ABSTRACT
MAMMARY ARTERIAL AND VENOUS CONCENTRATIONS

OF PROLACTIN, GROWTH HORMONE AND INSULIN
IN LACTATING COWS

BY
Neil F. G. Beck

Serum prolactin concentrations were determined in
mammary arterial and venous blood samples from six early
and six late lactating cows. In early lactators, serum
prolactin concentrations increased from an average of
28 ng/ml 30 min before milking to 48 ng/ml 0 to 20 min
after milking, then declined to 32 ng/ml 30 to 70 min post
milking. During these same periods in late lactators,
serum prolactin concentrations were lower (P < .05),
averaging 9, l7 and 14 ng/ml. Arteriovenous differences
_in serum prolactin concentrations averaged .20 ng/ml before
milking and .70 ng/ml after milking. Serum insulin con-
centrations in these samples remained constant, averaging
2.6 ng/ml in early and 3.5 ng/ml in late lactating cows
(P < .1). Arteriovenous differences in serum insulin
concentrations were different from zero (P < .05) in early
(.11 ng/ml) and late (.16 ng/ml) lactating cows between 0

and 20 min after milking.

Neil F. G. Beck

Serum prolactin concentrations were measured in
mammary arterial and venous blood samples before and after
intravenous administration of 200 ug thyrotropin releasing
hormone (TRH) followed by milking 30 min later. In early
lactators, serum prolactin concentrations averaged 29 for
30 min before and 175 ng/ml for 30 min after TRH. Between
0 and 20 min after milking serum prolactin concentrations
averaged 243 ng/ml. In six late lactators, serum pro-
lactin concentrations tended to be lower, averaging 13,
79, and 77 ng/ml during the same periods. Arteriovenous
differences in serum prolactin concentrations increased
from .4 to 7.7 ng/ml after TRH in early and from -.01 to
2.6 ng/ml in late lactating cows. Arteriovenous differ-
ences after TRH were different from zero (P < .05).
Growth hormone concentrations, determined in the same
samples, averaged 3.9 ng/ml for 30 min before TRH in early
and late lactating cows. During the 30 min interval after
TRH serum growth hormone concentrations increased to 14.9
in early and 11.4 ng/ml in late lactating cows. Between
0 and 20 min after milking, serum growth hormone averaged
7.3 in early and 5.1 ng/ml in late lactating cows.
Arteriovenous differences in serum growth hormone increased
from -.03 to .58 ng/ml after TRH in early and from -.05
to .73 ng/ml in late lactating cows (P < .05).

Five early lactating cows were treated with CB-154

(2-bromo-ergocryptine-methanosulphate) and five late

Neil F. G. Beck

lactating cows were treated with ergocryptine. Serum pro-
lactin concentrations decreased within 24 hr from 22.9 and
11.9 to 7.2 in early and 1.7 in late lactating cows. When
the ergot treated cows were injected during early lactation
with 500 ug TRH serum prolactin concentrations increased
from 9.3 ng/ml 30 min before, to 113 ng/ml for 0 to 20 min
after TRH; thereafter, declining to 90 ng/ml 30 to 70 min
post injection. Serum prolactin concentrations in late
lactation during the same periods were lower (P < .05),
averaging 2.0, 20.7 and 12.9 ng/ml. Arteriovenous dif-
ferences in serum prolactin concentrations increased from
-.14 to 6.3 ng/ml after TRH in early lactators and from
-.18 to -.24 ng/ml in late lactators. Serum growth
hormone concentrations averaged 5 ng/ml before, 20.3 ng/ml
0 to 20 min after TRH, and 7.4 ng/ml 30 to 70 min after
TRH in early lactating cows. In late lactating cows,
serum growth hormone concentrations were reduced to

(P < .05) 3.5, 12.8 and 5.2 ng/ml during the same periods.
Arteriovenous differences in serum growth hormone increased
from .08 ng/ml before to .6 ng/ml after TRH in early and
late lactating cows.

Prolactin concentrations in skim milk samples col-
lected from early and late lactating cows before adminis-
tration of TRH or ergot drugs averaged 24.6 and 11.4 ng/ml,
respectively. Administration of either 200 or 500 ug TRH

30 to 70 min before milking did not affect prolactin

Neil F. G. Beck

concentrations in skim milk. However, treatment of early
lactating cows with CB-154, and late lactating cows with
ergocryptine reduced skim milk prolactin to 11.9 and 7.0
ng/ml, respectively. Intramammary injection of 50 mg pro-
lactin or saline did not increase mammary venous prolactin
concentrations in cows treated with ergot drugs.

Within a stage of lactation, high ambient tempera-
tures (> 18 C) compared with low ambient temperatures
(< 18 C) were associated with greater serum prolactin con-
centrations, before and after milking or injecting TRH.

I concluded that greater intensity of lactation in
cows is associated with greater serum prolactin concen-
trations. Also, greater mammary uptake of prolactin and
growth hormone occurred at intervals when these hormones

were acutely increased in serum.

MAMMARY ARTERIAL AND VENOUS CONCENTRATIONS
OF PROLACTIN, GROWTH HORMONE AND INSULIN

IN LACTATING COWS

BY

L"
\<‘

I
‘ I-

Neil F? Gf Beck

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Dairy Science

1977

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
Drs. C. A. Lassiter and H. D. Hafs and the Department of
Dairy Science for providing the funds and facilities for
his graduate studies. He expresses a particular debt of
gratitude to his major professor, Dr. H. Allen Tucker,
for all the help and assistance throughout his graduate
program. Appreciation is also expressed for the assis-
tance and advice of his committee members, Drs. S. D.
Aust, J. Meites, R. S. Emery and ex officio member E. M.
Convey. A final debt of thanks is extended to Dr. W. D.
Oxender for performing the surgeries and Dr. R. Neitzel

for his computer programming.

ii

TABLE OF CONTENTS

LIST OF TABLES O O O O I O O O O O O O 0 O O 0

LIST OF FIGURES O O O O O O ' O O O O O O O O 0

INTRODUCTION 0 O O O O O O O O O O O O O O O 0

REVIEW OF LITERATURE . . . . . . . . . . . . .

A.

C.

Hormonal Requirements of the Lactating
Mammary Gland in Established Lactation

1. The Role of Prolactin in Established

Lactation . . . . . . . . . . .
2. The Role of Growth Hormone in

Established Lactation . . . .
3. The Role of Insulin in Established

Lactation . . . . . . . . . . . .

Mammary Binding of Prolactin, Insulin,
and Growth Hormone In‘Vitro and Its
Subsequent Effects . . . . . . . . . .
Application of the Arteriovenous
Difference Technique to Hormone
Utilization . . . . . . . . . . . . .

Summary . . . . . . . . . . . . . . . . .

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

A.
B.

D.

Animals . . . . . . . . . . . . .
Surgical Implantation of Pudic Artery
and Subcutaneous Abdominal Vein

Cannulae . . . . . . . . . . . . . . .

l. Cannulation of the Pudic Artery .
2. Cannulation of the Isolateral Sub-
cutaneous Abdominal Vein . . . . .

Common Experimental Protocol . . . . .
Hormone Assays . . . . . . . . . . . .

iii

Page
vi

vii

12
18

21
33
34
36

36

36
37
38

39
40

1. Validation of Prolactin Assay for
Skim Mi 1k 0 O O O O O O O O O O O

E. Experimental Objectives and Design . .

Experiment 1. Arterial and Venous
Concentrations of Prolactin and
Insulin During Milking . . . . . .

Experiment 2. Arterial and Venous
Concentrations of Prolactin and
Growth Hormone After the
Administration of TRH Prior
to Milking . . . . . . . . . . . .

Experiment 3. Arterial and Venous
Concentrations of Prolactin
After Milking in Cows Treated
With Ergocryptine . . . . . . . .

Experiment 4. Arterial and Venous
Concentrations of Prolactin and
Growth Hormone After Adminis-
tration of TRH in Cows Treated
With Ergocryptine . . . . . . . .

Experiment 5. Milk Concentrations of
Prolactin . . . . . . . . . . . .

Experiment 6. Serum Concentrations of
Prolactin in the Mammary Vein
Following Intramammary Infusion
of Prolactin . . . . . . . . . . .

Experiment 7. The Effect of Ambient
Temperature on Serum Prolactin
Concentrations Before and After
Milking or TRH . . . . . . . . . .

F. Statistical Methods . . . . . . . . .

RESULTS 0 O O O O O O O O O O I O O O I O O 0

Experiment 1. Arterial and Venous Concen-
trations of Prolactin and Insulin

During Milking . . . . . . . . . . . .

A. Prolactin . . . . . . . . . . . .
B. Insulin O O O O O O O I O O O O 0

Experiment 2. Arterial and Venous Concen-
trations of Prolactin and Growth
Hormone After the Administration
of TRH Prior to Milking . . . . . . .

iv

Page

41
42

42

43

44

4s
47

47

49

50

52

52

52
56

60

A. Prolactin . . . . . . . . . . . .
B. Growth Hormone . . . . . . . . . .

Experiment 3. Arterial and Venous Concen-
trations of Prolactin After Milking in
Cows Treated with Ergocryptine . . . .

Experiment 4. Arterial and Venous Concen-
trations of Prolactin and Growth
Hormone After Administration of
TRH in Cows Treated with
Ergocryptine . . . . . . . . . . . . .

A. Prolactin . . . . . . . . . . . .
B. Growth Hormone . . . . . . . . . .

Experiment 5. Milk Concentrations of
Prolactin . . . . . . . . . . . . . .

Experiment 6. Serum Concentration of Pro-
lactin in the Mammary Vein Following
Intramammary Infusion of Prolactin . .

Experiment 7. The Effect of Ambient Temp-
erature on Serum Prolactin Concen-
trations Before and After Milking
or TRH . . . . . . . . . . . . . . . .

DISCUSSION . . . . . . . . . . . . . . . .
SUMMARY AND CONCLUSIONS . . . . . . . . .

BIBLIOGMPHY O O O O O O O O O O O O O O O

Page

60
64

68

73
73
77
80

83

86
95
119
126

Table

LIST OF TABLES
Page

Milk weights and prolactin concentrations
in skim milk samples obtained from the
AM and PM milking of early and late
lactating cows throughout Experiments
1 to 4 . . . . . . . . . . . . . . . . . . . . 81

Arterial and venous serum prolactin con-
centrations before and after milking
at high and low ambient temperatures
in early and late lactating cows . . . . . . . 88

Arterial and venous serum prolactin con-
centrations before and after 200 ug
TRH, followed by milking at high and
low ambient temperatures in early
and late lactating cows . . . . . . . . . . . 90

Arterial and venous serum prolactin con-
centrations before and after 500 ug
TRH, at high and low ambient tempera-
tures in early lactating cows treated
with 80 mg CB-154 and late lactating
cows treated with 80 mg ergocryptine . . . . . 93

vi

LIST OF FIGURES
Figure Page

1. Prolactin concentrations in arterial and
venous serum samples before, during,
and after milking which began at 0
min and lasted 3 to 5 min . . . . . . . . . . 54

2. Insulin concentrations in arterial and
venous serum samples before, during
and after milking which began at 0
min and lasted 3 to 5 min . . . . . . . . . . 58

3. Prolactin concentrations in arterial and
venous serum samples of lactating
cows, before and after the injection
of 200 ug TRH (at -30 min), followed
30 min later by milking which began
at zero min and lasted 3 to 5 min . . . . . . 62

4. Growth hormone concentrations in arterial
and venous serum samples of lactating
cows, before and after the injection
of 200 ug TRH (at -30 min) followed
30 min later by milking which began
at zero min and lasted 3 to 5 min . . . . . . 66

5. Serum prolactin concentrations in arterial
and venous samples collected before,
during and after milking in five early
lactating cows treated with 80 mg
CB-154, 24 hr prior to experimentation
and five late lactating cows treated
with 80 mg ergocryptine, 24 hr prior
to experimentation. The animals were
milked for 3 to 5 min beginning at 0
min . . . . . . . . . . . . . . . . . . . . . 71

6. Prolactin concentrations in arterial and
venous serum samples before and after
injection of 500 ug TRH at 0 min to
five early lactating cows treated
with 80 mg CB-154 and five late
lactating cows treated with 80 mg
ergocryptine . . . . . . . . . . . . . . . . . 75

vii

Figure Page

7. Serum growth hormone concentrations in
arterial and venous serum samples
before and after injection of 500
pg TRH at 0 min to five early
lactating cows treated with 80 mg
CB-154 and five late lactating
cows treated with 80 mg ergocryptine . . . . . 79

8. Serum prolactin concentrations in
venous samples collected before and
after intramammary infusion of 50 mg
prolactin or saline at time 0 . . . . . . . . 85

viii

INTRODUCTION

Hormonal control of lactation has been investigated
for a number of years. The question of how changes in
availability of certain hormones control lactation has been
approached in a number of ways.

The most direct approach has been simple to remove
the source of origin of a particular hormone from the
lactating animal and observe the affect on milk production.
Then if removal of the hormone decreased milk production,
studies are usually made to determine whether replacement
of the hormone will restore lactation. This approach will
answer the question of whether or not the hormone is
needed by the mammary gland. However, it will not answer
the question, whether in the normal physiological situ-
ation, if variation in availability of a hormone to the
lactating mammary gland will result in a corresponding
change in milk production.

Initially, the only way to monitor changes in
hormone concentrations in givg was bioassays. However,
these assays were usually only sufficiently sensitive to
measure hormone concentrations at the endocrine gland

where the hormone was produced. Therefore, one could only

relate Changes in concentration of a hormone, within
endocrine glands with changes in lactational performance.
However, concentration of a hormone within an endocrine
gland is dependent upon at least two variables, i.e.,
synthesis and secretion rates, and drawing conclusions
from such studies was difficult.

With the advent of radioimmunoassays, it became
possible to determine very precisely hormone concentrations
in blood. Changes in serum hormone concentration in blood
could be related to changes in lactational performance.
Therefore, if serum concentrations of a hormone was
statistically related to milk production, one might infer
that this hormone is importantly associated with lactation.

In the past few years, it has been demonstrated
that protein hormones physically bind to their target organ
$2.!iEEQ' The mammary gland of the cow is a large target
organ for several hormones. We wished to determine
whether variation in serum concentration of prolactin,
growth hormone, and insulin, three polypeptide hormones
implicated in the control of lactation, was associated
with parallel changes in mammary uptake of these hormones
£g_gi!g. Furthermore, we wished to evaluate whether the
uptake of these hormones was effected by stage of lacta-
tion, a variable that importantly affects quantity of

milk produced.

REVIEW OF LITERATURE

A. Hormonal Requirements of the Lactating
Mammary Gland in Estéblished Lactation

 

 

It is‘now well established that the mammary gland
requires hormonal stimulation in order to function (Cowie
and Tindal, 1971; Convey, 1974; Meites, 1974; Tucker,
1974). The question of which hormones are required has
been approached in three major ways: (1) whether absence
of a certain hormone affects lactation; (2) following its
absence will subsequent replacement of the hormone restore
previous lactational performance; and (3) will increased
availability of the hormone alter lactational performance.
In spite of species variation, it has been generally
established that prolactin, growth hormone, thyroid
stimulating hormone (TSH) or thyroid hormones, adenocortico-
trophic hormone (ACTH) or glucocorticoids, oxytocin, and
to a lesser extent insulin and ovarian steroids all influ-
ence lactation. Since this research investigates the
Change in and mammary uptake of prolactin, growth hormone
and insulin during established lactation, these hormones
will be discussed in detail, initially by describing
their normal variation in their endocrine organ source,
in blood, and then how lactational performance is

3

affected when they are administered or removed from a

lactating animal.

1. The Roleiof Prolactin in
Established Lactation

 

 

In 1937, Reece and Turner observed that pituitaries
of lactating cows contained more prolactin than those
from nonlactating animals, and pituitaries from lactating
dairy cattle contained more prolactin than those from
lactating beef cows. More recently the use of radio-
immunoassays has established that the "milking" or "suck-
ing" stimulus temporarily increases blood serum prolactin
concentrations in rats (Amenomori et al., 1968), goats
(Bryant et al., 1968; Hart, 1975a), sheep (Lamming et al.,
1974), and cows (Johke, 1969 and 1970; Tucker, 1971;
Schams, 1972a; Koprowski and Tucker, 1973a). In cattle,
increased serum prolactin concentrations are observed
within 5 minutes of milking (Johke, 1969 and 1970; Schams
and Karg, 1970; Schams, 1972a; Koprowski and Tucker, 1973a).
Koprowski and Tucker (1973a) determined that serum pro-
lactin concentrations post milking do not return to their
pre-milking values for approximately 35 minutes. The
half life of serum prolactin in lactating cows is approxi-
mately 25 to 29 minutes (Johke, 1969; Schams and Karg,
1970; Tucker et al., 1972). Johke (1970) and Koprowski
and Tucker (1973a) observed that the amount of prolactin

released from the pituitary at milking declined with

advancing stage of lactation, but Schams (1972a) maintained
that the effects of stage of lactation on prolactin

release from the pituitary were minor compared with those
of season.

Schams and Karg (1970) previously reported that
blood serum prolactin concentrations were related more to
month of year than to stage of lactation or milk yield.
The effect of season on serum prolactin concentrations was
confirmed by Koprowski and Tucker (1973a) who observed
that basal concentrations of prolactin were higher in
summer than in winter. However, greater quantities of
prolactin were released in response to milking in winter
than in summer. Effect of season on prolactin concen-
trations are primarily associated with Changing day length
(Bourne and Tucker, 1975) and ambient temperature
(Wettemann and Tucker, 1974).

Koprowski and Tucker (1973a), observed that
within-stage-of-lactation correlations between serum
prolactin and milk yield were very low and sometimes
negative during the first 3 to 6 months of lactation.
However, during the remainder of lactation, correlations
were consistently positive and ranged from .08 to .48.
Average monthly milk production and average serum pro-
lactin concentrations measured immediately after milking
were highly correlated (.96). These results suggest

prolactin plays a role in milk production in cattle (but

it is not necessarily a direct, cause-effect type relation-
ship).

In addition to lactation, other physiological
states influence pituitary and blood serum prolactin con-
centrations in the bovine. After birth, pituitary pro-
lactin content of heifers increases, reaching a maximum at
nine months of age (Sinha and Tucker, 1969). These Changes
were associated with Changes in mammary development.

