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thesis entitled

EFFECT OF BOVINE SDMATOTROPIN AND BOVINE GROWTH
HORMONE-RELEASING FACTOR ON THE SOMATDTROPIC
CASCADE IN LACTATING DAIRY CATTLE

presented by

William Kenneth VanderKooi

has been accepted towards fuifiilment
of the requirements for

Masters Animal Science

degree in

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EFFECT OF BOVINE SOMATOTROPIN AND BOVINE GROWTH
HORMONE-RELEASING FACTOR ON THE SOMATOTROPIC
CASCADE IN LACTATING DAIRY CATTLE
By

William Kenneth VanderKooi

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirement
of the degree of

MASTER OF SCIENCE

Department of Animal Science

1993

 

ABSTRACT

EFFECT OF BOVINE SOMATOTROPIN AND BOVINE GROWTH
HORMONE-RELEASING FACTOR ON THE SOMATOTROPIC CASCADE
IN LACTATING DAIRY CA'I'I‘LE
By

William Kenneth VanderKOOi

Primiparous Holstein cows received 29 mg rbST/d (n=10) or 12 mg rbGRF/d
(n=10) by continuous i.v. (jugular) infusion or no infusion (controls, n=10) from
118 to 181 d postpartum. Cows treated with rbGRF tended to have more (P=.13)
serum ST at d 57 than cows treated with rbST. Despite this fact, rbGRF-treated
cows tended to have less (P=. 15) serum insulin-like growth factor-I (IGF—I), lower
(P<.Ol) liver IGF—I mRNA expression, less (P=.03) serum IGF—I binding protein-3
and less (P<.01) serum insulin at d 57 than rbST-treated cows. Also, rbST and
rbGRF increased (P=.02) mammary IGF-I receptor mRN A expression.

Results are consistent with the idea that rbST and rbGRF stimulate the
somatotropic cascade to increase milk yield. However, rbST stimulated the cascade
more than rbGRF. Perhaps, differences in ST variant profile and/or ST pulsatility
caused the different effects Of rbST and rbGRF on the somatotropic cascade.
Furthermore, both treatments increased milk yield similarly even though rbGRF did

not stimulate the cascade as much as did rbST. Perhaps, the galactopoietic effects

Of rbGRF are not mediated exclusively through the somatotropic cascade.

ACKNOWLEDGEMENTS

First, I would like to thank my major professor, Dr. Michael VandeHaar, for
his guidance and friendship. I am also appreciative of the generous hospitality
Mike and his family have extended tO me during the past two years.

TO the members Of my guidance committee: Drs. H. Allan Tucker, Michael
Allen and Dale Romsos, I am grateful for their contributions to my research project
as well as my graduate education as a whole.

I would especially like to thank Mario Binelli for his cooperation and
comradeship during the execution Of our research project and most importantly for
his friendship.

I thank Dr. Bal Shanna for his patience, assistance and instruction in the lab.
I also thank Mr. Larry Chapin for assistance with the execution Of my project and
analysis of my data. Furthermore, I thank Mr. Jim Liesman and Dr. Paula Gaynor
for their time and insight, including the participation in many thought-provoking
conversations. I thank Miss Miriam Webber, Michelle Payne and Mr. Jack
Heethouse for helping with the execution of my project. Also, thanks to Mr. Greg
DeMarco for assistance in the lab.

To fellow graduate students Rick Dado, Mary Beth Roe, Katharine

Knowlton and Christine Simmons; I extend thanks for their support and assistance

iv.

throughout graduate school.

I thank Drs. Mike Moseley and Jim Lauderdale from the Upjohn Company
for support Of my research. Also, I thank Bill Claflin and Glenn Alaniz for their
assistance with Vetport catheter surgeries, and Bud Krabill for sample analysis.

I express sincerest gratitude to my family and friends at home in Abbotsford.
TO my parents, Hank and Henrietta VanderKOOi, I extend thanks for their support
and love throughout not only my masters program but also for the last 24 years.
Also, thanks to my siblings, grandma McCorkell, the Kielstra family, Ken and
Henrietta VanderKOOi, Ross and Kelly Siemens, Steve and Paul Brandsma, D.R.
Vandraager and Kevin Zandberg for their encouragement and friendship.

Lastly, I thank God for everything.

 

 

TABLE OF CONTENTS

LIST OF TABLES ....................................... viii
LIST OF FIGURES ........................................ ix
LIST OF ABBREVIATIONS ................................. xi
INTRODUCTION .......................................... 1
LITERATURE REVIEW ..................................... 5
Effects of rbST on milk production .......................... 5
History .......................................... 5
Mammary cell numbers .............................. 5
Cellular metabolic activity ............................ 6
Direct action Of ST on the mammary gland ................ 8

Summary ........................................ 9 .
Physiological effects of rbST ............................. 10
ST characteristics .................................. 10
ST pulsatility ..................................... ll
Endocrine action Of IGF-I ............................ 12
Autocn'ne and/or paracn'ne action of IGF-I ................ 15
IGFBPs ........................................ 16
IGFBP-3 ........................................ l7
IGFBP—2 ........................................ l8
Insulin ......................................... 19
Effects Of rbGRF on milk production ........................ 20
Physiological effects of rbGRF ............................ 20

vi

MATERIALS AND METHODS ............................... 22

Design and Management ................................. 22
Blood Collection and Analysis ............................ 25
Tissue Collection and Analysis ............................ 28
Statistical Analysis ..................................... 30
RESULTS .............................................. 32
BW, DMI, SCM and energy balance ........................ 32
Serum ST ........................................... 32
Serum IGF—I ......................................... 34
Serum IGFBPs ....................................... 38
Serum Insulin ........................................ 38
Liver ST receptor and IGF-1 mRN A ........................ 42
Mammary IGF-I receptor mRN A ........................... 46
DISCUSSION ............................................ 48

Effects Of exogenous rbST and rbGRF on the STIIGF/BP cascade . . . . 48
The ST/IGFIBP cascade is stimulated more by rbST than rbGRF . . . . 52
Similar milk yield despite different effects on the

STIIGF/BP cascade .................................... 55
SUMMARY AND CONCLUSIONS ............................ 58
APPENDICES ............................................ 60

Appendix A (composition Of diets) ......................... 60

Appendix B (short-term effects of rbST and rbGRF) ............. 60

Introduction ...................................... 61
Materials and Methods .............................. 61
Results and Discussion .............................. 62
LIST OF REFERENCES .................................... 68

vii

LIST OF TABLES

Table 1 Least squares means BW, DMI, SCM and EB between
(I 50 and 62 Of treatment Of cows receiving no
treatment (n=10), 29 mg rbST/d (n=10) or 12 mg
rbGRF/d (n=10) for 63 d Of lactation (d 118 to 181
postpartum) .................................... 33

Table 2. Least squares means for baseline ST, number Of peaks,
amplitude, peak length and mean ST for d 57 of
treatment Of cows receiving no treatment (n=10), 29
mg rbST/d (n=10) or 12 mg rbGRF/d (n=10) for 63 d
of lactation (d 118 to 181 postpartum) ................. 35

Table 3. Ingredient composition Of diets fed during the treatment
period ........................................ 61

Table 4. Chemical composition Of diets fed during the treatment
period ........................................ 61

viii

LIST OF FIGURES

Figure 1. Schematic diagram of the endocrine somatotropic
cascade ........................................ 4

Figure 2. Concentration of serum ST for d 57 Of treatment of
cows receiving no treatment (n=10), 29 mg rbST/d
(n= 10) or 12 mg rbGRF/d (n=10) for 63 d of lactation
(d 118 to 181 postpartum) .......................... 36

Figure 3. Least squares mean concentration of IGF-I in semm for
d 57 Of treatment Of cows receiving no treatment
(n=10), 29 mg rbST/d (n=10) or 12 mg rbGRF/d
(n=10) for 63 d Of lactation (d 118 to 181 pospartum) ...... 37

Figure 4. Least squares mean concentration Of IGFBP-3 and
IGFBP-2 in serum for d 57 of treatment of cows
receiving no treatment (n=10), 29 mg rbST/d (n=10) or
12 mg rbGRF/d (n=10) for 63 d of lactation (d 118 to
181 postpartum) ................................. 39

Figure 5. Representative autoradiographs Of western ligand blots
for IGFBPs (blocks 8 and 9) ........................ 40

Figure 6. Least squares mean concentration of insan in serum
for d 57 Of treatment Of cows receiving no treatment
(n=10), 29 mg rbST/d (n=10) or 12 mg rbGRF/d
(n=10) for 63 d of lactation (d 118 to 181
postpartum) .................................... 41

Figure 7. Abundance Of mRN A for ST receptor and IGF-1 in
liver and IGF-1 receptor in mammary tissue for d 63 Of
treatment Of cows receiving 29 mg rbST/d (n=10) or 12
mg rbGRF/d (n=10) for 63 d Of lactation (d 118 to 181

postpartum) .................................... 43

Figure 8. Autoradiographs of northern blots for liver ST receptor,
IGF-I and p-actin mRNA for blocks 1 through 5 .......... 44

i}:

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Autoradiographs of northern blots for liver ST receptor,
IGF-I and B -actin mRN A for blocks 6 through 10 . .

Autoradiographs Of northern blots for mammary IGF-I
receptor and B—actin mRN A for blocks 1 through 10

Least squares mean concentration Of ST in serum for d
1 Of treatment Of cows receiving no treatment (n=10),
29 mg rbST/d (n=10) or 12 mg rbGRF/d (n=10) for 63
d Of lactation (118 to 181 postpartum) ..........

Least squares mean concentration Of IGF—I in serum for
d l of treatment Of cows receiving no treatment (n=10),
29 mg rbST/d (n=10) or 12 mg rbGRF/d (n=10) for 63
d Of lactation (118 to 181 postpartum) ..........

Least squares mean concentration of IGFBP-3 in serum
for d 1 Of treatment Of cows receiving no treatment
(n=10), 29 mg rbST/d (n=10) or 12 mg rbGRF/d
(n=10) for 63 d of lactation (118 to 181 postpartum)

Least squares mean concentration Of IGFBP-2 in serum
for d 1 Of treatment Of cows receiving no treatment
(n=10), 29 mg rbST/d (n=10) or 12 mg rbGRF/d
(n=10) for 63 d of lactation (118 to 181 postpartum)

....... 45

....... 47

....... 65

....... 66

....... 67

....... 68

BW
DMI
DNA
EB
GRF
IGFBP
IGF-I
mRNA

rbGRF

rbST
SCM

ST

LIST OF ABBREVIATIONS

Body weight

DMI

Deoxyribonucleic acid

Energy balance

Growth hormone-releasing factor
Insulin-like growth factor binding protein
Insulin-like growth factor-I

Messenger ribonucleic acid
Recombinant bovine growth hormone-
releasing factor

Recombinant bovine somatotropin
Solids-corrected milk

Somatotropin

INTRODUCTION

The agricultural industry is developing new methods to meet the
increasing demand for food in response to the world’s growing human
population. As the year 2000 approaches, current methods for promoting
productivity in agriculture will be enhanced by the employment of
biotechnology.

