the study. 
100% 
O m8 
ra 80% O m7 ..., 0 ..., D m6 .... 0 60% 
� • ms 0 
ra D m4 11) 40% ra D m3 u I:Q Cl:: 20% 
0% 
c C+ m65 m90 mr 
Diet 
Figure 22: The least squares mea ns of red b lood cel l  cha racteristic as a pe rcentage of tota l at 
the end of the study. 
87  
5.5 Serum Iron Concentration 
Ta ble 54 shows the s ig nificance of the effects of d ie ta ry  treatments, p ig s  withi n  d iets, weeks and 
week x d ie t  on serum iron concentration and body haemoglobin iron. The va riable sex, sex x d iet, 
and pig ( se x  x d iet) were a lso tested and were not sig nificant. The sta tistica l model was then re­
run without these variables . .  The r-sq u a red values ind icate how much of the variation between 
the treatments can be explained, by the sta tistica l model shown in section 3 .6 
Table 54: Significance levels of d ieta ry t reatment on serum iron . 
Group Sex Sex x group P ig ( sex x g roup) Week Week x g roup 
Iron * 
* *  
n s  n s  * * *  
* * *  
* * *  
*** 
* * *  
* * *  
0 . 64 
Fe 
n s :  not sig n ificant p > 0 .05; * p < O .O S ;  **  p <  0 . 0 1 ;  * * *  p < 0 . 0 0 1  
Statistically sig n ificant effects ( P <  0 .00 1 )  were observed between the g roups a nd there were a lso 
sign ificant d ifferences between pigs with in g roups, and between weeks. There wa s a lso a 
sig nificant interaction between week x g rou p .  Sex a nd the interaction between sex x g roup were 
not sign ifican t .  The results presented in Table 55 and a l so in Fig u re 23 show the least-squares 
means of the va lues for serum iron obta ined from each of the treatment g roups . 
Table 55:  The least-squares means of ser u m  iron content (!lmol / L) for each of the treatment 
groups. 
Group Week O Week 4 Change from Mean va lue Residual Standard 
week 0 to week 4 week 0 to week Deviation (RSD) 
C+ 
M 6 0  
M 9 0  
b e  a b  
a ,  b, and c means within a column, w�h a common superscript (or with no superscript) are not significantly different from each other (LSD; 
p<O.OS) 
I nitially the C+ g roup had serum iron levels that were sign ificantly d ifferent from the other 
treatment g roups this wa s an ticipated, and was d ue to the d iffe rence in a mounts of iron 
a d m i n i stered shortly after b i rth ( 20 0  mg vs. 60 mg) .  This was followed by a ra pid increase in 
serum iron (week 1 )  followed by an equally sharp decrease (weeks 2 and 3) but having se ru m  
8 8  
iron leve ls at the e nd of the study only ma rgina lly d ifferent from where they began. This 
h i g h l ights the find ings of Worwood ( 1 997) who reported that measures of serum iron only 
provide a snapshot of the iron status of a subject at the specific time when the blood sa mple is 
ta ken . And a lso that of Furugouri ( 1 9 7 1 )  who demonstrated a day-to-day physiolog ica l variation 
in se ru m  iron a nd TIBC capacity in pigs. 
The other g roups began the study with significantly lower leve ls of serum iron, but which cl imbed 
prog ressively over the study period . The greatest changes occurred in the pig lets consuming 
d iets conta in ing meat iron. 
Figure 23 : Least- sq uares mea n s ( ±  S. E.) of serum iron values (�tmol I L) for each of the 
treatment g roups. 
:1 6 +-------��------------------� 
.._ 
en S  
::::s 
� 4 +-��---------;�------------� 
c 
� 3 
§ 2 1-t=:��.,....-::.=-: 
"-� 1 
0 +----,�---.----,-----,---� 
0 1 2 3 4 
week of experiment 
5.6 Iron retention 
� c 
- c+ 
M60 
M90 
-..- Mr  
Statistica lly sig nificant effects ( P <  0 .0 0 1 )  were obse rved between th e  groups and there were a lso 
sig n ificant d iffe rences between pigs within groups, and between weeks. There wa s a lso a 
sig n ificant inte raction between wee k x g roups. Sex and the interaction between sex and g roup 
we re n ot significant. The results presented in Table 56 and a lso in Figure 24 show the least 
sq uare s  mea n s  of the values for body H G B  Fe obta ined from each of the treatment g roups. 
89 
Table 56: The least sq ua res means of body haemoglobin iron content ( mg )  for each of the 
treatment g roups. 
Group Week O Week 4 
c 104.9 a 2 04 . 4  a 
C+ 1 7 5 . 7  b 3 04 . 5  b 
M 6 0  1 0 5 . 2  a 3 0 9 . 8  b 
M 90 9 8 . 5 a 288 . 1 b 
M r  1 1 1 . 9 a 284 . 5  b 
Change from 
week 1 to week 4 
+ 99 . 5  a 
+ 1 2 8 . 8 a 
+ 2 0 4 . 6 c d 
+ 1 89.6 b d 
+ 1 7 2 . 6  b 
Mean va lue Residual Standa rd 
week 0 to week Dev iation (RSD) 
4 
1 3 5 . 7 a 2 5 . 1  
2 3 8 . 2  c 2 5 . 1  
1 89 . 9  b 2 5 . 1  
1 7 3 .0 b 2 5 . 1  
1 8 8 . 9  b 2 5 . 1  
a ,  b ,  and c means within a column, w ith a common superscript (or with no superscrirx) are not significantly different from each other (LSD; p<O.OS) 
350 
- 300 � 
E 250 -+- C ........ 
QJ 200 - c+ LL. 
a:l --. M60 C) 150 :I: M90 
> 100 "C ___...__ Mr 
0 
a:l 50 
0 
0 1 2 3 4 
week of experiment 
Figure 24: Least-squares means ( ±  S. E) of body Haemog lobin iron values ( mg )  for each of the 
treatment g rou ps. 
The regression ana lysis statistics for body H G B  Fe x FEI a re shown below 
Intercept ( a )  
Slope ( b )  
c- M R  
1 13 . 8a 
0 .2 6 5bcd 
M 6 5  
1 00 . 1a 
0 . 30 8bd 
M90 
9 3 . 3a 
0 . 254acd 
C+ 
188.0b 
0 . 1 94ac 
SE 
1 1 .7 
0 . 0 3 3  
a ,  b, and c means within a column, with a common superscript (or with no superscrirt) are not significantly different from each other (LSD; p<O.OS) 
S i m i la r  to Expe riment 1 the d iets containing meat iron had better rates of retention than those 
conta ining inorganic non-haem iron (C and C+ ) . 
90 
The slower initial rate of retention in the C+ g roup wou ld have resu lted from iron status, as they 
com menced the study with a positive balance of iron in the body, where the other g roups had a 
marginal state of iron defic iency, wh ich would have increased iron a bsorption from the sma l l 
intestine . Rapid increases in growth rate accompa n ied by active e rythropoiesis would have been 
respon sible for the continued retention in the C+ g roup, dieta ry  iron rep la cing dep leting body 
stores. The M60 g rou p saw body HGB Fe re plete more effic iently than either the m90 or m r  
g roups . Th is suggests that cooking a t  60-65°C was beneficia l .  Probably b y  increasing the 
d igestibi l ity of the meat a nd also the haem iron content of the meat was affected l ittle at this 
temperature. 
5.7  White cells 
Ta ble 57 shows the sig n ificance of the effects of groups, sex, sex x group, p ig ( sex x g roup) 
weeks and week x g roup on d iffe rentia l wh ite blood cell counts. The r-sq uared va lues ind icate 
how much of the variation between the treatments can be explained, by the statistica l model 
shown in section 3 .6 
(Where the d ifferential white blood cell counts a re those listed in column 1 of Table 5 7 . )  
Table 57: Sig n ifica nce levels o f  dieta ry treatment o n  d ifferential wh ite cel l  count. 
