LUTEOLYSIS, GROWTH HORMONE,- ewooconncoms,‘ ,.

PROLACTIN AND MILK PRODUCTION IN LACTATING
DAIRY COWS GIVEN PG F 21‘ '

Thesis for the Degree of M. S.

MICHIGAN STATE UNIVERSITY

RANDALL HARRELL REN EGAR
1977 '

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ABSTRACT

LUTEOLYSIS, GROWTH HORMONE, GLUCOCORTICOIDS,
PROLACTIN AND MILK PRODUCTION IN LACTATING
DAIRY COWS GIVEN PGFZa

By

Randall Harrell Renegar

The objectives of this thesis were to (1) determine
the minimal dose of prostaglandin an (PGFZa) which when
given (im) to lactating dairy cows results in an incidence
of luteolysis not significantly different from higher doses,
(2) characterize changes in growth hormone, prolactin and
glucocorticoids after PGFZa’ and (3) determine changes in
milk production after PGFZa'

Fifty-seven lactating (2-3 mo) Holstein cows were
given (im) 25 mgPGan 12 days before receiving at random a
second dose of either 0 (saline), 5, 15, 25 or 35 mg PGFZa‘
Blood samples were collected via the coocygeal vein twice
weekly from day ~12 to day 0 (day of treatment injection),
day 9 to 22 and days 2# and 26. Frequent blood samples were
collected (jugular cannulae) at .5-hr intervals from -.5 to
2 hr then at 2-hr intervals for 16 hr, h-hr intervals for
24 hr, Z-hr intervals for 5# hr and 12-hr intervals for

36 hours. Animals were observed for estrous behavior twice

daily from day 2 to day 26, and ovaries were palpated twice

Randall Harrell Renegar

daily for 5 days after treatment and twice weekly thereafter
to day 26. Milk samples were collected for protein and fat
determinations and milk yield was recorded for each cow at
AM and PM milkings 7 days before the first PGFZa injection
and for 7 days after treatment injection.

Luteolysis in response toIPGF2a (serum progesterone
less than 1.0 ng/ml and no palpable corpus luteum within
132 hr) occurred in O of 11 cows given saline, N of 10
given 5 mg PGFZa’ 9 of 11 given 15 mg, 8 of 10 given 25 mg
and 10 of 10 given 35 mg. Incidence of luteolysis in cows
given 15, 25 or 35 mg PGFZa was significantly greater than
that in cows given 0 or 5 mg. Incidence of luteolysis did
not differ among animals given 15, 25 or 35 mg PGFZa and
averaged 88%. Progesterone declined to less than 1.0 ng/ml
within 2# hr in animals with luteolysis after’PGFZa. In
animals given 5, 15 or 25 mg PGFZa not having luteolysis,
progesterone declined from 5.3 to 3.3 ng/ml within 12 hr,
but returned toward pretreatment concentrations within
24 hours. ’Progesterone was unaltered in animals given
saline. 'Following'PGFZa-induced luteolysis, estrus began
at 72 hr, peak of the LH surge occurred at 80 hr and
ovulation occurred at 100 hours.

Growth hormone increased from 1.7 to 3.3 ng/ml within
.5 hr after 35 mg PGan, but was unaltered in cows given 0,
5, 15 or 25 mg PGan' Glucocorticoid averaged 6.8 ng/ml

prior to injection and was unaltered by saline or 5 mg PGFéa

Randall Harrell Renegar

However, glucocorticoid increased to peaks of 22.3, 3h.9
and 39.0 ng/ml at 1.0 hr following 15, 25 and 35 mg PGan,
respectively. Prior to injection, prolactin averaged
37.8 ng/ml and was unaltered by saline or 5 mg PGFZa'
Prolactin increased to peaks of 56.8, 8#.5 and 95.6 ng/ml at
1.0 hr after 15, 25 and 35 mg PGFZa’ respectively. The peak
concentrations of glucocorticoid and prolactin were linearly
related to the dose of’PGF2a.

Average daily milk yield for the 7 days following PGFZa
was 22.# kg and did not differ among treatments. Percent
milk fat and percent total protein averaged 3.18 and 3.29,
respectively, unaltered by dose of'PGan.

These data indicate that 15 mg is the minimal
intramuscular dose of'PGFZa for lactating dairy cows result-
ing in an incidence of luteolysis not significantly different
from higher doses. 'PGFZQ also caused transient increases in
serum growth hormone, glucocorticoid and prolactin, three
hormones with important roles in galactopoesis and

lactogenesis. However, milk yield, percent milk fat and

percent total milk protein were unaltered following PGan.

LUTEOLYSIS, GROWTH HORMONE, GLUCOCORTICOIDS,
PROLACTIN AND MILK PRODUCTION IN LACTATING

DAIRY COWS GIVEN PGan

By

Randall Harrell Renegar

A THESIS

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

MASTER OF SC IENCE

Department of Dairy Science

1977

TO MARILYN

ii

ACKNOWLEDGMENTS

I would first like to thank the Department of Dairy
Science at Michigan State University for providing
facilities, faculty and financial support for this
graduate program. I

I would like to express my sincerest thanks to my
major professor, Dr. H. D. Hafs, for his encouragement and
guidance during the course of this program. I am grateful
for the advice of Dr. J. L. Gill and G. D. Riegle, who
served on my guidance committee. The able assistance of
Dr. R. Neitzel in computer programming is greatly
appreciated.

I wish to thank my colleagues, J. Stevenson, T. Kiser,
M. MCCarthy, J. Stellflug and L. Chapin, for their assis-
tance at the barn, and a special word of thanks to
'Dr. T. D. Carruthers and Dr. J. H. Britt for performing
rectal palpations. I would also like to thank E. Bostwich,
J. Martin and K. Wood for their assistance in performing
radioimmunoassays.

I would like to offer my esteemed gratitude to
Dr. B. H. Johnson for his encouragement in my pursuit of a

career in reproductive physiology.

iii

To my wife, Marilyn, to whom this thesis is dedicated,
I express my gratitude for her patience, encouragement and
financial assistance throughout my program. I am especially
grateful for the time and skill she contributed in the
typing of this thesis.

iv

BIOGRAPHICAL SKETCH

Randall Harrell Renegar was born on April 12, 1952,
in Winston-Salem, North Carolina. He attended primary
schools in Charlotte, North Carolina, graduating from
Independence High School in June, 1970. Pursuing his
interest in biological science, he enrolled at North
Carolina State University and completed requirements for
Bachelor of Science degrees in Animal Science and Zoology
in May, 1975. He accepted a research assistantship at
Michigan State University studying under the directorship
of Dr. Harold D. Hafs and completed the requirements for

a Master of Science degree in August, 1977.

TABLE OF CONTENTS

LIS T 0F TABIIE I I I I I I I I I I I I
LIS T OF FIGURE I I I I I I I I I I I
INTRODUCTION .5 . . . . . . . . . . .
REVIm 0F LITERATURE I I I I I I I I I I
The Normal Bovine Estrous Cycle . . . . .
Metestrus . . . . . . . . . . . .

D 16 Strus I I I I I I I I I I I I
Proestrus . . . . . . . . . . . .
Estrus I I I I I I I I I I I I I

Luteolysis . . . . . . . . . . . .
PrOStaglandins o o 'o o o o o o o o o
Luteolysis, Estrus and Fertility after PGF

Anterior Pituitary and Adrenal Cortical
Response to Exogenous PGFZa . . . . . .

Adrenal Corticotropin and.Glucocorticoids .
Growth Hormone . . . . . . . . . .
Prolactin . . . . . . . . . . . .

Milk Yield, Milk Fat and Milk Protein Response
to GH, Prolactin and Glucocorticoids . . .

Growth Hormone . . . . . . . . . .

Glucocorticoids . . . . . . . . . .
PralaCtin I I I I I I I I I I I I
MATERIALS AND METHODS . . . . . . . . .
Experimental Design . . . . . . . . .
Hormone Assays . . . . . . . . . . .

Determination of Percent Milk Fat and
Percent Total Protein . . . . . . . .