One of the first products of biotechnology developed for the dairy
industry is recombinant bovine somatotropin (rbST). The advances of
recombinant deoxyribonucleic acid (DNA) technology have made rbST both
abundant and affordable. Daily administration Of rbST to dairy cattle during
lactation substantially increases milk yield and the efficiency Of milk production
(Peel et al., 1983), resulting in greater economic return and efficient utilization
of resources.

Scientists throughout the world have conducted over 1,000 studies
examining the effects Of rbST on over 20,000 cows (Bauman, 1992). Scientists
from the US. Food and Drug Administration (FDA), after reviewing the
scientific literature, have concluded that the use of rbST in dairy cattle
Presents no increased health risk to consumers (J uskevich and Guyer, 1990).

Nevertheless, public perception is of paramount importance if rbST or any new

2

technology is to be effectively implemented (Bauman, 1992) and utilized as a
means for enhancing productivity in the dairy industry.

An elaborate regulatory system controls the secretion Of somatotropin
(ST) in viva. Two neuroendocrine hormones, namely growth hormone-
releasing factor (GRF) and somatostatin, are primary controllers of ST release
(F rohman et al., 1992). In vivo, GRF stimulates and somatostatin inhibits the
release Of ST from somatotrops in the anterior pituitary.

Administration of rbST to dairy cattle induces a series of coordinated
changes in whole-body metabolism that subsequently increase milk synthesis.
Current dogma suggests that insulin-like growth factor-I (IGF-I) in serum
partially mediates ST-induced increases in milk production. The IGF-I peptide
is predominantly synthesized and secreted from the liver in response to ST
binding with the hepatic ST receptor. The biological effects Of IGF-I are
further regulated by specific high-affinity binding proteins (IGFBPs) which
control access of IGF-I to target tissues. For the purpose of this thesis, I will
refer to the interactions of ST, IGF-I, and IGFBPs as the ST/IGF/BP cascade
(Figure 1).

As one might speculate, administration of recombinant bovine growth
hormone-releasing factor (rbGRF) stimulates milk yield in lactating dairy cattle
similar to that of rbST (Baile et al., 1985). Dahl et a1. (1993), however, found
that cows treated with rbGRF produced more milk than cows treated with

rbST even though concentration of ST and IGF-I were similar between the two

 

3

groups. Thus, we formulated the hypothesis that rbGRF stimulates the
ST/IGF-I/BP cascade more than does rbST in lactating dairy cattle.

The Objective Of this thesis is to quantify and compare the effects Of 63-
d infusion Of exogenous rbGRF and rbST on the endocrine ST/IGF/BP
cascade in lactating dairy cows. Certainly other facets, such as mammary
triiodothyronine, plasminogen, blood flow, and local autocrine and/or paracrine
action of IGF-I might also be important in mediating the galactopoietic effects
of rbST. Nevertheless, this thesis will focus specifically on the regulation and
interaction of the components of the endocrine ST/IGF/BP cascade in response
to rbGRF and rbST administration.

For the purpose Of this thesis, I will refer to endogenous growth
hormone as ST, exogenous recombinant bovine growth hormone as rbST,
endogenous growth hormone-releasing factor as GRF, and exogenous

recombinant bovine growth hormone-releasing factor as rbGRF.

 

 

bGRF bST
O pituitary
ST
liver
IGFBP's
IGF-I ¢‘/
mammary
gland

Figure 1. Schematic diagram Of the endocrine somatotropic cascade.

LITERATURE REVIEW

Effects of rbST on milk production
Hivory

The ability of ox pituitary gland extracts to increase milk yield of the
cow was first shown by Gruter and Stricker (1929) more than 60 years ago.
Since then, the specific hormone responsible for this action has been identified
as ST, and the genetic sequence has been elucidated to enable the synthesis
Of rbST (Seeburg et al., 1983). When cows are treated with rbST, typically
milk yield gradually increases within the first few days Of treatment but no
effect on milk composition and feed intake occurs in the short-term (Peel and
Bauman, 1987). Also, an increase in the concentration of IGF-I in serum is
characteristic within the first few days of rbST administration to dairy cows
(Davis et al., 1984). Although the effects Of rbST on lactation have been
extensively studied during the past several years, the precise mechanisms by
which rbST stimulates milk synthesis have not been established. Several
theories, however, have been proposed.
Mammary cell numbers

Brumby and Hancock (1955) proposed that ST stimulates growth of the

mammary gland by increasing mammary epithelial cell numbers or decreasing

6

mammary cell loss. The IGF-I peptide presumably mediates the effect of ST
on mammary cell numbers. Baumrucker (1989) Observed that IGF-I
stimulated [3H]thymidine uptake into DNA of lactating bovine mammary tissue
in vitra. Their study, however, revealed that ST had no direct effect on DNA
synthesis. Capuco et a1. (1989) found that rbST treatment did not increase
total mammary DNA in lactating cows, thus rbST presumably does not
increase mammary cell numbers directly in viva.

However, ST may prevent mammary cell loss. This idea is supported by
the low levels Of milk plasminogen Observed in milk of cows treated with rbST
(Politis et al 1990). Plasminogen is a serine-protease that is partially
responsible for mammary gland involution. Thus, low levels of plasminogen
in milk during rbST treatment suggest a reduction in mammary cell loss.

Nevertheless, increased cell numbers or decreased cell loss cannot
explain the rapidity of the rise in milk yield in response to rbST injections.
Milk yield increases substantially within the first few days Of rbST treatment
to dairy cows (Peel and Bauman, 1987). Thus, the galactopoietic effects of
rbST must be at least partly the result Of increased productivity of existing
secretory cells.

Cellular metabolic activity

Hutton (1957) proposed that the galactopoietic effects Of ST are due

mainly to increased metabolic activity of alveolar epithelial cells. Specifically,

these changes may result from an increase in mammary cell metabolism

 

7

resulting in a greater extraction rate of milk precursors by the mammary gland.
An increase in metabolic activity of mammary epithelial cells in response
to rbST is supported by the Observation that thyrordne-5’-monodeiodinase
activity increases in mammary tissue of cows treated with rbST (Capuco et al.,
1989). TherIdne-5’-monodeiodinase catalyzes the conversion Of thyroxine to
the more active triiodothyronine in the mammary gland; thus, an increase in
thyroxine-5’-monodeiodinase is associated with enhanced cellular metabolism.
Enhanced mammary epithelial cell metabolism involving triiodothyronine may
be a mechanism whereby rbST elicits galactopoietic effects in dairy cows.
Mammary blood flow is an important determinant Of the galactopoietic
response to the physiological concentration Of ST (Hart et al., 1980). An
increase in mammary blood flow results in greater availability of metabolites
to the mammary gland. Although rbST administration to cows increases blood
flow and subsequent nutrient supply, the blood flow response likely is caused
through other hormonal stimulation (Davis et al., 1983). Presumably, local
production of vasodilator substances decreases mammary vascular resistance
which subsequently increases blood flow. This vasodilation is likely linked to
mammary metabolism, because similar increases in udder blood flow and milk
yield were Obtained in thyroxine—treated cows (Davis et al., 1983).
Nevertheless, it remains to be elucidated whether or not the increase in
mammary gland blood flow is a cause Of, rather than a response to, the

increase in synthesis of milk during rbST and/or thyroxine administration

(Gluckman et al., 1987).

Insulin may be indirectly involved in mediating the effects of rbST on
activity of the mammary gland. Insulin coordination of glucose homeostasis
to alter activity of the mammary gland is evident with rbST treatment. Hepatic
glucose production increases and whole body glucose oxidation decreases in
response to rbST administration (Bauman, 1989). This coordination results in
greater availability of glucose to the mammary gland. These effects are
presumably mediated by insulin. Furthermore, Gallo and Block (1990) found
that rbST stimulates glucose uptake in the bovine mammary gland.

An increase in lipolysis and/or a decrease in lipogenesis appears to
mediate the galactopoietic effects of rbST. Cows in early lactation (negative
energy balance) increase mobilization of free fatty acids (FFA) in response to
rbST treatment (Peel et al., 1981). This may simply be a result of the
increased energy deficit evoked by rbST treatment (Gluckrnan et al., 1987).
Barber et al. (1992) reported that ST clearly regulates lipid metabolism in
adipose tissue in a manner which should favor nutrient utilization by the
mammary gland. Insulin resistance (decrease in lipogenesis) and increased
lipolysis provide a mechanism for preferential utilization of glucose and fatty
acids (FA) by the mammary gland Of cows treated with rbST. These effects,
however, are probably too small to account for the effects of ST on milk
production (Barber et al., 1992).

Direct action of ST on the mammary gland

 

9

The rbST compound may act directly on bovine mammary tissue to
stimulate milk yield. In situ hybridization results indicate that a low level of
ST-receptor mRNA is evenly distributed throughout the bovine mammary
gland (Hauser et al., 1990). In a similar study conducted by Glirnm et al.
(1990), ST receptor mRNA was identified and characterized in mammary
tissue from normal and rbST-treated lactating cows using northern and in situ
hybridization analyses. In situ hybridization revealed that the ST receptor gene
is primarily expressed in alveolar epithelial cells Of mammary tissue (Glimm
et al., 1990).

Despite these observations, the theory suggesting direct action of ST at
the mammary gland has been challenged because Of several findings.
McDowel et al. (1987) demonstrated that local arterial infusion of ST into the
udder of lactating ewes did not increase milk production of the infused udder
half. Also, mammary tissue seems devoid of functional ST receptor protein
(Keys and Djiane, 1988) and no direct effect Of ST on milk synthesis has been
demonstrated in lactating mammary tissue in culture (Gertler et al., 1983).
Summary

Somatotropin is a homeorhetic regulator that partitions nutrients
towards the mammary gland for increased milk synthesis (Bauman, 1992).
The primary factors mediating the ability of rbST to coordinate whole-body
metabolism and stimulate nutrient uptake and metabolism Of mammary cells

remain tO be elucidated.

 

10

Physiological effects of rbST
ST characteristics

The ST peptide, synthesized and secreted by the pituitary, is primarily
transported through the circulation to target tissues to cause a biological effect
through an endocrine pathway. Endogenous ST, produced by the anterior
pituitary, can either be 190 or 191 amino acids long, and can have either
leucine or valine at position number 126 in the protein sequence (Wood et al.,
1989). Therefore, four different variants Of ST are produced naturally.
Typically, pituitary production of ST involves approximately equal amounts of
the 190 and 191 amino acid proteins, and about two-thirds Of the total
produced has leucine at position 126, while the remaining one-third has the
amino acid valine at position 126 (Wood et al., 1989). Consequently, the ST
in serum Of cows treated with rbGRF presumably would consist of four native
variants in substantial amounts, although the profile may be different than
untreated cows. In contrast, the serum ST of rbST-treated cows would be
overwhelmingly the recombinant variant which is the 191 amino acid variant
with leucine at position number 126.