Group Sex Sex x Pig (sex x Week Wee k x R2 
g rou 
Wh ite blood cells ns ns ns ***  * * *  ns 0 . 57 
Neutraphi l  cells ns ns ns * *  * *  ns 0.50 
Lymphocyte cells ns * ns ***  * * *  ns 0 . 80 
Monocyte cells ns **  ns * * *  ns 0 . 54 
Eosi nophil ce lls ns ns ns n s  * * *  ns 0 . 55 
Ba soph il cel ls ns ns ns ***  ***  n s  0 . 57 
n s :  not sig nificant p > O . O S; * p < O .O S ;  **  p< 0 . 0 1 ;  * * *  p < O . O O l  
Statistically sig nificant effects ( P <  0 .0 0 1 )  were obse rved between pigs ( se x  x g roups) and 
between weeks. Group and sex, and the interactions between sex x g roup a nd week x g roup 
we re n ot sign ificant. The results presented in Table 58 a nd a l so  in Fig ure 25 show the least­
squares means of the values for white blood cells obtained from each of the treatment g roups. 
9 1  
Table 58: Lea st-sq uares mean s  of wh ite b lood cells (x 1 09 cells I L) for each of the trea tment 
g roups. 
Group Wee k O Week 4 Change from Mean va lue Residu a l  Standa rd 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 1 5 .4 1 2 . 9  - 2 . 5  a 1 6 . 3  4 . 86 
C+ 1 6 . 4  1 5 . 9  - o .s a 1 8 . 2  4 .86 
M 60 1 3 . 6  1 6 . 0  + 2 .4 a 1 7 . 1  4 .86 
M90 14.4 1 3 . 1  - 1 . 3 a 18.6 4 . 86 
M r  1 3 . 8  1 8 . 2  +4.4 b 1 9 . 1 4 . 86 
a ,  b, and c means within a column, w�h a common superscript (or with no superscriJ:t) are not significantly d ifferent from each ol:her (LSD; p<O.OS) 
Changes to wh ite b lood cells counts were mon itored over the experimenta l period, as the newly 
weaned p ig let is vulnerable to infection . Ind ivid ua l a n imals reported sporadic increases in white 
cel l  count but these increases were not associated with the specific d ietary treatments. 
Figure 25:  The least squa res means ( ±  S. E . )  of wh ite blood cells (x 1 09 ce lls I L) by week for 
each of the five treatment g roups. 
30 ,-------------------------. 
2 5  +-----------��---+--------1 � � ---� - ...I � '-20 +-------,r-"'�-.---- �1:;:-�---,-----l 
"C � o ­
..2 � 5 +-�"'---------L---�-+-- ---l 
.C Ol � �0 +---------------l 
.c >< 3: - s -----------------------l 
0 +------,-------,------,-------,,.-------l 
0 1 2 3 4 
Week of experiment 
5.7.1 Neutrophil 
---+- c 
--- c +  
M60 
M90 
� Mr 
Statistica lly sig n ificant effects (P< 0 .0 0 1 )  were observed between pigs (sex x g roup) a nd 
between weeks. Group and sex, and the interaction between sex x g roup and week x g roup were 
not significant. The results presented in Table 59 show the least-squa res means of the values for 
neutroph il cells obtained from each of the treatment g roups. 
92 
Table 59: Least-sq uare means of neutrophil cells (x 1 09 cells 1 L) for each of the treatment 
g roups. 
Group Week 0 Week 4 Change from Mean va lue Resid ua l Standa rd 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 2 . 5  4 . 1  1 . 6 a 3 . 6  3 . 6 1  
C +  3 . 2 4 . 3  1 . 1  a 4 . 2 3 . 6 1  
M 60 2 .9 4 . 9 2 a 4 . 1  3 . 6 1  
M 90 2 . 7  2 .4 - 0 . 3  a 5 . 2  3 . 6 1  
M r  1 . 7 6 . 0  + 4 . 3 b 5 . 5  3 . 6 1  
a ,  b, and c means within a column, w�h a common superscript (or with n o  superscri�) are not significantly different from each cther ( LSD; p<O.OS) 
5.7.  2 Lymphocyte 
Statistically sign ificant effects ( P <  0 .0 0 1 ) were obse rved between sex, pigs (sex x g roup) and 
weeks. Groups, a nd the interaction s between sex x g roup and week x g roup were not sign ifica nt. 
The resu lts presented in Table 60 show the least- squares means of the values for lymphocyte 
cel ls obta ined from each of the treatment g roups. 
Table 60: Least- sq uares mea ns of lymphocyte cells (x 1 09 ce l l  1 L) for each of the treatment 
g roups. 
Group Wee k 0 Wee k 4 Change from Mean va lue Residual Standa rd 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 1 0 . 9  7 . 6a - 3 . 3 a 1 0 . 9  1 .8 
C+ 1 0 . 8  1 0 . 0  b -0.8 b 1 1 . 9  1 .8 
M 60 9 .2 9 . 6 a b + 0 . 4  b 1 1 . 3  1 .8 
M90 9.6 9.3 a b - 0 . 3  b 1 1 . 3  1 .8 
M r  1 0 . 3  10.5 b + 0 . 2  b 1 1 . 7  1 .8 
a ,  b, and c means within a column, w�h a common superscript (or w�h no superscri�) are not significantly different from each cther (LSD; p<O.OS) 
5 .7.3 Monocytes 
Statistica lly sig n ificant effects ( P <  0 .0 0 1) were obse rved between sex, p ig s  (sex x g roup) and 
between weeks. The interactions between sex x g roup and week x group were not sign ificant. 
The re su lts p re sented in Table 61 show the least-square mean of the va lues for monocyte cells 
obtained from each of the treatment groups. 
93 
Table 61 :  Least-sq uare means of monocyte ce lls (x 109 cel ls I L) for each of the treatment 
groups. 
Group Week 0 Week 4 Change from Mean va lue Residu a l  Standard 
week 0 to week 4 week 0 to week Deviation ( RSD) 
4 
c 1 . 4 0 .4 - 1  0 . 9  0 . 58 
C+ 1 . 6  0 . 7 -0 .9 1 . 1  0 .58 
M60 0.9 0 . 7 -0 .2 0 .9 0 . 58 
M90 1 .6 0 .6 - 1  1 . 3  0 . 58 
a ,  b, and c means within a column, with a common superscript (or with no superscript) are not sign ificantly different from each other (LSD; p<O.OS) 
5.7.4 Eosinophils 
Statistica lly s ig n ifican t  effects (P< 0 .0 0 1 )  were obse rved between weeks. Group, a nd sex, and 
the interactions between sex x g roup, p ig (sex x g roup) and week x g roup we re not significant. 
The results p resented in Ta b le 62 and a lso in Figu re 68 show the least-squa re mean of the values 
for eosinop h i l  cells obtained from each of the treatment g roups. 
Table 62: Least-sq uare mea ns of eosinophil ce lls ( x  1 09 cells I L) for each of the treatment 
g roups. 
Group Wee k O Week 4 Change from Mean value Residual Standard 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 0 . 1 2 0 . 3 2  + 0 . 20 0 . 2  0 . 19 
C+ 0 . 1 3  0 .55 + 0 .42 0 . 3  0 . 19 
M 60 0 .0 2  0 . 33 + 0 . 3 1  0.2 0 . 19 
M90 0 .0 5  0 . 37 + 0 . 3 2  0 . 2  0 . 19 
a ,  b, and c means within a column, with a common superscript (or with no superscript) are not significantly different from each other (LSD; p<O.OS) 
5.7.5 Basophil 
Statistica lly sig n ificant effects ( P <  0 .0 0 1 )  were observed between pigs (sex x g roup) and 
between weeks. Group a nd sex, and the inte raction between sex x g roup and week x g roup were 
not significan t .  The results presented in Table 63 show the least- squares mea ns of the va lues for 
basophil cells obtained from each of the treatment g roups. 