Sinha and Tucker (1969) observed increased pituitary pro-
lactin concentrations in cattle around estrus. However,
reports on changes in blood serum concentrations during
the estrous cycle in cattle differ. Swanson et a1. (1972)
observed increased blood serum prolactin concentration
around estrus in nonlactating heifers, whereas Koprowski
and Tucker (1973a) and Wettemann and Hafs (1971) were
unable to detect any differences in serum prolactin con-
centrations attributable to changes in the estrous cycle
in lactating cows and heifers. Serum prolactin concen-
trations, in blood samples collected one hour before and
one hour after milking, were significantly greater in
pregnant than in nonpregnant lactating cows; however,
serum prolactin concentrations measured immediately after
milking were no different in pregnant compared with non-
pregnant lactating cows (Koprowski and Tucker, 1973a).
Wettemann and Hafs (1971) were unable to detect any

significant Changes in serum prolactin concentration

during early pregnancy in cattle, but increased serum
prolactin (Oxender et al., 1972) and pituitary prolactin
concentrations (Reece and Turner, 1937) have been observed
in cattle in late pregnancy. Marked increases in serum
prolactin concentrations, possibly associated with the
initiation of lactation, occur just prior to parturition
in cattle (Schams and Karg, 1970; Johke et al., 1971;
Ingalls et al., 1973).

Serum prolactin concentrations in lactating cattle
are affected by various other stimuli: washing the udder
and venipuncture (Tucker, 1971), washing the brisket
(Koprowski et al., 1971) and feeding (Johke, 1970; McAtee
and Trenkle, 1971). Stress increases blood serum pro-
lactin concentrations in rats (Krulich et al., 1974;
Riegle and Meites, 1976). Advancing stage of lactation
and season of the year affect prolactin release in goats as
in cows (Hart, 1975a). Serum prolactin concentrations
were greater post partum than prepartum period in ewes
(Arai and Lee, 1967; MacNeilly, 1971). Lactating ewes
had greater pituitary secretion rates, metabolic Clearance
rates, and blood serum concentrations of prolactin than
pregnant, anestrus, or ovariectomized animals (Davis and
Borger, 1973). Threfall et a1. (1974) observed that in
suckled lactating sows pituitary and blood serum prolactin

concentrations were approximately four times greater

than those of suckled agalactic animals at the same stage
post partum.

The author is unaware of any studies in which
hypophysectomy has been used as a means of removing pro-
lactin from lactating cattle. In goats surgically hypo-
physectomized during lactation, milk yield falls to
essentially zero (Cowie, 1964). Ovine prolactin alone, or
in combination with adrenal corticoids, or bovine growth
hormone in combination with glucocorticoids only partially
restored milk yield. However, a combination of ovine
prolactin, bovine growth hormone and adrenal glucocorti-
coids caused a substantial restoration of milk yield.

When ovine prolactin, bovine growth.hormone and adrenal
glucocorticoids were given in combination with tri-
iodothyronine, milk yield was fully restored to pre-
hypophysectomy levels. In hypophysectomized rabbits, milk
yield may be restored by prolactin alone (Cowie et al.,
1969). A combination of prolactin, growth hormone, and
glucocorticoids will partially restore milk yield in
hypophysectomized rats (Lyons, 1958).

Ergot alkaloids and their derivatives markedly
decrease serum prolactin concentrations in rats (Nagasawa
and Meites, 1970), sheep (Niswender, 1972), goats (Hart,
1973; McMurtry and Malven, 1974a), cows (Karg et al.,
1972; Fell et al., 1974; Smith et al., 1974), and women

(Brun del Re et al., 1973). Cows treated with CB-154

(2-bromo-a-ergocryptine-methanosulphate) in late pregnancy
and early lactation had depressed milk yields (Fell et al.,
1974; Karg and Schams, 1974). Although Schams and Karg
(1974) claimed to have observed Similar effects in cows
treated with CB—154 in established lactation, Smith et a1.
(1974) using a much larger number of experimental animals
were unable to detect any significant change in milk
yield, even though serum prolactin concentrations were
markedly decreased. Similarly, the administration of CB-154
to goats had no effect on milk yield (Hart, 1973). However,
in this species, CB-154 increases the quantity of growth
hormone released at milking, which may compensate for the
comparative absence of prolactin. In lactating rats
injected with ergot alkaloids, Shaar and Clemens (1972)
observed decreases in mammary weight and depressed litter
weight gains. Since CB-154 may also depress food intake,
Tomogane et a1. (1975) restricted food intake of the
control animals to that of the treated animals. Neverthe-
less litter weight gains of treated animals were still
less than those of controls. Administration of CB-154 to
rabbits (Taylor and Peaker, 1975) and women (Brun del Re
et al., 1973) caused an abrupt cessation of lactation.
In summary, CB-154 is a potent inhibitor of prolactin
and, except in ruminants, of lactation.

Since the availability of endogenous hormone may

be a limiting factor during established lactation,

10

exogenous hormone has been administered in an attempt to
enhance lactation. The effect of administration of ovine
prolactin to early lactating rats is dose dependent
(Tucker, 1974). Daily injections of one milligram of

ovine prolactin had no effect on litter weight gains but

3 mg per day did stimulate milk secretion, possibly through
increasing metabolism.h Neither injections of sheep pro-
lactin nor prolactin from isotransplanted pituitaries

will maintain litter weight gains in rats during extended
lactation (Tucker, 1974). Injections of prolactin to
lactating cows (Folley and Young, 1940; Cotes et al., 1949),
ewes (Denamur and Martinet, 1970; Morag et al., 1971) and
guinea pigs (Nagasawa and Naito, 1963) were without effect
on milk yields. But injections of ovine prolactin markedly
enhance lactation in rabbits (Cowie, 1969; Taylor and
Peaker, 1975). Hart (1975b) prevented the seasonal decline
in the amount of prolactin released at milking in lactating
goats by artificially extending daily photoperiod for three
months. But milk yields declined in a manner similar to
those of control animals which were exposed to natural
changes in daylength.

Synthetic thyrotropin-releasing hormone, pyro-
glutamyl-histidyl-proline amide (TRH), increases serum
concentrations of prolactin, growth hormone, and thyroxine
in lactating cows (Convey et al., 1973a), and growth

hormone and prolactin in dairy bulls (Convey et al., 1973a;

ll

Tucker et al., 1975) and prepubertal heifers (Vines et
al., 1976). Convey et al. (1973a) observed that in
lactating cows increases in serum prolactin and thyroxine
concentrations, following intravenous administration of
TRH, were not proportional to dose. In contrast growth
hormone increased proportionally to the dose of TRH. But
later Vines et a1. (1976) found that in prepubertal
heifers, the amount of prolactin and growth hormone
released following TRH administration was significantly
related to dose of TRH and baseline concentrations of
serum hormones. It was suggested by Vines et a1. (1976),
as a possible explanation for this discrepancy, that the
greater variation in serum prolactin concentrations in
lactating cows probably masked any dose-response effect.
Since TRH increases serum concentrations of pro-
lactin, growth hormone and thyroxine, three hormones
implicated as being important in lactation, Convey et al.
(1973b) investigated the galactopoietic potential of
this releasing hormone. Forty lactating cows receiving
twice daily injections of 50 ug TRH for 10 days had
significantly greater milk yields (on average 0.66 kg/cow/
day) during the last 5 days of treatment than when they
were injected with saline alone. Neither Kelly et a1.
(1973), nor Schams et a1. (1974) who gave greater doses
Of TRH to fewer animals over a shorter period of time

were able to detect any increase in milk yields. Since

12

TRH caused only a small increment in milk yield (Convey
et al., 1973b), it is not surprising that Kelly et a1.
(1973) and Schams et a1. (1974), using only four late
lactating cows, were unable to detect any change in milk
yield. However, Schams et a1. (1974) claimed that TRH
given during the peripartum period would increase subse-
quent milk production 10 to 50%. Adams et a1. (1973)
failed to stimulate milk production in rats treated with
TRH. In lactating women, Tyson et a1. (1972) were able
to cause breast engorement by injecting TRH intravenously,
but Zarate et a1. (1976) were unable to enhance lactation
in women given 20 mg of TRH orally, three times a day,
despite increased serum prolactin concentrations. Zarate
et a1. (1976) suggested that the difference between their
results and those of Tyson et a1. (1972) may have been
associated with route of administration. In concluding
this section, once TRH increases serum concentrations of
prolactin, growth hormone and thyroxine, its effect on
lactation is probably the result of the galactopoietic
action of all three of these hormones and not just the
single effect of one of them.

2. _The Role oflGrowth Hormone
in Established Lactation

 

Stimuli associated with suckling or milking
increase serum concentrations of growth hormone in rats

(Grosvenor et al., 1968; Tucker and Thatcher, 1968) and

13

goats (Hart and Flux, 1973), but not cows (Reynaert and
Peters, 1972; Koprowski and Tucker, 1973b). Serum con-
centrations of growth hormone decrease significantly
during the first 6 weeks of lactation in cattle (Smith

et al., 1976) and are negatively correlated with milk
yield (Koprowski and Tucker, 1973b). Koprowski and
Tucker (1973b) observed that in pregnant, lactating cows,
serum growth hormone concentrations remained constant
with time. However, the use of covariate analysis showed
that when effects of advancing stage of concurrent preg-
nancy are held constant, serum growth hormone concen-
trations decrease with advancing lactation. Conversely
when effects of advancing stage of lactation are held con-
stant, serum growth hormone concentrations increase with
advancing pregnancy. Vines (1976) observed that in cattle
TRH released significantly greater quantities of growth
hormone during the second month of lactation than at any
other stage. Hart et a1. (1975) observed that serum
growth hormone concentrations of lactating cows were
significantly greater in dairy cattle than in lower milk
yielding beef animals. Furthermore, Fitko et a1. (1969)

found that 131

I growth hormone disappeared at a slower
rate from blood of high producing cows than from blood of
low producing or dry cows. Overall these studies suggest
that greater serum concentrations of growth hormone are

associated with higher milk production in cows.

14

Other physiological states, apart from lactation,
influence pituitary and blood concentrations of growth
hormone in cattle. Pituitary growth hormone content is
greater in prepubertal cattle than in post pubertal cattle
(Armstrong and Hansel, 1956; Purchas et al., 1970).
Plasma growth hormone concentrations in cattle were
greater at birth than between 2 and 12 months of age,
after which time they remained stable (Purchas et al.,
1970). Therefore, growth hormone may be more actively
synthesized and secreted from the pituitaries of pre-
pubertal cattle during the first two months after birth.

Koprowski and Tucker (1973b) observed that serum
growth hormone concentrations were greater during the
estrogenic phase than during the luteal phase of an estrous
cycle in lactating cows. When effects of advancing stage
of lactation are held constant in a covariate analysis,
serum growth hormone concentrations increase linearly
with advancing pregnancy in cows (Koprowski and Tucker,
1973b). However, in unadjusted data, serum growth hor-
mone concentrations remain constant with advancing preg-
nancy in lactating cows (Oxender et al., 1972; Koprowski
and Tucker, 1973b). Serum growth hormone levels remain
constant throughout pregnancy in heifers (Koprowski and
Tucker, 1973b). Ingalls et a1. (1973) observed constant
serum growth hormone concentrations from 26 to 9 days

prepartum. Growth hormone concentrations began to

15

increase at 9 days prepartum and were maximum at parturi-
tion. In contrast, Reynaert et a1. (1976) were only able
to detect increased serum growth hormone concentrations,
in cows and heifers, during the act of parturition. The
cause of this abrupt increase in serum growth hormone
concentrations is debatable. In cattle, serum growth
hormone concentrations are inversely related to free fatty
acid concentrations (Reynaert et al., 1975a). Stress in
cattle causes a decline in blood free fatty acid concen-
trations and finally an increase in growth hormone con-
centration (Shirley et al., 1973; Reynaert et al., 1975b).
However, in cows and heifers, serum free fatty acid con-
centrations began to rise 1 day prepartum and remained
elevated for at least 5 days post partum (Reynaert et al.,
1976). Therefore, it is unlikely that a decrease in serum
free fatty acid concentrations was responsible for the
abrupt increase in serum growth hormone concentrations at
parturition. Reynaert et a1. (1976) suggested that
increased concentrations of prostaglandin in blood at
parturition may cause an increase in growth hormone con-
centrations.

In rats (Lyons, 1958) and goats (Cowie, 1964),
hypophysectomized during lactation, exogenous growth
hormone, in combination with other hormones, helped
restore lactation. Growth hormone is galactopoietic in

ruminants (Cowie, 1961; Meites, 1961). A number of workers

16

have observed increased milk production in response to
growth hormone injections in lactating cows. Hutton (1957)
gave single injections of growth hormone and found a sig-
nificant linear relationship between log weight of hormone
injected and increase in milk volume. Shaw et a1. (1955)
observed appreciable increases in milk production in cows
at different stages of lactation given daily injections of
100 mg growth hormone. A marked increase in milk pro-
duction occurred in twin cattle given subcutaneous
injections of 50 mg of growth hormone for a 12-week period
beginning at the peak of lactation and for a 4-week period
in late lactation (Brumby and Hancock, 1955). It is
unlikely that results of these earlier workers reflect
independent effects of growth hormone since preparations
injected were contaminated with prolactin and thyrotropin.
Therefore, Bullis et al. (1965) compared in cows the
galactopoietic potencies of commercial grade and highly
purified growth hormone preparations. Changes in milk
production during a 10-day injection period were 7.6,

3.4, and -4.4%, respectively, for commercial growth
hormone, highly purified growth hormone, and saline. The
increase in production caused by highly purified growth
hormone was not significantly different from that of the
saline control group. However, milk production in the
cows given highly purified preparation was greater than

that during the pre-injection period. In Contrast,

17

Machlin (1973) gave only half the dose of growth hormone
used by Bullis et a1. (1965) of a highly purified growth
hormone preparation for the same period of time and
obtained an 18% increase in milk production. In these
experiments, Machlin (1973) noted that milk production
continued to increase during the injection period and that
the increased production persisted for two days after the
final injection. Furthermore, the galactopoietic action
of growth hormone did not diminish in long-term experiments
where lactating cows were injected only three times a
week for 10 weeks. Growth hormone is not galactopoietic
in goats (Tomov, 1963), ewes (Denamur and Martinet, 1970)
and women (Lyons et al., 1968). However, conflicting
results have been obtained in rats (Tucker, 1974).

The effect of CB-154 or TRH, alone or in com-
bination, on serum growth hormone concentration varies
with species. In cattle, CB-154 has no effect on serum
growth hormone concentrations (Smith et al., 1974),
however, in goats (Hart, 1974) it increased the amount of
growth hormone released to the milking stimulus. In
normal women, CB-154 decreases growth hormone concen-
trations but in acromegalic women it increases them
(Camanni et al., 1975). In contrast, Benker et a1. (1976)
observed that CB-154 depressed serum growth hormone con-
centration but did not prevent TRH-stimulated release of

growth hormone in humans.

18

Investigations into the effect of TRH on serum
growth hormone concentrations in humans have produced
conflicting results. Anderson et a1. (1971) found no
change, whereas, Torjesen et a1. (1973) obtained increased
serum growth hormone concentrations in response to TRH in
normal humans. In acromegalic humans, Cryder et a1. (1973)
obtained increased serum concentrations of growth hormone
in response to TRH. TRH increases serum growth hormone
concentrations in normal rats (Takahara et al., 1974) and
in hypophysectomized rats bearing ectopic pituitaries
(Udeschinia et al., 1976). In lactating cows, the amount
of pituitary growth hormone released was in proportion to
the dose of TRH used (Convey et al., 1973a). In conclu-
sion, even though growth hormone has a potent galacto-
poietic action in cattle, its serum concentrations remain
remarkably constant throughout lactation.

3. The Role of Insulin in
Established LactatiOn

 

In cattle, serum insulin concentrations increase
during early lactation and are inversely related to serum
growth hormone concentrations (Koprowski and Tucker,
1973b; Hart et al., 1975; Hove, 1975; Smith et al., 1976).
Koprowski and Tucker (1973b) showed that serum insulin
concentrations increased 2 to 3 fold between weeks 4 to 12
of lactation and that blood samples collected during

milking contained greater serum concentrations of insulin

19

than those collected 2 to 4 hours before or one hour after
milking. That the milking stimulus may cause the pancreas
to release insulin is supported by recent evidence that
stimulation of the rat hypothalamus (Curry and Joy, 1974)
or stimulation of the vagus nerve of the dog pancreas
(Bergman and Miller, 1973) cause insulin release. Hart

et a1. (1975), using early lactating beef and dairy cows,
obtained an inverse relationship between serum concen-
trations of insulin and growth hormones, and noted that
insulin and glucose concentrations were significantly
lower in beef as compared with dairy cows. Hart et a1.
(1975) suggested that higher serum concentrations of
insulin and glucose, in the lower producing beef cows,
were associated with lower mammary uptake of glucose.

This hypothesis is consistent with the negative correlation
obtained between serum insulin concentrations and milk
yield obtained by Tucker and Koprowski (1973b). Glucose,
infused intravenously, increases serum insulin concen-
trations in cattle (McAtee and Trenkle, 1971). A signifi-
cant increase in plasma insulin and glucose concentrations
occurs during the first 3 to 5 weeks of lactation in cows
(Smith et al., 1976) and animals which had very low con-
centrations of plasma glucose and insulin in early lac-
tation tended to develop ketosis later (Schwalm and

Schultz, 1976).

20

Hove and Blom (1973) observed increased insulin
and decreased plasma glucose concentrations at feeding in
cows. Bassett (1974) observed, during feeding in sheep,
an increase in plasma insulin and a decrease in plasma
growth hormone concentrations which were unrelated to
changes in blood metabolite concentrations. He suggested
that the changes in insulin and growth hormone concen-
trations were caused by gastrointestinal hormones, reflexly
released at feeding. Hove (1975) and Halse et a1. (1976)
determined growth hormone, insulin and glucose concentra-
tions in blood plasma from lactating cows during the day
when blood concentrations of insulin, growth hormone and
glucose are effected by feeding and during the night when
they are affected to a lesser degree. In plasma samples
collected during the daytime, a negative correlation
existed between plasma insulin and glucose concentrations
but in samples collected during night time the correlation
was positive.