Statistical Analysis . . . . . . . . .

vi

Page

.viii

Ho
\OVVU‘NWUl—‘N

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O

12

12
13
14

1h

15
16

17

18
18

19

20
20

RESULTS AND DISCUSSION .

Reproductive Criteria

Luteolysis

Progesterone .

Intervals to Estrus, the Ovulatory Surge

of LH and Ovulation .

Milk Production.Criteria
Growth Hormone

Glucocorticoids .

Prolactin .
Milk Yield, Percent Milk Fat .and
Percent Total Milk Protein
GENERAL DISCUSSION

SUMMARY AND CONCLUSIONS

BIBLIOGRAPHY

vii

Page
21

21

21
23

27
30
30

37
38
45
48
so

LIST OF TABLES

Table Page

1. Luteolysis in lactating cows given
(1111) sza o o o o o o o o o I o o 22

2. Daily milk yield, milk fat and protein
during 7 days after PGF20L . . . . . . . 41

3. Daily milk yield, milk fat and protein
among cows with or without luteolysis

in response to PGan . . . . . . . . 43

viii

LIST OF FIGURES

Figure Page

1. Blood serum progesterone in lactating
cows given (im) 0, 5, 15, 25 or 35 mg
PGFZa' Standard errors ranged from
0.1 to 1.0 ng/ml and generally were
prOportional to the means . . . . . . . 26

2. Blood serum growth hormone in lactating
cows given (im) PGFZa' Standard
errors ranged from 0.2 to 1.9 ng/ml
and generally were proportional to
the means . .- . . . . . . . . . . 32

3. Blood serum glucocorticoids in lactating
cows given (im) PGan' Standard
errors ranged from 0.3 to 6.1 ng/ml
and were generally proportional to
the means . . . . . . . . . . . . 36

4. Blood serum prolactin in lactating cows
given (im) PGan. Standard errors
ranged from 4.7 to 24.5 ng/ml and
_were generally preportional to the
means . . . . . . . . . . . . . #0

ix

INTRODUCTION

Artificial insemination of dairy cattle has made major
contributions to improved milk production. However, some
believe progress from artificial insemination (AI) has been
retarded recently due to decreased estrus detection effi-
ciency brought about by increased herd size. The need for
accurate methods of estrus detection to enhance AI has
prompted development of methods of ovulation control and
estrus synchronization. Initial attempts to synchronize
estrus involved delaying estrus with a 15- to 18-day pro—
gestogen treatment until the corpus luteum regressed.
Subsequent removal of treatment resulted in reasonably
synchronous occurrence of estrus; however, the fertility of
an insemination following the synchronized estrus was low
compared to controls. Recently, the use of progestogens
'for more limited periods (9-12 days) in conjunction with
estrogens has been effective in synchronizing estrus with
no apparent reduction in fertility.

Another method of estrus synchronization involves the
use of prostaglandin FZa (PGFZa) to promote regression of
the corpus luteum. Injection, either intramuscularly (im)
or subcutaneously (so), or intrauterine (iu) infusion of

PGFéa results in demise of the corpus luteum. Normal

1

2
reproductive endocrine changes follow luteolysis, and estrus
occurs in 2-6 days. Fertility of an insemination following
estrus synchronization with PGFZa is comparable to fertility
following naturally occurring estrus, at least in heifers

and beef cattle. However, PGF has not been tested ade-

2a
quately in milking dairy cows because regulatOry agencies
required that milk be discarded for prolonged periods.
Recent regulatory changes have made this work possible.
Consequently, the research presented in this thesis was
designed primarily to determine the minimal dose of’PGF2a
which when given (im) to lactating dairy cows results in an
incidence of luteolysis not significantly different from
higher doses.

The second objective of this research was to
characterise changes in blood concentrations of growth
hormone, prolactin and glucocorticoids following PGan
treatment. Drastic shifts in secretion of these hormones
could reflect unwanted physiological changes produced by
exogenous PGan. Also, before it is approved for estrus
'synchronization in lactating dairy cows, we should know
whether PGFZa changes milk production and composition.
Therefore, the third objective of this work was to determine

milk yield, percent fat and percent protein in cows given

PGan.

REVIEW OF LITERATURE

The Normal Bovine Estrous Cycle

The bovine estrous cycle is approximately 21 days
although cycles with 19- to 22-day durations are considered
normal (Chapman and Casida, 1937; Kazama and Hansel, 1970;
Swanson gt gl., 1972). In this thesis, days of the estrous
cycle will be numbered 0 to 21 with day 0 representing the
day of estrus. Estrus averages 18 hr in duration ranging
from 12-24 hr (Desjardins and Hafs, 1968). In sequence,
the next three phases of the cycle are (l) Metestrus (day
l to 4): (2) Diestrus (day 4 to 18); and (3) Proestrus
(day 18 to 21). Slight variations may occur in the duration
of the cycle phases among animals (Chapman and Casida, 1937).
Here follows some of the major reproductive endocrine
changes during the bovine estrous cycle.

Metestrus. Metestrus is characterized by ovulation
and formation of the corpus luteum. The newly formed corpus
luteum contains very little progesterone on day 2 (Hafs and
Armstrong, 1968) but corpus luteum weight and function begin
to increase by day 2-3 (Mares 23 al., 1962; Erb gt al.,
1971; Glencross £3 91., 1973: Hansel gt gl., 1973). With
increased progesterone secretion in the developing corpus

luteum, the peripheral blood concentrations of progesterone

4

begin to increase on day 3-5 and continue to increase
reaching a peak on days 11—15 during diestrus (Stabenfeldt,
1968; Pope 33 31., 1969; Stabenfeldt 33 31., 1969; Hansel
and Snook, 1970; Hansel 33 31., 1973). Plasma estrogen
concentrations rise during metestrus from 2 pg/ml on day l
to 6-7 pg/ml on day 4 or 5 (Wettemann 33 31., 1972:
Glencross 35 31., 1973: Hansel 33 31., 1973). Hansel 33 31.
(1973) reported that estrone makes up the major rise in
estrogens during this period. Peripheral blood concentra-
tions of luteinizing hormone are low during metestrus
(Swanson 33 31., 1972; Hansel 33 31., 1973), ranging from
0.4 to 2.7 ng/ml (Schams 33 31., 1969; Wettemann.33 31.,
1972). Pituitary concentrations of LH decrease from day 0
to day 2 (Hackett and Hafs, 1969) but no change in blood
levels of’LH occurs (Swanson.3j 31., 1972). This would seem
to indicate that pituitary synthesis of luteinizing hormone
is depressed during this period. Pituitary FSH levels
decline on day 4. If this reflects released FSH, it may
stimulate the mid-cycle wave of follicle growth (Hackett
and Hafs, 1969). .

Diestrus. Corpus luteum growth and increased
progesterone secretion characterize diestrus. Diestrus .
ends upon the initiation of luteal regression, which may
occur anywhere from day 16 to 19 (Kazama and Hansel, 1970).

Corpus luteum growth continues until day 11-15 (Erb
31 31., 1971; Hansel 33 31., 1973; Hafs and Armstrong, 1968)

of the cycle with no further changes in corpus luteum weight

5
until luteolysis. Erb 33 31. (1971) reported three periods

of accelerated luteal growth which were associated with
increased secretion of urinary estrogens. Corpus luteum
concentration of progesterone increases significantly from
day 4 to 7 (Hafs and Armstrong, 1968) with subsequent
increases to day 17 (Mares 33_31., 1962). Plasma proges-
terone concentrations continue to increase during diestrus
to a peak on day 11 to 13 of 5-9 ng/ml and remain high until
luteolysis (Pope 33 31., 1969; Stabenfeldt 33 31., 1969:
Wettemann.33 31., 1972; Hansel 33 31., 1973). Estradiol
remains low during diestrus and averages about 3.6 pg/ml
(Wettemann.33 31., 1972). Hansel 33 31. (1973) reported a
rise in total estrogens on days 7 to 10 which may correspond
with the mid-cycle wave of follicular develOpment (Hackett
and Hafs, 1969). The mid—cycle follicle growth is reflected
by an increase in follicular wall weight during days 7 to 11
(Swanson 33 31., 1972). Plasma LH remains low during
diestrus (Swanson.3§ 31., 1972; Hansel 33 31., 1973) while
pituitary luteinizing hormone increases from day 2 to 20
(Hackett and Hafs, 1969). Pituitary follicle stimulating
hormone concentrations decline to mid-cycle but are restored
by days 10 to 13 with a subsequent decline prior to estrus
(Desjardins and Hafs, 1968).