94 
Table 63: Least-squares mean s  of basophil cells (x 1 09 cells 1 L) for each of the treatment 
g rou ps. 
Group Week 0 Week 4 Change from Mean va lue Resid ual Standard 
week 0 to week 4 week 0 to week Deviation (RSD) 
4 
c 0 . 55 0 .09 -0.46 0 . 29 0.26 
C+ 0 .39 0. 1 1  -0.28 0 .26 0 . 26 
M60 0 . 26 0 . 1 4  -0 . 1 2 0 .3 1  0.26 
M 9 0  0 .43 0 . 1 7  -0 . 26 0 . 29 0.26 
Mr 0 . 38 0 . 16 -0. 2 2  0 . 2 3  0 . 2 6  
a ,  b ,  and c means within a column, with a common superscript (or w�h no  superscriJX) are not significantly different from each cther ( LSD; p<O.OS) 
95 
Chapter 6 
6. Discussion 
Iron status effects 
A genera l  objective of this study was to determine the effect of iron status o n  iron a bsorption 
using both anaemic pig lets a nd p ig lets with a positive iron status. P ig lets are bom with l imited 
reserves of iron, which decline ra p id ly over the first few weeks of life . Anaemia is prevented i n  
the farmed p ig b y  the admin istration o f  supplementary iron shortly after b irth . 
Typica l ly the red cell mass conta ins a bout 80% of a ll absorbed iron, ( Finch and H uebers 1 99 1 )  
where it i s  present a s  haem, a sub stituted porphyrin with a centra l iron atom. Haem i s  u sed in 
the formation of haemog lobin, the protei n  found in red blood cells, which is used to tran sport 
oxygen, H+ ions and carbon d ioxide to and from body tissues. 
At th e  beg inn ing of experiment 1, the p hysiological difference in iron status had a l ready 
m a nifested itself in behavioura l  d ifferences between the groups of p ig lets. The a naemic pig lets in 
C, Cv250, CvSOO and meat groups were letha rgic and displayed inappetence . The non-anaemic 
p i g lets in the C+ g roup were generally l ivelier. 
Du ring the i n itia l days of the study (days 0-6) feed i ntake was low irrespective of treatment 
grou p .  The low intake resulted in negative rates of g rowth in the g roups conta ining iron deficient 
a n i ma ls; only p ig lets in the C+ g roup recorded sma l l  positive increases in l ivewe igh t .  
Poor inta ke a nd t h e  subsequent loss o f  weight and body condition a re n o t  uncommon in the 
immed iate post-weaning period . Eng l ish et a l .  ( 1984) reported seve ra l  studies that found that low 
feed inta ke a nd loss of body weight a nd condition at weaning could be as a resu lt of age a nd 
we ig ht at weaning, a nd a lso the change in environment and the transition from m i lk to a novelty 
d iet that takes place as a result of wea ning . 
Days 7 - 1 3  of the experiment saw the continuation of poor feed intake . Th is was despite the 
add ition of a raspberry flavour to the diet. H owever, Wise ( 1991 )  found that a ni ma l s  suffe ring 
fro m a n utrient d eficiency might reduce their food intake. This may provide a more probable 
explanation, as the C+ group that had been injected with iron shortly afte r birth , and were 
therefore not iron deficient, had sig n ificantly h ig he r  inta kes than those of the other treatment 
g roups throughout the study period . 
The d iets u sed in this study were u nlabelled ; th erefore the determination of iron b ioavailability 
was made from repeated measurements of some blood pa rameters and the calculation of iron 
96 
retention . The blood pa rameters were those l isted in Ta ble 14, and the calculation of iron 
retention shown in section 3.6.  
Changes t o  t h e  blood parameters, wh ich make u p  the red ce l l  mass, were genera l ly i n  line with 
cumu lat ive feed intake . Therefore u ntil days 14-27 of the experiment, when feed intake increa sed 
considerably, there was l ittle va riation in the composition of the red ce l l  mass. 
Unfortunately the poor feed intake prolonged the period of time that the pig lets were iron 
defic ie nt creating a no-win situation . As the iron deficiency, induced ina ppetence, wh ich would 
continue unti l pig lets o btained essentia l iron from the diet. 
As feed intake increased, the effect of iron status on the absorption of iron beca me more evident. 
An imals with a positive iron status (C+ g roup )  had relatively sta ble blood pa ra meters. This wa s 
beca use the blood para meters were a l ready within norma l physiological ra nges for healthy p ig s; 
a lso there wa s sufficient iron ava i lable with in the pig lets' bodies that enabled blood parameters to 
be m a intained within the normal physiolog ica l range throug hout the 2 8-day study. 
The anaemic p ig lets saw increases in the blood para meters occur only when feed consumption 
incre a sed . Th is demonstrates that in these an imals iron stores we re dep lete, a nd therefore iron 
was unava ilable to be u sed in the ma nufacture of red blood cells. 
The changes to blood pa ra meters we re a lso compa rative to feed intake. Piglets consuming the 
meat d iet had sign ificantly h igher feed intakes than those of the C, CV250, and CVSOO g roups; 
therefore iron intake in these animals was a lso higher. This resu lted in g reater changes in the 
blood para meters, a lso the rate at which the increa ses occu rred were more ra p id than those of 
the other anae mic pig lets. This demonstrates that in iron-deficient pig lets, d ietary iron was 
actively absorbed from the gastrointestinal tract, whe re as in non-iron deficient pig lets, the active 
absorption of d ietary i ron wa s not evident. 
Measures of iron rete ntion were calcu la ted using cu mulative iron intake (feed intake x iron 
content of the diet) correcting for any d ifferences that may have occu rred as a result of variation 
in feed intake. The accumu la tion and retention of body hae mog lob in iron increased in a l l  g roups 
from day 14 of the study. Of the iron deficient pig lets, the meat g roup had sig n ificantly higher 
retention rates than the C, CV250 or CVSOO groups, ind icating that there are d ifferences in 
bioava i la bi l ity between meat and non -meat iron . Meat iron is more bioavai la ble than non-meat 
iro n .  Iron retention a lso increased in the C+ g roup, in spite of the positive iron sta tu s .  This is 
beca use the p ig lets a re g rowing, the changes to the pig lets physiolog ica l sta te would see stored 
iron u sed to manufacture add itiona l red b lood cells, therefore body haemoglobin iron is 
incre a sed .  
97  
Iron bioavailabil ity for piglets 
In add ition to changes in the way iron is absorbed in iron deficient pig lets. The d iets con ta in ing 
either meat iron or non-meat iron i n  the vegeta ble ba sed d iet began to retu m p ig lets to 
haematologic norma l ity a t  d iffe rent rates. 
By day 21 of the study there were increases in a nu mber of the blood para meters in the Cv2 50, 
Cv500 and C g roups but the i ncrea ses in blood pa ra meters were most a pparent in the meat 
g roup. There were significa nt d ifferences betwee n  the groups consuming meat iron and those 
consuming non-meat iron in blood haemoglobin concentration, mean cell volume and mean ce l l  
haemoglobin . 
T h is pattern was not h oweve r repeated in the eva l uation of mean corpuscu lar haem 
concentration or in corpuscul a r  haemoglobin consta nt. There were no sig n ifica nt d ifferences 
between g roups consuming e ithe r meat iron or non- meta iron in mea su rements of these two 
pa ra meters. 
