A study of estrus synchronization with PGF2alpha in Brahman heifers

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A study of estrus synchronization with PGF2alpha in Brahman heifers proposal of a new system
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Cornwell, Deborah Gwen, 1953-
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Thesis (Ph. D.)--University of Florida, 1989.
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Includes bibliographical references (leaves 128-155).
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by Deborah Gwen Cornwell.
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Vita.

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A STUDY OF ESTRUS SYNCHRONIZATION WITH PGF2a IN BRAHMAN HEIFERS:
PROPOSAL OF A NEW SYSTEM















By

DEBORAH GWEN CORNWELL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1989


L

































... the barnyard was an expression of something that was real, vital, and
fluid, that ... was of natural and spontaneous growth, that ... turned with its
surroundings, that ... was a part of the life that offered itself to her.

Edith Summers Kelley
Weeds, 1923















ACKNOWLEDGEMENTS


During my years at the University of Florida there have been many people who

have graciously extended their friendship and help. I am certain I will be unable to

thank them all in this brief piece so I ask, in advance, their forgiveness for any possible

error of omission.

First, I must express my sincere appreciation to Dr. M. J. Fields, chairman of

my supervisory committee. Throughout this long and arduous ordeal he has shown an

extraordinary patience and has always been confident I would complete this

dissertation, even when I had doubts. His guidance and friendship have made this a

challenging and rewarding experience.

I would like to thank Dr. J.F. Hentges, Jr., for his years of counsel and

friendship. I am especially grateful for his understanding and his willingness to serve

on my committee even though he is "officially" retired. The opportunities and

encouragement he provided are the reasons I began and continued my graduate

studies.

I am grateful to Dr. M. Drost for his knowledge of applied reproductive

physiology and the nature of cows. It has been a real pleasure working beside him in

the cow pens. I would also like to thank Dr. L.H. Larkin and Dr. C.J. Wilcox for serving

on my committee. Dr. Larkin very graciously provided an outsider's insight (even

though he said he knew very little about cows) and Dr. Wilcox inspired me to design

and conduct my experiments correctly (even though I knew very little about statistics

before I took his course).









Special thanks go to Dr. Shou-mei Chang for her special humor and patience

when I took time out just to bug her. I would also like to thank Dr. Ciro Barros for his

recommendations and suggestions and for his very kind tolerance as I used his

computer to write this dissertation. I greatly value their friendship and experience.

Thanks go to Melody Stallings, the new blood in the lab, for her fresh

enthusiasm and ability to take a joke. She never lost her temper even when cleaning

up the acetic acid. She is a good friend and wiser than her years. I wish her success

in all her future endeavors.

Words are insufficient to express my gratitude for the friendship of Normandie

Vreeland. From our first day of class at the University of Florida it was as though we

were in each other's heads. When times were hard and my enthusiasm waning, she

encouraged me to "just get on with it." Through Normandie, I acquired other dear

friends. Marjorie, Norman and Meta Vreeland have taken me into their home and

made me feel like part of their family. It sounds trite to say that I am grateful for their

love, caring and kindness but still it must be said.

Finally, my deepest love and appreciation must go to my parents, Mae and

Paul Cornwell, and my siblings for all the years of patience and caring that allowed me

to pursue my studies in animal science. It is their respect for books and knowledge

that started me toward my college career. Even as I became their perpetual student

they believed I would eventually finish, and they still call to make sure I am eating and

sleeping. They are my anchor.















TABLE OF CONTENTS


ACKNOW LEDGEMENTS................................................................................................ iii

LIST OF FIGURES.......................................................................................................... vii

LIST OF TABLES.................................................................................................. ........ ix

LIST OF APPENDIX TABLES............................................................ ......................... x

ABSTRACT...................................................................................................................... xii

INTRODUCTION..... .............................................................................................................. 1

REVIEW OF THE LITERATURE.............................................................................. ....... 3

Artificial Insemination, Estrus Synchronization, and the Role
of the Corpus Luteum ................................................... .................... 3
Interrelationship of the Uterus and Ovary............................................ ......... 7
PGF2a as the Uterine Luteolysin..................................................................... 13
Theory of Action and Hormonal Regulation of PGF2a............................... 19
Hormonal Influences and Controls............................................. ................. 27
Practical Use of PGF2a for Estrus Synchronization.................................... 39
Synchronization and Al in the Brahman.............................................................. 44
Effect of Plasma Progesterone Concentrations on Pregnancy.................... 47

EXPERIMENTAL PROCEDURE............................ .......... .......................... 49

General Procedure............................................................................................. 49
Cattle Handling Facilities.................................................... ......... ................. 52
Radioimm unoassay for Plasma Progesterone.................................... ......... 52
Experimental Protocol....................................................................................... 56
Statistical Analysis of Data............................................................................ 61

RESULTS AND DISCUSSION....................................................................................... 63

SUMMARY................................................................................ ................................. 99

APPENDIX A RADIOIMMUNOASSAY......................................................................... 101

APPENDIX B RAW DATA............................................ .......................................... 110









APPENDIX C STATISTICS...................................................................................... 119

LITERATURE CITED.................................................................................................. 128

BIO GRAPHICAL SKETCH .............................................................................................. 156















LIST OF FIGURES


Figure Page

1 METABOLIC PATHWAY FROM ARACHIDONIC ACID TO THE
BIOLOGICALLY ACTIVE PROSTAGLANDINS PGE2, PGD2, AND
PG F2a.............................................................. ................................. 16

2 WEIGHT CHANGES FROM WEANING THROUGH EXPERIMENTAL
PERIOD FOR HEIFERS IN TRIAL 1 AND TRIAL 2........................... 51

3 CATTLE HANDLING FACILITIES WITH CORRAL CONSTRUCTED FROM
IN-GROUND SILAGE BUNKER................................. ............... 54

4 PLASMA P4 PROFILES FROM D 2 TO D 14 OF THE ESTROUS
CYCLE PRIOR TO PGF2a TREATMENT FOR ALL HEIFERS IN
TRIAL 1 (BY TREATMENT)........................................ ................... 65

5 PLASMA P4 PROFILES FROM D 2 TO D 14 OF THE INDUCED AND
CONTROL ESTROUS CYCLES AFTER PGF2a TREATMENT FOR
TRIAL 1 (BY TREATM ENT)................................................................ 67

6 PLASMA P4 PROFILES FROM D 2 TO D 14 OF THE INDUCED
ESTROUS CYCLE FOR HEIFERS THAT RESPONDED TO PGF2a
TREATMENT AND FROM D 6 TO D 18 AFTER THE PGF2a
INJECTION FOR NONRESPONDING HEIFERS (TRIAL 1)............... 74

7 P4 CONCENTRATIONS FOR HEIFER #59 (RESPONDER) AND HEIFER
#72 (NONRESPONDER) IN TRIAL 1.................................... ...... 76

8 PLASMA CONCENTRATIONS OF P4 FOR ALL NONRESPONDING
HEIFERS IN TRIAL 1................................................. ................... 78

9 PLASMA P4 PROFILES FROM PGF2a INJECTION FOR ALL INDUCED
AND CONTROL ESTROUS CYCLES IN TRIAL 2 (BY
TREATM ENT)........................................................ ......................... 85

10 PLASMA P4 CONCENTRATIONS (MEAN SE) FROM 1 D BEFORE
PGF2a INJECTION FOR ALL RESPONDING AND
NONRESPONDING HEIFERS IN TRIAL 2..................................... 87








Figure Page

11 PLASMA P4 CONCENTRATIONS FROM 1 D BEFORE PGF2a FOR ALL
NONRESPONDING HEIFERS AFTER PGF2a INJECTION ON D 7
OR D 10 OF THE ESTROUS CYCLE (TRIAL 2)............................. 89

12 PERCENT OF RESPONDING HEIFERS EXHIBITING ESTRUS ON
SPECIFIC DAYS AFTER PGF2a INJECTION ON D 7 OR D 7 AND
D 8 OF THE ESTROUS CYCLE (DEGREE OF SYNCHRONY)........ 94















LIST OF TABLES


Table Page

1 EXPERIMENTAL DESIGN FOR TRIAL 1: TO DETERMINE IF THE
PGF2a INDUCED CL PRODUCES LOWER CONCENTRATIONS
OF PLASMA P4 THAN THE SPONTANEOUSLY OCCURRING CL.. 57

2 EXPERIMENTAL DESIGN FOR TRIAL 2: TO FURTHER EVALUATE THE
EFFECT OF DAY OF CYCLE ON WHICH PGF2a IS GIVEN ON
THE EXPRESSION OF ESTRUS................................... ............... 59

3 EXPERIMENTAL DESIGN FOR TRIAL 3: TO DETERMINE IF TWO
INJECTIONS OF PGF2a GIVEN 24 HOURS APART INDUCE
ESTRUS MORE EFFECTIVELY THAN A SINGLE INJECTION.......... 60

4 MEANS OF PLASMA P4 CONCENTRATIONS (NG/ML) ON DAYS 2 TO
14 OF THE ESTROUS CYCLE BEFORE AND AFTER PGF2a
INJECTION (TRIAL 1)................................................ .................... 68

5 SYNCHRONIZATION AND PREGNANCY RATES OF PGF2a TREATED
AND CONTROL HEIFERS (TRIALS 1, 2, AND 3).......................... 72

6 MEAN DAY OF ESTROUS CYCLE AT THE TIME OF SECOND PGF2a
INJECTION (TRIAL 1)................................................ .................... 80

7 SYNCHRONIZATION RATES AND INTERVAL FROM INJECTION TO
ESTRUS ON D 7 OR D 14 OF THE ESTROUS CYCLE (TRIALS 1
AND 2 COMBINED).............................................. ........................ 82

8 ESTRUS RESPONSE AND INTERVAL FROM INJECTION TO ESTRUS
OF BRAHMAN HEIFERS TREATED WITH TWO INJECTIONS OF
PGF2a GIVEN 24 HOURS APART WITH THE FIRST ON RANDOM
DAYS OF THE CYCLE............................................. ..................... 97

9 CIRCUMSTANCE AND TIME OF DAY (AM OR PM) OF DETECTION OF
ESTRUS BEFORE AND AFTER PGF2a TREATMENT (TRIALS 2, 3
AND 4)...................................... ................. ..................................... 98















LIST OF APPENDIX TABLES


Table Page

1 ALIQUOT VOLUMES FOR P4 ASSAY (J).................................................... 106

2 VALIDATION FOR P4 ASSAY DR. L. FLEEGER ANTIBODY.................. 107

3 VALIDATION FOR P4 ASSAY VENEZUELAN ANTIBODY....................... 108

4 WEIGHTS OF HEIFERS FROM BIRTH THROUGH TRIAL 1 (LB)............... 109

5 WEIGHTS OF HEIFERS FROM BIRTH THROUGH TRIAL 2 (LB)............... 111

6 PLASMA P4 CONCENTRATIONS BEFORE TREATMENT (PG/ML) -
T R IA L 1 ................................................................................................... 112

7 PLASMA P4 CONCENTRATIONS AFTER TREATMENT (PG/ML) -
T R IA L 1 ................................................................................................... 113

8 PLASMA P4 CONCENTRATIONS AFTER TREATMENT (PG/ML) -
T R IA L 2 ................................................................................................... 114

9 ESTRUAL RESPONSE TO PGF2a TRIAL 3.......................................... 116

10 ESTRUAL RESPONSE TO PGF2a TRIAL 4............................................. 117

11 MODEL 1 USED TO TEST FOR HETEROGENEITY OF
REGRESSION (P4 DATA) TRIAL 1................................................ 119

12 MODEL 2 USED TO TEST FOR HETEROGENEITY OF
REGRESION (P4 DATA) DIFFERENCE DUE TO
TREATMENT TRIAL 2........................................................................ 120

13 MODEL 3 USED TO TEST FOR HETEROGENEITY OF
REGRESSION (P4 DATA) DIFFERENCE DUE TO
RESPO NSE TRIAL 2.......................................................................... 121

14 CHI-SQUARE ANALYSIS OF SYNCHRONIZATION RATES
TO PGF2a INJECTION ON D 7 OR D 14
(TRIALS 1 AND 2 COMBINED)..... ....................................................... 122








Table


15 MODEL 1 USED TO TEST FOR HETEROGENEITY OF 123
REGRESSION (P4 DATA) TRIAL 2...............................................

16 MODEL 2 USED TO TEST FOR HETEROGENEITY OF
REGRESSION (P4 DATA) DIFFERENCE DUE TO
TREATMENT TRIAL 2.................................................................. 124

17 MODEL 3 USED TO TEST FOR HETEROGENEITY OF
REGRESSION (P4 DATA) DIFFERENCE DUE TO
RESPO NSE TRIAL 2.................................................................... 125

18 CHI-SQUARE ANALYSIS OF SYNCHRONIZATION RATES
TO TREATMENT WITH EITHER 1 OR A SERIES OF
2 INJECTIONS OF PGF2a WITH THE SECOND
INJECTION GIVEN 24 H AFTER THE FIRST TRIAL 3.................. 126

19 CHI-SQUARE ANALYSIS OF SYNCHRONIZATION RATES
ON THE 7 D FOLLOWING EITHER 1 OR A SERIES OF
2 INJECTIONS OF PGF2a WITH THE SECOND
INJECTION GIVEN 24 H AFTER THE FIRST (DEGREE
OF SYNCHRONY) TRIAL 3........................................................... 127

20 T-TEST FOR INTERVAL FROM PGF2a INJECTION (LAST OR ONLY)
TO ESTRUS TRIAL 3.................................................................... 127


Page








Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


A STUDY OF ESTRUS SYNCHRONIZATION WITH PGF2a IN
BRAHMAN HEIFERS: PROPOSAL OF A NEW SYSTEM

by

Deborah Gwen Cornwell

December, 1989

Chairman: Dr. M. J. Fields
Major Department: Animal Science

A study was conducted to examine the efficacy of a natural prostaglandin F2a

(PGF2a) for synchronizing estrus in Brahman heifers. Estrous response rate and plasma

progesterone (P4) concentrations in heifers treated with PGF2a were compared to

determine whether induced corpora lutea (CL) produced lower concentrations of P4 than

spontaneously occurring CL.

Fewer heifers expressed estrus within 7 d following a single PGF2a injection on

d 7 of the estrous cycle than when injected on d 14 (61% vs. 100%, P<.05). Heifers

injected on d 7, d 10, d 14, or d 18 demonstrated a graduated response rate (50%,

67%, 100% and 100%, respectively). Importantly, PGF2a induced a normal functioning

CL since plasma P4 profiles did not differ between induced and spontaneous estrous

cycles. When plasma samples were collected from 1 d before injection to 3 d after

estrus (or to 6 d after injection in non-responders), P4 concentrations decreased by 12 h

after injection in all heifers. Although there was a precipitous decline in P4, heifers that

failed to express estrus had P4 concentrations that began to increase within 48 h after

injection and reached concentrations greater than in heifers exhibiting estrus (P<.001).








Two injections of PGF2a administered at a 24 h interval induced estrus more

effectively than a single injection (97% vs. 72%, P<.02). Heifers treated with two

injections at a 24 h interval were more tightly synchronized than heifers given a single

injection (P<.06), with 94% of the double injection heifers expressing estrus in a 36 h

period from 2.0 to 3.5 d after the first injection.