Binding Of pituitary and recombinant bST to receptors in bovine liver
membranes was investigated by Wood et a1. (1989). Both sources of rbST have
equivalent specific activities. Nevertheless, these results do not provide

evidence that either compound is more biologically active with regard to the

 

ll

somatotropic cascade in viva.

Although exogenous ST had a slightly longer half-life than endogenous
ST in rats (12.7 vs 10.5 min), this difference was not significant (Chapman et
al., 1990). Hence, ST secreted in response to rbGRF infusion and exogenous
rbST presumably have similar half-lives in viva in cattle.
ST pulsatility

The secretion Of ST in adult mammals is pulsatile in nature, but the
physiological significance Of such pulsatility is incompletely understood.
Recent studies in animals and man suggest a cooperative interaction of
hypothalamic somatostatin and GRF on somatotropic pituitary cells in the
generation of ST pulses (Hindmarsh et al., 1991). Differences in pulsatility Of
ST in serum may moderate the different in viva effects of rbST and rbGRF on
the ST/IGF/BP cascade when both are continuously infused.

Administration Of continuous ST in rats is more effective than pulsatile
administration for inducing ST-receptor in liver (Maiter et al., 1992). In
contrast, Mathews et al. (1989) Observed no effect of pulsatility on ST receptor
expression in rat hepatocytes. Hence, they suggested that the effects of ST on
its receptor abundance are manifested at a post-transcriptional level, because
ST receptor mRNA is not significantly affected by either hypophysectomy or
rbST treatment.

Maiter et al. (1992) demonstrated that pulsatile infusion of ST is more

effective than continuous infusion in stimulating rat liver IGF-I mRNA

 

12

expression, and that this effect is not mediated by an increase in ST-receptor
mRNA. In contrast, Bick et al. (1992) Observed that rats have greater
concentration of serum IGF-I after continuous ST treatment than after
pulsatile injections Of ST. However, the pulsatile treatment induced a greater
body weight gain than the same dose of ST infused continuously (Bick et al.,
1992). The reason for the discrepancy is not known, but local production Of
IGF-I in the target tissue is likely involved. In another study, the levels of
hepatic IGF-I mRNA were similar in rats given continuous or pulsatile ST
treatment, whereas the IGF-I mRNA levels in skeletal muscle and cartilage
were higher after pulsatile than after continuous ST treatment (Isgaard et al.,
1988). In light of the effects of ST pulsatility on growth, the effects Of serum
ST pattern on galactopoiesis may also be important.
Endocrine IGF-I action

Salmon and Daughaday (1957) initially reported that a serum factor,
now termed IGF-I, mediates the action of ST on bone cartilage. The IGFs are
a family Of polypeptides, related structurally to proinsulin, which have growth
promoting effects both in vitra and in viva. At physiological concentrations,
IGF-I is a potent mitogen of undifferentiated bovine mammary epithelial cells
cultured on collagen in serum-free media. Despite the information available,
significant gaps in our knowledge exist regarding the mechanisms by which
these peptides stimulate cell replication (Clemmons et al., 1989).

In cows, the expression Of ST receptor and IGF-1 mRNA are found in

 

13

several tissues, but predominately in liver tissue (Hauser et al., 1990). The
level Of IGF-I mRNA is regulated by ST and nuclear run-on assays were used
to show that ST regulation Of IGF-I is manifested at the transcriptional level
(Mathews et al., 1986). Thus, transcriptional mechanisms are predominately
responsrble for enhanced IGF-I expression elicited by rbST (Mathews et al.,
1986; Bichell et al., 1992).

During lactation, IGF-I may coordinate the metabolism of various
organs and tissues to support increased milk yield (Bauman, 1992). Treatment
with rbST increases concentration of ST and IGF-1 in lactating animals (Vicini
et al., 1991), whereas concentration of serum IGF-I during normal lactation is
inversely correlated with milk production and serum ST concentration. This
observation has perplexed scientists investigating the role of IGF-I on lactation,
however some theories have been postulated to explain this Observation.

The IGF-I peptide exerts its biological effects, like other peptides, by
binding tO cell surface receptors (Zapf et al., 1978). Polypeptide hormones
and growth factors are internalized by receptor-mediated endocytosis after
binding to cell surface receptors (De Diego et al., 1991). Onset of lactation
is associated with structural changes in the IGF-I receptor and the onset Of
lactation is associated with increases in the abundance of IGF-I receptors
(DeHOff et al., 1988). These changes in the receptor could play a role in
modulating the physiological effects of IGFs on mammary tissue and possibly

enhance clearance of serum IGF-I. The binding of IGF-I declines during the

14

prepartum period, increases 75% with the onset Of lactation, and then declines
during the postpartum period (Hadsell et al., 1990).

Stelwagen et al. (1992) suggested that low concentration of IGF-I in
serum during early lactation is important for mediating preferential nutrient
utilization by the mammary gland. The IGF-I peptide plays a role in several
different tissues throughout the body (D’Ercole et al., 1984). If serum IGF-I
increased during early lactation, the use of metabolites would increase in
several other tissues throughout the body and the mammary gland would have
to compete for these metabolites (Stelwagen et al., 1992). Low blood
concentration of IGF-I during this part of lactation, however, combined with
a selective increase of mammary receptors or mammary receptor affinity would
give the mammary gland an advantage over other tissues in utilization of the
available metabolite pool (Stelwagen et al., 1992).

Regardless of inconsistencies, considerable attention has been focused
on IGF-I as an important factor mediating the galactopoietic actions of ST.
The contribution of hepatic to total IGF-I varies markedly between tissues and
provides evidence that hepatic IGF-I may have specific endocrine functions in
selected tissues (Hodgkinson et al., 1991). Overall, similarity in the temporal
pattern between milk yield response and serum concentration of IGF-I is
consistent with a role for serum IGF-I in mediating a portion of the effects Of
rbST in lactating cows through an endocrine fashion (Cohick et al., 1989).

Lavandero et al. (1990) demonstrated IGF-I binding in rat mammary

-‘l.

 

15

tissue. In goats, infusion Of IGF-I into the mammary arterial supply Of a hemi-
gland enhances milk secretion and mammary blood flow Of the infused hemi-
gland in intact conscious goats (Prosser et al., 1990). The more pronounced
effect in the infused compared with non-infused hemi-glands suggest that IGF-
I acts directly on the mammary gland (Prosser et al., 1990). In contrast,
Shamay et al. (1988) concluded that galactopoiesis is not affected by IGF-I in
organ culture of bovine mammary tissue. Furthermore, the IGF-I peptide
stimulates induction Of carrier-mediated glucose transport activity in mouse
mammary epithelial cells (Prosser et al., 1987). However, no subsequent
increase in a-lactalbumin activity is observed. Despite the lack Of information,
it seems likely that, during lactation, IGF-I plays a specific role to enhance
mammary epithelial cell function in viva in response to rbST treatment.
Nevertheless, the mechanism by which IGF-I mediates the galactopoietic
actions Of ST remains to be elucidated.
Autocrine and/ or paracrine IGF-I action
Dai et al. (1992) demonstrated that the introduction and expression of
a IGF-I transgene in FRTL-5 cells create an autocrine and/or paracrine loop
with a non-transformed phenotype and that tropic hormones can synergize with
endogenous transgene IGF-I to regulate cell proliferation. Schlechter et al.
(1986) reported that ST stimulates long bone growth by inducing local
production of IGF-I, which in turn stimulates cell proliferation in an autocrine

and/or paracrine fashion. The autocrine and/or paracrine pathway also may

16

be important for IGF-I action in the mammary gland. IGF-I mRNA is present
and is localized in the stromal component of mammary tissue (Hauser et al.,
1990). Exogenous ST increases mammary IGF-I mRNA abundance in rats
(Kleinberg et al., 1990). Furthermore, bovine mammary explants were found
to synthesize and secrete IGF-I in lactating non-pregnant dairy cows (Campbell
et al., 1991)..

IGFBPs

The IGF peptides circulate bound to large molecular weight binding
proteins, which make measurement Of the IGFs and assessment Of their
biological role complicated (Hintz, 1984). Apart from their function as IGF-I
carriers, the binding proteins also play a modulating role in the interaction
between IGFs and their target cells (Hardouin et al., 1987; McCusker et al.,
1991).

As transporters Of IGF-I, IGFBPs regulate the rate of efflux Of IGF-I
from vascular space (Camacho-Hubner et al., 1991). Furthermore, COhick and
Clemmons (1991) suggested that IGFBPs may act to coordinately regulate
IGF-I transport from mesenchymal cells to epithelial cells within tissues.

At the tissue level, Campbell et al. (1991) reported that bovine
mammary explants from pregnant, non-lactating cows and lactating cows
synthesized and secreted IGFBPs. Similarly, McGrath (1991) reported that
normal bovine mammary cells secrete IGFBP-2 and IGFBP-3. In normal

mouse mammary epithelial cells, Fielder et al. (1992) demonstrated that

 

l7

IGFBPs are expressed and released into medium in response to both
lactogenic hormones and IGFs.
IGFBP-3

The major circulating binding protein is IGFBP-3, to which most of the
circulating IGF-I is bound (Vicini et al., 1991). Administration of rbST
increases serum levels of IGFBP-3 in dairy cows (Cohick et al., 1992). Zapf
et al. (1989) reported that IGF-I mediates much Of the ST-associated increase
in IGFBP-3 levels in viva. A similar study reported that insulin and IGF-1 are
stimulators Of IGFBP secretion and most likely mediate the change in serum
concentration of IGFBPs in response to rbST administration (McCusker and
Clemmons, 1988) .

Blum et al. (1989) concluded that IGFBP-3 acts as a reservoir, releasing
continuously small amounts of IGF-I and thereby creating a steady state
situation of receptor occupancy. This slow-release Of the IGF-I may be more
mitogenic than temporarily large concentrations Of free IGF-I (Blum et al.,
1989). Binoux and Hossenlopp (1988) concluded that IGFBP-3 complexes do
not cross the capillary barrier. This Observation provides further evidence that
IGFBP-3 acts as a reservoir and as a moderator of IGF-I action.

IGFBP-3 can be either soluble in serum or associated with a cell surface.
Soluble IGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I
receptor binding, whereas surface-associated IGFBP-3 enhances the growth-

promoting effects Of IGF-I in bovine fibroblasts (Conover et al., 1990). Cell-

18

associated IGFBP-3 enhances the presentation of IGF-I to its cellular receptor
and this heightens cellular receptor activity to IGF-I and related peptides
(Conover, 1992). The IGFBP-3 can also prevent IGF-I-induced IGF-I
receptor down-regulation (Conover and Powell, 1991), thereby maintaining
IGF-I effectiveness despite elevated concentration of serum IGF-I in response
to rbST administration.

