Endocrinology of the superovulated cow and subsequent prostaglandin F₂[alpha] regression of the multiple corpora lutea

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Endocrinology of the superovulated cow and subsequent prostaglandin F₂alpha regression of the multiple corpora lutea
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xiii, 105 leaves : ill. ; 28 cm.
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English
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Lopez Barbella, Sergio Rafael, 1945-
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Subjects / Keywords:
Beef cattle -- Breeding   ( lcsh )
Prostaglandins   ( lcsh )
Beef cattle -- Breeding   ( fast )
Prostaglandins   ( fast )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 94-104).
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Also available online.
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Typescript.
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Vita.
Statement of Responsibility:
by Sergio Rafael Lopez Barbella.

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ENDOCRINOLOGY OF THE SUPEROVULATED COW AND SUBSEQUENT PROSTAGLANDIN F2a REGRESSION OF
THE MULTIPLE CORPORA LUTEA









By

SERGIO RAFAEL LOPEZ BARBELLA
























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




UNIVERSITY OF FLORIDA


1977















ACKNOWLEDGEMENTS



In most cases, every research product is the result of multiple endeavors. This one is no exception. Since its very beginning, many persons and institutions have provided enormous contributions. Without them this dissertation would never have been possible. The list is long as deep is my indebtedness.

Special thanks go to every member of my Supervisory Committee: to Dr. Michael J. Fields, my chairman, for his help, patience, and encouragement not only during every phase of this research but throughout these years of graduate work; to Drs. Fuller W. Bazer and William W. Thatcher for always being available to share their knowledge and for their valuable comments; to Dr. Alvin C. Warnick, for providing his experience in the area through sound remarks; to Dr. Pejaver V. Rao, for statistical assistance; and to Drs. Daniel C. Sharp III and Robert J. Collier for reading and correcting the initials manuscripts of this study.

My appreciation to Universidad Central de Venezuela, Facultad

de Agronomia, for providing economical support throughout my graduate work.

I am also grateful to the Upjohn Company, especially to Dr. J. Lauderdale, for providing PGF2a and reviewing my experimental protocols. To Dr. J. L. Fleeger, Texas A&M University, and Dr. L. V. Estergreen, Washington State University, my gratitude is extended for


ii









their collaboration in providing the progesterone and estrogen antisera, respectively. Special thanks are also due to Dr. J. H. Hentges, University of Florida, and Mr. Ardeen Wiggins, University of Florida Foundation, for providing the Angus cows used in these experiments.

To my fellows graduate students Tomas H. Wise, Jorge Beltran, Daniel Hardin, and Thomas Thompson my indebtedness for their help at different phases of this study.

Finally, and above all, the author wishes to express gratitude and appreciation to his mother,Regina, to his wife, Lourdes, and to his children, Sergio and Ana, for their love in both good and difficult times, and to Mrs. Rossina Fernandez for her typing of this manuscript.

































i i
















TABLE OF CONTENTS



Page

ACKNOWLEDGEMENTS . . . . . . . . . . . .ii

LIST OF TABLES . . . . . . . . . . .. .. vi

LIST OF APPENDIX TABLES . . . . . . . . . vii

LIST OF FIGURES . . . . . . . . . . . ix

ABSTRACT . . . . . . . . . . . . xi

CHAPTER I: INTRODUCTION . . . . . . . . .

CHAPTER 11: LITERATURE REVIEW . . . . . . . 4

Prostaglandins and Reproduction . . . . . . 4
Prostaglandin F2a and Estrous Synchronization . 4 Luteolytic Effect of Prostaglandin F2 ....... 6 Prostaglandin F2a and Therapeutic Abortion . 8 Prostaglandin F2a and Superovulation . . 10
Gonadotropic Hormones and Limited Multiple Births . 11
Endocrinology of the Superovulated Cow . . 14
20-Dihydroprogesterone (4-Pregnen-206-ol-3-one). . 16