Data indicate a decreased estrual response to PGF2a when given early in the

estrous cycle. Injection of PGF2a on d 7 or d 10 initiates a decline in plasma P4 but

fails to pecipitate complete luteolysis in all heifers. A system is proposed that uses a

series of three injections (second injection given 11 d after the first and third given 24

h after the second) that would result in more animals in estrus and in a tighter

synchrony necessary for artificial insemination by appointment.















INTRODUCTION


The American Brahman and other Zebu breeds of cattle play a vital role in the

beef industry of tropical and subtropical areas of the world. Bos indicus cattle are

exceptionally well adapted to the hot, humid climate that typifies the tropics. Their

ability to flourish under conditions of heat extremes, insect infestations and endzootic

disease make use of Zebu cattle in purebred and crossbred operations in the tropics

highly desirable and, in many circumstances, essential.

One basic requirement of successful cattle production is continuous progress

towards genetic improvement in the herd. The most expedient way to hasten genetic

progress is to use artificial insemination (Al) to increase the proportion of cattle mated

to superior sires. Naturally bred Brahmans are reported to have lower pregnancy rates

when compared to Bos taurus breeds (Burns et al., 1959; Kincaid, 1962; Koger et al.,

1973; Crockett et al., 1978). Brahman producers have reported the pregnancy rate to

Al after prostaglandin F2a (PGF2a) estrus synchronization is also generally lower than

that expected in a Bos taurus herd. These reports were corroborated by Tucker et al.

(1982) and Landivar et al. (1985), but Adeyemo et al. (1979) found no difference in

pregnancy rates to Al between Bos taurus and Bos indicus cattle. Randel et al. (1984)

reported pregnancy rate was higher in Brahman cows than in Brangus cows that had

been synchronized with the PGF2a analog, alfaprostol.

Results of synchronization attempts with a PGF2a analog (cloprostenol) in

Brahman-crossbred females have been inconclusive (Hardin et al., 1980a,b). Hardin









2

and Randel (1982) reported Brahman cows treated with cloprostenol on d 8 to 12 of

the estrous cycle subsequently developed smaller corpora lutea (CL) with lower

progesterone (P4) content than the CL of untreated cows. In addition, serum P4 was

lower in Brahman cows from d 2 to 13 of a cloprostenol induced estrous cycle than in

naturally occurring cycles. The authors suggested impaired development of the

induced CL could be responsible for the lower fertility of Brahman and Brahman-type

females and that formation of this sub-functional CL might be one factor in the poor

estrus response to prostaglandin synchronization. More recently, Hansen et al. (1987b)

reported the CL formed following regression induced by the PGF2a analog alfaprostol

had fewer large and small luteal cells (in heifers) and lower in vitro P4 production in

response to LH when compared to the CL formed following spontaneous estrus (in

heifers and cows).

The studies presented here were designed to 1) examine the efficacy of a

natural PGF2a in synchronizing estrus in Brahman heifers, and 2) to determine whether

the PGF2a-induced CL produced lower concentrations of plasma P4 than

spontaneously occurring CL in Brahman heifers.















REVIEW OF THE LITERATURE


Artificial Insemination, Estrus Synchronization,
and the Role of the Corpus Luteum

Artificial insemination is the placement of spermatozoa in the female

reproductive tract by artificial instead of natural means. According to legend, the first

Al was perpetrated in 1322 when an Arab chieftain used artificial methods to breed a

valuable mare with semen surreptitiously collected from the sheath of a stallion

belonging to an enemy tribe (Ensminger, 1976). The first research in Al of animals was

conducted with dogs in 1780 by the Italian scientist Lazarro Spallanzani (Foote, 1986).

By the next century, American scientists were artificially inseminating mares that had

failed to settle by natural service (Ensminger, 1976) and by 1907 the Russian scientist

Ivanov was reporting success in the Al of mares, cows and ewes as a method for

widespread genetic improvement in livestock (Foote, 1986). Ivanov developed

procedures whereby semen was collected from the epididymides of slaughtered bulls,

diluted, and used to Al cows. If the cows were some distance from the abattoir, the

epididymides were refrigerated and spermatozoa were removed later for insemination.

This technique allowed the Al of cows at a distance of up to 2 h travelling time (Willet,

1956).

Al of cattle was first performed in the United States in 1937 and 1938 at the

Agricultural Experiment Station in Minnesota (Foote, 1986). Cattle producers were

quick to recognize the advantages of Al in allowing them access to superior sires that

previously were available exclusively to the owners. With the advent of technology for

3









4

preservation of semen by freezing, long-term storage and long-distance transport

became routine. By 1982, two-thirds of the 11,000,000 dairy cows in the U.S. were Al,

but only 4% of the beef cows (Foote, 1986).

While the benefits of Al are apparent, the implementation of a successful

program requires superior management of reproduction in a herd. In dairies it is a

relatively simple matter to Al the dairy cow at the proper time following detection of

estrual behavior (receptiveness of the cow to sexual overtures of the bull). This system

of observation and breeding is less readily accomplished in beef cattle herds. In

general, beef cattle are observed only occasionally, and if an Al program is desired,

special handling procedures and facilities must be established. The necessary detection

of estrus is time-consuming, difficult, and subject to human error. Systems of estrus

synchronization facilitate the prognostication of the time of estrus with a reasonable

degree of accuracy. This minimizes the amount of time a cattle producer must invest

in estrus detection and may, with some procedures, make it possible to Al at an

appointed time regardless of the manifestation of estrus (Hafez, 1987). Artificial

insemination is most successful when the time of estrus expression is regulated and

the hour and date of insemination precisely determined.

Estrus synchronization systems are ways in which times of estrus and ovulation

in a herd are regulated or "synchronized" so that all herd members are at the proper

stage of the estrous cycle for insemination at one time. Not only does synchronization

facilitate the Al program, but it produces the added benefit of more cows calving earlier

in the calving season.

In the cattle industry, as in most other businesses, "time is money." To cattle

producers this "time" is best measured by calving interval. Dairymen recognize that









5

milk per day of calving interval decreases as days open increase. This is because

additional days open result in more days in milk, which extend the low-producing part

of lactation, and in more days dry. Each additional day open results in 4.5 kg less

milk from heifers and 8.6 kg less milk from cows (Olds et al., 1979). Barr (1975)

determined that Ohio dairies increased calving interval by 14.7 d due to failure of the

cow to conceive once inseminated, but added 40.3 d due to failure of the herdsman to

notice estrus. Dairy cows and heifers treated with a PGF2a analog were Al sooner in

the season and became pregnant sooner than untreated controls (Seguin et al., 1983).

Estrus synchronization with PGF2a resulted in conception occurring 22 d earlier in

treated dairy cows than in cows that were Al without synchronization (Plunkett et al.,

1984). PGF2a-synchronized Al also reduced the interval to estrus after parturition in

Holstein cows (Lucy et al., 1986).

The value of synchronization is also evident in beef herds. Lambert et al.

(1976) reported fertility was greater in beef cows synchronized with PGF2a and that the

system resulted in more cows conceiving early in the breeding season. In one study,

beef cows were treated with 25 mg PGF2a on d 5 of the breeding season unless they

had previously been seen in estrus on d 1 to 4 and subsequently Al. All treated cows

were Al at the detected estrus until 80 h after injection at which time all remaining

cows in the groups were Al. Cows in control groups were not synchronized but were

Al at estrus throughout the breeding season. Estrus-synchronized cows conceived

earlier in the season, as 45.5% of the PGF2a treated cows vs 26.1% of the control

cows were pregnant to inseminations during the first 10 d of the breeding season

(Higgins et al., 1981).









6

One early method for altering length of estrous cycle and thus controlling time

of estrus was manual extirpation of the well-developed CL from the ovary. This

resulted in 90% of treated cows expressing estrus within 2 to 4 d (Willet, 1956). There

is some hazard associated with this method in that hemorrhage and(or) adhesions may

occur and possibly result in permanent damage to the treated cow. This rather crude

technique, however, results in the same outcome sought by hormonal controls, i.e.

destruction of the CL. Estrus synchronization methods seek to precipitate a premature

demise of the CL at a predetermined and synchronous time in a group of treated

animals.

The timing of estrus in domestic livestock is regulated by the production of the

steroid hormone progesterone (P4) from the CL (Hansel and Convey, 1983).

Progesterone prepares the uterus for reception and growth of the embryo. If

pregnancy does not occur the CL begins a natural regression at about d 17 of the

estrous cycle (estrus = d 0). Regression of the CL is accompanied by declining P4

concentrations and within 1 to 5 d of the beginning of this drop in P4 estrus results

and is followed by ovulation (Stabenfeldt et al., 1969).

Progesterone acts by employing a negative feedback control on luteinizing

hormone (LH) from the hypothalamus. As long as P4 concentrations are elevated the

pre-ovulatory surge of LH is suppressed. It is this LH surge that precipitates estrus

and ovulation (Hafez, 1987). Thus estrus synchronization is the control of the existence

of the CL (Hansel and Convey, 1983). An understanding of the processes by which

estrus synchronization is realized requires knowledge of the manner in which natural

luteolysis occurs.









7

Interrelationship of the Uterus and Ovary

Loeb (1923, 1927) was the first to document a link between the uterus and

ovarian function in a series of classic experiments in which guinea pigs were

hysterectomized. Removal of the uterus resulted in preservation and continued

function of the CL. The length of extended diestrus following partial hysterectomy was

inversely proportional to the amount of uterus remaining after surgery. He concluded

the uterus was implicated in control of the demise of the CL in the guinea pig.

The uterus appears to exert the same regulating influence in domestic livestock.

Wiltbank and Casida (1956) found that hysterectomy prolonged the life of the CL in

ewes and cows. Hysterectomy in heifers resulted in retention of the CL for at least 270

d (Anderson et al., 1962). It was suggested that CL regression was dependent on

stimulus from the uterus. In the sow, total hysterectomy before d 16 of the estrous

cycle results in protracted diestrus (Spies et al., 1960; Anderson et al., 1963), but if the

uterus is removed between d 16 and 18, estrus is usually expressed at the expected

time. Dissimilarly, regression of the CL, estrus and subsequent ovulation are prevented

by hysterectomy up to the last day of the cycle in ewes (Kiracofe et al., 1966). In the

mare, removal of the uterus results in persistence of luteal activity (Stabenfeldt and

Hughes, 1977), but functional luteolysis (decrease in P4) is often observed after 30 to

40 d (Ginther and First, 1971).

Destruction or damage of the uterine endometrium by corrosives (Anderson et

al., 1961) or infection (Coudert and Short, 1966; Ginther, 1968) causes the CL to be

maintained for extended periods. However, insertion of various intrauterine devices

(IUDs) into the uterine lumen shortens estrous cycle length in cows (Anderson et al.,

1965) and ewes (Ginther et al., 1966a). Distention of the uteri of cows with a sterile









8

gel results in premature estrus (Yamauchi et al., 1967). A plastic spiral coil placed in

one uterine horn in ewes inhibited sperm transport and ovum fertilization on both sides

of the tract. The effects of the IUD were therefore due to something other than

mechanical interference (Hawk, 1970). It became evident that destruction or

devastation of the endometrium prolonged CL lifespan, but irritation or distention

resulted in shortened cycles. Clearly the uterus was providing a luteolytic agent.

Extirpation of the uterus in sheep results in prolonged life of the CL (Moor and

Rowson, 1964). Unilateral hysterectomy, whether alone or in combination with

unilateral ovariectomy, consistently prolonged life of the CL on the ipsilateral ovary, but

failed to affect the CL on the contralateral ovary in the ewe (Inskeep and Butcher,

1966; Moor and Rowson, 1966) or in the heifer (Ginther et al., 1967). Insertion of a

plastic spiral coil into one uterine horn of ewes caused CL regression on the ovary

ipsilateral to the coil (Ginther et al., 1966a)

In related experiments, daily administration of oxytocin (OT) to cycling dairy

heifers during the first week of the estrous cycle resulted in a shortened estrous cycle

of 8 to 12 d (Armstrong and Hansel, 1959). Oxytocin causes premature expression of

estrus in heifers that have the uterus intact or have removal of the uterine horn

contralateral to the CL. This does not occur when OT is injected in heifers completely

hysterectomized or in which the ipsilateral horn is unilaterally removed (Ginther et al.,

1967; Brunner et al., 1969). These observations indicate luteolysis is somehow

mediated by the uterus in a local fashion in these species.

Since the ovary with CL must be in close proximity to the uterus for luteal

regression to occur, it would seem likely that surgical separation would result in

prolonged luteal function in these species. In fact, Goding et al. (1967) and McCracken








9

et al. (1971) reported that when the ovary or uterus in the ewe was autotransplanted to

the neck, this is precisely what happened. However, other researchers have reported

long and irregular cycles in cows following this procedure (Hansel and Snook, 1970) or

normal cycles when the uterus is transplanted to the momentum in ewes (Niswender et

al., 1970). Hansel and Snook (1970) attributed the irregular cycles in the cow to

removal of the ovary from local luteolytic influence of the uterus, but Niswender et al.

(1970) suggested maintenance of the CL in animals with uterus and ovary separated

may be an artifactual result of the surgical procedure. This seems unlikely as sham-

operated animals continue to cycle normally (Wiltbank and Casida, 1956; Ginther et al.,

1966b; Moor and Rowson, 1966; Lamond et al., 1973). In the study conducted by

Niswender et al. (1970), ewes with the entire uterus transplanted to the momentum had a

silicone rubber cannula placed in the cervical end of the uterus for drainage of uterine

effluent into the abdominal cavity. The possibility of a local effect of a uterine luteolytic

agent on the CL within this cavity can not be excluded. When both the ovary and

uterus were transplanted to the neck of ewes, normal luteal function appeared to

confirm that these organs must be contiguous for luteal regression and ovarian cyclicity

(McCracken et al., 1971).

Other species seem to be able to effect luteolysis by other than local means.

In the mare, the route by which the uterus exerts its influence on the CL appears to be

systemic. Total hysterectomy of the mare results in prolonged maintenance of the CL

but unilateral hysterectomy had no effect on cycle length and failed to indicate

involvement of a direct utero-ovarian relationship (Douglas et al., 1976). The uterus of

the pig may deliver its luteolytic agent by both local and systemic routes. Unilateral

hysterectomy does not affect cycle length in gilts (Ginther and First, 1971). In the gilt,









10

autotransplantation of the ovary to the body wall or abdominal muscles does not alter

length of the estrous cycle (Anderson et al., 1963; Hagen et al., 1981).

Some species do not appear to require uterine influence to control the CL.

Removal of the uterus has no effect on luteal function in the rhesus monkey (Burford

and Diddle, 1956), human (Beavis et al., 1969) or cynomolgus monkey (Castracane et

al., 1979).

