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BLOOD METABOLITE AND HORMONE CONCENTRATIONS 
OF DAIRY CALVES DIFFERING IN GENETIC POTENTIAL 
FOR MILK FAT PRODUCTION 
A THESIS PRESENTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS 
FOR THE DEGREE OF MASTERATE OF AGRICULTURAL SCIENCE 
IN ANIMAL SCIENCE AT MASSEY UNIVERSITY 
NEW ZEALAND 
GUO QIANG XING 
1985 
I 
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I 
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MASSEY UNIVERSITY 
ii 
ABSTRACT 
The present study was conducted at the Massey University Dairy 
Research Unit to investigate the effect of genetic merit for milk fat 
production on the physiology and metabolism of Friesian calves. 
Twenty four Friesian calves divided into four groups namely High 
Breeding Index (HBI) heifers, HBI bulls, Low Breeding Index (LBI) 
heifers, and LBI bulls were challenged with four different experimental 
treatments, ie. fasting, feeding, intravenous arginine infusion, and 
subcutaneous synthetic corticosteroid injection at ten to eighteen days 
of age. A total of eighteen blood samples were collected from each 
calf through an indwelling jugular cannula and the concentrations of 
plasma glucose, insulin, GH and cortisol were determined. 
Some statistically significant differences were found in plasma 
metabolite and hormone concentrations between the HBI and LBI groups. 
1. The basal glucose concentration in HBI group was significantly 
higher than that in LBI group (P<0.05). The basal plasma insulin 
concentration was also significantly higher in HBI group than in LBI 
group (P<0.01). The basal GH concentration in HBI calves was higher in 
HBI calves than in LBI calves, but the difference was not quite 
significance at 5% level (P=0.059). 
2. Following feeding, plasma insulin and GH concentrations in HBI 
group were significantly higher than those in LBI group (P<0.01, P<0.05 
respectively). 
3. Acute intravenous arginine infusion induced hyperinsulinemia 
and hypoglycemia in all calves. LBI calves had significantly higher 
increments of plasma insulin measured as a percentage of basal levels 
than HBI calves. The response of GH concentration to arginine 
challenge differed significantly in terms of level and pattern between 
HBI and LBI groups, with the HBI calves having more prolonged higher GH 
concentration than LBI calves (P<0.05). 
4. Subcutaneous injection of synthetic corticosteroid resulted in 
significant increments in plasma glucose and insulin concentrations, 
and a significant decrease in endogenous cortisol production in all 
calves. (P<0.01, P=0.05, P<0.01 respectively). But no significant 
differences were detected between HBI and LBI groups. 
iii 
Effects of sex on plasma metabolite and hormone concentrations 
were also found in the present study. Plasma insulin concentration was 
consistantly higher in bulls than in heifers and the differences were 
significant at the time of fasting, after feeding, and after arginine 
infusion (P<0.01). Plasma glucose concentrations following feeding 
were significantly higher in bulls than in heifers (P<0.05). GH 
concentration was slightly but not significantly higher in bulls than 
in heifers for most of the experiment. 
It was concluded that differences exist in some important 
metabolic and endocrinological characteristics between HBI and LBI 
calves, and these differences could become significant under certain 
physiological conditions and experimental treatments such as those 
applied in the present study. This study also showed the promise of 
identifying genetically superior Friesian dairy cattle at an early age 
by using physiological markers. However this possibility has yet to be 
tested by earring out measurements on calves for which breeding index 
value for milk fat production will be determined by methods such as 
progeny test. 
iv 
ACKNOWLEDGMENT 
I am grateful to my supervisors Professor D.S. Flux and Dr. D.D.S. 
MacKenzie for their invaluable guidance and encouragement during the 
period of the project. 
I also wish to thank the following : 
Dr. G.F. Wilson for help with the cannulation and sampling 
of the calves. 
Mr B. Parlane of the Animal Physiological Unit and farm staff of the 
Dairy Research and Development Unit for management of the calves. 
Mrs.J. Bridges for cortisol assay and Mrs.J. Rumbal for her technical 
assistance. 
Mr. D.J. Garrick for assistance in appling REG programmes 
for repeated measurments. 
Dr. S.N. McCutcheon for consrtuctive discussion. 
Miss C.Y.Z. Pang and Miss F.L.H. Chee for typing the thesis. 
The Animal Science Department for the fine environment and 
pleasant atmosphere. 
My friends for their support, encouragement, and challenge. 
The Chinese Educational Ministry for the financial support which has 
enabled me to undertake the present study. 
ABSTRACT 
ACKNOLEDGEMENTS 
LIST OF FIGURES 
LIST OF TABLES 
CHAPTER ONE 
CHAPTER TWO 
2. 1 
2. 1. 1 
2.1.1.1 
2.1.1.2 
2.1.1.3 
2.1.1.4 
2.1.2 
2.1.2.1. 
2.1.2.2 
2.1.2.3 
2.1.2.4 
2.1.2.5 
2.1.2.6 
2.1.3 
2.1.3.1 
2;1.3.2 
2.1.3.3 
2.1.3.4 
2.1.3.5. 
2.2 
2. 2. 1 
2.2.2 
2.3 
CHAPTER THREE 
3.1 
v 
TABLE OF CONTENTS 
page 
ii 
iv 
viii 
ix 
INTRODUCTION 1 
REVIEW OF THE LITERATURE 3 
Regulation of plasma insulin, growth hormone 3 
and cortisol concentrations 
Regulation of plasma insulin concentration 3 
Plasma metabolites and ions 3 
Enteric and pancreatic hormones s 
Other hormones and central nervous system 6 
Insulin receptor 8 
Regulation of plasma GH concentration 9 
Hypothalamic control 9 
Somatomedins 11 
Glucocorticoids 12 
Plasma metabolites 12 
Different physiological stage 14 
Secretion rhythm of GH 16 
Regulation of cortisol concentration 16 
ACTH and CRF 16 
Cortisol binding globulin 17 
Circadian fectors 19 
Stress 20 
Age 20 
Digestive metabolism and glucose homeostasis 20 
in young milk fed calves 
The Digestive Process 20 
Blood glucose homeostasis 21 
a) after feeding 21 
b) fasting 22 
Objectives of the present study 23 
MATERIALS AND METHODS 26 
The animals 26 
3.2 
3.3 
3.4 
3.5 
3.6 
3.7 
3.8 
3.9 
3. 10 
CHAPTER FOUR 
4.0 
4.1 
4.2 
4.3 
4.4 
4.4.1 
4.4.2 
4.5 
4.6 
CHAPTER FIVE 
5.1 
5.2 
5. 2. 1 
5.2.2 
5.2.3 
5.2.4 
Rearing of Calf 
Reconstituted milk 
Procedure for cannulation 
Blood sampling 
Measurement of Haematocrit 
Measurement of Immunoglobulin G1 
Glucose assay 
Radioimmunoassay of Insulin, GH and Cortisol 
1 )Hormones used in the RIAs 
2) Iodination 
3) Antisera 
4) RIA procedure for GH, insulin and cortisol 
5) RIA Data Transformation 
6) Parellelism in RIA 
Experimental procedure and statistical analysis 
1) Procedure 
2) Data analysis 
RESULTS 
General results 
Before feeding 
After feeding 
After arginine challenge 
After synthetic corticosteroids injection 
Comparison of pre-injection value with the 
after-injection value 
Comparison between calves of different BI and sex 
The relationship of BI and plasma metabolite 
and hormone concentrations 
Other physiological traits 
DISCUSSION 
General discussion 
Effect of fasting, feeding, arginine infusion 
and synthetic corticosteroid injection on 
plasma glucose, and hormone concentrations 
Effect of fasting 
Effect of feeding 
Effect of arginine challenge 
Effect of synthetic corticosteroid injection 
vi 
26 
29 
30 
30 
31 
31 
32 
32 
32 
32 
33 
33 
33 
35 
36 
36 
38 
40 
40 
40 
57 
60 
68 
68 
68 
70 
73 
75 
75 
76 
76 
77 
78 
79 
5.4 
5.5 
CHAPTER SIX 
APPENDICES 
REFERENCES 
vii 
Effect of genetic merit for milk production on 
plasma metabolite and hormone concentration 
1) Glucose 
2) Insulin 
3) GH 
Sex effect 
Estimation of calf genetic merit by using metabolite 
and hormone concentrations as gene markers 
SUMMARY AND CONCLUSIONS 
81 
81 
83 
84 
85 
86 
88 
91 
96 
LIST OF FIGURES 
Figure 
Control of Pituitary-adrenal function. 
2 Effects of fasting, feeding, and arginine 
infusion on plasma glucose concentrations 
18 
in groups of calves of different BI and sex. 41 
3 Effects of fasting, feeding, and arg1n1ne 
infusion on plasma insulin concentrations 
in groups of calves of different BI and sex. 42 
4 Effects of fasting, feeding, and arg1n1ne 
infusion on plasma GH concentrations in groups 
of calves of different BI and sex. 43 
5 Effects of fasting, feeding, and arginine 
infusion on plasma cortisol concentrations 
in groups of calves of different BI and sex. 44 
6 Effect of synthetic corticosteroid injection 
on plasma glucose concentrations in groups of 
calves of different BI and sex. 45 
7 Effect of synthetic corticosteroid injection 
on plasma insulin concentrations in groups of 
calves of different BI and sex. 46 
8 Effect of synthetic corticosteroid injection 
on plasma GH concentrations in groups 
of calves of different BI and sex. 47 
9 Effect of synthetic corticosteroid injection 
on plasma cortisol concentrations in groups 
of calves of different BI and sex. 48 
10 Relationship between breeding index of calves and 
the basal concentration of glucose, insulin, GH, 
and cortisol in the plasma within BI and sex group. 72 
11 Effect of arginine infusion on plasma 
glucose cocentrations in individual 
calves of different BI and. sex group. 61 
12 Effect of arginine infusion on plasma 
insulin concentrations in individual 
calves of different BI and sex group. 62 
13 Effect of arginine infusion on plasma 
GH concentrations in individual calves 
of different BI and sex group. 63 
14 Effect of arginine infusion on plasma 
cortisol concentrations in individual 
calves of different BI and sex group. 64 
viii 
LIST OF TABLES 
Table 
3.1 Number,Breeding Index,sex,date of birth, birth 
weight,body weight and age at cannulation 
of calves used in the experiment. 
3.2 Expected means, standard deviations, ranges and 
the standard errors in Breeding Index units 
of the HBI and LBI groups. 
3.3 General calf rearing procedure. 
3.4 The amount of milk intake of calves at each meal. 
3.5 Summary of experimental procedure. 
4.1 Analysis of variance of repeated measurements of 
basal glucose concentration between groups 
of calves of different BI and sex. 
4.2 Analysis of variance of repeated measurements 
of basal insulin concentration between groups 
of calves of different BI and sex. 
4.3 Analysis of variance of repeated measurements 
of basal GH concentration between groups of 
calves of different BI and sex. 
4.4 Analysis of variance of repeated measurements 
of basal cortisol concentration between groups 
of calves of different BI and sex. 
4.5 Analysis of variance of repeated measurements 
of after-feeding glucose concentrations between 
groups of calves of different BI and sex. 
4.6 Analysis of variance of repeated measurements 
of after-feeding insulin concentrations between 
groups of calves of different BI and sex. 
4.7 Analysis of variance of repeated measurements 
of after-feeding GH concentrations between 
groups of calves of different BI and sex. 
4.8 Analysis of variance of repeated measurements 
of after-feeding cortisol concentrations between 
groups of calves of different BI and sex. 
4.9 Analysis of variance of repeated measurements 
of glucose concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 
ix 
page 
27 
28 
28 
29 
37 
49 
49 
50 
50 
51 
51 
52 
52 
53 
Table 
4.10 Analysis of variance of repeated measurements 
of insulin concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 
4.11 analysis of variance of repeated measurements 
of GH concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 
4.12 analysis of variance of repeated measurements 
of cortisol concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 
4.13 Analysis of variance of repeated measurements 
of glucose concentrations in response to synthetic 
corticosteroid injection between groups of calves 
of different BI and sex. 
4.14 Analysis of variance of repeated measurements 
of insulin concentrations in response to synthetic 
corticosteroid injection between groups of calves 
of different BI and sex. 
4.15 Analysis of variance of repeated measurements 
of GH concentrations in response to synthetic 
corticosteroid injection between groups of calves 
53 
54 
54 
55 
55 
of different BI and sex. 56 
4.16 Analysis of variance of repeated measurements 
of cortisol concentrations in response to synthetic 
corticosteroid injection between groups of calves 
of different BI and sex. 56 
4.17 Analysis of covariance of repeated measuements 
of glucose concentration in response to feeding 
between groups of calves of different BI and sex. 58 
4.18 Analysis of covariance of repeated measurements 
of insulin concentrations in response to feeding 
between groups of calves of different BI and sex. 58 
4.19 Analysis of covariance of repeated measurements 
of GH concentrations in response to feeding 
between groups of calves of different BI and sex. 59 
4.20 Analysis of covariance of repeated measurements 
of cortisol concentrations in response to feeding 
between gro~ps of calves of different BI and sex. 59 
X 
Table 
4.21 Analysis of covariance of repeated measurements 
of glucose concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 66 
4.22 Analysis of covariance of repeated measurements 
of insulin concentrations in ~esponse to arginine 
challenge between groups of calves of different 
BI and sex. 66 
4.23 Analysis of covariance of repeated measurements 
of GH concentrations in response to arginine challenge, 
between groups of calves of different BI and sex. 67 
4.24 Analysis of covariance of repeated measurements 
of cortisol concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 67 
4.25 Mean plasma glucose, insulin, GH and cortisol 
concentrations of calves on day one before, 
and on day two after synthetic corticosteroid 
injection, together with the statistical 
significance of the difference. 69 
4.26 Correlations between BI and concentrations of 
glucose, insulin, GH and cortisol in calf plasma 
sampled at four different stages of the experiment. 71 
4.27 Mean values of birth weight, IgG1 concentration 
and haematocrit in calf groups of different BI and sex.74 
xi 
CHAPTER ONE: INTRODUCTION 
The Usefulness of physiological markers in dairy cattle breeding 
It has long been desired ,as recently discussed in several 
papers (Gorski, 1979; Kiddy 1979; Linstrom, 1982; Peterson et 
al,1982; Land et al, 1981) to be able to identify genetically 
superior dairy cattle from their contemporaries at an early stage of 
their life by using physiological or biochemical markers which are 
genetically correlated with milk production. 
A dairy breeding system using such markers would have advantages 
over the conventional breeding technique, progeny test, in several 
respects:1) reduced breeding cost, 2) more intensive selection of 
bulls, as a large population of animals can be tested for the marker, 
3) shorter generation intervals, due to the early intensive use of 
the identified bulls in the artificial insemination scheme. 
Animal breeding programmes for dairy cattle, so far, have been 
based on the principles of quantitive genetics. These programmes are 
expensive and genetic improvement is slow. In New Zealand, contracts 
are made each year by the Dairy Board with individual farmers to 
purchase about 150 newborn dairy bulls (including both Friesian and 
Jersey breeds) for progeny testing. All these bulls are kept in the 
breeding centre till they are 5 years old, waiting the assessment of 
their daughters production. Thereafter only a few of the proven 
bulls are used intensively in the AI scheme while others are culled. 
Nevertheless this conventional breeding method has proved reliable 
and rewarding in dairy herd improvement as witnessed by the great 
improvement in milk production of Friesian cattle both around the 
world (Jasiorowski et al, 1983) and within New Zealand (Bryant et al, 
1981; Davey et al, 1983; Wickham et al, 1978). This provides the 
opportunity to compare the physiological differences between two 
groups of genetically diversed Friesian cattle, a study of which may 
lead to the identification of suitable gene markers which could ' in 
turn, aid in further improvement of dairy cattle production. 
Previous work on early identification of genetically superior 
dairy cattle 
2 
Though the idea might not be new, the literature contains very 
few reports on early identification of dairy cattle which are 
genetically superior for milk production. A study reported by 
Tilakaratne et al (1980) indicated that dairy calves with different 
genetic merit might be identified by measuring the concentrations of 
certain plasma metabolites because they found significant differences 
in the concentrations of total protein and urea between the high and 
low genetic merit calves. 
Osmond et al (1981) showed however, that simple spot estimates 
of plasma concentrations of tri-iodothyronine (T3), thyroxine (T4) 
and insulin from 2 to 7 years old Friesian bulls did not supply 
useful predictors of their daughters milk production. 
Studies with lactating Friesian cows of known genetic merit in 
this Department showed that high breeding index (HBI) cows differed 
from low breeding index (~BI) cows in certain important metabolic 
aspects (Flux et al, 1984). Plasma glucose,insulin and growth 
hormone (GH) concentratioris in HBI cows were significantly higher 
than in LBI cows when feed intake was restricted. 
The present study 
The aim of the present study was to investigate the 
physiological attributes of young pre-ruminant HBI and LBI calves 
with similar genetic backgrounds to those of the cows used by Flux et 
al (1984).The means of the breeding indices based on ancestry were 
134.8 and 104.3 for HBI and LBI calves respectively. Plasma 
concentrations of glucose,insulin,GH,and cortisol were chosen as the 
markers to be examined , not only because they are of great metabolic 
importance to calf development in general,but also due to the fact 
they are at least equally important to the metabolic economy of 
lactating cows (Cowie et al, 1980). Thus differences in the 
physiological attributes between the HBI and LBI calves may also 
persist in the adult animals. Therefore the review of the literature 
is focused on the mechanisms controlling the plasma concentration of 
the metabolite and hormones of particular interest to the present 
experiments. 
• 
3 
CHAPTER TWO: REVIEW OF THE LITERATURE 
2.1 Regulation of plasma insulin, GH, and cortisol concentrations 
2.1.1 Regulation of plasma insulin concentration 
2.1.1.1 Plasma metabolites and ions: 
Changes in plasma glucose concentration are of recognised 
importance in regulating insulin secretion in ruminants (Bassett 
1975,1981), as well as in other species (O'Connor et al, 1977). The 
exact mechanism by which glucose stimulates insulin secretion is 
still unknown. Two mechanisms so far have been proposed: 1) glucose 
may act by interacting with specific membrane receptors on the B-cell 
from which a secondary signal may originate , regardless of the 
further glucos_e metabolism within B-cell. 2)glucose may act as a 
substrate, the stimulus for insulin release being due to a metabolite 
or cofactor arising during its metabolism within the B-cell 
(Belfiore, 1980). Studies with isolated pancreatic B-cells show that 
prolonged stimulation with glucos~ induces a biphasic insulin 
secretory response characterised by an initial rapid phase lasting 
about 5 minutes during which about 2-3 percent of the pancreatic 
insulin content is released, followed by a slow increase in insulin 
secretion which results in the liberation of up to 20 percent of 
total pancreatic insulin content (O'Connor et al, 1977). It has been 
suggested that two, one small and one larger, pancreatic components 
of insulin are responsible for the minor and major insulin secretion 
peaks respectively (Belfiore, 1980). Glucose besides its stimulating 
effect on insulin release, also stimulate insulin synthesis probably 
at the transcriptional and post-transcriptional levels but most of 
the insulin released initially upon glucose stimuli comes from the 
hormone stored in the secretory granul~s (Randal and Hales, 1972). 
Some amino acids (AA) especially arginine,lysine, leucine, and 
phenylalanine, are very effective in stimulating insulin secretion in 
several species (reviewed by Parman, 1979) including ruminants 
(Hertelendy et al, 1970, Davis, 1972; Stern et al, 1971; Bohn 
1978). Amino acids do not appear to act as insulin secretagogues by 
4 
serving as fuels forB-cells (Hellman et al, 1971) but probably by 
potentiating glucose-stimulated insulin release (Basabe et al, 1971). 
It has been found amino acids greatly stimulate lactate formation 
from glucose (Pace et al, 1975). This effect may play a significant 
role in regulating the extent to which glucose serves as a stimulus 
for insulin secretion by pancreatic islets. 
Free fatty acids (FFA) in man enhance the secretory response of 
insulin to glucose (Balasse and Oans, 1973). Medium chain 
triglycerides also induced insulin secretion in dogs (Bach et al, 
1974). In adult and young ruminants, volatile fatty acid (VFA), 
especially propionic acid and butyric acid are better stimuli for 
insulin release than glucose (Bassett, 1975; Manns and Boda, 1967; 
Horino et al, 1968; Ambo et al, 1973; Bohn, 1978). 