Several tissues express the IGFBP-3 gene (Albiston and Herington,
1992). It has been suggested by Ernst and Rodan (1990) that locally produced
IGFBP-3 may function by keeping IGF within tissue compartments, thereby
increasing the bioactivity of IGF-I. Furthermore, cell-derived IGFBP-3 may
function in a buffering capacity to restrict IGF-I and target cell interaction,
thereby modulating the biological response to changes in local IGF-I levels
(Conover and Powell, 1991).

In addition to synthesis of IGFBP-3, its degradation may also play an
important role for regulating IGFBP-3 concentration as well as concentration
Of unbound IGF-I in serum (Davenport et al., 1992). Evidence suggests that
there is a specific protease present in serum which can degrade IGFBP-3 to
enhance IGF-1 clearance as well as increase the amount Of free IGF-I in
serum (Davies et al., 1991; Davenport et al., 1992). Davies et al. (1991)
concluded that the presence Of a circulatory IGFBP-3 protease may be an
adaptive response to increase the bioavailability of the IGFs.

IGFBP-2

 

19

Administration of rbST decreases concentration Of serum and lymph
IGFBP-2 in dairy cows (Cohick et al., 1992). Binoux and Hossenlopp (1988)
concluded that IGF-I complexed with IGFBP-2 can cross the capillary barrier.
The IGFBP-2 can leave the microcirculation and distribute to subendothelial
tissues (Booth et al., 1990). Plasma IGFBP-2 levels are relatively stable, and
therefore may serve as a reservoir Of IGF-I that is available for IGF-I transport
across the capillary barrier to tissues (Clemmons et a1. 1991). The IGFBP-2
can bind with IGF-I and act as an inhibitor or a potentiator of IGF-I mediated
DNA synthesis (Bourner et al., 1992). Thus, IGFBP-2 may transport IGF-I
from circulation to their target cells as well as modulate IGF-I activity.
Insulin

Effects Of exogenous ST on circulating insulin concentration have been
puzzling. Treatment of dairy cows with rbST chronically increased circulating
insulin concentration in some, but not all studies (Peel and Bauman, 1987).
The reasons for this inconsistency are not identified, but rbST-induced
increases in milk yield occur regardless of whether a chronic hyperinsulinemia
is Observed (McGuire et al., 1992).

Nevertheless, Vicini et al. (1991) speculated that an insulin response to
rbST treatment is probably due to coordinated metabolic changes. The rbST
treatment has three main effects on metabolism: 1) increased glucose
irreversible loss, which reflects increased liver gluconeogenesis (Cohick et al.,

1989); 2) decreased whole body glucose oxidation (Bauman et al., 1988); and

 

20

3) reduced glucose response to insulin (Sechen et al., 1989).

Exogenous insulin can also increase IGF-I mRNA expression, indicating
that insulin can act as a regulator of the IGF-I system in viva (Salamon et al.,
1989). Furthermore, in hepatocyte primary culture, insulin appears to regulate

IGF-I synthesis at the mRNA level (Phillips et al., 1991).

Effects of rbGRF on milk production

It was not until 1982 that GRF was isolated and characterized from
extracts of pancreatic tumors of two acromegalic humans (Rivier et al., 1982).
Following its isolation, the GRF peptide was described for several species
including cattle (Esch et al., 1983), sheep and goats (Brazeau et al. 1984). The
galactopoietic activity Of exogenous GRF administration were first reported in
lactating ewes (Hart et al., 1985) and lactating dairy cows (Baile, 1985) less
than ten years ago. The administration Of exogenous GRF tO lactating dairy
cows stimulates an increase in concentration Of serum ST and a subsequent
increase in concentration of serum IGF-I (Enright et al., 1986; Lapierre et al.,
1990; Dahl et al., 1993). Recently, rbGRF has been expressed in E. coli
(Kirschner et al., 1989), and this 1-45 (Leu27, Hse‘s) rbGRF was used in the

experimental trial conducted for this thesis.

Physiological effects of rbGRF

The GRF peptide binds to the plasma membrane of the somatotrops in

21

the anterior pituitary (Morel, 1991). After GRF binding, the area occupied by
secretory granules in the somatotrops decreases, reflecting a release of ST
(Morel, 1991). In vitra, secretion Of ST induced by GRF occurs immediately
and continues for duration of the exposure (Cronin et al., 1983). In
somatotrops, the GRF peptide increases total immunoreactive ST
concentration (Fukata et al., 1984), ST mRNA (Gick et al., 1984; Tanner et
al., 1990), transcription rate of the ST gene (Baringa et al., 1983), and
translocation Of ST mRNA from the nucleus to the cytoplasm (Morel, 1991).

Studies investigating the effects of rbGRF administration on subsequent
synthesis and secretion of IGF-I have been limited. The increases in
concentration of serum IGF-I following rbGRF administration are presumably
a result of the increase in concentration of serum ST that, in turn, stimulates
IGF-I synthesis and secretion predominantly from the liver.

NO studies, to date, have been published which investigated the effect
of rbGRF administration on concentration of serum IGFBPs, expression of
liver ST receptor, liver IGF-I, or mammary IGF-I receptor. However, GRF
likely effects these components Of the ST/IGF/BP cascade in a manner similar

to rbST.

 

MATERIALS AND METHODS

Design and Management

Thirty primiparous Holstein cows were used in a randomized complete
block design. Ten blocks Of three cows were formed based on calving date.
Within each block, cows were assigned randomly to treatment (10
cows/treatment).

Treatments began 118 d postpartum and were: 1) continuous i.v.
(jugular) infusion of 29 mg/d rbST; 2) continuous i.v. (jugular) infusion of 12
mg/d Of rbGRF (1-45) homoserine lactone; and 3) no infusion (controls) for
63 d.

Eight days before treatment began, VETport' (Thermedics, Worburn,
MA) infusion catheters were implanted surgically into cows assigned to rbST
and rbGRF treatments. In preparation for surgery, hair was clipped over the
neck and shoulder areas, these areas were washed, and the cow was given a
sedative i.m. (Rompun, Mobay Co., Shawnee, KN). A vertical path
approximately 6 cm wide along the neck, and areas approximately 12 cm in
diameter at the top of the shoulder and at the base of the neck over the right
jugular were anesthetized by s.c. injections of lidocaine (American Veterinary

Products Inc., Fort Collins, CO). The cow was immobilized on an operating

22

23

table, drapes were laid around the anesthetized areas, and the anesthetized
areas were scrubbed with aseptic soap. A 3 to 5 cm incision was made at the
top Of the shoulder and over the right jugular vein. Catheter tubing was
inserted aseptically at the shoulder, routed s.c. using a stainless steel rod and
inserted into a jugular vein using a needle. Upon completion both incisions
were stapled closed. The neck of the cow was wrapped with stretch wrap after
final cleansing with hydrogen peroxide.

Infusion pumps (AS-2BH, Autosyringe, Inc., Hooksett, NH) were
connected to the catheters with a .20-um pore Sterile Acrodisc syringe filter
(Gelman Sciences, Ann Arbor, MI) placed between the syringe, containing
either rbST or rbGRF solutions, and the infusion catheter. The infusion
pumps were fixed in back-packs that were mounted and secured with
adjustable straps on the withers of a cow. Control cows did not wear a
backpack and were not infused with any solution.

Bottles of rbGRF and rbST were stored at 4° C and solubilized on the
day of use with 21 ml of sterile distilled water. Syringes were changed daily
at 0700 h. Infusion volume was 12 ml/d for both rbGRF or rbST. Infusion
catheters were coated initially with 1% bovine serum albumin dissolved in
sterile water. Cows received rbGRF or rbST in pulses every 3.75 min.
Treatments began at 1700 h on day 0.

Cows were housed in tie stalls and exposed to 24 h of light per day.

Cows receiving rbGRF were milked at 0700, 1600, and 2330 h, and cows

1 I . 5m}.—

 

 

24
receiving rbST or no infusion were milked at 0545, 1430, and 2200 h. Milk

production was recorded daily, and milk samples were collected weekly
beginning at 2 wk before treatment began. Fat, protein, and lactose
concentration in milk were measured using an infrared analyzer (Multispec,
Wheldrake, UK) at Michigan DHIA (East Lansing). Yield of solids-corrected
milk (SCM) and output of energy in milk were calculated: SCM (kg) = [27.1
(kg fat) + 14.4 (kg solids-not-fat) - .166 (kg milk)] (Tyrrell and Reid, 1965);
milk energy (Meal) = [milk yield x (.0921 x % fat + .0490 x % solids-not-fat -
.0563], (Tyrrell and Reid, 1965).

A total mixed ration (TMR) was fed ad libitum. The TMR was
formulated (Appendix A) to provide adequate nutrition for a cow (590 kg BW)
producing 38.5 kg/d Of milk containing 3.5% fat and assuming an intake of 22.7
kg/d dry matter (DM) (NRC, 1989). Feed was offered daily at 0330 and 1630
h. The amount of feed Offered and refused was recorded daily. Samples of
feed Offered were collected weekly and chemically analyzed for DM, crude
protein (CP) and neutral detergent fiber (NDF). Net energy Of lactation (NE')
value of feed was calculated (NRC, 1989). DM content was determined
gravimetrically after drying samples at 105°C for a minimum of 12 h. The
percentage of NDF in the TMR samples was determined in duplicate
according to Goering and Van Soest (1970) with omission Of
decahydronapthalene, sodium sulfite, the substitution of triethylene glycol for

2-ethoxyethanol, and inclusion of a-amylase (Robertson and Van Soest, 1977).

 

25

Nitrogen content Of fractions was determined using a modified Kjeldal
procedure (Watkins et al. 1987). The CP content Of samples was determined
by multiplying the nitrogen content of the fractions by 6.25. All laboratory
analyses were performed by a single technician. Standard samples were carried
through all laboratory analyses to ensure consistent measurements.

Body weight (BW) Of cows was recorded at 1030 h on two consecutive
days at 2-week intervals beginning on experimental wk -1. NEl requirement
for maintenance Of cows was calculated based on body weight (.08 x BW°75,
NRC 1989).

Energy balance (EB) was calculated: EB (Meal NEI/d) = [feed energy
input (Meal NE,/d, NRC, 1989) - maintenance energy (Meal NE,/d, NRC,

1989) - milk energy output (Meal NE/d, Tyrrell and Reid, 1965)].

Blood Collection and Analysis

An indwelling jugular catheter (16 gauge; lea-Rally, Palo Alto, CA)
was inserted into the left jugular vein contralateral to the rbGRF- or rbST-
infused vein on d 55 Of the experiment. The catheters were coated with
heparin (TDMAC Heparin, Polyseiences Inc., Warrington, PA) to reduce fibrin
accumulation and sterilized with ethylene oxide. Catheter pateney was
maintained by flushing with 3.5% Na citrate and 50% sucrose in sterile water
after collection of each sample.

Blood samples were collected from cows at 20-min intervals for 6 h

26
(0900 to 1500 h) at d 57. Blood samples were stored at 4 ° C for approximately

24 h, and then serum was harvested by centrifugation for 30 min at 1550 x g
and frozen at -20 ° C until assayed. Serum ST (Moseley et al., 1982) and IGF-1
(Dahl et al., 1990), insulin (Villa-Godoy et al., 1990) and IGFBPs (Hossenlop
et al., 1986) were quantified in blood collected at d 57.