The evidence is quite clear that the uterus governs the existence of the CL on

the ovary and consequently the estrous cycle (at least in livestock species). But how

does it deliver its luteolytic messenger? Both oviductal and neural routes have been

suggested and ruled out. Transection of the oviduct in the ewe did not influence life or

death of the CL (Baird and Land, 1973). Moore and Nalbandov (1953) demonstrated

IUDs failed to induce estrus in ewes when they were placed in denervated sections of

the uterine horn ipsilateral to the CL. Observation that ewes continued to cycle

normally after separation of the uterus and ovary (Niswender et al., 1970) argues

strongly against either pathway.

The only other apparent route would be via the blood supply. McCracken et al.

(1971) proposed a "counter-current" mechanism in species with local control of the

ovary by the uterus. By this mechanism, there would be a direct venoarterial pathway

between a uterine horn and its adjacent ovary. In those species that have a local route

of control there is close apposition of the uteroovarian vein (primary vessel of uterine

drainage) to the uteroovarian artery. Numerous contacts between vein and artery

occur along the tortuous course of the artery over the vein in sheep (Mapletoft and

Ginther, 1975) and swine (Oxenreider et al., 1965). There is very little association

between these vessels in the mare, a species in which the uterine influence is systemic











(Del Campo and Ginther, 1972; Del Campo and Ginther, 1973). In the cow, as in

sheep and swine, the uteroovarian vein and the ovarian artery are very closely

associated (Ginther, 1974; Mapletoft et al., 1976). Ginther and Del Campo (1974)

reported the uteroovarian arterial anastomosis was significantly more prominent in the

side ipsilateral to the CL than on the contralateral side in cattle.

Barrett et al. (1971) infused the luteolytic substance prostaglandin F2a (PGF2a)

into the ovarian artery of sheep that had ovaries autotransplanted to the neck. This

caused CL regression, except in sheep in which the uterine vein was separated from

the ovarian artery. Strong support for the hypothesis of transport of the uterine

luteolytic agent by the uteroovarian vein is offered by Ginther and Bisgard (1972).

Anastomosis between ipsi- and contralateral uteroovarian veins in sheep resulted in CL

regression when an IUD was placed in the uterine horn contralateral to the ovary with

CL. In similar experiments, Ginther et al. (1973) demonstrated a local uteroovarian

venoarterial pathway for uterine induced luteolysis in ewes. In these studies unilateral

hysterectomy ipsilateral to the CL bearing ovary resulted in luteal maintenance, but

surgical anastomosis of either the main uterine vein or the ovarian branch of the

ovarian artery from the intact side to the corresponding vessel on the hysterectomized

side resulted in CL regression on the hysterectomized side. When this experiment was

conducted with cows the results were the same (Mapletoft et al., 1976).

Interestingly, it has been suggested this local uteroovarian route may also be

the mode by which a blood borne luteotrophin from the gravid horn in ewes could

reach the ovary and effect its unilateral inhibition of the uterine luteolysin during

pregnancy. When the main uterine artery on one side was surgically anastomosed to

the corresponding vein on the opposite side (gravid to nongravid in one group and








12

nongravid to gravid in the other), blood from the gravid side resulted in maintenance of

the CL on the nongravid side. Likewise, blood supplied from the nongravid side

resulted in luteolysis on the gravid side (Mapletoft et al., 1975).

Coudert et al. (1974a) could find no direct physical connections between uterine

venous and ovarian arterial vessels in sheep. In a histological study of the

uteroovarian vascular pedicle in sheep no channels between vein and artery could be

found (Del Campo and Ginther, 1972). Even though no direct connections have been

elucidated, Douglas and Ginther (1973) demonstrated that injection of a relatively small

dose (2 mg) of PGF2a locally into the lumen of the uterine horn ipsilateral to the CL in

the ewe was more effective than a systemic injection (i.e., there was a local constituent

of its transport to the CL). Larger doses worked systemically. This is also true for

cows. Only 10 mg PGF2a infused into the uterine lumen of cows resulted in a

decrease in plasma progestin concentrations, but a 30 mg injection was required if

PGF2a was administered intramuscularly (Chenault et al., 1976).

Hixon and Hansel (1974) reported a selective increase in ovarian artery

concentrations (higher amounts than in carotid artery or jugular vein) of the same

luteolysin (PGF2a) following intrauterine administration in cows. They attributed this to

the preferential transfer of PGF2a from the uteroovarian vein to the ovarian artery. In

contrast, other researchers reported when the ovarian artery (in ewes) was sectioned

distal to the region where transfer of the uterine luteolytic agent is believed to take

place, there was no interruption of the estrous cycle. It was proposed that local

transfer could not be the only mechanism by which the luteolysin reached the CL

(Lamond and Drost, 1973). In another study, Lamond et al. (1973) reported PGF2a

injected into the uterine lumen of cows with sectioned ovarian arteries caused CL








13

regression. Thus PGF2a was transported by an alternate route (other than local) to the

ovary.

Coudert et al. (1974b) could find no transfer of infused 3H-PGF2a from the

uterine vein to the ovarian artery in the ewe. They concluded there was no evidence

of active local transport from the uterus to the ovary. McCracken et al. (1972),

however, did find that 3H-PGF2a infusion into the uterine vein, at a point before it joins

the uteroovarian vein, was followed by an increase in 3H-PGF2a in the ovarian arterial

blood. More recently, Einer-Jensen and McCracken (1981) found evidence for P4

counter-current transfer in sheep by infusing labelled P4 into the uteroovarian vein

close to the hilus of the ovary. Radioactivity levels were higher in the ovarian artery

than in the aorta, with an apparent .5% to 1% efficiency of transfer to the ovarian

artery. Wolfenson et al. (1985) estimated a 1% transfer efficiency of blood PGF2a from

the uterine vein to ovarian artery in cycling cows. Knickerbocker et al. (1986) were also

able to demonstrate increased concentrations of PGF2a in the ovarian artery as

compared to a peripheral artery in response to estradiol-17B (E2-17p) in cattle.

PGF2a as the Uterine Luteolysin

From the previously mentioned research, it seems evident the endometrium

produces a luteolytic substance that is then conveyed to the ovary by either local or

systemic means where it acts on the CL to effect luteolysis. Much of the early work

on this substance characterized its actions and predicted its existence, but efforts to

obtain luteolytic extracts from uterine contents or venous blood have had variable

results.

Injections of ether-soluble extracts or lyophilized homogenate of sheep uteri at

various stages of the estrous cycle (d 0 or d 4 to 7) failed to promote luteal regression








14

in hysterectomized ewes (Kiracofe et al., 1966), but aqueous endometrial extracts from

the diestrual stage in cows (d 14 and 16) and ewes (d 14 and 15) caused luteal

regression in pseudopregnant hysterectomized hamsters (Anderson et al., 1969). Lipid

extracts from hamster uteri also caused luteal regression in hamsters (Lukaszewska et

al., 1972). Uterine flushings from sows on d 12 to 18 of the estrous cycle caused

destruction of pig granulosa cell cultures (i.e., the medium was luteolytic). Flushings

from sows on d 1 to 10 or d 20 of the cycle had no effect (Schomberg, 1967).

Caldwell and Moor (1971) reported that freeze-dried uterine venous plasma infused into

the ovarian artery of ewes precipitated a decrease in ovarian vein P4 and shortened

estrous cycle lengths when blood was collected on d 14 but not on d 8.

Babcock (1966) was the first to suggest the luteolytic agent from the uterus

might be a prostaglandin. These substances were first isolated from human seminal

plasma in the early 1930s (Kurzrok and Lieb, 1930; von Euler, 1934). They were

described as producing strong vasodilation and contractions of smooth muscle. Von

Euler (1935) gave them the name "prostaglandin" because he erroneously thought that

they originated in the prostate gland. They are actually secreted by the seminal vesicle

in the male (Setchell, 1977).

Prostaglandins are a family of 20 carbon, monocarboxyllic, unsaturated fatty

acids (Walpole, 1975). Position of the oxygen groups) on the pentane ring determines

the series to which a prostaglandin belongs (figure 1). The biologically active

prostaglandins are members of the D, E and F series. The number of double bonds in

the side chains are indicated by the numerical subscripts 1, 2 or 3. Prostaglandins are

found in numerous tissues and mediate a myriad of often contradictory actions (Katz

and Katz, 1974).

























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The first report of exogenous PGF2a causing CL regression was by Pharriss et

al. (1968). Pharriss and Wyngarden (1969) proposed that the uterine luteolytic agent

was PGF2a.

Exogenous PGF2a is luteolytic in hysterectomized guinea pigs (Blatchley and

Donovan, 1969), rats (Gutknecht et al., 1969; Pharriss and Wyngarden, 1969), hamsters

(Lukaszewska et al., 1972), pregnant rabbits (Koering and Kirton, 1973), sheep

(McCracken et al., 1970), pigs (Moeljono et al., 1976), horses (Douglas and Ginther,

1972; Allen and Rowson, 1973; Oxender et al., 1974), water buffalo (Kamonpatana et

al., 1979), and cattle, either reproductively intact (Lauderdale et al., 1974) or

hysterectomized (LaVoie et al., 1975). Species in which the uterus is not necessary for

luteal control respond to exogenous PGF2a only under specific conditions. Rhesus

monkeys experienced luteolysis when PGF2a was administered for five consecutive

days with the first injection on d 11, 12 or 13 of the menstrual cycle (no effect when

given on d 7 to 11 or d 4 to 10) (Kirton et al., 1970). In the human, infusion of PGF2a

over a 4 h period on d 21 of the cycle resulted in a sharp decline in plasma P4 by 48

h after treatment. By 72 h after infusion plasma P4 concentrations were less than 1

ng/ml and menstruation ensued (Lehmann et al., 1972). Wentz and Jones (1973)

observed that PGF2a caused only a transient decline in plasma P4 concentrations

when infused for 8 h beginning on d 3, 4, 6, 7, 8, 9, 10, 11 or 12 of the cycle in

humans. Plasma P4 concentrations declined by 50% within 12 h after infusion of

PGF2a in pregnant women (12 to 14 weeks of gestation) and abortion was induced

(Lehmann et al., 1972).

Support for the hypothesis that PGF2a was the luteolysin supplied by the uterus

came from observations that distention of guinea pig uteri in vitro (Poyser et al., 1971)









18

and sheep uteri in vivo (Pexton et al., 1975) resulted in release of PGF2a. Wilks et al.

(1972) found PGF2a is synthesized by rabbit uterine tissue in vitro and there is an

increase in release rate in tissue obtained from animals during estrus. In the pig,

PGF2a concentrations in uterine flushings increase during the luteal phase, reaching

peak levels around d 16 of the cycle (Frank et al., 1978; Zavy et al., 1980). Additional

evidence for PGF2a as the luteolysin is that ewes and cows passively immunized

against PGF2a exhibit prolonged estrous cycles (Fairclough et al., 1981).

Although PGF2a has been shown to be synthesized by the uterus and to be

luteolytic when injected in vivo it is not always luteolytic in cultures of luteal cells.

Speroff and Ramwell (1970) stimulated P4 production in bovine CL slices incubated

with PGF2a and suggested the luteolytic effect of PGF2a when administered in vivo

was due to an indirect inhibition of luteal steroidogenesis. PGF2a stimulated P4

secretion and morphological luteinization in rhesus monkey granulosa cell cultures

(Channing, 1972) but when rabbit CL tissue was incubated with PGF2a, O'Grady et al.

(1972) reported an inhibition of P4 synthesis. Henderson and McNatty (1975) found

that small amounts of PGF2a inhibit secretion of P4 by bovine granulosa cells in vitro.

They hypothesized that PGF2a initiates functional luteolysis (inhibition of P4 synthesis)

by inhibiting synthesis of cyclic adenosine monophosphate (cAMP) in the luteal cell.

Whether PGF2a is luteotropic or luteolytic in vitro seems to depend on the milieu of the

culture media.

Luteinizing hormone and arachidonic acid are luteotropic when incubated alone

in bovine CL cultures (Shemesh and Hansel, 1975b). Prostaglandin F2a increases P4

accumulation in the absence of, but not in the presence of, LH in cultures of bovine

luteal cells (Hixon and Hansel, 1979). This is also true for rat luteal cells (Thomas et









19

al., 1978). Pate and Condon (1984) found no effect of PGF2a on basal concentrations

of P4 but reported that PGF2a was able to inhibit LH-stimulated P4 production in vitro

cultures of bovine CL. It appears the functional luteolytic effect of PGF2a on cells in

culture is upon the agonist-induced P4 production and not the inhibition of basal P4

concentrations.

Theory of Action and Hormonal Regulation of PGF2a

Precisely how PGF2a effects luteolysis is not known. It was first suggested that

PGF2a acted indirectly by causing vasoconstriction of vessels to the ovary and the CL

subsequently died of ischemia (Pharriss and Wyngarden, 1969). Niswender et al.

(1976) found blood flow to the luteal ovary in sheep declined concurrently with P4

concentrations by 6 h after PGF2a treatment. Intravenous administration of PGF2a in

ewes was followed by reduced blood flow to the ovary with CL and lower

concentrations of systemic P4 (Nett et al., 1976). Other researchers, however, could

find no evidence of diminished blood flow despite marked decreases in P4 secretion in

response to PGF2a (Behrman et al., 1971; McCracken and Einer-Jensen, 1979). One

excellent argument against vasoconstriction as the means of luteolysis is the fact that

PGF2a causes functional luteolysis in vitro (Henderson and McNatty, 1975; Hixon and

Hansel, 1979).

Alternative theories for the mechanism by which PGF2a precipitates luteolysis

involve alteration of receptor number or orientations. Receptors for both LH and

PGF2a are found on the plasma membrane of the CL (Powell et al., 1974; Haour and

Saxena, 1974; Behrman et al., 1979).

Luteinizing hormone and its receptor form a complex unit that stimulates P4

synthesis by luteal cells. The binding of LH with its receptor causes an increase in









20

intracellular cAMP concentrations (Henderson and McNatty, 1975). As cAMP

concentrations increase, P4 synthesis also increases (Lahav et al., 1976; Behrman et

al., 1979; Wakeling and Green, 1981). This increase in P4 synthesis results through

the activation of adenylate cyclase, the enzyme that converts adenosine triphosphate

(ATP) to cAMP. Cyclic AMP then phosphorylates protein kinases which, in turn,

activate the enzymes necessary for P4 synthesis, such as cholesterol esterase (Caffrey

et al., 1979). Garverick et al. (1985) reported addition of LH to cultures of bovine luteal

tissue (collected on d 7, 10, 13 and 16 of the estrous cycle) increased adenylate

cyclase activity relative to basal activity. Activation of adenylate cyclase requires the

continued occupation of LH receptors. Dissociation causes deactivation but occupation

of only a fraction of the available receptor sites is adequate to cause maximal

production of cAMP by the luteal tissue (Koch et al., 1974). Luteinizing hormone

increased adenylate cyclase activity in cultures of bovine luteal cells as a result of

increased cAMP (Marsh, 1970). Prostaglandin F2a may act to dissociate LH from its

receptors, thus causing a decline in cAMP concentrations (Koch et al., 1974) and this,

as previously mentioned, may result in the initiation of functional luteolysis (Henderson

and McNatty, 1975).

Other ways in which PGF2a could function would be by causing a decrease in

LH receptors or blocking their occupation. Hichens et al. (1974) reported PGF2a

treatment produced a fall in the binding capacity of rat luteal tissue for human

chorionic gonadotropin (hCG) without changing the affinity constants of LH receptors.