High and normal extracellular calcium ion concentrations 
stimulate insulin release by increasing the amount of calcium ions 
entering into the B-cell (reviewed by Wollheim, 1981). The full 
development of the insulin response to physiological stimuli requires 
the presence of extracel1ular calcium. It is inferred that glucose 
increases the cytosolic calcium ion concentration of the B-cell by 
stimulating a rapid entry of calcium ions across the plasma membrane 
into the cell after an imbalance of calcium ion flux across the 
membrane. It is proposed that the biphasic pattern of 
glucose-induced insulin secretion can be accounted for by a biphasic 
changes in the concentration of calcium across the B-cell membrane. 
The amino acids (AA) also require the presence of extracellular 
calcium to stimulate insulin release (Wollheim, 1981). 
The physiological significance of insulin responses to nutrient 
entry into the circulation lie in that insulin is the major anabolic 
hormone. Its initial short-term action is to maintain nutrient 
homeostasis by promoting uptake of glucose, FFA, and AA by the 
peripheral tissue (cells), especially muscle, adipose and connective 
tissues, and increasing the synthesis of lipid and protein in these 
tissues. It also minimizes the endogenous production of glucose, FFA 
and AA by antagonizing the catabolic effect of many other hormones, 
thus leading to a decrease in the plasma concentration of the 
metabolites. 
5 
2.1.1.2 Enteric and pancreatic hormones 
Gut hormones: Many gastrointestinal hormones have been 
demonstrated to have a stimulatory effect on insulin release 
following feeding. These include gastrin, cholecystokinin (CCK), 
secretin, gut-glucagon, cerulein, vasoactive intestinal peptide 
(VIP), and gastric inhibitory polypeptide (GIP) (Misra, 1980; 
Bertaccini, 1982 Adrain and Blam, 1982). However so far only GIP has 
been shown to be physiologically significant. The mechanisms as to 
how these gut hormones affect insulin release is still poorly 
defined. Neurotensin has also been considered a very potent 
regulator of insulin and glucagon secretion in the calf (Blackburn et 
al, 1 980) . 
Somatostatin (SLI): Soon after the discovery of the presence of 
somatostatin (SLI) in the hypothalamus, it was found that SLI is also 
present in many other tissues. It is now established that SLI has a 
widespread influence on the ov~rall metabolism of the animal by its 
\ 
inhibitory effect on the secretion of many hormones, including GH, 
thyrotropin, prolactin, insulin, glucagon, and most of the gut 
hormones (Gerich, 1981). It has been proposed that the insulin 
release in response to a meal may be attenuated by SLI (Schusdziarra, 
1980) to avoid abrupt metabolic disturbances which could be induced 
by hyperinsulinemia during a meal. SLI may also decrease insulin 
response to a meal by inhibiting the secretion of gut hormanes that 
control the digestion and absorption of nutrients. Thus reducing gut 
motility, gastric and pepsin secretion, pancreatic fluid, electrolyte 
and enzyme production, splanchnic blood flow as well as affecting 
many other vital processes (Vale et al, 1975; Gerich , 1977; 
Gerich, 1981; Effendic et al, 1978). At present, however, there is 
a lack of knowledge concerning the role of SLI in the regulation of 
insulin secretion in ruminants. 
Glucagon: The role of glucagon as an antagonist of insulin 
action in blood glucose homeostasis has been discussed in several 
papers (Bassett, 1975, 1978, 1980; McDowell 1983; Unger et al, 
1977; Lefebvre and Luyckx, 1979). It is established firmly that 
glucagon has a direct stimulatory effect on insulin secretion (Samols 
6 
et al, 1965; Mayhew et al, 1969). Insulin release in response to 
glucagon infusion has also been observed in ruminants (Bassett,1971). 
It is proposed that there is a insulin-glucagon negative feedback 
mechanism in which insulin suppresses glucagon while glucagon 
stimulates insulin secretion (Samols et al, 1972). A recent 
morphological study of the islets of Langerhans suggested that 
insulin release from the B-cell may be less inhibited by SLI from the 
D-cell than glucagon fran the A-cells (Unger and Orci 1976). So far, 
however, it is not clear to what extent insulin secretion is 
stimulated by levels of circulating nutrients and by glucagon. 
2.1.1.3 Other hormones and the central nervous system (CNS): 
GH: Whether the naturally occuring sudden rise in GH release is 
capable of stimulating pancreatic insulin secretion has not been 
established. It has been reported that perfusion of an isolated rat 
pancreas preparation with.bovine or ovine GH produced a transient 
insulin and glucagon secretion. (Tai and Pek, 1976). Acute pancreatic 
insulin and glucagon release was also observed in dogs following 
intravenous injection of GH (Sirek et al, 1979). Insulin 
concentration was raised in cows f.oll owing GH treatment in one study 
(Bines et al, 1980), but not in another (Peel. et al, 1983). Another 
study showed that highly purified hGH did not affect insulin release 
(Larson et al, 1978). Thus there is no conclusive evidence that GH 
has a direct effect on the endocrine pancreas. There is the 
possibility that contaminants in the GH administered to the animals 
in the above experiments may be responsible for the observed 
pancreatropic effect. Somatanedins should be ruled out from these 
contaminants as it requires several hours to attain a measurable 
increase in its activity (Wyk et al, 1973). It is also possible GH 
can affect insulin concentration by regulating the affinity of 
insulin receptors (Lwasaki, 1982). Thus the mutual relationship 
between GH and insulin is still poorly understood as it appears that 
certain amino acids stimulate the secretion of both insulin and GH, 
but for insulin the relationship to acute energy substrate levels may 
be more overiding than for GH. High insulin concentrations were 
found in acromegalic patients whose GH level was high (Yallow and 
Berson, 1960). Higher insulin and GH levels were also found in 
energy restricted HBI cows than in LBI cows (Flux et al 1984). In 
these cases insulin secretion may be stimulated to antagonize the 
diabetogenic and lipolytic effect of GH. 
7 
Cortisol: It is known that alteration in adrenal cortical 
function can dramatically perturb insulin secretion in experimental 
animals and in man. Decreased concentration of glucocorticoids are 
associated with decreased insulin secretion while either endogenous 
or exogenous hyperadrenocorticoidism is associated with increased 
insulin secretion (Kinash and Hasi t, 1954; Malaisse et al, 1967a, 
1967b; Rastogi and Campell, 1970; Arkerblom et al, 1973; Curry and 
Bennett, 1973; Bassett and Wallace, 1967) and these alterations in 
insulin concentration could be due to either a change in insulin 
secretion or a change of insulin binding to its receptor on 
peripheral tissue. Recent studies have demonstrated the presence of 
steroid receptors in islet cells (Green et al, 1978; Tesone et al, 
1979). While other expertments have demonstrated the direct effects 
of glucocorticoids on irisqlin .secretion (Billaudel et al, 1978; 
\ --
Brunstedt et al, 1979, 1981; Borelli et al, 1982). Insulin 
secretion may be stimulated by corticosteroids through the following 
ways:1) increased glucose metabolism in B-cells. 2) increased 
calcium uptake by B-cells. 3) increased CAMP production by B-cells. 
4) hyperglycemia. Glucocorticoids may also affect insulin 
concentration by acting on insulin receptors on peripheral tissue. 
Catecholamines: It is now firmly established that a-adrenergic 
activation has an inhibitory control, and s-adrenergic activation has 
a stimulatiory effect on insulin secretion (Mayhew et al, 1969; 
Rossini and Buse, 1973). Noradrenalin is an almost pure a-adrenergin 
agonist, while adrenalin can stimulate both a- and s-adrenergic 
receptors in the islets equally well. Both catecholamines are 
directly under sympathetic nervous control. However the 
physiological relevance of both adrenalin and noradrenalin released 
from nerve terminals in response to sympathetic nervous activity is 
thought to be negligible except during acute muscle exercise,or in 
response to stress when the dual effect of stimulating glucagon and 
inhibiting insulin would be expected to encourage hepatic glucose 
release. Because of the short half-life of catecholamines in plasma, 
their effect on the pancreatic endocrine function may also be a 
temporary one. 
8 
Central Nervous System (CNS): Recent studies show that the 
functions of the islets are under the integrative control of the CNS 
(Pearse and Polak, 1978). The islets of Langerhans are extensively 
innervated by adrenergic, cholinergic and peptidergic nerve fibers. 
It has been suggested that the first phase of insulin secretion in 
response to a meal or oral glucose is due to the cooperativity 
between sympathetic and parasympathetic systems (Burr et al, 1976). 
However, the CNS involvement in regulation of pancreatic secretion is 
yet to be fully investigated. 
2.1.1.4 Insulin receptor: 
The insulin receptor is defined as the molecular structure on 
the cell surface which is ·capable of recogniszing and binding insulin 
in a specific manner. Ins,ulin and the receptor then forms a complex 
which is involved in the initiation of the stimulus-response 
sequence. Insulin binding to the receptor is a necessary but not 
sufficient condition for insulin action. The hormone receptor 
complex must be linked to an effector system for a biological 
response to be expressed (Kahn, 1976). It is now believed that the 
number and affinity of insulin receptors can be affected by several 
factors. Insulin and analogoes can directly down regulate the number 
of insulin receptors (Baret al, 1979; Olefsky and Reaven, 1977; 
Olefsky and Ciaraldi, 1981). The binding characteristic of remaining 
receptors are unaltered. Short term fasting leads to a striking 
increase in insulin binding to its receptors (Olefsky, 1976) due to 
an increase in receptor affinity. In both man and animals, a high 
carbohydrate intake will result in a decreased affinity of insulin 
receptors on adipose tissue (Olefsky and Saekow, 1978). It was also 
found that in the long term insulin binding to the receptor may be 
controlled by altering the receptor number with binding affinity 
returning to normal (Kolterman et al, 1980; Yasuda and Kitabchi, 
1 980). 
It is proposed that maximal insulin stimulation of glucose 
uptake occurs when only a small fraction ,about ten percent, of total 
9 
adipose insulin receptors are occupied (Kono and Barharm, 1971; 
Gammeltoft and Gliemann, 1973). This suggests that the cellular 
response to insulin increases with receptor occupancy until a certain 
level of occupancy is reached (10% of available receptors) and that 
further increases in receptor occupancy will not lead to further 
increases in cellular.response. This also suggests that increased 
plasma insulin level may be either due to an increase in insulin 
secretion or a decrease in insulin binding to the receptor. Thus 
some factor which can affect insulin concentration may act on the 
regulation of insulin receptor affinity. Decreased insulin binding 
has been found in animals treated with glucocorticods (Olefsky et al, 
1975; Kahn et al, 1978). In acromegaly insulin binding to its 
receptor may be enhanced by an elevated GH level (Muggeo et al, 
1977). 
2.1.2 Regulation of plasma GH concentration 
2.1.2.1 Hypothalamic control: 
The mechanism controlling GH secretion is complex and work in 
this field is far from conclusive for any species, but it is now 
accepted that hypothalamic control is mainly responsible for the 
rhythmic release of GH and similar mechanisms exist in all species 
(reviewed by Chiodini and Liuzzi, 1979; Sonksen and West, 1979; 
Cockram et al, 1983). 
GH secretion from the anterior pituitary is regulated by a dua 
of hypothalamic control. Growth hormone releasing factor (GHRF) 
stimulates and GH release inhibitory factor (GHRIF) or somatostatin 
(SLI) inhibits GH secretion. The basal GH level is thought to be 
regulated by SLI while the episodic spike of GH release occurs when 
GHRF predominates over the inhibitory action of SLI which has a short 
half life in the circulation. Besides supressing GH release SLI has 
also been found to have widespread inhibitory effects on the release 
of other hormones, such as thyrotrophin, (TSH), prolactin, insulin, 
glucagon and many gut hormones which might indirectly influence GH 
secretion (Gerich, 1981). GH concentration can be depressed by SLI 
treatment in sheep (Redekopp et al, 1980). Depressing SLI 
concentration with antibody against SLI has resulted in increased GH 
1 evel in lambs. (Varner et al , 1980) • It was believed that SLI 
regulates GH secretion by a short loop negative feedback mechanism. 
10 
The neural mechanisms which control GH secretion via their 
influence on hypothalamic release of GHRF and SLI has also been 
extensively reviewed in the literature (Sonksen and West 1979; 
Cockram et al, 1983; ,Chiodin and Liuzzi, 1979). In summary, the 
high concentration of monoamines (noradrenalin, dopamine, and 
serotonine) in the hypothalamus, particularly in the median eminence 
region, suggested that these may be important in the regulation of 
hypothalamo-hypophysiotrophic hormones. In man and primates there is 
a stimulating effect of the central noradrenergic system operating 
through a-adrenergic receptors, while s-receptors appear to be 
inhibitory. The central noradrenergic system appears to represent 
the major drive for GH secretion in the primate and rat. Recent 
evidence in rats suggest the noradrenergic system mediates GH 
secretion by stimulating GHRF release rather than by inhibiting SLI 
release (Eden et al, 1981 L 
The central serotonin~rgiC. system also has a stimulatory effect 
I 
on GH release and it is suggested that the serotoninergic system is 
involved in the physiological regulation of GH as blockade of 
serotonin receptors in man suppress both sleep related GH secretion 
(Chihara et al, 1976) and GH release in response to insulin 
hypoglycemia (Smythe and Lazarus, 1974). 
The role of dopaminergic system is less clearly defined. L-dopa 
which crosses the block-brain barrier, generally evokes GH secretion 
although it may reduce the GH response to hypoglycemia, suggesting a 
dual action in GH release. The dopaminergic system possibly acts to 
augment basal and reduce stimulated GH release (Cockram et al, 1983). 
Vasoactive intestinal polypeptide (VIP) now widely accepted as a 
gastrointestinal hormone has been found in the hypothalamus of many 
species. Evidence shows that VIP secreted from the hypothalamus 
possibly acts as a hypothalamic neurohormone. GH release can be 
stimulated by VIP both at the hypothalamic and anterior pituitary 
levels (reviewed by Nicosia et al, 1983). This stimulating effect of 
VIP may due to its inhibition of SLI release as shown in rat 
(Epelbaum et al, 1979). However the physiological role of VIP in 
regulating GH release is yet to be defined. 
Other hypothalamic hormones may also influence GH release 
directly or indirectly. Thyrotrophin releasing hormone is a potent 
stimulus for GH release. Overall it appears that GH secretion is 
regulated by the integrated action of may hypothalamic hormones. 
2.1.2.2 Sanatanedins (SM): 
11 
Considerable evidence suggests the existence of short loop 
negative feedback regulation of GH secretion. Though SLI may 
regulate GH release by such a mechanism at the hypothalamic level 
(Molitch and Hlivyak, 1980), many studies suggested that sanatomedins 
(SM) may be partly responsible for the negative feedback control of 
GH secretion (Berelowitz et al, 1981; Abe et al, 1983; Tannenbaum 
et al, 1983). Low concentrations of the SM and high concentration of 
GH are found in Lars'()n Dawrfism, in which GH does not stimulate SM 
jj/ 
production. Sanatanedin C not only stimulates SLI release fran the 
rat hypothalamus, but also acts directly on the anterior pituitory 
cells to suppress GH release (Berelowitz et al, 1981). In these 
studies insulin-like growth fac.tor II was less potent. It is also 
\ 
suggested that SM may be generated locally in the pituitary or 
hypothalamus which is involved in the feedback control of GH release 
(Abe et al, 1983). GH also appeared to exert negative feedback 
effects on its own secretion. It is proposed .that short term 
autoregulation is via GH effects on hypothalamus SLI or possibly GHRF 
secretion, whereas longer term effects may be via the somatamedins 
(Abe et al, 1983). 
12 
2.1.2.3 Glucocorticoids: 
The effect of glucocorticoids on GH secretion has been reviewed 
by Chiodin and Liuzzi (1979). In summary, administration of 
glucocorticoids can impair the secretion of GH in humans as well as 
in other species. Chronic administration of cortisol resulted in 
impaired release of GH in response to insulin hypoglycemia, and 
smaller dosage of cortisol resulted in less consistent inhibition. 
Large and acute doses of cortisol can also suppress GH release 
induced by hypoglycemia. Sleep-related GH secretion is not affected 
by acute cortisol administration but is impaired by chronic 
treatment. There is at present no satisfactory explanation for the 
inhibitory activity of glucocorticoids on GH secretion. Cortisol in 
low concentration stimulated GH release from normal human pituitary 
in vitro (Bridson and Kohler 1970) but inhibited GH release from rat 
pituitary when given in high concentrations (Birge et al, 1967). It 
is not know at what stage GH concentration is affected by cortisol, 
synthesis, secretion or af~er secretion events. 
2.1.2.4 Metabolites: 
Energy substrate: In relation to plasma levels other than amino 
acids GH appears to be diametrically opposed to insulin. Whereas 
insulin is secreted in response to high glucose concentration, GH is 
often stimulated by hypoglycemia including that induced by 
administration of insulin. It is noted that a rapid decrease in 
blood glucose was always effective in inducing GH release, regardless 
of the absolute glucose levels involved. (Roth et al, 1964). 
Similar responses were observed in ruminants (Stern et al, 1971; 
Wallace and Bassett., 1970). It is suggested that there is a 
hypoglycemic threshold for GH release (West and Sonksen, 1977). 
Conversely, hypoglycemia has been found to inhibit basal GH secretion 
and arginine or exercise induced GH secretion, but does not block 
sleep or stress induced GH release. It is believed that the 
hypothalamus contains areas which act as glucoreceptors and are 
important in relation to GH secretion. It has been observed that 
some adaptation of glucoreceptors occurs in relation to chronic blood 
13 
glucose changes during recovery from hyperglycemia even though 
absolute blood glucose levels are high (Irie et al, 1967). Glucose 
perfusion of the median eminence region has blocked GH response to 
insulin hypoglycemia in monkeys (Blanco et al, 1966) and induction of 
intracellular glucopenia in the hypothalamus has stimulated GH 
secretion in monkeys ~Himsworth et al, 1972). These data suggest 
that the lateral hypothalamus is sensitive to a fall in blood glucose 
and the ventro medial nucleus to a rise in blood glucose 
concentration. 
Prolonged fasting in many species is characterized by rises in 
plasma concentration of free fatty acids (FFA) and ketone bodies. It 
is currently held that the increased FFA concentration in starvation 
results from the lipolytic effect of the raised GH concentration and 
less of the antilipolytic action of insulin due to reduced insulin 
secretion. So far, however, there is no convincing evidence that FFA 
concentration directly affects GH release. Some evidence shows that 
an inverse relationship exists between plasma concentration of FFA 
and GH (Quabbe et al·, 1977; H~rtelendy and Kipnis, 1973). It is 
thought that the inhibitory effect of FFA on GH release may be 
transferred through serotonin. 
Increased GH secretion as a result of low plasma energy 
substrate levels has been found in fasted laborotroy animals 
(Gonzales et al, 1981). In ruminants limited energy intake caused 
higher GH levels in control animals than in animals fed ad lib 
(Purchas et al, 1971; Hove and Blom, 1973; Flux et al, 1984). The 
main chronic effect of GH on energy substrate is to decrease the 
transport of glucose into cells and to promote lipolysis perhaps by 
increasing the responsiveness of the adipose tissue to other 
lipolytic agents (Peel et al, 1981, 1983). Thus plasma energy 
substrate 1 evels are raised as a result of GH secretion occuri ng when 
they were low. The action of GH in this respect has been found to be 
biphasic. It initially exerts a relatively short-term insulin like 
effect prior to exerting its insulin antagonistic effect (Altszuler 
et al, 1968; Ahren and Hjalmarsson, 1968). The other effect of GH 
in maintenance of plasma energy level is thought to be lipolytic. 
Studies showed that GH stimulated lipolysis in animals under certain 
conditions notably with the fact that it synergised with 
glucocorticoids, resulting in far more lipolysis than the additive 
14 
effect of each alone (Caldwell and Fain, 1970; Goodman et al, 1970). 
GH stimulates lipolysis in the absence of insulin, such as in 
fasting, to prevent energy substrate from decline. It is also known 
that the lipolytic effect of GH can be inhibited by insulin. 
Amino acids: Intr.avenous infusion of certain amino acids, 
particularly arginine, which stimulate insulin secretion, also 
stimulate GH release, but larger doses are required for a GH 
response. In cases where the concentrations of both hormones were 
estimated (Hertelendy et al, 1970; Davis, 1972; Bohn, 1978), the 
insulin peak preceded the GH peak by 15 or 30 minutes. It is 
possible that GH release is induced by the insulin hypoglycemia. It 
was suggested that arginine may have a direct stimulatory effect on 
GH release (Bohn, 1978), but the mechanism is still unknown. The 
physiological significance of the GH response to amino acid challenge 
may lie in the fact that GH is the major anabolic hormone which 
promotes the transport of amino acids into cells and increases 
protein and nucleic acid sy:nthe.sis causing sana tic growth (Hart, 
\ 
1980; Bauman and McCutcheon, 1984). 