CHAPTER Ill: MATERIAL AND METHODS . . . . . . 21

EXPERIMENT I. CHARACTERIZATION OF PLASMA PROGESTERONE
(P), 20B-DIHYDROPROGESTERONE (206-P), TOTAL ESTROGENS (E), AND LUTEINIZING HORMONE (LH) IN PMSG SUPEROVULATED COWS SYNCHRONIZED WITH PGF2a-THAM SALT. . 21 Surgical Description of Supravaginal Laparotomy 23
EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN
PMSG SUPEROVULATED BEEF CATTLE FOLLOWING ADMINISTRATION OF 40 MG PGF2a-THAM SALT. 23 EXPERIMENT 3. LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN BRED VS CYCLING BEEF COWS PREVIOUSLY TREATED WITH PMSG . . . . . 25
Radioimmunoassay of Steroid Hormones . . . . 27
Cleaning of Glassware . . . . . . . 27
Organic Solvents . . . . . . . 27
Preparation, Use, and Storage of Radioactive
Steroids . . . . . . . . . 27


iv










TABLE OF CONTENTS Continued


Page

Conversion of Progesterone (3p) to 208-Dihydroprogesterone (3H-2O0 -P). . ... . . . . 28
Use and Storage of Antibody . . . . 29 Serum Extraction and Chromatography . . . 29 Conversion of 3H-2OB-P to 3p . . . . . 33
Preparation, Use, and Storage of Assay Buffer. 33
Preparation, Use, and Storage of Charcoal
Suspension . . . . . . .... . . . 34
Preparation and Use of Counting Solution . 34
Preparation, Use, and Storage of Standard
Steroids . . . . . . . . . .34
Radioimmunoassay . . . . . . . . 35
Calculations . . . . . . . . . 40
Separation by LH-20 Column Chromatography. . 40 Precision . . . . . . . . . 41
Statistical Analysis . . . . . . . . 41

CHAPTER IV: RESULTS AND DISCUSSION . . . . . 43

EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE
(P) 208-DIHYDROPROGESTERONE (20B-P), ESTROGENS (E), AND LUTEINIZING HORMONE
(LH) IN THE PMSG SUPEROVULATED COW SYNCHRONIZED WITH PGF2a-THAM SALT . . 43 Reproductive Response . . . . . ... ..43
Endocrine Response . . . . . . . 48
Progesterone . . . . . . . . . 48
Estrogens . . . . . . . . . 53
Luteinizing Hormone . . . . . . 54
208-Dihydroprogesterone . . . . . 55
EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN THE SUPEROVULATED BEEF COW FOLLOWING ADMINISTRATION OF 40 MG PGF2u-THAM SALT 57 Reproductive Response . . . . . . 57
Endocrine Response . . . . . . . 58
EXPERIMENT 3. LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN
THE PMSG BRED VS NONBRED BEEF COW . 62 Reproductive Response. . . . . . . 62
Endocrine Response . . . . . . . 65

CHAPTER V: SUMMARY AND CONCLUSIONS . . . . . 72

APPENDIX . . . . . . . . . . . . 78

LITERATURE CITED . . . . . . . . . . 94

BIOGRAPHICAL SKETCH . . . . . . . . . 105


v














LIST OF TABLES



TABLE Page

I EFFICIENCY OF 3H-P CONVERSION TO 3H-20-P AT
VARYING LEVELS OF COFACTORS. . . . . .. .31

2 STANDARD CURVE DILUTIONS . . . . . .. .. 36

3 ADDITIONAL TUBES USED TO SUPPLEMENT RIA ANALYSIS . 37

4 DISTRIBUTION OF ANIMALS SHOWING ESTRUS AFTER THE
SECOND PGF2a ADMINISTRATION AND SUBSEQUENT OVARIAN
RESPONSE OF ANIMALS TREATED WITH PMSG IN EXPERIMENT I . . . . . . . . . . . 44

5. CONCEPTION RATES FOLLOWING ARTIFICIAL INSEMINATION
POST-TREATMENT WITH PMSG AND PGF2a AND EXPOSURE
TO THE BULL FOR 90 DAYS IN EXPERIMENT 1. . . . 47

6 DISTRIBUTION OF ESTRUS AND OVULATION RATE IN COWS
AFTER A DUAL INJECTION OF PGF2a-THAM SALT AND
PMSG . . . . . . . . . . . 59

7 DISTRIBUTION OF ESTRUS IN COWS AFTER A DUAL 33.5
MG SYNCHRONIZING INJECTION OF PGF2a-THAM SALT
AND ONE 40 MG ABORTIFACIENT DOSE OF PGF2 *...... . 63

