Agriculture
Canada

■ * m Agriculture

Canada

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Research Branch
Technical Bulletin 1993-6E

Somatocrinin
in cattle

Canada

w6

Cover illustration

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to improve the long-term competitiveness of the Canadian

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Designed by Research Program Service.

Illustration de la couverture

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®

Somatocrinin
in cattle

H. LAPIERRE, D. PETTTCLERC, and G. PELLETIER

Research Station
Lennoxville, Quebec

Technical Bulletin 1993-6E

Research Branch

Agriculture Canada

1993

Copies of this publication are available from

Director

Research Station

Research Branch, Agriculture Canada

P.O. Box 90, 2000 Route 108 East

Lennoxville, Quebec

JIM 1Z3

©Minister of Supply and Services Canada 1993
Cat. No. A54-8/1993-6E
ISBN 0-662-20664-9

Egalement disponible en francais sous le titre
La somatocrinine chez le bovin

TABLE OF CONTENTS

Page

Acknowledgments iv

Summary v

Resume vi

1. Introduction 1

1.1 The endocrine system ..... 1

1.2 Physiology of lactation 1

1. 3 Hormones 2

2 . Somatotropin 3

2.1 Somatotropin in the body 4

2.2 Methods of increasing blood concentrations

of somatotropin 4

2.3 Somatotropin and lactation 5

3 . Somatocrinin in cattle . 6

3.1 Effect on somatotropin secretion 6

3.2 Effect on milk production 7

3.3 Effect on the development of the mammary gland 12

3.4 Effect on growth and metabolism 12

4. Conclusion 16

Bibliography 17

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of all those who
participated in the experiments discussed in this document, particularly the
staff of the dairy cattle section of the Lennoxville Research Station for
their excellent animal care, the technicians for their enormous enthusiasm in
the collection and painstaking analysis of thousands of blood samples, the
graduate students who assisted with the project and, finally, Drs P. Brazeau,
P. Dubreuil, P. Gaudreau, Y. Couture and J. Morisset for the opportunity to
work with them. In addition, the authors wish to express their gratitude to S
Gagne-Giguere for assistance in revising the document and to L. Cote for its
typing and preparation.

IV

SUMMARY

Somatocrinin (or growth hormone -re leasing factor, GRF) is the
hypothalamic factor which stimulates the secretion of somatotropin (ST, or
growth hormone, GH) by the pituitary gland. Since the early 1980s, when it
became possible to produce ST through the use of recombinant DNA technology, a
number of studies have demonstrated its value in improving the animal
performance of cattle. GRF was first sequenced in the early 1980s. Shortly
thereafter, the Lennoxville Research Station developed a method involving the
administration of exogenous GRF to increase endogenous secretion of ST as a
mean of improving animal performance. This document describes the progress
achieved to date by researchers at Lennoxville working on GRF in cattle since
its characterization in 1982. First, a number of experiments were conducted to
determine the optimal route and dose of GRF required to produce significant
increases in ST concentrations. The next step was to determine whether the
increase in ST concentrations following the administration of GRF was
sufficient to increase milk production. The initial 10-day studies on milk
production showed increased yields of 15%, or 2 to 3 kg of milk per day. These
initial studies were conducted using the original GRF molecule, which contains
44 amino acids. Research on rats had shown that the fragment containing the
first 29 amino acids had the same biological activity as the parent molecule.
The GRF(1-29)NH2 fragment was therefore selected for study in cattle. In cows,
the (1-29)NH2 fragment was as potent as (1-44)NH2 on ST secretion and milk
production. Therefore, in an effort to reduce the doses required and to
increase ST concentrations still further, an analogue containing three amino
acid substitutions was used: it proved to be 16 times more potent than
GRF(1-29)NH2 in stimulating ST secretion and milk production. Finally, if it
is to be of commercial value, GRF must induce ST secretion over extended
treatment periods. Studies involving the administration of GRF for 60 or 182
days showed that it maintained its effect on ST secretion and increased milk
production.

In growing cattle, GRF increased dietary digestibility and nitrogen
retention. It did not, however, affect the animal's total energy retention;
instead, it diverted this energy towards protein and away from fat. In
heifers, GRF increased the volume of parenchymal epithelial tissue in the
mammary gland.

In little more than 10 years, GRF has advanced from the stage of
biochemical characterization to a demonstrated potential for use in improving
animal performance of cattle. Further research will be required to determine
the exact mechanisms by which it works and the various signals involved in the
metabolic coordination permitting these increases in production efficiency.