Additiona lly red blood cel l  counts, in piglets in the meat g roup we re not sign ifica ntly d ifferent to 
those of pig lets cons u m ing the Cv500 d iet, but were sig nificant from the C and Cv250 g roups. 
Increased feed intake a t  this time would have resulted in the d ietary i ron being present in the 
ga strointestina l tract. The negative iron status would see iron actively absorbed and incorporated 
into blood products conta in ing iro n .  As other variables have been excluded, diffe rences in the 
rate of repletion cou ld be a ttrib uted to the d iffe rence in bioava i labil ity between the meat iron and 
the inorg a n ic non-haem iron found in the vegetable based d iet. 
T h is would be consistent with the findings of South et al. (2000) who established that in pigs' 
meat iron is more read ily a bsorbed ( 1 5-35%) than non-haem sources of iron (2-20%) .  Bjom ­
Ra smussen et a l .  ( 1974) proposed that the d ifferences in bioava i la b ility were because d ietary iron 
forms two pools withi n  the intestinal l u men, a haem pool and a non-haem iron pool. Also the two 
sources of iron a re absorbed using d iffe rent pathways (Con rad et a l .  1 999) .  The meat iron 
consisting mostly of organic haem iron is more read ily a ssimi lated as it taken up d irectly by the 
ente rocyte cells of the sma l l intestine as an intact porphyrin . Whereas the inorgan ic non-haem 
iron has to be solubil ised before it can be absorbed . These pathways a re described i n  section 2 . 3 .  
The effects of vitamin C: Iron bioavailability in iron deficient piglets consuming non­
haem iron sources, supplemented with vitamin C. 
I n  add ition to d iffe rences i n  the bioava ilabil ity of meat iron and non-meat sources of iron, a 
furthe r  general objective of the study was to dete rm ine the effects of vita min C on the a bsorption 
of non-meat iron . The study esta blished that, of the three g roups consuming d iets conta ining 
98 
non-meat iron in vegetab le  based diets; there were sig n ificant d ifferences in some b lood 
pa ra meters resu lting from the add ition of Vita min C .  
Vitamin C has been shown to enhance the bioava ila bi l ity o f  non-haem iron i n  iron deficient 
subjects, con su m ing e ither se m i-synthetic or experimenta l diets. Th is is shown in Table 2. The 
role of vita min C in enhancing non-haem iron from a comp lete d iet is less clea r. Th is study u sed 
food cho ices from a typ ical human d iet and found that overal l  there were no sig nificant 
d iffe rences between nu mber of red b lood ce lls and haematocrit between the C and CV250 d iet, 
containing 0, a nd 250 pp m of vita min C, yet the nu mbers observed in the CV500 we re 
sig n ificantly d iffe rent from both the C and CV250 d iets. This patte rn was not however repeated in 
the eva luation of mean cel l  vol u me ,  mean cel l  haemog lobin, mean corpuscu lar haem 
concentration or in corpuscular haemoglobin constant. The blood haemog lobin concentration in 
pig lets consu m ing the CV500 d iet wa s sig nificantly h ig h e r  than for the C d iet d u ring week fou r  of 
the study but not overa l l .  
T h is suggests that the vitamin C may b e  utilized in the transfer o f  iron s o  that i t  can b e  used in 
the production of red b lood cel ls rather than increasing b ioa va i labil ity in the sma ll intestine . In 
wh ich ca se the vita m in C has to be included in the d iet a t  high leve ls. U nder these experimental 
cond itions 500 ppm (as fou nd in the CV500 d iet) appears to be the most beneficia l .  
Red blood cells a re p rod uced i n  the bone marrow a nd pass through several stages of 
development before being re leased in to the circu latory system (as shown in Appendix 5). In 
addition to the number per volume of blood, red blood cells in circu lation ca n a lso be 
characterised, by volume (V) and haemog lobin concentration (HC) with in the cells, u sing a 3 x 3 
matrix using the ab breviations m 1-m9. 
A blood sa mple taken from a hea lthy non-iron deficient piglet wou ld have blood ce lls with a HC 
read ing between 1 10 - 1 70 g I L with a correspond ing V measure ment of 50-68 fl correspond ing 
to the m5 position .  Val ues below these parameters ( m l -m4) ind icate that sma l l  im matu re 
reticulocytes were prematu re ly entering the circ u latory system, where as values a bove these 
levels ( m6-m9) suggest that cells a re la rger, as protoporphyrin accumu lates in the ce l ls. 
A change in categorisation of the red blood ce lls wa s not evident a s  a result of feed ing the 
control d iet supplemented with 0, 2 50 or 500 ppm of vita min C. There were no sig n ificant 
d iffe rence between the nu mbers of cells placed in categories m l ,  and m3-5. The Cv500 g roup 
had a greate r nu mber of ce lls p laced in the m 2  and m6-m9 categories than either the C or Cv250 
group, cha ra cteristic of prolonged iron deficiency. The increase in the number of red ce lls in the 
matrix categories ( m6-m9) suggests that high levels of vita min C may play a role in the 
accu mulation of protoporphyrin in cells in the a bsence of sufficient iron. 
99 
Measu re me n ts of body haemog lobin iron a lso show that in iron deficient p ig lets, consuming non­
meat iron supplemented with vita min C, iron accumulates more slowly tha n in those consu ming 
meat iron . Add itiona lly there were no sign ifican t  d ifferences between the various levels of vita min 
C and th e  rate at w hich i ron accu mulated in the red cell mass. 
Iron bioavailability in non-iron deficient piglets 
Having received an iron injection shortly after b irth, p iglets in the C+ group were not i ron 
deficient and an evaluation of the red b lood cells from these a n i ma ls showed that app roximately 
90% of red cel ls were classified in the optim u m  m5 position . There was little variation in the 
nu mbe rs of red cells in th is category th roughout the study showing that in these p ig lets there 
were sign ificant quantities of iron a va ilable in the body to susta in active erythropoiesis. 
This was s upported by measurements of seru m  iron concentration, and unsaturated iron binding 
capacity. There we re sma l l deviations in the serum iron concentration measurements taken 
throughout the study; Worwood ( 1997) reported that this was because serum iron pools a re 
tu rned ove r so ra p id ly ( 1 0-20 times per day). H owever, used in conj unction with measurements 
of u n saturated binding capacity, it can be determined that there wa s l ittle variation in the 
proteins a b ility to b ind iro n .  This provided further evidence that in non-iron deficien t  pig lets iron 
wa s not being actively absorbe d .  
The effectiveness of meat and non-meat sources of i ron in returning iron deficient 
piglets to haematologic normality. 
At th e  be g i nn in g  of the study the extent of the iron deficiency, a nd its role in the prod uction of 
healthy red b lo od  cells is a l ready evident, as a l l  iron deficient p iglets in the C, Cv250, Cv500 and 
the meat g roups had la rger numbers of red b lood cel ls in the lower matrices category. This is 
consistent with the findings of Pippard ( 1995) , who reported that iron deficiency impa ired 
haemog lobin synthesis a nd as a result the red blood cells tended to be sma l l  a nd poorly 
haemog lobin ised . H o wever as the natural life of red b lood cells is approximate ly 1 20 days, the 
28-day study would see on ly 23% of the initia l red blood cel ls turned over. Therefore on ly sma l l 
changes in classification would be evident. Nevertheless, by week 4 of the study there were 
sig n ifica nt d iffe rences between piglets cons u m ing meat and non-meat iron, and the placement of 
red blood cells with in the matrix. The meat g roup had increased the n u mber of red b lood cells in 
the optimu m m5 categ ory. In contrast pig lets consu ming non-meat sources of iron (C, Cv250 and 
Cv500 g roups) had decreased n u mbe rs of red cells in th is position . Th is supports the earlier 
find ings of th is study, i n  that meat iron is more bioavailable than non-meat sources of iron, 
resulting in a more rapid re tu m  of iron-deficient p ig lets to haematolog ic normality ove r those 
l OO 
consu ming non-meat sou rces of iron. Measureme nts of body haemoglobin iron retention a lso 
show the same d ifference in the effectiveness of meat iron and non-meat iron in returning iron 
defic ie nt pig lets to haematologic norma lity. The body haemoglobin iron accu mulated more rap i d ly 
in the iron deficient pig lets consu ming the meat iron than those con su m ing the inorga n ic non­
meat iron a lternative sou rce of iro n .  