8 OVULATION DISTRIBUTION POST-PGF2a INDUCED ESTRUS
IN COWS IN EXPERIMENT 3 . . . . . . 66

9 OVULATION RATES IN COWS TREATED TWICE WITH PMSG
AT FOUR MONTH INTERVAL . . . . . . . 67













VI














LIST OF APPENDIX TABLES



TABLE Page

10 PREPARATION AND STORAGE OF ASSAY BUFFER . . . 78 11 PREPARATION OF NADH IN 0.1 M TRIS BUFFER ..... 79

12 PLASMA CONCENTRATIONS OF PROGESTERONE,20-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING MORE THAN THREE CORPORA LUTEA AT
LAPAROTOMY . . . . . . . . . . 80

13 PLASMA CONCENTRATIONS OF PROGESTERONE, 20-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN
COWS HAVING TWO OR THREE CORPORA LUTEA AT LAPAROTOMY 82

14 PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN
COWS WITH ZERO OR ONE CORPORA LUTEA AT LAPAROTOMY. 83

15 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES
FOR PROGESTERONE . . . . . . . . 84

16 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
ESTROGENS AND 20a-DIHYDROPROGESTERONE (208-P). . 85

17 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
LUTEINIZING HORMONE . . . . . . . 86

18 COEFFICIENTS OF CORRELATION BETWEEN PLASMA HORMONAL
LEVELS AND NUMBER OF CORPORA LUTEA . . . . 87

19 PLASMA CONCENTRATIONS OF PROGESTERONE AFTER 40 MG
PGF2ct IN COWS TREATED WITH OR WITHOUT PMSG . . 88

20 SPLIT-PLOT ON TIME ANALYSIS OF VARIANCE OF PROGESTERONE IN EXPERIMENT 2 . . . . . . . 89

21 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTERONE . . . . . . . . . . 90

22 ANALYSIS OF VARIANCE IN EXPERIMENT 3 . . . . 91

23 PLASMA CONCENTRATIONS OF PROGESTIN AFTER 40 MG PGF2a
IN COWS SUPEROVULATED WITH PMSG AND EXPOSED TO
BREEDING . . . . . . . . . . . 92

vii










LIST OF APPENDIX TABLES Continued


TABLE Page

24 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTIN . . . . . . . . . . 93














































viii















LIST OF FIGURES



FIGURE Page

I Treatment protocol for experiment I involving
endocrine characterization following PMSG and
PGF2a . . . .................. . .. 22

2 Treatment protocol for experiment 2 evaluating
the effectiveness of PGF2. to regress multiple
corpora lutea in nonbred cows . . . . . 24

3 Treatment protocol for experiment 3 comparing
the efficacy of PGF2a to regress multiple
corpora lutea in the bred cow . . . . 26

4- LH-20 profile of 3H-1,2 progesterone (P) conversion to 3H-,I 2-20B-dihydroprogesterone
(20B-P) using hexane:benzene:methanol (80:15:5). 30

5 Standard curve of progesterone. (Each point
represents the mean of triplicates on 20
determinations.) . . . . . . . . 38

6 Standard curve of estradiol. (Each point represents the mean of triplicates on seven
determinations.) . . . . . . . . 39

7 Concentration of progesterone ('**), 20-dihydroprogesterone (---), luteinizing hormone (---),
and estrogens (--) in plasma from PMSG treated
cows with four or more corpora lutea . . . 49

8 Concentration of progesterone (.. ), 208-dihydroprogesterone (---), luteinizing hormone ---),
and estrogens ---) in plasma from PMSG treated
cows with two or three corpora lutea . . . 51

9 Concentration of progesterone (.-- ), 20B-dihydroprogesterone (---), luteinizing hormone (--),
and estrogens (---) in plasma from PMSG treated
cows with zero or one corpora lutea . . . 52



ix










LIST OF FIGURES Continued


FIGURE Page

10 Effectiveness of PGF2a to regress corpora lutea and
reduce plasma progesterone in PGFea synchronized
cows treated with PMSG (-) and without PMSG (---). 61