It is also know that cAMP and calcium ion are effective in 
stimulating GH release and probably act directly on the pituitary 
sanatotrophs. However highly purified GHRF did not increase cAMP 
within the pituitary but increased cGMP levels which in turn promote 
calcium uptake into the pituitary causing GH release (Peake, 1973). 
Metabolic factors are less important in the day to day 
regulation of GH secretion than the complex neuroendocrine mechanisms 
although metabolic factors may act in concert with neuroendocrine and 
other endocrine secretions in emergency. 
2. 1 • 2. 5 Different physi ol ogi cal stage: 
Age:It is has been observed in the study of human and other 
species that plasma GH concentration are high at birth and decrease 
rapidly during the first few weeks of life and then more slowly 
towards the end of weaning (Cornblath et al, 1965; Machlin et al, 
1968; Tsushima et al, 1971; Tucker et al, 1974; Reyneart et al, 
1976; Roy et al, 1983; Bassett and Alexander, 1971; Hertelendy et 
al, 1969) and young adults appear to have higher GH concentrations 
15 
than old adults. These changes are due to differences in secretion 
rather than metabolism of GH because of the absence of a change in 
the metabolic clearance rate of GH with increasing age (Taylor et al, 
1969). As the young animal reaches puberty, there is evidence that 
the GH secretion is again enhanced (Finkelstein et al, 1972; Dudl et 
al, 1973).The pubertal increase in GH level may be directly related 
to increased levels of gonadal steroids. It has been observed that 
both androgens and oestrogens have an enhancing effect on GH 
concentration (Illig and Prader, 1970). 
Sex: The literature contains sane reports that females have 
higher plasma GH concentration than males in some species, but 
studies with laboratory animals indicated that plasma GH levels bear 
little relationship to pituitary GH concentration (Schindler et al, 
1972). Thus higher plasma GH level in the females may result from 
the inhibitory effect of oestrogen on GH removal rather than by 
stimulation of secretion. ·. But studies in human (Merimee et al, 1969) 
suggested that oestrogen may h?ve a stimulatory effect on GH 
i 
secretion. 
Pregnancy: GH levels were found to increase progressively in 
maternal plasma as pregnancy advanced (rat: Saunders et al, 1976; 
cow: Koprowski and Tucker, 1973). The GH content of the pituitary 
of the fetus of various animal species undergoes a continuous 
increase throughout intrauterine life, reaching a maximum just before 
birth (Contopoulos et al, 1967). The plasma concentration of GH in 
fetus is also greatly in excess of the maternal plasma GH 
concentration at comparable stages of gestation. This may be due to 
a relatively poorly developed hypothalamic controlling system in the 
fetus. 
Lactation: While prolactin is the major pituitary hormone 
responsible for the maintenance of established lactation in 
non-ruminants it is now clear that GH is more important than 
prolactin as a lactogenic hormone in ruminants (Cowie et al, 1980; 
Bines and Hart, 1982; Bauman and McCutcheon, 1984). The higher GH 
concentration found in early lactation (Hart, et al, 1978) and in 
high yielders (Flux et al, 1984; Hart et al, 1978) may lie in the 
16 
fact that the lactogenic and lipolytic effect of GH favour the milk 
synthesis in the mammary gland by increasing the availability of 
precursors for synthesis. However the precise mechanisms of how GH 
acts on the different target tissue (eg. adipose tissue, muscle and 
mammary gland) is still poorly defined. 
2.1.2.6 Secretory rhythm of GH 
The episodic secretion of GH has been well documented in the 
literature. For example, GH release occurs with a cyclicity of 3.3 
hr in rats, independent of other physiological factors (Tannenbaum 
and Martin, l976a, 1976b). Similar rhythmic secretion has also been 
found in other species, but the biological significance is still ill 
defined. In man, a most striking increase in GH secretion occurs 
during sleep and appears to be linked closely to slow wave sleep 
(SWS) (Pawel et al, 1972). It is not known to what extent the sleep 
induced GH release is facilitated by the nocturnal state of fasting, 
though central nerve system (CNS) certainly plays a dominant role in 
) 
GH release. GH concentration also varies greatly during the day in 
ruminants. It may be due to the fluctuation in plasma energy status 
as GH level is normally minimized .after the start of feeding (Blam et 
al, 1976; Bassett, 1975). 
2.1.3 Regulation of cortisol concentration 
2.1.3.1 ACTH and CRF 
Corticortropin (ACTH) has long been known to be intimately 
involved in the regulation of plasma cortisol levels in animals. In 
response to increased levels of ACTH, the adrenals increases in size, 
adrenal blood flow increases and biosynthesis of corticosteroids and 
androgens in the adrenals increases. The response of the adrenal to 
ACTH is very rapid, occuring in a few minutes. It is believed that 
increased cAMP production and increased permeability of the membrane 
to certain ions are responsible for the rapid response (Fahmy 1983). 
In addition to its immediate effect, ACTH also exerts a pronounced 
trophic effect on the adrenal cortex. ACTH is necessary for normal 
growth, development and maintenance of the adrenal cortex which 
l7 
atrophies very quickly after removal of the pituitary unless ACTH is 
administered. Thus ACTH regulates glucocorticoid concentration by 
increasing glucocorticoid production rather than by affecting its 
secretion because adrenal steroid hormones are released as soon as 
they are synthesized and only trace amounts can be detected in the 
gland (reviewed by Gower, 1979; Fahmy 1983, Hardy 1981; Yates, 
1967) . 
It is also established that secretion of ACTH is under 
hypothalamic control. Stimulation of higher neural areas by various 
stresses such as mental disturbance; infection, insulin hypoglycemia, 
cold and other enviromental challenges can cause the release of 
corticortropin releasing factor (CRF) from the hypothalamus which 
stimulates the release of ACTH. ACTH secretion is also regulated by 
both fast and delayed feedback systems (Jones, 1979). Increased 
cortisol and cortisone are fast feedback agonists whereas other 
adrenal steroids and many synthetic glucocorticoids have little or no 
effect on this system. Ho~ever higher concentrations of the latter 
may be involved in the delayed_feedback mechanism. The fast feedback 
l 
reduces the amount of CRF released whereas the delayed feedback 
reduces the synthesis of CRF. Fig .1 shows the control of 
hypothalamic - pituitary - adrenal function. 
2.1.3.2 Cortisol binding globulin (CBG) 
Corticosteroid circulating in plasma are largely bound to plasma 
protein, particularly to albumin, but cortisol and cortisone are also 
bound by a specific cortisol- binding globulin (CBG). In human more 
than eighty percent of circulating of cortisol is bound to CBG, ten 
percent to albumin, and ten percent as free steriod (Fahmy, 1983). 
The binding percentage in ruminant is not clear. The biological 
activity of cortisol is solely accounted for by the free cortisol as 
it is only form which can be taken up by the target tissue. The high 
affinity of cortisol for CBG may be of importance in buffering the 
high amplitude fluctuation of the "free" steriod due to the episodic 
secretion of cortisol (Fahmy, 1983). 
Figure 1 Control of hypothalamus-pituitary-adrenal function 
Stimulation of higher neural areas by stresses, mental 
disturbance, insulin- induced hypoglycemia etc. 
Hypothalamus 
Control of 
1 CRF '\ \ ? I 
I Control of 
CRF 
secretion 
(fast feedback) 
\ 
Anterior pituitary; 
/ 
..... ACTH-
~ Adrenal cortex 
.~ 
CRF 
synthesis 
(delayed feedback) 
1) Plasma free cortisol and cortisone 
2) Other adrenal steroids and synthetic 
corticosteroids 
(Adapted from Gower, 1979) 
18 
19 
2.1.3.3 Circadian factors 
Glucocorticoids secretion shows a diurnal fluctuation in many species 
(man: Rose et al, 1972; pig: and horse: Bottans et al, 1972; 
rats: Guileman, 1959i cat: Krieger et al, 1968; cattle: Macadam 
and Eberhart, 1972); with the maximal production occuring in the 
early morning and the minimal production in the late evening; 
Patterns are sanetime difficult to discern. The diurnal fluctuation 
of glucocorticoids concentration involves a parallel fluctuation in 
ACTH and CRF release though the source of the rhythm is not clearly 
defined. It is possibly due to the changes in plasma energy 
substrate levels as it is known that glucocorticoids play an 
important role in the control of carbohydrate metabolism. 
Administration of glucocorticoids has a hyperglycemic effect in 
animals (Ford, 1971; Bassett and Wallace, 1967). This has been 
attributed to both an increased endogenous glucose production and 
impaired peripheral glucose metabolism (Reilly, 1971), as well as 
\ 
antagonism of insulin action by cortisol (Bassett and Wallace, 1967). 
Glucocorticoids stimulate glucose production by two separate 
gluconeogenic effects. First they stimulate the breakdown of protein 
in skeletal muscle and the release of amino acids, leading to 
increased plasma precusor levels for hepatic and renal 
gluconeogenesis. Secondly, they induce the formation of specific 
proteins in the liver, such as the transaminases which catalyse the 
transfer of a-amino moieties to a-ketoglutarate, thereby providing a 
pathway for conversion to carbohydrate (Gower, 1979; Hardy 1981). 
Thus there is a simultaneous increase in the release of amino acid 
from skeletal muscle and the rate at which the amino acid are trapped 
by the liver and converted to carbohydrate. 
The anti-insulin effect of glucocorticoids in peripheral tissue 
may lie in that glucocorticoids can effectively inhibit glucose 
uptake especially by adipose tissue (Belfiore, 1980) probably by 
reducing the affinity of insulin receptors for insulin (see section 
2.1.1.4). Therefore the maximal glucocorticoid production in the 
early morning as reported in the literature can be partly explained 
by the fasting hypoglycemia which is sensed by the CNS, causing the 
release of CRF and ACTH. 
20 
2.1.3.4 Stress: 
Increased cortisol secretion has been observed in animals under 
various stress conditions, such as fear, cold, insulin hypoglycemia 
etc. (Falconer,1976;_ Johnson and Vanjonack,1976; Dooley and 
Williams, 1975). It is believed that CNS senses these stressful 
stimuli and the activity of the higher nerve area causes the release 
of CRF ,thus the release of ACTH. 
2.1.3.5 Age: 
Young animals are normally born with high cortisol levels, but 
these decline rapidly after birth within the first few weeks of life. 
Morphologically, newborn animals have a large adrenal relative to 
body weight which is due to the trophic stimuli fran the fetal 
pituitary (Orti 1979; Neville and O'Hare, 1982). However , the 
adrenals involute rapidly after birth with a concomitant fall in 
\ plasma cortisol concentration. This pattern resembles that of GH but 
the evolutionary significance has not been defined. 
2.2 Digestive metabolism and glucose homeostasis 
in young milk fed calves 
2.2.1 The digestive process: 
The digestive system of a young calf fed solely milk is 
essentially monogastric since the milk by-passes the rumen-reticulum 
and its gastric digestion commences in the abomasum. Once in the 
abomasum, the milk is quickly coagulated by renin to form a clot of 
casein and fat. The whey is released, assisted by the motility of 
the abomasom into the duodenum. The passage of the whey into the 
duodenum, which is the major absorption site of the nutrients, 
decreases over time and complete passage of whey may require about 7 
to 9 hours. The breakdown products from the casein clot are released 
slowly during the first 6 hours after feeding but thereafter at a 
more rapid rate as the curd disintegrates (Roy, 1980; Thi vend et al, 
21 
1979). 
2.2.2 Blood glucose homeostasis: 
a) After feeding: In the pre-ruminant calf, blood glucose 
concentration after f~eding rises rapidly to a peak of about 130 
mg/dl at 1-2 hr after feeding and then declines to pre-feeding values 
5-6 hr after feeding (Dollar and Porter, 1957). The rapid increase 
in glucose concentration is due to the rapid entry of nutrients from 
the gut. In contrast, blood glucose concentrations are more stable 
in the ruminating calf, a slight depression occurs about 3 hr after 
feeding followed by a gradual rise to maximum values 8 - 12 hr after 
feeding. (Preston and Ndumber, 1 961) • 
It is established that insulin is the most important anabolic 
hormone involved in the post-prandial regulation of glucose 
homeostasis. It inhibits gluconeogenesis and glycogenolysis, 
promotes glucose uptake by insulin-dependent tissues, such as muscle, 
adipose and connective tissue., As a result of these actions insulin 
\ lowers the circulating glucose concentration. The predominant 
insulin secretion together with hyperglycemia in the post-prandial 
period also antagonize the action of many other hormones such as 
glucagon, glucocorticoids, catecholamines, an9 GH, which promote 
endogenous glucose and fatty acid production (Bassett 1975, 1981; 
Belfiore 1980; Marks and Ross 1981; McDowell, 1983). 
Blood glucose homeostasis,in the post-prandial period, no doubt 
is also regulated by some gut hormones and somatostatin from the 
D-cells present both in pancreas and gut. It is well accepted that 
several of the gut hormones and peptides have a more or less clearly 
defined physiological role in digestion and absorption as well as 
metabolism, while the majority of these gut hormones still await 
further characterization of their physiological role (Adrain and 
Bloom, 1982). Increased rate of digestion and absorption of the 
nutrients and changed motility of the gut will certainly affect the 
nutrient entry rate into the circulation. It is also recognized that 
gut hormones and SLI may indirectly regulate metabolic homeostasis by 
stimulating or inhibiting the secretion of glucagon and insulin. GIP 
now appears to be more important in regulating insulin and glucagon 
secretion than other gut hormones such as CCK, gastrin, secretin etc. 
22 
(Adrain and Bloom, 1982). The mechanism by which gut hormones affect 
insulin and glucagon is still not clear. Somatostatin (SLI) has also 
been proposed to be a major factor in integrating information fran 
ongoing digestive and metabolic events and providing a fine tuning of 
metabolic homeostasis in the way that certain nutrients enter. the 
organism in balance with their rate of disposal (Reviewed by 
Schusdziarra 1980). There is evidence to shown that SLI slows 
intestinal carbohydrate absorption (Wahren and Felig, 1976, 
Bratusch-Marrain et al, 1981), while the inhibitory effect of SLI on 
the secretion of pancreatic, pituitary and gut hormones is well 
established (in section 2.1) . It should be emphasised that most of 
the work concerning gut hormones and SLI so far has been conducted on 
laboratory animals but they are assumed to have similar roles in 
ruminants. 
b) Fasting: In the fasting animal when the supply of glucose 
from the gut is below the ·requirement of the body, the hormonal 
regulation of metabolic homeostasis would favour the endogenous 
\ 
production of glucose. It is well known that insulin concentration 
is diminished in fasted animals and hormones with antagonizing 
effects on insulin action, such as glucocorticoids and GH, appear to 
peak when plasma energy substrate levels decline. A historical study 
(Long et al, 1940) shows that glucocorticoid hormones are essential 
for maintaining blood glucose levels during fasting, and this appears 
to be achieved by increasing the rate of protein degradation and 
mobilising amino acid from skeletal muscle while decreasing protein 
synthesis in tissues (Tischler, 1981). In fasting, a fall in insulin 
concentration and a rise in the plasma molar ratio of glucocorticoids 
to insulin may be important for expression of the glucotropic effect 
of the corticoids. Glucocorticoids may also have a permissive 
influence on the insulin antagonizing actions of other hormones such 
as GH and glucagon. 
GH has also been considered a hormone of fasting as its plasma 
concentration is inversely related to energy status of the animal 
(Trenkle 1971, 1976; Bassett, 1974). Though there is no evidence 
that GH promotes gluconeogenesis in the fasting animal, it may help 
to increase plasma glucose concentration by 1) promoting mobilization 
and oxidation of FFA from the adipose tissue. 2) reducing glucose 
23 
uptake and utilization by peripheral tissue to antagonize the effect 
of insulin on glucose metabolism. 
The principle role of glucagon is to balance the effect of 
insulin in maintaining glucose homeostasis. It acts primarily to 
increase hepatic glucose output by promoting gluconeogenesis from 
amino acids and other.glucose prcursors, such as lactate and 
propionate, and by promoting hepatic glycogenolysis. The importance 
of glucagon in regulating glucose homeostasis may also lie in the 
fact it is a fasting hormone, when insulin secretion is suppressed or 
glucagon secretion is increased, lipolysis in adipose tissue may also 
be faciliated (Trenkle, 1981). The molar ratio of insulin and 
glucagon has been considered of greater physiological significance 
than the absolute concentration of the two hormones in maintaining 
glucose homeostasis in both ruminants (Bassett, 1975) and non 
ruminants (Unger, 1971). A recent study in lactating sheep indicated 
that the decreased molar ratio of insulin to glucagon during fasting 
is due to a decreased insulin concentration rather than increased 
glucagon value (Gow et al,1981).. 
-- i 
The literature contains few reports concerning the regulation of 
blood glucose homeostasis in young milk fed calves. It may be 
expected that such a regulating system in the young calf would bear 
more resemblance to the one of monogastrics rather than of adult 
ruminants. 
2.3 Objectives of the present study 
In the present study, 24 young milk fed Friesian calves of HBI 
and LBI groups were studied to investigate the effect of genetic 
divergence for milk production on metabolic and endocrinological 
status of the calves, and the possibility of finding some suitable 
markers that can be used in early identification of superior dairy 
cattle. 
Four different experimental treatments i.e. fasting, feeding, 
arginine challenge and synthetic corticosteroid injection were 
applied to all calves in order to find out the differences in the 
metabolite and hormone concentrations between HBI and LBI calves. 
It is well known that in fasting, the metabolic homeostasis of 
24 
animals favours endogenous production of energy substrates and 
mobilization of body energy reserve such as glucose and FFA through 
endocrine system (Section 2.2.2) 
Studies with lactating cows suggested that the differnce in milk 
fat production between HBI and LBI cows might be due to the 
difference in their apility to mobilize body energy reserve when 
chronic negative energy balance occurred in lactation (Davey et al, 
1983). And this was most probably paralleled by difference in 
metabolite and hormone concentrations in the blood as significant 
differences were found in plasma metabolite and hormone 
concentrations between HBI and LBI cows (Flux et al, 1984) when 
energy intake was restricted. So it appeared that it was worthwhile 
,in the present study, to investigate the effect of fasting which 
could partly mimic the status of negative energy balance incurred by 
dairy cows in peak lactation on the metabolite and hormone 
concentrations in the calves. 
The effect of feeding· on metabolic homeostasis has been 
intensively studied by nutritignists in many species, and the 
\ 
knowledge in the literature is abundant. However, it might be also 
rewarding to investigate its effect from the viewpoint of breeding, 
as Hart et al (1978) found that a low milk yielding breed had a more 
vigorous insulin response to feeding than a high yielding breed. 
Thus, the effect of feeding was studied in the present experiment. 
Arginine challenge is a good stimulus for the release of GH and 
insulin, as well as other hormones in many species, including dairy 
cattle. It seemed appropriate to measure the response of the calves 
of different breeding index to arginine injection since GH and 
insulin secretion and their responsiveness to metabolic signals are 
essential factors in the control of metabolism. 
The role of cortisol in lactating dairy cattle has not been 
clearly defined. Cortisol may be a determinant factor for the 
established lactation (Cowie et al, 1980). Furthermore, Johnson and 
Vanjonack (1976) found that blood cortisol level was higher in higher 
yielding cows than in low yielding cows and it was spectulated that 
the difference in plasma glucose concentrations between HBI and LBI 
cows was caused partly by a difference in plasma cortisol 
concentration (Flux et al, 1984). Thus the cortisol concentration 
and the responsivenesses of plasma glucose, insulin and GH 
25 
concentration to synthetic corticosteroid injection were investigated 
in the present study. 
26 
CHAPTER THREE: MATERIALS AND METHODS 
3.1 Animals 
A total of 2~ Frtesian calves aged from 10 to 18 days old were 
used in this experiment. Eleven of the 2~ calves were classified as 
high breeding index (HBI) calves the other 13 calves were low 
breeding index (LBI) calves. Of the 11 HBI calves six were bulls 
(HBIB) and 5 were heifers (HBIH). Seven of the 13 LBI calves were 
bulls (LBIB) and 6 were heifers (LBIH). Some general information 
about these calves ie., date of birth, birth weight, breeding index 
(BI), age and body weight at the begining of cannulation and sex are 
given in Table 3-1. The BI for each calf was estimated from the Bls 
of the calf's parents. The mean values, the standard deviations, and 
the standard errors of the mean of the breeding index for both HBI 
and LBI groups are presented in Table 3-2. 
All the calves were born pn the Dairy Cattle Research Unit at 
i 
Massey University in Spring 1983 (from 31st July to 8th September). 
Only healthy calves were used and twin calves or calves with birth 
weights under 30kg were rejected from this experiment. 