RESUME

La somatocrinine (GRF) est le facteur hypothalamique qui stimule la
secretion de la somatotrophine (ST ou hormone de croissance) par l'hypophyse.
Depuis le debut des annees 80 ou il est devenu possible de fabriquer la ST par
ADN recombinant, plusieurs experiences ont demontre que son utilisation permet
d'augmenter les performances zootechniques des bovins . Au debut des annees 80,
la sequence du GRF a ete caracterisee . A la Station de recherches de
Lennoxville, nous avons alors developpe l'approche utilisant l'apport exogene
de GRF afin d'augmenter les secretions endogenes de ST comme moyen d'ameliorer
les performances des animaux. Le present bulletin decrit le cheminement que le
groupe de scientif iques de Lennoxville oeuvrant sur le GRF chez le bovin a
suivi, depuis que le GRF a ete caracterise en 1982 jusqu'a maintenant.
Quelques experiences ont tout d'abord ete realisees afin de determiner la voie
et la dose de GRF a administrer afin d'augmenter de facon significative les
concentrations de ST. II a ensuite fallu determiner si 1' augmentation de ST
suite a 1 'administration de GRF etait suffisante pour augmenter la production
laitiere. Les premieres experiences sur la production laitiere, d'une duree de
10 jours, ont entraine des augmentations de production de l'ordre de 15 %,
soit de 2 a 3 kg de lait par jour. Ces premieres experiences ont ete
effectuees avec la molecule originale de GRF qui contient 44 acides amines.
Des travaux chez le rat avaient demontre que le fragment contenant les 29
premiers acides amines etait biologiquement aussi actif que la molecule mere.
Le fragment GRF(1-29)NH2 a ainsi ete etudie chez le bovin. Chez la vache , le
fragment (1-29)NH2 a ete aussi puissant que le (1-44)NH2 sur la secretion de
ST et sur la production laitiere. Aussi, afin de diminuer les doses utilisees
et d'augmenter encore davantage les concentrations de ST, un analogue
comportant trois substitutions d'acides amines a ete utilise: il a ete 16 fois
plus puissant que le GRF(1-29)NH2 pour stimuler la secretion de ST et la
production laitiere. Finalement, pour avoir un interet pratique, le GRF doit
pouvoir stimuler la secretion de ST pendant de longues periodes de traitement.
Le GRF administre pendant 60 ou 182 jours a maintenu son effet sur la
secretion de ST et a augmente la production laitiere.

Chez le bovin en croissance, le GRF a augmente la digestibilite de la
ration et la retention azotee . II n'a cependant pas influence l'energie totale
retenue par 1 'animal mais a modifie la repartition de cette energie vers les
proteines au detriment des lipides. Chez la genisse, le GRF a augmente le
volume du tissu epithelial parenchymateux de la glande mammaire.

Ainsi, depuis a peine dix ans , le GRF est passe de la caracterisation
biochimique a la demonstration de son potentiel d 'utilisation pour ameliorer
les performances zootechniques des bovins. II reste encore a preciser ses
mecanismes d'action et les differents signaux qui participent a la
coordination metabolique permettant ces augmentations de l'efficacite de la
production.

VI

1. INTRODUCTION

USE OF SOMATOCRININ - PHYSIOLOGICAL BASIS

The regulation of body functions is controlled by the nervous and
endocrine systems. In general, the nervous system serves to regulate those
functions requiring rapid adjustment, while the endocrine system controls
long-term processes, such as growth, reproduction and lactation.

1.1 THE ENDOCRINE SYSTEM

The endocrine system consists of a variety of glands located in
different parts of the body and specializing in the production of hormones. By
definition, hormones are chemical agents synthesized in specific parts of the
body, usually specialized glands producing internal secretions. Following
synthesis, the hormones are transported by the blood to the specific target
organs or tissues on which they act. Specific receptors on each target cell
ensure that each hormone acts in a very specific manner. The receptors of the
protein hormones, such as prolactin and somatotropin, are located on the
external surface of the cell membrane. Once formed, the hormone -receptor
complex becomes a single entity responsible at each binding site for the
biological actions characteristic of the hormone, such as cell multiplication,
organelle formation, synthesis of DNA, RNA or proteins and protein
phosphorylation .

A certain functional coordination exists between the nervous system and
the endocrine system. This function is performed by the neuroendocrine system.
The central organ of the neuroendocrine system is the part of the brain known
as the hypothalamus, which acts directly on the anterior and posterior
pituitary. The neurons of the hypothalamus at the base of the brain synthesize
stimulatory or inhibitory hormone factors. Some of these factors are then
released into the hypothalamo-pituitary portal circulation which supplies the
anterior pituitary, thus regulating its hormone secretion to the peripheral
circulation. For example, secretions of somatotropin (or growth hormone) are
controlled primarily by two hypothalamic factors: somatostatin, which inhibits
the release of somatotropin by the somatotrophic cells of the pituitary gland,
and somatocrinin, which stimulates it. We shall return to this subject later
in the text.

1.2 PHYSIOLOGY OF LACTATION

A cow's milk production potential is dependent on the development of its
mammary gland (mammogenesis) , the milk- synthesizing capacity of the secretory
cells (lactogenesis) and the cow's ability to sustain an existing milk flow
(galactopoiesis) . Numerous hormones are involved in the control of mammary
development in ruminants. The most significant are prolactin and somatotropin
secreted by the pituitary gland, estrogens and progesterone secreted by the
ovaries and the lactogenic hormone produced by the placenta. Estrogens and
somatotropin stimulate the growth of the lacteal ducts, while progesterone and

prolactin promote the growth of lobulo-alveolar tissue. Without the pituitary
hormones, steroids are completely ineffective. The corticosteroids secreted by
the adrenal gland increase the potential effects of these mammogenic hormones.
However, the mammary development produced by a combination of all these
hormones is comparable only to that of mid-gestation. It is the lactogenic
placental hormone which is believed to stimulate the maximal development of
the mammary gland in the second half of gestation. This action on the part of
the lactogenic placental hormone is believed to be due to biological
properties similar to those of prolactin and somatotropin.