The udder of the lactating cow utilizes 60 to 85%
of glucose entering the blood system (Annison and Linzell,
1964; Kronfeld and Emery, 1970), and it is therefore of
little surprise that hypoglycemia, resulting from adminis-
tration of insulin causes a depression in milk yields in
lactating cows (Cowie, 1961; Kronfeld et al., 1963;

Schmidt, 1966). However, if blood glucose concentrations

21

are maintained constant, infused exogenous insulin stimu-
lates lactation.

The role of insulin in lactation in rats is also
important because surgical removal of the pancreas or
administration of the diabetogenic agent alloxan reduces
litter weight gain in lactating rats (Tucker, 1974).

B. Mammary Binding of Prolactin, Insulin,

and Growth Hormone in Vitro and
Its Subsequent Effects

 

 

 

It is pertinent to consider whether hormonal main-
tenance of lactation is the result of the direct action
of hormones on the mammary gland; the result of indirect
extramammary effects or a combination of both. That hor-
mones have a direct effect on the mammary gland has been
established in studies where the mammary gland is isolated
in an ig_!it£g culture system (Juergens et al., 1957;
Wood et al., 1975). However, this does not dismiss the
possibility that extramammary effects, e.g., stimulation
of other target organs or increased blood metabolite con-
centrations, are involved. Evidence that prolactin,
growth hormone and insulin bind specifically to target
organs other than the mammary gland will be presented at
the beginning of this section. Later, mammary binding of
prolactin, growth hormone and insulin in vitrg, will be
considered, and finally metabolic stimulation of mammary
cells by protein hormones and excretion of prolactin in

milk will be discussed.

22

Recently prolactin, growth hormone, and insulin
have been found to bind specifically to tissues other than
the mammary gland. For example, 1251 labelled prolactin,
growth hormone, and insulin, bind specifically to liver,
adrenal and heart tissue from many species (Kelly et al.,
1974; Posner et al., 1974; Aragona and Friesen, 1975;
Donatsh and Richardson, 1975; Marshall et al., 1975; Saito
and Saxena, 1975; Rolland and Hammond, 1975; Posner,
1976). Liver membranes from a number of species specific-

ally bind 125

I labelled prolactin, growth hormone and
insulin (Kelly et al., 1974; Posner, 1976; Gelato et al.,
1975). The quantity of those hormones specifically bound
to liver varied with physiological state. Membranes from
fetal and immature female rat livers bound much less human
growth hormone and ovine prolactin than those of adult
females (Kelly et al., 1974). Furthermore, Specific
binding of human growth hormone and ovine prolactin
increased markedly at puberty and pregnancy. In male rats,
Kelly et a1. (1974) observed that the liver membranes of
adults bound more human growth hormone and ovine pro-
lactin than liver membranes from fetal or immature animals.
Quantitatively, the liver membranes of male rats specific-
ally bound less hormone than liver membranes from female
rats, irrespective of stage of development. Specific

125

binding of I labelled insulin to liver membranes was

quantitatively identical in both sexes and at all stages

23

of development (Kelly et al., 1974). Gelato et a1. (1975)
observed that prolactin binding capacity is depressed in
liver membranes from thyroidectomized, ovariectomized or
thyroidectomized-ovariectomized female rats compared with
those of intact controls, and hormone therapy with
thyroxine or estrogen alone or in combination will restore
the specific binding capacity of prolactin to control
values.
125I-labelled prolactin, growth hormone and
insulin binding to liver membranes has also been investi-
gated in rabbits, guinea pigs and mice. Kelly et a1.
(1974) observed that liver membranes from fetal or immature
rabbits bound significantly less human growth hormone and
ovine prolactin than liver membranes from mature female
adults. In contrast, liver membranes from pregnant
rabbits bound significantly more human growth hormone and
ovine prolactin than liver membranes from mature females.
Unlike rats, rabbit liver membranes from both sexes bound
equal amounts of ovine prolactin and human growth hormone.
The amount of 1251 insulin which binds to rabbit liver
membranes is constant for all stages of development.
Liver membranes from guinea pigs did not bind significant
amounts of human growth hormone or ovine prolactin, but
did bind a significant quantity of insulin. Liver mem-
125

branes from pregnant animals bound significantly more I

insulin than liver membranes from animals in other

24

physiological states. Posner (1976) Observed that mouse
liver membranes bound significant quantities of human
growth hormone, ovine prolactin and insulin. Compared with
liver membranes from adult female mice, liver membranes
from fetal and male mice bound less, and liver membranes
from pregnant mice bound more ovine prolactin and human
growth hormone. Furthermore the specific binding of bovine
growth hormone to mouse liver membranes was negligible,
except during pregnancy when liver membranes bound a
significant amount of this hormone. The specific binding

of 125

I labelled insulin to mouse liver membranes was
constant at all stages of development.

Marshall et a1. (1975) investigated the effect of
different physiological conditions on the specific binding
of prolactin to rat kidney membranes. Compared with con-
trol animals, specific binding of prolactin to kidney
membranes was unaffected by water deprivation, salt
loading or unilateralnephrectomy. In the same study, both
salt loading and nephrectomy significantly increased the
specific binding of prolactin to adrenal tissue.

Collectively, the above studies suggest not only
that binding of prolactin, growth hormone and insulin to
their various target organs is of physiological signifi-
cance (binding changes with physiological state), but also

that galactopoietic actions of these three hormones may

involve target organs in addition to the mammary gland.

25

A number of workers have investigated protein
hormone binding to receptors in the membrane fraction
isolated from the mammary glands of laboratory species.
However, the author is unaware of similar work in rumi-
nants. Frantz et a1. (1974) isolated prolactin receptors
from mammary glands of pregnant or lactating mice. These

1251_

receptors, specifically bound biologically active
ovine prolactin. Unlabelled ovine prolactin and to a
lesser extent human growth hormone and placental lactogen
competitively displaced labelled prolactin from its

125I-ovine prolactin to the

receptors. Binding of the
receptor occurred over a range of physiological Concen-
trations and took 3 minutes to complete. Compared with
normal receptors, receptors pretreated with trypsin,

bound 60% less 125

I-prolactin, suggesting a component of
the receptor is protein. The number of specific, saturable
binding sites of labelled prolactin was greater in lactating
mammary tissue than in mammary tissue from mid-pregnant
mice, indicating the specific binding of prolactin Changes
with the physiological state of the gland.

Shiu and Friesen (1974) investigated the physical
properties of prolactin receptors from rabbit mammary
gland. These receptors bound human, simian, ovine, bovine,
and murine prolactin, human growth hormone and human

placental lactogen. The binding was dependent on pH,

temperature and the ionic composition of the incubation

26

media. Unlike observations in mice that specific mammary
binding of prolactin was greater in lactation than in
pregnancy (Frantz et al., 1974), binding capacity of
rabbit mammary tissue for prolactin did not change from
pregnancy to lactation. Furthermore, under the same
physical conditions, the time it took for the binding
process to be half completed, was considerably longer in
the rabbit (5 hr) than in the mouse (3 min). Enzymic
pretreatment with either trypsin or phospholipase C
reduced the binding capacity of rabbit mammary receptors
for prolactin. This suggested that the receptor contained
both a protein and a phospholipid component. Holcomb

et a1. (1976) studied specific prolactin binding to rat
mammary tissue. Under optimal physical conditions the
binding process took 3 hr to reach equilibrium. Rat mam-
mary receptors were specific for prolactin and their
binding capacity was completely abolished by pretreatment
with trypsin and reduced 51% by phospholipase C. Specific

binding of 125

I-labelled ovine prolactin to rat mammary
slices was consistently low during pregnancy but increased
3 to 6 fold after parturition. Holcomb et a1. (1976)
maintained that low mammary binding of ovine prolactin
during pregnancy was the result of high serum concen-
trations of placental lactogen "masking" the prolactin

binding sites, since removal of the gravid uterus and

ovaries during pregnancy resulted in a large increase in

27

binding. However, I question this hypothesis since if rat
prolactin and placental lactogen are competing for the
same binding sites, then in vitro the placental lactogen

125I-labelled pro-

should be competitively displaced by
lactin. The only possible way placental lactogen could
"mask” prolactin binding sites is by irreversibly binding
to the prolactin receptor.

Qualitative studies using autoradiography on mice,
rats, and rabbits indicated that biologically active
125I-prolactin binds to the apical membrane of the mammary
cell (Birkinshaw and Falconer, 1972; Rajaniemi et al.,
1974). However, those workers were unable to detect any
hormone inside the cell. Turkington (1970) demonstrated
that prolactin bound to an inert carrier (Sepharose beads)
was as biologically active as free hormone in stimulating
mammary cell metabolism. Since prolactin bound to
Sepharose is unable to enter the cell, this work is evi-
dence for a prolactin receptor located in the surface
membrane of the mammary cell. Collectively, the above
studies support the concept that mammary cells have
specific membrane receptors for prolactin and therefore
the hormone does not need to enter the cell to be active.
However, the mechanism of action for prolactin may not be
exclusively through receptors at the cell surface since

Chomczynski and Topper (1974) demonstrated that placental

lactogen and prolactin stimulated ribonucleic acid

28

synthesis in isolated nuclei from lactating rat and mouse
mammary cells, but were without effect on nuclei from
kidney or liver cells. Insulin failed to stimulate
ribonucleic acid synthesis in any of the isolated nuclei,
which is consistent with evidence that insulin acts via a
cell membrane receptor (Cuatrecassas, 1969). More recently,
Nolin and Witorsch (1976) using a horse radish peroxidase
immunoprecipitation technique discovered the presence of
endogenous immunoreactive prolactin inside rat mammary
epithelial cells during lactation. It would therefore
seem possible that prolactin may not only act through a
cell membrane receptor but also through an intracellular
receptor.

Studies using insulin bound to an inert carrier
(Sepharose) indicate that the mechanism of action of
insulin is through a brief interaction with a membrane
receptor on the surface of the mammary cell (Cuatrecassas,
1969; Oka and Topper, 1972). However, more recently Kalb
et a1. (1975) claimed that insulin dissociated from its
carrier and therefore its mechanism of action may not be
exclusively a surface phenomena. O'Keefe and Cuatrecassas

(1974) investigated 125

I-insulin binding to mammary
epithelial cells from pregnant and lactating mice. Com-
pared with fat cells, mammary cells exhibited very

similar physical binding Characteristics and constants for

125I-insulin. Cells from fat tissue and cells from

29

mammary tissue of pregnant and virgin animals each pos-

sessed approximately 1000 binding sites for insulin. The
number of binding sites per cell increased four fold for
lactating mammary tissue, compared with tissue from preg-

125

nant or virgin mice. I-labelled insulin stimulated

3H-uridine incorporation into nucleic acid and physio-
logical concentrations of unlabelled native hormone

lzsI-labelled

competitively displaced receptor bound
insulin.

The above evidence indicates that protein hormones
bind specifically to receptors located on the surface mem-
brane of mammary cells. The hormone receptor interaction
probably results in a change in the concentration of some
intracellular messenger. For example, Rillema (1975, 1976)
observed a stimulatory effect of prolactin on ribonucleic
acid and subsequent casein synthesis in mouse and rat
mammary explants which could be mimmicked by increasing
tissue concentrations of cyclic guanosine monophosphate
or prostaglandins of the F series. Conversely, the
stimulatory effect of prolactin on cell metabolism could
be blocked by increasing intracellular concentrations of
cyclic adenosine monophosphate or prostaglandins of the A
or E series. Since the effects of prolactin, cyclic

mononucleotides and prostaglandin were nonadditive,

Rillema (1975, 1976) suggested that cyclic mononucleotides

30

and prostaglandins probably act as intracellular messen-
gers in the mechanism of action of prolactin.

Speake et a1. (1975, 1976) showed that insulin,
glucocorticoids and prolactin were essential for syn-
thesis, activity and degradation of the enzyme fatty acid
synthetase in mammary explants from mid-pregnant rabbits.
Prolactin caused a rapid decrease in the rate of degra-
dation of this enzyme. The enzyme lactose synthetase is
also responsive to prolactin. Following hypophysectomy,
there is a four fold decline in lactose synthetase activity
in the mammary gland of the lactating rabbit (Jones and
Cowie, 1972). However, this may be reversed by adminis-
tration of prolactin, but not growth hormone. Prolactin
will normally induce milk secretion in pseudopregnant
rabbits but when administered simultaneously with pro-
gesterone, secretion is blocked and the expected increase
in lactose synthetase activity prevented (Assairi et al.,
1974). However, prolactin-stimulated-RNA-synthesis was
not affected, suggesting that prolactin may stimulate
mammary metabolism by a number of intracellular routes,
one of which is unaffected by progesterone. Zinder et a1.
(1974) observed that in lactating rats, lipoprotein lipase
activity was much greater in mammary than in adipose tissue
and that hypophysectomy of lactating rats caused a marked
decrease in mammary lipOprotein lipase activity and

increased adipose lipoprotein lipase activity. However,

31

treatment of hypophysectomized animals with prolactin
restored mammary lipoprotein lipase activity and decreased
adipose lipoprotein lipase activity. Field and Coore
(1976) showed that insulin and prolactin withdrawal
decreased mammary gland pyruvate dehydrogenase activity
and activity was restored with insulin and prolactin
replacement therapy.

In addition to its effects on nucleic acid syn-
thesis and enzyme systems, prolactin affects milk com-
position by changing mammary epithelium permeability.
Linzell et a1. (1975) observed that exogenous prolactin
restored ion and lactose concentrations in milk, and

14C-sucrose of late

permeability of mammary epithelium to
lactating rabbits to that normally found in early lacta-
tion. Prolactin stimulates sodium transport, and conse-
quently water movement across the cells of the amniotic
membrane (Holt and Perks, 1975), intestine and gall
bladder (Mainoya et al., 1974; Mainoya, 1975).

The fate of the prolactin molecule after it has
reacted with its receptor is as yet unknown. Since its
binding to mammary tissue is competitive, it may be dis-
placed by another prolactin molecule or possibly metab-
olized after binding to liver and kidney cells. In the
lactating animal, one route of excretion of prolactin is

into milk. Concentrations of prolactin, similar to those

in blood, have been determined in milk of rats, goats,

32

cows (MCMurtry and Malven, 1974a; McMurtry and Malven,
1974b; McMurtry et al., 1975), and humans (Gala et al.,
1975). Unless there exists a very rapid transcellular
transport system for prolactin in the mammary gland,
evidence to date indicates that prolactin enters milk,
from the blood stream, via a paracellular route. Since
administration of exogenous ovine prolactin to rats
(McMurtry and Malven, 1974a) or increased endogenous pro-
lactin concentrations in goats (McMurtry and Malven,
1974b) results in a prompt increase in milk prolactin con-
centration. The rapid increase in milk prolactin concen-
trations in response to increased blood prolactin concen-
trations suggests that prolactin enters the milk by simple
diffusion between the mammary cells. It is noteworthy,
however, that milk prolactin concentrations remained high
after serum prolactin concentrations had returned to their
original values (McMurtry and Malven, 1974b; Grosvenor

and Whitworth, 1976). Why the return passage of prolactin
from the milk into the blood is restricted is unknown.

It is possible that the prolactin in milk becomes
physically bound to a milk component or that passage of
prolactin between blood and milk is more than a process of
simple diffusion. Prolactin in milk may be chemically
different from prolactin elsewhere, because Grosvenor and
Whitworth (1976) observed that time of incubation of rat

milk had no effect on endogenous concentrations of

33

immunoreactive prolactin in milk. However, when they
incubated pituitary prolactin or prolactin purified from
pituitary cell culture media with milk, the measurable
prolactin concentrations decreased.

C. Application of the Arteriovenous

Difference Technique to
Hormone Utilizatibn

 

 

 

Arteriovenous difference technique has been used
extensively to measure mammary utilization of blood
metabolites, for example, glucose, amino acids and volatile
fatty acids (see chapters by Annison, Linzell and Mepham
in Falconer, 1970). The same technique has been employed
to determine mammary uptake of steroid hormones in
ruminants (Heap and Linzell, 1966; Paterson and Linzell,
1971; Heap et al., 1975a; Heap et al., 1975b). Essentials

3 labelled hormone was infused

of the technique were that H
intravenously at a rate which maintained its blood concen-
tration constant. When a steady state was obtained, blood
samples were obtained simultaneously from the exteriorized
carotid artery and the subcutaneous abdominal vein (mammary
or milk vein). Mammary uptake of hormone (uCi/min) was
calculated as the product of the arteriovenous difference
in hormone concentration (uCi/litre) and the mammary blood
flow (liters/min). Paterson and Linzell (1971) observed
that mammary tissue extracted 50% of the 3H-cortisol

entering the gland in pregnant and in nonpregnant lactating

goats. A similar study in pregnant and nonpregnant

34

lactating cows (Heap et al., 1975b) gave a range of mammary
extraction of 3H progesterone of 0 to 24% of its arterial
concentration.

Cicmanec and Niswender (1973) attempted to find if
arteriovenous differences existed in gonadotrophin concen-
trations across the ovary of sheep during different repro-
ductive states. Prolactin, luteinizing hormone and
follicle stimulating hormone concentrations were deter-
mined in blood samples obtained simultaneously through
indwelling cannulae in the dorsal aorta and ovarian vein.
No consistent differences were observed between arterial
or venous blood concentrations of gonadotrophins. Burd
et a1. (1976) were unable to detect any uterine uptake
of prolactin, estradiol-l7B or cortisol in ewes under-
going induced or spontaneous labor. However, progesterone
concentrations in blood samples obtained from the uterine
vein were consistently greater than those obtained from

the jugular vein and mammary artery.

Summary

In general, increased or decreased concentrations
of prolactin, growth hormone and insulin in blood should
result in increased or decreased mammary utilization of
those hormones accompanied by appropriate changes in rate
of synthesis and secretion of milk and ultimately milk
yield. However, the literature indicates that increased

or decreased availability of protein hormones in the

35

lactating animal, does not necessarily elicit a mammary
response. This is surprising since following total removal
of many of those hormones, lactation is generally inhibited
and replacement therapy is required to re-establish lac-
tational performance. The specific binding of prolactin,
growth hormone and insulin to mammary tissue suggests a

role for those hormones in galactopoeisis.

MATERIALS AND METHODS

A. Animals

 

A total of 12 lactating, nonpregnant Holstein-
Fresian cows, six of which were in early lactation (5 to
12 weeks) and six of which were in late lactation (37 to
57 weeks), were used in these studies. During the experi-
mental period, the cows were kept in stanchions, with free
access to water and were fed daily 40 lb of corn silage,
10 lb of hay and 1 lb of concentrate per 2.5 lb of milk
produced.