1 0 1  
Experiment 2 
The effects of cooking temperature on iron bioavailability in meat. 
The g e ne ra l  objective of experiment 2 was to determine the effects of cooking temperature on 
iron b ioava ilability in meat, using 100% visua l lea n bull beef that rema ined uncooked ( mr) or that 
had been subject to cooking in th ermostatical ly controlled c i rculating wate r baths at either 60°C 
( m60) and 90°C (m90) a nd incorporated into the d iet at 25% of the tota l d ietary ing red ients (on 
a n  a s-fed basis ) .  
As in experiment 1 ,  measures of bioava ilab ility were made b y  measuring the rate of repletion of 
some of the elements of the red ce l l  mass a nd by the calculation of iron retention . 
P ig lets in the m60 and m90 groups consistently had significantly bette r rates of repletion than the 
mr g roup, in nu mber of red b lood cel ls pe r volume of blood, level of hae matocrit, blood 
haemoglobin concentration, mean corpuscu la r haemoglobin concentration a nd corpuscu lar 
haemog lobin con stant. This is in a g reement with Newsome ( 1 980 ) who reported that the net 
effect of heat processing was positive, by increasing the nutrient a nd I or d igestibility of food 
components. I n  this case the cooking p rocess would have i n itiated the denatu ration of some of 
the meat prote ins; enabling the p orphyrin complex, contain ing the iron to be relea sed . Whereas 
pig lets con su ming the mr d iet conta i n ing the raw uncooked meat protein would rely solely upon 
e n zymatic hydrolysis that takes p lace within the d igestive tract to release the iron from the meat. 
The lack of clarity between the m60 and m90 g roups, desp ite d ifferences in cooking temperature 
could be becau se  meat con ta in s  both haem a nd non-haem fractions of iro n .  Purchas et a l .  ( 2 0 0 1  
u n p u b l ished data) ca lculated th a t  in u ncooked meat these va lues were 85% a nd 1 5% 
respectively, for haem and non -h a e m  iron . 
Kriste n se n  a nd P urslow ( 2 0 0 1 ) ,  Schricker and M i l ler ( 1983) and P u rchas et a l .  ( 2 00 1 unpublished 
da ta )  h a ve demonstrated that heat p rocessing of meat produced an incremental decrease in 
haem iron content a s  a function of heating te mperature .  So that the haem iron content of meat 
beg i n s  to decrea se when process i ng temperatures exceeds 55°C. This is because processing heat 
a ffects the therma l stability of the m u scle myog lobin, causing denaturation, which releases a nd 
causes d eg ra dation to the haem molecule ( Kristensen and P urslow 200 1 ) .  
T h e  meat used i n  the m60 diets wa s subject t o  relatively mild heat processing that would have 
i n itiated the denaturation of some of the meat protei n s, facilitating the release of the porphyrin 
complex .  H owever, the heat treatment (60°C) may not have been h a rsh enough to see a 
s ig n ificant reduction in the haem iron content (Schricker and M il ler 1983 ) .  
I n  contrast, t h e  m 9 0  d iet con ta in ing meat that h a d  been subjected t o  a much h igher cooking 
temperature would have seen a significant decrease in the haem iron content as it was converted 
i nto non-haem iron . Therefore, the tota l  iron in the m90 diet would be the sa me as that of the 
1 02 
m60 d iet, but as a result of cooking, a g reater proportion of the iron would be non-haem iron 
( 78 . 2 5% vs. 68.6%) . 
Da ta from experiment one showed that non -meat iron has a lower bioava i la b i l ity tha n that of 
meat iron. Although the absorption of n on-meat iron can be enhanced by specific a m ino acids 
found in meat as outl ined in section 2. 7 .  It may be that the presence of these amino acids 
increased the bioavai la bil ity of the non-haem iron fraction so that overa ll, iron bioava ilabil ity 
between the m60 and m90 g roup we re n ot sign ifica ntly d ifferent. 
In itially se rum iron levels in the mr, m60 and m90 groups reflected d iffe rence in iron 
ad min istered shortly afte r birth ( 60 mg vs. 200 mg administered to the C+ g rou p ) .  The levels of 
serum iron increased as the study prog ressed so that by week 4 of the study serum iron 
concentrations in the mr group eq ual led that of p ig lets in the C+ g roup. This shows clearly the 
effectiveness of meat iron in retu rn ing anaemic piglets to haematolog ic norma lity, desp ite an 
i n it ia l  140 mg iron deficit. Diets conta i n ing cooked meat a lso had changes in serum iron 
concentration that were sig nifica ntly h ig her than pig lets consuming non -meat iron . 
Similar to expe riment one, the accu mulation and rete ntion of body haemoglobin iron increased in 
a l l  g roups throughout the study. Of the iron deficient pig lets, the g roups consuming meat d iets 
had sign ificantly h igher retention rates than the C g roup; ind icating the d iffe rence in 
bioava i la b i l ity between meat and non-meat iron . Of the g roups consuming the meat d iets, 
retention rates were highest in the m60 g roup .  This ind icates that cooking at 60°C was 
beneficia l .  Probably by faci l itating the release of the porphyrin complex, as the muscle prote ins 
were denatured and add itiona lly the haem iron content of the meat would be least affected at 
t h i s  tempe ratu re .  
Iron retention a lso increased in the C +  g roup, but re mained below that of the g roups consuming 
meat. Aga in increases in iron retention in pig lets that initia l ly had a positive iron status is 
beca use the pig lets a re g rowing rapidly, the changes to the pig lets physiolog ica l state wou ld see 
stored iron used to ma nufacture add itional red blood cells, therefore body haemoglobin iron is 
i ncreased . H owever, blood haemog lobin concentrations of p ig lets in the C+ g roup declined from 
week one of the study indicating that there was insufficient stored iron to meet the piglets' 
requ irements. Also anima ls in this g roup a re a lso consu ming a d iet conta ining non -meat iron that 
has lower bioava ilabi l ity than that of meat iro n .  So that overa l l  the accumulation a nd retention 
rates of these pig lets were lower tha n that of the m60, m90 a nd mr g roups. 
1 03 
The effectiveness of meat iron in bringing about a return of anaemic piglets to 
haematologic normality. 
The 3 x 3 matrix described the changes in haemog lobin concentration a nd cel l  volume of the red 
blood cells over the experimental period . 
To prevent the lethargy d isplayed by the iron deficient pig lets in experiment 1 ,  in add ition to the 
2 0 0  mg of supplementa ry iron ad min iste red shortly after b i rth to pig lets in the C+ g roup, 
60 mg of supplementa ry iron was admin istered to p ig lets in the C, m60, m90 and m r  g roups 
provid ing a ma rg in a l  state of iron deficiency. 