11 Plasma progestin profile in PMSG-BRED (----) and
PMSG-CYCLING (---) cows after 40 mg PGF2C. . ..... 68

12 Plasma progestin in BRED (-) and CYCLING (---)
cows after 40 mg PGF2 . *............... . 69






































x












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




ENDOCRINOLOGY OF THE SUPEROVULATED COW AND SUBSEQUENT PROSTAGLANDIN F2a REGRESSION OF THE MULTIPLE CORPORA LUTEA

By

Sergio Rafael Lopez Barbella

December 1977

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

The endocrinology of the Pregnant Mare Serum Gonadotropin (PMSG) superovulated beef cow was studied in one experiment in which estrous synchronization of 15 Angus cows was achieved with a dual injection of 33.5 mg prostaglandin F28 (PGF2a) at a 12 day interval. Ovarian stimulation was achieved by injecting these cows with 2,000 i.u. PMSG 24 hr prior to the second PGF2a administration. Cows were artificially inseminated three times with Brahman semen at 0, 12, and 24 hr after detection of the PMSG-PGF2a-induced estrus. Progesterone, 20-dihydroprogesterone, luteinizing hormone and estrogen levels were monitored by RIA of plasma from cows bled twice daily for 21 days starting 2 days prior to PMSG administration. Ovulation rate, determined by supravaginal laparotomy 13 days after PMSG administration, indicated that 66% (10/15) of the animals had more than one corpus luteum (CL). Cows with the longest interval from treatment to estrus displayed the lowest superovulatory response. This suggested a relationship between length


xi









of the interval from PMSG-PGF2a to estrus and superovulation. This possibility was supported by observed changes in the plasma endocrine patterns.

When the endocrinology of the superovulated cow was studied by

partitioning animals according to number of CL and progesterone levels, it was found that hormonal trends were similar with respect to postPMSG levels of progesterone, but estrogen concentrations were higher in superovulated cows (P<.1O). Although no significant relationship was detected between plasma LH concentrations and ovulation rate, the surge of LH was 12 hr earlier in cows having more than four CL when compared to treated cows having only one CL.

In an attempt to reduce fetal wastage from the superovulatory

response, cows having more than three CL, at laparotomy, were treated with 40 mg PGF2a. Although there was a significant (P<.Ol) decline in plasma progesterone levels and an absence of palpable CL during the 7 days post-40 mg PGF2a, cows continued to carry the fetuses throughout gestation. The question thus arose as to whether the fetus was rescueing the CL or the dose of PGF2a was indadequate for the increased CL mass. In an attempt to answer these questions, two experiments were conducted to test the efficacy of PGF2a to regress multiple CL in bred vs nonbred cows.

In the second experiment estrous was synchronized in 13 Angus cows with a dual injection of 33.5 mg PGF2a at a 12 day interval. Ten cows received 2,000 i.u. PMSG 24 hr prior to the second PGF2a injection. Cows were not bred. Determination of ovulation rate and treatment was as previously described. Cows were bled daily for 11 days, starting 2 days prior to laparotomy and plasma was analyzed for progesterone xii









concentration by RIA. Forty milligrams of PGF2a was effective in regressing multiple CL in these PMSG-treated nonpregnant cows based on a precipitous decline in plasma progesterone levels, the absence of palpable CL on day 7 post-40 mg PGF2a and the resumption of recurring estrous cycles.

In experiment 3, the inclusion of mating post-treatment resulted in a 2x2 factorial with PMSG and MATING as treatments for comparison. The accumulated data from this trial supported the resul'ts from the previous experiment. Therewereno observed differences between mated and nonmated animals in response to 40 mg PGF2a.

In conclusion,, these series of experiments confirmed that the ovarian and endocrine response of the bovine female is affected by PMSG. -When PMSG is used in an attempt to attain multiple fetuses, the high abortion rate.associated with the superovulation effect might be minimized by regressing the multiple CL with PGF2a and rebreeding the cow in a short breeding season. This possibly could lead to a number of cows with multiple fetuses at birth which would increase the percent calf crop.



















xiii















CHAPTER I
INTRODUCTION



In presert-day beef production in the United States the trend-is

towards a reduction in the life cycle by finishing cattle at an earlier age. Steady progress can be expected towards eliminating the long holding periods enabling a faster turnover of capital and a finished beef animal that will be more acceptable to both butcher and consumer. As age of slaughter is reduced by advances in breeding and feeding, fertility must assume an even greater importance in the economy of production.