Proestrus. Regression of the corpus luteum

characterizes proestrus, beginning between day 16 to 19 in

6

cows with 21-day cycles (Kazama and Hansel, 1970).
Stabenfeldt 33 31. (1969) also reported a significant
variation in day at which luteal regression occurs.

Progesterone concentrations fall precipitiously from
peak values of 4-9 ng/ml about 3 days before estrus to less
than 1.0 ng/ml 1-2 days before estrus (Pope 33 31., 1969;
Stabenfeldt 33 31., 1969; Erb 33 31., 1971). Ayalon and
Shemesh (1974) reported a proestrus peak of progesterone
(2.3 ng/ml) at about 16 hr prior to the onset of estrus,
but a subsequent study did not confirm this (Chenault 33 31.,
1975). Pituitary FSH concentration decreases markedly from
day 18-0 (Hackett and Hafs, 1969) coincided with a major
increase in the number and size of follicles on the ovaries
(Swanson 33 31., 1972)-.

Plasma estradiol concentrations begin to increase 3-4
days prior to estrus (Wettemann.33 31., 1972: Chenault
33 31., 1975). Chenault 33 31. (1975) reported slowly
increasing estradiol concentrations (2.0 pg/ml to 4.0 pg/ml)
from -12 to -4 hr with a more rapid increase up to a peak of
'7.4 pg/ml at or about the time of the peak of the ovulatory
surge of LH. Estradiol then decreases to baseline by 14 hr
after the peak. Wettemann 33 31. (1972) and.G1encross
33 31. (1973) reported similar estradiol changes. The
ovulatory peak of LH occurs at or shortly before the begin-
ning of estrus (Hansel and Snook, 1970; Swanson and Hafs,
1971) and remains above baseline for 6-10 hr (Schams, 1969;
Swanson and Hafs, 1971). This peak is believed to be

7

stimulated by the increase in estradiol prior to the LH
surge (Chenault 33 31., 1975). Swanson and Hafs (1971)
reported maximum LH concentrations of 25.9 ng/ml. Snook
33 31. (1971) and Chenault 33_31. (1975) reported similar
values. Chenault 33 31. (1975) reported a 22—hr interval
from the peak of the ovulatory surge of LH to ovulation;
however, Swanson and Hafs (1971) reported an interval of
32 hours. The variance in these reported intervals may
reflect dissimilarity in the interval between consecutive
palpations used for determining the time of ovulation.

Estrus. Estrus is the 12- to 24—hr period during which
the cow is sexually receptive. Progesterone concentrations
are less than 1 ng/ml during estrus, evidence of no func—
tional corpus luteum (Stabenfeldt 33 31., 1969; Henricks
33 31., 1971: Glencross 33 31., 1973; Chenault 33 31.,
1975). Estradiol concentrations decline from the peak at
outset of estrus to baseline values well before the end of
estrus (Chenault 33 31., 1975). Plasma LH also falls
precipitously from the peak at the outset of estrus to basal
'values well before the end of estrus (Hansel 33 31., 1973;

Chenault 33 31., 1975).

Luteo1ysis

Luteolysis is the process by which the corpus luteum
becomes incapable of synthesis of progesterone and aids in
returning the animal to sexual receptivity after a period

of anestrum. Research principally on sheep indicated that

8

luteolysis is initiated by a blood born substance synthesized
in the uterus (Caldwell 33 31., 1969; Goding 33 31., 1972).
McCracken 33 31. (1972) autotransplanted ovaries to a loca-
tion (neck) distant from the uterus of sheep, and found that
the corpus luteum was retained for an extended period.
Ovaries autotransplanted with accompanying uterine horn
resulted in normal cyclic corpus luteum regression. When
blood from the venous supply of a uterine horn 13 3133 in
one ewe was transfused to the arterial supply of an auto-
transplanted ovary in another ewe, luteolysis occurred in a
normal cyclic pattern. Thus, the luteolytic influence of
the uterus appears to act only when the uterus and ovary
are located near each other. The uterus also is required
for normal cyclic luteal regression in the cow (Wiltbank and
Casida, 1956; Anderson 33 31., 1969). The local pathway for
transport of the uterine luteolysin is venoarterial in
nature (Ginther, 1976). Close apposition of the uterine
vein and its adjacent ovarian artery in sheep (Ginther,
1974) and cattle (Ginther and Del Campo, 1974) allows
'diffusion of the luteolytic factor from the uterine drain-
age into arterial blood supplying the ovary (Mapletoft and
Ginther, 1975; Mapletoft 33 31., 1976).

The mechanism of action of the uterine luteolytic
agent is unknown. Pharriss (1970) purposed the hypothesis
that the uterine luteolysin is acting to restrict blood flow
to the ovary leading to the demise of the corpus luteum.

Additional evidence indicates that luteolysis is brought

9
about by action of the luteolysin upon responsiveness of
the corpus luteum to LH (Armstrong and Black, 1966; Lahav
33 31., 1976).

The identity of the substance responsible for
luteolysis in cows is not known with certainty, although
evidence strongly suggests that PGFZa is the physiological
luteolysin in sheep (Goding 33 31., 1972; McCracken 33 31.,
1972). Recently, Scaramuzzi and Baird (1976) reported that
sheep autoimmunized against PGFZa fail to undergo a normal

pattern of cyclic luteal regression.

£33333g1andins

Prior to reviewing the literature concerning PGFéa-
induced luteolysis and estrus synchronization, I feel that
a brief summary of work leading up to recognition of the
potential of'PGan in reproductive physiology is needed.

The prostaglandins are a family of biologically active,
unsaturated fatty acids widely distributed among animal
tissues. Kurzrok and Lieb (1930) were the first to demon-
strate the action of prostaglandins. They found that fresh
human semen could cause either contraction or relaxation of
uterine tissue 13'31333. Goldblatt (1933) and von Euler
(1935) demonstrated the ability of seminal fluid to cause
smooth muscle contraction. Prostaglandin E1 and prosta-
glandin F2 were isolated from sheep vesicular gland in pure
crystalline form by Bergstrom and Sjovall in 1957. Subse-

quently other prostaglandins were isolated and their

, 10
structures defined (Bergstrom 33 31., 1962a; Bergstrom
33 31., 1962b), thus stimulating increased effort in under-
standing the biological properties of the prostaglandins.
It was not until Pharriss and'Wyngarden (1969) demonstrated
the luteolytic property of'PGan in pseudopregnant rats
that its potential regarding application to controlled

reproduction was recognized.

Luteolysis, Estrus and Fertility after PGan

A number of reviews of literature have recently

 

appeared concerning the use of PGFZa in estrus synchroniza-
tion and controlled breeding (Inskeep, 1973; Arriola, 1975:
Hafs 33 31., 1975a; Lamming 33 31., 1975; Manns and Hafs,
1976). For this reason, the following will be a brief
summary of the literature including references to both
reviews and original references.

Discovery of the luteolytic property of’PGFéa in rats
(Pharriss and Wyngarden, 1969) prompted efforts to determine
the effect of PGFZa on luteal function in other species.