3.2 Rearing of calves 
After birth all calves were left with their dams for three days after 
which they were transported to the calf rearing shed and fed by 
bucket. Normally 2 or 3 days were required before they learnt to 
suck milk from a bucket through a teat properly, then the calves were 
transported to the Animal Physiology Unit (APU) and isolated from 
each other by being housed in individual pens. All calves spent 3 
days in these pens, and were fed regularly twice daily at 9.00 and 
16.00 hr. This feeding practice was observed until the end of the 
experiment. Once in the APU the diet was gradually changed from 
tank-stored colostrum to reconstituted milk made of Ancalf milk 
powder (The New Zealand Co-Operative Dairy Co.Ltd., Hamil ton). 
A mixture of half colostrum and half reconstituted milk was fed 
at each meal for the first 3 days in the APU. They also received a 
sulphonamides tablet (Scourban Bolus, Vetco Products Ltd) at each 
27 
Table 3.1 Number,Breeding Index,sex,date of birth,birth weight, 
body weight and age at cannulation of calves used in 
the experiment. 
Calf BI Sex Birth Birth Body AGE 
No Date Wt kg cannulated cannulated 
(kg) (days) 
6 134 M 6-8-83 38.6 43.0 11 
13 138 M 8-8-83 45.9 46.5 13 
16 133 M 9-8-83 38.1 41.8 15 
33 130 M 21-8-83 44.0 43.0 15 
40 137 M 27-8-83 51.8 58.0 17 
42 130 M 30-8-83 43.6 47.0 13 
5 135 F 5-8-83 41.8 47.3 12 
7 137 F 6-.8-83 37.7 40.0 11 
12 133 F 7-8-83 36.3 38.3 14 
~ 
34 132 F 21..J8-83 43.1 44.5 15 
50 142 F 8-9-83 46.4 46.5 11 
1 103 M 31-7-83 47.2 49.5 14 
8 105 M 7-8-83 43.0 47.0 14 
10 103 M 7-8-83 46.3 48.5 10 
14 99 M 8-8-83 45.0 44.5 13 
17 101 M 10-8-83 44.0 45.5 14 
18 105 M 11-8-83 40.0 47.0 18 
43 108 M 30-8-83 45.9 48.5 13 
15 106 F 9-8-83 42.7 44.8 15 
20 106 F 13-8-83 44.5 49.0 16 
21 103 F 13-8-83 41.8 44.3 16 
26 105 F 16-8-83 37.7 43.8 14 
29 107 F 17-8-83 36.3 41.5 13 
31 102 F 19-8-83 36.0 40.0 17 
28 
Table 3.2 Expected means,standard deviations, ranges and the 
standard errors in Breeding Index units of the HBI 
and LBI group. 
Calf Number of Mean of Standard Range for Standard 
group calves BI deviation 95% calves error of 
{individuals} the mean 
HBI 11 134.8 7-3 120.2-149.4 2.2 
LBI 13 104.3 7.3 89.7-118.9 2.0 
Table 3.3 General calf rearing procedure. 
Stage Approx age Rearing Feeding 
regime 
1 
2 
3 
4 
5 
of calves 
(da s) 
1-3 
4-6 
7-9 
10-12 
13-15 
approx 
age at 
cannulation 
site, 
with dam 
on 
pasture 
in calf 
shed. 
Colostrum ad lib. 
---
Tank-stored colostrum fed twice 
daily using bucket and teat. 
Individual Feeding time: 9.00 and 16.00 hr. 
craters in Diet: half tank-stored colostrum 
APU plus half reconstituted milk. 
Intake: see Table' 3.4. 
Individual Feeding time: 9.00 and 16.00 hr. 
pens in 
APU 
as above 
for days 
10-12 
Diet: reconstituted milk only. 
Intake: as above for days 7-9. 
as above for day 0-12. 
29 
evening meal for the first 3 days to prevent the occurence of calf 
diarrhea. By the end of the 3rd day the calves were fed completely 
with reconstituted milk and they were transferred to separate pens in 
another room where they stayed for 6 days till the end of the 
experiment. The general calf rearing procedure was summarised in 
Table 3-3. 
Throughout this experiment good hygiene practices were strictly 
observed: each calf had its own bucket and teat which were rinsed 
and disinfected after each meal. Calf pens were washed down after 
every evening meal. The health of the calves appeared very good 
during the experiment. 
3.3 Reconstituted milk: 
The reconstituted milk was made up of Ancalf milk powder. The 
nutritive composition of Ancalf is listed in appendix I. Before 
feeding, a condensed reconstituted milk was made up by mixing 4.5kg 
milk powder to 1 Okg water and ~tored in a refrigerated room. The 
\ 
condensed milk was diluted with warm water in the ratio 1:1 for each 
meal. 
The calf intake at each meal was calculated according to Table 
3-4. 
Table 3-4 Milk Intake of Calves at Each Meal 
weight condensed warm total 
of calf milk water reconstituted 
(kg) (g) (g) milk (g) 
30 806 806 1612 
35 903 903 1806 
40 1000 1000 2000 
45 1095 1095 2190 
50 1182 1182 2364 
30 
3.4 Procedure for cannulation of the jugular vein. 
The cannulae (E-ZCATH, 16 gauge,90 em long, Desert 
Pharmaceutical Co.,Inc, Sandy, UTAH 84070) were prepared before 
cannulation with adhesive stuck to the cannulae 15-20cm from the tip. 
The neck and shoulder of the calves were clipped and then washed 
using disfectant solution. The position to be cannulated was marked 
with twink and injected with a local anesthetic (Xylocaine). 
The jugular vein of the calf was enlarged by pressing a thumb on 
it and preventing blood flow to the heart. The tip of cannula was 
aimed at the centre of the vein and pushed down with an appropriate 
pressure. Once the blood was obtained from the vein, the needle 
within the cannula was withdrawn gradually and the cannula inserted 
into the vein about 15-20cm. Before the start of suturing, sampling 
was tested and 2ml of heparinised saline injected via the cannula 
into the vein, then the cannula was plugged. Adhesive tape was 
sutured to the skin above ··the cannulating site to prevent the cannula 
from folding and slipping-out., Another 2 or 3 suturings were also 
I 
necessary on the neck and back of the calf to support the cannula. 
Finally a puff of antibiotic powder (Aureomgcin, Cyanamid of New 
Zealand Ltd., Auckland) was applied at places of trauma to prevent 
infection. 
In some cases a 13 or 14 gauge hypodermic needle was inserted 
into the jugular vein when difficulty was experienced with the usual 
procedure. The cannula was passed into the jugular vein through the 
needle. Calves 29, 31, 43 and 50 were cannulated in this way. They 
usually lost more blood than calves cannulated with the normal 
procedure. However, these two different cannulation methods were not 
expected to have a substantial effect on the physiological status of 
the calves and the results of the experiment because all calves were 
sampled on the following day. Twenty four hours was assumed to be 
adequate for recovery from the surgery. 
3.5 Blood sampling 
Each calf had its own sampling and flushing syringes. Clean 
centrifuge tubes were labelled according to calf numbers, sample 
number, and date of sampling and filled with 5ul of heparin (100,000 
31 
iu/ml) prior to_the start of sampling. The first 2 ml of mainly 
heparinised saline was discarded at each sampling then a 10 ml sample 
of blood was withdrawn. The cannula was then flushed with 2 ml 
heparinised saline from the flushing syringe. The blood sample was 
immediately transferred to the centrifuge tube containing 5ul of 
heparin.The tube was topped with parafilm and inverted gently, mixing 
the sample with the heparin. Then the sample tube was put into ice 
bucket or centrifuged immediately after sampling. 
The supernatant plasma from the centrifuged sample was removed 
by using a transfer pipette and aliquoted into 3 vials appropriately 
labelled. The vials were than capped and stored in a deep freezer. 
It is imperative to have all samples processed quickly and kept cold 
to minimize the degradation of plasma components. 
3.6 Measurment of haematocrit 
Haematocrit were meas~ed,on sample 1 and 15 from each calf, 
\ 
using microhaematocrit tubes, centrifuge and reader 
(International-micro-capillary-reader,International Equipment 
Company,Boston 35, Massechusetts) following the manufacturers 
instructions. 
3.7 Measurement of Immunoglobulin G1 (IgG1) 
Immunoglobulin G1 (IgG1) concentration in the plasma of each 
calf was determined in a sample collected one day ahead of 
cannulation at 8.30 am before feeding, using the radial 
immunodiffusion method described by Fahey and McKelvey (1965). The 
plasma samples from most calves were diluted 1:40 before assay. 
Calves 20,29,31, and 43 had low IgG1 concentrations and no measurable 
precipitate rings were found even when their plasma samples were 
diluted 1:10. 
32 
3.8 Plasma glucose assay: 
Plasma glucose concentrations were measured with a Yellow 
Springs Instrument Model 27 Industrial Analyzer (Yellow Springs 
Instrument Co. Inc. Ohio). The YSI model 27 was calibrated using 
five concentrations o! standard glucose solutions (25, 50, 75, 100 
and 150 mg/dl).The instrument 27 was recalibrated using the standard 
of 150 mg/dl after every five samples. All samples were assayed 
twice for glucose concentration with an interval of approximate 
week between duplicate assays. The result, from the two assays 
matched each other closely and the mean values of glucose 
concentration were used for later statistical analysis. 
3.9 Radio immunoassays (RIA) of plasma insulin, GH and cortisol. 
1) Hormones used in RIA for iodination, standards and raising 
antibodies. 
\ GH: BGH NIH GH B8 donated to Prof.Flux by Prof. A.E. Wilhelimi 
(National Institute of Health, U.S.A) was further purified on DEAE 
cellulose to remove prolactin (Flux, et al, 1984). 
Insulin: Bovine crystal insulin, Sigma Chemical Co. no I-5500 
10t 121 C-1350 (26.4 iu/mg). No further purification was made. 
Cortisol: Stock 1, 2- 3 H cortisol(New England Nuclear no 
NET-185). 
2) lodination 
The bovine GH and insulin were iodinated by the chloramine T 
method (Greenwood et al, 1963). The BGH was dissolved in 2M urea at 
room temperature for 1 hr. before iodination. The iodinated GH was 
purified from the iodide on a Sephadex G50 Column and was used within 
two weeks of iodination. A further purification on a Sephadex G100 
column was carried out for iodinated GH used more than two weeks 
after iodination. 
33 
3) Antisera 
Professor Flux raised the 1st antibody to BGH in guinea pigs in 
1974 and the second antibody in Romney sheep in 1980, and in 1982 
(personal communication). Antisera was stored at -15°C. 
The antisera to ~nsulin was raised in guinea pigs by Bohn (1978) 
using bovine insulin polymerised with diethyl-pyrocarbonate. The 
second antibody was raised in sheep. Cortisol antiserum no F3-314 
was purchased from Endocrine Science (18418 Oxnard St.,Tarzana, 
Cal if or ni a, 91 3 56) . 
The GH RIA showed no significant cross-reaction with any of the 
following anterior pituitary hormones, ACTH, FSH, LH, prolactin and 
thyrotropin. The insulin assay showed a small cross-reaction with a 
glucagon preparation. Glucagon has approximately 0.03 percent the 
effect of insulin, with a non-parallel dose-response line. This 
degree of cross reaction was considered unimportant (Flux et al, 
1984). The cross-reaction of various steroids with cortisol 
antiserum F3-314 is given ~n Appendix II. 
4) RIA procedures for GH, insulin and cortisol 
Plasma GH concentration was determined by RIA using the method 
of Hart et al (1975). Insulin concentration was determined by a 
double-antibody RIA (Hales and Randle, 1963). Cortisol level was 
assayed by the method recommended by the suppliers of the antiserum 
(Endocrine Science, 18418, Oxnard Street,Tarzana, California,91356, 
1982). The detailed RIA procedures for GH,insulin and cortisol are 
presented in Appendix III. 
5) RIA Data Transformation 
The original RIA results expressed as counts/min were further 
processed on a computer (Sord M222), using a program written by Prof. 
Munford based on a method developed by Burger et al (1972). The 
untransformed standard carve data were represented by the equation: 
A 
Y=---------- + e 
c + xE 
Y --amount of radioactive hormone bound by 1st antibody. 
X --amount of non-radioactive hormone present in the tube. 
A, C & E --constants specific for each assay. 
e - randan error. 
34 
A,C, and E were determined by an iterative technique and 
the estimate of the- amount of hormone present in each sample. (X) 
was calculated from the equation: 
A 
X (---- - C)E 
y 
X - estimate of amount of hormone present in each of the 50 or 
100 ul sample. 
The estimate of GH concentration fran the Sord printout required 
two further processing steps: 
1) Since each sample had two estimates one from the two 50ul 
subsamples, and the other fran the two 100ul subsamples the estimate 
from the Sord subsamples was doubled to make it comparable with that 
\ 
fran the 100 ul subsamples. But doubling the estimate from the Sord 
subsample also doubles the error. To cope with this a weighted 
estimate (W) of the unknown hormone concentration in the 50 and 100 
ul samples were calculated according to: 
Y50 + Y100 
w ----------
1 • 5 
where Y 50 and Y 100 are the estimate of hormone concentration from 
the 50 and 100 ul subsamples respectively. 
2) The Sord estimates were satisfactory as far as parallelism 
was concerned, but the standard curve based on the GH standards is 
displaced because the standard samples have less plasma in them than 
in the unknown samples. GH assay 82 and 83 both included a set of 
samples in which known amounts of GH were added 
sanatostatin treated sheep. The correlation 
to plasma from a 
between the Sord 
estimations for the GH concentration in the "normal" plasma samples 
with known amounts of GH added and these estimations for the GH in 
the somatostatin treated sheep plasma was high, r = 0.987 for assay 
82 and r = 0.978 for assay 83 when GH concentrations were expressed 
35 
in log units (pg/ml). So a simple regression was used to adjust the 
Sord estimate of GH to compare with the added GH in somatostatin 
treated sheep plasma. The adjusted Sord GH estimations were used in 
further statistical analysis. 
Since each cortisol sample only had two equal volume subsamples 
the mean of them were.used for further statistical analysis. 
6) Parallelism in RIA 
Logically the estimate from the 100 ul subsample should be 
double that from the 50 ul subsamples. This can be checked by 
plotting the levels of the unknown sample estimates adjusted to the 
standard curve so that they are the same distance apart on the X 
axis. If the slope of the line joining the 50 and 100 ul estimate is 
not parallel to the standard curve such a deviation from parallelism 
can indicate that: 
a) The immunoreactivity of the standard hormone differs from 
that of the natural hormones in the plasma sample; 
b) Or some factor in the assay buffer is depressing or 
stimulating the affinity of the antisera to the natural hormone to a 
different degree compared to the standard hormone (Bohn 1978); 
c) The suppressing or stimulating effect.of some assay factors 
on the affinity of 50 ul and 100 ul subsamples to the antisera 
differs; 
d) The standard curve in the assay is not capable of providing 
equal precision over the whole range of the curve as it is often 
observed that the precision is much reduced at both ends of a 
standard curve, where the regression line curves away quickly from 
the major central section (Snedecor and Cochran, 1980). 
If lack of parallelism occurs and its cause is detected the 
problem can be minimised, for example, problem a) can be solved by 
using a suitable standard hormone; problems b) and c) can be 
minimised by eliminating or balancing the disturbing factors, or if 
the disturbing effect is systematic , the estimate from 50 ul and 100 
ul may still be valid; problem d) can be tackled by diluting the 
first antibody at a different ratio, a high ratio increases the 
sensitivity of the standard curve at low concentrations while a low 
ratio increases the sensitivity of the standard curve at high 
36 
concentrations. 
Par allelism for 
experiment (Appendix 
GH 
IV). 
and insulin RIA 
Samples used 
were checked in this 
for checking were chosen 
randomly and as no serious parallelism problem was encounted, no 
further investigation was necessary. 
10 Experimental procedure and Statistical Analysis 
1) Procedure 
Before the start it had been planned to used 24 young 
pre-ruminant calves in this experiment with 6 calves in each of the 
four groups, HBIB HBIH, LBIB and LBIH. However, 6 calves in HBIB, 5 
in HBIH, 7 in LBIB and 6 in LBIH were actually used as there were 
only 5 healthy HBI heifers available. 
The aim of the experiment was to examine the metabolic and 
endocrinological differences between the high breeding index (HBI) 
and low breeding i'ndex (LBI) groups of Friesian calves under 
I different physiological ·conditions and experimental challenges. 
Special attention was given to the bull calves. 
Four treatments were applied to each calf and 18 blood samples 
were collected from each calf during the experiment. The timing of 
the treatments (fasting, feeding, acute arginine infusion, and 
cortisol injection) and samplings are presented in Table 3-s. On day 
one, the following treatment were given to the calves and fourteen 
samples were collected. 
Fasting: Each calf was fasted for approximately 16 hrs before 
two blood samples were collected in the morning at an interval of 30 
minutes. The calves were fed immediately after the second sample. 
Feeding: Calves were fed reconstituted milk according to their 
body weight (section 3.3). Six samples were collected after feeding 
at unequal intervals with more intense sampling immediately after 
feeding as indicated in Table 3-5. 
Arginine infusion: Three and a half hours 
feeding each calf received an intravenous 
after the start of 
infusion of 0.04g 
arginine/kg body weight. Another 6 samples were collected from each 
calf at unequal intervals (see table 3-5). 
Corticosteroid injection: Subcutaneous injection of the 
37 
Table 3.5 Summary of experimental procedure. 
Treatment Time of Sample Time intervals 
sampling number (minutes) 
Day One 
Fasting 16 (hours) 
8:30 -30 
9:00 2 -1 
Fed 9:00 to 9:03 0 
9:05 3 5 
9:10 4 10 
9:20 5 20 
9:40 6 40 
11:00 7 120 
1'2:25 8 205 (or -5) 
Infuse arginine 
(0.04g/kg bwt) 12:30 0 
12:35 9 5 
12:40 10 10 
12:50 11 20 
13:10 12 40 
13:50 13 80 
15:55 14 205 (or -5) 
Inject Betsalan 
(50ug/kg bwt) 16:00 0 
Day Two 
8:30 15 -30 
Fed 9:00 0 
9:20 16 20 
9:40 17 40 
16:00 18 380 
38 
synthetic corticosteroid (Betsalan,Glaxo,New Zealand Ltd.) was given 
to each calf at 16:00hr. The dosage was 0.05g/kg body weight. Four 
samples were collected on the following day at the time 8:30, 9:20, 
9:40 and 16:00 hr respectively. 
Plasma glucose, insulin, GH and cortisol concentration were 
analysed using the methods described in sections 3.8 and 3.9. 
2) Data analysis: 
The data of plasma metabolite and 
this experiment were analysed using 
computing package (REG) (Gilmour, 1983). 
hormone concentration from 
a generalized linear model 
Multivariate analysis was 
conducted to measure the variance between the repeated observations 
(i.e. samplings) within each calf and the variance between the main 
factors, BI and sex, assuming an underlying linear model, which 
included BI, sex and the appropriate interaction effects. 
The form of the model used to describe the observations 
pertaining to the first sample measured in the BI and belonging to 
the sex was: 
U overall mean together with the effect 
of the pth sampling. 
A effect of the ith BI in the pth sampling. 
B effect of the jth sex in the pth sampling. 
AB effect of the interaction of the ith BI and jth sex 
in the pth sampling. 
e random residual effect specific to pij combination 
of BI and sex in the pth sampling. 
In the analysis pertaining to the repeated-measurement models, 
null hypotheses were tested using the Chi-squared statistic, the 
basis for so doing being fully described in section 5 - 8 of Morrison 
(1976). 
The following abbreviation are used to denote the significant 
level of analysis results. 
NS not significant or P>0.05 
39 
* P<0.05 
** P<0.01 
*** P<0.001 
40 
CHAPTER FOUR: RESULTS 
4.0 Results: 
Mean values of ~lasma glucose, insulin, GH and cortisol 
concentrations for each group of different BI and sex in samples 
collected before and after feeding, and after arginine infusion are 
plotted in Figures 2-5 • Mean values of these metabolite and hormone 
concentrations in samples collected after synthetic corticosteroid 
(Betsalan) injection are plotted in Figures 6-9 • Results of the 
statistical analysis of the plasma metabolite and hormone 
concentrations are as follows. 
4.1 Before feeding (fasting status) 
Results of statistical analysis of the plasma metabolite and 
hormone concentrations in _pre~eeding samples (sample 1 and 2) are 
presented in Tables 4.1-4.14 which show that: 
a) HBI calves had significantly higher basal plasma glucose 
concentration than LBI calves (P < 0.05 ) (103 vs 93, 102 vs 93 mg/dl 
for sample 1 and 2 respectively)(Fig.2). 
b) HBI calves had significantly higher basal plasma insulin 
concentration than the LBI calves (P < 0.01) (22 vs 12, 21 vs 14 
ng/ml, for samples 1 and 2 respectively). Basal insulin concentration 
in bull calves were also significantly higher than in heifers (P < 
0.01) (20 vs 13, 20 vs 12 ng/ml, for samples 1 and 2 respectively). 