Briefly, concentration of ST in serum was assayed by the Upjohn CO.
using a double antibody radioimmunoassay procedure described by Niswender
et al. (1969). It was modified using 1251 in place Of 131I and 1% bovine serum
albumin in phosphate buffer was used instead Of egg whites. The reference
standard was NIH-B17-GH. Antisera against purified GH (NIH-B17) was
prepared in guinea pigs. The second antibody was raised in sheep against
guinea pigs gamma globulin.

Concentration Of IGF-I in serum was assayed by Upjohn using a glycl-
glysine HCl extraction and a double antibody radioimmunoassay.
Recombinant DNA-derived human IGF-I (Amgen Corp., Thousand Oaks, CA)
was used for standards and radioiodination. Concentration serum IGF-I was
measured by radioimmunoassay using rabbit anti-hIGF-I (IMC, Terre Haute,
IN). After overnight incubation Of samples and standards with the first
antibody, labeled IGF-I (15,000 epm/tube) was added and samples were
incubated for an additional 48 h. The IGF-I complexed with the antibody was

precipitated with goat anti-rabbit gamma globulin (Antibodies Inc., Davis, CA).

 

27

Concentration Of insulin in serum was assayed using a double antibody
radioimmunoassay procedure as described by Villa-Godoy et al. (1990).
Bovine insulin (5 ug) was iodinated using the chloramine T method. An
insulin standard solution (.02564 ng/ul in preparation was diluted with .05
phosphate buffer, .85% NaCl, pH 8.6/Bl buffer in a 1:1 ratio) was used to
quantitate measurements. Concentration Of serum insulin was measured by
radioimmunoassay using guinea pig anti-bovine insulin (diluted to 1:30,000).
After overnight incubation of samples and standards with the first antibody,
labeled insulin (20,000 cpm/tube) was added and samples were incubated for
an additional 48 h. The insulin complexed with the antibody was precipitated
with sheep anti-guinea pig gamma globulin.

Serum IGFBP-2 and -3 were quantified in blood collected at d 57 using
western ligand blotting (Hossenlop et al., 1986). Briefly, serum (2 ul) diluted
at 1:8 using .05M Tris buffer pH 7.4 was mixed with sample buffer containing
.42M tris-ehloride and 13.3% sodium-dOdeeyl-sulfate-polyacrylamide (SDS).
After heating at 60°C for 30 min, samples were electrophoresed on SDS-
polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE was
performed in a 12.5% resolving, 4% stacking gel using constant current
(approximately 20 volts applied overnight) in a Vertical Gel System (Bethesda
Research Laboratories, Gaithersburg, MD). Proteins were transferred to
nitrocellulose filters (Schleicher and Sehuell Inc., Keene, NH) in a wet

electroblot system (Hoeffer Scientific Instruments, San Francisco, CA).

28

Hybridization Of membranes was as described by Davenport et al. (1990) using
125I-IGF-I iodinated by the chloramine T method. Following overnight
hybridization, blots were washed and exposed to autoradiography film (X-
OMAT AR film; Eastman Kodak Co., Rochester, NY). After 24 to 48 h, film
was developed and the relative concentration of serum IGFBP-3, -2, -1 and -4
were determined by densitometry (Model FB934 densitometer; Fischer
Biotech, Fischer Scientific) with software from Biomed Instruments Inc.,
Fullerton, CA of the 36 and 43, 33, 27 and 23 kd bands, respectively.

After ligand blotting analysis, nitrocellulose filters were incubated for 90
min with a dilution Of 1:1000 of bovine IGFBP-3 antiserum and then for 2 h
with alkaline phosphatase-conjugate goat antirabbit irnmuoglobin G (GAR-AP;
Bio-Rad Chemical Division, Richmond, CA). Color development substrates
from a GAR-AP Bio-Rad kit were used to detect alkaline phosphatase activity

to depict the presence of the IGFBP-3 bands in the membrane.

Tissue Collection and Analysis

At (1 63 of the treatment period, liver and mammary tissue were
removed from a cow within 5 min of slaughter. A 10 g piece of liver and
several 10 g pieces of mammary tissue from the left hemi-gland were removed
and immediately placed in liquid nitrogen. After freezing, tissue was stored
at -70°C.

Total RNA was isolated from liver and mammary tissue (Chirgwin et al.,

29

1979). Briefly, 1 g of tissue was homogenized in 24 ml guanidinium
thiocyanate (4 M) and then layered over 5.7 M cesium chloride in an
ultracentrifuge tube. The tubes were centrifuged at 100,000 x g for 16 h. The
RNA pellet was recovered and purified further by chloroform-phenol
extraction.

After isolation of total RNA, expression of mRNA for ST-receptor,
IGF-I and p-aetin in liver and IGF-1 receptor and p-actin in mammary tissue
were determined by northern blot hybridization. Briefly, total RNA (20
ug/sample for liver and 40 ug/sample for mammary tissue) was denatured in
50% formamide, 2.2 M formaldehyde at 55 C for 15 min. The sample mixture
was electrophoresed in an agarose-formaldehyde gel (1.2% agarose) made with
1x MOPS buffer (.04M 3-N-morpholinO-propane-sulfonic acid, IOmM sodium
acetate, lmM ethylenediamine tetraacetie acid) pH 7.0 and 4.45% v/v
formaldehyde. Following electrophoresis, RNA was transferred onto a
GeneScreen Plus membrane (NEN Research Products, Boston, MA) using an
electroblot system. The membrane was then baked 2 h at 80° C to reverse the
formaldehyde reaction. Nucleic acids in the membrane were hybridized at
42°C in a solution containing 50% formamide, 10% dextran sulfate and 1%
SDS in 1 M NaCl. Overnight hybridization was completed with a 32P-labeled
eDNA probe (500,000 epm/ml). For IGF-I, the probe was made by random-
priming Of a human IGF-I cDNA clone (Bell et al., 1984); homology of bovine

and human IGF-I mRNA is 93% (Fotsis et al., 1990). For ST receptor, a

 

30

rabbit cDNA clone Obtained from Dr. William Wood was used (Genetech Inc.,
San Francisco, Ca; Leung et al., 1987). For IGF-I receptor, a cDNA clone was
used (American Type Culture Collection, #59294). For fi-actin, a cDNA clone
Obtained from Dr. William Helfrich was used (Michigan State University, East
Lansing, MI). Following hybridization, membranes were washed at medium
stringency and then exposed to autoradiography film for 2 to 5 d. Band
intensities Of developed film were subjected to densitometry for quantification.
Membranes were rehybridized with the respective probes. Between
hybridization with each probe, the membranes were stripped by soaking in

boiling solution (.01% SDS, .01% SSC) for 2 h.

Statistical Analysis

Characteristics of ST in serum (baseline, number Of peaks, peak
amplitude, peak length and mean) during the 6-h sampling period at d 57 were
determined using a pulse analysis program (PULSAR; Merriam and Watcher,
1982). To minimize heterogeneous variance; number Of peaks, peak amplitude
and peak length were transformed to natural logarithms for analysis (Gill,
1978).

All data were subjected to randomized complete block AN OVA. Mean
comparisons were made using orthogonal contrast (C1= Control vs rbST +
rbGRF and C2= rbST vs rbGRF).

When significant (P> .10), initial BW, DMI, SCM and BB were included

31
in the model as a covariate for analysis of BW, DMI, SCM and EB.

RESULTS

BW, DMI, SCM and EB

Initial measures of BW, DMI, SCM and EB, which were measured 7 d
before treatment began, were significant when tested as a covariate; therefore
BW, DMI, SCM and EB reported for d 50 to 62 were adjusted by covariance
(Table 1). Average covariate-adjusted BW, DMI, SCM and EB are reported
for the 13 (1 period before slaughter (d 50 to 62). Body weight was not
different among treatment groups. Dry matter intake for rbGRF-treated cows
(23.4 kg/d) tended to be greater (P=.08) compared with rbST-treated cows
(22.2 kg/d). There was no difference in SCM between rbST- (34.1 kg/d) and
rbGRF-treated cows (33.1 kg/d). Hormone treatments (rbST and rbGRF)
increased (P<.01) SCM by 18% compared with controls (28.5 kg/d).
Calculated EB was positive for all treatments. However, the rbST-treated cows
(3.8 Meal/d) tended to have lower (P=.07) EB compared with rbGRF treated
cows (6.3 Meal/d). Also, average EB for rbST-treated and rbGRF-treated

cows was lower (P<.01) than controls (8.6 Meal/d).

Serum ST

Results for serum hormone concentrations are from d 57 of the

32

 

33

Table 1. Least squares means for BW, DMI, SCM and EB between d 50 and 62
of treatment of cows receiving no treatment (n=10), 29 mg rbST/d (n=10) or 12
mg rbGRF/d (n=10) for 63 d of lactation (d 118 to 181 postpartum).

 

 

 

 

Pr > F
Con rbST rbGRF SEM C1' C2”
BW, kg 544 543 548 6 .82 .54
DMI, kg/d 22.7 22.2 23.4 .9 .92 .08
SCM, kg/d 28.5 34.1 33.1 1.2 .01 .52
EB‘, Meal/d 8.6 3.8 6.3 .7 .01 .07

 

 

 

 

a Orthogonal contrast of means for control vs. average of rbST- and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.
C Energy balance, calculated as {feed energy input [NE], NRC, 1989] -

maintenance energy [.08 x BW-75, NRC, 1989] - milk energy output [milk yield x
(.0921 x % fat + .0490 x % solids-not—fat - .0563),Tyrrell and Reid, 1965]}

34

treatment period. Concentration of ST in serum increased from 3.3 ng/ml for
controls to 14.2 and 18.3 ng/ml for rbST- and rbGRF-treated cows, respectively
(Table 2). The pooled effect of rbST and rbGRF treatments increased
(P<.01) concentration of ST in serum compared with controls. Also, cows
treated with rbGRF tended to have greater (P=.13) concentration of ST in
serum than cows treated with rbST at d 57.

Parameters of ST pulsatility were adjusted by log-transformation to
minimize heterogeneous variance for statistical analyses. Infusion Of rbGRF
increased (P=.05) number Of peaks/d and amplitude (ng/ml) and tended to
increase (P=.10) peak length (min) compared with rbST-infused cows (Table
2). Figure 2 illustrates the pulsatility profile Of a representative cow from

each treatment for d 57.

Serum IGF-I

Concentration Of IGF-I in serum at d 57 increased (P<.01) from 110
ng/ml for controls to 257 and 211 ng/ml for rbGRF- and rbST-treated cows,
respectively (Figure 3). Cows treated with rbGRF tended to have greater
(P= .15) concentration of IGF-I in serum compared to cows treated with rbST.
The greater concentration of IGF-I in serum for rbST-treated cows occurred
despite the fact that concentration of ST in serum was slightly lower, not

higher, in rbST- treated cows compared with rbGRF-treated cows.