Subsequent work demonstrated the luteolytic capacity
of’PGFZa in sheep (McCracken 33 31., 1972; Mellin and Busch,
1976), guinea pigs (Charchareon, 1974), horses (Hafs 33 31.,
1974), beef cows (Hafs 33 31., 1975b) and dairy heifers '
(Louis 33 31., 1973; Louis 33 31., 1974: Elving 33 31.,
1975; Stellflug 33 31., 1976). Louis 33 31. (1974) reported
that changes in progesterone, estrogen and LH in heifers

during and after PGan-induced luteolysis were not

11

significantly different from changes occurring after
luteolysis in untreated heifers, and the interval from
luteolysis to estrus or to the ovulatory surge of LH was not
significantly different. Furthermore, the cycle following
PGFZa treatment did not differ endocrinologically from normal
cycles in cows (Louis 33 31., 1974). This information indi-
cates that PGFZa has no residual effect on the endocrine
events of the estrous cycle and probably modifies the cycle
by effecting only on luteolytic mechanisms. Louis 33 31.
(1973) gave PGFZa to heifers during different phases of the
estrous cycle and found that luteolysis occurred in heifers
treated during diestrus but not metestrus or proestrus. A
functional (progesterone secretion) corpus luteum (day 5 to
18 in the cow) is necessary for PGFZa to initiate luteolysis.
Hafs 33 31. (19750) found a substantial degree of synchrony
in interval to estrus following PGFZa—induced luteolysis in
dairy heifers, with 88% of the animals in estrus 2-4 days
after treatment. Lauderdale 33,31. (1974), Elving 33,31.
(1975) and Kaneda 33,31. (1976) recorded similar results.

Lauderdale 33,31. (I974) treated beef cows and dairy
heifers with.PGF2a to induce luteolysis and estrus with AI
at either (1) 12 hr after observed estrus or (2) 72 and 90
hr after PGFZa injection. Control cattle were inseminated
12 hr after observed estrus. Pregnancy at 35-60 days after
AI did not differ among the three groups. Hafs,33,31.
(19750) also found no difference in fertility in animals

inseminated at 72 and 88 hr when compared to controls.

12
Recent evidence reported by Stellflug 33 31. (1976) suggests
that the minimum dose of’PGFZa to obtain maximum luteolytic
response in dairy heifers is 15 mg when given (im) as
Tromethamine salt.

In overview, reported evidence indicates that PGFZa may
be of great value for ovulation control to increase the use
and efficiency of AI by eliminating the prerequisite of
estrus detection. Aftificial insemination at a predeter-
mined time should decrease the time necessary for estrus

detection.

Anteriop Pituitary and Adrenal Cort13al Responge to
Exogenous PGan

Agrenal CorticotrOpin and Glucocorticoids. In 1969,
de Wied 33 31. demonstrated the ability of prostaglandins of
the E series but not F series to increase blood corticos-
terone in rats. Peng 33,31. (1970) found that the increase
in adrenal corticoids associated with PGF1 injection did not
occur in hypophysectomized rats. In addition, PGE1 injec-
tions given animals pretreated with morphine did not result
'in an adrenal cortical response (Peng 33 31., 1970).
Stereotaxic injections of'PGE1 and.PGFéa into the median
eminance area of the hypothalamus gave a far greater
corticosterone response than injections into the anterior
pituitary (Hedge and Hanson, 1972). In contrast, cortico-
tropin releasing factor injections result in maximum response
when given in the anterior pituitary. Louis 33,31. (1974)

demonstrated that (im) injections or (iv) infusion of BGan

13
caused increased glucocorticoids in dairy heifers. This
evidence indicates that prostaglandins of both the E and F
series are capable of increasing adrenal corticoids via
increased release of ACTH. The action of these prostaglan—
dins appears to be on the hypothalamus, and not directly on
the anterior pituitary (Hedge and Hanson, 1972).

Growth Hormgne. Schofield (1970) incubated anterior
pituitary slices with PGEl and reported increased growth
hormone secretion.‘ Cooper 33 31. (1972) reported that
adenosine 3'5' monophosphate increased prior to increases
in growth hormone in ox pituitaries incubated with PGEZ.
Single injections of PGE1 given to pentobarbitol treated
rats increased blood growth hormone concentrations (Hertelendy
33,31., 1972). Hertelendy,33 31. (1972) also reported that
single injections of’PGEi increased growth hormone from
2 ng/ml to 14.4 ng/ml within 20 min in castrate male sheep.
Louis 33,31. (1974) reported that 30 and 60 mg of PGF?“
given (im) to dairy heifers cause 7— and 26-fold increases
in blood growth hormone levels, respectively. Tucker 33 31.
(1975) observed similar increases in growth hormone with
PGFZa infusions into heifers at a rate of 30 mg/hour. In
summary, prostaglandins Bland FZa increase growth hormone
secretion,13,3133 and,13,11333. Evidence reported for PGE1
suggests that prostaglandins exert their effect directly on
the pituitary via cAMP. Prostaglandin F2a is a potent

inducer of growth hormone release 13 vivo in heifers.

14

Prolactin. Sato,33,31. (1974) first demonstrated the
ability of prostaglandin E1, E2 and F2a to increase serum
prolactin in rats. Subsequently, Louis 33 31. (1974) showed
that PGFZQ given (im) to heifers causes an increase in blood
prolactin concentrations from 26 ng/ml prior to injection to
81 ng/ml by 10 min after injection. Tucker 33 31. (1975)
demonstrated that infusions of’PGFZa dramatically increase
blood serum prolactin in heifers. I

In overview, the published evidence indicates that
PGFZa given in doses sufficient to cause luteolysis also
elicits increased secretion of three hormones (glucocorti-
coid, growth hormone and prolactin) important in regulating
intermediary metabolism (Hafs, 1975). 'Whether changes in
these hormones induced by'PGFéa may affect milk production
is unknown.

Milk11eld, M11k Fat and Milk Ppotein Response to GH,
Prolactin and G1ucocorticoids

 

Based upon the evidence outlined above, PGFZa is an
effective method of synchronizing estrus for controlled
breeding in cattle. Before PGan is widely used for con-
trolled breeding in lactating cows, the possibility of
undesirable side effects should be investigated. This is
of special interest when one considers the increased release
of growth hormone, prolactin and glucocorticoids following

PGF a treatment to induce luteolysis (Hafs, 1975).

2
The following reviews relationships of growth hormone,

prolactin and glucocorticoids with galactopoesis.

15

Growth Hormone. Asimov and Krouze (1937) first
reported that anterior pituitary preparations relatively
pure in growth hormone caused increased milk production in
lactating cows. Chung 33 31. (1953) found that six daily
injections of 100 mg of growth hormone caused a 50% increase
in milk production (accompanied by substantial increases in
percent milk fat) by the sixth injection in late lactating
cows. In addition, milk production was maintained and per-
cent milk fat increased in the face of a 40% reduction in
TDN intake when accompanied by eight daily 100 mg injections
of growth hormone. Wrenn and Sykes (1953) also reported
increases in milk production after growth hormone treatment
in heifers which had been hormonally induced into lactation.
Bullis 33,31. (1965) compared a commercial preparation of
growth hormone with a highly purified preparation and found
increased milk production with both treatments although the
reSponse associated with the commercial product was signifi-
cantly greater. All milk production increases were accompanied
by increased percent fat and total solids. The reported
work of Bullis 33,31. (1965) may indicate that the dramatic
increases seen by Chung 33,31. (1953) and'Wrenn and Sykes
(1953) were partially due to contamination with other
hormones in the preparations used, although it does not
rule out the galactopoietic effect of growth hormone.
Subsequent study with highly purified growth hormone indi-
cated a significant galactopoietic effect (Machlin, 1973).

16
Machlin (1973) also reported that growth hormone induced
increases in milk production without accompanying increases
in feed consumption.

Glucocorticoids. In 1956 Shaw reported decreases in
milk production and increases in blood glucose in cows
during glucocorticoid treatment. Link 33,31. (1957) and
Vigue (1958) reported similar results. Braun,33,31. (1970)
administered (im) various types of synthetic glucocorticoids
for 10 days to normal lactating Holstein cows. Milk produc—
tion decreased over the 10-day period with the largest
decrease occurring in the animals with the greatest increase
in blood glucose. Bassett (1963) reported that cortisol fed
to sheep caused increased blood glucose without increases in
ketone bodies and plasma free fatty acids. This evidence
may indicate that glucocorticoids decrease milk production
by decreasing glucose utilization. In contrast, Swanson
and Lind (1976) reported that prolonged glucocorticoid
treatment (day 4 postpartum to end of lactation) resulted in
increases in milk production when 5-10 pg was given orally
each day. Higher doses of 20 pg and 50 pg caused no change
and depression, respectively. In addition milk production
was unchanged when 5 or 10 pg was given daily for 25 days.
Head,33,31. (1976) reported no change in milk production
but an increase in percent protein with 10 pg given orally
from week 4 to week 44 of lactation.