Of the four groups of calves, HBIB had the highest basal plasma 
insulin concentration followed by HBIH, LBIB and LBIH (Fig3). 
c) Basal GH concentrations in the HBI calves were also higher 
than in LBI calves (Fig 4, Table 4.3) at a significance level of 5.1% 
( 11.4 vs 10.5, 11.2 vs 10.3 Lnpg/ml for samples 1 and 2 respectively) 
though the variance within each group was large. 
d) Basal cortisol concentrations in HBI calves were slightly but 
not significantly higher than that in LBI group (P > 0.05) (9.9 vs 
8.9, 8.2 vs 7.7 ng/ml for sample 1 and 2 respectively) (Table 4.4). 
-~ 
.......... 
a» 
E 
-
15'1 T 
125l 
1001 I 
. I r -.fn:r .. J. 
I 75 1111 I 
1 001-_n.cr i 
, 
, 
75 I ~ .. 4P 
-30 0 20 
I 
I 
- HBIH 
--- LBIH <f 
If' ... , 
I ' 
I 'l 
I 
120 
---l 
I £----
_t, // 
1111 r 
L HBIB I ---- LBIB .. 
Bulls 
---- Heiferst 
---
-HBI 
----· LBI 
\ ------1 ~ /r-
, / 
I~ If I 
-5 20 80 205 
Time from treatment (minutes) 
Figure 2 Effects of fasting, feeding, and arginine 
infusion on plasma glucose concentrations 
in groups of calves of different BI and sex. 
(Vertical bar = standard error of the mean) 
(1) = Fed (2) = arginine infusion (3) = steroid injection 
41 
CD 
50 l 
25 
I -r -~..c--.r 
® 
J.-----'i 
- r--- -{ 
HBI H -- ­
LBI H---....; 
42 
-
0 ~~~~-----.--------~-r--.---------~ 
E 
! 50 ·t 
c u ~li ~ I f\ 
HBI B - ­
LBI 8 ----
~ 25 -- - T - - - -1 \ c J- -p:r.c--1'"" r---:c------ -1 
e 
(,) 
c 
0 
(,) 
c 
-
-::J 
• c 
G 
E 
• G 
-a. 
50 
25 
' ----\-r---
.L- -.s~:x.r-r , : 
0 I. Ill I 
t 
Ill 
I 
Bulls 
Heifers 
HBI calves 
LBI calves 
1 
_ - - - r - - - -1 
.r-1.Ilr-r 
-r-------:1 
0 I 1l1°1 1 
-30 0 40 
I 
120 
0 40 
I II I I 
-5 20 
I 
80 
Time from treatment (minutes) 
'• 
' 
Figure3 Effects of fasting, feeding, and arginine 
infusion on plasma insulin concentrations 
in groups of calves of different BI and sex. 
(Vertical bar = standard error of the mean) 
(1) = Fed (2) = arginine infusion (3) = steroid injection 
I 
-5 
Figure 4 
43 
12l ~ ~ 
11-l / 1_Jru J// ' 
',,J 
1 I ~ 
' ~--
--HBIH 
-
--- LBIH 
E 
-........ ~ ~ Q. 
c 
.J 11i ]I ; 
-c ' 11//; 
.2 
-
10 
co 
... 
-
9 c --HBIB 
G) 
(,) --- LBIB 
c 
0 
(,) ~ G) 12 
c 
0 
E 
... 
0 
.c 1 
.c 
-~ 
0 --au• 
... 
~ --- Helfera 
co B 
E 
U) 
ca 12 
-Q. 
l- - -- 1llt J:. 11-l 
10 
1<1~11_...." "' ,1-/ '-,J 
9 - HBICalvea 
--- LBicalvea 
8 0 
-30 0 40 120 -5 
Time from treatment (minutes) 
Effects of fasting, feeding, and arginine 
infusion on plasma GH concentrations in groups 
of calves of different BI and sex. 
(Vertical bar = standard error of the mean) 
(1) = Fed (2) = arginine infusion (3) = steroid injection 
15 
10 I 
r-yl 5} 't--_r- __ 
0 ~ 1111 
= 15 l t E 
" C) 
c 
-
10 
it I I 
l 
HBIH­
LBIH---
HBIB-
® 
t 
.__J\ LBIB 
! : -+-l ~~-\--_--.-t -_-_-,..,..,~-,.-L----rl-----____,-: 
~ 15 l 
(,) 
t'G 
E 
(I) 
t'G 
-Q. 
10 
5 
Bulls -
Heifers---
0 ~~~-------.------.. n-.--.----------, 
15 
10 
5 
0 
l ~ 
5 40 
II I I I 
-30 0 20 
Time 
I 
120 
HBI calves - ­
LBI calves ---
0 -~-- --:~ 
1111 I I 
-5 20 40 80 
(205) 
-5 
(205) 
from treatment (minutes) 
Figure 5 Effects of fasting, feeding, and arginine 
infusion on plasma cortisol concentrations 
in groups of calves of different BI and sex. 
(Vertical bar = standard error of the mean) 
(1) = Fed (2) = arginine infusion (3) = steroid injection 
44 
•HBIB 
•LBIB 
• HBIH 
• LBIH 
] t ~ 150 ~r=:::::::::::::::=======!~  -=={t 
~ 125i l . i L------------·--' 
_2 100 »-- _J~f~--- =- ::-.:_--:-:_:-_·=-·--:...:.-=-'-~ ~-:-·-·1.f .. iT·-·-.l·' ----------~ 
- j/ .----(Q .{ .1 ------~ '',(l ~ 75 ~I~.~,-----------------~ 
c 175l 1 · •HBI calves l+ • Bulls 8 + 11===-• LBI calves . T 1 • Heifers ~ ----- T y ~ 
0 150 ~ ==== !A. 
0 ~ u . 
t 125~ ~---------:~~=-:-c.~- I L- ------ =---7-~--I ... 
(Q ,AL _1i --------- v --------
0:: 100•1--r-r·------ tr---:_1~---
7 5 I • •,o • 415 lf--...r,2:r~.,...., ---------------.--
-30 40 -30 40 
Time from feeding (minutes) 
Figure 6 Effect of synthetic corticosteroid injection on plasma 
glucose concentrations in groups of calves of different 
BI and sex 
Solid line ( -- ) represents values obtained on day 2 
following betsalan injection. Broken line (---) represents 
values obtained on day l before betsalian injection. 
~ = fed 
445 
+­
\Jl 
50- t •HBIB •LBIB 
::::- ... ~~ 0) 2 5 - - - - - - - ---.l,A 
- ..,- -.c'- - - - -- -- - _r .. c - --
.!! I ------i 
- 0 I 11 
C'a 
... 
-c CD 
(,) 
c 
0 
(,) 
.5 1 • HBI calves 
~ 50 l ir • LBI calves 
·~ • -L--1J T .._ 
I 25~-- _______ :r. fl) 
C'a 
0:: 
~ ~ 
er--.J..~-- ----------"1· 
0 I I •4•0 I 
-30 20 445 
6 HBIH 
• LBIH 
------~· 
1 .._ Bulls 
• Heifers ~ J.& 
AJ T ------) y_.... ---~ :l: .l. --c- -.:a..-:a.-
1 40 
-, 
-30 20 445 
Time from feeding (minutes) 
Figure 7 Effect of corticosteroid injection on plasma insulin concentrations 
in groups of calves of different BI and sex. 
Solid line ( -- ) represents values obtained on day 2 
following betsalan injection~ Broken line (---) represents 
values obtained on day l before betsalian injection. 
i = fed 
+o-
0'\ 
-E 
'12-C) 
Q. 
c 
d 11-r 
... 
A.HBIB 
• LBIB 
~ - .. 
c 
0 1'-
.l. r--=------------- -,• 
':ii10] ,: ' ~ ~· ~ 
- ~ c 
Q) (J Q , I I I 
c 
0 
CJ 
Q) 
c 
~ 12-' ~ • HBI calves 
0 •L..L.:r eLBI calves i 11R - 1 
0 ..J. ', 
: 1 o J 
0 
r r --- -- ----------1• 
0 9 Ill I 
.! -30 40 445 
0.. 
• HBIH 
• LBIH 
Pf-----~ --- -r: 
...... 
_, '"' --,..,_ __ _ ... 
.l.j 
.L. 
_,. 
I 
..... 
r--.-~T-- I 
~ • Bulls l • Heifers 
·~ J. ' ~~ •J 
'i - -- - - - = .r· L.~-- I 
-30 40 445 
Time from feeding (minutes) 
Figure 8 Effect of synthetic corticosteroid injection on plasma GH concentrations 
in groups of calves of different BI and sex 
Solid line ( -- ) represents values obtained on day 2 
following betsalan injection. Broken line (---) represents 
values obtained on day 1 before betsalian injection. 
i = fed 
+­
-.._] 
j' 15l • HBIB 
• LBIB 
• HBIH l 1 l • LBIH ......... CJ) 
c 
- \' Jl.'· ~ c 10-:-l .... . 2 .... 
(U 
... 
.... 
c 
G) 
(,) 
c 
0 
(,) 
0 
tn 
.... 
... 
0 
(,) 
G) 
> 
·-.... 
(,) 
(U 
G) 
... 
0 
c 
::J 
E 
E 
(U 
E 
tn 
(U 
-0. 
., ' \ \T e . j \ "{ . 1 l' J 5 \ \ - --·. ·, "'-\ I -------- ' ·-·-·-·---· \. .t.. ---------·---· ·l l'r-- --·-·-----=:=-· -· -·-. -- --· =1T 
5 
1'·-~.::.----·-· -· -· -·-·-· -- -·-. -·--r ·------ -·-· 
1 
• HBI calves 
• LBI calves 
'f= I ff )t 
• Bulls 
• Heifers l L 
~-
,, _l 
\>.J ·-·-·-·=:=·-=- 1. \ "T ·- -·.:r't -·---·- ----
\ ·--- - -·-·-
.'L.-·-·-
~·Iff 1 ~ I if ~ 
-30 0 40 445 -30 0 40 445 
Time from feeding (minutes) 
Figure 9 Effect of synthetic corticosteroid injection on plasma cortisol 
concentrations in groups of calves of different BI and sex 
Solid line ( -- ) represents values obtained on day 2 
following betsalan injection. Broken line (---) represents 
values obtained on day l before betsalian injection. 
-t = fed 
+-
00 
49 
Table 4.1 Analysis of variance of repeated measurements of 
basal glucose concentration between groups of 
calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 1 2.57 NS 
BI 1 4.16 * 
Sex 1 0.09 NS 
BI x Sex 1 1.26 NS 
Time x BI 0.74 NS 
Time x Sex 1 0.44 NS 
Time x BI x Sex 1 0.08 NS 
Number of observations 24 
at per stage 
Table 4.2 Analysis of variance of repeated measurements of 
basal insulin concentration between groups of 
calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 0.17 NS 
BI 10.33 ** 
Sex 6.91 ** 
BI X Sex 1 0.05 NS 
Time X BI 1 1. 57 NS 
Time x Sex 1 0.28 NS 
Time x BI x Sex 1 0.01 NS 
Number of observations 24 
at per stage 
50 
Table 4.3 Analysis of variance of repeated measurements of 
basal GH concentration between groups of calves 
of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 4.15 * 
BI 1 3.57 NS (P<5.9%) 
Sex 1 0.19 NS 
BI X Sex o.oo NS 
Time x BI 0.08 NS 
Time x Sex 1 0.51 NS 
Time x BI x Sex 1 0.13 NS 
Number of observations 24 
at per stage 
Table 4.4 Analysis of variance of repeated measurements of 
basal cortisol concentration between groups of 
calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 1 1.96 NS 
BI 1 0.10 NS 
Sex 0.00 NS 
BI X Sex 1 0.50 NS 
Time X BI 0.07 NS 
Time x Sex 0.84 NS 
Time x BI x Sex 1 0.21 NS 
Number of observations 24 
at per stage 
Table 4.5 Analysis of variance of repeated measurements of 
after-feeding glucose concentrations between 
groups of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 5 42.00 *** 
BI 2.98 NS (P<8.5%) 
Sex 3.80 NS (P<5.2%) 
BI x Sex 1.57 NS 
Time x BI 5 1.20 NS 
Time x Sex 5 9.45 NS 
Time X BI x Sex 5 1. 74 NS 
Number of observations 24 
at per stage 
Table 4.6 Analysis of variance of repeated measurements of 
after-feeding insulin concentrations between 
groups of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 5 16.81 ** 
BI 1 8.14 ** 
Sex 1 8.45 ** 
BI x Sex 0.91 NS 
Time x BI 5 6.83 NS 
Time x Sex 5 1.56 NS 
Time x BI x Sex 5 9.52 NS 
Number of observations 24 
at per stage 
51 
Table 4.7 Analysis of variance of repeated measurements of 
after-feeding GH concentrations between groups 
of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 5 21.71 *** 
BI 1 4.01 * 
Sex 0.48 NS 
BI X Sex 1 0.03 NS 
Time X BI 5 7.60 NS 
Time x Sex 5 5.03 NS 
Time x BI x Sex 5 1.92 NS 
Number of observations 24 
at per stage 
Table 4.8 Analysis of variance of repeated measurements of 
after-feeding cortisol concentrations between 
groups of calves of different BI and sex. 
Source of Variation 
Time 
BI 
Sex 
BI x 
Time 
Time 
Time 
Sex 
X BI 
x Sex 
X BI 
Number of 
x Sex 
observations 
at per stage 
DF 
5 
1 
5 
5 
5 
24 
Chi Significance 
square 
24.17 *** 
0.19 NS 
2.36 NS 
3.29 NS 
1. 80 NS 
4.01 NS 
7.69 NS 
52 
Table 4.9 Analysis of variance of repeated measurements of 
glucose concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 
Source of Variation OF Chi Significance 
s uare 
Time 5 32.06 *** 
BI 1 o. 72 NS 
Sex 1 0.19 NS 
BI x Sex 1 0.36 NS 
Time x BI 5 2.70 NS 
Time x Sex 5 5.29 NS 
Time x BI x Sex 5 3.80 NS 
Number of observations 24 
at per stage 
Table 4.10 Analysis of variance of repeated measurements 
of insulin concentrations in response to 
arginine challenge between groups of calves of 
different BI and sex. 
Source of Variation OF Chi Significance 
s uare 
Time 5 34.69 *** 
BI 1 1. 94 NS 
Sex 1 4.72 * 
BI X Sex 0.21 NS 
Time X BI 5 1.98 NS 
Time x Sex 5 7.92 NS 
Time X BI x Sex 5 9.19 NS 
Number of observations 24 
at per stage 
53 
Table 4.11 Analysis of variance of repeated measurements 
of GH concentrations in response to arginine 
challenge between groups of calves of different 
BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 5 17.69 ** 
BI 5.46 * 
Sex 1 0.04 NS 
BI x Sex 1 0.09 NS 
Time x BI 5 12.95 * 
Time x Sex 5 4.64 NS 
Time X BI x Sex 5 2.19 NS 
Number of observations . 24 
at per stage 
Table 4.12 Analysis of variance of repeated measurements 
of cortisol concentrations in response to 
arginine challenge between groups of calves of 
different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 5 6.65 NS 
BI 1 0.01 NS 
Sex 1 0.27 NS 
BI x Sex 1 0.05 NS 
Time x BI 5 1.22 NS 
Time x Sex 5 3.50 NS 
Time X BI x Sex 5 0.97 NS 
Number of observations 24 
at per stage 
54 
Table 4.13 Analysis of variance of repeated measurements 
of glucose concentrations in response to 
synthetic corticosteroid injection 
between groups of calves of different BI and sex. 
Source of Variation 
Time 
BI 
Sex 
DF 
3 
BI x Sex 1 
Time x BI 3 
Time x Sex 3 
Time x BI x Sex 3 
Number of observations 24 
at per stage 
Chi Significance 
s uare 
6.55 NS 
0.06 NS 
1.38 NS 
0.73 NS 
0.46 NS 
5.87 NS 
6.02 NS 
Table 4.14 Analysis of variance of repeated measurements 
of insulin concentrations in response to 
synthetic corticosteroid injection 
between groups of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 3 12.55 ** 
BI 1 2.88 NS 
Sex 1 5.67 * 
BI x Sex 1 0.85 NS 
Time X BI 3 1.69 NS 
Time x Sex 3 1. 79 NS 
Time X BI x Sex 3 3.25 NS 
Number of observations 24 
at per stage 
55 
Table 4.15 Analysis of variance of repeated measurements 
of GH concentrations in response to synthetic 
corticosteroid injection between groups of 
calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 3 10.62 * 
BI 3. 51 NS (P<6.2%) 
Sex 1 0.03 NS 
BI x Sex 1 0.01 NS 
Time x BI 3 3.83 NS 
Time x Sex 3 7.67 NS (P<5.4%) 
Time X BI x Sex 3 2.66 NS 
Number of observations -- 24 
at per stage 
Table 4.16 Analysis of variance of repeated measurements 
of cortisol concentrations in response to 
synthetic corticosteroid injection 
between groups of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Time 3 2.38 NS 
BI 1 0.41 NS 
Sex 1 0.29 NS 
BI X Sex 0.30 NS 
Time X BI 3 1.76 NS 
Time x Sex 3 0.92 NS 
Time x BI x Sex 3 0.16 NS 
Number of observations 24 
at per stage 
56 
57 
4.2 After feeding: 
Results of the statistical analysis of the plasma metabolite and 
hormone concentrations in the after feeding samples (samples 3 - 8) as 
presented in Tables 4.5-4.8 show that: 
a) HBI calves still maintained higher glucose concentrations than 
LBI calves during the time after-feeding (117.9 vs 109.6 mg/dl for 
means only) but the difference was significant only at the 8% level 
(Table 4.5). The mean value of plasma glucose concentration in bulls 
was 117.8 mg/dl which is significantly higher than that of the heifers 
of 108.3 mg/dl (P<0.05). In response to feeding there were notable 
fluctuations of blood glucose homeostasis in most of the calves with 
both elevated and depressed glucose concentrations recorded in the 
first 20 minutes, but after 20 minutes hyperglycemia developed in all 
calves with a peak occuring 2-3 hours after feeding. 
b) The post-prandial insulin secretion pattern was bi-phasic with 
a first minor peak occuring within half an hour from the start of 
feeding, and a second large peak occuring 2-3 hours after feeding. 
For most of the calves, their second insulin peaks matched the 
post-prandial glucose apogee very well. 
Although the pattern of insulin concentration following feeding 
was similar between groups (Fig 3), the mean ~oncentration of insulin 
remained significantly higher in HBI and bull calves (29.3 and 28.4 
ng/ml respectively) than those in LBI and heifer calves (16.6 and 15.4 
ng/ml respectively) (P< 0.01, Table 4.6) following feeding. 
c) It was observed that GH concentration was stablized or reduced 
in the time immediately after feeding in most of the calves. It 
appeared that GH concentration pattern resembled those of glucose and 
insulin in response to feeding in that GH concentration in most calves 
was at its highest about 3 hours after feeding. 
GH concentration after feeding w~s ~ignificantly higher in HBI 
calves (11.3 Ln pg/ml) than in LBI calves (10.4 Ln pg/ml) (P < 0.05,). 
However, no such difference was detected between bulls and heifers ( 
11 vs 10.7 Ln pg/ml) (P > 0.05). 
d) There was a rapid response of plasma cortisol concentration to 
feeding in most of the calves. The mean plasma cortisol 
concentrations in HBIH, LBIB and LBIH were increased, but decreased in 
the HBIB group (Fig.5). Then cortisol concentration was depressed in 
Table 4.17 Analysis of covariance of repeated measuements 
of glucose concentrations in response to feeding 
between groups of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Basal 1 9.76 
Time 5 41.95 *** 
BI 1 0.57 NS 
Sex 1 5.38 * 
BI x Sex 0.52 NS 
Time x BI 5 3.96 NS 
Time x Sex 5 9.76 NS 
Time X BI X Sex 5 2.98 NS 
Number of observations 24 
at per stage 
Table 4.18 Analysis of covariance of repeated measurements 
of insulin concentrations in response to feeding 
between groups of calves of different BI and sex. 