 

35

Table 2. Least squares means for baseline ST, number of peaks, amplitude, peak
length and mean ST for d 57 Of treatment Of cows receiving no treatment (n=10),
29 mg rbST/d (n=10) or 12 mg rbGRF/d (n=10) for 63 d of lactation (d 118 to 181
postpartum).

 

 

 

 

 

 

 

Pr > F

Con rbST rbGRF SEM (31a C2”
Baseline ST, ng/ml 2.7 13.1 16.0 1.9 .01 .28
Number of peaks, 6 h .61 .25 .72 .16 ~54 -05
(natural log)
Amplitude‘mg/ml 1.0 .8 1.8 .4 .50 .05
(natural log)
Peak length, min .71 .44 .99 .22 .98 .10
(natural log)
Mean ST, [lg/ml 3.3 14.2 18.3 1.8 .01 .13

 

a Orthogonal contrast of means for control vs. average of rbST- and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.

C Calculated relative to baseline ST concentrations.

36

 

 

 

50
S 40
erum
30
ST, 20
ng/ml 10
O M. ' "H '50“. ‘N
Control
50
40
Serum 30
ST. 20 W
ng/ml 10
0
rbST
50
40
Serum 3O
21"] 20
n m 10
0
rbGRF

Figure 2. Concentration of ST in serum for d 57 of treatment of cows receiving
no treatment. (n=10), 29 mg rbST/d (n=10) or 12 mg rbGRF/d (n=10) for 63 d of
lactation (d 118 to 181 postpartum). Each graph depicts the pulsatility profile of a
representative cow from each treatment. Each point represents one Of ninteen
samples taken at 20 min intervals over a 6 h period.

 

37

 

250 —

: C13P<.01

200 :— C2” P=.15
Serum I
IGF'Is 150 :-
ng/ml
100 :—
50 j—
0 _

 

 

 

 

 

Con rbST rbGRF

Figure 3. Least squares mean concentration of IGF-I in serum for d 57 of
treatment of cows receiving no treatment (n=10), 29 mg rbST/d (n=10) or 12 mg
rbGRF/d (n=10) for 63 d of lactation (d 118 to 181 postpartum). Pooled standard
error of the mean among treatments was 22 ng/ml.

a Orthogonal contrast of means for control vs. average of rbST— and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.

 

38
Serum IGFBPs

Serum IGFBP-3 had two forms at 43 and 36 kd as shown by
autoradiographs of western ligand blots in Figure 5. Irnmunoblots using
bovine IGFBP-3 antiserum indicated that these two bands were indeed
IGFBP-3. At d 57 IGFBP-3 in serum increased (P<.01) from 6.0
densitometric units for controls to about 16 densitometric units hormone-
treated cows, respectively (Figure 4). Also, cows treated with rbST had
greater (P=.03) concentration of IGFBP-3 in serum compared to cows treated
with rbGRF. Furthermore, the ratio Of IGFBP3:IGF-I was not difference
among groups.

Serum IGFBP-2 had one band at 33 kd as shown by autoradiographs Of
western ligand blots in Figure 5. IGFBP-2 in serum at d 57 decreased from
11.1 densitometric units for controls to 8.9 densitometric units for rbST
treatment and 7.2 densitometric units for rbGRF treatment (Figure 4). Both
treatments together decreased (P=.03) IGFBP-2 in serum compared with
controls. There was no difference (P>.27) between rbST and rbGRF

treatment on serum IGFBP-2.

Serum insulin
Concentration Of insulin in serum on d 57 increased from .41 ng/ml for
controls to .72 and .46 ng/ml for rbST- and rbGRF-treated cows, respectively

(Figure 6). Hormone-treated cows tended to increase (P=.08) concentration

 

 

39

      

 

C1” P<.01
C2b =.03 '2' C0“
20 M rbST
- rbGRF
15 C1P=.03
Densitometric C2 P>.27
UnIts 10
5
0

IGFBP-3 IGFBP-2

Figure 4. Least squares means of IGFBP-3 and IGFBP-2 in serum for d 57 of
treatment of cows receiving no treatment (n=10), 29 mg rbST/d (n=10) or 12 mg
rbGRF/d (n=10) for 63 d of lactation (d 118 to 181 postpartum). Pooled standard
error of the mean among treatments was 2.7 and 1.1 densitometric units for
IGFBP-3 and IGFBP-2, respectively.

a Orthogonal contrast of means for control vs. average of rbST- and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.

40

kd Control rbST rbGRF
5012345012345
. ~ ,

01234
E. ‘1';

43
36
33
27

23

   

kd Control rbST rbGRF
012345012345012345

         

43
36

33
27
23

bp3
bp3

' bp2
bpl
. bp4

Figure 5. Representative autoradiographs of western ligand blots for IGFBP's (blocks
8 and 9). Numbers 0 through 5 represent d 0, 1, 2, 3, 29, and 57 of treatment,
respectively.

41

0.7

0.6 C 1a P=.08

b
Serum C2 P<.01

Insulin,
ng/ml 0.4

 

0.3

0.2
0.1

Tlllllllllllllllllllllllllllllll

 

 

 

 

0

 

Con rbST rbGRF

Figure 6. Least squares mean concentration of insulin in serum for d 57 of
treatment of cows receiving no treatment (n=10), 29 mg rbST/d (n=10) or 12 mg
rbGRF/d (n=10) for 63 d of lactation. Pooled standard error of the mean among
treatments was .08 ng/ml.

a Orthogonal contrast of means for control vs. average of rbST— and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.

 

42

of insulin in serum compared with controls. Also, concentration Of insulin in
serum was greater (P<.01) for rbST- compared to rbGRF-treated cows.
Cows treated with rbST had heavier (P < .01) livers (wet-basis) compared
with cows treated with rbGRF. Perhaps, greater insulin concentration in
serum resulted in increased glycogen storage in the liver of rbST- compared

with rbGRF-treated cows.

Liver ST receptor and IGF-1 mRNA

Results for tissue mRNA are for d 63 Of the treatment period, and have
been adjusted for the amount of ,B-actin mRNA expression in liver.
Abundance of mRNA for ST receptor was measured from autoradiographs Of
northern blots using densitometry (Figure 8 and 9). The band which
hybridized to the ST receptor probe was measured at 4.5 kb. Expression Of
mRNA for ST receptor in liver was not different among treatments (Figure 7).

Expression of mRNA for IGF-I in liver, measured at 7.5 kb (Figure 8
and 9), was 290% and 191% of control for rbST- and rbGRF-treated cows,
respectively (Figure 7). Hormone treatments increased (P<.01) expression of
IGF-I mRNA in liver compared to controls. Also, rbST-treated cows
increased (P<.01) expression of IGF-I mRNA in liver more than did rbGRF-
treated cows. The results for expression of IGF-I mRNA in liver are

consistent with concentration of IGF-I in serum for the three treatment

groups.

 

 

43

 
 
  
   
        

 

  

€112.01
_ 2P<.01
300 u rbST A
250 3. rbGRF I
: |
mRNA 200 :— C1P=.02 ?
Abundance, E (3121525 (:2 p>.93 :.
% of Control 150 :— C2 P>' , '
100 :—
50 :—
0

 

   

Liver ST Liver Mammary
receptor IGF-I IGF-I
receptor

Figure 7. Abundance Of mRNA for ST receptor and IGF-1 in liver and IGF-1
receptor in mammary tissue for d 63 of treatment of cows receiving 29 mg rbST/d
(n=10) or 12 mg rbGRF/d (n=10) for 63 d of lactation (d 118 to 181 postpartum).
Each bar represents the average of abundance of mRNA (least squares means) as a
percent of the abundance of the respective mRN A for control cows (n=10).

a Orthogonal contrast of means for control vs. average of rbST- and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.

44

Liver mRNA Abundance

kb Bla B2 B3 B4 B5
CbSchCSGCSGCSGCSG
ST I . ..
31

 

receptor

4.5

IGF-I

 

Figure 8. Autoradioraphs of northern blots for liver ST receptor, IGF-I and B—Bctin
mRNA for blocks 1 through 5.

a Block, b Control 0 rbST treatment d rbGRF treatment

45
Liver mRNA Abundance

kb B6a B7 B8 B9 B10
CbSCGdCSGCSGCSGCSG

 

ST
receptor
4.5 a - - - 5‘ 9‘ -- .. U.
mm J .
15 ON *‘fi O “W
i
, 1
1

at

zcudflqafigaflflflmbfifi

B-actin . p l
C34

Figure 9. Autoradioraphs of northern blots for liver ST reCeptor, IGF-I and B-actin
mRNA for blocks 6 through 10.

a Block b Control 0 rbST treatment d rbGRF treatment

 

46
Mammary IGF-I receptor mRNA

The predominant type-I IGF receptor mRNA species in mammary tissue
was 11.3 kb (Figure 10). Expression of p-actin mRNA in mammary tissue was
used tO adjust the measurement of IGF-I receptor mRNA in mammary tissue.
The IGF-I receptor mRNA expression was 146% and 144% (P=.02) of
control for rbST and rbGRF treatments, respectively (Figure 7). Expression
Of IGF-I receptor mRNA in mammary tissue was not different between rbST-

and rbGRF-treated cows.

 

47
Mammary mRNA abundance
kb Bla B2 B3 B4 B5
CbSCGdCSGCSGCSGCSG
IGF-I —- A- A. A.
receptor

 

li-actin

kb B6 B7 B8 B9 B10
CSGCSGCSGCSGCSG

IGF-I ’ “'- '1; g; - g i " ,, '27, ’
receptor ' ‘~ ~ ‘; .
11.3

   

 

v 1'..." 7

             

' . 5:»; , -. we;
B-actin '

m a EU .adfiOO
Figure 10. Autoradioraphs of northern blots for mammary IGF—I receptor and B-actin
mRNA for blocks 1 through 10.

a Block P Control ‘3 rbST treatment d rbGRF treatment

DISCUSSION

Effects of exogenous rbST and rbGRF on the ST/IGF/BP cascade

In the present experiment, hormone treatments (rbST and rbGRF)
increased concentration Of ST in serum compared with controls. The sources
of ST in response to rbST and rbGRF treatments were presumably the
exogenous rbST itself and endogenous ST from the anterior pituitary,
respectively. Although our goal was to have equal elevations of ST in serum
for the two hormone-treated groups, concentration of ST in serum tended
(P=.13) to be greater for rbGRF- compared with rbST-treated cows. If
concentrations Of ST were different, the difference was likely dose-related.
Doses of rbST and rbGRF used in current study were selected based on the
results Of a previous trial conducted by Dahl et al. (1993). There were,
however, some changes in design between the two experiments. Cows from
the previous trial were multiparous whereas cows from the present experiment
were primiparous. The previous trial was conducted during late-lactation (175
to 235 d postpartum) whereas the present experiment was conducted during
mid-lactation (118 to 181 d postpartum). Despite these differences in parity
and stage of lactation between the two trials, the responses in serum ST were

similar with only a trend for greater response with rbGRF in the present study.