Milk yield and composition responses to exogenous

glucocorticoids appear to be related to synthetic preparation,

1?
dosage, length of administration and possibly mode of
administration. Although the results are not conclusive,
evidence supports the view that large doses of glucocorti-
coids are detrimental to milk production while moderate
levels of glucocorticoids are without effect or are slightly
stimulatory when given chronically. Limited evidence sug-
gests milk protein may be increased with moderate levels of
glucocorticoid treatment.

Prolactin. Prolactin is galactopoietic in the rat
(Johnson and Alfredson, 1958; Kumaresan et a1., 1966), sheep
(Cowie, 1969), rabbit (Cowie, 1969) and goat (Meites, 1961),
but has minor galactopoietic activity in the cow (Karg and
Schams, 1974). Experimentally induced reduction of prolactin
secretion during lactation does not alter milk production
significantly in cows (Karg and Schams, 1974; Smith 33,31.,
1974). Similarly, Wrenn and Sykes (1953) found no change in
milk yield in heifers given prolactin during hormonally
induced lactation. Koprowski and Tucker (1973) found blood
prolactin concentrations 2-4 hr before milking were not
'related (r = -.03) with milk yield. In contrast, Karg
33 31. (1972) reported depressed onset of lactation in cows
given CB-l54 (a prolactin release inhibitor) prior to lacta-
tion, indicating that prolactin is necessary for lactogenesis.
Conclusive studies involving the galactopoietic effects of
prolactin are not available in cows. However, the present
evidence indicates that prolactin has no significant effect
on milk production during established lactation in cows, but

is required for initiation of lactation.

MATERIALS AND METHODS

This research was conducted primarily to determine the
minimal intramuscular dose of prostaglandin FZa (PGan)
required for luteolysis among lactating cows. Prolactin,
growth hormone and glucocorticoid (indicator of adrenocorti-
cotropin release) responses to PGFZa were characterized for
4 hr after treatment. Additional information was obtained
to determine acute (7 days following treatment) changes in
milk production, percent milk fat and percent total protein

following PGan.

Egperimental Desigg

Fifty-seven lactating (2 to 3 mo) Holstein-Friesian
cows were assigned at random to be given 0, 5, 15, 25 or
35 mg of’PGFZa. All animals were given 25 mg PGFZa 12 days
prior to treatment injection to insure the existence of a
functional corpus luteum at treatment. PGFZa was diluted in
5 ml of .85% saline and given intramuscularly. Blood
samples were collected from all animals via the tail vein
twice weekly from day -12 to day 0 (day of treatment injec-
tion), day 9 to 22 and days 24 and 26. A limited number of
animals (n = 30) were randomly selected to be bled at
frequent intervals after treatment to determine anterior
pituitary hormone and glucocorticoid changes after PGan

18

19
and to detect the ovulatory surge of'LH. These blood
samples were collected through indwelling jugular cannulae
at .5-hr intervals from -.5 to 2 hr after treatment, then at
2-hr intervals for 16 hr, 4-hr intervals for 24 hr, 2-hr
intervals for 54 hr and 12-hr intervals for 36 hr (last
sample 132 hr after treatment). The remaining 27 animals
were bled via tail vein at 0, 6, 12, 24, 48, 72, 96, and 132
hours. Blood samples were held at room temperature for 2-6
hr following collection and then held at 4 C for 48-72 hours.
Following centrifugation at 3000 rpm for 30 min, serum was
decanted and stored at -20 C until assayed. Animals were
observed for estrous behavior twice daily beginning on day 2
and continuing to day 26. The ovaries and uterus were
palpated twice weekly in each cow from day -12 to day 26 to
detect changes in the size of the corpus luteum, ovulation
and signs of estrus. Composite milk samples were collected
for each cow at AM and PM milkings for 7 days prior to the
first PGFéa injection (pre-treatment samples) and 7 days
after the treatment injection (post-treatment samples).

‘Milk production was recorded for each milking.

Hormone Assays

Serum luteinizing hormone, prolactin and growth hormone
were analyzed by double antibody radioimmunoassays (RIA)
reported by Oxender 33,31. (1972), Tucker,33,31. (1971) and
Purchas 33 31. (1970), respectively. The analysis of serum

progesterone was by the single antibody radioimmunoassay

20
method described by Louis 33,31. (1973). Serum glucocorticoid

concentrations were measured by protein-binding as reported

by Smith 33 31. (1973) .

Determinat1ongf Percent Milk Fat and Pegcent Tota1 Protein
Milk samples were analyzed for percent milk fat using

a Milko-Tester MK III by the method described by McGann

33 31. (1970). Total milk protein was measured using the

dye-binding technique reported by Udy (1971).

Statistical Analysis

Treatment differences in the number of animals
responding to PGan (measured by luteolysis within 132 hr)
were determined by chi-square statistics. Progesterone,
prolactin, growth hormone and glucocorticoid data were
analyzed by multivariate analysis of trends as described
by'Gill and Hafs (1971). Repeat—measure designs may result
in heterogeneous variance of samples among periods within
treatment group, and the statistical procedure used
accounted for higher correlation between samples taken at
“close intervals than for samples taken more distant in time.
Orthogonal contrasts were determined to locate significant
differences among treatments. The difference between pre—
treatment average and individual post-treatment data for
milk yield, percent milk fat and total milk protein was
analyzed by univariate split-plot.- Interval to estrus was
analyzed by one-way analysis of variance with orthogonal

contrasts to test treatment differences.

RESULTS AND DISCUSSION

Reproductive Criteria

Five animals had serum progesterone less than 1.0 ng/ml
and no palpable corpus luteum at the time treatment injec-
tions were given. Since they could not respond, they were
eliminated from the analyses for luteolytic response,
progesterone, and intervals to estrus, the ovulatory LH
surge and ovulation.

Luteolysis. Luteolysis was determined by blood serum
progesterone concentrations and ovarian palpation. I
defined PGFZa-induced luteolysis as a decline in progester-
one to less than 1.0 ng/ml and no palpable corpus luteum
within 132 hr after treatment injection. No cows given
saline had luteolysis within 132 hr following injection
.(Table l). Luteolysis occurred in 4 of 10 cows given 5 mg
'PGFZa, 9 of 11 cows given 15 mg, 8 of 10 cows given 25 mg
and 10 of 10 cows given 35 mg. The incidence of luteolysis
in cows given 15, 25 or 35 mg PGFZa was significantly
greater (P<.05) than in cows given 0 or 5 mg PGFZa. No
significant difference was detected among the cows given
15, 25 or 35 mg; however, there was some evidence (P<.l5)
that 35 mg PGFZa was more effective in causing luteolysis.

The average incidence of luteolysis for all animals given

21

22

Table 1. Luteolysis in lactating cows given (im) PGFZa'

 

 

1’6an W
(mg)
0 I 0/11a
5 4/10b
15 9/11°
25 8/1o°
35 10/1o°

 

a,b,c Ratios with different superscripts differed

significantly (P<.025)

23
15, 25 and 35 mg:PGF2a was 88%. Incidence of luteolysis in
these lactating dairy cows is similar to reported incidences
of 79 and 88% for suckled beef cows given (im) 30 or 60 mg
PGFZa and dairy heifers given (im) 20, 30 or 40 mg PGFZa’
respectively (Hafs 33 31., 1975c). In contrast, Stellflug
,33,31. (1976) found 100% incidence of luteolysis when six
heifers were given (im) 15 mg PGFéa; however, the signifi-
cance of this value is limited due to the number of animals
used. Comparison of data from this thesis and previous work
indicate that incidence of luteolysis for lactating dairy
cows is similar to that reported for heifers and suckled
beef cows, but conclusive comparisons must be done within
one trial.