Unfortunately, the beef cow is one of the least efficient of all the meat animals, i.e., the animal is maintained year round to produce only one useful product, a calf. Failure of the cow to wean a calf leaves the maintenance costs to be borne by the productive members of the cow herd. On the other hand, one-cow-one-calf per year is often a tenuous economical position when applied to land which offers profitable alternative utilization. A primary means available for efficiently improving production in the beef cow is to wean a greater number of calves each year. With little or no increase in cow numbers and only a limited rise in production costs, a substantial increase in net income could possibly be achieved. One way in which this could be accomplished would be to increase the rate of twinning in the beef

cow.



I




2




Unfortunately, the heritability and incidence of natural twin

births is low. Therefore, some alternative means for increasing the twinning rate needs to be developed, i.e., embryo transfer or hormonally induced controlled ovulation rate.

Superovulation in beef cattle has been induced by various extracts of the'anterior pituitary; however, pregnant mare serum gonadotropin (PMSG) has the advantage of being more readily available. In addition, PMSG is not as readily destroyed by the body which allows the use of a single injection for ovarian stimulation. Pituitary extracts require a series of injections; however, in either case ovulation rate is highly variable.

Almost without exception, researchers in the field of superovulation have reported high death losses among triplet and larger litters, with a lower mortality rate among twins. Therefore, in the case of the superovulation effect from exogenous gonadotropins, it is proposed that Prostaglandin F2. (PGF2a) may be introduced into the hormonal regimen to induce premature regression of multiple corpora lutea (CL) resulting from an excessive ovulation rate in an attempt to reduce fetal wastage. Thus, it was the objective of this study to determine if PGF2. can effectively regress multiple CL in the pregnant cow.

It was important that the gonadotropin injections be timed rather precisely with reference to the occurrence of the previous estrus. Therefore, it was of interest to determine if synchronization of the estrous cycle could be accomplished with a series of two injections of PGF2a and if this would minimize the necessity of determining the exact stage of the estrous cycle for giving the injections of PMSG.









Since several reports indicate that ovulation, egg transport,

embryo transport, and fertilization in the superovulated cow might be affected by an imbalance in the hormonal pattern, the effect of synchronizing estrouswith PGF2a when used in conjunction with PMSG was evaluated through studying the levels and interrelationships of the reproductive hormones.














CHAPTER II
LITERATURE REVIEW



Prostaglandins and Reproduction


Prostaglandins have been intimately linked with reproduction since their first extraction from human semen and sheep vesicular glands by von Euler in 1934. Studies in basic reproductive endocrinology with prostaglandins were stimulated by the report of Pharris and Wyngarden (1969) who demonstrated the luteolytic action of PGF2a in the rat. To date, prostaglandins have been shown to be involved in nearly all phases of the endocrine system regulating reproductive function. The following review attempts to highlight the effect of PGF2a in synchronizing estrus and as an abortifacient agent in the bovine. Prostaglandin F2a and Estrous Synchronization


The successful agent for synchronization of estrus should allow effective synchronization of estrus and normal fertility of the synchronized estrus. In addition, the method has to be practical enough to be used under range conditions.

After the initial report of PGF2a induced luteolysis in pseudopregnant rats (Pharris and Wyngarden, 1969), others observed that treatment with a single injection of PGF2a (Rowson et al., 1972; Lauderdale, 1972; Inskeep, 1973; Oxender et al., 1974; Rodriguez, 1974; Roche, 1974; Fields et al., 1975; Thatcher and Chenault, 1976) or synthetic analogues




4









of PGF2a (Tervit etal., 1973; Cooper, 1974; Fields et al., 1977b) was effective in causing luteolysis in heifers and cows except for the first 5 days post-estrus (Rowson et al., 1972; Louis et al., 1973).

In cattle, a single injection of PGF20 given during the responsive stage of the estrous cycle is followed by an ovulation and normal fertility. There are, however, several factors associated with the uie of a single injection of PGF2c which limits its practical usefulness for ovulation control. It is well documented that PGF2a is effective only after day 5 of the estrous cycle when a mature corpus luteum (CL) is present (Lauderdale, 1972; Cooper, 1974). Administration of PGF2a to animals with a palpable CL resulted in 65% of the treated animals displaying visual signs of estrus within 7 days post-injection (Lauderdale et al., 1974). Others have reported similar results following PGF2. treatment (Louis et al., 1973; Louis et al., 1974a; Chenault et al., 1976).