Incubation with PGF2a did not interfere with binding of LH to membranes containing

gonadotropin receptors. They suggested PGF2a may act indirectly via an effect on

synthesis, conformation, or breakdown of LH receptors. In sheep, the number of luteal








21

LH receptors is correlated with luteal weight, P4 content and serum P4 throughout the

estrous cycle. The number of LH receptors is lower during early (d 2 to 6) and late (d

16) phases of the cycle than during mid-luteal phase (d 10 to 14). Affinity constant

was the same on all days of the cycle (Diekman et al., 1978). Number of luteal LH

receptors and P4 concentrations during the estrous cycle are also correlated in cattle

(Rao et al., 1979, Spicer et al., 1981). Other reports, however, dispute the concept that

luteolysis is preceded by a decrease in LH receptor populations.

In cattle, plasma P4 concentrations are positively correlated with unoccupied LH

receptor concentration, basal adenylate cyclase activity, and LH-activated adenylate

cyclase activity on d 4, 7, 10, 13, 16 and 19 of the estrous cycle but not with occupied

LH receptor concentrations (they remain essentially the same from d 10 through luteal

regression). Total occupied LH receptor content, however, is positively correlated with

mean plasma P4 concentrations on d 4, 7 and 10, with total occupancy of LH

receptors increasing fourfold from d 4 to 10 of the estrous cycle. Total LH receptor

occupancy remained unchanged during the rest of the cycle and did not decrease after

luteal regression began (Garverick et al., 1985).

The hCG binding capacity of rat CL following injection of PGF2a was not

depressed by 2 h after treatment, but serum P4 concentrations were reduced by 70%.

The drop in P4 concentrations occurred before the decline in LH receptor number

(Grinwich et al., 1976). In ewes, P4 decreased by 7.5 h after PGF2a injection. There

were no changes in luteal weight, luteal P4 concentration, total number of LH receptors

or number of receptors occupied until 22.5 h post injection. Secretion of P4 by CL

decreased well before decreases in occupied or unoccupied LH receptors could be

detected (Diekman et al., 1978). This was also the case in cows (Fitz et al., 1980). In









22

rats, PGF2a caused a decrease in P4 concentrations within 2 h, but LH binding

capacity was unchanged. However, removal of the source of gonadotropin releasing

hormone (GnRH) by hypophysectomy causes complete and immediate CL regression

(Kaltenbach et al., 1968) and reduces LH binding capacity and P4 concentrations within

48 h, indicating the presence of fewer receptors (Behrman et al., 1978).

Alteration in LH receptor numbers may not initiate luteolysis, but it is likely that

it is involved in the final destruction of the CL. Progesterone concentrations decrease

before any significant morphological changes occur in the CL following treatment of

rabbits with PGF2a (Koering and Kirton, 1973). Luteolysis appears to occur in two

stages. The first is functional luteolysis in which the CL loses its ability to secrete P4

and the second is structural luteolysis which involves leukocytic infiltration, cellular

degeneration and eventual resorption of the CL (Baird and Scaramuzzi, 1975; Behrman

et al., 1979). As previously mentioned, decline in LH receptor number and binding

affinity comes at some point after decrease in P4 concentrations. Grinwich et al.

(1976) proposed the ultimate decline in LH receptors is a mechanism to insure

luteolysis continues once started (structural luteolysis). Rao et al. (1984), however,

suggested the receptor number decline during bovine luteal regression was an artifact

associated with the general deterioration of the cell structural, functional and metabolic

integrity.

Luteinizing hormone and its receptors are slow to dissociate once bound

(Haour and Saxena, 1974; Henderson and McNatty, 1975), but the effect of PGF2a on

P4 secretion by the CL is rapid. Treatment with PGF2a reduced plasma P4 and hCG

binding by the CL within 30 min in rats. This led Behrman and Hichens (1976) to

suggest PGF2a caused luteolysis by blocking LH uptake. This block on LH binding









23

could be expected to cause a decrease in adenylate cyclase activity in the luteal cell.

There was little effect of PGF2a on adenylate cyclase stimulation in homogenates of

bovine CL (Marsh, 1971), but adenylate cyclase activity and cAMP accumulation were

inhibited in intact luteal cells (Thomas et al., 1978).

The impact PGF2a has on LH-dependent adenylate cyclase activity is probably

not by binding to LH receptors. There are receptors specific for PGF2a on the plasma

membrane and there is little cross-reactivity of PGF2a and LH with the non-

homologous receptor (Rao, 1975, 1976; Rao et al., 1979).

How then does PGF2a initiate luteolysis? Henderson and McNatty (1975)

proposed that PGF2a initiated functional luteolysis by interfering with LH stimulation of

cAMP formation. This could result in decreased P4 synthesis. Lahav et al. (1976)

reported PGF2a prevented a LH-stimulated rise in cAMP if they were both added to

cultures of rat CL at the same time. This also occurred in cultures of human CL

(Hamberger et al., 1979). A decrease in luteal adenylate cyclase activity was

associated with a decrease in plasma P4 concentrations in sheep (Agudo et al., 1984)

and cattle (Fitz et al., 1980) during PGF2a-induced luteolysis.

Other researchers, however, suggest the influence of PGF2a on P4 synthesis

occurs at a step after the formation of cAMP. Pate and Condon (1984) reported

PGF2a has no effect on basal P4 concentrations in cultures of bovine CL, and P4

synthesis was stimulated by LH or dibutyryl cAMP. The presence of PGF2a in the

culture inhibited the LH-stimulated P4 production. This occurred at a site beyond the

accumulation of cAMP because dibutyryl cAMP did not increase P4 in the presence of

PGF2a. Phosphodiesterase is an enzyme located in the cytosol and membranes of

most tissues and is involved in regulation of cAMP concentrations by hydrolyzing cAMP


I








24

to 5'AMP (Thompson and Strada, 1978). Garverick et al. (1985) reported an increase

in phosphodiesterase activity in bovine luteal tissue on d 19 of the estrous cycle, a

time at which adenylate cyclase activity was declining. This relationship between the

two enzymes was also reported in sheep within 2 h after PGF2a injection, and the

changes in enzyme activity occurred before a decrease in plasma P4 concentrations. It

was suggested that a decrease in adenylate cyclase activity (necessary for cAMP

synthesis) and an increase in phosphodiesterase activity (responsible for cAMP

catabolism) may act in concert to decrease intracellular cAMP concentration and that

this decrease in cAMP may be an early event resulting in lowered P4 concentrations

during PGF2a-induced (or naturally occurring) luteolysis (Agudo et al., 1984).

Calcium (Ca"+) also appears to be involved in the process of luteolysis. There

are two groups of PGF2a receptors on the CL, one of which has high affinity binding

and the other low affinity binding. Plasma membranes of bovine luteal cells cultured in

buffer with no Ca++ contain low affinity receptors, but lack high affinity receptors.

Addition of Ca++ to the media results in the appearance of high affinity receptors.

When Ca++ is then removed from the culture they disappear. Only high affinity

receptors are dependent on Ca++, as low affinity receptor numbers remained constant

(Rao, 1975). Rao et al. (1979) suggested CL sensitivity to PGF2a during the cycle is

controlled by modulating PGF2a receptor affinity. In bovine CL, there are large

numbers of PGF2a receptors present by d 13 of the estrous cycle, but their binding

affinity was 203 times lower than at d 20 (about the time CL regression occurs).

Buhr et al. (1979) suggested regression of the CL may involve phase changes

in the phospholipid bilayer of cellular membranes. Prostaglandin F2a may be reducing

fluidity and increasing permeability of the microsomal and plasma membranes, resulting









25

in disruption of intracellular enzyme complexes. A gel-phase lipid can be detected in

plasma membranes of bovine CL that were removed 24 h after PGF2a injection

(Goodsaid-Zalduondo et al., 1982). Corpora lutea from cows in the luteal phase of the

estrous cycle had microsomal membranes with all membrane lipids in the liquid-

crystalline stage, but samples prepared from regressing CL revealed a phase transition

in which some of the lipid bilayer was gel-phase (i.e., less fluidity). Coincident with this

physical change was a decline in P4 secretion (Carlson et al., 1982). Carlson et al.

(1984) used fluorescence polarization and x-ray diffraction to determine the structural

properties of membranes from rat luteal cells. Membrane fluidity was observed to

decrease during luteolysis, and this was correlated with a decrease in P4 secretion.

This alteration in membrane structure occurs in cells of either spontaneously regressing

or PGF2a-regressing CL. Treatment of in vitro cultures of rat CL with PGF2a produces

a change similar to that found during spontaneous luteolysis. Polarization increases,

which indicates a decrease in membrane fluidity (Riley and Carlson, 1985).

Riley and Carlson (1987) suggest the decreases in fluidity are caused by a

synergistic effect of Ca++ and hydrolysis products of phospholipase A activity. This

decrease in fluidity is probably due to a deterioration of the methylation process.

Milvae et al. (1983) suggested methylation of phospholipids within the plasma

membranes of luteal cells was an important regulatory step in LH-stimulation of P4

synthesis. They proposed that LH binds to its receptor and stimulates methylation,

which in turn, increases membrane fluidity. This increase in fluidity results in the

unmasking of more receptors which increases LH binding. An increase in membrane

fluidity may increase the probability of the LH-receptor complex interacting with

adenylate cyclase.









26

Phospholipase A2 governs the concentration of arachidonic acid (precursor of

PGF2a) in human platelet cells. This process requires the influence of Ca'+ for

maximum activity (Wong and Cheung, 1979). Phospholipase A2 is a water soluble

enzyme that catalyzes the hydrolysis of phosphoglycerides to yield a lysophosphotide

and an unsaturated fatty acid (typically arachidonic acid) (Riley and Carlson, 1985).

Calmodulin and PGF2a stimulate the activity of phospholipase A2 in the presence of

Ca'" (Moskowitz et al., 1983). In some cells, phospholipid methylation blocks Ca"+

influx into the cell which results in a decrease in phospholipase A2 activity and

arachidonic acid synthesis (Hirata and Axelrod, 1980). Prostaglandin F2a acts to

stimulate phospholipase A2, which precipitates an increase in arachidonic acid

concentrations. This precursor for PGF2a may enhance the production of the luteolytic

substance (via the cyclooxygenase system), which might further accelerate regression

in a positive feedback manner (Riley and Carlson, 1985).

Prostaglandin F2a influence on intracellular concentrations of Ca" may also act

directly to affect enzyme activity. Calcium will decrease adenylate cyclase activity in

luteal cells (Berridge, 1975; Dorflinger, 1978), while it activates phosphodiesterase

through calmodulin in brain, heart, lung and testes tissue (Cheung, 1981; Beavo et al.,

1982). In rat luteal cell cultures, ovabain (digitalis) and monensin inhibit the acute

stimulation of cAMP by LH, probably as a result of influx of Na' into the luteal cell.

This increase in intracellular Na+ does not directly inhibit adenylate cyclase activity but

appears to induce a secondary influx of Ca"+ which in turn inhibits activation of

adenylate cyclase at a site involved in coupling of the receptor to the enzyme.

Prostaglandin F2a may act in the same manner as Na'. Maintenance of CL function

by LH may result in part by processes that maintain low Ca" levels in the luteal cell









27

(Gore and Behrman, 1984). Dorflinger et al. (1984) concluded that an acute increase

in intracellular Ca'+ inhibits activation of adenylate cyclase by LH but that this inhibition

by PGF2a is not dependent on an influx of extracellular Ca+*, but rather is due to an

increase in intracellular Ca++ by other mechanisms. They suggested intracellular Ca++

may increase by the sequestering of Ca++ in mitochondria and endoplasmic reticulum

or by a decrease in expulsion to the exterior of the cell as well as by an increase in

influx from the extracellular medium.

Hormonal Influences and Controls

Substances other than PGF2a have been shown to be luteolytic. Daily injection

of estradiol (E2) from d 2 to 12 of the cycle in dairy heifers caused precocious CL

regression (Greenstein et al., 1958). Injections of E2 valerate or a natural estrogenic

product also caused early CL regression in beef heifers (Wiltbank et al., 1961). The

luteolytic properties of E2 are mediated through stimulus of PGF2a release from the

uterus. If heifers are hysterectomized (i.e., no PGF2a source), E2 causes a decline in

plasma P4 concentrations and P4 content of the CL (Kaltenbach et al., 1964) but does

not result in total regression or expression of estrus (Brunner et al., 1969). Estradiol

cypionate is an effective luteolytic agent in the intact but not the hysterectomized ewe

(Bolt and Hawk, 1975) or heifer (Watson et al., 1980). When estrogen is injected early

in the cycle (d 1 to 6) there is no apparent effect on weight or morphology of the CL

in the ewe, but when injected on d 9 and 10 of the cycle, CL weight was reduced

(Hawk and Bolt, 1970). In the ewe, E2 injection on d 10 of the cycle into the CL

caused a decrease in P4 but no change in CL weight (Cook et al., 1974).

During early and mid-cycle the PGF2a concentrations in the bovine

endometrium are low (Shemesh and Hansel, 1975a). When PGF2a concentrations are








28

low or non-existent (due to hysterectomy), E2 influence on CL function is probably

through a negative feedback mechanism that decreases the concentrations of

circulating luteotropins. However, the decreased concentrations are inadequate to

cause total CL regression. During the later phases of the estrous cycle, PGF2a is

present in significant quantity in the uterus and E2 stimulates its release and increased

production (Bartol et al., 1981; Knickerbocker et al., 1986). Injections of E2 into cycling

heifers caused plasma concentrations of 15-keto-13,14-dihydro PGF2a (PGFM) to

increase with the resultant CL regression (Thatcher et al., 1986). Plasma PGF2a is

inactivated during passage through the pulmonary circulation (probably by 15-

hydroxyprostaglandin dehydrogenase) and forms the metabolite, PGFM (Piper et al.,

1970). If indomethacin, a substance that inhibits PGF2a synthesis by the endometrium

(Lewis and Warren, 1977), is injected in heifers along with E2 benzoate, it prevents the

expected induced CL regression. This would suggest the luteolytic action of estrogen

is by increased PGF2a synthesis and its release from the uterus (Warren et al., 1979).

Some researchers have demonstrated that the E2 stimulated synthesis and

release of PGF2a from the endometrium must be preceded by P4 priming of the uterus

(Caldwell et al., 1972; Barcikowski et al., 1974; Scaramuzzi et al., 1977). Spontaneous

E2 peaks occur throughout the cycle, but it is not until the time of CL regression that

peaks of PGF2a are correlated with E2 peaks. PGF2a is released only in late luteal

phase from an autotransplanted uterus following injection of E2. This would indicate

P4 priming is necessary to PGF2a synthesis (Roberts et al., 1975).

Estrogen appears to have a role in spontaneous CL regression. Destruction of

all visible follicles (the source of endogenous estrogen) on both ovaries in ewes

resulted in delayed CL regression following IUD insertion into the uterus of ewes.