Th is was evident in the p lacement of cells within the matrix . I nitially p ig lets in the m60, m90 a nd 
m r  g roups had la rger n umber of cells in the lower matrix categories. This is symptomatic of th e  
red cel l  m a s s  conta ining large n u mbers of sma l l  i m mature red b lood cells, b u t  is consistent with 
P ippa rd ( 1995) who reported that iron deficiency impa ired haemoglobin synthesis and as a result 
the red b lood cel ls tended to be small a nd poorly haemog lobi n i sed . But u n l ike experiment 1, the 
smal l a moun t  of supple mentary iron admin istered to the m60, m90 and m r  g roups shortly after 
b i rth -p revented inappetence . Consequently feed intake was m uch h igher than in experiment 1 ,  
averag ing 552 ± 75g I day a t  the beg inning o f  the study and increasing to 1 147 ± 33  g I day at 
the end of the study .  So despite the re latively sma l l turnover of i n itia l red blood cells, at the end 
of the study there was an improvement in the p lacement of cel ls within the matrix. 
Pig lets consu ming the d iets conta ining meat increase the numbe r  of cells placed in the optimu m 
m S  category, whereas th e  piglets in the C and C+ g roup saw a reduction in the nu mber of cel l s  
placed here . The increased n u mber o f  cells p laced i n  the opti m u m  category show that t h e  meat 
iron was read ily assi m i lated, ma i n ta i n ing active erythropoiesis when body weight was i ncrea sing . 
Iron bioavailability in anaemic and non-anaemic piglets consuming non-meat iron. 
Aga in using the matrix to a na lyse the volume a nd haemog lobin concentration of red b lood cells. 
I n itial ly the C +  g roup had a g reater p roportion of red cells placed in the "idea l "  m5 category. Th is 
reflected , that having received 2 0 0  mg of supplementary iron shortly after b irth that the p ig lets 
were n ot iron deficient and that the bone marrow had sufficient iron to manufacture red cells. 
The positive iron status of the pig lets in th is g roup would have i n h i b ited fu rther i ron absorption 
from the sma l l intestine until iron reserves were depleted . 
P i g lets u sed in Experiment 2 saw g rowth rates increase by 266 ± 47 g I p ig let/day d u ring the 28-
day study period, therefore despite the administration of su pple mentary i ron, pig lets in the C +  
g roup had insufficient stores o f  iron to ma i n  active erythropoiesis d uring changes i n  physiological 
state , which would have increased the d e mand for iron. This i s  seen in the fluctuating serum iron 
levels; the change co incides with changes in ce llu la r  demand for iron as the anima l g rows 
( Fu rugouri a nd Kawa bata 1 976) .  Changes in the reg u latory processes that would have enabled 
1 04 
the pig lets to increase iron absorbed from the gastrointestinal tract take p lace over time and 
therefore a re not a pparent. 
In contrast, the negative iron sta tus of piglets in the C g roup would have seen iron actively been 
absorbed from the sma ll intesti ne . Unfortunately pig lets in this group were fed a d iet conta ining 
non-meat iron, wh ich has a lower ava ilability tha n  that of meat iron (South et a l .  2 00 0 ) .  
Therefore iron was n o t  ava i la ble i n  sufficient quantities to support the a ctive e ryth ropoiesis taking 
pla ce during the physiolog ica l state of growth . Therefore red blood ce l ls produced a re in the 
lower matrix categories and ce l l  tu rnover wou ld  decrease the number of cells placed in the 
optimal posit ion. 
Body haemog lob in iron in these two groups increased, yet at a slower rate of repletion than that 
of the mr, m60 and m90 g roups. In the C+ g roup the lower rate of repletion would be due to 
iron sta tus, a s  they commenced the study with a positive bala nce of iron in the body whereas the 
other groups had a marg i nal state of iron deficiency. The lower rate of depletion in the C grou p 
than that of othe r anaemic pig lets resulted from the d ifference in bioava ilabi lity of iron . Meat iron 
whether raw or cooked is more bioava i lable than non-meat iron . 
The m60, m90 and mr g roups began the study with marg inal leve ls of iron deficiency having 
received on ly 60 mg supple mentary iron shortly after birth . These g roups saw blood haemog lobin 
concentrations incrementa lly increase throughout the study. The an ima ls in two of these g roups 
the m60 and m90, fi n ished the study with haemoglobin concentrations statistica lly h igher than 
the C+ g roup. This occurred despite the initia l 140 mg iron deficit. 
Of the 3 groups consuming haem iron, the increases in blood hae mog lobin concentration were 
greatest in the m60 and m90 g roups. 
An a ssumption can be made that not all food processing is necessa rily detrimental to the nutritive 
value of food as the blood haemoglobin concentrations were higher in the groups consu ming 
processed meat than that of the mr g roup that consu med raw unprocessed meat. 
The d iffe re nces between the p rocessed a nd unprocessed meat are in genera l ag reement with 
Han et al ( 1 993) who measured the haem iron content of beef as a function of heating 
temperature and found that an inverse re lationship exists between haem and non-hae m iron 
content of the meat. 
In this case the heat treatment would have caused changes to the ion isation states of the a m i no 
acids causing the protein to denature, facil itating the release and deg radation of the iron 
conta ining porphyrin complex. At the sa me time the non-haem iron content wou ld have increased 
due to the conversion of haem iron into non-ha e m .  
1 05 
Concluding d iscussion of experiments 1 and 2 
Repeated h ematologic measure ments were used in th is series of experiments to identify the rate 
of repletion in severa l blood pa ra meters following the consu mption of meat, vegetable and milk 
based d iets over a 28-day period . Table 64 shows a summa ry of the changes over the 4-week 
period in the b lood para meters between the treatment groups in experiment 1 .  
Table 64: A comparison of changes over the 4-week period i n  blood pa ra meters for experiment 
1 .  
Blood parameter Control Control + 250 ppm Control + 500 ppm Meat 
Vit C Vit C 
Red blood Cel l s  + 1 .37 + 1 .22 + 1 .93 + 1 . 95 
( x  1 012 cel l s/ L) 
Haemoglobin (g I L )  + 4 .4 +4.0 + 10 . 4  + 2 2 . 0  
Haema tocrit ( L  I L) + 0 .02 + 0 .02 + 0 . 05 +0 . 09 
Mean Ce l l  Volu m e  (fl) -7 -7 -5 +2 
Mean Ce l l  -2 -2 -2 0 
A d irect comparison of the 4 trea tment g roups that had equ iva lent iron status shows that at the 
end of the tria l p ig lets consuming the meat d iet had h igher n u mbers of red cells, blood 
haemoglobin concentration , haematocrit, mean cel l  volume, a nd mean ce l l  haemoglobin than 
those consum ing vegetable based d iets. As the d iets were comparable i n  d igestible energy (DE), 
D E :  lysine ratio a nd the p ig lets i n  these fou r g roups had equivalent iron status, the results 
i n d icate that the d iffere nces in the blood para meters were a result of the two d ietary forms of 
iron being consumed by these groups. The h a e m  iron found mainly in meat was sig nificantly 
more b ioavai lble th a n  the n on -meat iron found in the vegetab le  based d iets. T h i s  is consistent 
with the find i n g s  of South et al (2000) who a lso observed d ifferences in bioava ilability when pigs 
we re fed meat a nd non-meat sou rces of i ron . .  
I n  add ition to the q ua ntitative d iffe rence i n  th e  blood pa rameters the deg ree to which th e  
haematologic pa ra meters changed were a lso o f  inte rest. The meat group had a h igher rate of 
repletion tha n th ose consum i ng the vegetable d iets conta in ing non-haem iron . H owever, of the 
a n ima ls consuming non-haem iron the g roups consu m ing non-haem iron that had been fortified 
with vita m in C at 500-ppm (CV500) had h igher ra te s  of repletion than those con su m ing either 
2 5 0-pp m of vita m i n  C (Cv250 g roup) or the C g roup consum i ng solely non-haem iron. The 
d ifferences in the degree of repletion point to the d iffe rences in the way the two forms of iron 
a re pooled a nd then tra n sported in to the body using the hae m  uptake pathway a nd the DCT- 1 
path way for haem and non-haem iron, respective ly . 