B. Surgical Implantation of Pudic Artery

and Subcutaneous Abdominal
Vein Cannulae

 

 

 

Prior to surgery each cow was deprived of food and
water for 24 hr. On the day of surgery the cow was
restrained on a tilting surgical table, and anesthesia
was induced with 0.3% sodium thyiamylal and 5.0% glycerol
guaiacolate solution given intravenously at a dose rate
of 0.25 ml/kg body weight. The animal was then intubated
and further anesthesized with halothane (4 to 5%) and
oxygen (8 to 10 l/min) until the required surgical plane
was reached. Anesthesia was then maintained at 2 to 3%
halothane and 6 l/min of oxygen throughout surgery.

36

37

l. Cannulation of the
Pudic Artery

 

 

Following surgical preparation of the area, a 10
to 12 cm incision was made over the inguinal region
between the medial thigh and lateral surface of the udder.
The pudic artery was then located by blunt dissection and
the vessel was freed of connective tissue. A purse string
suture was placed in the wall of the pudic artery in the
region where the cannula was to be inserted.

The cannula was constructed from a 110 cm length
of polyvinyl tubing (V-8, Bolab, Inc., Derry, NH) encased
in a outer sheath of polyvinyl tubing (.156" x .25",
Portex Limited, Hythe, Kent, England) 100 cm long; thus,
10 cm of the inner tubing was exposed. At the point
where the 10 cm length of inner tubing left the outer
sheath, a silicone sponge (1 cm x 1 cm x 0.5 cm, Bellco
Glass Inc., Vineland, NJ) was attached with plastic glue
(Woodhill Chemical Sales, Cleveland, OH). The cannula
was filled with heparinized saline (10 units/ml, Sigma
Chemical Co., St. Louis, MO). The vessel was then occluded
by hand with umbilical tape and an incision made in the
center of the region surrounded by the purse string suture.
The exposed 10 cm of inner polyvinyl tubing was pushed
through the incision in the pudic artery wall. The
incision was closed by tightening the purse string suture
and the cannula sutured to the vessel wall through the

silicone sponge. The cannula outside of the vessel was

38

then brought through the peritoneal cavity to the surface
through an incision in the skin of the animal's flank.
The end of the cannula was then stored in a pouch which
was sutured to the animal's flank.

2. Cannulation of the Isolateral
Subcutaneous Abdominal Vein

 

 

Following surgical preparation, a 5 cm incision
was made in the skin on the ventral surface of the sub-
cutaneous abdominal vein at a point approximately 30 cm
from where the vein leaves the front quarter of the udder.
The vessel was exposed by blunt dissection and cannulated
in a manner identical to that described for the pudic
artery. The cannula was implanted subcutaneously between
the vein and the surface of the skin of the flank. The
venous cannula was brought through the same incision as
that for the arterial cannula and stored in the same
pouch. In the event of the subcutaneous abdominal vein
cannula failing to function during the experimental period,
it was replaced with a single wall polyvinyl cannula
(V—10, Bolab, Derry, NH) installed by venipuncture.

Following closure of incisions, each animal was
maintained on oxygen until the pharyngeal reflexes
returned. Immediately following surgery the cannulas were
flushed every 2 hr, for 12 hr, with heparin (10 units/ml).
Thereafter, the cannulas were flushed every 12 hr for 3

days. Antibiotics (Procaine-Penicillin, Pfizer, NY) were

39

injected (20 ml every 12 hr) intramuscularly for 5 days.
Experimentation was delayed until 3 to 5 days post
surgery. Experimentation was begun when milk production

approximated that prior to surgery.

C. Common Experimental Protocol

 

Certain procedures were common to all experiments.
All experiments were performed during the afternoon.

This enabled the experimental animals to be milked at their
normal afternoon milking time of 1500 hr._ The complexity
of the sampling schedule and the number of experiments
meant that we could perform experiments on only one animal
at any given time. Consequently, experiments were per-
formed on different animals during different months of the
year. Therefore, the day, month and the ambient tempera—
ture of each experiment were recorded to enable the data

to be adjusted for variation caused by changing season of
the year.

On any given experimental day the cannulae were
opened 2 hr prior to each experiment and blood samples
collected and discarded every 15 min until the beginning
of each experiment. This procedure accustomed the experi-
mental animal to periodic disturbance and minimized changes
in blood hormone concentrations caused by stress (Tucker,
1971). Between each blood sample the cannulae were
flushed with a sterile anticoagulant solution of 0.85%

sodium chloride containing 3.5% sodium citrate. Between

40

each experiment cannulae were filled with an anticoagulant
solution of 0.85% sodium chloride containing 3.5% sodium
citrate and 50% dextrose. After each experiment, blood
samples were left to coagulate at room temperature (25 C)
for 2 hr, stored in a cold room (5 C) for 24 to 36 hr and
then centrifuged at 2,500 x g for 15 min. Blood serum was
then stored at -20 C prior to hormone assay.

Each animal was milked twice daily throughout the
series of experiments. The weight of milk obtained at
each milking was recorded and a sample of milk representative
of each AM and PM yield collected. Milk samples were
stored at 5 C for approximately 12 hr and then skimmed by
centrifugation at 2,500 x g for 15 min. Skim milk samples

were then stored at -20 C prior to assay for prolactin.

D. Hormone Assays

 

Radioimmunoassays were used to determine serum
hormone concentrations. The assay procedures used for
prolactin (Tucker, 1971; Koprowski and Tucker, 1971),
growth hormone (Purchas et al., 1970), and insulin
(Koprowski and Tucker, 1973b) have been described else-
where. Following validation, prolactin concentrations
in skim milk were determined by the same radioimmunoassay
used for blood serum. Hormone concentrations were deter-

mined in duplicate, and accepted when accuracy was 15%.

41

1. Validation of Prolactin
Assay for Skim Milk

 

 

Prolactin concentrations in skim milk were deter-
mined by radioimmunoassay (Tucker, 1971; Koprowski and
Tucker, 1971). As greater volumes of skim milk were added
to the "total precipitate tubes" of the prolactin assay,
radioactivity bound to the antibody decreased progressively,
parallel to the standard curve. Addition of 5, 10 or 15
ng of NIH-B3 prolactin to tubes containing 20 ul of a
skim milk sample gave quantitative recoveries of 90, 100
and 100%. Measurement of prolactin in 165 milk samples
prior to and after skimming indicated that 30.9 i 1.2% of
the prolactin was associated with milk fat. The percentage
of prolactin lost from the milk after skimming did not
change with advancing stages of lactation. Prolactin
concentrations in skim milk samples were significantly
correlated with whole milk (r = .73; P < .001). Prolactin
concentrations were determined in eight samples of fresh
milk after freezing (-20 C) and thawing (25 C) three times
(conducted over a 3 day period). Prolactin averaged 13.8 t
1.8 and 13.3 i 2.5 ng/ml (P > .05) for fresh and frozen-

thawed skim milk samples.

42
E. Experimental Objectives and Design

Experiment 1. Arterial and
Venous Concentrations of
Prolactin and Insulin
DuringiMilking

 

 

 

 

The objectives of this experiment were to deter-
mine whether increased blood serum concentrations of pro-
lactin (Johke, 1969, 1970; Tucker, 1971; Schams, 1972a)
and insulin (Koprowski and Tucker, 1973b) during milking
were associated with a greater mammary uptake compared
with the period immediately before milking when blood
serum concentrations of those hormones would be relatively
constant. This was accomplished by measurement of arterio-
venous differences in concentrations of prolactin and
insulin across the mammary gland before, during and after
milking. The second objective was to determine whether
stage of lactation affected mammary uptake of prolactin
and insulin.

This experiment was repeated on three consecutive
afternoons, beginning on day 1 of experimentation. The
time of the PM milking was designated as time 0. Simul-
taneous arterial and venous blood samples were collected V
at 30, 25, 20, 15, 10, 8, 6, 4, 2 and 0 min before milking.
Approximately 15 to 20 sec prior to milking the cows
udder was washed and at time 0 the milking machine was
attached to the animal. Milking required approximately 3

to 5 min. Blood samples were then collected at 2, 4, 6,

43

8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, and 70 min

after milking.

Experiment 2. Arterial and
Venous Concentrations of Pro-
lactin and Growth Hormone
After the Administration of
TRH Prior to Milking

 

 

 

The primary objectives of this experiment were,
firstly, would elevated prolactin concentrations prior to
milking saturate mammary prolactin receptors and thereby
prevent uptake of prolactin normally associated with
increased prolactin concentrations at milking. Secondly,
since the milking stimulus does not release growth hormone
in cows (Reynaert and Peters, 1972; Koprowski and Tucker,
1973b) but growth hormone is galactopoietic in cows.
Whether TRH stimulated release of growth hormone (Convey
et al., 1973a) would be associated with a greater mammary
uptake of this hormone compared with that associated with
basal concentrations of this hormone. Thirdly, I wished
to determine if stage of lactation affected the data.

This experiment was repeated three times on 12
cows following the completion of Experiment 1. To mini-
mize carryover effects the replicates of Experiment 3 may
have had on each other, a 48-hr interval was allowed
between replicates.

Blood sampling was begun 60 min prior to milking.
Arterial and venous blood samples were drawn simultane—

ously 60, 55, 50, 45, 40, 35, and 30 min before milking

44

to determine basal hormone concentrations. Immediately
after the samples at 30 min had been drawn, 200 ug of TRH
(Abbott Laboratories, Chicago, IL) in 2 m1 of .85% NaCl
was injected intravenously to stimulate release of pro-
lactin and growth hormone. Arterial and venous samples
were then drawn at 28, 26, 24, 22, 20, 18, 16, 14, 12, 10,
5 and 0 min before milking. Each cow was milked immedi-
ately after the 0 min samples were collected for approxi-
mately 3 to 5 min. Blood samples were collected at 2, 4,
6, 8, 10, 12, l4, l6, l8, and 20 min after the start of
milking.

Experiment 3. Arterial and

Venous Concentrations of

Prolactin after Milking

in Cows Treated with
Ergocryptine

 

 

 

 

Ergocryptine markedly reduces bovine serum pro-
1actin concentrations and prevents the milking-induced
release of pituitary prolactin (Schams and Karg, 1974).
Therefore, following administration of ergocryptine, the
quantity of serum prolactin available to the mammary gland
should be severely reduced. The objective of Experiment 3
was to determine if there is a mammary uptake of pro-
lactin, before, during and after milking when serum pro-
lactin concentrations are chronically depressed, and
whether this uptake is affected by stage of lactation.

This experiment was performed in triplicate follow-

ing the completion of Experiment 2 on five early and five

45

late lactating cows. The reduction in the number of
animals from 12 to 10 was the result of the arterial
cannulae in two animals becoming nonfunctional. The
replicates were performed on three consecutive afternoons.
Ergocryptine was administered to the five early
lactating cows as the salt 2-bromo-a-ergocryptine-
methanosulphate (CB-154, Sandoz, Basle, Switzerland) and
to the five late lactating as pure ergocryptine (Sigma
Chemical Co., St. Louis, MO). Each cow was injected sub-
cutaneously with 80 mg of either CB-154 or ergocryptine
dissolved in 5 ml 95% ethanol, 24 hr prior to Experiment 3.
Simultaneous arterial and venous samples were
collected 30, 25, 20, 15, 10, 8, 6, 4, 2, and 0 min before
milking. After washing the udder and attaching the milking
machine at 0 min, blood samples were collected 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60 and 70 min later.
Experiment 4. Arterial and
Venous Concentrations of
Prolactin and Growth
Hormoneiifter Adminis-

tration of_TR§ in Cows
Treated with Ergocryptine

 

 

 

 

The quantity of prolactin released in response to
TRH is severely diminished in cows treated with ergo-
cryptine, and greater doses of TRH are required to release
quantities of prolactin comparable to those released in
untreated cows (Schams, 1972b). The objective of this

experiment was to determine if prolonged exposure to low

46

serum concentrations of prolactin as a result of ergo-
cryptine would potentiate the mammary uptake of serum
prolactin following administration of 500 ug of TRH. I
also wished to know whether mammary uptake of prolactin
under these conditions was affected by stage of lactation.
Furthermore, since the effects of ergocryptine on TRH-
induced growth hormone release were unknown in cattle,
serum growth hormone concentrations were also determined.

Experiment 4 was conducted in triplicate after
Experiment 3 on five early and five late lactating cows.
Serum prolactin concentrations in the 10 cows were still
depressed during Experiment 4 by the CB-154 or ergocryptine
given prior to Experiment 3. To minimize effects the TRH
treatment might have on succeeding replicates, the three
experimental replicates of Experiment 4 were conducted on
alternate afternoons, thus allowing a 48-hr period between
them.

Simultaneous arterial and venous blood samples
were collected 30, 25, 20, 15, 10, 8, 6, 4, 2, and 0 min
before injection of TRH. Immediately after the collection
of the 0 min samples, 500 ug of TRH in .85% NaCl was
injected intravenously. Samples were then collected 2, 4,
6, 8, 10, 12, 14, l6, 18, 20, 30, 40, 50, 60 and 70 min
after injection of TRH. The animals were milked following

the collection of the 70 min samples.

47

Experiment 5. Milk Concen-
trations of Pro1actin

 

 

 

Malven and McMurtry (1974a and b) observed that
alterations of blood serum concentrations of prolactin in
goats and rats resulted in parallel changes in the pro-
lactin concentrations of milk. This evidence would sug-
gest that passage of prolactin from blood into milk is
unrestricted. The treatments in Experiments 1, 2, 3 and
4 should have resulted in both acute and chronic changes
in blood serum prolactin concentrations. Therefore, I
obtained skim milk samples from each cow for AM and PM
milkings throughout Experiments 1, 2, 3 and 4. The
changes in serum prolactin during the different experiments
were compared with prolactin concentrations in skim milk.
Experiment 6. Serum Concen-
trations of Prolactin in
the Mammary_Vgin Following

intramammary Infusion of
Prolactin

 

 

 

 

 

To investigate further the relationship between
prolactin in blood and milk, the experimental animals
were given intramammary injections of prolactin. The
objective of this study was to determine if a large
quantity of prolactin infused via a streak canal into the
milk contained in one quarter of the udder would increase
venous blood concentrations of prolactin.

This experiment was performed after Experiment 4

was concluded. To facilitate detection of any increase in

48

prolactin concentration in mammary venous blood resulting
from the intramammary injection of prolactin, 40 mg of
CB-154 in 95% ethanol was given subcutaneously to each of
the five early lactating cows and 40 mg of ergocryptine

in 95% ethanol was given subcutaneously to each of the
five late lactating cows, 24 hr prior to the experiment.
Use of CB-154 and ergocryptine would reduce concentrations
of endogenous prolactin and minimize release of endogenous
prolactin as a result of manipulation of the teat during
injection of prolactin.

The experiment was performed on three consecutive
afternoons. On the first two afternoons 50 mg of pro-
lactin (National Institutes of Health, BB), dissolved in
approximately 20 ml of alkaline 0.85% NaCl was injected
into the gland cistern of one quarter via the streak canal
of the teat. On the third afternoon, 20 ml of alkaline
0.85% saline was injected into the same quarter as a con-
trol experiment. Each experiment was performed 90 min
before milking when the udder was full of milk and the
quarter was massaged immediately after injection to
facilitate distribution of the prolactin throughout the
milk of the injected quarter.

Mammary venous blood samples were collected 30,
25, 20, 15, 10, 5 and 0 min before the injection of either
prolactin or saline and 5, 10, 15, 20, 30, 40, 50, 60 and

90 min after injection.

49

Experiment 7. The Effect of
Ambient Temperature on Serum
Prolactin Concentrations
Before and After Milking
or TRH

 

 

 

Increasing ambient temperature increases basal
serum prolactin concentrations in rats (Mueller et al.,
1974) and heifers (Wettemann and Tucker, 1974; Tucker and
Wettemann, 1976). Furthermore, higher ambient temperatures
are associated with increased prolactin release in
response to TRH (Tucker and Wettemann, 1976). The
objective of this experiment was to determine if ambient
temperature affected serum prolactin concentrations in
arterial and venous samples before and after milking or
TRH.

When the blood samples were collected for each
experimental replicate of Experiments 1, 2 and 4, the
ambient temperature on that particular afternoon was
recorded from a mercury-in-glass thermometer. From the
temperatures recorded for each experimental replicate, a
mean overall ambient temperature was determined for each
experiment for each cow. Then, cows were allocated,
within stage of lactation, to either a higher or lower
temperature group for a particular experiment. The higher
temperature group was composed of cows in which experi-
ments were performed at greater than 18 C and the lower
temperature group was composed of cows in which experiments

were performed at less than 18 C. This particular

50

temperature was chosen since the cows within a particular
stage of lactation naturally fell into two equal groups

on either side of 18 C.

F. Statistical Methods

 

The statistical analyses described below were
calculated with the aid of a CDC 6500 computer.

Average hormone concentrations at each sampling
time were calculated for each cow from the three replicates
of each experiment. From these values, a mean value and
a standard error was calculated for each sampling time for
each stage of lactation. The average values for arterial,
venous, and arteriovenous difference in hormone concen-
trations, for each sampling time, for each cow, were then
used in a split plot analysis of variance. For the
analysis each experiment was divided into three time
periods, which were for Experiments 1, 3, and 4: the 30-
min prior to milking or injection of TRH, the 20-min
period immediately after milking or TRH, and the last 50
min of each experiment. For Experiment 2 the three time
periods were: the 30-min period prior to injection of
TRH, the 30-min period immediately after the injection of
TRH, and the 20-min period beginning at milking. During
those time periods, overall means and overall standard
errors of arterial and venous concentrations and arterio-
venous differences in hormone concentration were cal-

culated.

51

From the split plot analysis of variance the
effects of experimental treatment, period within an
experiment, and stage of lactation on arterial, venous
and arteriovenous differences in hormone concentrations
were determined. The F-test was used to determine if two
overall means were different, and the t-test was used to
determine if overall mean arteriovenous differences for a
period were different from zero.

In Experiment 7, the results of Experiments 1, 2,
and 4 were re-analyzed at each stage of lactation accord-
ing to whether the average ambient temperature when each
cow was sampled was high (> 18 C) or low (< 18 C). The
same split plot analysis was used except that temperature

was entered as an additional variable.