Lactogenesis is the process of cell differentiation by which the
alveolar cells of the mammary gland acquire the ability to synthesize milk.
The term lactogenic describes the factors responsible for the initiation of
milk secretion in late gestation and at parturition. The minimum hormonal
requirements for lactogenesis are increased secretions of prolactin,
corticotropin, which stimulate secretion of the glucocorticoids, and
estrogens, and a relative absence of progesterone.

The secretion of milk, or lactation, involves the intracellular
synthesis of the components of milk in the alveolar epithelial cells and their
subsequent passage from the cellular cytoplasm to the alveolar luminal space.
The milk is then discharged from the mammary gland during lactation or
milking. Continued lactation, or galactopoiesis , requires maintenance of the
number and synthetic activity of the alveolar cells, together with the
efficiency of the milk ejection reflex. The hormones required to maintain the
number and activity of the alveolar cells are prolactin, somatotropin, the
glucocorticoids, the thyroid hormones, insulin and the parathyroid hormones.
Oxytocin, in turn, is essential for milk ejection.

1 . 3 HORMONES

The word hormone has an unfortunate connotation among consumers. It is
associated with hormone -treated chicken, doping in amateur sport, sex, etc.
Hormone-treated chicken is merely an expression, since chickens today are not
given hormones to stimulate their growth, folk wisdom to the contrary.
Hormones are not alone in having a poor reputation among consumers. They are
followed, in decreasing order, by antibiotics, residues and pathogenic
organisms as the factors most feared by consumers in their food. When
researchers are asked the same question, the order of priority is reversed:
pathogens are at the top of the list, with hormones a distant last.

In fact, in all mammals, including man and all domestic species,
hormones are merely organic chemical substances synthesized by specialized
cells within the body, whose role is to coordinate and regulate cell activity
Without hormones, there would be no body growth, no milk production ... no
life.

2. SOMATOTROPIN

Somatotropin (ST), also known as growth hormone, is a natural protein
present endogenously in all animal species, including man. The secretion of ST
by the pituitary into the bloodstream is regulated by the brain
(hypothalamus), which releases two control factors: somatocrinin or growth
hormone-releasing factor (GRF) , which stimulates the secretion of ST, and
somatostatin, which inhibits it (figure 1). GRF acts specifically on ST, while
somatostatin influences the secretion of a number of hormones. Somatostatin, a
powerful inhibitor of ST secretion, was first discovered by Krulich and
collaborators (1968) and subsequently characterized by Brazeau and
collaborators (1973). Guillemin, Brazeau et al. (1982) used tumours of the
human pancreas to isolate and sequence three molecules showing GRF activity:
GRF(l-37)OH, GRF(1-40)OH and GRF(1-44)NH2. In 1984, Guillemin and
collaborators indicated that the GRF from human hypothalamus cells was
identical to the GRF(1-44)NH2 molecule isolated from tumours of pancreatic
cells of human origin (Guillemin et al. 1984).

Cattle brain, sagittal section

Fig. 1

Regulation of somatotropin secretion

2.1 SOMATOTROPIN IN THE BODY

ST plays a vital role In ensuring that the nutrients absorbed are
efficiently used for muscular growth in the young animal or for milk
production in the cow.

ST can be visualized as the manager of a processing plant (the dairy
cow) who must ensure that inputs (feed) are directed to a processing unit
(mammary gland) at the optimum rate to maximize production (milk) while
maintaining or making judicious use of stocks (body reserves) during periods
of supply deficit (beginning of lactation). The manager's drive, however, on
which the plant's productivity is directly dependent, is influenced by the
board of directors, particularly two powerful members, GRF and somatostatin.

Animals with greater potential for growth and milk production have, in
fact, higher blood concentrations of ST during growth and lactation than
animals with low genetic potential. Blood concentrations of ST are high when
lactation begins and show a marked decline from the beginning to the end of
lactation as milk production declines.

Results obtained in basic research thus suggested the idea that ST
supplements might improve lactation performance in dairy cows.

2.2 METHODS OF INCREASING BLOOD CONCENTRATIONS OF SOMATOTROPIN

Blood concentrations of ST can be increased in five ways: (1) by genetic
selection, which permits slight gradual increases in milk production, and thus
ST, over a period of years; (2) by the administration of exogenous ST; (3) by
stimulation of endogenous secretion of ST through the administration of
exogenous GRF; (4) by vaccination against the inhibiting effect of
somatostatin; or (5) by insertion in the genetic code of additional copies of
the genes responsible for the synthesis of GRF or ST.

Bovine ST, a protein consisting of 190 or 191 amino acids in a sequence
specific to the species, is currently produced by genetic engineering, like
insulin, a hormone used in the therapeutic treatment of human diabetes. GRF, a
much smaller molecule, can be produced in the laboratory by simple chemical
synthesis. In recent years, the administration of exogenous ST or GRF has been
tested intensively in lactating cows. Research is still continuing and these
two molecules are not yet commercially available in Canada. Research on
vaccination against somatostatin or manipulation of the genetic code is still
in its infancy. The researchers at the Lennoxville Research Station have
chosen to work on the GRF and somatostatin approaches.