In an attempt to by-pass the unresponsive days I to 5 of the

estrous cycle, animals can be injected twice with PGF2a at a 10 to 12 day interval, as suggested by Inskeep (1973). Cooper (1974) reported that this dual injection technique, with an ICI analogue of PGF20 (ICI 80,996), resulted in only 2 of 175 animals failing to respond to the second PGF2. treatment. Furthermore, 90% of the animals were in estrus between 48 and 72 hr after the second treatment and fertility of this second estrus was normal. Using this technique, in a large field study with heifers and cows, Hafs (1975) found 68% of the heifers and 62% of the cows were in estrus 48 to 84 hr after the second PGF2a injection. Several researchers have shown this synchronized estrus to be fertile (Inskeep, 1973; Lauderdale et al., 1974; Roche, 1974;








Rodriguez, 1974; Cooper and Jackson, 1975; Hafs et al., 1975b; Turman et al., 1975; Ellicott and Thompson, 1976; Fields et al., 1977b; Moody and Lauderdale (1977).


Luteolytic Effect of Prostaglandin F2U


The sequence of changes in the reproductive tract and plasma levels of gonadotropic and ovarian hormones that occur during synchronization of estrus with PGF2a are similar to those occurring around natural estrus. The induction of estrus is rapid and precise and the induced CL has a normal life-span.

Louis et al. (1972a) injected 5 mg PGF2a-Tham Salt into the uterine horn ipsilateral to the CL and reported the interval to estrus was 72 hr, to LH peak 71 hr, and to ovulation 96 hr. An intramuscular injection of 30 mg PGF2a during diestrus resulted in a 60% decrease in plasma progesterone within 12 hr with the initial progesterone concentration of 4 ng/ml declining to .8 ng/ml by 24 hr (Louis et al., 1972a, 1973). The interval to onset of estrus, LHI peak and ovulation was 74, 77, and 104 hr post-PGF2a treatment, respectively. Similar results in declining plasma progesterone were reported by Oxender et al. (1974) evenwhen PGF2a was given intramuscularly in varying numbers of injections. The spaced double injection regimen produced precise synchronization of estrus in all the heifers responding to treatment (Dobson et al., 1975). After both injections, the CL showed rapid morphological regression that was similar to that observed for a single injection and was associated with a significant fall in plasma progesterone concentration 6 hr post-treatment with basal values being reached within 24 hr. Rapid follicular growth and secretion of estradiol




7




ensued with a return to estrus 48 to 96 hr after the first, and 48 to 55 hr after the secondPGF2a treatment. A preovulatory surge of LH occurred 62 to 103 hr after the first, and 48 to 62 hr after the second PGF2, treatment, and was followed by ovulation. Similar changes have been reported by Stellflug et al. (1973), Louis et al. (1973), and Louis et al (1974a,b).

Chenault et al. (1976) reported that a single injection of PGF2a produced a rapid decline in plasma progestin to estrus concentrations by 24 hr post-treatment, whereas, estradiol concentrations slowly increased and apparently stimulated an ovulatory surge of LH at 72 21 hr post-treatment. 'Ovulation occurred at 99.5 19 hr after PGF2a administration. Gimenez et al. (1976) reported plasma progesterone in the uterine vein decreased from 1,000 ng/ml to 2 ng/ml within 5 days after PGF2a intrauterine treatment and 24 hr after intramuscular injection. In the same study, plasma estrogens concentrations ranged from 25 to 100 pg/ml prior to treatment with no observed change in estrogens levels occurring after giving PGF2a when compared to the salinetreated cows.

The mechanism by which PGF2a initiates CL regression is unknown. Novy and Cook (1973) and Thornburn and Hales (1972) demonstrated that PGF2a may redistribute intraovarian blood flow, reducing the amount of blood flowing to the CL and increasing that to the stroma and follicular component of the ovary. Morphologically, this will result in both a functional termination in progesterone secretion and a structural regression or physical destruction of the luteal cell.