29

Cauterization of the follicles on only one ovary did not result in delayed regression

(Ginther, 1971). Cook et al. (1974) reported injected E2 caused CL regression in ewes

when ovaries had all follicles destroyed, but only if injected into the ipsilateral ovary

and not the contralateral one. Progesterone concentrations were maintained past the

expected time of luteolysis in ewes (Hixon et al., 1975) and in cows that had all follicles

on the ovaries destroyed at some time during mid-cycle (Fogwell et al., 1985). It was

suggested that E2 initiated luteal regression, possibly by involvement in PGF2a release.

The other factor involved in luteolysis is oxytocin (OT). Armstrong and Hansel

(1959) found that administration of OT by subcutaneous or intravenous injections daily

from d 0 to 7 of the estrous cycle in dairy heifers shortened cycle length to 8 to 12 d.

They concluded OT caused inhibition of CL function possibly by interfering with the

secretion of a luteotrophic hormone from the pituitary. However, as in the case of

estrogen, the luteolytic effect of OT is probably mediated through PGF2a release from

the uterus. Administration of exogenous OT shortened estrous cycle length if heifers

were reproductively intact or if the contralateral uterine horn was removed. Removal of

the ipsilateral horn prolonged the estrous cycle (Ginther et al., 1967).

Like estrogen, OT injection early (d 0 to 4) or late (d 15 to 22) in the estrous

cycle has no effect on cycle length, but injection during the luteal phase results in CL

regression (Hansel and Wagner, 1960; Black and Duby, 1965). It is likely that

injections of OT administered earlier than d 5 of the cycle, as in a study by Armstrong

and Hansel (1959), are superfluous. A later study (Hansel and Wagner, 1960)

demonstrated that injections given on d 0 to 2 or d 0 to 4, inclusive, failed to shorten

the estrous cycle in dairy heifers. Administration of OT injections on d 3 to 6 was as

effective as injections given from d 0 to 7.








30

When physiological amounts of OT are infused into the arterial supply of the

uterus in ewes, the tone of the uterus and amplitude of contractions increase and are

associated with a simultaneous release of PGF2a (Roberts et al., 1975). A single

injection of OT resulted in an increase in plasma PGFM when given to ewes on d 14 of

the estrous cycle but not on d 3 or 8 (Fairclough et al., 1984). Lafrance and Goff

(1985) reported a single injection of OT on d 17, 18 or 19 of the cycle in heifers

precipitated an increase in PGFM but had no effect when the injections were given on

d 6 or 13. In contrast, multiple injections of OT did elicit PGF2a release earlier in the

cycle. Treatment of heifers with OT on three consecutive days beginning on d 3 of the

estrous cycle resulted in increased concentrations of PGF2a in the peripheral blood

supply (Newcomb et al., 1977). Daily OT injections on d 4, 5 and 6 or d 5 and 6

caused shortened estrous cycles and increased uterine venous PGF2a concentrations

in heifers (Milvae and Hansel, 1980). Injections given twice daily from d 2 through 6 of

the estrous cycle resulted in increased plasma PGFM concentrations on d 2 and 3 and

a shortened cycle in two of six cows treated. All cows treated in this manner had a

slower P4 increase through d 8 of the estrous cycle than controls (Oyedipe et al.,

1984). Administration of OT on d 3 to 6 of the cycle in goats also results in elevated

plasma PGFM concentrations with accompanying P4 decline (Cooke and Homeida,

1982).

Flint and Sheldrick (1985) reported continuous infusion of OT between d 13 and

21 of the estrous cycle in ewes delayed return to estrus by 7 d. Progesterone also

remained high, indicating luteal regression was prevented. Continual infusion of OT

during this phase of the cycle prevented the rise in uterine OT receptors which

normally precedes estrus, possibly by down-regulation. This may result in an inhibition









31

of PGF2a synthesis or release from the endometrium. However, continuous infusion of

cattle with OT from d 14 to 22, d 15 to 18, or d 16 to 19 did not significantly affect

luteolytic events (Kotwica et al., 1988).

Oxytocin enhanced PGF2a release from cultures of endometrium. The number

of high affinity OT receptor sites on the endometrium and myometrium were at their

peak in cultures of these tissues from ewes at estrus (Roberts et al., 1976). Mean OT

receptor concentrations in caruncular and intercaruncular endometrium and

myometrium increased from d 10 to estrus in cycling ewes. This increase in receptors

coincided with luteolysis and the concomitant decrease in P4 (Sheldrick and Flint,

1985). As in sheep, endometrial OT receptor concentrations in heifers are low during

the luteal phase of the estrous cycle, but increase rapidly during luteolysis and reach a

maximum at estrus (Meyer et al., 1988). Myometrial plasma membranes bound nearly

ten times more OT when the tissue was collected on d 21 of the cycle than when

collected on d 7 (Soloff and Fields, 1989).

Endogenous OT concentrations increase following PGF2a injection in ewes (Flint

and Sheldrick, 1983) and cows (Schams and Karg, 1982; Schallenberger et al., 1984).

When production of endogenous PGF2a in vitro was suppressed with indomethacin,

the myometrium responded normally to OT, demonstrating that increased synthesis of

PGF2a is not essential for activation of the myometrium by OT (Roberts and

McCracken, 1976). Tritschler et al. (1983) found OT promoted luteolysis in all cows

treated and this effect was not blocked by indomethacin, suggesting that increases in

uterine PGF2a synthesis may not be responsible for OT-induced luteolysis, but that OT

may act to initiate release of PGF2a. In contrast, Cooke and Knifton (1981) reported

subcutaneous injections of OT caused induction of estrus in goats, but administration








32

of meclofenamic acid (a prostaglandin synthetase inhibitor) inhibited this luteolytic

effect. Active immunization of ewes against OT prolonged the luteal phase of the

estrous cycle (Sheldrick et al., 1980; Schams et al., 1983).

Oxytocin is a nonapeptide hormone generally thought of as being produced by

the hypothalamus and released from the posterior pituitary (Wathes and Swann, 1982).

Early in this century, Ott and Scott (1910) reported the corpora lutea of goats

contained an oxytocic-like substance. More recently, extracts of ovine (Wathes and

Swann, 1982; Theodosis et al., 1986) and bovine (Fields et al., 1983; Wathes et al.,

1983) CL have been shown to contain OT. Large quantities of mRNA for OT exist in

the bovine CL during mid-luteal phase of the estrous cycle. This mRNA for luteal OT is

very similar to mRNA for hypothalamic OT, but an active CL produces approximately

250 times more OT mRNA than a single hypothalamus (Ivell and Richter, 1984). The

CL is the primary site of ovarian OT (Flint and Sheldrick, 1982), but Ivell et al. (1985)

reported finding mRNA for OT detectable at low concentrations in mid-cycle follicles.

Other researchers reported the measurement of immunoreactive OT in the follicles of

cycling cattle (Wathes et al., 1984; Kruip et al., 1985; Schams et al., 1985; Wise et al.,

1986).

Corpora lutea from ewes (Fitz et al., 1982) and cows (Priedkalns and Weber,

1968; Koos and Hansel, 1981; Weber et al., 1987) contain two populations of luteal

cells. One population consists of large cells (> 23 gm in diameter) and the other of

small cells (12 to 23 ;m in diameter). Interestingly, immunoreactive OT or OT-

associated neurophysin is contained in large luteal cells and not small cells of cycling

ewes (Rodgers et al., 1983; Fields and Fields, 1986) and cows (Guldenaar et al., 1984;

Fields and Fields, 1986). Only the large cell of bovine CL contains mRNA for OT (Fehr








33

et al., 1987). Immunoreactive OT and OT-associated neurophysin could not be found

in the large luteal cells of pregnant cows (Guldenaar et al., 1984).

These same large cells also contain the majority of receptors for PGF2a (and

coincidently, PGE2) and the fewest receptors for LH/hCG when compared to small

luteal cells in cycling ewes (Fitz et al., 1982). Large cells contain and secrete most of

the P4 produced by the CL, but small cells demonstrate an increase in P4 synthesis

and secretion in response to LH challenge in cultures of luteal tissue from the mid-

cycle cow (Ursely and Leymarie, 1979; Koos and Hansel, 1981) and ewe (Fitz et al.,

1982). Harrison et al. (1987) reported the basal P4 production by large cells of mid-

cycle ovine CL was 6 to 8 times higher than that of small cells. Addition of LH to

separate cultures of these cells stimulated P4 production by small cells, but not large

cells. However, when small and large cells were recombined in a single culture the

effect of addition of LH was synergized and the combined culture produced more P4

than cultures of the small cells alone.

Gemmel et al. (1974) reported granules were present in the cytoplasm of luteal

cells of the ewe and their numbers increased as the estrous cycle progressed. This is

correlated with the rise and decline of P4 during the cycle (Heath et al., 1983). The

peptide hormones neurophysin and OT have been demonstrated to be present in

electron dense granules within the large luteal cell (Fields and Fields, 1986; Theodosis

et al., 1986; Fields et al., 1989). It has been theorized that these or other electron

dense granules may also contain sequestered P4 and that this is probably the method

of P4 release from the large luteal cells (Gemmel and Stacy, 1979; Quirk et al., 1979).

Rice et al. (1986) demonstrated approximately 30% of total P4 in ovine CL is

associated with subcellular granules, but that the particle associated P4 does not have








34

similar physical or biochemical characteristics to OT containing granules. Luteal

granules that do contain OT displayed physical and biochemical characteristics similar

to those reported for neurohypophysial OT granules except that luteal granules were

1.3 times larger in diameter (Rice, 1988). Injection of PGF2a in sheep (Stacy et al.,

1976) or cattle (Heath et al., 1983; Braun et al., 1988) resulted in decreases in the

relative percentages of cytoplasm occupied by granules in large luteal cells, but not

small luteal cells. Similar observations were made when bovine luteal slices were

incubated with PGF2a (Chegini and Rao, 1987).

Wathes and Swann (1982) hypothesized the OT in the peripheral plasma could

be of luteal origin because its increase and decrease correspond to growth and

regression of the CL. Flint and Sheldrick (1982) demonstrated that injections of PGF2a

in sheep produced a secretion of OT into the uteroovarian vein. Pulses of OT,

neurophysin and PGF2a were measured in blood samples collected at hourly intervals

from the uteroovarian vein draining the CL in sheep on d 13 to 16 of the estrous cycle

(Hooper et al., 1986). In addition, plasma OT concentrations decrease with

ovariectomy and episodic release is not detected during seasonal anestrus in sheep

(Sheldrick and Flint, 1981; Schams et al., 1982).

Oxytocin and estrogen are closely aligned in their luteolytic effect on the CL. In

ovariectomized ewes, OT alone could not effect PGF2a release from the uterus.

Injection of E2 alone increased PGF2a concentrations 3 fold, but when OT was injected

into E2 primed ewes, PGF2a concentrations rose 30 fold (Sharma and Fitzpatrick,

1974). As previously mentioned, P4 priming also appears to be necessary for the

synthesis and release of PGF2a from the endometrium (Roberts et al;, 1975). Oxytocin

injections caused increases in plasma PGFM in ovariectomized heifers after 7, 14 or 21









35

d of P4 priming. The OT induced PGFM increase after 14 or 21 d of P4 priming was

higher at 6 h after E2 injection than before the injection. It was suggested that under

the influence of P4, E2 enhances the OT-induced release of PGF2a and that there was

a possible synergistic action of these hormones in the induction of luteolysis in heifers

(Lafrance and Goff, 1988).

This synergism between estrogen and OT may be mediated through estrogen

and(or) OT receptor regulation. Increases in estrogen produce increases in OT

receptors on the endometrium. As these receptors become occupied with OT, which is

present at basal levels in the peripheral circulation, they induce PGF2a release from the

uterus, which may result in initiation of luteolysis. Prostaglandin F2a causes the

release of OT from the CL and this OT may reinforce the further secretion of PGF2a

from the uterus. Receptors for OT may be down-regulated by the release of OT. As

the receptors for OT are regenerated they may cause the further episodic releases of

PGF2a from the endometrium (McCracken et al., 1984). Luteolysis is accompanied by

a decline in P4 concentrations, which would result in decrease of the negative feedback

control of P4 on estrogen receptors. Increasing occupation of the estrogen receptors

would elicit increased numbers of OT receptors and the resultant occupation of those

receptors with subsequent PGF2a release could cause the final luteolysis of the CL

(Leavitt et al., 1985).

Other researchers suggest that measurements of plasma OT at about the time

of luteal regression do not support the theory of increased release of PGF2a in

response to peripheral OT. Webb et al. (1981) reported plasma OT concentrations in

the ewe (in blood samples collected every 3 h) decreased around the time of CL

regression, preovulatory gonadotropin surge and beginning of the next luteal phase.








36

This was in contrast to increased concentrations of PGFM occurring during luteal

regression. Sheldrick and Flint (1981) reported an increase in basal concentrations of

OT in the ewe (in blood samples collected once a day), but they suggested it was

unlikely to cause the rapid increase in uterine release of PGF2a at the end of the

estrous cycle. Oxytocin concentrations in bovine ovaries increased from d 1 to 10 of

the cycle and then declined from d 11 to 20, before a decline in P4 occurred (Wathes

et al., 1984).

But release of OT from the CL occurs in a pulsatile fashion and is associated

with the release of PGF2a from the endometrium in the ewe (Flint and Sheldrick, 1983)

and cow (Schams et al., 1985). Fairclough et al. (1983) reported coincident surges of

OT-associated neurophysin and PGFM in plasma during luteal regression in ewes.

However, in a subsequent study, injection of OT in ewes on d 14 of the cycle

produced a rise in PGFM concentrations but no consistent increase in OT-associated

neurophysin (Fairclough et al., 1984). They concluded that because only 1 of 4 ewes

had a significant rise in OT-associated neurophysin following OT injection the data did

not support the view that endometrial release of PGF2a stimulated OT release from the

CL. Conversely, daily injections of indomethacin (a prostaglandin synthetase inhibitor)

on d 11 to 16 of the estrous cycle in goats suppressed the decline in basal

concentrations of OT and the pulsatile appearance of OT and PGFM in peripheral

circulation. This would suggest PGF2a may stimulate the pulsatile release of OT at

luteolysis (Cooke and Homeida, 1984). Abdelgadir et al. (1987) demonstrated that

PGF2a did induce OT release by bovine CL in vitro if the CL was collected on d 8, but

not d 12 to 16, of the estrous cycle. The addition of PGF2a to cultures of ovine CL,

however, had no effect on secretion of OT (Hirst et al., 1986, 1988). Hooper et al.









37

(1986) found most PGF2a pulses measured in plasma samples collected at hourly

intervals from d 13 to 16 in cycling ewes coincided with pulses of OT. Hixon and Flint

(1987) reported the administration of E2-17/ on d 9 and 10 of the estrous cycle in

ewes raised OT receptor concentrations in caruncular endometrium and myometrium

by 12 h, followed by an increase in peripheral plasma OT by 26 3 h, an increase in

plasma PGF2a by 35 3 h, and a decrease in plasma P4 by 42 3 h.