2.3 SOMATOTROPIN AND LACTATION

Numerous studies over the past 50 years have demonstrated that bovine ST (bST)
has a galactopoietic effect; that is, it can amplify an existing milk
production. Azimov and Krouze (1937) were the first to stimulate milk
production in dairy cows through the administration of a crude anterior
pituitary extract in physiological saline. Dairy cows receiving subcutaneous
(s.c.) injections of these extracts every two days for a period of three weeks
produced 20% more milk (Folley and Young 1945). However, the galactopoietic
effect of the pituitary extract, initially attributed to prolactin (PRL) , was
due instead to its ST content (Brumby and Hancock 1955; Cotes et al. 1949;
Hutton 1957). In 1973, Machlin clearly demonstrated that a preparation of bST
relatively free of PRL and thyrotropin increased milk production by 187. and
consumption by 19% when injected every day for a period of 10 days. He also
observed an increase in milk production of 5 kg per cow per day (35%) in cows
receiving daily injections of a preparation of ST for eight weeks. However,
the limited supply of bST available until very recently precluded the
possibility of commercial applications of bST to improve the milk production
of dairy cows.

The recent discovery of the recombinant DNA technique has made it
possible to synthesize large quantities of recombinantly-derived bST . In a
long-term study (84 to 272 days of lactation), Dale Bauman of Cornell
University studied the effect of pituitary bST and recombinantly-derived bST
on milk production and feed intake of high producing dairy cows (Bauman et al .
1985). Methionyl-bST increased milk production from 27.9 kg/day (controls) to
34.4, 38.0 and 39.4 kg/day in cows receiving daily s.c. injections of 13.5,
27.0 and 40.5 mg per injection, respectively. The increase in milk production
varied from 23 to 41%, with no change in milk composition; unstimulated
anticipated milk yield of these cows, based on pre-treatment estimates, was
over 9600 kg. Milk production following injections of pituitary bST was lower
than that produced by the administration of an equal dose of recombinantly-
derived bST. The biological basis for this difference is not clear, according
to Bauman et al. (1985), and remains unexplained at present. The cows
receiving bST injections were able to maintain their physical condition over
the entire treatment period, and increased their feed consumption to sustain
the higher milk production. In addition, the cows treated with bST converted
feed to milk more efficiently (8 to 24%), as indicated by the higher ratio of
the number of kilograms of milk corrected for milk fat per megacalorie of feed
consumed. On the whole, these results are extremely impressive. While there is
certainly a plateau on milk production, there is no indication that it has yet
been reached in dairy cows. In general, however, subsequent studies showed
that treatment with bST increased milk yield by an average of 3 to 5 kg per
day (review: Chilliard 1988).

3. SOMATOCRININ IN CATTLE

Since somatocrinin (GRF) increases endogenous secretions of somatotropin
(ST), the biological effects of GRF should be similar to those of ST.

3.1 EFFECT ON SOMATOTROPIN SECRETION

It has been noted that the original molecule of GRF was discovered in
man (Guillemin et al. 1982). The first step was to determine whether human (h)
GRF(1-44)NH2 was biologically active in cattle, since it was not until two
years later that bovine GRF was sequenced (Guillemin et al. 1984). First of
all, experiments were conducted to determine the optimal dose, frequency and
route of injection and the nature of the peptide required to produce a
satisfactory biological response in lactating cows (Petitclerc et al. 1985).

In an initial experiment, various doses of hGRF(l-44)NH2 were compared
for the ST secretion response. Nine Holstein cows between 30 and 60 days of
lactation received an intravenous (i.v.) bolus of 0.5 mg of hGRF(l-44)NH2 (0.2
nmol/kg body weight) .

One week later, the same cows received an i.v. bolus of 2.0 mg of
hGRF(l-44)NH2 (0.8 nmol/kg). With the 0.2 nmol/kg dose, serum ST levels peaked
at 10.8 ng/mL 120 minutes following the injection of hGRF(l-44)NH2. Similarly,
the injection of 0 . 8 nmol/kg raised serum ST levels to 13.4 ng/mL 180 minutes
following the injection. The area under the ST curve, however, was different
for the two doses (table 1) .

Table 1 Areas under the ST curve (ng.min/mL) following injection of
hGRF(l-44)NH2 in dairy cows

Experiment No Dose (/ig/kg body weight)

Intravenous Intramuscular

injection injection

1 560 1250

2 523

3 735 (10:00 a.m.)

468 (4:00 p.m.)

1 The 1 MgAg dose of hGRF(l-44)NH2 represents 0.2 nmol/kg body weight

We can thus conclude that the higher dose of 0.8 nmol/kg, representing 4
fig of GRF(l-44)NH2/kg body weight, permits greater secretion of ST than the 1
/ig/kg dose after an i.v. injection.