Because structural regression was preceded by accumulation of

lipid droplets, Stacy et al. (1976) postulated functional CL regression




8




was most likely due to a blockage in one or more stages in steroidogenesis. Finally, Henderson and McNatty (1975) presented a biochemical hypothesis by which PGF2a may initiate CL regression through a direct or indirect action at the adenylate cyclase catalytic site at the cellular membrane level.


Prostaglandin F2a and Therapeutic Abortion


An effective means of inducing parturition in the cow could reduce calving losses and labor costs by decreasing the calving period to a shorter and predictable period. Parturition can be induced in the cow by treating with estrogens (Spears et al., 1974), corticoids (J6chle, 1973), and PGF2. (Lauderdale, 1972). In addition, PGF2a can be used to terminate unwanted pregnancies.

The abortifacient property of PGF2a was initially demonstrated in laboratory species when pregnancy was terminated in 100% of rats given three daily injections of PGF2a (Gutknecht et al., 1969). In humans, PGF2U has been used, with varying degrees of success, to terminate pregnancy at different stages of gestation (Henricks, 1972). In farm animals, vascular infusions or systemic injections of PGF2a have been reported to terminate pregnancy in porcine (Diehl and Day, 1973), caprine (Currie and Thorburn, 1973), equine (Douglas et al., 1974), and bovine (Lauderdale, 1972, 1974; Fields et al., 1977a) species. Administration of PGF2a either intramuscularly or systemically at early stages of gestation in the bovine resulted in a dramatic decline in plasma progesterone within 24 hr and behavioral estrus 2 to 16 days post-treatment (Zerobin et al., 1973; Douglas et al., 1974).









Louis et al. (1974a) injected 5 mg PGF2. into the uterine horn ipsilateral for the CL In cows 11 days postmating and reported plasma progesterone at 0, 24, and 48 hr declined from 3.6 0.3 to 1.7 0.2 ng/ ml and then to 1.0 0.1 ng/ml, respectively. Plasma estradiol concentrations increased from 5.0 1.0 pg/ml to 6.1 0.4 pg/ml, to 11.3

0.7 pg/ml, and 12.7 1.3 pg/ml at 0, 12, 24, and 48 hr post-PGF2atreatment. The LH peak was detected at.71 4 post-treatment followed by estrus at 72 5 hr and ovulation at 95 5 hr post-treatment.

Although hormonal patterns resulting from PGF2a induced CL regression of pregnant vs nonpregnant cows were similar, the expression of estrus and ovulation appear to be dependent on stage of gestation. Douglas et al (1974) reported cows at 80 to 90 days of pregnancy to be in estrus 2 days following abortion and ovulating 6.5 days postabortion. In contrast, cows at 160 to 180 days of gestation showed estrus and ovulated 15.9 and 29.3 days post-abortion. In addition the late pregnant cow had a high incidence of retained placenta. Similar observations have been made by Henricks et al. (1977).

With the use of cloprostenol, a PGF2a analog, for the termination of pregnancy, Jackson and Cooper (1977) reported that cows in the first, second, and third trimester of gestation aborted with 58% abortions in less than 7 days, 25% between 7 and 14 days, and 13% in more than 14 days post-treatment, even though the composite plasma progesterone concentration declined to basal levels of 1.5 0.6 ng/ml within 48 hr post-cloprostenol. No retained placentas were observed. Fields et al. (1977a) reported 250 to 500 vg cloprostenol aborted 100 to 107 heifers between 60 and 120 days of pregnancy, with no complications.




10




Prostaglandin F2a and Superovulation


Prostaglandin F2a has been used in conjunction with Pregnant Mare Serum Gonadotropin (PMSG) for the induction of superovulation in beef cattle. Cupps et al. (1976) reported that- the administration of PGF2 24 hr post-PMSG, to previously synchronized heifers, shortened the interval and reduced the variability to onset of estrus and increased the number of animals responding behaviorally (Cupps et al., 1976). Dobson et al. (1975) suggested that the second PGF2c-induced luteolysis may allow the wave of growing f-llicles that originated after the first PGF2a-induced estrus to progress and ovulate rather than become atretic. Rajakoski (1960) reported such a wave of follicular growth approximately

4 days after estrus culminating in a single follicle growing to day 10 or 12 of the cycle. He suggested the presence of the CL and thus the absence of an ovulatory surge of LH resulted in the follicle becoming atretic. This might account for the observation that the induction of estrus was more rapid and precise after the second PGF2a administration.