Concentrations of PGF2a in the uteroovarian vein of ewes during luteolysis

began to increase before concentrations of OT and OT-associated neurophysin

increased (by an average of 17 min) (Moore et al., 1986). This supports the theory

that endometrial PGF2a initiates the release of ovarian OT during luteolysis. If this is

the case, OT may provide positive feedback on PGF2a release and cause down-

regulation of uterine OT receptors to fine tune PGF2a pulses so they can cause CL

regression more efficiently (Schramm et al., 1983). Luteal OT probably reaches the

endometrium in the same local transfer manner that results in transport of endometrial

PGF2a to the CL. Radioactively labelled OT (1251-OT) was exchanged locally from the

uteroovarian vein to the ovarian artery in sheep with a transfer rate of approximately

1% (Schramm et al., 1986). Currently, evidence from the above data is unable to

prove conclusively that ovarian OT precipitates luteolysis by initiating PGF2a release

from the endometrium or, alternately, that PGF2a initiates the release of ovarian OT to

achieve luteal regression.

Some researchers have suggested OT in the CL may be involved in limiting

luteal P4 secretion by a local mechanism (Flint and Sheldrick, 1982; Wathes et al.,

1983). Cultures of bovine luteal cells responded to low levels of OT with an slight

enhancement of P4 production. Higher concentrations of OT, however, resulted in an









38

inhibition of basal and hCG stimulated P4 production (Tan et al., 1982). Flint et al.

(1989), however, suggest it is possible that impurities in OT preparations are responsible

for the stimulatory and inhibitory effects reported for in vitro cultures. They suggest

evidence supporting a systemic role of oxytocin in the control of luteolysis is the fact

that concentrations of oxytocin receptors in caruncular and inter-caruncular

endometrium rise as plasma P4 concentrations fall during luteolysis in ewes (Sheldrick

and Flint, 1985) and that either OT receptor concentrations rise as a result of the

declining P4 concentrations or the rise in OT receptor concentrations is a cause of

luteal regression (Flint et al., 1989).

In addition to the peptide hormone OT, the CL of sheep have been shown to

contain PGF2a (Patek and Watson, 1974; Rexroad and Guthrie, 1979). Shemesh and

Hansel (1975c) reported in vivo injection of arachidonic acid into the bovine CL

produced a decline in P4 and increase in PGF2a and estrogen concentrations in the

ovarian vein draining the CL, indicating the synthesis of PGF2a by either the ovarian or

luteal tissue. In culture, the bovine ovary synthesizes PGF2a in both follicular and

luteal tissue (Shemesh and Hansel, 1975b). It has been suggested that local

production of PGF2a by the CL may result in its ultimate regression (Patek and

Watson, 1976; Rothchild, 1981). Chronic intraluteal administration of PGF2a caused

luteolysis in rhesus monkeys, leading the authors to suggest the data supports the

hypothesis that local production of PGF2a initiates normal CL regression (Auletta et al.,

1984).

As previously discussed, exogenous PGF2a is luteolytic in domestic livestock

species. Regardless of the exact mechanism by which PGF2a induces luteolysis,









39

practical application of its effect has been used to synchronize estrus in cattle

production enterprises.

Practical Use of PGF2a for Estrus Synchronization

Soon after the first report that exogenous PGF2a was luteolytic in

pseudopregnant rats (Pharriss and Wyngarden, 1969) researchers began exploring its

potential use for control of the estrous cycle in cows. Administration of PGF2a by

subcutaneous or intramuscular injection (Lauderdale et al., 1974) or by infusion into the

uterus (Louis et al., 1974) of the cycling cow or heifer resulted in premature expression

of estrus in most of the treated animals. Similar results were obtained when

synthetically produced analogs were used (Tervit et al., 1973; Cooper, 1974). Cycling

animals treated with PGF2a generally expressed an induced estrus by 68 to 80 h after

administration of the drug (Louis et al., 1974; Henricks et al., 1974; Chenault et al.,

1976; Stellflug et al., 1977; Renegar et al., 1978). Some researchers, however,

reported shorter average intervals to estrus of 40 to 62 h (Galina et al., 1982; Gonzalez

et al., 1985; Graves et al., 1985). Part of this difference may be due to the subjective

determination of time of estrus, but interval to estrus is also influenced by stage of the

estrous cycle at which PGF2a is injected. Cows and heifers injected at an early point

in their estrous cycle have shorter intervals to estrus than those injected late in the

cycle (Macmillan, 1978, 1983; Tanabe and Hann, 1984; Watts and Fuquay, 1985).

Fertility to artificial insemination following a single injection of PGF2a did not

differ or was slightly higher than controls (Roche, 1974; Day, 1977; Gonzalez et al.,

1985, Wahome et al., 1985) when animals were Al according to the AM/PM rule first

proposed by Trimberger (1948). This rule requires that all animals expressing estrus in

the morning (AM) be Al in the evening (PM, approximately 12 h later) and, likewise, all









40

animals exhibiting estrus in the PM be Al during the following AM. Use of this system

is labor intensive as it makes imperative a careful visual appraisal of the treated

animals at least twice daily during the anticipated breeding period.

An alternative would be the Al of all treated animals at an appointed time after

injection of PGF2a. One obstacle to this system was evident from the first studies

using PGF2a in cattle. Cows or heifers that were on d 0 (estrus) to 5 of the estrous

cycle failed to demonstrate response to an injection of PGF2a by exhibiting a

premature estrus (Inskeep, 1973; Henricks et al., 1974; Ellicott et al., 1975). Therefore,

in a group of randomly cycling cows, approximately 25% (at any one time) will be at a

point in their cycle when an injection of PGF2a is ineffective. One method for

circumventing this problem was to inject only those cows that were known to be on d

6 or later of the cycle (as determined from date of previous estrus). Another was to

treat only those animals with a CL of adequate size to be rectally palpable (Lauderdale

et al., 1974) or to produce concentrations of P4 indicative of diestrus (Turman et al.,

1975). When animals were treated selectively, some researchers reported no difference

in pregnancy rates between cows Al according to estrus or those Al at pre-set times

after injection (Lauderdale et al., 1974; Plunkett et al., 1984). Others indicated a

tendency for lower pregnancy rates to timed Al when compared to Al by the AM/PM

rule (Turman et al., 1975; Hardin et al., 1980b).

Even when Al by appointment was successful, this system required a great deal

of time and effort to assure animals were at the proper phase of their estrous cycle to

achieve response to an injection of PGF2a. Roche (1974) proposed a system using

two injections of PGF2a given with a 10 to 12 d interval between injections.

Theoretically, this system would make sure that all cycling animals treated would be at








41

the proper stage of the estrous cycle to respond to a second injection. As previously

mentioned, randomly cycling cows at d 0 to 5 would not respond to the first injection,

but cows on d 6 to 21 could be expected to express estrus (either natural or induced)

within 3 to 4 d after initial treatment. Ten to 12 d later, at the time of the second

injection, cows which had not responded to the first injection would be at d 10 to 17 of

the cycle (a phase during which they should respond) and cows that had expressed

estrus after the first injection would be at approximately d 6 to 9, again at a stage of

the cycle when they should be responsive to PGF2a. Some researchers have reported

outstanding success with this system. In these cases over 90% of treated animals

(cattle that had functional CL prior to treatment) expressed a synchronized estrus after

the second injection (Cooper, 1974; Dobson et al., 1975; Leaver et al., 1975; Adeyemo

et al., 1979; Jochle et al., 1982; Kiracofe et al., 1985; Adeyemo, 1987).

Other studies produced response rates that were lower than should be

expected when treating only cycling animals. These studies reported that 11 to 36% of

those treated failed to express estrus after the second injection (King and Robertson,

1974; Britt et al., 1978; Burfening et al., 1978; Ansotegui et al., 1983). Field trials of

the two injection protocol also yielded lower response rates of 52 to 73% (Lauderdale

et al., 1981). A somewhat lower response rate would not be unexpected as field trials

involve treatment of entire herds which would contain both cycling and non-cycling

cows. However, lack of response by non-cycling animals may not entirely account for

a low response rate after the second injection. When cows were treated using the two

injection system with a 12 d interval 62% responded to the second injection. Of the

cows that were treated, 15% were found to be non-cycling and 23% were cycling but

failed to be synchronized (Hafs and Manns, 1975). Donaldson et al. (1982) reported 93









42

of 237 treated cows showed estrus after the first injection, but 37.6% of those

responding to the first injection failed to respond to the second. It can legitimately be

argued that estrus detection is a subjective system for measuring response rate (and

therefore subject to errors of interpretation) or that cows may experience luteal

regression without expression of estrus, but when P4 concentrations were used as an

indicator of CL function in dairy cows, 23 of 176 (13%) with high concentrations of P4

failed to experience luteal regression after the second injection (Stevenson et al., 1987).

In other words, these animals "should" have responded, but did not (Lucy et al., 1986).

The apparent difficulty in synchronization with the two injection system is not

limited to cows. Smith et al. (1984) used injections of PGF2a to synchronize Holstein

heifers, all of which were cycling prior to treatment. They reported a significant number

(16%) of treated animals were not observed in estrus after the second injection. When

these non-estrual heifers were Al at 80 h post-injection only one conceived. Overall

pregnancy rate of the PGF2a treated and timed Al heifers (52%) was lower than in

controls bred at a naturally occurring estrus (73%). Differences in the pregnancy rates

were attributed to 1) poor synchrony of estrus, 2) failure of a significant number of

heifers to respond to the second injection and(or) 3) improperly timed inseminations

rather than to reduced fertility in the treated heifers.

As in the case of breeding by appointment after a single injection of PGF2a,

pregnancy rates to timed Al following use of the two injection system varied greatly.

Some researchers reported no difference in rates between animals bred by the AM/PM

rule and those Al at set times (Hafs et al., 1975; Manns et al., 1976; Waters and Ball,

1978; Roche and Prendiville, 1979; Kazmer et al., 1981; Jdchle et al., 1982).

Conversely, others reported timed Al after synchronization with two injections of PGF2a








43

(with an interval of 10 to 12 d between injections) resulted in lower pregnancy rates

than Al according to estrus (Ellicott et al., 1975; Roche, 1976; Moody and Lauderdale,

1977; Donaldson, 1977; Hardin et al., 1980a; Graves et al., 1985; Stevenson et al.,

1987). Short et al. (1978) concluded inseminating at predetermined times following

synchronization lowered pregnancy rates, but when breeding was done in relation to

estrus, pregnancy rates after PGF2a are similar to unsynchronized Al and natural

service. Other studies reported no difference in pregnancy rates to Al to estrus after

synchronization using the two injection system and Al to a natural estrus if

insemination was performed according to estrus expression (King and Robertson, 1974;

Lauderdale et al., 1980; Hardin et al., 1980a; Lauderdale et al., 1981; Neuendorff et al.,

1984; Kiracofe et al., 1985). Macmillan and Day (1982) and Macmillan (1983) went so

far as to suggest PGF2a enhanced fertility if Al was performed according to estrus.

Dairy cows treated with two injections of PGF2a at an 11 d interval had pregnancy

rates of 69% compared to 60% in untreated herdmates.

As suggested by Smith et al. (1984), unsatisfactory results to timed insemination

may result from a lack of synchrony after treatment. Johnson (1978) and Refsal and

Seguin (1980) reported synchrony of estrus was more precise after the second injection

than after the first in a two injection scheme. The interval to estrus after a second

injection is also shorter (Johnson, 1978; Burfening et al., 1978; Hardin et al., 1980b).

Although this is what is desired in a timed insemination program, the increase in

degree of synchrony may not be adequate to assure successful timed Al. Interval to

estrus is shorter in cattle injected in early diestrus than in those injected during late

diestrus (by approximately 12 h) (King et al., 1982; Stevenson et al., 1984). Jackson et

al. (1979) reported cows injected on d 7 to 8 or d 15 to 16 had shorter intervals from









44

injection to LH peak and estrus than animals injected on d 12 to 14. This effect of

stage of cycle at time of treatment on interval to estrus could result in fewer animals in

a herd being at the correct stage of estrus for timed Al. Donaldson et al. (1982)

reported that only 55.7% of the cows that responded to injections of PGF2a expressed

estrus in the time frame necessary for Al by appointment.

Synchronization and Al in the Brahman

The Brahman was developed in the U.S. just after the turn of the century by

crossing four breeds of Bos indicus cattle. The four breeds, Kankrej (Guzerat), Krishna

Valley, Ongole (Nellore), and Gir, were imported from India and Brazil largely between

the years 1900 and 1946 (Brockett, 1977). The American Brahman Breeders

Association (ABBA) was established in 1924. Its first secretary, J. W. Sartwelle,

proposed the name Brahman for the breed (Saunders, 1980).

The Brahman and other Zebu breeds are used in purebred and crossbreeding

programs in the tropics and sub-tropics because of their ability to adapt to hot, humid

climates and to flourish under conditions of insect infestations and enzoodic diseases

that prove fatal to many Bos taurus breeds (Fowler, 1969). The southern states were

once considered the poorest beef producing region in the U.S. because of this type of

environment. Expansion of improved pastures and use of Brahmans in crossbreeding

programs have been credited with the extensive increase in beef production in this

section of the country (Fowler, 1969). Frequently, Al is the method by which

crossbreeding has been accomplished.

Naturally bred Brahmans have been reported to have lower pregnancy rates

when compared to Bos taurus breeds (Burns et al., 1959; Kincaid, 1962; Koger et al.,

1973; Crockett et al., 1978). This has also been reported in Brahmans that have been









45

Al after PGF2a synchronization. Tucker et al. (1982) found the pregnancy rate to Al at

estrus following PGF2a synchronization was lower in purebred Brahmans than in

commercial Angus, Hereford or Simmental cows (20.8% vs 61.5%, 66.6% and 61.5%,

respectively). Zebu cows treated with PGF2a had lower pregnancy rates to Al than

untreated controls, but treated cows were Al by appointment at 80 h after injection and

controls were Al according to the AM/PM rule (Landivar et al., 1985). The poor

pregnancy rate after PGF2a may have resulted from improper timing of Al as the

responding treated cows exhibited estrus at 46 to 54 h after injection. Gilson et al.

(1981) reported a higher pregnancy rate to Al at 8 to 16 h after induced estrus than to

timed Al at 80 h in high percentage Brahman crossbred cows. The average interval to

estrus following a single injection of alfaprostol (a PGF2a analog) during the luteal

phase of the cycle (approximately d 12) in Brahman heifers and cows was 89 h. A

tight synchrony of estrus did not result and only 13% of the treated animals would

have been in the correct time frame for optimum fertility to timed Al (Hansen et al.,

1987a).

Low pregnancy rates to timed Al after PGF2a synchronization may result from a

poor response (as measured by rate of estrus) after treatment. In Zebu cows, only

59% of animals with a palpable CL prior to a single injection of PGF2a expressed

estrus following treatment (Orihuela et al., 1983). Other studies have indicated a poor

response rate in Zebu or Zebu crossbred animals (Galina et al., 1982; Landivar et al.,

1985). Use of the two injection system of PGF2a synchronization has also resulted in

inadequate response rates. Purebred cycling Brahmans expressed estrus 46.0% and

46.4% of the time after the first and second injection, respectively (Neuendorff et al.,

1984). Nagaratnam et al. (1983) reported response rates of 47% and 76% following









46

the first and second injections in cycling White Fulani and Sokoto Gudali cattle. Estrus

expression may not be the best indication of actual response to PGF2a treatment.