Source of Variation 
Basal 
Time 
BI 
Sex 
BI x 
Time 
Time 
Time 
Sex 
X BI 
x Sex 
X BI 
Number of 
x Sex 
observations 
at per stage 
DF 
1 
5 
5 
5 
5 
24 
Chi Significance 
s uare 
26.98 
18.15 *** 
0.18 NS 
1.56 NS 
2.37 NS 
5.71 NS 
2.30 NS 
8.94 NS 
58 
Table 4.19 Analysis of covariance of repeated measurements 
of GH concentrations in response to feeding 
between groups of calves of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Basal 43.60 
Time 5 26.45 *** 
BI 0.67 NS 
Sex 1 1.52 NS 
Bl x Sex 1 0.25 NS 
Time x BI 5 4.62 NS 
Time x Sex 5 4.93 NS 
Time X BI x Sex 5 1. 83 NS 
Number of observations 24 
at per stage 
Table 4.20 Analysis of covariance of repeated measurements 
of cortisol concentrations in response to feeding 
between groups of calves of different BI and sex. 
Source of Variation 
Basal 
Time 
BI 
Sex 
BI x 
Time 
Time 
Time 
Sex 
x BI 
x Sex 
X BI 
Number of 
x Sex 
observations 
at per stage 
DF 
5 
1 
5 
5 
5 
24 
Chi 
s uare 
20.85 
27.04 
0.62 
3.83 
3.19 
2.13 
3.89 
6.69 
Significance 
*** 
NS 
* 
NS 
NS 
NS 
NS 
59 
60 
all groups reaching a nadir when the plasma glucose and insulin peaked 
i.e. about 2-3 hours after feeding. LBI and heifer calves had 
slightly higher cortisol concentrations (5.3 and 6.2 ng/ml 
respectively) than HBI and bull calves (4.9 and 4.2 ng/ml 
respectively). However neither of the differences were significant (P 
> 0.05). 
e) The post-prandial plasma glucose, insulin, GH and cortisol 
concentrations were further analysed by covariance analysis, using the 
value of sample 2 as a covariate. The adjusted results by covariance 
analysis are presented in Tables 4.17-4.20 • Comparison of this 
result with the unadjusted results in Tables 4.5-4.8 shows that: 
i) After adjustment, glucose concentration remained significantly 
higher in bulls than in heifers (P < 0.05), i.e. feeding per~ 
produced a relative hyperglycemia in bulls when both bulls and heifers 
were fed at the same level. 
ii) The apparently significantly higher insulin concentrations 
after feeding in HBI calves and bulls were due to their higher basal 
insulin concentrations. Feed~ng per~ did not produce an additional 
\ 
significant difference in insulin concentration between different BI 
groups and between different sex groups as there was no significant 
difference in the adjusted results. 
iii) Feeding per ~ did not produce additional differences in 
plasma GH concentrations between calves of different BI and sex. The 
significant difference between HBI and LBI calves which existed in the 
unadjusted result for the post-feeding period resulted from the 
differences existed before feeding. 
iv) After adjustment by covariance analysis, the difference in 
plasma cortisol concentrations between bulls and heifers become 
statistically significant (P = 0.053) with heifers having higher 
cortisol concentrations than bulls in response to feeding. 
4.3 After arginine infusion 
The patterns of the mean plasma metabolite and hormone 
concentrations in response to arginine infusion in different calf 
groups are presented in Figures 2 and 5, and the responses of 
individual calves within each group are plotted in Figures 11-14 • 
Results of statistical analysis of the mean values are presented in 
-
"0 
......... 
CJ) 
E 
-c 
0 
..... 
«J 
.. 
..... 
c 
G) 
(.) 
c 
0 
(.) 
G) 
0 
0 
(.) 
:::s 
-CJ) 
«J 
HBIH 
5----
7 
20 l 12-·-·-·-. -· -·-34 ................... .. so--------------
-:;;..:.~;.: ;=--- .:-;;:' .- -::..: 
.-... -------~·· .. ··~ 10 
75 
504-~~~--~--------~ 
200 1 
150 
125 
HBIB 
e -------
18-----
13 -·-·-· -·-·-·-· 33 _,_ .. __ , -· ·-42 ___________ _ 
40 ...................... .. 
l 
LBIH 
15-------· 
2Q .................. .. 
21-----
26-·-·-·-·-·-·-29-.. .:.. .. _ .. _ .. -31 ____________ _ 
·\ 
.I . ·;.<~?\:. ',_--- -- - --- - .:::-·-':::. 
.. ~\....... . . .-- _ .. -·· 
t 
........ 
\ .. ...... • JJ.' ...... :::: •. r.:.: ...... :-:-: 
" ........ r:--------- ------
·' .· ," ~ 
,\"""'.,."' . 
,.. / 
.......... 
LBIB 
1 -----
,0---,---------
14 -·-·-·-·-·-·-17 .................... .. 
18- .. -··- .. - .. _ .. . 
E 100 
0 
\:£ ,. ~. . .;.! ':..:.:: ;.:,.:.;.:.:~ =..:.. .... ;:;;.:.'...:: --\. ·""""--~-=-·"' ,,~:-.. . . ~ :;:..~" < r:::.:..::-.-. -- ---
«J 
a: 75 
\ '>, ,.;.~·· .. _ .. _ 
-..:.....'.'"' .. ' ··- .. 
..::/ -·-
5 0-1-_;!.,~--,----.-------,-
-55 40 80 205 -5 5 40 80 
Time from Arginine infusion (minutes) 
Figure 11 Effect of arginine infusion on plasma glucose concentrations 
in individual calves of different BI and sex group 
+ = arginine infusion 
205 
()\ 
-' 
-E 
'-.. 
m 
c 
-
c 
0 
-cu 
.. 
-c 
G) 
(,) 
c 
0 
(,) 
c 
·­
-::;, 
Cf) 
c 
·-
cu 
E 
Cf) 
cu 
-Q. 
t 
HBIH 
5 
7 ------
12---------
34---- --. 
so-·-·-·-·-·-
HBIB 
6 
13---16-------- -· I,. ~g:.:::..:.:·.:.:.~.:-.~.~:-.-:-.-:-. 
·-'\'. 42 __ -·-·-· 
;I' -' " -. 
r . " ' \ .... ...... ~· ' ................... ':.'i ·.~-. .:: . .._:~;;.;-,;;,-.:::-_;.;_-~,; 
----
0 
IT-o'' ---1 
-5 20 80 205 -5 20 
LBIH 
15-·-· -· -·-·-
20 -·--- --- --- --
21------
26-----
29----31''' .................. . 
LBIB · 
1 ..................... . 
8 
1 o-·-· -·-· -· -·-. 
14------
17---------· 
18-----· 
43_ .. _,_, ·-· ·-
--
------
.. -.. ·-··-··-··-··-·· 
80 205 
Time from Arginine Infusion (minutes) 
I 
Figure 12 Effect of arginine infusion on plasma insulin concentrations 
in individual calves of different BI and sex group 
+ = arginine infusion 
(J'\ 
f\) 
-E 
....... 
CJ) 
Q. 
CJ) 
_. 
-c 
0 
·-
-cu .. 
-c G) 
u 
c 
0 
u 
G) 
c 
0 
E 
.. 
0 
s:; 
s:; 
-~ 
0 
.. 
CJ) 
cu 
E 
f/) 
.! 
0.. 
6 
5 
4 
3 
6 
5 
4 
3 
l HBIH 
----50 34~ "-.,.. .. . ··::.···.:.·.-.::· . ·········:..:_-·~· ....... -·-rr .. -:-:.734 1
2- . -=-·""----.=---------- ---.12 5 0 .. ·-)~------- -·-·-·-·-·-. 5 
5 ~ I -·-7 .... ,,. 
~ .. HBIB 
13 ---·-· ..... . 
33 : --:::::..: 3 
\
,f ,- ..,._.._____ 1 1 .. -<.~ , .. .-:.,.?-·- ... :..:-----~.:::--- v 
1 I' .~..:..--· ·.. -·-. -. --...······ 42 42 . ............... - ---.33 
40 ~:· ·-·-·-·-·-·-8 
•, -··-40 
...... - .. _ .. __ .. - .. - .. - .. -·· 
-5 10 40 80 205 
~ LBIH 
21 
31 
21 • ~--, 1 
1 '· •. ' -----------· 
"· - -.-.--·-·~ .. '-':.:.·.:·.:::,: ...... ~-~·-·-280 
··- ' <· ·-·-·--'<>·· ···-· ---._ ·, -<·· --·-·- ----.•• -,: ..... :.-:;: ..:::. --.-
20 ·.. .;;:.. ·-·--·--28-.·:-:.::--:-:.:-
·.· 
--
--
--
-- 15 
~ LBIB 
8......_ 
--- . 
10 ----17~-. ---18 
1'4 ~·~:::.--, ,- ., ·-·-· -· -·-. _:-..::::.~-.::: :...---18 
1 - ... .._..,.,. ' '· 14 
43-, 
'\ 
- .. -··-··- .. - .. _ .. _"- .. - .. _ .. _ 1 
_...-- -----
-- -' 
--
-
--- 43 
-5 10 40 80 205 
Time from arginine Infusion (minutes) 
Figure 13 Effect of arginine infusion on plasma GH concentrations in 
individual calves of different BI and sex group 
+ = arginine infusion 
0'1 
\)J 
-E 
......... 
en 
c 
-c 
0 
·-
-«S 
... 
-c G) 
(,) 
c 
0 
(,) 
-
·o 
fl) 
·-
-
... 
0 
(,) 
«S 
E 
fl) 
«S 
-0. 
20 ~ HBIH ~ :'1 / . . I 
I . 
LBIH 
15 : i ! 
. I 
I 
10 
5 
0 
j l I 
: " \ 
21/ I I 16 
\ ....-28 
I I ·,_ ----
31• \ ~).,--------------._-20 
15 ·,~--><- .. -·-· -·31 I ,_.-...., ,.>...·. 20j''i ·,_.- ·-- .. _ 29 
28 'If· ......... ::-:-.., 21 
7 
'---- --12 
-----50 
20 HBIB LBIB 
1 
15 
10 - 1 1 lr, :.~ ~ ......... I, I\ 43, ......._ / , 14 / 
5 
0 
.\; ',....._ '-.../ 
42- ,· ... '/... ......... ...... ......... \ t . / ...... 
a -i!' '-·-·-·-~:~-Ius 1o "' -"' _,__.-~ -......., 43 
18 :·· I -----··-·· .... 42 1A·' :,_;;:;.--"" -··- .. - 18 
. . -- ,..-· --- .,- , r.' ··-. .... 9 <·J ,_ ...... ---- ······· 40 17-·\· .· ·.' . ----'-'---··-17 
: · ... ,!~~·. . ............ - -· 33 18 'f.J.~/ ~:-...... ----- 10 
: ,.. , ~ • • • • • • • . • .. of' 1..-- - ·\ 8 
3 /'/ . . 81 -------- -II I I I I I 
-5 20 80 205 -5 20 80 205 
Time from arginine infusion (minutes) 
Figure 14 Effect of arginine infusion on plasma cortisol concentrations 
in individual calves of different BI and sex group 
i = arginine infusion 
(}\ 
+ 
65 
Tables 4.9-4.12 which shows that: 
a) Significant hypoglycemia developed in all calves following 
arginine challenge, reaching a nadir about 40 minutes after infusion. 
It was noted that most LBI calves established a short-lived increment 
in glucose concentration before the occurence of hypoglycemia while 
HBI calves did not s~ow a similar response. Nevertheless no 
significant differences were found between calves of different BI and 
different sex in the plasma glucose concentration and its pattern (P > 
0.05) in response to arginine challenge. 
b) Following arginine infusion, there was a rapid insulin release 
in all calves. Peaks were ~etected within 5 to 10 minutes of arginine 
infusion. When compared with the pretreatment concentration, peak 
concentration of insulin in HBI group increased by 40%, in LBI group 
by 120%, in bulls by 60% and heifers by 100%, and fell to the 
pre-infusion concentration about 40 minutes after infusion when the 
glucose concentration was also at its nadir. Though there was no 
significant differences in plasma insulin concentration and insulin 
pattern between different ,BI ~d between different sex groups, the 
I 
percentage increment of plasma insulin concentration in the LBI group 
was significantly higher (120%) than in the HBI group (40%) (P < 
0.05). 
c) The response of GH concentration to arginine infusion was 
significantly different between HBI and LBI calves in terms of pattern 
and absolute concentration. LBI calves showed rapid decreases in GH 
concentration following arginine infusion, while GH concentrations in 
HBI calves were slightly increased and remained stable for about 30 
minutes before falling (Figure 13). 
d) Cortisol concentration fluctuated after arginine infusion 
(Figure 14) in all calves but no significant differences were found 
between HBI and LBI calves or between bulls and heifers. 
e) Covariance analysis was also conducted on the plasma 
metabolite and hormone concentrations following arginine treatment, 
using the value of sample 8 (pre-treatment sample) as a covariate. 
The adjusted results are presented in Tables 4.21-4.24 which show 
that: 
i) Arginine challenge produced a significant difference in GH 
concentration between HBI and LBI calves (P < 0.01) with HBI calves 
having the higher concentration. Arginine stimulation resulted in a 
66 
Table 4.21 Analysis of covariance of repeated measurements 
of glucose concentrations in response to arginine 
challenge between groups of calves 
of different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Basal 1 2.87 
Time 5 35.81 *** 
BI 0.34 NS 
Sex 1 0.01 NS 
BI x Sex 1 0.29 NS 
Time x BI 5 4.75 NS 
Time x Sex 5 5. 72 NS 
Time x BI x Sex 5 4.11 NS 
Number of observations 24 
at per stage 
Table 4.22 Analysis of covariance of repeated measurements 
of insulin concentrations in response to arginine 
challenge between groups of calves of 
different BI and sex. 
Source of Variation 
Basal 
Time 
BI 
Sex 
BI x Sex 
DF 
5 
Time x BI 5 
Time x Sex 5 
Time x BI x Sex 5 
Number of observations 24 
at per stage 
Chi 
s uare 
11.65 
30.87 
0.15 
0.95 
o.oo 
5.29 
5.80 
8.41 
Significance 
*** 
NS 
NS 
NS 
NS 
NS 
NS 
Table 4.23 Analysis of covariance of repeated measurements 
of GH concentrations in response to arginine 
challenge ,between groups of calves of 
different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Basal 32.12 
Time 5 21.03 *** 
BI 8.10 ** 
Sex 1 0.30 NS 
BI x Sex 1 2.20 NS 
Time x BI 5 13.59 * 
Time x Sex 5 4.42 NS 
Time x BI x Sex 5 6.64 NS 
Number of observations 24 
at per stage 
Table 4.24 Analysis of covariance of repeated measurements 
of cortisol concentrations in response to 
arginine challenge between groups of calves of 
different BI and sex. 
Source of Variation DF Chi Significance 
s uare 
Basal 1 2.72 
Time 5 6.29 NS 
BI 0.01 NS 
Sex 1 0.01 NS 
BI x Sex 1 o.oo NS 
Time x BI 5 1.16 NS 
Time x Sex 5 2.54 NS 
Time X BI x Sex 5 1.36 NS 
Number of observations 24 
at per stage 
67 
different GH response pattern between HBI and LBI groups (P < 0.05). 
ii) Arginine infusion per ~ did not result in a significant 
diffe~ence in plasma glucose, insulin, and cortisol concentrations 
between HBI and LBI or between bull and heifer calves. 
4.4 After synthetic corticosteroid (Betsalan) injection 
Mean values of plasma glucose, insulin, GH and cortisol 
concentrations of different calf groups before and after Betsalan 
injection are plotted in Figs 6-9. 
4.4.1) Comparison of pre-injection value with the after-injection 
value: 
68 
Results of the statistical analysis of the corresponding means of 
metabolite and hormone concentration on day one (before injection) and 
day two (after injection) are in Table 4.25 which show: 
a) The mean plasma glucos~ concentration were significantly 
elevated following Betsalah injection for all groups of calves (P < 
o. 01). 
b) Plasma insulin concentration was also significantly raised by 
Betsalan injection (P=0.054) for all calves but the increment in 
individual groups was not significant even though glucose 
concentration was increased by 41% in the HBI group; 63% in the LBI 
group; 52% in bulls and 44% in heifers. 
c) GH concentrations were also enhanced in all group of calves 
following Betsalan treatment. However none of the increments were 
significant (P > 0.05). 
d) Endogenous cortisol production was significantly suppressed 
after the injection of synthetic corticosteroid (Betsalan) in calves 
of different BI and sex group. (P < 0.01). 
4.4.2 Comparison between calves of different BI and sex 
Results of statistical analysis of the plasma metabolite and 
hormone concentration in the samples collected after Betsalan 
injection are presented in Tables 4.13-4.16 which shows that: 
a) HBI calves still had a higher GH concentration than LBI calves 
69 
Table 4.25 Mean plasma glucose, insulin, GH and cortisol 
concentrations of calves on day one before, 
and on day two after synthetic corticosteroid 
injection, together with the statistical 
significance of the difference. 
Hormones Group before After Significance 
Injectio Injection 
Insulin Overall 18.5 27.8 * 
(ng/ml) HBI 23.7 33.6 NS 
LBI 14. 1 23.0 NS (P<6.5~) 
Bulls 22.9 34.9 NS 
Heifers 13.4 19.4 NS 
Glucose Overall 103.4 151.0 *** 
(mg/dl) HBI 107.8 152.0 *** 
LBI 99.6 150.1 *** 
Bulls 105.0 155.0 *** 
Heifers 101.4 146.1 *** 
GH Overall 10.7 11.0 NS 
(ln pg/ml) HBI 11.3 11.4 NS 
LBI 10.2 10.6 NS 
Bulls 10.8 11.0 NS 
Heifers 10.6 10.9 NS 
Cortisol Overall 5.6 0.5 *** 
(ng/ml) HBI 5.6 0.4 *** 
LBI 5.6 0.6 *** 
Bulls 5.3 0.6 *** 
Heifers 5.9 0.5 *** 
(11.4 vs 10.6 ~n pg/ml; P = 0.06) following Betsalan injection and 
the interaction of sampling and calf sex was significant at the 5.3% 
level. 
10 
b) HBI calves still maintained higher, but not at the 5% 
significance level, plasma insulin concentrations than LBI calves 
(33.6 vs 23 ng/ml P= _Q.09) and the bulls had significantly higher 
insulin concentrations than the heifers (34.9 vs 19.4 ng/ml P< 0.05). 
c) Plasma glucose concentrations were slightly higher in HBI and 
bull calves (152 and 155 mg/dl respectively) than those in LBI and 
heifer calves (150 and 146 mg/dl respectively), but the difference 
were not significant (P > 0.05). 
d) Cortisol concentration was lower in all groups of calves and 
no significant difference was found between HBI and LBI calves or 
between bulls and heifers. 
4.5 The relationship between calf BI and plasma metabolite 
and hormone concentrations: 
The correlation coeff~cients of calf BI estimated from ancestry 
and the plasma metabolite and hormone concentrations estimated at four 
different stages of the experiment for different BI and sex groups are 
presented in Table 4.26 and the simple regres~ion lines for the BI and 
basal (pre-feeding) metabolite and hormone concentrations are plotted 
in Figure 10 • It is noted that: 
a) The correlation coefficients of BI and basal insulin 
concentration were significant for all calves (r=0.54, P< 0.01); and 
for the HBIB group (r=0.82, P < 0.05). The value of the correlation 
coefficient between BI and basal insulin were not significant for LBIB 
(r=0.74, P: 0.07), for HBIH (r = 0.59, P>0.05) and for LBIH (r=0.37, 
P>0.05). 
The correlation coefficients of the estimated BI and basal 
glucose, GH and cortisol concentrations were not significant for all 
group of calves with the exception of the relationship between BI and 
basal glucose in LBIB and basal GH in LBIH. The correlation 
coefficient of BI and basal glucose value was significant for LBIB (r 
= -0.78, P = 0.05). The correlation coefficient of the estimated BI 
and basal GH was significant for LBIH (r=-0.81,P<0.05). 
b) The correlation coefficients of the estimated BI and plasma 
71 
Table 4.26 Correlation coefficients between BI and concentrations 
of glucose, insulin, GH and cortisol in calf plasma 
sampled at four different stages of the experiment. 