RESULTS

Experiment 1. Arterial and Venous
Concentrations of Prolactin and
Insulin During Milking

 

 

 

A. Prolactin

 

Overall mean prolactin concentrations, for
arterial and venous serum samples collected from the six
early lactating cows during the 30-min pre-milking period
were 28.3 t 11.2 and 27.6 i 11.0 ng/ml (Figure 1).
Immediately following application of the milking machine,
serum prolactin concentrations increased to maximum
arterial concentrations of 55.8 t 11.8 to 56.2 t 11.9 ng/ml
within 6 to 12 min (P < .01). Serum prolactin concen-
trations in venous samples changed in a similar manner,
reaching maximum concentrations of 56.5 i 12.1 to 54.1 1
10.2 ng/ml 8 to 10 min after initiation of milking (P <
.01). Thereafter arterial and venous prolactin concen-
trations returned to pre-milking concentrations within
50 min averaging 29.4 i 11.5 and 28.9 i 10.9 ng/ml,
respectively.

For the 30-min pre-milking period, overall mean
serum prolactin concentrations in arterial and venous
samples were 9.1 t .7 and 9.3 i .6 ng/ml, respectively,

52

53

Fig. 1.--Prolactin concentrations in arterial and venous
serum samples before, during, and after milking
which began at 0 min and lasted 3 to 5 min.

[I- [lMean prolactin concentrations of arterial
serum samples from three replicates in each of six early
lactating cows.

+ - + Mean prolactin concentrations of venous
serum samples from three replicates in each of six early
lactating cows.

A - A Mean prolactin concentrations of arterial
serum samples from three replicates in each of six late
lactating cows.

‘ x - x Mean prolactin concentrations of venous
serum samples from three replicates of each of six late
lactating cows.

54

 

 

 

 

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55

for the six late lactating cows (Figure 1). Following
application of the milking machine at time 0, serum pro-
lactin concentrations increased, reaching maximum concen-
trations of 19.7 i 3.4 to 20.5 i 4.1 ng/ml in arterial
samples 6 to 10 min after the start of milking, and 18.7 t
3.1 to 19.3 i 5.5 ng/ml in venous samples 8 to 14 min
after milking was initiated (P < .01). Thereafter serum
arterial and venous prolactin concentrations declined to
values of 14.0 i 4.7 and 13.8 i 4.1 ng/ml, 70 min post
milking. Throughout the experiment, late lactating cows
had lower serum prolactin concentrations (P < .05) than
early lactating cows.

Prior to milking, the overall mean arteriovenous
difference for serum prolactin concentrations was .68 t
.53 ng/ml for the early lactating cows and -.16 i .14 ng/
ml for late lactating cows. Neither of those values was
different from zero (P > .10). From 0 to 20 min post
milking, the mean arteriovenous difference in serum pro-
lactin concentrations in early lactating cows (1.05 t .65
ng/ml) approached statistical significance (P 2 .10),
whereas in late lactating cows the arteriovenous difference
was .35 i .37 ng/ml (P > .10). From 30 to 70 min post
milking the mean arteriovenous difference decreased to
.25 i .57 in early lactating and increased to 2.18 i 2.28

ng/ml in late lactating cows.

56

The apparent increase in arteriovenous differences
in serum prolactin of late lactating cows at 50 and 60 min
post milking was due to two of the six cows releasing a
large quantity of prolactin on one of the three days of
the experiment for unknown reasons. This large increase
in serum prolactin resulted in an abnormally large arterio-

venous difference.

B. Insulin

 

Serum insulin concentrations were relatively
stable prior to milking in early lactating cows (Figure 2).
During this period overall mean serum arterial and venous
insulin concentrations were 2.6 i .65 and 2.5 i .61 ng/ml,
respectively. Serum insulin concentrations remained con-
stant during and after milking. For the 20-min period
beginning at milking serum arterial and venous insulin
concentrations ranged from 2.6 i .61 to 3.0 i .71 ng/ml
and 2.4 i .57 to 3.0 i .77 ng/ml, reSpectively. Overall
mean serum insulin concentrations from 30 to 70 min post
milking were 2.6 i .66 for arterial and 2.4 i .64 ng/ml
for venous samples.

Prior to milking the six late lactating cows,
overall mean serum concentrations of insulin were 3.7 i
.28 and 3.5 i .21 ng/ml for arterial and venous samples,
respectively (Figure 2). During the 20-min period
beginning at milking, serum arterial and venous concen-

trations averaged 3.6 i .26 and 3.4 i .27 ng/ml. Thus,

57

Fig. 2.--Insulin concentrations in arterial and venous
serum samples before, during and after milking
which began at 0 min and lasted 3 to 5 min.

[l- E]Mean insulin concentrations of arterial
serum samples from three replicates in each of six early
lactating cows.

+ - + Mean insulin concentrations of venous serum
samples from three replicates in each of six early lactating
cows.

A - A Mean insulin concentrations of arterial
serum samples from three replicates in each of six late
lactating cows.

x - x Mean insulin concentrations of venous serum
samples from three replicates in each of six late lactating
cows.

58

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milking did not affect serum insulin concentrations.

From 30 to 70 min post milking, mean arterial and venous
serum insulin concentrations were 3.5 t .40 and 3.3 i .30
ng/ml. Overall concentrations of serum insulin through-
out the entire experiment were greater in late than in
early lactating animals (P < .10).

For the 30 min prior to milking, the mean arterio-
venous difference for serum insulin was .13 i .04 ng/ml
for the six cows in early lactation. This value was
different from zero (P < .05). However, during this
period in late lactating cows, the arteriovenous differ-
ence (.21 i .08 ng/ml) was not different from zero (P >
.05). During the 20-min period beginning at milking,
arteriovenous differences in serum insulin concentration
in both early (.11 i .03 ng/ml) and late (.16 i .04 ng/ml)
lactating cows were different from zero (P < .05).
Between 30 and 70 min post milking arteriovenous differ-
ences in serum insulin concentrations in early (.20 1
.09 ng/ml) and late (.23 i .14 ng/ml) were not different
from zero (P > .05). A split plot analysis of variance of
the data revealed that neither stage of lactation nor the
period during the experiment when the samples were col-
lected significantly affected arteriovenous differences
of insulin. Overall arteriovenous differences from -30
to 70 min after milking averaged .13 ng/ml but this value

was not different from zero (P > .05).

60

Experiment 2. Arterial and Venous
Concentrations of Prolactin and
Growth Hormone After the
Administration of TRH

_—Pfior to Milking

 

 

 

 

 

A. Prolactin

 

During the 30-min period prior to injection of
200 ug TRH, overall mean prolactin concentrations in
arterial and venous serum samples from the six early
lactating cows were 29.0 i 9.8 and 28.8 i 10.3 ng/ml,
respectively (Figure 3). Following administration of 200
ug TRH 30 min before milking, concentrations of arterial
serum prolactin increased to a maximum 210 i 52 ng/ml
after 12 min (P < .01). Concentrations of prolactin in
venous serum samples increased to a maximum of 212 t 51
ng/ml at 14 min after injection of TRH (P < .01). There-
after serum prolactin concentrations remained relatively
constant until milking. Overall mean prolactin concen-
trations for the 30-min period beginning with the injection
of TRH and ending at milking were 179.0 1 43.0 for
arterial samples and 171.0 i 40.0 ng/ml for venous samples.
Following initiation of milking at time 0, there was a
further increase in serum prolactin concentrations until
10 to 12 min after milking commenced. Maximum post milk-
ing concentrations of serum prolactin were 269.0 : 60.0
ng/ml in arterial samples and 264.0 : 63.0 ng/ml in venous
samples. Overall mean serum prolactin concentrations for

the 20-min post-milking period were 248.0 t 55.0 and

61

Fig. 3.--Prolactin concentrations in arterial and venous
serum samples of lactating cows, before and after
the injection of 200 ug TRH (at -30 min),
followed 30 min later by milking which began at
zero min and lasted 3 to 5 min.

E]- ClMean prolactin concentrations of arterial
serum samples from three replicates in each of six early
lactating cows.

+ — + Mean prolactin concentrations of venous
serum samples from three replicates in each of six early
lactating cows.

A - A Mean prolactin concentrations of arterial
serum samples from three replicates in each of six late
lactating cows.

x - x Mean prolactin concentrations of venous
serum samples from three replicates in each of six late
lactating cows.

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238.0 1 51.0 ng/ml for arterial and venous samples,
respectively.

Overall mean serum prolactin concentrations during
the 30-min pre—injection period were 9.1 1 .6 ng/ml in
arterial samples and 9.3 1 .6 ng/ml in venous samples from
six late lactating cows (Figure 3). Following adminis-
tration of 200 ug TRH 30 min before milking, serum pro-
lactin concentrations increased to a maximum prolactin of
90.0 1 25.0 (P < .01) in arterial samples and 89.0 1 28.0
(P < .01) ng/ml in venous samples. These peaks occurred
10 to 12 min post injection, respectively. Overall mean
serum prolactin concentration for the 30 min between
injection of TRH and milking were 80.0 1 24.0 ng/ml in
arterial samples and 77.0 1 23.0 ng/ml in venous samples.
After attaining maximum concentrations, serum arterial
and venous prolactin concentrations remained relatively
constant until milking when a slight increase (18%) in
serum prolactin concentrations occurred. Serum prolactin
concentrations during the 20 min after the start of milk-
ing averaged 77.0 1 20.0 ng/ml in arterial samples and
76.0 1 20.0 ng/ml in venous samples.

Split plot analysis of variance indicated that the
six early lactating cows had greater serum prolactin con-
centrations prior to TRH injection (P < .05) and released
a greater amount of prolactin (P < .05) to the TRH stimu-

lus, compared with late lactating cows. Furthermore, after

64

injection of 200 ug TRH, early lactating cows released
more prolactin to the milking stimulus than did late
lactating cows (P < .01).

Prior to injection to TRH, mean arteriovenous
differences in prolactin concentrations for early (.40 1
.77 ng/ml) and late (-.01 1 .20 ng/ml) lactating cows
were small and not different from zero (P > .10). However,
mean arteriovenous differences during the 30-min post
TRH injection period increased to 7.7 1 3.1 ng/ml for
early lactating and 2.6 1 1.0 ng/ml for late lactating
cows. These values were greater (P < .05) than zero, but
stage of lactation did not affect these arteriovenous
differences. During the 20 min beginning with milking
the mean arteriovenous difference increased slightly in
the early lactating cows to 10.3 1 6.7 ng/ml. Conversely,
in the six late lactating cows the mean arteriovenous
difference decreased from 2.6 1 1.0 to 1.0 1 1.1 ng/ml
in the 20-min post-milking period. However, neither of
the mean arteriovenous differences in serum prolactin
during the post-milking period were different from zero

(P > .05) .

B. Growth Hormone

Overall mean serum growth hormone concentrations
in arterial and venous samples from six early lactating
cows collected during the 30-min pre-injection period were

4.0 i .2 and 4.0 i .2 ng/ml, respectively (Figure 4).

65

Fig. 4.--Growth hormone concentrations in arterial and
venous serum samples of lactating cows, before
and after the injection of 200 ug TRH (at -30
min) followed 30 min later by milking which began
at zero min and lasted 3 to 5 min.

[I - D Mean growth hormone concentrations of
arterial serum samples from three replicates in each of
six early lactating cows.

+ - + Mean growth hormone concentrations of
venous serum samples from three replicates in each of
six early lactating cows.

A - A Mean growth hormone concentrations of
arterial serum samples from three replicates in each of
six late lactating cows.

x - x Mean growth hormone concentrations in
venous serum samples from three replicates in each of six
late lactating cows.

66

 

 

 

 

 

 

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Serum growth hormone concentrations increased (P < .01) to
maximum concentrations of 18.6 1 3.1 in arterial and 18.5 1
3.3 ng/ml in venous samples 12 min after intravenous
injection of 200 ug TRH (P < .01). Thereafter, serum
growth hormone concentrations declined in arterial and
venous samples to values of 12.1 1 1.4 and 11.7 1 1.7 ng/
ml at 30 min after injection of TRH, respectively.

Overall mean arterial and venous concentrations of growth
hormone during the 30-min post TRH injection period were
15.2 1 2.3 and 14.6 1 2.4 ng/ml. Milking was without
effect on serum growth hormone concentrations and they
continued to decline to values of 7.7 1 .8 (arterial) and
7.0 1 .6 ng/ml (venous) at 20 min after milking.

Before injection of TRH in six late lactating cows,
mean serum concentrations of growth hormone in arterial
and venous samples were 3.7 1 .6 and 3.8 1 .7 ng/ml
(Figure 4). Injection of 200 ug TRH increased markedly
(P < .01) serum growth hormone concentrations. Maximum
arterial (14.8 1 2.7 ng/ml) and venous (13.1 1 2.3 ng/ml)
concentrations occurred 12 min after injection of TRH.
Overall mean arterial and venous concentrations of growth
hormone during the 30-min post TRH injection period were
11.8 1 2.0 and 11.1 1 1.9 ng/ml, respectively. Although
the late lactating cows appeared to release less growth
hormone in response to TRH than early lactating cows,

the difference was not significant (P > .05). Milking

68

these cows at 30 min after injection of TRH did not affect
serum growth hormone concentrations. Serum arterial and
venous growth hormone concentrations continued to decline
from values of 8.6 i 1.9 and 8.6 i 1.6 ng/ml at milking
(time 0) to 5.7 i .81 and 4.6 i .81 ng/ml 20 min after
milking. The overall mean arteriovenous difference in
serum growth hormone concentration during the 30-min pre—
TRH injection period was -.03 i .08 ng/ml for early and
—.05 i .10 ng/ml for late lactating cows. These values
were not different from zero (P > .05). During the 30-min
post TRH injection period, the mean arteriovenous differ-
ence was .58 i .22 ng/ml in early and .73 1 .18 in late
lactating cows. These values were different from zero

(P < .05) and greater than arteriovenous differences in
any other period. The overall mean arteriovenous dif-
ference (-.06 i .20) during the 20-min period beginning at
milking was not different from zero in early lactating
cows (P < .05). However, during this period in late
lactating cows the mean arteriovenous difference was sig-
nificant (.33 i .12 ng/ml; P < .05). Stage of lactation
did not affect arteriovenous differences during any

experimental time period.

69

Experiment 3. Arterial and Venous
Concentrations of Prolactin After
Milking in Cows Treated
with Ergocryptine

 

 

 

 

 

The administration of 80 mg CB-154 to five early
lactating cows reduced (P < .01) serum prolactin concen-
trations from an average of 28.0 ng/ml just prior to
treatment to 7.0 ng/ml 24 hr later. Similarly, adminis-
tration of 80 mg ergocryptine to five late lactating
cows reduced serum prolactin concentrations from an
average of 9.0 ng/ml immediately prior to treatment to
2.0 ng/ml 24 hr later (P < .01).

Overall mean serum prolactin concentrations in
five early lactating cows averaged 7.0 1 2.0 ng/ml for
arterial and 7.4 1 2.1 ng/ml for venous samples during the
30-min period prior to milking (Figure 5). After appli-

cation of the milking stimulus maximal serum prolactin

H-

concentrations were 9.1 1 3.2 ng/ml in arterial and 10.0
2.5 ng/ml in venous samples. However, the mean arterial
(7.7 1 2.4 ng/ml) and venous (8.0 1 2.5 ng/ml) prolactin
concentrations were not different from pre-milking serum
prolactin concentrations (P > .05). Between 30 and 70 min
after milking mean serum prolactin concentrations changed
little, averaging 7.2 1 1.8 and 6.9 1 1.7 ng/ml for
arterial and venous samples, respectively.

During the 30-min pre-milking period of five late
lactating cows serum prolactin concentrations were 1.7 1

.2 ng/ml in arterial samples and 1.7 1 .2 ng/ml in venous

70

Fig. 5.--Serum prolactin concentrations in arterial and
venous samples collected before, during and after
milking in five early lactating cows treated with
80 mg CB-154, 24 hr prior to experimentation and
five late lactating cows treated with 80 mg
ergocryptine, 24 hr prior to experimentation.

The animals were milked for 3 to 5 min beginning
at 0 min.

E]- []Mean prolactin concentrations in arterial
samples from three replicates in each of five early
lactating cows.

+ - + Mean prolactin concentrations in venous
samples from three replicates in each of five early
lactating cows.

A - A Mean prolactin concentrations in arterial
samples from three replicates in each of five late
lactating cows.

x - x Mean prolactin concentrations in venous
samples from three replicates in each of five late
lactating cows.

71

 

 

 

 

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72

samples (Figure 5). Milking did not affect serum pro-
lactin concentrations in arterial or venous samples.
During the 20-min post-milking period serum prolactin
concentrations averaged 2.0 1 .3 ng/ml in arterial and
2.0 1 .4 ng/ml in venous samples. From 30 to 70 min after
milking, prolactin concentrations were 1.7 1 .20 ng/ml in
arterial serum samples and 1.7 1 2.3 ng/ml in venous serum
samples. Prolactin concentrations were greater (P < .05)
in early lactating compared with late lactating cows.
Arteriovenous differences in serum prolactin con-
centrations were small and not different from zero in
either early or late lactating cows. During the 30-min
pre-milking period mean arteriovenous difference in serum
prolactin concentration was -.38 1 .11 ng/ml in early and
-.02 1 .03 ng/ml in late lactating cows. The mean
arteriovenous difference from 0 to 20 min post milking
was -.28 1 .18 in early and 0.02 1 .03 ng/ml in late
lactating cows. Thereafter, the mean arteriovenous dif-
ference in serum prolactin from 30 to 70 min post milking
was .34 1 .21 and .00 1 .06 ng/ml for early and late

lactating cows, respectively.

73

Experiment 4. Arterial and Venous
Concentrations of—Prolactin and
Growth Hormone After Adminis-
tration of TRH in Cows
Treated With
Ergocryptine

 

 

 

 

 

 

 

A. Prolactin

 

Serum prolactin concentrations remained depressed
during this experiment as a result of the 80 mg of CB-154
given to the five early lactating cows, and the 80 mg of
ergocryptine given to the five late lactating cows, just
prior to the start of Experiment 3.

Serum prolactin concentrations during the 30-min
pre-injection period averaged 9.2 1 1.5 in arterial and
9.4 1 1.5 ng/ml in venous samples from five early lactating
cows (Figure 6). Serum prolactin concentrations increased
(P < .01) rapidly following administration of 500 ug TRH
to maximum values of 154 1 29 ng/ml in arterial and 143 1
25 ng/ml in venous samples at 12 to 14 min. Thereafter,
serum prolactin concentration declined. Overall mean
serum prolactin concentrations were 116 1 25 and 110 1 21
ng/ml, respectively, in arterial and venous samples during
the 20-min post-TRH injection period. From 30 to 70 min
after injection of TRH prolactin concentrations in arterial
and venous samples continued to decline; overall mean con-
centrations during this period were 91 1 12 and 89 1 11

ng/ml.