Moreno et al. (1986) observed estrus in only 47% of treated Zebu cattle in one

experiment and 60% of those in a second experiment, but palpation of the ovaries and

plasma P4 at 70 h after injection indicated most animals had experienced CL

regression. They concluded that PGF2a was luteolytic in Zebu cattle although estrus

expression after treatment was poor. Some researchers have reported excellent

response rates in Zebu cattle to PGF2a treatment (Adeyemo et al., 1979; Gilson et al.,

1981).

Still, as previously mentioned, pregnancy rates to either timed Al or Al to estrus

have been reported to be low in Brahman or other Zebu animals. Brahmans have

been reported to have smaller CL, less P4/CL, and lower plasma P4 concentrations on

d 2 to 11 of the estrous cycle than Herefords (Irvin et al., 1978; Randel, 1984).

Brahman cows also have lower serum P4 concentrations on d 7 to 17 of the cycle than

Angus cows (Segerson et al., 1984). When Brahman cows were injected with

cloprostenol (a PGF2a analog) the CL that resulted after the induced estrus was

smaller and contained less P4 than the CL formed after a natural estrus (Hardin and

Randel, 1982). A single luteolytic dose of cloprostenol administered at mid-cycle (d 8

to 12) reduced the weight and total P4 content of the subsequently developing CL in

Brahman cows when compared to the CL after a natural estrus. Plasma

concentrations of P4 on d 2 to 13 after a cloprostenol induced estrus were lower than

in controls (Hardin and Randel, 1982).

Hansen et al. (1987b) also reported the formation of a subfunctional CL in

Brahman heifers and cows following treatment with another PGF2a analog, alfaprostol.









47

Brahman females were given a single injection of alfaprostol on d 12 0.2 of a

spontaneous estrous cycle and the corpora lutea were removed on d 13 of the induced

estrous cycle. All of the females treated with a dose of 2.25 mg/100 kg bodyweight

had lower serum P4 concentrations on d 3, 4, 10, 11, and 12 of the induced estrous

cycle when compared with controls. Corpora lutea formed following treatment with

alfaprostol produced lower in vitro P4 concentrations in response to LH than corpora

lutea formed after a spontaneous estrus. It was suggested the low fertility in Brahman

or Brahman crossbred cows could be caused by impaired CL development or other

direct ovarian effects.

Effect of Plasma Progesterone Concentrations on Preqnancy

Could low concentrations of P4 during the estrous cycle following breeding

result in poor conception and pregnancy rates? Direct evidence of this is hard to

obtain as a developing pregnancy may influence the concentrations of circulating P4.

However, plasma P4 concentrations are generally higher during the cycle before

breeding in fertile dairy cows than in infertile cows (Folman et al., 1973; Erb et al.,

1976; Fonseca et al., 1983). Rosenberg et al. (1977) reported ineffective inseminations

were preceded by cycles in which the peak of P4 concentration was reached 8 to 11 d

before Al vs the P4 peak being reached 4 to 7 d before Al (i.e., the shape of the P4

curve was important not P4 concentrations per se).

Mean concentrations of P4 were higher in pregnant cows than in cows returning

to estrus after breeding (Henricks et al., 1970). Over the first 15 d after mating

pregnant heifers had about 1.7 times more P4 in the plasma than those that returned

to estrus (Henricks et al., 1971). Progesterone concentrations were lower in infertile

cows following Al than in fertile cows (Erb et al., 1976). Concentrations of plasma P4









48

after Al were higher for Holsteins that conceived compared to those that did not

(Fonesca et al., 1983). Beef females with normal developing embryos after Al had

higher serum P4 at d 3 to 6 than females with abnormal embryonic development

(Maurer and Echternkamp, 1982). It is impossible to say whether the low P4 caused or

was a result of the abnormal embryos, but Holstein heifer embryo transfer recipients

had lower pregnancy rates when P4 concentrations were low than when concentrations

were high at time of transfer (Remsen and Roussel, 1982). In contrast, Sreenan and

Diskin (1983) found no difference in P4 concentrations in heifers pregnant to embryo

transfer and nonpregnant heifers until d 16 of the cycle (when the CL begins to regress

in non pregnant animals).

It would seem possible from the aforementioned data that low P4

concentrations may influence conception and that use of PGF2a (or its synthetic

analogs) may precipitate diminished pregnancy rates in Brahman or Brahman

crossbred females.














EXPERIMENTAL PROCEDURE


General Procedure

In trial 1, trial 2, and trial 4, purebred Brahman heifers from 24 to 27 months of

age were randomly assigned to treatment with a natural PGF2a' or used as untreated

controls. In trial 3, the research population consisted of 22 2-year-old, six 3-year-old,

and five 4-year-old purebred Brahman females. In all trials the animals were non-

lactating and had displayed at least one estrus prior to initiation of each trial (i.e., they

were cycling). Throughout the studies the diet of research animals consisted of coastal

bermudagrass hay, molasses-based liquid supplement (16% CP equivalent) and

complete mineral mix offered ad libitum plus 1.36 to 1.82 kg (3 to 4 Ib) of ground corn

per head per day. Overall average daily gain for heifers in the first two trials was .36

kg (.8 Ib) (figure 2). Heifers during all trials in the study were considered to be in

superior condition. In each trial, all heifers were pastured together and moved as a

herd regardless of treatment group. During a 1 month period before initiation of trial 1

and trial 2 heifers were trained to walk through the holding pens and Al chute twice

daily (AM and PM) prior to feeding of ground corn. This was to acquaint the heifers

with the facility in an effort to minimize stress during the blood collection phase of the

trials.

Prostaglandin F2a was administered by intramuscular injection with 3.81 cm (11/2

inch) 20 gauge needle into the gluteobiceps. Brahman heifers were monitored for


'Lutalysee, UpJohn Co., Kalamazoo, MI.

49







































FIGURE 2. WEIGHT CHANGE FROM WEANING THROUGH EXPERIMENTAL PERIOD FOR
HEIFERS IN TRIAL 1 AND TRIAL 2.














TRIAL 1


600


500


400


EXPERIMENTAL
PERIOD


100 200


300 400


500 600 700


TRIAL 2


600 L


500


400


EXPERIMENTAL
PERIOD


300


200 K


CU
0_
v:


100
0


300


200


100


0 100 200 300 400 500 600 700 800

DAYS AFTER WEANING


i


]









52

estrus using teaser bulls (surgically deviated penis) equipped with chin ball markers.

Records were made of time of day estrus was first observed and circumstance of

estrual behavior determination (stood to be mounted by bull, by other heifers, or no

longer standing to be mounted but previously marked by bull).

Blood was collected in heparinized Vacutainer tubes (Becton Dickson and

Company, Rutherford, NJ) by coccygeal venipuncture and immediately placed in ice

water until processed to yield plasma. Plasma was stored at -200C until assayed for

P4.

Cattle Handling Facilities

All experiments in this study were conducted at the Purebred Beef Unit

(Sandhill) of the Animal Science department, University of Florida, Gainesville. An

animal handling facility was constructed using an in-ground concrete silage bunker as

the primary corral area (figure 3). Heifers were rotated between the north and south

pastures, depending on the available forage. This facility was used strictly for

administering PGF2a injections, blood collection and Al of the heifers following

treatment. Any other routine handling of the cattle, such as administering of

antihelmintic medication or vaccinations, was conducted at a separate corral area.

Again, this was to minimize the stress associated with the facility used for treatment

and Al.

Radioimmunoassay for Plasma Proqesterone

Trial 1. Plasma P4 was determined according to the radioimmunoassay

procedure of Abraham et al. (1971) as modified for this laboratory by Lopez-Barbella et

al. (1979). An aliquot of sample plasma (500 ul) was placed in a screw top glass tube

and spiked with 100 lI of 1000 cpm [1,2-3H]-P4 (New England Nuclear, Boston,












54














--------










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1 D











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55

MA.,SA = 53.4 Ci/mmol) in .1 M phosphate buffered saline with .1% gelatin (PBSG).

The tube was vortexed and 10 ml ethyl ether was added. The solution was revortexed

and the tube plunged into liguid nitrogen for a period of time sufficient to freeze the

plasma to a pellet. The ether was decanted into 16 x 100 mm borosilicate tubes and

evaporated under nitrogen gas. Five milliliters of PBSG was added to the tube and

vortexed to resusupend the dried ether extract of the plasma sample.

Progesterone antiserum, provided by Dr. L. Fleeger of Texas A & M University,

College Station, was developed in a rabbit against P4 conjugated to bovine serum

albumin (BSA). Progesterone concentrations were determined against a linear standard

curve of P4 from 1000 to 31.25 pg/ml using the procedure described in Appendix A.

This assay was validated by adding to five replicates 1000, 500, 250, 125, 61.5,

and 31.25 pg P4/ml plasma (from an ovariectomized cow). A linear regression

equation of added vs measured P4 described differences among concentrations [Y =

2.66 + 1.05X; Y = amount of P4 measured (pg/ml) and X = amount of P4 added

(pg/ml); R2 = .98]. Recovery of the extracted P4 spiked samples was 90%. Intra- and

inter-assay coefficients of variation for sample assays, determined by assay of standard

plasma collected during diestrus, were 4.3% and 10.0%, respectively.

Trial 2. Plasma P4 concentrations were determined by a procedure similar to

the one outlined for trial 1. Progesterone antiserum for these assays was provided by

Dr. Juan Troconiz and Dr. Megalay de Manzo from the Universidad Central Venezuela,

at Maracay, Venezuela. This antiserum was generated in sheep against P4 conjugated

to BSA. In this procedure an aliquot of sample plasma (500 nl) was extracted with 5

ml benzene and hexane (1:2), frozen, and the solvent decanted (Louis et al., 1973).

The decanted solvent was then evaporated under nitrogen gas and the assay









56

proceeded as described above. Validation for this assay was conducted as for the

previous assay and a linear regression equation described differences among

concentrations (Y = 2.63 + 1.01X; Y = amount of P4 measured (pg/ml) and X =

amount of P4 added (pg/ml); R2 = .93]. Recovery of the extracted P4 spiked samples

was 97%. Intra- and inter-assay coefficients of variation for the sample assays,

determined by assay of standard plasma collected during diestrus, were 8.9% and

14.6%, respectively.

Experimental Protocol

Trial 1. Prior to trial 1, all heifers were monitored for estrus and then bled daily

from d 2 to d 14 of the first spontaneously occurring estrous cycle after March 1. This

established a base of data demonstrating the plasma P4 concentrations of normally

cycling Brahman heifers during the luteal phase of the estrous cycle.

After this initial blood collection period, trial 1 was initiated to determine whether

the PGF2a induced CL was different from the spontaneously occurring CL in Brahman

heifers in terms of plasma concentrations of P4. Treatment heifers were not

synchronized as a group but treated were individually and injected intramuscularly

either once or twice (with 11 d interval between injections) with 25 mg PGF2a. Heifers

in the control group received no injection but were handled in the same manner as

treated heifers. The first injection was given on either d 7 or d 14 of the estrous cycle

(estrus = d 0) and blood was collected once daily (0700 h) on d 2 to d 14 of the

induced or naturally occurring estrous cycle (table 1). If a heifer did not express estrus

after treatment with PGF2a the bleeding regimen was started on d 6 after the final

injection and continued for 13 d. Artificial insemination was delayed to 12 h (AM/PM















TABLE 1. EXPERIMENTAL DESIGN FOR TRIAL 1: TO DETERMINE IF THE
PGF2a INDUCED CL PRODUCES LOWER CONCENTRATIONS OF PLASMA P4
THAN THE SPONTANEOUSLY OCCURRING CL



No. of Day of cycle at Estrous cycle of
Group heifers Treatmenta first PGF2a injection bleed (d 2 to 14)


C 8 no PGF2a --- 1st cycle

1A 6 1 x PGF2a 7 1st cycle post PGF2a

1B 6 1 x PGF2a 14 1st cycle post PGF2a

2A 6 2 x PGF2a 7 1st cycle post 2nd
PGF2a injection

2B 6 2 x PGF2a 14 1st cycle post 2nd
PGF2a injection


a Heifers were injected with 25 mg PGF2a intramuscularly either once or twice
with the second injection given 11 d after the first.








58

rule) after the first naturally occurring estrus following blood sample collection to avoid

pregnancy confounding the P4 data.

Trial 2. A second experiment was devised to further evaluate the effect of day

of cycle when PGF2a is administered on expression of estrus. Plasma P4 was

monitored in an attempt to elucidate PGF2a effect in non-responding heifers. Brahman

heifers were randomly assigned to treatment as shown in table 2.

Heifers were treated individually with 25 mg PGF2a and not synchronized as a

group. They were bled twice daily (0700 h and 1900 h) from 1 d before injection to 3

d after the induced estrus (or from d 16 of the natural cycle in the case of untreated

animals). If a heifer failed to express estrus following an injection, she was bled twice

daily until 6 days after injection. Control heifers received no injections but were moved

through the corral and chute with the treated heifers at the time of injection and blood

collection. Heifers in this trial were Al at the PGF2a induced estrus.

Trial 3. A third trial was conducted to determine if two injections of PGF2a

given 24 h apart would induce estrus more effectively than a single injection. Non-

lactating Brahman heifers and cows were monitored for estrus and then assigned to

one of two treatment groups. Heifers were given either a single intramuscular injection

of 25 mg PGF2a on d 7 of the estrous cycle or two 25 mg injections with the first on d

7 and the second on d 8 of the cycle. A split plot design was used and each heifer

was treated twice (phase 1 and phase 2) during the study (table 3). Animals were Al

12 h after the onset of the last induced estrus (AM/PM rule) following treatment in the

second phase of the study.

Trial 4. In the fourth year of this study a preliminary trial was conducted to

assess the possibility of incorporation of double injections at a 24 h interval into the















TABLE 2. EXPERIMENTAL DESIGN FOR TRIAL 2: TO FURTHER EVALUATE THE
EFFECT OF DAY OF CYCLE ON WHICH PGF2a IS GIVEN ON
THE EXPRESSION OF ESTRUS



No. of Day of cycle at
Group heifers Treatmenta PGF2a injection Time of bleeding


1 6 no PGF2a --- d 16 to estrus + 3 d

2 6 1 x PGF2a 7 d 6 to estrus + 3 d

3 6 1 x PGF2a 10 d 9 to estrus + 3 d

4 6 1 x PGF2a 14 d 13 to estrus + 3 d

5 6 1 x PGF2a 18 d 17 to estrus + 3 d



a Heifers were injected once with 25 mg PGF2a given intramuscularly.















TABLE 3. EXPERIMENTAL DESIGN FOR TRIAL 3: TO DETERMINE IF TWO
INJECTIONS OF PGF2a GIVEN 24 HOURS APART INDUCE ESTRUS
MORE EFFECTIVELY THAN A SINGLE INJECTION



No. of Day of cycle at
Phase heifers Treatmenta PGF2a injection


1 16 1 x PGF2a d 7
17 2 x PGF2a d 7 and d 8



2 no. PGF2a
injections
in Phase 1

1 8 1 x PGF2a d 7
2 8 1 x PGF2a d 7

1 8 2 x PGF2a d 7 and d 8
2 9 2 x PGF2a d 7 and d 8


either once or


a Heifers were injected with 25 mg PGF2a intramuscularly
twice with the second injection given 24 h after the first.