48

 

49

Similar to the findings of Dahl et al. (1993), hormone treatments
increased concentration Of IGF-I in serum compared with controls. However,
concentration of IGF-I in serum tended (P=.15) to be greater for rbST-
compared to rbGRF-treated cows. The greater concentration Of IGF-I in
serum of rbST-treated cows occurred despite the fact that concentration of ST
in serum tended to be lower, not higher, for rbST- compared to rbGRF-
treated cows. Possible explanations for the IGF-I response to rbST and
rbGRF treatment will be discussed in the next section Of this discussion.
Negative EB has been associated with low concentration of IGF-I in serum for
lactating dairy cows (McGuire et al., 1992). Even though hormone treatments
decreased EB compared to controls, and rbST-treated cows tended (P=.07)
to have lower EB compared to rbGRF-treated cows, all cows were in positive
EB. Thus, EB probably did not play a role in regulating concentration of IGF-
I in serum.

Administration of rbST increases serum concentration Of IGFBP-3 in
dairy cows (Cohick et al., 1992). Cows treated with rbST increased (P=.03)
concentration of IGFBP-3 in serum compared with rbGRF-treated cows. Zapf
et al. (1989) reported that IGF-I mediates much of the ST-associated increase
in IGFBP-3 levels in viva. Consequently, greater concentration of IGFBP-3
in serum for rbST- compared to rbGRF-treated cows was likely related to the

trend for greater concentration of IGF-I in serum Of rbST- compared with

 

50

rbGRF-treated cows.

The exact role for IGFBP-3 has not yet been defined. However,
IGFBP-3 can prevent IGF-I-induced IGF receptor down regulation (Conover
and Powell, 1991), thereby maintaining the effectiveness Of the elevated
concentration of serum IGF-I during rbST administration. Blum et al. (1989)
suggested that IGFBP-3 acts as a reservoir for IGF-I, continuously releasing
low amounts Of IGF-I and thereby creating a steady state of receptor
occupancy. Binoux and Hossenlopp (1988) concluded that IGFBP-3 complexes
do not cross the capillary barrier, which provides further evidence that IGFBP-
3 acts as a reservoir and as a moderator of IGF-I action.

In the present experiment, exogenous hormone treatments decreased
concentration of IGFBP-2 in serum. Cohick et al. (1992) found that rbST
administration decreases IGFBP-2 concentration in serum. The physiological
implications of this decrease in IGFBP-2 is not well understood. Binoux and
Hossenlopp (1988) conclude that IGFBP-2 complexes cross the capillary
barrier. Thus, IGFBP-2 may be involved in the transport Of the IGFs from
circulation to their target cells. The IGFBP-2 can leave the microcirculation
and distribute to subendothelial tissues (Booth et al., 1990). Furthermore,
plasma IGFBP-2 levels are relatively stable, and therefore may serve as a
reservoir Of IGF-I available for transport (Clemmons et al. 1991). Bourner
et al. (1992) found that the IGFBP-2 can bind with IGF-I and act as an

inhibitor or a potentiator of IGF-I-mediated DNA synthesis.

 

51

Although expression of ST receptor mRNA in liver was not different for
the three groups, the results do not necessarily suggest that the activity or
abundance of ST receptors in the liver was affected. Mathews et a1. (1989)
suggested that, in rat hepatocytes, any effect of ST on receptor expression is
manifested at a post-transcriptional level, because ST receptor mRNA was not
significantly affected by either hypophysectomy or rbST treatment. It is also
possible that the hormone treatments alter the affinity or turnover of the
receptors to increase their biological activity and abundance. Somatotropin
binding studies utilizing liver from cows from the present experiment are
currently being conducted in another laboratory (R. Michael Akers, Virginia
Polytechnic Institute and State University). Results from these binding studies
will reveal whether or not there is increased activity or abundance Of the ST
receptor despite the fact that mRNA expression was not altered.

Expression of mRNA for IGF-I in liver was substantially increased by
both treatments and to a greater extent for rbST treatment. In
hypophysectomized rats, exogenous ST increases abundance Of hepatic IGF-I
mRNA (Bichell et al., 1992). Furthermore, nuclear run-on assays were used
to show that ST regulation of IGF-I is manifested in part at the transcriptional
level (Mathews et al., 1986). Thus, transcriptional mechanisms are
predominately responsible for enhanced IGF-I expression elicited by rbST
(Roberts et al., 1986; Bichell et al., 1992).

Expression of mRNA for IGF-I receptor in mammary tissue increased

 

52

similarly for hormone treatments compared to controls. These results are
different from those of Glimm et al., (1992) in which mammary IGF-I receptor
mRNA was decreased in two out Of three rbST-treated cows, but increased for
the third cow. The third cow was in negative EB because her feed intake
dropped significantly during the experiment. Glimm et al. (1992) speculated
that the response for the third cow may reflect a high nutrient priority afforded
to mammary tissue function during lactation. Stelwagen et al. (1992) suggested
that cows in early lactation (usually in negative energy balance) have a
selective increase Of mammary IGF-I receptors or IGF-I receptor affinity,
giving the mammary gland an advantage over other tissues with regard to IGF-
1 action and subsequent utilization of the available metabolite pool. Cows in
the present experiment were in lower EB compared with controls.
Consequently, the increase in expression of IGF-I receptor mRNA in
mammary tissue may be a mechanism by which the mammary gland can
maintain maximum functional capacity in early lactation or during rbST or

rbGRF treatment.

The ST/IGF/BP cascade is stimulated more by rbST than rbGRF

Results of the present experiment are consistent with the hypothesis that
rbST and rbGRF treatments stimulate the ST/IGF/BP cascade. However, the
extent to which the hormone treatments stimulated the cascade was different.

Even though concentration of ST in serum tended to be higher for cows

 

53

treated with rbGRF compared with cows treated with rbST, concentration of
IGF-I in serum tended to be lower, not higher, for rbGRF treatment
compared with rbST treatment. Cows treated with rbST also had greater
expression Of IGF-I mRNA in liver than cows treated with rbGRF. In
agreement with concentration of IGF-I in serum, increased IGF-I mRNA
expression indicates that IGF-I protein synthesis in the liver was greater for
rbST- than rbGRF-treated cows. Furthermore, rbST treatment increased
IGFBP-3 to a greater extent than rbGRF treatment. Thus, compared with
rbST, rbGRF resulted in greater (P<.01) liver IGF-I mRNA expression, a
trend for greater (P=.15) serum IGF-I and greater (P=.03) serum IGFBP-3.
Perhaps the difference in serum IGF-I was less significant than the other two
measures because IGFBPs may interfere with IGF-I radioimmunoassay, and
because the IGFBP profiles were different for the two hormone treatments.
Nevertheless, these results indicate that the endocrine ST/IGF/BP cascade was
less responsive to endogenous ST secreted in response to rbGRF than it was
to exogenous rbST. Possible reasons why the ST/IGF/BP cascade was less
responsive to rbGRF than to rbST include their differential effects on ST
variants, pulsatility profiles and/or concentration Of insulin in serum.
Endogenous ST, produced in the anterior pituitary, can be either 190 or
191 amino acids long, and can have either leucine or valine at position number
126 in the protein sequence (Wood et al., 1989). Typically, pituitary

production of ST involves approximately equal amounts of the 190 and 191

 

54

amino acid proteins, and about two-thirds of the total produced has leucine at
position 126, while the remaining one-third have the amino acid valine at
position 126 (Wood et al., 1989). Treatment with rbST leads to a predominant
variant in circulation which is similar to the 191 amino acid native variant with
leucine at position 126 in the protein sequence. Cows treated with rbGRF will
likely stimulate release of all variants of ST from the anterior pituitary. It is
possible that the exogenous rbST variant stimulates IGF-I mRNA expression
to a greater extent than the combination Of variants secreted in response to
rbGRF. Thus, the increase in IGF-I mRNA in the liver and subsequent serum
IGF-I concentration in the present experiment may be a result of differences
in circulating ST variant profiles in response to rbST vs rbGRF treatment.
Pulsatility of ST in circulation may also be an important determinant Of
ST action in viva. We found that cows treated with rbGRF had greater
pulsatility (number Of peaks/d, amplitude and peak length) of ST in serum
than rbST-treated cows. In fact the rbST-treated cows exhibited little
pulsatility Of ST in serum. Perhaps, ST at steady concentrations is a more
effective secretagogue for liver IGF-I than ST at pulsatile concentrations. Bick
et al. (1992) found that rats have greater concentration of IGF-I in serum after
continuous (similar to continuous rbST) than after pulsatile (similar to
continuous rbGRF) administration Of the same daily dose of ST. The
mechanism to explain increased concentration Of IGF-I in serum for

continuous verses pulsatile infusion is unclear. However, it is possible that

 

55

differences in pulsatility may alter the binding characteristics or abundance Of
the ST receptor in the liver and subsequently increase synthesis and secretion
of IGF-I.

In the present experiment, cows treated with rbST tended (P=.10) to
have greater concentration Of insulin in serum than cows treated with rbGRF.
Perhaps this greater concentration of insulin in serum is the reason that IGF-I
mRNA was stimulated to a greater extent in rbST- than rbGRF-treated cows.
Exogenous insulin increases IGF—I mRNA expression in rats, thus insulin
regulates the IGF-I system in viva (Salamon et al., 1989). In hepatocyte
primary culture, insulin also appears to regulate IGF-I release at the mRNA
level (Phillips et al., 1991). Thus, it is possible that insulin was a factor in
stimulating IGF-I mRNA expression and subsequent synthesis and secretion
of IGF-I and IGFBP-3 in liver tissue to a greater extent in rbST- compared

with rbGRF-treated cows.

Similar milk yield despite different effects on the STIIGF/BP cascade
Despite the fact that rbST stimulated the endocrine ST/IGF/BP cascade
to a greater extent compared to rbGRF, both treatments stimulated milk yield
similarly. Thus, it is possible that the galactopoietic effects of rbGRF are not
mediated exclusively through the endocrine ST/IGF/BP cascade.
One possible reason to support this speculation is that the four different

ST variants produced in response to rbGRF stimulate galactopoiesis through

 

T'VT .

 

 

56

an autocrine/paracrine pathway as well as the endocrine ST/IGF/BP pathway.
The combination of these variants may stimulate production Of IGF-I and its
binding proteins in target tissues such as the mammary gland to a greater
extent than the single rbST variant. Thus, less stimulation Of the endocrine
cascade by rbGRF treatment may be supplemented by an autocrine and/or
paracrine pathway resulting in similar milk yield for rbST- and rbGRF-treated
cows. In further support, Krivi et al., (1989) reported differences in
galactopoietic activity Of ST variants in lactating cows.