The second experiment was designed to determine whether intramuscular
(i.m.) injections of hGRF(l-44)NH2 could increase ST secretion as well. Nine
Holstein cows between 150 and 175 days of lactation received a saline solution
or i.m. bolus of 5 mg of hGRF(l-44)NH2 (1.8 nmol/kg body weight). Serum ST
concentration peaked at 10.5 ng/mL 15 minutes following the injection. The
area under the bST curve was 523 ng.min/mL (table 1). It should be noted that
the quantity of ST secreted with the i.m. dose of 9 fig/kg is similar to that
secreted with an i.v. injection of 1 /ig/kg; in other words, an i.m. dose nine
times larger than the i.v. dose produced the same ST response.

3.2 EFFECT ON MILK PRODUCTION

The third experiment was designed to determine whether the increase in
ST obtained following the administration of GRF could increase milk
production. Fifteen cows at an average of 186 days of lactation received 1
/ig/kg i.v. injections of saline or GRF(1-44)NH2 twice a day, at 10:00 a.m. and
4:00 p.m., for 10 consecutive days. ST concentrations rose at both injection
times, from 6.4 to 12.9 and 9.6 ng/mL, respectively (table 1). The cows' milk
production averaged 16.6 kg/day for the last five days of the injection
period. The administration of GRF(1-44)NH2 permitted a marginal increase in
milk production of 4.8% after correction for persistency of lactation
(Lapierre et al. 1985).

At the time, the difficulty involved in obtaining sufficient quantities
of hGRF(l-44)NH2 to perform the tests on dairy cows represented a major
problem, since hGRF(l-44)NH2 was very costly and very rare. A search therefore
began for biologically active molecules which would be less expensive to
produce. As a result, it was demonstrated that a fragment of GRF,
hGRF(l-29)NH2, had the same biological activity as hGRF(l-44)NH2 on ST
secretion in growing pigs and heifers (Petitclerc et al. 1987). The subsequent
study was designed to compare the effect of hGRF(l-44)NH2 and a fragment,
hGRF(l-29)NH2, on milk production (Pelletier et al. 1987). The GRF was
administered for 10 days, six times a day, at a dose of 0.2 nmol/kg. The cows
used were at an average of 158 days of lactation. The administration of
hGRF(l-44)NH2 or hGRF(l-29)NH2 produced increases in milk production of 16.6
and 12.4%, respectively (table 2).

Table 2 Comparison of intravenous injections of hGRF(l-44)NH2 and

hGRF(l-29)NH2 in doses of 0 . 2 nmol/kg six times a day for 10
consecutive days in dairy cows

Variable

Treatment-

Control

hGRF(l-44)NH2 hGRF(l-29)NH2

Milk production
(kg/day)

Consumption of dry
matter (kg/day)

Feed conversion
(kg milk/kg dry
matter consumed)

23.4

19.8

1.19

27.3

19.8

1.38

26.3

19.9

1.33

1 The 0.2 nmol/kg dose represents 1 Mg/kg for hGRF(l-44)NH2 and 0.66 /xg/kg
for hGRF(l-29)NH2.

The total yield of fat and protein increased with the hormone treatments
as well. The fat and protein content of the milk also changed slightly in
response to the treatment, since the cows received injections for only ten
days and their metabolism did not have time to adjust. It is known, however,
that prolonged injections of ST in dairy cows do not affect milk composition
(review: Chilliard 1988).

In the dairy cow, hGRF(l-29)NH2 and hGRF(l-44)NH2 demonstrated similar
biological activity. The intrinsic biological activity of hGRF(l-44)NH2 on the
release of ST thus lies in the sequence of the first 29 amino acids of the
peptide. This experiment proved extremely valuable for subsequent studies
since it justified the use of hGRF(l-29)NH2, which was easier to synthesize
and less expensive to produce than hGRF(l-44)NH2. In addition, milk production
increased in this experiment by 15 to 18% , compared to barely 5% in the
previous experiment. It thus appears that twice-daily i.v. injections of
hGRF(l-44)NH2 at a dose of 0.2 nmol/kg body weight do not stimulate ST
secretion enough to produce any substantial increase in milk production. Six
i.v. injections a day, however, would be impractical.

The next step was to determine whether a single daily s.c. injection,
even if it required more GRF than multiple i.v. injections, could produce a
satisfactory biological response. The following experiment was therefore
performed on 12 cows at an average of 209 days of lactation. Each of these
cows received one daily s.c. injection of gelatin or 10 mg of hGRF(l-29)NH2
(18 /ig/kg) in gelatin for 10 days. The results were conclusive. The milk
production of the treated cows increased by 3 kg/day (14.3%) for the last five
days of treatment, with no change in milk fat or protein composition. Maximum

8

ST concentrations following injection of GRF averaged 34.1 ng/mL, compared to
2.9 ng/mL for the control cows (Lapierre et al. 1988a). This study
demonstrated that a single daily injection of GRF could stimulate the
endogenous secretion of ST enough to increase milk production.

Until this point, all the studies conducted using GRF at the Lennoxville
Research Station had involved treatment periods of ten days. The next step was
to determine whether long-term treatment was possible. Would continuous
stimulation by the administration of exogenous GRF lead to desensitization of
the pituitary gland? Previous studies with GRF did not suggest any such
possibility, but the answer to this question held the key to future
development of this technology in the area of animal production. The next
experiment was therefore designed to determine the effect of GRF treatment for
two months on 17 cows averaging 252 days of lactation at the beginning of the
treatment. On completion of the treatment period, the cows were dried off. A
preliminary test was conducted to determine whether the dose used in the
preceding experiment produced optimal ST secretion. Doses of 5, 10 or 20 /ig/kg
produced statistically similar increases in ST concentrations (Lapierre et al.
1988b). Because the results produced by the 5 fig/rag dose were numerically
slightly lower, however, the 10 /ig/kg dose was selected.