Prggesterone. Average changes in serum progesterone
among treatments were confounded with incidences of
luteolytic response within teratments. Preliminary analysis
indicated that serum progesterone changes for cows responding
with luteolysis did not differ among treatments. 'Therefore,
progesterone values for all cows (n = 31) responding with
luteolysis were pooled. Progesterone data for the 10 cows
without luteolysis following injection of 5, 15 or 25 mg
PGFZa likewise were pooled, as preliminary analysis indi7
cated no difference in progesterone trend among treatments.
Cows given saline (0 mg PGFéa) were designated as a control
group (n = 11). Progesterone concentration did not differ

among treatments prior to injections: it averaged 5.0,: 0.3

ng/ml.

24

Serum progesterone was unaltered for 72 hr after saline
injection, and it increased (P<.05) above pretreatment con-
centrations by 132 hr after treatment. Thus, the function
of the corpus luteum was not reduced following saline
injection.

Progesterone declined from 5.3 to 3.3 ng/ml (P<.05)
within 12 hr after PGan injection in cows without
luteolysis, but returned toward pretreatment concentration
by 24 hr following PGFZa (Figure l). Evidently, animals
which did not have complete luteolysis following PGan did
have partial luteolysis. However, luteal function in these
cows returned to near normal within 24 hours. Stellflug
33 31. (1976) reported similar changes in progesterone
concentrations in heifers given doses of PGFZa not suffi-
cient for complete luteolysis. They found that progesterone
concentrations declined by 12 hr following PGFZa in
heifers without complete luteolysis, but remained stable
or increased during the remaining portion of a lO8-hr
sampling period.

Blood progesterone declined 50% (P<.05) from 4.9 to
2.4 ng/ml within 6 hr in cows with complete luteolysis after
PGan. Then progesterone decreased (P<.05) to .8 ng/ml at
24 hr after injection and remained less than 1 ng/ml for
the remainder of the 132-hr sampling period. Progesterone
values for lactating cows with complete luteolysis in this

thesis agree with previous work in heifers reported by

Louis,33,31. (1973). They reported a 60% decrease in

25

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mam; haawpmcom cam HE\wc o.H op H.o Scam vmwsmn muonum cumvcmpm

.H musmflm

26

5:02.: ES“. 9:3:
mm. N» we em N. m 0 ml

 

 

 

fl\‘..:.. q \TlT—I . KXI. -
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:35
8."...

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("11/ bu) euclegsebmd

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27

progesterone within 12 hr after PGan, and progesterone
concentrations of less than 1 ng/ml within 24 hours. Louis
33 31. (1974) reported similar changes after PGFZa treatment
(im) in non-lactating dairy cows. Thus, the progesterone
data for lactating cows in this thesis agree with previously
reported data for non-lactating dairy cows and heifers.

In33gyals to Estrusl3the Ovulatory Surge of LH and
Ovulation. All cows were observed twice daily for estrus
behavior at approximately 10- to l4-hr intervals from day 1
to day 26 following injection. The beginning of estrus was
assumed to have occurred at the time half way between the
observation at which estrus was first detected and the pre-
ceding observation. In this experiment estrous behavior was
considered a result of PGan-induced luteolysis only if it
occurred within 6 days after treatment. One animal not
observed in estrus (but with progesterone falling from 2.5
to .9 ng/ml within 12 hr) was eliminated from this analysis.
No cows given saline were observed in estrus during the 6
days following injection. Estrus in response to PGFZa was
observed in 4 of 11 cows given 5 mg, 9 of 11 given 15 mg,
8 of 10 given 25 mg and 9 of 9 given 35 mg. Incidences of
observed estrus were greater for cows given 15, 25 or 35 mg
PGFZa' There were no significant differences in incidences
of estrus among animals given 15, 25 or 35 mg PGFZa; however,
there was some evidence (P<.l5) that incidence of estrus was
greater in cows given 35 mg PGFZa' The average interval to

estrus was 72.8 i 3.7 hr for all animals (n = 30) in estrus

28

as a result of'PGFéa injection. Animals (n = 11) not in
estrus within 6 days of treatment (5, 15 or 25 mg PGFZa)
averaged 272.3 1 17.9 hr to the onset of estrus and this did
not differ from the mean interval (280.9 3 26.2) for saline
treated controls (n = 10). The interval to estrus in these
data is similar to that previously reported for heifers by
Stellflug 33 31. (1975). They reported a 78-hr interval
from.PGF2a injection to onset of estrus for heifers given

5, 10 or 15 mg (im) of PGF Louis,33,31. (1973) reported

2a'
a similar interval (74 hr) for heifers given 30 mg PGan.
Following intrauterine infusion of'PGFZa, Welch 33 31. (1975)
reported that a majority of the suckled beef cows returned
to estrus between 56 to 88 hr following treatment.

Interval from PGFZa injection to peak of the ovulatory
surge of luteinizing hormone (LH) was determined for cows
with frequent blood sampling for 132 hr (n = 30). Only
cows with luteolysis after PGFZa had an.LH surge within
132 hr (n = 18). Cows not responding to PGan (n = 12)
were designated as having an ovulatory surge greater than
132 hr after injection. The mean interval to the LH surge
for cows with luteolysis_after injection was 82.3 1 6.1
hours. This value includes two cows which developed cystic
follicles following injection. Excluding these two cows,
the average interval was 80.3,: 5.3 hours. The mean inter-
val in this work is slightly longer than the interval
(64 hr) previously reported for heifers given (im) 30 mg

PGan (Louis 33 31., 1973), or the interval (67 hr) reported

29
for beef cows given (iu) 1 mg PGFZG (Welch 33 31., 1975).
Louis 33 31. (1974) reported an average interval to the LH
surge of 71 hr in six non-lactating cows given (iu) 5 mg
PGFZa’ similar to the interval reported in this thesis. The
mean interval to the LH surge reported in this work is not
significantly greater than the interval from injection to
the beginning of estrus (72.8 i 4 hr) and agrees with
previous reports that the ovulatory surge of LH occurs
coincident with or shortly before the beginning of estrus
(Hansel and Snook, 1970; Swanson and Hafs, 1971).

Ovulation was determined by palpation of the ovaries
via the rectum. The time of ovulation was assumed to be
midpoint between the last palpation without an ovulation and
the first palpation indicating ovulation had occurred. The
average interval from injection to ovulation for cows
responding to PGFZa injection was 100.1 i 3.8 hr (n = 29).
Two animals that developed persistent follicles following
injection were not included in this calculation. Louis
33 31. (1973) reported an average interval to ovulation of
104 hr for non-lactating cows given (im) 30 mg injections of
PGF

2a
similar intervals for lactating beef cows and dairy heifers

. Welch 33 31. (1975) and Louis 33 31. (1974) reported

given intrauterine PGFZa infusion, respectively. Cows in
the present study not responding with luteolysis following

PGF ovulated on the average 301.8 i 13.7 hr after treatment

2a

(n 21).

30

Ovulation in all cows occurred on the average 28.3 i
3.7 hr after the onset of estrus (n = 48). Two cows with
persistent follicles following luteolysis and one cow not
detected in estrus were not included in the calculation.
The interval from the beginning of estrus to ovulation
reported in this work is similar to the interval of 32 hr
reported by Swanson and Hafs (1971) for nontreated dairy
heifers. Louis 33_31. (1973) and Elving 33 31. (1975)
reported an average interval of 30.0 and 29.8 hr, respec-

tively, for PGF’ -treated heifers.

2a
The data presented in this section indicate that PGFZa
is an effective method of synchronizing estrus and ovulation
in lactating dairy cows. In addition, the intervals to
estrus, the ovulatory surge of LH and ovulation resemble

the intervals reported for treated dairy heifers, or from

comparable values in untreated cattle.

Milk Production C31teria

The remaining portion of these results and discussion
will be concerned with (l) the intermediary metabolic
hormones; growth hormone, glucocorticoids and prolactin,
following PGan, and (2)' the effect of PGFZOL on milk yield
and milk composition.