 

74

Fig. 6.--Prolactin concentrations in arterial and venous
serum samples before and after injection of 500
ug TRH at 0 min to five early lactating cows
treated with 80 mg CB-154 and five late lactating
cows treated with 80 mg ergocryptine.

E]- ClMean prolactin concentrations in arterial
samples from three replicates in five early lactating cows.

+ - + Mean prolactin concentrations in venous
samples from three replicates in five late lactating cows.

A - A Mean prolactin concentrations in arterial
samples from three replicates in five late lactating cows.

x - x Mean prolactin concentrations in venous
samples from three replicates in five late lactating cows.

'75

 

 

 

 

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76

Serum prolactin concentrations averaged 1.9 1 .2
for arterial and 2.1 1 .2 ng/ml for venous samples in five
late lactating cows during the 30-min pre-injection period
(Figure 6). Following injection of TRH, serum prolactin
concentrations increased to maximum values of 24.9 1 8.7
ng/ml in arterial and 24.0 1 8.3 ng/ml in venous samples
8 to 10 min after injection of TRH (P < .01). Between 0
and 20 min post-TRH injection, mean serum prolactin con-
centrations were 20.5 1 6.7 in arterial and 20.8 1 6.7
ng/ml in venous samples. Serum prolactin concentrations
averaged 13.3 1 3.9 in arterial and 12.6 1 3.7 ng/ml in
venous samples collected between 30 and 70 min after
injection of TRH. Serum prolactin concentrations in
serum samples collected in each period from early lactating
cows were greater (P < .01) than from late lactating cows.

Mean arteriovenous differences for serum prolactin
were -.14 1 .18 for early lactating and -.18 1 .16 ng/ml
for late lactating cows, prior to injection of TRH. From
0 to 20 min after injection of TRH, arteriovenous differ-
ences averaged 6.3 1 4.5 ng/ml in early lactating and
-.24 1 .40 ng/ml in late lactating cows. Prolactin arterio-
venous differences averaged 2.4 1 1.5 ng/ml in early and
.68 1 .27 ng/ml in late lactating cows between 30 and 70
min after TRH injection. None of the above mean arterio-

venous differences were different from zero (P > .10).

77

B. Growth Hormone

 

Mean serum growth hormone concentrations during
the pre-TRH injection period averaged 4.9 1 .9 ng/ml in
arterial samples and 5.0 1 .9 ng/ml in venous samples
collected from cows treated with CB-154 (Figure 7).
Injection of 500 ug TRH caused an increase in arterial and
venous serum growth hormone concentrations to peaks of
25.2 1 4.5 and 24.7 1 4.5 ng/ml 12 min after injection of
TRH, respectively (P < .01). During the 20-min post-TRH
injection period arterial and venous concentrations of
serum growth hormone averaged 20.6 1 3.6 and 19.9 1 3.5
ng/ml. Between 20 and 70 min after TRH injection, serum
arterial and venous growth hormone concentrations declined
from 24.1 1 3.8 and 21.5 1 2.9 to 7.1 1 1.1 and 7.7 1 1.8
ng/ml, respectively.

Serum growth hormone concentrations before
injection of TRH in arterial (3.6 1 .7) and venous (3.4 1
.7) samples of late lactating cows were not different from
those of early lactating cows (P > .05). Injection of
500 ug of TRH increased serum growth hormone concentrations
to maximum values in arterial (15.7 1 2.6) and venous
(15.1 1 2.7) samples within 12 min (P < .01). Overall
mean concentrations of serum growth hormone for arterial
(13.1 1 2.0 ng/ml) and venous (12.6 1 2.1 ng/ml) samples
during the 20-min post-injection period were lower (P < 0.01)

than those obtained during the same period in early

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78

Fig. 7.--Serum growth hormone concentrations in arterial
and venous serum samples before and after
injection of 500 ug TRH at 0 min to five early
lactating cows treated with 80 mg CB-154 and
five late lactating cows treated with 80 mg
ergocryptine.

E]- Mean growth hormone concentrations in
arterial samp es from three replicates in five early
lactating cows.

+ - + Mean growth hormone concentrations in
venous samples from three replicates in five early
lactating animals.

A - A Mean growth hormone concentrations in
arterial samples from three replicates in five late
lactating cows.

x - x Mean growth hormone concentrations in
venous samples from three replicates in five late
lactating cows.

79

 

 

 

 

 

 

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lactating cows. From 30 to 70 min post TRH injection,
serum concentrations of growth hormone in arterial and
venous samples declined from 14.0 1 2.0 and 14.9 1 2.4
ng/ml to 5.4 1 .8 and 4.9 1 .7 ng/ml, respectively.

During the 30-min pre-TRH injection period arterio—
venous differences for both early (-.04 1 .14) and late
(.20 1 .11 ng/ml) lactating cows were small and not
different from zero. In the 20-min period immediately
following administration of TRH arteriovenous differences
increased to .7 1 .36 (P > .10 relative to zero) in early
lactating cows and .5 1 .12 ng/ml in late lactating cows;
however, those arteriovenous differences were not different
from each other (P > .10). Arteriovenous differences in
serum growth hormone concentration from 30 to 70 min post—
TRH injection decreased to .52 1 .29 in early and .16 1
.30 in late lactating cows but these values were not
different from zero (P > .10).

Experiment 5;; Milk Concentrations
of Prolactin

 

 

In neither early nor late lactating cows was milk
production affected by experimental treatment (P > .10;
Table 1). Early lactating cows produced approximately
92% more milk than late lactating cows throughout
Experiments 1 to 4.

The data given in Table l are mean values for pro-

lactin concentrations in skim milk samples collected over

81

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82

each experimental period. Prolactin concentrations in
skim milk samples from early lactating (AM = 25.6 1 8.7
and PM = 23.9 1 7.4 ng/ml) and late lactating cows (AM =
10.4 1 .8 and PM = 12.4 1 1.2 ng/ml) collected during
Experiment 1, were quantitatively similar to serum concen-
trations (27.6 and 9.3 ng/ml, respectively) of prolactin
collected before milking (Table l and Figure 1).

Since any increase in prolactin concentrations in
skim milk following the administration of TRH may not be
apparent in the overall means given in Table 1, specific
means for the prolactin concentrations in skim milk
samples collected immediately after and 12 hrs after TRH
were calculated and are given below.

The elevation of serum prolactin in Experiment 2
in early lactating cows as a result of injecting TRH
(Figure 3) did not increase skim milk concentrations of
prolactin approximately 30 min later in the PM milking
(19.8 1 3.6 ng/ml), above those found in the milk during
Experiment 2 (i = 24.8 ng/ml). Similarly, in late lac-
tating cows, concentrations of prolactin in skim milk
collected 30 min after injection of TRH (12.8 1 2.1 ng/ml)
were no different from concentrations found in those
cows during Experiment 1 (i s 11.4 ng/ml).

The administration of 80 mg CB-154 to five early
lactating cows and 80 mg of ergocryptine to five late

lactating cows reduced basal serum prolactin concentrations

83

from 28 and 9 ng/ml, respectively, in Experiment 2
(Figure 3), to 7 and 2 ng/ml in Experiment 3 (Figure 5).
Prolactin concentrations in skim milk samples were also
reduced following the administration of CB-154 to early
lactating cows or ergocryptine to late lactating cows
(Table 1).

Administration of 500 ug TRH to five early lac-
tating cows treated with CB-154 and to five late lactating
cows treated with ergocryptine increased serum prolactin
concentrations from 9.0 to 113.0 and 2.0 to 20.0 ng/ml,
respectively. Approximately 70 min later the cows were
milked. Similar to the results obtained in Experiment 3,
CB-154 and ergocryptine reduced skim milk concentrations
relative to those observed in Experiment 1. Moreover,
prolactin concentrations in skim milk samples collected
from that PM milking, were similar in both early (11.9 1
2.9), and late (4.7 1 .08) lactating cows to the pro-
lactin concentrations in the skim milk samples collected
during Experiment 3 in which no TRH was given (Table 1).

Experiment 6. Serum Concentration of
Prolactin in the MammaryxVein

Fbilowing Intramammaxy
Infusion of Prolactin

 

 

 

 

Concentrations of serum prolactin in the mammary
vein before injection of saline or 50 mg prolactin ranged
from 10.0 1 1.1 to 13.2 1 4.0 ng/ml in the five early

lactating cows (Figure 8). The apparent increase in serum

84

Fig. 8.--Serum prolactin concentrations in venous samples
collected before and after intramammary infusion
of 50 mg prolactin or saline at time 0.

o - 0 Mean prolactin concentrations of five early
lactating cows before and after the injection of 50 mg
prolactin.

+ - + Mean prolactin concentrations of five early
lactating cows before and after the injection of saline.

[]- [)Mean prolactin concentrations of five late
lactating cows before and after the injection of pro-
lactin.

x - x Mean prolactin concentrations of five late
lactating cows before and after the injection of saline.

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prolactin concentrations to 20.3 1 9.4 ng/ml, 10 min post
injection was not significant (P > .05). Thereafter serum
prolactin concentration varied between 19.7 1 8.1 and

13.8 1 4.3 ng/ml for the remainder of the experiment.
There was a great deal of variation in serum prolactin
between individual animals during the post-injection
period. Prior to the injection of saline, serum pro-
lactin concentrations ranged from 11.2 1 4.0 to 15.8 1 5.4
ng/ml (Figure 5). From 5 to 50 min post injection serum
prolactin concentrations ranged from 11.7 1 4.3 to 16.5 1
8.3 ng/ml. However, at 60 and 90 min post injection
serum prolactin concentrations increased unexpectedly to
23.3 1 14.2 and 24.9 1 13.5 ng/ml.

Prior to intramammary injection of 50 mg prolactin,
serum prolactin concentrations ranged from 1.5 1 .17 to
1.7 1 .21 ng/ml in five late lactating cows (Figure 8).
During the 90 min post-injection period serum prolactin
concentrations remained constant ranging from 1.7 1 2.5 to
3.0 1 1.3 ng/ml.

Experiment 7. The Effect of Ambient
Temperature on Serum Prolactin

ConcentratiOns Before and
After Milking or TRH

 

 

 

 

Blood samples were collected from three early
lactating cows when the ambient temperature was high (20
to 28 C) and from three early lactating cows when the

ambient temperature was low (3 to 8 C) before, during and

87

after milking. In the higher ambient temperature group,
concentrations of serum prolactin in arterial and venous
samples were 41.2 and 40.8 ng/ml, 30 min before milking
(Table 2). From 0 to 20 min after milking, serum pro-
lactin concentrations were 63.5 in arterial and 61.8 ng/ml
in venous samples, thereafter declining (between 30 to
70 min after milking) to 44.5 ng/ml in arterial and venous
samples. In the low temperature group, serum prolactin
concentration averaged 15.6 in arterial and 14.5 ng/ml in
venous serum samples, 30 min before milking (Table 2).
For 20 min after milking serum prolactin concentrations
averaged 33.6 in arterial and 33.0 ng/ml in venous serum
samples. From 30 to 70 min after milking serum prolactin
concentrations were 20.1 and 19.3 ng/ml, respectively,
in arterial and venous samples.

The ambient temperature was high (21 to 26 C) when
three late lactating cows were sampled, and low (1 to
17 C) when three other late lactating cows were sampled
before, during and after milking. In the higher ambient
temperature group, serum prolactin concentrations aver-
aged 9.6 ng/ml in both arterial and venous samples 30 min
before milking, increasing to 16.2 and 15.2 ng/ml,
respectively, during the 20 min after milking (Table 2).
From 30 to 70 min after milking, serum prolactin concen-
trations averaged 11.2 ng/ml in arterial and 10.9 ng/ml

in venous samples. For 30 min before milking serum

88

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ocm smHs am mcaxaas amamm ocm ma0mmn mcoaamuacmocoo cwaomaoum asumm msocm> pom HmaamaH¢n1.~ manme

89

prolactin concentrations averaged 8.7 in arterial and
9.1 ng/ml in venous samples, in the low ambient tempera-
ture group. During the 20 min after milking, serum pro-
lactin concentrations averaged 20.0 in arterial and 19.7
ng/ml in venous blood samples. From 30 to 70 min post
milking, serum prolactin concentrations were 19.3 in
arterial and 16.6 ng/ml in venous blood samples.

The differences in serum prolactin concentrations
between high and low temperature groups, within a stage
of lactation were not significant (P > .05). This is
probably due to a large amount of variation associated
with the small number of animals within each temperature
group.

Blood samples were collected from three early
lactating cows when the ambient temperature was high (20
to 29 C) and from three early lactating cows when the
ambient temperature was low (11 to 12 C) before and after
TRH, which was followed 30 min later by milking. In the
high temperature group, serum prolactin concentrations
were 43.8 in arterial and 43.6 ng/ml in venous samples,
30 min before administration of 200 pg TRH (Table 3).

For 30 min after 200 ug TRH serum prolactin concentrations
were 243 in arterial and 233 ng/ml in venous samples.

The milking stimulus applied 30 min after TRH increased
serum prolactin concentrations to 286 in arterial and

268 ng/ml in venous samples. In the lower ambient

90

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91

temperature group, serum prolactin concentrations were
14.6 in arterial and 13.9 ng/ml in venous serum samples,
30 min before 200 pg TRH. After TRH serum prolactin
concentrations increased to 115 in arterial and 110

ng/ml in venous blood samples. Milking, 30 min after TRH,
increased serum prolactin concentrations to 209 in
arterial and 205 ng/ml in venous samples.

In three late lactating cows, from which blood
samples were collected when the ambient temperature was
high (18 to 23 C), serum prolactin concentrations aver-
aged 17.1 in arterial and 17.4 ng/ml in venous samples
before TRH (Table 3). For 30 min after 200 ug TRH, serum
prolactin concentrations averaged 119 in arterial and
115 ng/ml in venous samples. For 20 min after milking,
which occurred 30 min after TRH, serum prolactin concen-
trations averaged 104 in arterial and 101 ng/ml in venous
samples. In three late lactating cows, from which blood
samples were collected when the ambient temperature was
low (1 to 9 C), serum prolactin concentrations were 9.0
in arterial and 8.8 ng/ml in venous samples. For 30 min
after TRH, serum prolactin concentrations averaged 41.7
in arterial and 40.7 ng/ml in venous samples. For 20
min after milking, serum prolactin concentrations were
50.2 in arterial and 50.8 ng/ml in venous samples.

Within a stage of lactation, serum prolactin

concentrations were not statistically different (P > .05)

92

between the high and low temperature groups, again because
of a large amount of variation and small number of animals
within a group.

The ambient temperature was high (22 and 23 C),
when samples were collected from two early lactating
cows treated with CB-154, before and after 500 pg TRH.
For 30 min before TRH, serum prolactin concentrations
were 7.7 in arterial and 7.6 ng/ml in venous samples
(Table 4). For 20 min after TRH, serum prolactin concen-
trations in arterial and venous samples were 75.5 and
76.9 ng/ml, respectively. Serum prolactin concentrations
averaged 65.7 in arterial and 66.8 ng/ml in venous samples
30 to 70 min post injection. In two early lactating cows
treated with CB-154, from which blood samples were
collected when the ambient temperature was low (6 and 7 C),
serum prolactin concentrations were 10.6 in arterial and
11.1 ng/ml in venous samples 30 min before 500 pg TRH
(Table 4). For 20 min following 500 pg TRH, serum pro-
lactin concentrations were 171 in arterial and 155 ng/ml
in venous blood samples. Thirty to 70 min after TRH
serum prolactin concentrations were 113 in arterial and
107 ng/ml in venous blood samples.

The ambient temperature was high (19 C) when
blood samples were collected before and after 500 pg TRH
in two late lactating cows treated with ergocryptine.

Thirty min before TRH, serum prolactin concentrations

93

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94

(ng/ml) were 2.0 in arterial and 2.4 ng/ml in late
lactating cows. Following TRH, serum prolactin concen-
trations increased to 35.9 in arterial and 36.1 ng/ml in
venous samples. For 30 to 70 min after TRH, serum pro-
lactin concentrations were 22.3 in arterial and 21.1
ng/ml in venous samples. Ambient temperature was low

(2 and 5 C) in two late lactating cows treated with
ergocryptine when blood samples were collected before and
after the administrations of 500 pg TRH (Table 4). Serum
prolactin concentrations were 1.9 in arterial and 2.1
ng/ml in venous samples before 500 pg TRH. Following TRH,
serum prolactin concentrations increased to 6.6 in arterial
and 6.3 ng/ml in venous samples. Thirty to 70 min after
TRH serum prolactin concentrations were 5.0 in arterial
and 4.5 ng/ml in venous samples.

Within the early stage of lactation, the two
animals treated with CB-154 and sampled during the higher
ambient temperatures had significantly lower serum pro-
lactin concentrations after 500 pg TRH, compared with the
two animals sampled at the higher ambient temperature
(P < .05). Conversely, within the late stage of lactation,
animals treated with ergocryptine and sampled at the
lower temperature had significantly greater serum pro-
1actin concentration following the administration of

500 pg TRH (P < .05).

DISCUSSION

In the present study basal serum prolactin con-
centrations before application of the milking stimulus or
administration of TRH were approximately 300% greater in
early lactating than in late lactating cows. Throughout
this series of experiments early lactating cows produced
approximately twice as much milk per day as did late
lactating animals. In lactating ewes, basal serum prolactin
concentrations were greater than in pregnant ewes (Arai and
Lee, 1967; MacNeilly, 1971; Borger and Davis, 1973).
Similarly, lactating pigs had greater basal concentrations
‘of serum prolactin than agalactic sows at the same stage
post partum (Threfall et al., 1974). KOprowski and Tucker
(1973b) obtained estimates of basal serum prolactin con-
centrations in lactating cows from single samples, collected
once a month, 2 to 4 hr before milking. Basal serum
prolactin concentrations increased for the first 3 months
of lactation, after which they remained relatively constant.
Basal serum prolactin concentrations were not associated
with milk yield for the first 3 months, but they were

consistently and positively correlated in later lactation.