Pulsatility Of ST in circulation may also be an important determinant Of
the galactopoietic effects for the hormone treatments. As mentioned
previously, cows treated with rbGRF had greater pulsatility (number of
peaks/d, amplitude and peak length) of ST in serum than rbST-treated cows.
Perhaps the greater pulsatility with rbGRF treatment stimulated galactopoiesis
at an autocrine/paracrine level more than did rbST treatment. Bick et al.
( 1992) found that pulsatile infusion (similar to rbGRF treatment) induced a
greater body weight gain than the continuous infusion (similar to rbST
treatment). In another study the levels of hepatic IGF-I mRNA were similar
in rats given continuous and pulsatile ST treatment, whereas the IGF-I mRNA
levels in skeletal muscle and cartilage were higher after pulsatile than after
continuous ST treatment (Isgaard et al., 1988). Thus, it is possible that more
pulsatile secretion Of ST, which was a result Of rbGRF treatment, may enhance

the galactopoietic effects of ST either directly or by stimulating autocrine

 

 

 

57

and/or paracrine action of IGF-I at target tissue even though circulating
concentration of IGF-I tended to be lower in rbGRF-treated compared to
rbST-treated cows.

It may also be possible that the increased immunoassayable IGF-I was
not reflected in increased biological activity Of IGF-I for rbST- compared with
rbGRF-treated cows. Although immunoassayable IGF-I concentration in
serum was greater with rbST treatment, IGFBP-3 was also greater; thus, more
of the serum IGF-I was likely bound to IGFBP-3 in serum Of rbST-treated
cows. Consequently, the two treatments may have had a similar pool size of
unbound biologically active IGF-I even though the pool size of bound IGF-I
and IGFBP-3 was greater for rbST than rbGRF treatments. Nevertheless, it
is difficult to determine whether this was the case or not because current
methods do not allow us to measure the amount Of unbound verses bound
IGF-I in serum.

Lastly, it is possible that the reason why milk yield was similar, despite
less stimulation Of the endocrine ST/IGF/BP cascade by rbGRF, is that the
rbGRF compound may have direct effects at tissues which support increased

milk yield.

 

SUMMARY AND CONCLUSIONS

The main Objective Of this thesis was to quantify and compare the long-
term effects Of exogenous rbST and rbGRF on the endocrine ST/IGF/BP
cascade in lactating dairy cows. Results from the present experiment are
consistent with the idea that treatment with rbST and rbGRF stimulate the
endocrine ST/IGF/BP cascade to increase milk yield. However, rbST
treatment stimulated the cascade to a greater extent compared with rbGRF
treatment. It is likely that differences in the profile Of ST variants, pulsatility
of ST and/or concentration of insulin in serum may have facilitated the
differences in the effects of rbST compared to rbGRF on the somatotropic
cascade.

However, both rbST and rbGRF treatments increased milk yield to the
same extent compared to controls. Possibly, the galactopoietic effects of
rbGRF are not mediated exclusively through the endocrine ST/IGF/BP
cascade.

Lastly, results from the present experiment indicate the complexity of
the coordination of physiology in response to rbST and rbGRF treatment. The
precise mechanisms by which these hormone treatments enhance galactopoiesis

remains to be elucidated. In the future, whether or not IGF-I is the sole

58

59

mediator of the galactopoietic actions Of rbST and rbGRF needs to be
investigated. One possible experiment would be to investigate the effects of
rbST or rbGRF with concurrent administration of an IGF-I antibody.

In addition to the investigation Of the endocrine actions of IGF-I, the
effects of rbST or rbGRF treatment on autocrine and/or paracrine action of
IGF-I and its binding proteins in various tissues need to be investigated. This
may be achieved by quantifying the expression Of ST receptor, IGF-I and
IGFBP mRNA in tissues (such as mammary gland, liver and adipose) which

support enhanced milk yield in response to hormone treatments.

 

 

 

APPENDICES

 

60

APPENDIX A

Table 3. Ingredient composition Of diets fed during the treatment period.

 

Ingredient percentage, %DM
Alfalfa haylage 21.5
Corn silage 20.5
High moisture shell corn 19.0
Ground shell corn 10.5
Soybean meal 11.7
Whole cotton seed 10
Mega-Lac .3
Energy balancer 1.5
Bypass protein 2.0
Mineral/Vitamin mix 2.0

 

Table 4. Chemical composition of diets fed during the treatment period.ab

 

Dry matter, % 59
Crude protein, % 18
Neutral detergent fiber, % 31
Acid detergent fiber, % 19
Net energy lactation, Meal/d .79

 

a Calculated values from NRC book values and forage report values.

P Average percentage of CP and NDF analyzed from weekly TMR samples were
17.4 and 33.5, respectively.

61

APPENDIX B

Introduction
A second Objective of this experiment was to investigate the short-term

effects of rbST verses rbGRF treatment on the endocrine ST/IGF/BP cascade.

Materials and Methods

Additional blood samples were collected for analysis of the short-term
effects. Blood samples were collected from cows at 6 h intervals for 2 d (800
h on d 0 to 2000 h on d 1). Blood samples were also collected at 24 h
intervals for 2 d (1400 h on d 2 to 1400 h on d 3). Samples of blood were
stored at 4°C for approximately 24 h and then serum was harvested by
centrifugation for 30 min at 1550 x g and frozen at -20 ° C until assayed. Blood
samples within a day were pooled into one sample for each day.

Concentration Of serum ST (Moseley et al., 1982) was quantified in
blood collected at d 1. Concentration Of IGF-I, IGFBP-2 and IGFBP-3 in
serum were quantified in blood collected at d 0, 1, 2 and 3 using the same
methods as described in the previous materials and methods section.

All data was subjected to randomized complete block AN OVA. Short-
term effects Of the treatments on ST was analyzed with mean comparisons

using orthogonal contrast (C1= Control vs rbST + rbGRF and C2= rbST vs

62
rbGRF). Short-term responses for concentration of IGF-I, IGFBP-2 and

IGFBP-3 in serum were analyzed using linear orthogonal contrast (CL1=

Linear; Control vs rbST + rbGRF and CL2= Linear; rbST vs rbGRF).

Results and Discussion

Concentration of ST in serum was increased (P<.01) for hormone
treatments compared with controls (Figure 11). Treatment with rbST
increased (P<.01) concentration Of ST to a greater than did rbST treatment
at d 1. Since the long-term effects Of rbST increased ST to a lesser extent
than rbGRF treatment, the acute increase of ST for rbST compared to rbGRF
is not likely dose related. The reason for the greater increase in concentration
of ST in serum at d 1 is likely that the ST system in viva had not yet
equilrbrated in response to rbST infusion.

Hormone treatments increased (P<.01) concentration of IGF-I from d
0 to 3 compared with controls (Figure 12). These result are consistent with
the literature since an increase in the concentration of IGF-I in serum is
characteristic within the first few days of rbST administration to dairy cows
(Davis et al., 1987). The linear response for serum IGF-I between rbST and
rbGRF treatment was different (P< .01). The slope for concentration of IGF-I
in serum at d 1 rose more rapidly for rbST- compared to rbGRF-treated cows.
The faster response by rbST treatment can likely be attributed to the greater

concentration Of ST in serum at d 1.

 

63

Hormone treatments increased (P<.01) concentration of IGFBP-3 from d
0 to 3 compared to controls (Figure 13). Consequently, these greater
concentration Of IGFBP-3 in serum can be attributed to the elevated
concentration of IGF-I in serum since it has been reported that IGF-I
- mediates much Of the ST-associated increase in IGFBP-3 levels (Zapf et al.,
1989. Also, similar to the IGF-I response, concentration Of IGFBP-3 rose
faster in rbST-compared to rbGRF-treated cows. The linear response for
IGFBP-3 between rbST and rbGRF treatment was different (P=.03). Thus,
the more rapid increase in concentration Of IGF-I may, in turn, be attributed
to the rapid rise in concentration of IGF-I from d 0 to 3. The linear response
for the concentration of IGFBP-2 between rbST and erRF treatment was not

different (P>.65) (Figure 14).

 

 

 

 

30 _—

Cla P<.01

25 3' C2b P<.01
Serum :
ST, 20 _—
ng/ml
15 _-
l0 _—
5 3-

Con rbST rbGRF

Figure 11. Least squares mean concentration of ST in serum for d l of treatment
of cows receiving no treatment (n=10), 29 mg rbST/d (n=10) or 12 mg rbGRF/d
(n=10) for 63 d of lactation (d 118 to 181 postpartum). Pooled standard error Of
the mean among treatments was 1.6 ng/ml.

a Orthogonal contrast of means for control vs. average of rbST- and rbGRF-
treated cows.

b Orthogonal contrast of means for rbST treatment vs. rbGRF treatment.

65

 

 

1000 —
: -0- Con
800 :- -a- rbST
Serum I -I- rbGRF
IGF-I, 600 _-
400 —
200 }- CILa P<.01
; C2Lb P<.01
0 I l l

 

 

0 1 2 3

Time Of treatment, (I

Figure 12. Least squares mean concentration of IGF-I in serum of cows receiveing
no treatment (n=10), 29 mg rbST/d (n=10) Of 12 mg rbGRF/d (n-10). Each point
represents the average of a treatment group at d 0, l, 2 and 3. Standard error of the
mean among treatments was 49 ng/ml.

a Orthogonal contrast of control vs. average of rbST- and rbGRF- treated cows for
linear contrast of means at d 0 to 3.

b Orthogonal contrast of rbST treatment vs. rbGRF treatment for linear contrast of
means at d 0 to 3.

66

160 C1L3P<.0l
140 C2Lb :03

Serum 120
IGFBP-3, 100
densitometric 80

 

   

 

 

 

40 -a- rbST
20 -I- rbGRF
0 l l I I
0 1 2 3

Time Of treatment, (I

Figure 13. Least squares mean concentration of IGFBP-3 in serum of cows
receiveing no treatment (n=10), 29 mg rbST/d (n=10) of 12 mg rbGRF/d(n-10).
Each point represents the average of a treatment group at d 0, 1, 2 and 3. Standard
error Of the mean among treatments was 9.6 densitometric units.

a Orthogonal contrast of control vs. average Of rbST- and rbGRF- treated cows for
linear contrast of means at d 0 to 3.

b Orthogonal contrast Of rbST treatment vs. rbGRF treatment for linear contrast Of
means at d 0 to 3.

1 00

Serum 80

IGFBP-2, 60
densitometric

unrts 40

20

0

-

 

67

 

CILa P>.18
C2Lb P>.65

-0- Con
-B- rbST
-- rbGRF

Time Of treatment, (I

Figure 14. Least squares concentration of IGFBP-2 in serum of cows receiveing
no treatment (n=10), 29 mg rbST/d (n=10) of 12 mg rbGRF/d (n-lO). Each point
represents the average of a treatment group at d 0, 1, 2 and 3. Standard error of the
mean among treatments was 5.5 densitometric units

a Orthogonal contrast of control vs. average Of rbST- and rbGRF- treated cows for
linear contrast of means at d 0 to 3.

b Orthogonal contrast of rbST treatment vs. rbGRF treatment for linear contrast of

means at d 0 to 3.

 

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