Growth HOpmpne. Blood serum growth hormone did not
differ significantly among treatment groups prior to PGan

injection; it averaged 1.7 ng/ml (Figure 2). The average

value was similar to baseline values previously reported for

31

Figure 2. Blood serum growth hormone in lactating cows
given (im) PGFZa' Standard errors ranged from 0.2
to 1.9 ng/ml and generally were proportional to
the means.

 

 

Growth Hormone (ng/ml)

 

32

 

‘i5

O+5mq
O-CIHqu
A~1|25wm
o~<>35nm

I

1 l L I l #H
O .5 L0 IJS 2i) 4£>

Hours From Injection

33
lactating cows by Tucker,33,31. (1975). In contrast, Tucker
33 31. (1975) and Louis 33 31. (1973) reported baseline
growth hormone concentrations in heifers of 5.7 and 6.7
ng/ml, respectively.

Growth hormone was unaltered for 4 hr after injection
of saline or 5, 15 or 25 mg PGFZa' However, growth hormone
increased (P<.05) 74% to a peak of 3.3 ng/ml at .5 hr in
cows given 35 mg PGFZa. Growth hormone returned to near
pretreatment concentration by 2 hours. The growth hormone

increase in lactating cows after 35 mg PGF reported in

2d
this study was much less than previously reported in heifers
given similar doses of PGFZa' Louis 33 31. (1974) gave (im)
30 mg of’PGFZa to heifers and reported 7-fold increases in
growth hormone within 30 minutes. In the same study, an
intravenous injection of 5 mg increased growth hormone
approximately 4-fold within 30 minutes.

Previous work by Hertelendy 33 31. (1972) indicates
that prostaglandin-induced increases in growth hormone are
mediated through the pituitary. They incubated ox pitui-
taries with PGEl and reported increased growth hormone
synthesis. Thus, data from this thesis and earlier work
suggest that changes accompanying lactation reduce PGFZa-
induced growth hormone increases by decreasing pituitary
response.

Glucocorticoids. Blood serum glucocorticoids averaged

6.8 ng/ml prior to injection. This agrees with earlier work

by Koprowski and Tucker (1973), who measured glucocorticoid

34
concentrations in 26 cows at 2 to 4 hr prior to milking for
an entire lactation. Glucocorticoids in samples taken from
8 to 20 weeks after calving (the approximate stage of
lactation for cows sampled in this experiment) averaged
4.5 to 9.0 ng/ml.

In the current work, when cows were given saline or
5 mg PGFZa’ glucocorticoid concentrations were not altered
significantly (Figure 3). However, glucocorticoids increased
rapidly to peaks of 22.3, 34.9 and 39.0 ng/ml at 1.0 hr
following injection of 15, 25 and 35 mg PGFZa' respectively.
Peak glucocorticoid concentrations were linearly related to
dose of PGFZa given. Glucocorticoids remained above pre-
treatment values until 4 hr after injection. In previous
work, Louis 33 31. (1974) gave (im) 35 mg PGan to heifers;
glucocorticoids increased to 100 ng/ml at .5 hr and returned
to pretreatment concentrations by 2.0 hr after injection.
Hafs (1975) reported 2- and 4-fold increases of glucocorti-
coids (over basal concentrations of 3.0 ng/ml) in bulls
given 20 and 40 mg of’PGFZa, respectively. Peak concentra-
tions were obtained at 1.0 hr after PGFZa and glucocorticoids
remained above pretreatment values for 4 hours.

The duration of this PGFZa-induced increase of blood
serum glucocorticoids in lactating dairy cows is similar to
that reported for bulls (Hafs, 1975), but is longer than
values reported for heifers (Louis 33 31., 1974). Perhaps,
glucocorticoids are cleared more readily in heifers than

lactating cows, or the difference may be directly related

Figure 3.

35

Blood serum glucocorticoids in lactating cows
given (im) PGFZa' Standard errors ranged from
0.3 to 6.1 ng/ml and were generally proportional
to the means.

 

Glucocorticoids (ng /ml)

 

36

 

O .5 LC I5 20,—.

Hours From Injection

37
to ACTH secretion. In addition, the PGFZa-induced increase
of glucocorticoids in lactating cows is intermediate in
magnitude to reported increases for bulls and heifers after
similar doses of PGFZa'

Work by Peng 33 31. (1970) indicates that PGFZa-induced
increases in glucocorticoids are mediated by the hypothala-
mus in rats. Recent work in heifers (Stellflug 33 31.,
1977) and bulls (Haynes 33 31., 1977) indicates that PGFZa
acts on the anterior pituitary or hypothalamus but not on
the adrenal gland. Thus, the reduced glucocorticoid
increase in lactating dairy cows observed in the present
study as compared to that reported for heifers may be the
result of a lactation-induced alteration of hypothalamic or
anterior pituitary response to PGF2a' In bulls, steroid
environment may have a role in modulating PGan-induced
changes in blood glucocorticoids.

Prolactin. Prior to PGan injection, blood serum
prolactin averaged 37.8 ng/ml. In contrast, Tucker,33 31.
(1975) reported pretreatment prolactin concentrations of
14.9 to 16.1 ng/ml in lactating cows 4 months post—partum.
This dissimilarity is not*surprising, since baseline pro-
lactin concentrations are influenced by temperature
(Wettemann and Tucker, 1974), photoperiod (Bourne and
Tucker, 1975) and handling stress (Tucker, 1971); all
factors which may have differed in these studies.

In this study, when cows were given saline or 5 mg

PGan, prolactin concentrations were not altered within

38
4 hr (Figure 4). However, prolactin increased rapidly to
peaks of 56.8, 84.5 and 95.6 ng/ml at 1.0 hr following
injection of 15, 25 and 35 mg of PGFZa’ respectively. As
with glucocorticoids, peak prolactin concentrations were
linearly related to the dose of PGFZa' Prolactin returned
to pretreatment concentrations within 4 hr after injection.
These changes in prolactin represent 1.5—, 2.0— and 2.5-fold
increases over pretreatment values. Louis 33 31. (1974)
reported 5-fold increases in prolactin when heifers were
given (im) 15, 30 or 60 mg PGFZa; prolactin increased from
26 ng/ml to approximately 150 ng/ml .5 hr after PGF

20.

injection. Hafs (1975) gave (im) 40 mg PGF to bulls and

2a
reported a 2-fold increase in prolactin, from approximately
10 ng/ml to 20 ng/ml, within 1.0 hour. However, prolactin
did not increase in bulls given 5 or 20 mg PGFZa'
As with glucocorticoids and growth hormone, differences
in prolactin secretion among cows, heifers and bulls in
response to PGan may be the result of physiological changes
accompanying lactation or differences in steroid environ-
ment. In addition, photOperiod and temperature effects on
prolactin complicate comparisons among separate reports.
Milk Yield; Percent M1lk Fat and Percent Total Milk
Protein. The average post-treatment milk yield, adjusted
by covariance for pretreatment milk yield, was 22.4 kg/day
(Table 2). PGFZa treatment did not significantly alter
milk yield during the 7 days after injection. Adjusted

post-treatment percent milk fat and percent total protein

Figure 4.

39

Blood serum prolactin in lactating cows given
(im) PGFZa’ Standard errors ranged from 4.7 to
24.5 ng/ml and were generally proportional to
the means.

Proloctin(ng/ml)

I00

80

60

4O

20

 

40

 

I

-.5 o .5 LC I.5 2.0' 4.0

Hours From Injection

41

Table 2. Daily milk yield, milk fat and protein during
7 days after PGF

 

 

2a'
PGF2 Milk Fat Protein
0:

(mg) (kg) (%) (7»)

0 22.2 i .881 3.09 1 .06 3.29 i .03

5 22.7 1 .8 - 3.23 1 .06 3.25 i .03
15 22.4 i .5 3.18 i .05 3.29 1 .02
25 22.7 i .8 3.21 1 .06 3.32 1 .03
35 21.8 I .8 3.17 3: .06 3.31 1 .03

 

a Entries are mean.i standard error for n = 11 to 13,
adjusted for pretreatment values.