95

96

Vines (1976) observed in cows that basal serum prolactin
concentrations declined from 22 ng/ml at the 2nd month of
lactation to 12 ng/ml at the 8th month of lactation, after
which they increased to 16 ng/ml at the 10th month of
lactation. Collectively, greater basal concentrations of
serum prolactin may be associated with greater intensity of
milk production.

Administration of CB-154 to early lactating cows,
or ergocryptine to late lactating cows caused a precipitous
decline in basal prolactin concentrations. Similar observa-
tions have been recorded in rats (Nagasawa and Meites,
1970), sheep (Niswender, 1972), goats (Hart, 1973; McMurtry
and Malven, 1974a), cows (Karg et al., 1972; Fell et al.,
1974; Smith et al., 1974), and women (Brun del Re et al.,
1973). However, in my study ergocryptine was somewhat more
effective in depressing basal serum prolactin concentrations
in late lactating cows (90% reduction) than was CB-154 in
depressing basal prolactin concentrations in early lactating
cows (75% reduction). After ergot treatment, serum pro-
lactin concentrations in late lactating cows averaged less
than 2 ng/ml whereas early lactating cows averaged approxi-
mately 7 ng/ml. It is possible that the difference is
pharmacological since the two compounds are chemically
different and may have different potencies. However, the
pituitaries of early lactating, higher milk—producing cows
contain greater quantities of prolactin compared with

pituitaries of lower-producing animals (Reece and Turner,

97

1937). Furthermore, pituitaries of lactating sheep
secrete prolactin at a faster rate than those of non—
lactating sheep (Borger and Davis, 1973). Therefore, on
the assumption that CB-lS4 and ergocryptine have similar
potencies in cattle, then CB-154 may have been less
effective in reducing basal serum prolactin concentrations
because of physiological differences between early and
late lactating cows. Coincident with the decrease in basal
serum prolactin concentration there is also a decrease in
the quantity of prolactin released to the milking or TRH
stimulus with advancing stage of lactation. There are a
number of possible explanations for this, for example the
pituitary may gradually become refactory with advancing
lactation, gradually releasing less prolactin both before
and after the milking or TRH stimulus. Alternatively, as
lactation advances, the quantity of prolactin synthesized
by the pituitary may decline with advancing lactation, so
that there is less prolactin available for release either
before or after stimulation. A third possibility would be
that the nerve tracts conveying the impulses to the brain,
become less sensitive to the milking stimulus with advancing
lactation. Obviously, further research is needed to solve
this problem.

The milking or suckling stimulus releases prolactin
not only in cows (Johke, 1969 and 1970; Tucker, 1971;
Schams, 1972a; Koprowski and Tucker, 1973a) but in rats

(Amenomori et al., 1968), goats (Bryant et al., 1968; Hart,

98

1975a; Bryant and Chameley, 1976), and sheep (Lamming et al.,
1974). These observations were confirmed in Experiment 1

of the present study. Quantitatively serum prolactin con-
centrations of cows during early lactation increased an
average of 20.3 ng/ml whereas late lactators increased

8.7 ng/ml. The greater release of prolactin in early as
compared with late lactating cows agrees with the work of
KOprowski and Tucker (1973a) in cows and that of Hart

(1975a) in goats.

Vines (1976) observed that cows at two months of
lactation tended to release more prolactin to the TRH
stimulus than cows in later stages of lactation. In the
present study, I observed that early lactating cows in-
creased 150 ng/ml and late lactating cows increased
67 ng/ml following 200 ug of TRH. In cows pretreated with
ergots early lactators increased 108 ng/ml whereas late
lactators increased 19 ng/ml following 500 ug TRH.

Since milking and TRH released greater quantities
of prolactin in early as compared with late lactators, this
may suggest that the absolute quantity of prolactin
released may be associated with lactational intensity.
Indeed, Convey et al., (l973a,b) observed that TRH not only
stimulated prolactin release from the pituitary but also
caused a small but significant increase in milk yield. The
increase in milk yield may well have been due partially to

increased prolactin concentrations.

99

Although the quantities of prolactin released to
milking or TRH were different between stages of lactation,
the percentage differences between mean basal concentrations
and mean post-milking or post-TRH concentrations were very
similar in early and late lactating cows. Since milk yields
were about 100% more in early as compared with late lacta-
tors, it seems doubtful that percent change in serwm pro-
lactin after milking or TRH is related to milk production.

The milking and TRH stimuli probably act to release
pituitary prolactin by different mechanisms. Support for
this concept is evident by comparing the results of Experi—
ments 3 and 4. In cows treated with CB-154 or ergocryptine,
milking did not release prolacting, whereas TRH did release
prolactin. Ergot inhibition of milking-induced release of
prolactin has been reported previously in goats (Hart,

1973) and cows (Smith et al., 1974). That TRH will over-
come ergot inhibition was reported previously in goats
(McMurtry and Malven, 1974a) and cows (Schams, 1972b).
Collective evidence from the literature suggests that
prolactin secretion from the pituitary is under chronic
inhibition from release inhibiting factor(s) secreted from
the hypothalamus (Meites and Clemens, 1972). The milking
stimulus probably acts to reduce this inhibition on the
pituitary, thus, allowing prolacting to be released. TRH
acts directly on the pituitary to release prolactin (Smith
and Convey, 1975). Furthermore, Hill-Salmi and MacLeod

(1975) observed that ergocryptine blocked release of

100

prolactin from rat pituitaries in vitro, but TRH would
overcome this inhibition. I postulate, in explaining the
results of Experiments 3 and 4, that the milking stimulus
in cattle may block release of the physiological prolactin
release inhibiting factor(s) of hypothalamic origin, in
Experiment 3, but because of the presence of an exogenous
prolactin release inhibiting factor (CB-154 or ergocryptine)
prolactin is not released. However, in Experiment 4,
administration of a prolactin releasing factor, TRH, over-
comes the inhibition of prolactin secretion by CB-154 or
ergocryptine and releases prolactin.

Further evidence that TRH and the milking stimulus
act by different mechanisms to release pituitary prolactin
is provided in Experiment 2. Application of the milking
stimulus following administration of 200 pg TRH increased
serum prolactin concentrations an additional 39% in early
lactating cows, and an additional transitory 18% in late
lactating cows. A similar observation was reported by
Tucker et a1. (1975), who were able to obtain additional
releases of prolactin by application of the milking stimulus
to lactating cows being continuously infused intravenously
with TRH. If in the above experiments, the pituitary is
responding maximally to prolactin releasing factor stimula-
tion (TRH), then the additional prolactin release following
milking is probably caused by a different mechanism.

Although TRH released prolactin after ergot drug

therapy and milking stimuli did not, the TRH-induced

101

release of prolactin was much reduced. For example,
lactating cows treated with CB-154 and then given 500 pg
TRH (Experiment 3) released only 65% as much prolactin as a
lower dose (200 pg TRH) released in the same animals in the
absence of CB-154 (Experiment 4). Similarly in late
lactating cows treated with ergocryptine, 500 pg of TRH
released only 25% as much prolactin as was released by

200 pg TRH in the same animals before ergocryptine treat-
ment. These results agree with those of Schams (1972b) who
observed a marked reduction in the ability of TRH to
release prolactin in lactating cows pretreated with CB-154.
Thus, whether the failure of the milking stimulus to
release prolactin in the face of ergots truly represents a
difference with TRH in mechanism of action, or merely a
difference in potency of these two prolactin-releasing
stimuli will require additional research.

The rate of disappearance of prolactin from the
blood, following milking or TRH stimulus was slower
especially in late lactating cows than would have been
expected on the basis of past reports. Other workers
determined the half life of serum prolactin in lactating
cows to be 25 to 29 min (Johke, 1969; Schams and Karg,
1970; and Tucker et al., 1972). Tucker (1971) determined
that serum prolactin concentrations post milking returned
to pre-milking values within approximately 35 min. In the
present study, following milking in early lactating cows,

serum prolactin concentrations returned to pre-milking

102

values 50 min later. In late lactating cows prolactin
concentrations had not returned to their pre-milking values
70 min after milking, and serum prolactin concentrations
only declined 12% during the 70 min post-milking period.
Davis and Borger (1973) found that lactating sheep had
greater pituitary secretion rates and metabolic clearance
rates for prolactin compared with non-lactating animals.
Since early lactating cows were producing twice as much
milk as cows during late lactation, perhaps the secretion
and clearance rates of prolactin were reduced during late
lactation in my study. Therefore, greater serum concentra-
tions of prolactin, resulting from milking in the early
lactating cows, would decline at a faster rate compared
with those of the late lactating cows.

A rather slow disappearance rate for serum prolac-
tin, following the administration of TRH, is evident in
early lactating cows treated with CB-154 and late lactating
cows treated with ergocryptine. For example, administration
of TRH caused a 10 to 13-fold increase in serum prolactin
concentrations, however, during the 70 min post-injection
period, serum prolactin concentrations only declined 21
to 35%. Malven and McMurtry (1974a) observed in two goats
pretreated with CB-154 that serum prolactin concentrations
following TRH remained elevated for 6 hr. These slow
disappearance rates during ergot therapy are consistent
with reduced clearance rates for serum prolactin. Further-

more, since absolute quantities of serum prolactin are

103

also reduced, I suggest that ergots also may reduce
secretion rates of prolactin.

The results of Experiment 7 suggest that, in
addition to stage of lactation, another important factor
which affects serum prolactin concentrations in cows is
ambient temperature. Within a stage of lactation, basal
(before milking or TRH) prolactin concentrations of serum
in cows were on average 270% greater, but not significantly
different, during high (>18 C) ambient temperatures com-
pared with low (<18 C) temperatures. Although on average
there was little difference in basal prolactin concentra-
tions before milking between the high and low ambient
temperature groups during late lactation (Table 2), this
may be explained by one cow in the "low" temperature group
being sampled during a relatively "high" (17 C) ambient
temperature.

Within a stage of lactation, the groups subjected
to high ambient temperature had quantitatively greater
serum prolactin concentrations following milking or TRH
plus milking. Serum prolactin concentrations were 100%
greater after milking in early lactating cows milked at
higher ambient temperatures compared with early lactating
cows milked at lower ambient temperatures. Similarly,
serum prolactin concentrations were respectively 211% and
175% greater in groups during high temperatures compared
with groups during low temperatures after administration

of 200 pg TRH followed by milking. That serum prolactin

104

concentrations are quantitatively greater in lactating cows
at higher ambient temperatures, before and after TRH agree
with previous reports in heifers (Wettemann and Tucker,
1974; Tucker and Wettemann, 1976), lactating cows (Vines,
1976) and rats (Mueller et al., 1974). Furthermore, serum
prolactin concentrations were greater before and after
milking in goats (Hart, 1975b) and cows (Schams, 1972a;
Koprowski and Tucker, 1973b) during warmer months.

Although absolute serum prolactin concentrations
were generally greater before and after milking in the
higher temperature groups the quantity of prolactin
released after the milking stimulus (post-milking concen-
trations minus pre-milking concentrations) was not markedly
affected by ambient temperature. In contrast, Koprowski
and Tucker (l973b) observed greater quantities of prolactin
released after milking during low temperatures as compared
with high temperatures. However, in their study prolactin
estimates were determined on single serum samples collected
2 to 4 hr before milking, 5 min after or 1 hr after
milking. The design of the present study was based on
multiple samples collected before and after milking and is
in my opinion more representative of the changes which occur
in serum prolactin at milking during high versus low
ambient temperatures.

A discrepancy was observed in cows pretreated with
ergots. Within a stage of lactation, basal serum prolactin

concentrations were similar at either high or low ambient

105

temperatures. However, following 500 pg TRH, serum pro-
lactin concentrations were 230% greater in the low tempera-
ture group compared with high temperature in early lactators
pretreated with CB-154. In contrast, in the late lactating
cows pretreated with ergocryptine serum prolactin concen-
trations following 500 pg TRH were 540% greater during low
ambient temperatures compared with high ambient temperature
group. Furthermore, changes in the quantities of prolactin
released after TRH (post TRH minus pre TRH concentrations)
parallelled the absolute concentrations. A possible
explanation for the difference in response between stages
of lactation at the same ambient temperature may be pharma-
logical, since CB-154 and ergocryptine are chemically
different.

I conclude that within a stage of lactation, higher
ambient temperatures are associated with greater serum
prolactin concentrations, both before and after milking or
TRH. Furthermore, since greater prolactin concentrations
are associated with increased water movement across
membranes in lower animals, it may be speculated that
greater serum prolactin concentrations in lactating cows
during the warmer, drier summer months are associated with
water conservation by the animal.

Prolactin binds to mammary glands of laboratory
species in gitrg (Frantz et al., 1974; Shiu and Friesen,
1974; Holocomb et al., 1976). Considering that mammary

glands of lactating cows are very large target organs for

106

protein hormones, it seemed reasonable to suppose that
binding of prolactin to bovine mammary cells might be
apparent as a difference in serum hormone concentrations
before and after blood has passed through the gland.
Furthermore, since early lactating cows quantitatively
released more prolactin to TRH and milking, one might
suspect that mammary utilization or uptake would also be
increased compared with that found in late lactating cows.
Therefore, the arteriovenous differences in hormone con-
centration reported here are assumed to be representative
of mammary binding or uptake of prolactin.

Arteriovenous differences tended to be small and
not different from zero when serum prolactin was at basal
concentrations. The average arteriovenous difference for
basal serum prolactin concentrations was 0.1 ng/ml for all
cows across all experiments. However, following TRH or the
milking stimulus, average arteriovenous differences in-
creased to approximately 3.0 ng/ml, and on average remained
at this value for at least 50 min. However, the increases
in arteriovenous differences for serum prolactin in Experi-
ments 1 and 4 were not significant, although the increase
in the arteriovenous difference for early lactating cows
in Experiment 1 following milking approached significance.
The absence of significant arteriovenous differences in
Experiments 1 and 4 was caused by a large amount of between-
animal variation. A particularly obvious example of this

occurred in late lactating cows 30 to 70 min post milking

107

in Experiment 1. Here, arteriovenous differences were
large and positive as the result of a large, unexplained
release of prolactin in two of the six cows. This occurred
after the milking-induced release of prolactin in one
particular replicate of that experiment. Arteriovenous
differences tended to be small and negative throughout
Experiment 3 in which serum prolactin concentrations
remained constant and markedly depressed because of ergot
treatment. For 30 min after TRH in Experiment 2, arterio-
venous differences for prolactin became large and positive
for both early (7.7 ng/ml) and late (2.6 ng/ml) lactating
cows.

The increase in arteriovenous differences associated
with increased serum prolactin concentration may be indi—
cative of prolactin binding to the mammary gland. This
contention is supported by in vitro studies, in which the
specific mammary binding of prolactin increased with
increasing concentrations of prolactin, until all the
binding sites are saturated (Frantz et al., 1974; Shiu and
Friesen, 1974; Holcomb et al., 1976). Possibly, as serum
prolactin concentrations increase in vizg, more prolactin
binds to specific mammary receptors, and in order to
saturate the binding sites large serum concentrations are
required. This contention is supported by the results of
Experiment 2 where concentrations of serum prolactin were
greatest and arteriovenous differences, which were differ-

ent from zero, were largest. Furthermore, in cows treated

108

with CB-154 or ergocryptine, in which serum prolactin con—
centrations remained low, arteriovenous differences also
remained small and never differed from zero.

Early lactating cows tended to have larger arterio-
venous differences in serum prolactin after application of
TRH or milking stimuli than late lactating cows. This may
have been due to the early lactating cows releasing larger
quantities of prolactin which may have resulted in more
mammary binding sites taking up prolactin. Birkinshaw
and Falconer (1972) showed that in rabbits, mammary receptors
for prolactin are located on the epithelial cell membrane.
If this is also true for cows, the mammary glands of cows
during early lactation should have a larger number of
prolactin receptors compared with late lactating cows,
since they have a larger number of epithelial cells. The
possibly larger number of prolactin receptors may explain
the tendency for the mammary glands of early lactating
cows to take up greater quantities of prolactin following
milking or TRH.

The approximate blood flow, estimated from milk
yield (using a 500:1 multiplication factor) through the
mammary glands of early lactating cows is 81/min and in
late lactating cows is 4l/min. Therefore, mammary uptake
of prolactin, calculated from blood flow times arterio-
venous difference of prolactin concentrations could be at
most approximately 9 pg/min in early and 1.5 pg/min for

late lactating cows for the 20 min beginning at milking.

109

Similarly, during the 30 min after 200 pg TRH, maximum
mammary uptake of prolactin could be 64 pg/min in early
and 11 pg/min in late lactating cows. Therefore the mammary
glands of the early lactating cows could be taking up sub-
stantially greater quantities of prolactin compared with
those of late lactating cows, even though arteriovenous
differences are not markedly different between the two
stages of lactation. I speculate the possible increased
uptake of prolactin in early lactating cows may be associ-
ated with their greater milk production.

In_git£g studies have indicated that the number of
specific binding sites for prolactin in the mammary gland
is limited, and becomes saturated at higher concentrations
of prolactin (Frantz et al., 1974; Shiu and Freisen, 1974;
Holcomb, 1976). For 30 min following the TRH stimulus,
arteriovenous differences were large and positive in both
early and late lactating cows. When the milking stimulus
was applied 30 min after TRH, serum prolactin concentra-
tions increased 39 and 18% in early and late lactating
cows. However, the mean arteriovenous differences after
the application of the milking stimulus were not different
from zero. This may indicate that mammary binding became
saturated within 30 min of elevated serum prolactin con-
centrations.

It has been observed in rats (McMurtry and Malven,
1974b; Grosvenor and Whitworth, 1976) and goats (McMurtry

and Malven, 1974a) that increased in plasma prolactin

110

concentrations are rapidly followed by increased in con-
centrations of prolactin in milk. This may imply that
passage of prolactin from blood into milk is unrestricted.
It is therefore pertinent to consider whether arteriovenous
differences in serum prolactin concentrations reflect active
uptake and Specific binding of prolactin by the mammary
gland or is indicative of a simple diffusion process in
which prolactin leaks from blood into milk.