Experimental Calf 
stages 
Pre-feeding 
After­
feeding 
After 
arginine 
infusion 
After 
Bets alan 
injection 
group 
Overall 
HBIB 
HBIH 
LBIB 
LBIH 
Overall 
HBIB 
HBIH 
LBIB 
LBIH 
Overall 
HBIB 
HBIH 
LBIB 
LBIH 
Overall 
HBIB 
HBIH 
LBIB 
LBIH 
Glucose Insulin Cortisol 
(mg/dl) (ng/ml) (ng/ml) 
0.33 
-0.04 
0.17 
-0.78* 
-0.25 
0.13 
0.25 
-0.10 
-0.25 
-0.06 
0.11 
0.23 
0.33 
-0.29 
0.21 
0.01 
0.83* 
-0.11 
-0.29 
0.05 
0.54** 
0.82* 
0.59 
~~""--_, 
o. 74* 
0.57 
0.38* 
0.38 
0.46 
0.49 
-0.40 
0.11 
0.38 
0.43 
0.27 
-0.03 
0.21 
0.41 
0.36 
0.42 
-0.13 
o. 11 
0.31 
-0.12 
0.21 
-0.17 
0.03 
0.03 
-0.15 
0.20 
-0.24 
0.02 
-0.16 
0.34 
0.27 
-0.38 
-0.11 
0.33 
-0.34 
-0.17 
-0.38 
GH 
Eln pg/ml) 
0.31 
_0.56 
0.46 
0.47 
-0.81* 
0.27 
0.27 
0.39 
0.42 
-0.71 
0.34 
-0.36 
0.45 
0.35 
-0.68 
0.29 
-0.62 
0.38 
-0.09 
-0.64 
Note: the concentrations of insulin from calf 20 and GH from calf 
43 (both were LBI calves) were not used in the calculation of 
correlation coefficients as their concentrations were anomalously 
lower than the corresponding means of the LBI group. 
a) Glucose concentration b) Insulin concentration 
(mg/dl) (ng/ ml) 
120 30 ... 
... 
... 
... 
...~ . 100 0 ..... 20 
• • •• 
... 
.... 
... 
80 
... 10 
oo 
... 0 
70 & I I 
c) GH concentration d) Cortisol concentration 
14 
(Ln pg/ml) 
30 
(ng/ml) 
13 
... !:. 
12 • 20 !:. ... ... 
... .... • 11 
!:. wtt ... 
10 aro 10 • ~ !:. • 9 0 • !:. 
110 120 130 140 110 120 130 140 
Calf breeding Index Value HBIBA 
HBIHe 
LBIB!:. 
LBIHO 
Figure 10 Relationship between breeding index of calves and the basal 
concentration of (a) glucose, (b) insulin, (c)GH, and (d) 
cortisol in the plasma within BI and sex group 
72 
73 
glucose, insulin, GH and cortisol concentration after feeding were not 
significant in any group of calves. 
c) The correlation coefficients of the estimated BI and plasma 
glucose, insulin, GH and cortisol concentration after arginine 
infusion and after Betsalan injection were not significant for all 
groups with the exception of a significant relationship between BI and 
glucose concentration following Betsalan injection in the HBIB group 
(r=0.83, P<0.05). 
4.6 Other physiological traits 
Mean values of birth weight, blood immunoglobulin (Ig) G1 
concentration and haematocrit for different calf groups are presented 
in Table 4.27. Statistical analysis of these results shows that the 
birth weight of bulls (44 kg) was significantly higher than that of 
heifers (40 kg) (P<0.05). IgG1 concentration in bulls was 
significantly higher than in heifers (22.5 vs 15.3 mg/ml,P<0.05). 
Four LBI calves 20, 29, 31, 43 had such low IgG1 concentrations in 
I 
their blood that it was below the sensitivity of the assay. When 
these low IgG1 values were included in the statistical analysis IgG1 
concentration in HBI calves was significantly higher than in LBI 
calves (19.5 vs 14.5 mg/ml, P<0.05) but no such difference was present 
after the rejection of these values from LBI group (19.5 vs 20 mg/ml 
for HBI and LBI respectively). Mean value of haematocrit in HBI group 
was slightly higher than that in LBI group (38.9 vs 37.8), and these 
value agreed well with other studies (Bradley et al, 1982). 
74 
Table 4.27 Mean values of birth weight, IgG1 concentration and 
haematocrit in calf groups of different BI and sex. 
Variable Mean BI of calves Sex of calves 
HBI LBI significance M F Significance 
(HBI-LBI) (M -F) 
Number 24 11 13 
Birth Weight 42.4 42.5 42.5 NS 44.1 40.4 * 
(kg) 
IgG1 (mg/ml) 19.7 19.5 20 NS 22.5 17.3 * 
Haematocrit 38.3 38.9 37.8 NS 37.5 39.3 NS 
(%) 
75 
CHAPTER FIVE: DISCUSSION 
5.1 General discussion 
All calves appe~red to have acquired adequate Immunogloblin G1 
(IgG1) from their dam's colostrum after birth except 4 calves in the 
LBI group whose IgG1 concentration was less than 2 mg/ml. It would 
appear that there was a failure in the transfer of immunoglobulins 
from dam to circulation in these four calves. Further studies would 
be needed to ascertain if this was affected by breeding index of the 
cow and the calf. Even so no problems of calf health was incurred 
during the experiment possibly due to the strict hygiene practice 
applied. It is hard to explain the higher IgG1 concentration observed 
in the bulls. It could be caused by a greater consumption of 
colostrum by the bulls or higher IgG1 concentration in the colostrum 
of bull's dam at the time when the calf was born. Differences in the 
closing time of the intestinal barrier for IgG1 absorption and in the 
I 
turnover rate of IgG1 may be responsible for the different IgG1 
concentrations between the bulls and heifers. It is not known if the 
endogenous IgG1 production differs in quantity between bulls or 
heifers, but it is normally accepted that endogenous IgG production 
will not become dominant until many weeks after birth (Roy, 1980; 
Butler, 1969). 
The greater birth weight of the bulls observed in this study 
agreed with the report of others (Appleman et al, 1974). 
The concentration of the various hormones and metabolite in the 
plasma samples collected in the present experiment were within the 
range of values reported in the literature. Thus comparable values 
have been measured for glucose (Bradley et al, 1982), insulin and GH 
(Bohn, 1978; Roy et al, 1983; Flux et al, 1984) and cortisol 
(Venkalaseshu and Estergreen, 1970). 
5.2 Effects of fasting, feeding, intravenous arginine infusion, 
and subcutaneous synthetic corticosteroid injection on 
plasma glucose and hormone concentrations 
5.2.1 Effect of fasting 
76 
After 16 to 17 hours fasting, the basal plasma glucose and 
insulin concentrations were significantly lower than those observed in 
subsequent samples collected after feeding (Figures 2-5). This is in 
agreement with the established knowledge that plasma energy substrate 
concentrations, especially glucose concentration, are the main stimuli 
for insulin secretion. In fasting animals decreased absorption of 
nutrient from the gut will lead to hypoglycemia, thus hypoinsulinemia. 
The prefeeding plasma cortisol concentrations in the calves were 
significantly higher than those after feeding (Figure 5). Maximal 
cortisol production has also been reported to occur in the early 
morning in many species (see section 2.1.3) but the pattern is less 
discernable in adult ruminants. Glucocorticoids are known to have a 
major role in glucose homeostasis during fasting (Trenkle, 1981; 
McDowell, 1983, Tischler, 1981). They promote endogeneous glucose 
production by facilitating gluconeogenesis and antagonize the anabolic 
action of insulin probably by reducing the affinity of insulin 
receptors (Kahn et al, 1978), thus leading to increased blood glucose 
concentration. 
Increased GH concentration as a result of low plasma energy 
concentrations has been found in fasted animals of many species (Roth 
et al, 1964; Machlin et al, 1968; Mcintyre and Odell, 1974; Purchas 
et al, 1971; Hove and Blom, 1973). However in the present study, no 
such increment has been found in basal GH concentrations when compared 
to the value in the subsequent samples. This may due to the 
infrequent samplings over the pre-feeding time. Great variation were 
found in the basal GH concentrations in all calves which is consistent 
with an episodic release of GH. 
77 
5.2.2 Effect of feeding 
Changes in both the glucose and insulin concentrations in 
response to feeding in the present study were bi-phasic (Figures 2-3). 
This was very apparent in bull calves. These patterns are almost 
identical to those of non-ruminants (Mayhew et al, 1969) and similar 
responses were observed in young ruminants (Stern et al, 1971; Bohn, 
1978). Though increased plasma glucose concentration is primarily due 
to the absorbed digestive products from the gut, the initial glucose 
response to feeding may also be endogenous caused by feeding-induced 
glucagon and catecholamines release. 
Insulin response in some calves preceded the changes in glucose 
concentration. Similar observations were also made in steers (Chase 
et al, 1977). It was suggested that a direct neural stimulation of 
the pancreatic B-cells or the release of some gut hormones such as 
GIP, or CCK on the ingestion of food were responsible for the rapid 
insulin release (Bassett, 1981; Parman, 1979). Another explanation 
for the post-prandical insulin secretion pattern was proposed by 
Schusdziarra (1980) who suggested that feeding-induced insulin release 
was modulated by somatostatin (SLI) secreted from the gastrointestinal 
and pancreatic area. In spite of many other factors, the 
post-prandial insulin concentration is primarily controlled by the 
increased glucose concentration as observed in the present study in 
which the major insulin peak coincided with the maximal plasma glucose 
concentration. 
The observed fall of GH concentration after feeding (Figure 4) is 
consistent with other reports (Bassett, 1974, 1975; Blom et al, 1976; 
Hove and Blom, 1973). The diminished episodic GH secretion after 
feeding does not correlate with the energy concentration, a case which 
might occur in animals sustaining chronic energy deficit. In fact GH 
concentration peaked in most calves about 3 hr after feeding when 
plasma glucose concentration was still significantly elevated in 
response to feeding. So far the literature contains very few 
satisfactory explanations for the stablized GH concentration 
immediately after feeding. Bassett (1975) proposed that the secretion 
of hypothalamic GHRF and SLI may be influenced by feeding. However, 
there is no direct evidence to support that statement. Recent 
evidence shows that plasma SLI concentration was elevated in dogs 
78 
following feeding (Shusidziarr et al, 1978a, 1978b) due to the 
secretion of SLI from gastrointestinal and pancreatic area and it is 
established that increased blood SLI concentration has a powerfull 
inhibitory effect on GH secretion (Fernandez-Durango et al, 1978; 
Siler et al, 1973; Mortimer et al, 1974). So it is_postulated here 
that the suppressed GH concentration after feeding may be due to the 
inhibitory effect of SLI released from gastrointestinal and pancreatic 
area rather than from the hypothalamus. However as reviewed in 
chapter two, the regulation of GH secretion is complex, it would not 
be suprising if many other factors are involved in the regulation of 
post-prandial GH release. 
Cortisol concentration was depressed in all calves following 
feeding with a nadir developed 2 - 3 hr later (Figure 5) when plasma 
glucose and insulin reached their maximal values. The reduced 
cortisol production may be due to the inhibition of 
hypothalamus-pituitary release of CRF and ACTH which is sensitive to 
various stresses and hypoglycemia. The stimulatory effect of 
hypoglycemia on CRF-ACTH release, thus cortisol production, may be 
antagonized by the hyperglycemia developed following feeding as 
observed in the present study. Rapid short-lived cortisol release was 
also recorded in some calves during the time immediately after 
feeding. This was probably caused by the frequent handling of the 
calf during sampling or in response to drinking. 
5.2.3 After arginine infusion 
Arginine infusion triggered a burst of insulin release in all 
calves (Figure 12). This was consistent with other studies (Bohn, 
1978, Mayhew et al, 1969; Hertelendy et al, 1970; Davis, 1972). The 
mechanisms by which the pancreatic B-cell is stimulated by arginine 
and other amino acids is still unknown, possibly by potentiating the 
effect of glucose on insulin release. In the present studies, 
arginine was infused into the calf at a time when the glucose 
concentration was still high following feeding. It is known that the 
combination of glucose and certain amino acids is a better stimulus 
for insulin release then either of them alone (Pagliara et al, 1974). 
Following arginine infusion none of the calves showed significant 
increases of blood glucose concentration (Figure 11). In fact, 
79 
glucose concentration declined steadily in most calves. This result 
was in contrast to the reports of others who observed increased 
glucose concentration following arginine infusion (Bohn 1978, Gerich 
et al, 1974; Pagliara et al, 1974) and attributed it to the effect of 
concurrent release of glucagon. The discrepancy in the results 
between this study and others may be due to the lower dosage of 
arginine used in the present experiment (0.04g/kg bwt vs 0.5g/kg bwt). 
A significant hypoglycemia development in all calves soon after 
arginine challenge (Figure 11). This probably was a secondary effect 
of the hyperinsulinemia as the insulin response preceded the occurence 
of hypoglycemia. A similar observation was also made by Bohn (1978). 
Arginine infusion has been shown to stimulate GH release in 
animals in many studies (Hertelendy et al, 1970; Stern et al, 1971; 
Davis, 1972; Bohn,1978). However, GH response to arginine challenge 
was not consistent in the calves of present study (Figure 13 ). This 
may due to the lower dosage of arginine given. The apparent 
differential effect of arginine on GH release between calves of 
different BI will be discussed later (in secton 5.3). 
No significant cortisbl responses to arginine challenge were 
found in the present study (Figure 14). The small fluctuation of 
cortisol concentration may be due to the restraint of the calves 
during the period of intensive sampling or due to the insulin 
hypoglycemia as increased plasma corticosteroid concentration was 
found in sheep with insulin hypoglycemia (Dooley and Williams, 1975). 
5.2.4 After injection of synthetic corticosteroids (Betsalan) 
Plasma glucose concentration in all calves were significantly 
elevated after Betsalan injection (Figure 6). This effect of 
synthetic corticosteroids on glucose concentration may be achieved by 
two ways: a) an increase in the rate of endogenous glucose 
production; b) a decrease of peripheral utilization of glucose. 
Studies by Ford (1971) indicated that the increased rate of 
gluconeogenesis in the liver and kidney of sheep are responsible for a 
betamethason injection induced hyperglycemia and the peripheral 
utilization of glucose was not reduced at the time 24 hr after 
injection. A study by Bassett (1963) suggested that a progressive 
impairment of glucose utilization relative to the blood glucose pool, 
80 
but not a reduction in total glucose utilization occurred in sheep 
after cortisol injection. There are other data which suggested that 
glucose utilization by muscle (Morgan et al, 1959) and adipose tissue 
(Feldman, 1977) were inhibited by administration of corticosteroids. 
The increased insulin concentration following Betsalan injection 
in the present study was consistent with the observation by Bassett 
and Wallace (1967). The elevated insulin concentrations may be caused 
by: a) cortisol induced hyperglycemia as high glucose concentration 
is well known as a good stimulus for insulin release and synthesis; 
b) direct effect of cortisol on B-cell. Recent studies showed 
increased insulin response ~o oral glucose and intravenous tolbutamide 
infusion in man ( Parley et al, 1968) and to intravenous glucose 
administration in sheep (Bassett and wallace, 1967) after cortisol 
treatment. More recent investigations have demonstrated the presence 
of steroid receptors on the islet cells (Green et al, 1978; Tesone et 
al, 1979) and the direct influence of insulin secretion by circulating 
glucocorticoids (Borelli et al, 1982); c) increased half-life of 
plasma insulin due to the red~ced binding to receptor in the 
I 
peripheral tissue. Decreased insulin binding has been observed in 
animals following glucocorticoid treatment (Olefskey et al; 1975, 
Kahn, 1976). 
Endogenous immunoreactive cortisol concentration fell 
significantly in all calves following Betsalan injection (Figure 9). 
This fall was most probably accompanied by corresponding decreases in 
CRF and ACTH release as it is well known that cortisol production in 
adrenal cortex is under the direct control of ACTH whose secretion in 
turn is under the regulation of CRF (section 2.1.3). It is suggested 
that the synthetic corticosteroids (in the present study Betsalan) 
regulate ACTH secretion probably through a delayed feedback system, 
i.e. by reducing the rate of CRF synthesis, while endogenous cortisol 
and cortisone regulate ACTH release through a fast feedback system 
i.e. by regulating CRF release (Jones, 1979). 
GH concentration was not significantly affected following 
Betsalan injection. This result is not conclusive. In view of the 
episodic nature of GH release the number of samples taken after the 
injection of Betsalan were insufficient for an accurate assessment of 
GH concentration. It is currently believed that administration of 
glucocorticoids impair GH release (Chiodin and Liuzzi, 1979). 
5.3 Effect of genetic merit for milk fat production on plasma 
metabolite and hormone concentrations 
81 
The impact of genetic divergence for milk production on the 
plasma metabolite and hormone concentrations of the calf has been 
clearly demonstrated by the present study. Calves of high genetic 
merit (HBI) had higher plasma glucose, insulin, and GH concentrations 
than LBI calves for most of the time of the experiment (Figures 2-9 ). 
Similar difference were also found between HBI and LBI cows with 
similar genetic background to the calves when feed intake of the cows 
was restricted (Flux et al, 1984). The present experiment for the 
first time showed that such differences also exist between bull calves 
of different genetic merit. 
1) Glucose: Glucose concentrations were significantly different 
between HBI and LBI calves after 16 hr fasting (p < 0.05), but the 
difference was minimized after. feeding, arginine infusion and 
\ 
synthetic corticosteriod injection ( P > 0.05). One recent study in 
England (Tilakaratne et al, 1980) also showed that glucose 
concentration concentration was constantly higher in calves of high 
genetic merit than in calves of low genetic merit for milk production. 
The prolonged fasting (30 to 40 hours) in that experiment also 
resulted in a significant difference in plasma glucose concentrations 
between the two groups of calves. Studies with the lactating cows 
showed that LBI cows had significantly lower plasma glucose value than 
HBI cows when the animals were underfed, while the underfed HBI cows 
maintained glucose concentration comparable to that of better fed cows 
(Flux et al, 1984). This evidence suggests that dairy cattle of high 
genetic merit have the ability to maintain higher glucose 
concentrations than that of low genetic merit especially under 
conditions of energy deficit, regardless of the age of animals. It is 
known that glucose is a limiting factor in determining milk yield of 
lactating ruminant, as glucose is the precusor of lactose in milk • 
The higher basal glucose concentration in HBI calves may be due 
to either an increased endogenous glucose production or decreased 
peripheral utilization of glucose. As gluconeogenesis is stimulated 
by glucagon and glucocorticoids, both hormones might be involved in 
82 
th~ present study. Administration of cortisol to sheep (Bassett and 
Wallace, 1967) and Betsalan to calves (present experiment) resulted in 
hyperglycemia and hyperinsulinemia. The increased glucose 
concentration is caused by increased gluconeogenesis from amino acids 
in liver and decreased glucose turnover (Reilly and Black, 1973). In 
the present study, however, basal cortisol concentration were only 
slightly and not significantly higher in HBI calves than in LBI 
calves. Thus it is unlikely that the higher basal glucose 
concentration in HBI calves can be attributed to an increase of 
glucose production stimulated by cortisol, but the role of glucagon 
requires further investigat~on. Administration of glucagon to sheep 
also resulted in a significant increase in blood glucose concentration 
(Bassett, 1971). 
A decreased glucose utilization in HBI calves in comparison with 
LBI calves could be caused by: 
a) Increased GH concentration: Chronic administration of 
exogenous GH increased blood glucose concentration in non-lactating 
animals (Wagner and Veenhuizen, 1978, Wagner et al, 1970, Wallace and 
I 
Bassett 1966; Bassett, 1978) and insulin is less active in GH treated 
sheep (Wagner et al, 1970). In the fasting state the effect of GH is 
though to be lipolytic. It promotes the mobilization and oxidation of 
FFA, thus reducing the peripheral utilization of glucose. In the 
present study basal GH concentrations were significantly higher in HBI 
calves though great variation existed within both BI groups. It may 
be the major factor accounting for the higher glucose concentration 
found in the HBI group. High GH and glucose concentrations were also 
found in HBI cows (Flux et al, 1984) when feed intake was 
restricted.The lactogenic effect of GH in ruminant has been 
excellently reviewed (Cowie et al, 1980; Hart, 1981; Bauman and 
McCutchen, 1984). 
b) Increased molar ratio of glucagon to insulin due to a large 
decrease in the insulin concentration in the fasting state. Glucagon 
action on adipose tissue is also lipolytic when insulin concentration 
is kept low (Brockman, 1976). Both of these would facilitate 
gluconeogenesis and reduce peripheral utilization of glucose. 
c) Increased energy substrate concentrations other than glucose. 