1 06 
Haem iron is transported d irectly into the enterocyte using the haem upta ke pathway. This is in 
contrast to non-haem iron that in itia lly has to be red uced and ma i ntained in its ferrous form prior 
to absorption and also has to ove rcome problems of competitive i n h ib ition, as the transporters 
a re sha red with other d iva lent ions (a s shown in section 2 .3 ) .  
Therefore the resu lts a re ind icative o f  the red uction poten tia l of vita min C ,  however a h ig h  level 
( 500 ppm) is necessary to maintain a pH in the sma ll intestine that would enable the non-haem 
iron to be red uced to a more soluble fe rrous for m .  
Th is i s  consistent with the find ings o f  Clydesdale ( 1 982) w h o  found th a t  b ioava i labil ity o f  non­
haem iron is directly correlated to acid so lubil ity; the acid ity in creasing ion isation a nd favouring 
the ferrous form which has a g reater solubil ity at the pH of the intesti ne . 
The re su lts presented a lso esta b l ish the basis of the intrinsic relationship between iron sta tus and 
iron absorption . A comparison of the C and C+ g roup, wh ich d iffe r solely in iron status show that 
in the iron deficient subject iron is actively absorbed . 
In comparison hematologic para mete rs in the C+ group that showed less move ment, ind icative 
of nu m ber of red cells and blood haemoglobin concentrations that a re a ppropriate for the age of 
the a n imal and have not been impa ired by iron deficiency. 
The hypothesis of experiment 2 built on the find ings from the in itial experiment; that meat iron is 
more b ioavai lble than non-meat iron . Experiment 1 used raw meat; the 2nd experiment therefore 
took the next logical step, the effect of processing heat or cooking on the bioava i labil ity of haem 
iro n .  
Aga i n  using a repletion study, the rates o f  repletion to a n u m be r  of blood pa ra meters were 
measu red over a 28-day study period . 
Ta ble 65 shows a compa rison of the changes over the 4-week study period in the blood 
pa ra meters between th e  treatment g roups in experiment 2 
1 07 
Table 64: A comparison of changes over the 4-week study period in blood pa ra meters 
experi ment 2 .  
Blood pa ra meter Control Raw Meat Meat cooked a t  Meat cooked at 
60°C 90°C 
Red blood Cel l s  + 1 .4 + 2 .5 + 3 . 5  + 3 . 4  
( x  1 012 cel l s/ L )  
H a emoglobin (g I L )  - 1  + 1 8  + 2 9  + 3 0  
H a ematocrit ( L  I L )  0 +0 . 1  + 0 . 1  +0. 1 
Mean Cel l  Vol u me (fl) - 1 1  -7 -7 -7 
-3 -2 -2 -1 
The n u m be r  of red cells, blood haemog lobin concentration, level of hematocrit, mean cel l  volume 
and mea n cell haemoglobin were h ighest in the p i g lets consuming heat processed meat. 
Although the re wa s a d istinction between raw a nd heat processed meat d iets, there was little 
d ifference between the levels of repletion of the blood parameters between meat cooked at 60 oc 
and 90°C as contained in the M60 a nd M 90 d iets respective ly. 
The results show that the d ifference in repletion rate between raw a nd cooked meat cou ld be 
attributed to d igestibility. The cooking process causes some of the meat p rote ins to denature, 
wh ich may fac ilitate the release of porphyrin complex, which may increase its d igestibility. This i s  
Consistent with Newsome ( 1980) w h o  reported that the n e t  effect of food p rocessing was 
positive; increasing the nutrient content a nd/ or d ig estibil ity of the food component. 
The lack of clarity between the two heat-processed d iets is more d ifficult to expla i n .  Work by 
Kriste n se n  a nd P urslow ( 20 0 1 )  and H a n s  et a l .  ( 1993) both show that heat processing in pork, 
chicken and beef decreases the haem iron content of the meat. 
The m60 d iet was heat processed to 60° C; thi s  coincides with the temperature necessa ry to 
denatu re the myog lobin molecule . Meat exposed to processing temperatures in excess of 80° see 
further reductions in haem iron content d ue to the degradation of the porphyrin complex. 
Therefore the expectation was that the anima ls consuming the m60 d iet would have better rates 
of repletion than those consu ming the m90 d iet. 
U n expecte d ly an inve rse rela tionshi p  exists between haem and non-haem iron, a s  a function of 
heating temperature (Han et a l 1 99 3 ) .  Therefore a s  p rocessing heat increases some of the haem 
iron i s  converted to non-haem iron, thereby increasing the non-haem iron content. 
Therefore it wou ld seem likely that the m90 d iet conta ining beef processed to gooc, would have 
decreased its haem iron content, but the non-haem iron content of the meat would have actually 
increased . Non-haem i ron has lower bioava ila b i l ity than haem iron, but its b ioava ilab i l ity can be 
e n ha nced by some a mino acids found in meat. Therefore an assumption can be made that the 
bioava ilab i l ity of conve rted non -haem iron ha s been increased in a similar way in which vitamin C 
1 08 
enha nced the absorption of non-haem iron in experiment 1 .  The m60 d iet conta ined meat heat 
processed to 60°C h igh enough to denature the myoglobin molecu le but n ot harsh enough to 
s ig n ificantly reduce the haem iron content of the meat. Therefore the overa ll iron bioava ilability 
could be a l ike. Thereby providing a possible solution to the similar rates of rep letion in the 
an imals consuming the m60 and m90 d iets. 
One of the a i ms of this study was to va lidate the pig let a s  a model that could be used to eva luate 
iron bioava ilabi lity. Althoug h direct compa risons of existing stud ies in iron bioava i la bil ity between 
pig lets a nd h u ma n s  are d ifficu lt to ca rry out as they a re measured using d ifferent haematologic 
pa rameters. A review of severa l experiments that investig ated the relationship between d iet type 
and iron bioava ilab il ity showed that Ma ssey U n iversity resea rch re su lts (this stu dy) using the 
pig let as an an imal model reflected the find ings of the h u man stud ies therefore provid ing further 
evidence that the p ig let is a good model for future human iron bioava i la bi l ity stud ies. 
Table 65 shows a summa ry  of the findings. 
Table 65: A comparison of changes in blood parameters in the human and piglet mode l .  
Diet Species HGB RBC Serum Repletion Absorption Refe rences 
Fe 
M i lk P ig t t = t Ma ssey Un iversity 
unpub l .  
H uman Hertrampf et a l .  
( 1998) 
Vit C P ig t t Ma ssey Un iversity 
(th is study) 
H u man t t Cook et al ( 2 00 1 )  
Cooking P ig t t t Ma ssey Un iversity 
(th is study) 
H uman t Ma rtinez et a l  ( 1998) 
Haem P ig t t t Ma ssey Un iversity 
(th is study) 
H u man t Roughead and Hunt 
2000 
1 09 
Chapter 7 
7. The utilisation of findings. 
The a i m  of this study was to eva luate a model, using piglets that could be used to evaluate the 
bioava ilability of iron from the whole d iet for humans. 
Bioava ilabi l ity is d ifficu lt to evaluate, a s  it is not a n  absolute va lue . Iron is extremely sensitive to 
the p H  changes that occur a l ong the digestive tract. Add itionally iron can form complicated 
associations with other d ietary nutrients that ca n  change the way in which it is a ssimi lated by the 
body. 
Iron deficient pig lets were used as h u ma n  models in a repletion study, wh ich evaluated the rate 
of regeneration of a nu mber of blood para meters . Following the consumption of d iets conta i n ing 
i n g redients typical of the human d iet. 