Following administration of TRH, serum prolactin
concentrations were elevated for 30 to 70 min immediately
before the PM milking. However, prolactin concentrations
in skim milk samples collected at this milking or at the
AM milking 12 hr later were not different from the pro-
lactin concentrations in skim milk samples collected before
administration of TRH. These results argue against the
concept that simple diffusion is the mechanism responsible
for the passage of prolactin from blood into milk. If
simple diffusion was the mechanism responsible, one would
expect prompt elevation in milk prolactin concentrations
following TRH. Although administration of CB-154 or
ergocryptine reduced milk concentration of prolactin 53%
in comparison with milk from untreated cows, prolactin
concentrations in skim milk samples remained elevated 71%
above serum prolactin concentrations. McMurtry and Malven
(1974a) observed that prolactin concentrations in whole
milk samples from goats treated with CB-154, were greater

than in plasma samples. Following intravenous infusion of

111

exogenous prolactin in rats, milk prolactin concentration
remain elevated after serum prolactin returned in pre-
infusion concentrations. Collectively, the above results
support the concept of an active transport system which
moves prolactin molecules from lower concentrations in the
blood to higher concentrations in the milk.

Further evidence that passage of prolactin from
blood into milk is not a simple process is provided by the
results of the intramammary injections of saline and pro—

1actin. Intramammary injection of 50 mg prolactin into

late lactating cows did not increase blood serum concentra-

tions of prolactin. Therefore, even when large quantities
of prolactin are present in milk, there was no measurable
passage of prolactin into the blood. The results of the
intramammary injection of prolactin or saline in early
lactating cows is more complex. Following intramammary

injection of prolactin in early lactating cows there was a

50% increase in serum prolactin concentrations 10 min later.

However, this was the result of increases in serum pro-
lactin concentrations in two of five cows. I Speculate
that the increase in serum prolactin in these two cows was
not the result of intramammary injection of prolactin, but
caused possibly by the manipulation of the udder or
possibly a non-specific release of prolactin from the
pituitary. The possibility of a non-specific increase in
serum prolactin concentration is likely since, following

intramammary injections of saline, serum prolactin

 

112

concentrations remained low and relatively constant until
50 to 60 min post injection when they increased sub-
stantially for no apparent reason. I, therefore, suggest
that the arteriovenous differences observed in the above
experiments are probably a reflection of active transport
of prolactin from blood to milk. Whether this process is
part of the mechanism of action of prolactin on mammary
cells is questionable, but after binding to a receptor at
the surface of the cell, the prolactin molecule may be
transported through the cell and excreted on the luminal
side into the milk. Evidence to support this hypothesis
has been presented by Nolin and Witorsh (1976) who found
prolactin inside rat mammary epithelial cells.

On average the quantity of prolactin contained in
the milk removed at one milking was approximately 288 pg
for early and 72 pg in late lactating cows. Theoretically,
in both early and late lactating cows, the mammary uptake
of prolactin during the 30 min after milking or 6 min after
TRH could account for all the prolactin found in milk.
However, mammary uptake of prolactin continued for 30 min
following TRH, and was in excess of the total amount of
prolactin found in milk. This may indicate that a certain
proportion of the prolactin taken up by the gland, is not
immediately transferred to the milk or is metabolized in
the mammary cells en route to the milk. The large arterio-
venous differences which occurred in two late lactating

cows following an unexplained increase in serum prolactin

..__n"

.1 - an-uiwmpnfi‘h'zn ' .' . q

E- s“

113

concentrations, 40 to 50 min after milking, suggests that
there may be a greater uptake of prolactin when the mammary
gland is empty, after milking.

It is pertinent to consider that there are certain
limitations in using the arteriovenous difference technique
to determine mammary utilization of protein hormones. For
example, not all the hormone passing from the blood
capillaries into the extracellular fluid may be bound by
the mammary cells. Excess hormone might be tranSported
away by the lymphatic system, which would mean that the
arteriovenous differences would yield an exaggerated esti-
mate of hormone utilization. Conversely, the arterio-
venous difference technique might underestimate mammary
uptake of hormone. This is possible since in binding to a
mammary receptor, a hormone may be competitively displacing
a molecule of the same hormone already occupying the recep-
tor. If the displaced molecules after binding to the
receptor are biologically inactive, but still immunologi-
cally active, then any mammary uptake of hormone will be
masked, since the radioimmunoassays used to determine
serum hormone cannot distinguish between biologically
active or inactive hormone.

The observations that basal concentrations of serum
growth hormone are neither affected by stage of lactation,
CB-154 nor ergocryptine, agree with previous reports
(Oxender et al., 1972; Koprowski and Tucker, 1973b; Smith

et al., 1974). Following administration of 200 pg TRH,

c mdpm‘AA-ir'l A“

‘LJNWA

-l

114

serum growth hormone concentrations increased 250% in

early and 192% in late lactating cows. Injection of 500 pg

TRH into early lactating cows or late lactating cows
treated with ergot increased serum growth hormone approxi-
mately 280%. Thus, I conclude that ergot drugs did not
effect markedly the TRH-induced release of growth hormone.
This conclusion agrees with the finding of Benker et a1.
(1976) who observed that CB-154 did not effect TRH-
stimulated release of growth hormone in humans.

The larger dose of 500 pg TRH released more growth
hormone than the 200 pg dose. Convey et a1. (l973a)
observed that the quantity of growth hormone released in
lactating cows was proportional to dose. Following 200 pg
and 500 pg TRH growth hormone concentrations in serum
increased 32 and 58% more in early than in late lactating
cows. Vines (1976) found that cows in their second month
of lactation released more growth hormone to the TRH
stimulus than cows at any other stage of lactation. The
greater quantity of growth hormone released to TRH by the
early lactating cows of my study, may indicate that at the
earlier stages of lactation, the pituitary is capable of
releasing more growth hormone to other physiological
stimuli. Furthermore, since growth hormone is galacto-
poeitic in cows (Hutton, 1957; Shaw et al., 1955; Brumby
and Hancock, 1955; Bullis et al., 1965; Machlin, 1973),
the larger quantities of growth hormone available for

release in the pituitaries of early lactating cows may be

115

associated with the greater intensity of lactation at this
stage.

That growth hormone plays an important role in milk
production is suggested by the arteriovenous difference
data. Prior to administration of TRH, arteriovenous
differences in growth hormone were small (0.1) and not
different from zero. However, associated with increased
serum growth hormone concentrations after TRH, arterio-
venous differences became larger (0.6 ng/ml), positive and
significant. This may suggest that at higher serum con-
centrations, growth hormone binds to the mammary gland.
Furthermore, since the estimated blood flow through the
mammary glands of the early lactating cows is approximately
twice that of the late lactating cows, mammary uptake of
growth hormone after TRH would be approximately 5 pg/min
in early and 2.6 pg/min in late lactating cows. Suggesting
a greater mammary utilization of growth hormone in early
lactating cows. In lactating cows growth hormone concen-
trations are remarkably constant. For example in the
present study the milking stimulus did not increase serum
growth hormone after TRH administration. This finding is
in agreement with those of Reynaert and Peters (1972) and
KOprowski and Tucker (1973b). However, growth hormone is
responsive to changes in blood metabolite concentrations
(Reynaert et al., 1975a). Therefore, mammary uptake of

serum growth hormone may be dependent upon increased

-5-“ 3'1‘K.‘

‘L-ml.

116

growth hormone concentrations resulting from changes in
blood metabolite concentrations.

Serum insulin concentrations were greater in the
late lactating cows compared with the early lactating cows.
This agrees with previous reports in which serum insulin
concentrations increased with advancing lactation (KOprowski
and Tucker, 1973b; Smith et al., 1976). Serum insulin con-
centrations are negatively correlated with milk yield
(KOprowski and Tucker, 1973b; Hart et al., 1975). My
observation of greater serum insulin concentrations in cows
in late lactation which are producing 50% less milk come
pared with the early lactating cows would agree with these
reports.

Serum insulin concentrations in both early and late
lactating cows remained constant for a period of time
beginning 30 min prior to milking until 70 min post milking.
In other words, milking did not cause release of insulin
in my cows. In contrast, KOprowski and Tucker (1973b)
observed greater serum concentrations of insulin in samples
collected within 5 min of milking, compared with samples
collected 2 to 4 hr before or 1 hr after milking. However,
in the experiment of KOprowski and Tucker (1973b) serum
insulin concentrations were determined in single samples,
collected at monthly intervals, which may not have been
truly representative of the actual serum insulin concentra-

tions during that period of time.

“I

 

ml

117

Although serum insulin concentrations tended to be
greater in late lactating cows, arteriovenous differences
for early and late lactating cows were not significantly
different from each other. However, from 30 min before
milking until 20 min post milking in early lactating cows
and for the 20-min period beginning at milking for the
late lactating cows, arteriovenous differences for insulin
were greater than zero. I speculate that the arteriovenous
differences for insulin observed during and after milking
are associated with an increased uptake of glucose and
other metabolites at this time, since removal of milk
probably stimulates uptake of metabolites by the mammary
cells for the further synthesis of milk products. However,
the mammary glands of early lactating, higher producing
cows probably utilize more glucose and other metabolites
than those of late lactating cows. Therefore, it is
surprising that the arteriovenous differences for insulin
were similar at both stages of lactation. However, esti-
mated mammary blood flow is approximately twice as great
in early compared with late lactating cows. Therefore,
from 30 min before, until 70 min after milking, mammary
uptake of insulin would approximate .10 pg/min in early
and .05 pg/min in late lactating cows. This suggests that
early lactating cows would have a greater overall mammary
uptake of insulin. Plasma insulin concentrations increase
with plasma glucose concentrations (McAtee and Trenkle,

1971; Hove, 1975) and when administered simultaneously with

.3

It- -nmnIn—nms}
4‘.

Vi;

118

glucose, insulin is galactopoeitic in cows (Cowie, 1961).
Perhaps a better time to determine the mammary utilization
of insulin would have been obtained when both insulin and
glucose concentration were increasing.

I conclude that greater intensity of lactation in
cows is associated with greater serum prolactin concentra-
tion, and lower serum insulin concentrations. Also, that

greater mammary uptake of prolactin and growth hormone

 

occurred at intervals when these hormones were acutely Li

increased in serum. The uptake of insulin by the mammary
gland which occurs at milking may be associated with in-

creased mammary uptake of metabolities.

.4.‘

SUMMARY AND CONCLUSIONS

1. In six early (5 to 12 weeks post partum) lactating
cows, serum prolactin concentrations in arterial and venous
samples, collected simultaneously, averaged 28 ng/ml for
30 min before milking, 48 ng/ml for 20 min beginning at
milking and 32 ng/ml for 30 to 70 min after milking. For
the same three periods, serum prolactin concentrations
averaged 9, l7, and 14 ng/ml in six late (37 to 57 weeks
post partum) lactating cows; values which were less than
correSponding averages in early lactating cows. Arterio-
venous differences in serum prolactin concentration were
.68 in early and -.16 ng/ml in late lactating cows during
the 30 min pre-milking period. For the 20 min beginning at
milking, arteriovenous differences increased to 1.05 in
early and .35 ng/ml in late lactating cows. None of the
arteriovenous differences were different from zero but the
arteriovenous difference during the 20 min beginning at
milking approached significance (P=.1). The generally
greater concentrations of serum prolactin and the greater
arteriovenous differences following milking in early
lactating cows may be associated with greater intensity of

lactation characteristic of this physiological state.

119

an!

 

~I

120

2. Serum prolactin concentrations in arterial and
venous samples collected 30 min before administration of
200 pg TRH averaged 29 ng/ml in six early and 13 ng/ml in
six late lactating cows. Following administration of
200 pg TRH, serum prolactin concentrations averaged 175
ng/ml in the early lactating and 79 ng/ml in late lacta-
ting cows. Application of the milking stimulus 30 min
later, caused a further increase in serum prolactin con-
centrations to 243 ng/ml in the early lactating cows, but
had little effect on serum prolactin concentrations in late
lactating cows (77 ng/ml). Arteriovenous differences,

30 min prior to administration of TRH were not different
from zero in early (.40 ng/ml) or late lactating cows

(-.01 ng/ml). However, during the 30 min after TRH,
arteriovenous differences in serum prolactin increased to
7.7 ng/ml in early and 2.6 ng/ml in late lactating cows
(P<.05). The greater concentrations of serum prolactin and
greater arteriovenous differences after TRH in early
lactating, as compared with late lactating cows may be
reflective of greater intensity of lactation.

3. Twenty-four hr following administration of 80 mg
CB-154 to five early lactating and 80 mg ergocryptine to
five late lactating cows, serum prolactin concentrations
declined from 29.0 to 7.2 and 13.0 to 1.7 ng/ml, respec-
tively. Thirty min prior to milking, serum prolactin
concentrations in arterial and venous blood samples

averaged 7.2 in early and 1.7 ng/ml in late lactating cows.

 

“t. ‘A'H'AHZk'JQ . ' . ..

121

Seventy min following milking, serum prolactin concentra-
tions remained constant, averaging 7.5 and 1.8 ng/ml,
respectively. Overall mean arteriovenous differences from
30 min before, until 70 min after milking, averaged -.1
ng/ml in early, and -.02 in late lactating cows.

4. Prior to administration of 500 pg TRH, serum pro-
lactin concentrations in arterial and venous blood samples
averaged 9.3 ng/ml in five early lactating cows pre-treated
with CB-154, and 2.0 ng/ml in five late lactating cows pre-
treated with ergocryptine. For 20 min after injection of
500 pg TRH serum prolactin concentrations averaged 113
ng/ml in early and 20.7 ng/ml in late lactating cows.
Thereafter, serum prolactin concentrations declined,
averaging 90 ng/ml in early and 12.9 ng/ml in late lacta-
ting cows 30 to 70 min post TRH injection. During the 30
min pre-injection period arteriovenous differences in serum
prolactin concentration were -.14 in early and -.18 ng/ml
in late lactating cows. Arteriovenous differences increased
to 6.3 ng/ml in early and averaged -.24 ng/ml in late
lactating cows during the 20 min post-injection period. I
speculate that greater serum prolactin concentrations and
arteriovenous differences following administration of TRH
in early lactating cows was associated with greater inten-
sity of lactation during this physiological state.

5. Prolactin concentrations in skim milk samples,
collected at milking, averaged 24.6 ng/ml in five early

lactating and 11.4 ng/ml in five late lactating cows.

 

122

These values were similar to basal serum concentrations of
prolactin in early (27.6 ng/ml) and in late (9.3 ng/ml)
lactating cows. Administration of CB-154 to early or
ergocryptine to late lactating cows reduced prolactin con-
centrations in skim milk samples from 22.9 to 11.9 and from
12.0 to 7.0 ng/ml, reSpectively. However, the decrease in
prolactin concentrations in skim milk was not as great as
in serum, in which prolactin concentrations decreased from
28.8 to 7.4 in early and 13.0 to 1.7 ng/ml in late lactating
cows. Thus, prolactin is transported from blood into milk
against a concentration gradient.

6. Five early lactating cows, pre-treated with CB-154
were given intramammary injections of 50 mg prolactin.
Prior to injection, serum prolactin concentrations in
mammary vein samples averaged 11 ng/ml. Serum prolactin
concentrations increased to 20 ng/ml, 10 min post injection,
but because of a large amount of between animal variation
this change was not significant. In control experiments,
in which an equal volume of vehicle was injected, serum
prolactin concentrations averaged 13.2 ng/ml from 30 min
before until 50 min after injection. At 60 and 90 min post
injection, serum prolactin concentrations increased un-
expectedly to 23 and 24 ng/ml, respectively.

For 30 min prior to intramammary injection of 50 mg
prolactin or saline serum prolactin concentrations averaged
1.7 ng/ml in five late lactating cows treated with ergo-

cryptine. During the 90 min after injection, serum

#E
___J

u. -. -. .4.‘ “Hum MAI-I5.-.- “AI .'

_._.

T:7

:I

I

123

concentrations remained constant at 1.7 ng/ml. I conclude
that prolactin passes from blood into milk but not from
milk to blood.

7. Serum prolactin concentrations tended to be greater,
within a stage of lactation, before and after milking or
TRH, when blood samples were collected during high ambient
temperatures in comparison with serum collected at low
ambient temperatures.

8. Serum growth hormone concentrations in arterial and
venous samples collected 30 min before administration of
200 pg TRH averaged 4.0 ng/ml in six early lactating cows
and 3.8 ng/ml in six late lactating cows. For 30 min after
TRH, serum growth hormone concentrations averaged 14.9 in
early and 11.4 ng/ml in late lactating cows. Application
of the milking stimulus 30 min after TRH did not affect
serum growth hormone concentrations, and 20 min post milking
serum growth hormone concentrations averaged 7.3 ng/ml in
early and 5.1 ng/ml in late lactating cows. Arteriovenous
differences in early (-.03 ng/ml) and late (-.05 ng/ml)
lactating cows were not different from zero 30 min before
TRH. For 30 min following administration of TRH, arterio—
venous differences were different from zero increasing to
.58 ng/ml in early and .73 ng/ml in late lactating cows.
The significant arteriovenous differences following
administration of TRH may be indicative of mammary binding

of growth hormone.

._ . "'1'

n' It

It *E_ _in—:- ".‘g

124

9. Serum growth hormone concentrations in arterial and
venous blood samples averaged 5 ng/ml in five early lacta—
ting cows pre-treated with CB-154, and 3.5 ng/ml in five
late lactating cows pre-treated with ergocryptine, 30 min
prior to administration of 500 pg TRH. For 20 min after
TRH, serum growth hormone concentrations averaged 20.3
ng/ml in early lactating cows. During the same period,
serum growth hormone concentrations were significantly
lower in late lactating cows averaging 12.8 ng/ml. Seventy
min after TRH, serum growth hormone concentrations declined
to 7.4 in early and 5.2 ng/ml in late lactating cows.
Arteriovenous differences were -.04 in early and .20 ng/ml
in late lactating cows before administration of TRH. After
TRH, arteriovenous differences increased to .7 in early and
.5 ng/ml in late lactating cows. Whether the greater serum
growth hormone concentrations in early lactating cows
following TRH was associated with a greater responsiveness
of the anterior pituitary in general must await further
investigation.

10. From 30 min before until 70 min after milking,
serum insulin concentrations remained constant averaging
2.6 in six early and 3.5 ng/ml in six late lactating cows.
Serum insulin concentrations in arterial and venous samples
were greater in late lactating cows (P<.10) compared with
early lactating cows. During the 20 min post-milking
period, arteriovenous differences were different from zero

in both early (.11 ng/ml) and late (.16 ng/ml) lactating

125

cows. The greater concentrations of serum insulin in late
lactating cows, compared with early lactating cows, may be

associated with a lower intensity of lactation.

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