61

traditional PGF2a management protocol (two injections at an 11 d interval). To this

end, 23 Brahman heifers were monitored for estrus and then treated, as a group, with

two injections of 25 mg PGF2a given 24 h apart. Stage of estrous cycle at time of

injection was recorded for each animal. Fertile Brahman bulls equipped with chin ball

markers were placed with the heifers at time of PGF2a treatment, making time of estrus

also the time of breeding in this trial. Pregnancy was determined by rectal palpation at

65 d after PGF2a treatment.

Statistical Analysis of Data

Plasma P4 data from trial 1 and trial 2 were analyzed using the least squares

analysis of variance and polynomial regression of the General Linear Model (GLM)

procedure of the Statistical Analysis System (SAS, 1985). Data in trial 1 were analyzed

by comparing a model consisting of treatment, response, treatment by response,

animal within treatment by response, and day up to the third order as sources of

variation with models in which the day variable was replaced with either day by

treatment or day by response to the third order. Possible differences in regression

relationships due to treatment and response were tested by examining the

heterogeneity of slopes (appendix tables 11, 12, and 13). Plasma P4 data in trial 2

were analyzed in a similar manner with the model consisting of treatment, response,

treatment by response, animal within treatment by response, and period to the third

order as sources of variation (appendix tables 15, 16, and 17).

The Catmod (Chi-square analysis) procedure of the SAS (1985) was used to

compare the effect of treatment and day of treatment on rates of estrual response in

trials 1, 2, and 3 (appendix tables 14 and 18). Chi-square analysis was also used to

determine if there was a difference in degree of synchrony of estrual response









62

following treatment with a single 25 mg injection of PGF2a on d 7 of the estrous cycle

as compared to a series of two 25 mg injections of PGF2a with the first given on d 7

and the second given on d 8 (trial 3, appendix table 19). Two-sided t-tests were used

to test for a possible effect of treatment on interval from PGF2a injection to induced

estrus (appendix table 20).















RESULTS AND DISCUSSION


Trial 1

The administration of either a single injection or a series of two injections (the

second given 11 d after the first) of a natural PGF2a (25 mg) did not adversely affect

the plasma P4 concentrations on d 2 to d 14 of the induced estrous cycle indicating

formation of a normal functioning CL after treatment (figure 5). Heifers given a single

injection of PGF2a on d 7 of the cycle (treatment 1A) had slightly higher P4

concentrations than heifers in the control group (treatment C, P<.01). This was largely

due to the influence of heifer #28. Because there were two nonresponders in this

group the third order regression curve was based on the data from only four heifers.

Heifer #28 had plasma P4 concentrations much higher than the other three heifers in

this treatment group with a peak of 13.04 ng/ml on d 12 of the estrous cycle. The

mean P4 concentration for the other three heifers on the same day the cycle was 7.78

1.31 ng/ml (mean SE). Progesterone concentrations in this same heifer on d 12

during the estrous cycle before treatment (appendix table 6) peaked at 12.84 ng/ml

while P4 at the same time for the other three heifers was 7.24 1.43 ng/ml. Although

the third order regression curve for treatment 1A was not parallel with the curve for the

controls, it was concluded the difference was not due to PGF2a treatment. For all

other treatments plasma P4 concentrations during an induced estrous cycle were

similar to those of the controls and to those of all heifers during the estrous cycle prior

to treatment with PGF2a (figures 4 and 5 and table 4).




















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69

Jim6nez et al. (1985) also reported no difference in P4 concentrations of Brown Swiss

or Indubrazil cows before and after treatment with a natural PGF2a.

In contrast, other researchers have reported that Brahman cows and heifers

treated with the PGF2a analog cloprostenol had lower serum P4 concentrations on d 2

to 13 of the induced estrous cycle than in naturally occurring estrous cycles. In

addition, treatment with cloprostenol on d 8 to 12 of the estrous cycle resulted in

development of a smaller CL which contained lower concentrations of P4 (Hardin and

Randel, 1982). Similarly, use of the PGF2a analog alfaprostol in Brahman heifers

resulted in lower P4 concentrations during the induced estrous cycle and produced a

CL with fewer small and large luteal cells which had lower in vitro P4 production in

response to LH challenge when compared to CL formed following spontaneous estrus

(Hansen et al, 1987b).

One explanation for the lower P4 concentrations reported in the previously

mentioned studies might be the effect of stress induced by the intensive handling of

the research animals necessary for blood sample collection. While the effects of stress

on P4 concentrations is poorly documented it has been suggested stress elicits a

release of corticosteroids from the adrenal glands which in turn results in an increase

in P4 release from the adrenals (Wagner et al., 1972). Holstein heifers that were

stressed had increased corticosteroid concentrations (Stoebel and Moberg, 1982a).

Administration of adrenocorticotropin hormone (ACTH) on d 1 to 8 of the estrous cycle

in heifers produced elevated corticosteroid concentrations as well as transient increases

in plasma P4 on d 1 to 5 followed by a significant decrease in P4 concentrations on d

8 to 10 (Wagner et al., 1972). The researchers suggested the increase in P4

concentrations was due to secretion of corticosteroids by the adrenals and that the









70

subsequent decrease in plasma P4 was due to a negative feedback on the

hypothalamus or pituitary which might have been sufficient to block normal LH

production. Stoebel and Moberg (1982b) reported use of ACTH caused increased P4

secretion by the adrenal cortex which resulted in elevated plasma P4 concentrations in

dairy cows. Heat stress of cows caused lower basal and peak LH concentrations

(Madan and Johnson, 1973). Stressed heifers had no LH surge following estrus but

unstressed heifers did (Stoebel and Moberg, 1982a). Hardin and Randel (1982)

reported the handling of Brahman females prior to estrus had detrimental effects on the

endocrine changes during the periestrous period but that frequent sampling during the

luteal phase did not alter the reproductive cycle. In the study presented here much

effort was exerted to minimize the amount of stress imposed on the research animals

(through training, facility use, and method of blood collection). It is believed that the

effects of stress on estrual response and P4 concentrations were negligible. During

these trials all heifers (treated and controls) were handled in exactly the same manner.

Presumably, if there was an effect of stress present it influenced all treatment groups

equally.

Another explanation for dissimilarities in the previously mentioned studies

(Hardin and Randel, 1982; Hansen et al., 1987b) and the data presented here is the

possibility that use of the PGF2a analogs had an adverse effect on the subsequently

forming CL. Hansen et al. (1987b) suggested artificial shortening of the estrous cycle

may alter selection of the ovulatory follicle and differentiation of the granulosa and

theca internal cells to luteal cells which might result in the formation of a subfunctional

CL. Since treatment with a natural PGF2a did not result in lower P4 concentrations it









71

is conceivable the use of these PGF2a analogs, instead of shortening of the cycle per

se, could result in lower P4 production.

The intent of this study was to determine whether the PGF2a induced CL

produced lower concentrations of plasma P4 than spontaneously occurring CL in

Brahman heifers. Days on which PGF2a was to be administered were selected to test

response when treated in the early and mid luteal phase. Unexpectedly, only 67% of

the heifers injected with PGF2a on d 7 of the cycle expressed estrus within 7 d after

injection while 100% of those injected on d 14 exhibited estrus (table 5). This was

reflected in the plasma P4 profiles for heifers that expressed estrus within 7 d after

treatment responderss) vs those that did not (nonresponders, figure 6; P<.001). Blood

sample collection was initiated on d 6 after injection in heifers that failed to exhibit

estrus. Plasma P4 profiles for two representative animals given a single injection on d

7 are shown in figure 7. Heifer #59 responded to the 25 mg of PGF2a and displayed

estrus on the second day following injection. Heifer #72 did not respond to the PGF2a

and expressed estrus 10 d after injection (or 17 d after the previous estrus a normal

cycle). Graphs of P4 concentrations for all nonresponders (figure 8) demonstrate the

diverse patterns of P4 for these heifers. Heifers P4 profiles were dependent on the

length of the individual estrous cycle. Heifer #1 (lower panel), for example, expressed

estrus 10 d after the first PGF2a injection (a 17 d estrous cycle) and so received the

second injection on d 1 of the estrous cycle. The heifer then exhibited estrus 2 d after

the end of the bleeding regimen (a 20 d estrous cycle). Heifer #83 (upper panel)

expressed estrus on the third day of the regimen (a 17 d estrous cycle).

Some researchers have reported a lower response rate when heifers are

injected with PGF2a or its analogs early in the cycle (Roche, 1974; Macmillan, 1978;



























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FIGURE 6. PLASMA P4 PROFILES FROM D 2 TO D 14 OF THE INDUCED ESTROUS
CYCLE FOR HEIFERS THAT RESPONDED TO PGF2a TREATMENT AND FROM D 6 TO D 18
AFTER THE PGF2a INJECTION FOR NONRESPONDING HEIFERS (TRIAL 1).












1ST PGF2a ON

12 DAY 7
12- 1TPFaO


RESPONDERS






NONRESPONDERS


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DAY 14


RESPONDERS


2ND PGF2a ON
DAY 7.71.1


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79

King et al., 1982; Macmillan, 1983; Wahome et al., 1985; Watts and Fuquay, 1985)

while others reported good response to treatment at this time (Edqvist et al., 1975;

Gonzalez et al., 1985). The failure of PGF2a treatment to induce estrus in all heifers

when injected on d 7 is especially troublesome when the two injection system of

PGF2a synchronization (second injection given 10 to 12 d after the first, Roche; 1974)

is used. In trial 1, heifers that were given a first PGF2a injection on d 7 or d 14 of the

estrous cycle followed by a second 11 d later demonstrated estrus 67% of the time

following the second injection (table 5). In view of the low response rate to a first or

only injection on d 7 of the cycle, this would not be unexpected as heifers that

responded to the first injection on d 7 or d 14 were at 7.75 d and 7.00 d, respectively,

of the induced estrous cycle at the time of the second injection (table 6). Indeed, this

system of PGF2a synchronization depends on a majority of animals being at d 7 to 8

of the estrous cycle at the time of the second injection.

Artificial insemination was postponed in this trial until the first naturally occurring

estrus after final PGF2a injection to avoid pregnancy confounding the P4 data. There

was an overall pregnancy rate of 60% with a first service pregnancy rate range of 33 to

67% (table 5). Trial 1 was designed to examine the effect of PGF2a on plasma

progesterone concentration and so the number of heifers in each treatment group were

limited. The small number of heifers in each treatment group did not allow a valid

statistical analysis of the response or pregnancy data.

Trial 2

A second trial was conducted to further evaluate the effect of day of cycle when

PGF2a is administered on expression of estrus. In trial 2, as in previous trial, the rate

of estrous response differed with day of injection (table 5). Only 50% of the heifers















TABLE 6. MEAN DAY OF ESTROUS CYCLE AT THE TIME OF
SECOND PGF2a INJECTION (TRIAL 1)


Day of cycle Day of cycle
at 1st injection at 2nd injection Range


7 7.75 7 9

14 7.00 5 9









81

injected on d 7 of the estrous cycle and 67% injected on d 10 expressed estrus within

7 d following treatment with PGF2a. All heifers injected on d 14 responded to the

PGF2a. Likewise, all heifers injected on d 18 of the cycle expressed estrus within 7 d.

When data from trials 1 and 2 were combined, significantly fewer heifers expressed

estrus after the first or only PGF2a injection on d 7 (61%) than those given a first or

only injection on d 14 (100%) (P<.05; table 7).

In this same combined data set the interval from PGF2a to estrus tended to be

shorter for heifers injected on d 7 than for heifers injected on d 14 (2.95 d vs 3.64 d,

respectively; P<.09; table 7). Similar findings for either natural PGF2a or PGF2a

analogs were reported by other researchers (Jackson et al., 1979; Refsal and Seguin,

1980; King et al., 1982; Stevenson et al., 1984). Jackson et al. (1979) suggested the

shorter interval to estrus when PGF2a is injected early in the cycle may be attributed to

an early wave of follicular growth and the resultant increase in plasma estrogen.

Pierson and Ginther (1984), using ultrasonography, determined there were two follicular

waves during the estrous cycle of the cow with the first large follicle in the first wave

regressing around mid-cycle. Sirois and Fortune (1988), however, indicated the

ultrasonography of the ovaries in heifers showed three waves of follicular development

with the first beginning on d 1.9, the second on d 9.4, and the third on d 16.1. The

effect that developing follicles may have on interval to estrus following PGF2a treatment

is probably due to the peaks of estrogen which follow the same wavelike pattern of

follicular growth (Hansel and Echternkamp, 1972; Shemesh et al., 1972; Dobson and

Dean, 1974; Glencross and Pope, 1981). As previously discussed estrogen, OT and

PGF2a act in concert to effect luteolysis. High plasma concentrations of estrogen may

act to drive the luteal regression initiated by a PGF2a injection.
















TABLE 7. SYNCHRONIZATION RATES AND INTERVAL FROM INJECTION
TO ESTRUS ON D 7 OR D 14 OF THE ESTROUS CYCLE
(TRIALS 1 AND 2 COMBINED)



Day of cycle Number of In estrus by Days from injection
at 1st injection heifers 7 d post-inj., % to estrus SE


7 18 61 2.95 .41


14 18 100 3.64+ .24


P<.05


+ P<.09








83

Heifers in this trial were Al to the induced estrus after PGF2a (AM/PM rule).

There was an overall pregnancy rate of 63% with a first service pregnancy rate range

of 33 to 50%. As in trial 1, the purpose of trial 2 was to examine the effect of PGF2a

on P4 concentrations. The small number of heifers in each treatment group precluded

valid statistical analysis of the effect of treatment on response and pregnancy rate

within trial 2.

Plasma P4 was measured in trial 2 as an attempt to further elucidate PGF2a

effect in nonresponding heifers. Regression curves of plasma P4 concentrations, from

time of PGF2a injection, for responding and control heifers are shown in figure 9.

Progesterone profiles from the time of injection differed due to treatment with untreated

heifers in the control group having a slower rate of P4 decline (P<.01). Figure 10

shows the means SE of plasma P4 concentrations for heifers that either expressed

estrus within 7 d after PGF2a responderss) or did not (nonresponders). All treated

heifers demonstrated a precipitous decline in plasma P4 by 12 h after injection and P4

continued to decline until 24 h after injection. The depressed concentrations of P4,

however, began to increase within 48 h after PGF2a in the heifers that failed to express

estrus after treatment. Analysis of the third order regression curves for these data

indicate plasma P4 concentrations for nonresponders continued to increase (P<.001)

and reached concentrations approximately three times greater than in heifers exhibiting

estrus by 6 d after PGF2a injection. The drop in plasma P4 by 12 h after injection and

the subsequent increase is common to all animals in trial 2 that failed to express estrus

after treatment (figure 11). Plasma P4 concentrations for nonresponders following a

PGF2a injection on d 7 (upper panel) and d 10 (lower panel) are shown in figure 11.




















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