For a period of 56 days, the cows received daily s.c. injections of
saline or 10 /ig/kg of hGRF(l-29)NH2. After two months of treatment, the
maximum concentration of ST achieved following the injection of GRF was higher
than on the first injection, at 46.8 compared to 25.2 ng/mL respectively,
while the maximum concentrations achieved in the control cows averaged 3 . 6
ng/mL. In addition, at the end of the treatment period, all the cows received
an injection of GRF (1 /ig/kg i.v.) to determine clearly whether two months of
treatment had influenced the cows' ability to respond to GRF injections. The
cows which had received no previous injections of GRF produced maximum
concentrations slightly lower than those which had received daily injections
of GRF for two months, at 21.8 compared to 32.2 ng/mL; the areas under the ST
response curve, however, showed no difference (Lapierre et al. 1988b). In
terms of milk yield following this treatment, treatment with GRF led to a less
rapid decline in production at the end of lactation, resulting in a mean
increase of 1.9 kg/day over the treatment period, with the control cows
producing 13.9 kg/day over their 60 days of lactation. On the subsequent
lactation, calf weight and milk production were recorded. GRF treatment at the
end of the previous lactation did not affect these variables (Lapierre et al.
1988c) . This experiment clearly demonstrated that long-term treatment with GRF
was possible with no risk of pituitary desensitization, since the cows
responded just as well after two months of injections as on the initial
administration, if not better.

As mentioned previously, endogenous secretion of ST following the
injection of GRF reaches a plateau at high doses of GRF. This means that,
above a certain dose of GRF, 10 /ig/kg s.c. in the dairy cow, the quantity of
ST secreted will not increase even if the quantity of GRF administered is
increased. It had been demonstrated that hGRF(l-44)NH2 was degraded in one
minute to hGRF(3-44)NH2, a peptide with only 1/100 the activity of the parent
molecule (Frohman et al.1986). Substitution of amino acids could produce an
analogue resistant to the peptide degradation of amino acids 1-3. As a result,

some study was given to the substitution of amino acids to improve the helical
amphiphilic structure of GRF . An analogue of hGRF(l-29)NH2 containing three
substitutions was then synthesized (Felix et al. 1986). First of all, tyrosine
and alanine, in positions 1 and 2 respectively, were replaced by a
desamino- tyrosine and a D-alanine to reduce the peptide's sensitivity to
peptidases. Secondly, the glycine in position 15 was replaced by an alanine,
to permit better insertion in the hydrophobic region of the molecule. The use
of a GRF analogue permitted more effective stimulation of endogenous ST
secretion. It remained to be determined how this analogue would behave in the
dairy cow and whether increases in ST secretion beyond those previously
obtained would be reflected by corresponding increases in milk production.

The next step was to determine the dose of the analogue to be used. We
compared the ST response at different doses of hGRF(l-29)NH2 (3.3 and
10 /ig/kg) and at different analogue doses (0.37, 1.11 and 3.33 /ig/kg). The
responses to the different GRFs are shown in figure 2. By deduction, an
analogue dose of 0.6 /ig/kg should produce endogenous secretion of ST similar
to the administration of 10 /ig/kg of hGRF(l-29)NH2. This dose was therefore
selected for the next experiment on milk production, together with a dose
three times as large (1.8 /ig/kg) , and compared with our usual dose, 10 /ig/kg
of hGRF(l-29)NH2. For ten days, twenty-four cows received daily s.c.
injections of saline, hGRF(l-29)NH2 (10 /ig/kg) or aT* analogue containing three
substitutions (0.6 or 1.8 /ig/kg; n = 6 per treatment). The ST concentrations
in response to the different GRF treatments were similar, with mean maximum
concentrations of 40.1 ng/mL, compared to maximum concentrations of 3.3 ng/mL
in the control cows (figure 3). It should be noted, however, that even after
eight hours of blood sampling, ST concentrations in the cows receiving the
highest dose of the analogue had not returned to the basal level. Milk yields
increased by 1.8, 2.2 and 3.1 kg/day in response to hGRF(l-29)NH2 and GRF
analogue in doses of 0.6 /ig/kg and 1.8 /ig/kg respectively, with the control
cows producing 18.8 kg/day (figure 3). The increases obtained in response to
treatment with hGRF and with the lower dose of the analogue were similar,
while the higher dose of the analogue resulted in a further increase in milk
production (Lapierre et al. 1990a). This experiment indicates that the
analogue with the three substitutions generates the same increase in dairy
production as hGRF(l-29)NH2, but with 1/16 the dose.