42
were 3.18 and 3.29, respectively. As with milk yield, PGFZa
did not significantly alter milk fat or total protein.

The data from this thesis do not provide significant
evidence that PGan has deleterious effects on milk yield,
percent milk fat or percent total milk protein. Although
serum concentration of glucocorticoids, growth hormone and
prolactin were altered in lactating dairy cows given.PGF2a,
apparently these acute changes in hormone concentrations do
not significantly alter milk production. Brush (1960)
reported that glucocorticoids decreased milk production, but
only when serum glucocorticoids reached very high concentra-
tions (200 ng/ml). Likewise, Machlin (1973) reported
increased milk production in lactating cows given growth
hormone, only after relatively high doses (30 mg)
administered for extended periods (10 days).

These milk production data also were analyzed to compare
cows with luteolysis to cows with no luteolysis (Table 3).
All cows with luteolysis exhibited estrous behavior, but no
cows without luteolysis were observed in estrus during the
7-day milk sampling period. The data in Table 3 led to the
conclusion that luteolysis,and subsequent estrus did not
significantly alter milk yield, percent milk fat or percent
total milk protein.

McClandish (1926) reported a decrease in milk
production on the day of estrus but a compensatory produc-
tion 2 days prior to estrus. Similarly, Hooper and Brown

(1919) reported decreased milk production on the day of estrus.

43

Table 3. Daily milk yield, milk fat and protein among cows
with or without luteolysis in response to PGFéa.

 

Response Milk Fat Protein

 

(kg) (7») (%)
No Luteolysis (n=21) 22.7 3 .5 3.06,: .06 3.25,: .03
Luteolysis (n=3l) 22.4 + .4 3.19,: .05 3.20 3 .03

 

a Entries are mean,: standard error adjusted for
pretreatment values.

44

However, in both studies some cows had no change in milk
production on the day of estrus or had slight increases.
Copeland (1929) found slight increases in milk production

on the day of estrus. In addition, he reported an equally
small decrease in butter fat. Thus, previously reported

data indicate conflicting evidence of the effect of estrus

on milk yield and composition. The data presented in this
thesis suggest that any changes in milk yield and composition
occurring at estrus are not sufficient to substantially alter

average daily milk yield or milk composition.

GENERAL DISCUSSION

The results from this study indicate that 15 mg is the
minimal dose of PGan which when given to lactating dairy
cows results in an incidence of luteolysis not significantly
different from higher doses. Complete luteolysis occurred
in 88% of the cows given 15, 25 or 35 mg PGFZa' This agrees
with previously reported values for luteolytic response in
suckled beef cows (Hafs 33 31., 1975b) and heifers (Louis
33 31., 1973). Analysis of the incidence of luteolysis
for lactating dairy cows in the present study suggested
(P<.l5) that cows given 35 mg of PGan had higher incidences
of estrus. Additional work is necessary to determine the
validity of this observation.

Blood serum progesterone in cows with complete luteolysis
declined approximately 50% within 6 hr after injection.
Following luteolysis in these cows (1) estrus began at
72.8,: 3.7 hr, (2) the peak of an ovulatory surge of LH
occurred at 80.3 1 5.3 hr, and (3) ovulation occurred at
100.1 1 3.8 hours. These results are similar to those
previously reported for heifers by Louis 33 31. (1973) and
E1ving,33,31. (1975). Some cows given 5, 15 or 25 mg PGFZa
did not have completeluteolysis within 132 hours.

Progesterone concentrations in these cows declined within

45

46
12 hr, but returned to normal values within 24 hr after

PGF Stellflug 33 31. (1976) reported similar progester—

2a'
one changes when partially luteolytic doses of PGFZa were
given to heifers. Apparently, luteal biosynthesis of pro-
gesterone is reduced transiently following even non-luteolytic
doses of'PGFZa. In support of this observation, Lahav 33 31.

(1976) reported that PGF a blocked LH stimulated cyclic AMP

2
accumulation in isolated rat ovaries.

The results of this thesis indicate that PGF is an

2d
effective method of estrus synchronization in lactating dairy
cows. Hafs,33 31. (1975b) reported that fertility of heifers
and suckled beef cows to inseminations at 70 and 88 hr after
PGFZa is comparable to fertility of animals inseminated at
observed estrus. Since reproductive events following luteo-
lysis (interval to estrus, LH surge and ovulation) in
lactating dairy cows are similar to those reported for
heifers and beef cows, breeding at a predetermined interval

following PGF is feasible for lactating dairy cows.

2a
Predetermined breeding would eliminate the need for estrous
detection and encourage dairymen not using artificial
insemination to do so.

PGF —induced increases of growth hormone,

2a
glucocorticoids and prolactin in lactating cows were less
than increases reported for heifers (Louis 33,31., 1973),
but greater than increases reported for bulls (Hafs, 1975)
following similar doses of’PGFZQ. Differences in growth

hormone, prolactin and glucocorticoid secretion among

lactating cows, heifers and bulls following PGan injection

47
may be due to altered hypothalamic or pituitary responses to
PGFZa' Another possible explanation for the variance in
reported PGFZa-induced secretion of these hormones is that
weight differences among animals given similar doses of
PGan alter the concentration of PGFZa that reaches the
hypothalamus or pituitary. In addition, differences in
reported peak concentrations of these hormones after PGF20L
treatment may be due to differences in clearance rate. Data
from this thesis and previous work are not sufficient to
determine factors responsible for these differences in.PGF2a-
induced changes of growth hormone, glucocorticoid and
prolactin.

The results of this study indicated that milk yield,
percent milk fat and percent total milk protein were
unaltered following various doses of PGFZa' Apparently,
the acute increases in growth hormone and glucocorticoids
were insufficient to cause changes in milk production
reported following large chronic doses of these hormones
(Machlin, 1973; Braun, 1970).

PGFZa given to lactating dairy cows is an effective
method of synchronizing estrus to facilitate the use of
artificial insemination. (The acute changes in growth
hormone, glucocorticoids and prolactin concentration
following PGan treatment fail to alter milk yield, percent

milk fat or percent total milk protein.

SUMMARY AND CONCLUSIONS

Earlier reports indicate that PGFZa is an effective
luteolysin in dairy heifers and suckled beef cows. In

addition, PGF is an effective method of synchronizing

2a
estrus and ovulation for controlled breeding. PGFZa given
to heifers induces increases in blood serum growth hormone,
glucocorticoids and prolactin; three hormones necessary for
the initiation and maintenance of lactation. The purpose
of this thesis was to (1) determine the minimal dose of
PGFZa which when given to lactating dairy cows results in
an incidence of luteolysis not significantly different from
higher doses, (2) characterize changes in blood serum
growth hormone, glucocorticoids and prolactin after various
doses of PGFZa and (3) determine the acute response of milk
yield, percent milk fat and percent total protein to various
doses of PGFZa'
The results obtained in this thesis indicate that
15 mg is the minimal intramuscular dose of'PGF2a for
lactating dairy cows resulting in an incidence of luteolysis
not significantly different from higher doses. Following

PGan-induced luteolysis the mean interval from injection to

the beginning of estrus, peak of the ovulatory surge of LH

48

49

and ovulation were 72.8, 80.2 and 100.1 hr, respectively.
The average interval from the beginning of estrus to
ovulation was 28.3 hours.

Blood serum glucocorticoids and prolactin increased at
1.0 hr following injection of 15, 25 or 35 mg of PGFZa'
Growth hormone increased slightly within .5 hr after
injection of 35 mg PGFZa' All hormones returned to pre-
treatment concentrations within 4 hr, so this effect of
PGFZa is transitory. Perhaps the short duration of the
elevations in hormones explains why various doses of PGFZa
failed to significantly alter milk yield, percent milk fat
or percent total milk protein in lactating dairy cows.

I conclude that PGFZa is an effective method of estrous

synchronization for lactating dairy cows, without deleterious

effects on milk yield or gross milk composition.

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