The results show that regene ra tion in the n umber of red cells, b lood haemoglobin concentration, 
mean ce l l  volume a nd mean ce l l  haemog lobin concentration a nd plasma iron is g reatest in g roups 
consuming a meat-based d iet (conta i ning 2 5 %  beef) . Comparative vegetab le - based d iets that 
contained either non-haem iron or non-haem iron that had been fortified with the organ ic acid 
vita min C had lower rates of regeneration . 
The d iffe rences in the rates of regeneration a re  ind icative of the d ifferences in the bioava ilabil ity 
of the two forms of d ietary iron . Meat iron is more bioava i lable than non-meat iron found in 
vegetable materia l .  This trend is maintained even when the bioava ilabil ity of non-meat iron is 
enha nced by the strategic use of vita m in C. 
Th is suggests that the most effective way of reducing the incidence of iron deficiency is to 
include meat a s  pa rt of d iet. Unfortunately a large proportion of the world s popu lation consume 
a vegetable based d iet . Therefore this g roup should be encouraged to increase the consumption 
of food s  conta i n ing vita min C the reby enha ncing the b ioava i la b i l ity of the non-meat i ro n .  
Although its use appears to b e  l imited, increasing red blood cel l  production rather than iron 
retention . 
Having established that the haem iron found mainly in meat regenerates the b lood para meters 
more effectively; a second experiment then evaluated the effects of p rocessing heat as most 
meat is cooked prior to consu m ption . Again the bioavailability of iron was assessed using a 
repletion study . 
The findings suggest that iron deficient subjects consuming cooked meat d iet have regeneration 
rates that a re h igher than those consuming e ither a d iet contai n ing raw uncooked meat .  Which i n  
turn has regeneration rates that a re h ig her than a d iet conta i ning milk p rote ins .  T h i s  suggests 
1 1 0 
that the cooking p rocess is in some way beneficia l, possibly by increasing the nutrient ava ilabil ity 
and d igestibi l ity of the d iet. 
I l l  
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Appendices 
Appendix 1 
The production of red blood cells 
[ . . .  ] th e  prod uction of red blood cells ( e rythropoiesis) takes place in the bone ma rrow. Red cell 
go through severa l stages of development before being released into th e  circu latory system. 
• Proerythroblast 
l 
• Basophilic erythroblast 
l 
• Polychromatoph ilic erythroblast 
Haemog lobin synthesis beg ins. 
l 
• Acidophilic erythroblast 
Haemoglobin synthesis is at maxim u m .  The Acidophilic erythroblast ejects its n ucleus 
a nd becomes a reticu locyte . 
l 
• Reticulocyte 
This ce l l  has the characteristic biconcave shape of the red b lood cell .  It contains a bout 
34% haemoglobin and reta in s  some mitochondria, ribosomes and endoplasmic reticul u m .  
They pass from t h e  red bone marrow into the b lood stream b y  squeezing between the 
endothelial cel ls of blood cap illaries. Deve loping into eryth rocytes or mature red blood 
cells 1-2 days after their re lease from the red bone marrow. 
l 
• Erythrocyte ( mature red blood cel l ) .  [ . . .  ] Totara and Reynolds- Grabowski 1996. 
1 1 8 
Append ix 2 
E ryth ropoiesis regu lation .  (Ada pted from Tota ra and Reynolds- Grabowski 1996) 
Normally e ryth ropoiesis a nd red blood ce l l  destruction p roceed at the sa me pace . If the oxygen 
carrying capacity of the blood fa l ls and oxygen delivery to the tissue is red uced then a negative 
feed back syste m increases red b lood ce ll prod uction . 
Homeostasis is d isru pted . 
l 
Oxygen d e l iver to tissues is reduced . 
l 
• Receptor cel ls located in the kidneys detect low oxygen level . 
l 
• Cells secrete erythropoietin ( EPO) a hormone that circulates to the red bone ma rrow . 
l 
• Erythropoietin is detected in the red bone ma rrow. 
l 
Erythropoietin induces proeryth robla sts in the red bone ma rrow to matu re more q u ickly 
into reticulytes. 
1 
Increased nu mbers of reticulytes enter the circulatory syste m .  
l 
• Thereby increasing the numbers of red blood cells in circu latio n .  
l 
Oxygen del ivery to the tissues is increa sed . 
l 
Return to homeostasis. 
1 1 9 
Appendix 3 
The red blood cell l ife cycle 
Red b lood cells have a l imited l ife span of a pproximately 120 days as they lack a nucleus and 
other orga nelles that a re needed to reproduce and ca rry out extensive metabolic activities. 
Worn out red ce lls a re removed from the circulation and destroyed by fixed phagocytic 
macrophages in the spleen a nd l iver a nd the breakdown prod ucts are recycled . 
Fig ure 26 shows the forma tion and destruction of red blood cells a nd the recycling and the 
brea kdown products. 
1 .  Macrophages in the splee n, l iver or red bone ma rrow phagocytize worn out red blood 
ce lls. 
2 .  The g lobin and haem portions of th e  haemoglobin a re split apart. 
3.  Globin i s  b roken down into amino acids. 
4. The a m ino acids a re reused in the synthesis of other proteins. 
5 .  T h e  iron is removed from t h e  haem portion . 
6 .  T h e  iron forms a n  association with a plasma protein called tra nsfe rrin, which 
tra nspo rts iron in the b lood . 
7. I n  muscle fibres, liver cells a nd macrophages of the spleen and liver, iron detach 
from transferrin and attaches to the iron storage proteins fe rritin a nd haemosideri n .  
8 .  U pon release from the storage site or a bsorption from the ga strointestin a l  tract iron 
attaches to transfe rrin . 
9. It i s  then tra nsported to the bone ma rrow, where red blood cell precu rsors take it up 
th rough receptor-mediated e ndocytosis. 
1 0 .  Where it is then used in the production of new haemoglobin molecu les. 
1 20 
1 1 .  Erythropoiesis in the red bone marrow results in the production of red blood cells, 
wh ich enter the circu lation syste m. 
12. The non-iron portion of the haem is converted to biliverd in a g reen pigment. 
13. Bi l iverdin is then converted into bi l iru b in, a n  orange pigment. 
14. Bi l iru b in enters the b lood and is transported to the liver. 
15. With in the liver b il iru b in is secreted by l iver cells into b ile, which is passed into the 
sma 11 intestine. 
16. I n  the la rge intestine bacteria convert b il iru bin into u rob ilinogen . 
1 7 .  Some urob ilinogen is absorbed back into the blood, conve rted into u robilin, a yellow 
pig ment a nd excreted in urine . 
1 8 .  Most u robil inogen is elim inated in faeces in the form of a brown p ig ment ca l led 
ste rcob i l i n .  
1 2 1  
Figure 26 : Formation and destruction of red blood cells and recycling of the haemoglobin 
components ( From The principles of anatomy a nd physiolog y gth ed ition . Tota ra and Reynolds­
Grabowski. Pg 560 . 
Red blood cell 
death and 
phagocytosis 
Macrophage in 
spleen, liver or 
red bone marrow 
Circulation for about 
1 20 days 
_ ... 
� c � Amino 
Globin 
Biliverdin -
acids 
,. 
\ 
Kidney 
fUrobilin 
I 
Reused for 
protein synthesrs Fe'+- Transferrin 
Ferritin and 
hemosiderin 
I 
Brlrrubln 
B1hrubm 
Small 
intestine 
Urobii�Bacteria 
, 
Stercobilin 
� L rge mtestine 
Unne Feces 
Erythroporesrs 111 
red bone marrow 
Key: 
in blood 
in bile 
1 22