10

6-,

ST (ng.min/mL) ('000)

-•- ANA

-+- hGRF(1-29)NH2

0.37 1.11
Dose of GRF (ug/kg)

3.33

10.0

Fig. 2 Effect of different doses of somatocrinin (hGRF(l-29)NH2) or an

analogue (ANA) on the secretion of somatotropin (ST) in dairy cows

In a final experiment, cows received long-term treatment for a period of
182 days, from 120 days of lactation to drying off. They received daily
injections of hGRF(l-29)NH2 (10 /ig/kg, s.c). GRF increased milk production by
9.57. and feed efficiency by 6.1% with no change in milk composition (Lacasse
et al. 1991a). The ST response to GRF injections persisted and even increased
over the injection period (Lacasse et al. 1991b).

In summary, following a demonstration of hGRF(l-44)NH2 activity in dairy
cattle, it was established that the fragment containing the first 29 amino
acids of the original molecule was equipotent to the parent molecule on ST
secretion and milk production. In addition, the effect of GRF on ST secretion
and, indirectly, on milk production, persisted over long-term treatment
periods varying from 60 to 182 days. Finally, synthesis of a GRF analogue
containing substitutions of amino acids to improve the half-life and structure
of the GRF permitted the use of much smaller doses of peptides and even larger
increases in milk production. GRF has thus been shown to be a tool with the
potential to increase milk yield and feed efficiency in dairy cows.

11

ST (ng.mln/ml_) ('000)

Milk (kg/day)

Control GRMOug/kg Ana-0.6 jig/kg Ana-l.8ug/kg

Fig. 3 Effect of somatocrlnin (GRF) or an analogue (ANA) on the secretion of
somatotropin (ST) and milk production in dairy cows

3.3 EFFECT ON THE DEVELOPMENT OF THE MAMMARY GLAND

The weight or volume of parenchymal epithelial tissue in the mammary
gland can be increased by 30 to 45% by injecting prepubertal heifers with
bovine ST (Sejrsen et al. 1986; Sandles and Peel 1987) or with GRF to increase
ST concentrations (Ringuet et ale 1989). These treatments are accompanied by a
significant reduction in the quantity of adipose tissue in the mammary gland.
Milk production in heifers receiving this treatment does not increase,
according to Sandles and Peel (1987); in this study, however, the heifers
growth over the treatment period was very low (less than 400 g/day) . Sejrsen,
on the other hand, reports an increase in milk production of approximately 8%
in heifers with feeding levels permitting growth rates of 700 or over 1000 g
per day (personal communication) .

3.4

EFFECT ON GROWTH AND METABOLISM

ST brings about much more spectacular biological responses in lactating
cows than in growing cattle. In general, ST treatment increases nitrogen
retention (Moseley et al. 1982, 1987; Crooker et al. 1990) by diverting energy
towards protein and away from fat (Eisemann et al. 1986). Except in one
experiment (Moseley et al. 1982), dietary digestibility was not affected.

12

The effect of GRF on growing cattle was studied first in grain-fed
calves. A grain-fed calf is a young bovine which receives milk-replacer for
approximately six weeks and is then weaned; it receives cereal-based
concentrates and protein supplements ad libitum from the age of two weeks
until slaughter. After weaning, 30 male dairy calves with a mean live weight
of 70 kg received two s.c. injections a day of saline or hGRF(l-29)NH2, at a
dose of 5 /ig/kg per injection, until slaughter, at a live weight of 220 kg
(123 days of treatment). The ST response to GRF injections declined with age.
This decline was not due to the treatment with GRF since, after 87 days of
treatment, each animal received an injection of GRF and the control animals
showed exactly the same response as those which had been previously treated
(Lapierre et al. 1990b). In contrast to the observations noted with ST,
dietary digestibility increased with GRF treatment. Nitrogen retention also
increased slightly. This increase in nitrogen retention was not reflected,
however, in a change in body composition or an increase in the animals'
growth. The feed efficiency of animals treated with GRF and those which were
untreated was identical (Lapierre et al. 1991). GRF treatment of young growing
cattle, while it increased the quantity of nitrogen retained by the animal,
did not affect their growth performance or body composition.

In a second experiment on growing cattle, we studied the effect of GRF
on energy and splanchnic metabolism (gastrointestinal system and liver) . Beef
steers, weighing an average of 339 kg, received two daily s.c. injections of
saline or hGRF(l-29)NH2 (10 /xg/kg per injection) and one of two feeding
treatments (a moderate quantity just above maintenance level and a level 1.8
times as high); treatment continued for 21 days. Catheters were inserted in
the portal and hepatic veins and caudal artery to permit simultaneous blood
sampling at these three sites, and blood flow was measured by an infusion of
para-aminohippuric acid.

Treatment with GRF increased nitrogen retention by 108% in the animals
receiving the moderate feed level and by 83% in those receiving the high feed
level. As observed earlier, GRF increased dietary digestibility. The energy
required for maintenance was not affected by GRF. The efficiency with which
the animal uses the metabolizable energy for retention in its tissues did not
change. The amount of energy retained by the animals was not affected by GRF
but the partitioning of this energy changed significantly, with energy being
diverted towards protein and away from fat (figure 4; Lapierre et al. 1992).
Catabolism of amino acids and production of urea by the liver, like the
urinary excretion of nitrogen, were reduced by GRF treatment (Reynolds et al.
1992) . This experiment clearly indicates the coordination between the
different organs in responding to the metabolic changes brought about by
treatment with GRF.