Increased FFA concentrations were found in calves of high genetic 
merit in prolonged fasting (Tilakaratne et al, 1980). In lactating 
cows high FFA concentrations were also found to be positively 
associated with milk yield (Hart et al, 1978). It may be inferred 
from these studies that basal FFA concentrations were also higher in 
HBI calves than in LBI calves in this experiment. A higher FFA 
concentration together with high GH concentration in the HBI group 
would reduce the requirement for glucose by the peripheral tissue. 
d) Decreased anabolic action of insulin: This may be due to the 
decreased binding of insulin to its receptor on the peripheral 
tissues. High insulin concentration does not necessarily mean an 
increased insulin action, as the affinity of the insulin receptor is 
down regulated by the amount of circulating insulin (Olefsky and 
Reaven, 1977; Olefsky, 1981). 
e) Increased glucocorticoid production: Administration of 
cortisol was found to cause hyperglycemia in animals (Bassett and 
Wallace, 1967) by impairing glucose utilization. This is probably 
achieved by altering the affinity and number of insulin receptors on 
the target tissue (Kahn et al, 1978). However, mean cortisol 
concentration was only slightly higher in the prefeeding samples in 
HBI calves than in LBI cal~es (Figure 5). 
83 
2) Insulin: Plasma insulin concentration was consistently higher 
in HBI calves than in LBI calves (Figures 3 and 7) and this difference 
was significant during fasting, after feeding and after synthetic 
corticosteroid (Betsalan) injection. Higher insulin concentrations 
were also found in HBI cows which were under fed (Flux et al, 1984). 
This is hard to explain. The higher insulin concentration may be 
caused by higher glucose concentration observed in HBI calves and HBI 
cows as it is well accepted that glucose is the most efficient 
stimulus for B-cell function. Higher GH concentration found in HBI 
calves might also contribute to the difference in insulin 
concentration between HBI and LBI groups. Acute pancreatic insulin 
release has been observed in dogs following intravenous GH injection 
(Sirek et al, 1979). GH treatment of cows caused elevation of insulin 
secretion in one study (Bines et al, 1980) but was not recorded in 
another (Peel et al, 1983). So far however, there is no conclusive 
evidence to show that GH can stimulate insulin release from the 
B-cells as purified hGH did not affect insulin secretion (Larson, 
1978). It is still possible that GH may affect plasma insulin 
concentration by changing the affinity of insulin receptors, probably 
by promoting the production of somatomedins, and insulin like growth 
factors. Recent studies implied that some of the peptide hormones may 
compete with insulin for insulin receptors in the peripheral tissues 
(Czech et al, 1983). Investigation in this field is still going on. 
Injection of cortisol_has been found to increase the concentration of 
insulin as well as glucose in ruminants (sheep: Bassett and Wallace, 
1967; calf: the present experiment). However,it is unlikely that 
the consistently higher insulin concentrations found in HBI calves 
were caused by the cortisol , as there was no significant difference 
in plasma cortisol concentra~ion between the HBI and LBI groups. The 
higher insulin concentration found in HBI calves was most probably 
accompanied by a higher glucagon concentration as parallel changes 
were found in plasma insulin and glucagon concentrations of sheep 
following feeding and injection of energy substrates (Bassett, 1972). 
Glucagon infusion also resulted in a rapid increase in insulin 
concentration in sheep (Bassett, 1971). Further investigation of 
glucagon concentration in HBI and LBI calves would be desirable. 
3) Growth Hormone: Higher plasma GH concentration in HBI calves 
in the present study were consistent with the observation in HBI cows 
(Flux et al, 1984). Higher GH concentrations were also found in a 
high milk yielding breed than in a low yielding breed (Hart et al, 
1978). It is postulated here that the hypothalamic GHRF and SLI 
secretion may differ between HBI and LBI calves. In the present study 
the higher GH concentration in HBI group was apparently not caused by 
insulin hypoglycemia as the concentration of insulin and glucose were 
consistently higher in HBI calves. Glucagon, again, may play a role 
in determining GH concentrations in calves as glucagon administration 
to sheep increased plasma GH, insulin and glucose concentrations 
(Bassett, 1971). 
In response to arginine challenge the differences in plasma GH 
concentrations between HBI and LBI groups become significantly 
different • HBI calves not only had higher GH concentrations but 
maintained such concentrations for a longer time than LBl calves 
(Figures 4 and 13). The biological relevance of this might be very 
important in that HBI calves could release more GH than LBI calves 
when the same amounts of amino acid are available to the animals. GH 
84 
85 
is known to be a major hormone for the preservation of body protein, 
particularly during periods of energy deficit by stimulating the 
incorporation of amino acids into protein while directing glucose away 
from tissue deposition and by possibly stimulating lipolysis (Raben, 
1973). The different responses of GH concentration to arginine 
challenge of a relatively low dosage under ~he condition of the 
present study might result from a difference in hypothalamic 
sensitivity to arginine challenge between HBI and LBI calves. 
However, a possibility that the episodic nature of GH release caused 
the different GH response still exists. An accurate estimate of GH 
may not have been obtained ~ith the infrequent sampling regime used in 
the present study, and further experiments are required to reach a 
reliable conclusion. 
5.4 Sex effect: 
The present study showed that bull calves had consistently higher 
insulin concentrations than heifers (Figures 3 and 7), and the 
. 
differences were significant throughout the experiment. Glucose 
concentration was also significantly higher in bulls than in heifers 
during the post-prandial period (Figure 2). Cortisol concentration, 
however, was significantly lower in bulls than in heifers following 
feeding (adjusted result Table 4.20, Figure 5). 
The effect of sex on plasma metabolite and hormone concentrations 
has been less investigated in calves. Tilakaratne et al (1980) 
reported that sex had little effect on plasma concentrations of BHBA, 
glucose, FFA, urea, total protein and albumin, only globulin 
concentration was significantly higher in bulls (P<0.05). In the 
present study, higher glucose concentration developed in bulls which 
coincided with the lower cortisol concentration occuring in bulls at 
the same time. This suggests that glucose entry rate from the gut 
might be faster in bulls than in heifers. It is not clear if the 
lower cortisol concentration in bulls after feeding was caused by the 
higher glucose concentration in bulls or by mechanisms other than 
hyperglycemia. 
The observed higher insulin concentrations in bulls is also hard 
to explain. This is probaly caused by the sex steroids which might 
act on the pancreatic B-cells and peripheral insulin receptor (Tesone 
86 
et al, 1979). This is yet to be tested. It may be inferred from this 
experiment that pancreatic B-cells were more mature in bulls than in 
heifers at the time of the study. This is indirectly supported by the 
fact that the body weight of bulls was significantly higher than that 
of heifers. 
GH concentration was slightly but not significantly higher in 
bulls than in heifers as observed in the study (Figures 4 and 8). 
This suggested insulin instead of GH is the major anabolic hormone for 
the early development of the young calf. 
5.5 Estimation of calf genetic merit by using metabolite 
and hormone concentrations as gene markers 
1l The primary aim of this experiment was to investigate the 
metabolic and endocrinological response to various treatments in HBI 
and LBI calves with particular emphasis on the early identification of 
genetically superior bulls by using the metabolite and hormones as 
markers to predict the BI of the calf. As the BI of each calf in the 
present study was estimated from their ancestry BI (instead of true BI 
from a progeny test), some discrepencies between the estimated and 
true Bis for each calves are expected. This is because: 
a) for a calf born to a sire which had been used very widely in 
AI and to a dam itself born to genetically superior parents and with a 
good lactation performance the upper limit for accuracy of BI 
estimated from ancestry would be about 0.4. The standard deviation of 
the BI for each calf would be about 7 BI units (Table 3.2). 
b) For a large number of calves all with estimated Bis of 130, 
95% of their actual Bis would be expected to lie in the range of 116 
to 144. Thus a high correlation between a measurement of hormone or 
metabolite concentration and the estimated BI could not be expected 
even if there was actually a high correlation between the hormone or 
metabolite measurement and the true BI. 
c) The number of calves used in this experiment was not very 
large and the estimated Bis are not evenly distributed within each BI 
groups. 
87 
£L Nevertheless results of the present study suggest that basal 
glucose and insulin concentration may be used as markers to 
discriminate between HBI and LBI calves in general. GH concentration 
and pattern after arginine challenge may also be useful criteria for 
selecting HBI calves. All of these will be discussed in the following 
section. 
i) Basal glucose: Though fasting glucose concentration was 
significantly higher in the HBI group than in the LBI group (102 vs 93 
mg/dl, P< 0.05), the correlation coefficients of the estimated BI and 
basal glucose concentration for all calves was not significant (r = 
0.33,P> 0.05, Table 4.26, and Figure 10). Calves with the highest 
estimated BI did not have the highest fasting glucose concentration 
within each group. The correlation coefficients of the BI and basal 
glucose within each group is either small or negative. Thus it 
appears that glucose would not serve as a good marker to predict the 
estimated BI of individual calves, though it appears that basal 
glucose concentration may be used to discriminate HBI and LBI calves 
as a group. 
ii) Basal insulin: Basal insulin in the preliminary results 
showed promise as a gene marker to estimate the genetic merit of the 
young Friesian calf as the correlation coefficents of BI and basal 
insulin were significant for all calves (r=0.54, P<0.01); for HBIB 
(r=0.84, P<0.05); for LBIB (r=0.78, p=0.07) (Figure 10, Table 4.26 ). 
However, the value of this result in the early identification of 
superior dairy bull has yet to be tested using bulls whose BI has been 
estimated in the progeny testing programme. 
iii) GH response to arginine challenge: In response to arginine 
challenge GH concentration differed significantly in terms of absolute 
level and pattern between HBI and LBI calves (Figure 4, Tables 4.11 
and 4.23). It appears that HBI calves can be discriminated from LBI 
calves by their differing GH response to arginine challenge. In 
general HBI calves had higher GH concentration and remained elevated 
after arginine infusion while GH concentration fell rapidly in LBI 
calves after arginine infusion. 
88 
CHAPTER SIX: SUMMARY AND CONCLUSION 
Some important metabolic and endocrinological differences were 
found between HBI and LBI calves in the present study. 
HBI calves were characterized as having higher plasma glucose, 
insulin and GH concentrations than LBI calves. Similar obseryations 
were also made in mature cows of similar genetic background (Flux et 
al, 1984). These differences may be associated with the differences 
in genotype for milk fat production. 
1. After 16 hours fasting basal plasma glucose and insulin 
concentrations were significantly higher in HBI calves than in LBI 
calves (P < 0.05, P < 0.01, P= 0.59 respectively). GH concentrations 
were also higher in HBI calves but not at 5% significance level 
(P<0.06). 
2. The changes in concentrations of glucose , GH and insulin 
with time in response to feeding was very similar for each group 
calves of different BI and sex. However,insulin concentration was 
significantly higher in HBI calves than LBI calves (P<0.01). GH 
concentration in HBI group was also significantly higher than in LBI 
group (P<0.05). But the differences in GH and insulin concentrations 
between HBI and LBI group resulted from differences which existed 
before feeding rather than caused by feeding per se because no 
significant differences were found when the results were adjusted by 
the prefeeding values. Cortisol concentration declined rapidly 
following feeding in all calves but this could have been related to 
the circadin nature of cortisol secretion rather than be a response 
to feeding. No significant difference in cortisol concentration was 
found between HBI and LBI calves. 
3. Acute intravenous arginine infusion induced significant 
hypoglycemia and hyperinsulinemia in all calves and the former was 
apparently due to the effect of the later. Following arginine 
challenge calves in the LBI group showed a greater absolute increment 
of insulin concentration and a significantly higher percentage 
increase of insulin release than HBI calves (P<0.05). But insulin 
concentration was not significantly different between HBI and LBI 
groups. Neither was glucose concentration. 
89 
The response of GH concentration to arginine challenge was 
significantly different between HBI and LBI calves. In the HBI group 
GH concentration was slightly increased and remained elevated whereas 
in the LBI group GH fell rapidly after arginine challenge • The 
biological relevance of the different response is not clear. 
Plasma cortisol concentration fluctuated to a small extent 
within each group of calves following arginine infusion, but no 
significant difference was detected between HBI and LBI groups. 
4. Subcutaneous injection of synthetic corticosteroid 
(Betsalan) caused significant elevations of plasma glucose and 
insulin concentrations and a significant decrease of endogenous 
immunoreactive cortisol concentration in all calves, but none of the 
metabolite and hormone concentrations were significantly different 
between HBI and LBI calves. GH concentration was slightly elevated 
in most calves but the change in concentration was not significant. 
Following synthetic corticosteroid treatment, GH concentration was 
higher in the HBI group than in the LBI group, but the difference was 
not quite significant at the five percent level (P=0.061). This 
difference was similar to that observed before Betsalan injection, 
which suggests that the difference in GH concentration is not due to 
the effect of Betsalan treatment. 
5. The significant positive correlations between BI and fasting 
plasma insulin concentration for all calves, for HBIB and LBIB groups 
indicate that basal insulin concentration could be used as a gene 
marker for the early identification of genetically superior Friesian 
dairy cattle. Basal GH concentration was also positively though not 
significantly correlated with BI for all calves. As the spot 
estimation of basal GH concentration may not represent the real 
status of GH concentration in a calf the possibility that the mean 
concentration in a intensive series of samples may serve as a useful 
marker in identifying superior dairy cattle could not be ruled out. 
Basal glucose concentrations were poorly correlated with the BI of 
each group, possibly because the blood glucose levels were maintained 
by homeostasic mechanisms within a relatively narrow range. Very 
90 
small differences in mean basal plasma cortisol concentrations were 
found between HBI and LBI calves in the present study and no 
significant correlation existed for basal cortisol level and calf BI. 
6. Effect of sex on plasma metabolite and hormone 
concentrations were found in the present study. Plasma insulin 
concentration was consistently higher in bulls than in heifers and 
the differences were significant at the time of fasting, after 
feeding, and after arginine infusion. Following feeding, glucose 
concentration was significantly higher and cortisol concentration was 
lower but not at a significantly level in bulls than those in heifers 
respectively. GH concentration was slightly not significantly higher 
in bulls than in heifers during most of the experiment. 
It was concluded that HBI and LBI calves differ significantly 
from each other in certain metabolic and endocrinological aspects and 
there is a promise of identifying superior Friesien dairy cattle at 
an early age by measuring the concentrations of some metabolites and 
hormones as gene markers. 
Appendix I Analysis of Ancalf milk powder 
component quantity component quantity 
~/100~ (amountlk!:';) 
Lipid 18 Vitamin A 22,000IU 
Protein 27 Vitamin D 6,600IU 
Lactose 43 Vitamin c 66mg 
NaCl 1.5 Vitamin E 22IU 
Fibre 0 
note: calcium, iron, phosphorous, iodine,manganese, 
cobalt, and zinc were also blended in ancalf 
by the manufacturer. 
Appendix II Percent cross-reaction of various steroids 
with antiserum F3-314 
Compound 
Corticosterone 
Cortisone 
Deoxycorticoserone 
Estradiol 
Estriol 
17-hydroxyprogesterone 
Prednisone 
Pregesterone 
Testosterone 
Tetrahydrocortisone 
percent 
cross-reactivity 
0.015 
3.3 
< 0.01 
< 0.01 
< 0.01 
0.23 
3.1 
< 0.01 
< 0.01 
0.3 
From Endocrin Science, 18418 Oxnard Street, Tarzana, 
California 91356 (1982). 
91 
92 
Appendix III 
RIA procedures for GH, insulin and cortisol 
i) GH RIA: Assay buffer: 0.01M pH 7.5, phosphate buffered saline 
(PBS) made 0.01M with respect to NaEDTA and 2% with respect to inert 
protein (plasma from a somatostatin (SLI) treated sheep and filtered 
using a millipore filter before use) (MPBS). 
Plastic tubes (Luckhams LP3) were used in both GH and insulin 
assays. Thirty tubes were required for the standards of GH ( 0, 0.5, 1, 
2, 4, 8, 16, 32, 64, and 128 ng/ml) and 4 tubes for each unknown sample 
2 tubes for 100 ~1 plasma samples, 2 tubes for 50 ~1 plasma samples). 
The antisera to bGH (1st antibody) was diluted at 1:16,000 in PBS 
solution. Guinea-pig-gamma globulin (gpg-r-globulin) was also made to 
ug/ml in PBS. This is the final make-up of PBS (FPBS). 
The FPBS reagents were added to LP3 tubes in the following order: 
1) 100 ~1 of FPBS cotitaining first antibody and gpg-r-globulin to 
each tube; 
2) 100 ~1 of standard GH solution or 100 ~1 of a sample, or 50 ~1 of 
a sample plus 50 ~1 of MPBS. 
Then the reagents were mixed on a vortex mixer and incubated at 
laboratory temperature for 24 hours. Then 50 ~1 per tube of 125-I 
labelled GH (about 6,000 counts/min) was added, mixed and followed by a 
further 24 hours incubation at room temperature. 
On the third day, 50 ul/tube of the second antibody (to 
gpg-r-globulin raised from a sheep and diluted at 1:40 before use) was 
added and mixed on a vortex mixer, then the tubes were transferred to a 
refrigerator and incubated at 4°C for 72 hours. 
After that the tubes were centrifuged at 2,000 rpm for 30 min at 
4°C. The supernatant was removed with a pipet attached to a vaccumm, 
then the radioactivity of the precipatate on the bottom of the tube was 
counted in a NE 1600 gamma counter. 
ii) Insulin RIA: The assay buffer was the same as that used in the 
GH RIA: 0.01M pH 7.5 PBS with respect to 0.01 NaEDTA (NaSalt) with 2% 
added plasma from a somatostatin treated sheep. 
93 
To each tube were added : 
1) 100 ~1 of assay buffer containing 1st antibody; 
2) 100 ~1 standard insulin solution or 100 ~1 sample or 50 ~1 of a 
sample plus 50 ~1 assay buffer. The tubes were incubated at room 
temperature for 24 hours. 
Then 50 ~1 125-I labelled insulin (6,000 counts/min) were added to 
each tube and incubated for a further 24 hours at room temperature. On 
the third day 50 ~1 of ovine antiserum to gpg-r-glubolin diluted at 1:40 
in assay buffer was added to each tube, and mixed and incubated for 72 
hours at 4°C. 
The remaining steps of centrifuging and counting were identical to 
those for the GH RIA. 
iii) Cortisol RIA: Three days were required for each batch of 
cortisol assays. 
Day One: Extraction of plasma cortisol: To each tube were added: 
1) 100 ~1 sample; 
2) 2 ml Dichlorometh~ne (DCM). 
All tubes were mixed on a vortex mixer for 30 seconds and frozen 
overnight. 
Day Two: The DCM containing the extracted cortisol was poured into 
glass tubes and dried down under air flow. The extract was redissolved 
in 1 ml ethanol and mixed for 5 seconds on a vortex mixer (to minimize 
evaporation, only 10 tubes were processed at a time). A set of standards 
containing five concentrations of cortisol (0.3125, 0.625, 1.25, 2.5 and 
5 ng/ml) were also suspended in ml ethanol. 2 x 100 ~1 eliquots of 
each ethanol sample or standard were dispensed into assay tubes and then 
dried under a flow of air. 
Antibody was diluted as recommended by the suppliers of the 
antiserum (Endocrine Science, 18418 Oxnard street, Tarzana, California 
91356,1982). 200 ~1 reagent mixture containing borate buffer, stock 
tritiated cortisol (6,000-7 ,000 counts/min), bovine gamma globulin (BGG), 
bovine serum albumin (BSA), and antiserum were added to each tube and 
incubated at room temperature. 
94 
Day Three: Separation of bound radioactivity: From this point every 
step was done at 4°C. Tubes and reagents were kept on ice or in a 
referigerator. 
A suspension of charcoal/dextran mixture (7.~ug/ml) was stirred and 
500 ~1 of the suspension was added to each of the tubes. All tubes were 
centrifuged at 2,500 rpm for 20 minutes at 4°C. Immediately after 
centrifuging, 500 ~1 of supernatant was transferred into scintillation 
vials. 5 ml of scintillation fluid was added to each vial. Vials were 
placed in the counter for several minutes before counting. Each vial was 
counted for 2 minutes in the counter. 
95 
Appendix IV Standard curves of GH and Insulin RIAs and check of parallelism 
.. 
E 
' ~ 
.... 
c 
::::J 
0 
0 
3600 
2800 
2000 
1200 
0 
3400 
2600 
1800 
1000 
0 
GH RIA 82 
·\ 
\ 
' \ 
'· 
\ 
\ 
\ \ \\ . 
\ '. 
\ 
\ 
\ \ 
\ \ 
\\. 
,, 
',, 
'~ 
1 4 16 64 
GH RIA 83 
' ' 
\ 
\ 
\ 
' '. 
0 1 4 16 64 
Standard GH (ng/ml} 
Insulin RIA 77 
\ 
\ 
\ \ 
,, 
\ \ 
\ 
' \ " \\ ,, 
"' \\\ \\ 
Insulin RIA 79 
' 
' 
\ ,, 
\ \ 
\ \ . ,, \ 
' ' ~~ 
\"' \. 
0. 1 4 16 64 0 1 4 16 64 
Standard Insulin (ng/ml} 
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