13

Energy retained (Meal/day)

Control GRF

moderate

Control GRF

high

Fig. 4 Effect of somatocrinin on energy retention in the tissues and on
energy partitioning in growing steers fed two different intake
levels, moderate or higher

It has been noted that the secretion of ST is controlled by two
hypothalamic factors: GRF, which stimulates the secretion of ST, and
somatostatin, which inhibits it. The studies presented above involved the use
of GRF to increase endogenous secretions of ST and thus animal performance.
The degree of improvement in performance appears to be related to the level of
the increase in ST as a result of GRF injections.

However, the injection of GRF, which acts as an accelerator, does not
eliminate somatostatin, which acts as a brake. The following hypothesis was
therefore proposed. If the brake (somatostatin) could be released and the
accelerator (GRF) activated, ST secretion might increase even more in response
to lower doses of GRF. In addition, inhibition of the brake alone might be
enough to increase ST secretion. Studies by other investigators indicate that
this may be a promising approach. The basic principle of immunoneutralization
of somatostatin is similar to that of vaccination against disease, except that
the antibodies which the animal produces are directed against endogenous

14

somatostatin rather than a virus or bacteria.

An experiment was conducted on grain-fed calves to determine the effect
of GRF infusion and active immunization against somatostatin on animal growth,
carcass quality, weight of the digestive organs and hormone secretion.
Thirty-two male calves were divided into two uniform groups, with half being
immunized against human alpha-globulin and the other half against somatostatin
conjugated with human alpha-globulin, one week after weaning (79 kg). Since
somatostatin is a small molecule, it must be combined with an antigen to
encourage the formation of antibodies. Boosters were administered on days 14,
28, 56 and 73 of the experimental period. The calves immunized against
somatostatin developed significantly more antibodies specifically against
somatostatin than the control calves. Between days 79 and 111, half of the
calves from each of the immunization groups were infused with hGRF(l-29)NH2 at
a dose of 3.33 ng.min/kg of body weight. The animals were slaughtered at days
119 and 128 to obtain data on carcass quality and the weight of various
organs .

The desired results were not achieved: immunization against somatostatin
reduced the concentrations of ST (8.3 compared to 16.1 ng/mL) and insulin-like
growth factor 1 (126.0 compared to 150.9 ng/mL). Two possible explanations
were proposed. First, the clearance speed of these hormones may have increased
as a result of immunization against somatostatin, thus reducing blood
concentrations, as Kenison (1987) has suggested. Another possibility is that
immunization against somatostatin led to the development of antiidiotypes,
substances acting as antibodies against the antibodies which inhibit
somatostatin. Their biological effect could even be similar to that of
somatostatin (image-like antiidiotypes), which inhibits the secretion of ST.
As in earlier studies, GRF treatment caused an increase in blood
concentrations of ST and insulin-like growth factor 1 (Roy et al. 1989).

The effect of the hormone treatments on the calves' performance is
related to the concentrations of ST and insulin-like growth factor 1. In fact,
GRF increased the daily gain in body weight, while immunization against
somatostatin reduced it. Carcass composition and the weight of various organs
(reticulo-rumen, omasum, abomasum, duodenum, small and large intestines,
lungs, kidneys, spleen, thymus and testicles) were not affected by the
treatments, with the exception of liver and pancreas weight, which decreased
following immunization against somatostatin (Roy et al. 1990).

This experiment demonstrated that GRF specifically increases the
activity of pancreatic amylase (Roy et al. 1991). This action may have a
beneficial effect on the digestion of starch in ruminants.

GRF thus influences the metabolism of growing cattle by increasing the
quantity of energy retained in the form of protein without affecting total
energy retention. In addition, GRF increased dietary digestibility in two
experiments and did not affect it when infused. This effect, which differs
from that of ST, requires further study. Immunization against somatostatin did
not prove, at least initially as promising.

15

4. CONCLUSION

We at the Lennoxvllle Research Station have chosen to concentrate on the
use of somatocrinin to increase somatotropin concentrations in order to
improve production efficiency in growing or lactating cattle. Somatocrinin was
first characterized barely ten years ago. In this technical document, we have
described the progress which we have made in this short time, moving from the
newly discovered molecule to a final demonstration of its potential.

The mechanisms involved in the increased milk production in dairy cows
and nitrogen retention in growing cattle following treatment with somatocrinin
have not yet been fully explained. However, it is becoming increasingly clear
that the entire organism is implicated in this metabolic change: the various
organs or tissues act together to divert nutrients towards sites of increased
demand and to reduce the use of these nutrients by other sites. The
somatotropic axis appears to play a leading role in the orchestration of the
metabolism for high producing animals. The insulin-like growth factor 1, which
show higher blood concentrations following GRF treatment (Abribat et al. 1990;
Lapierre et al. 1990a, 1991, 1992b), but which have now been shown to display
paracrine activity as well, complete the series of events associated with the
somatotropic axis. However, their exact mode of action and the effect of their
binding proteins require further clarification. It also remains to be
determined whether the effect of somatocrinin in increasing dietary
digestibility is indeed real and, if so, why it acts differently from
somatotropin in this context. Research is still required for a better
understanding of the mechanisms involved in the metabolic coordination
required of animals to increase their bioenergetic efficiency.

16

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20

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