In line with this concept, Tervit et al. (1973) reported that a

prostaglanding F2a analog (ICI 79,939) given to cattle with large PMSGinduced follicles demonstrated a shorter interval to estrus. Archbald (1976) reported excellent synchronization of estrus when a second injection of PGF2a was given 48 hr post-PMSG with 67% of the cows in estrus within 48 to 72 hr. Menino and Wright (1977) reported the administration of 2,000 i.u. PMSG 24 hr prior to the hormonal induced-estrus resulted in 81% of the cows responding behaviorally within 48 to 132 hr following treatment.









Gonadotropic Hormones and Limited Multiple Births


Among the several methods available to increase ovulation rate in the bovine, the most promising are hormonal treatments with Pregnant Mare Serum Gonadotropin (PMSG) and pituitary Follicle Stimulating Hormone (FSH). Different gonadotropins, alone or in combination, have been used by many researchers with consistent results. Casida et al. (1943) and Dowling (1949), in early trials, reported successful, induction of multiple ovulations in mature animals from injecting FSH and PMSG during the follicular phase of the estrous cycle. Schilling and Holm (1963) injected 1,000 to 1,500 i.u. PMSG to 11 cows on day 5 following estrus.- On day 16 to 18, the CL was enucleated, followed by an injectionof 2,000 i.u. PMSG. In an attempt to synchronize ovulation, an intravenous dose of 4,000 i.u. of LH was given at estrus. In these trials, more than 70% of the cows ovulated the desired number of 2 to

3 eggs. Turman et al. (1969, 1971) used this same hormonal protocol and obtained a 109% weaned calf crop, from treated animals.

Kidder et al. (1952) and Dawson (1961) reported cows with double ovulations gave birth to a very low number of twins. Gordon et al. (1962) in an extensive study of induced multiple births, used various levels of PMSG in one injection on day 16 or 17 of the estrous cycle. Six weeks following artificial insemination of the 416 treated cows, 76% were pregnant. In one trial, 33% of the cattle treated with 1,600 i.u. of PMSG carried multiple fetuses while 32 of 67 double ovulating cows for all treatments possessed twins. There was further noted an increase in fetal survival, from 29 to 62%, when the eggs were shed by both ovaries rather than one. Cows with three ovulations had a greater ability to sustain twins in a single horn (46%) than those









with double ovulations from a single ovary (29%). The precise relationship between the number of ovulations per ovary and maintenance of pregnancy to term remains to be established. The minimal intrauterine migration (1:200) in the bovine (Perkins et al., 1954; Gordon et al., 1962; Rowson et al., 1971; Scanlon, 1972a),does pose some serious problems to limited multiple births through the use of exogenous administration of gonadotropin hormones as an avenue to increase calf crop. The mechanism of intrauterine migration in the bovine remains undetermined.

Bellows et al. (1969) reported 6,25 mg of FSH injected twice daily for 5 days, in heifers synchronized by feeding 180 mg medroxyprogesterone acetate (MAP) daily for 9 to 11 days and injected with 5 mg estradiol valerate on day 2, resulted in a controlled ovulation rate. When the FSH injections were begun on day 8 of MAP treatment, it resulted in

8 cows with 17 ovulations (Bellows et al., 1970). When this treatment was combined with breeding by natural service, twins were produced by 5 of 43 heifers. Vincent and Mills (1972) in a similar study reported

6.3 to 12.5 mg FSH, given with norethandrolone injections for preventing simultaneous estrus, resulted in 49% of all treated cows with multiple ovulations. Only 5 of 84 cows were estimated to have more than three CL. There were, however, no significant differences in ovulation or pregnancy rates between levels of FSH. Furthermore, calving rates for cows responding to PMSG treatment were similar (121%) to those reported by Bellows et al. (1969), but considerably lower than the calving rate of 173% with PMSG treatment reported by Turman et al. (1971).

Reynolds et al. (1970), Vincent and Mills (1972), and Smith et al. (1973) attempted to prolong the action of FSH by using a 1% sodium carboxymethyl cellulose and polyvinylpirrolidone. It was concluded that




13




these diluents, used for one or two injections of FSH, were not as satisfactory as a series of injections.

According to Laster (1972, 1973) and Smith et al. (1973), the

discrepancy observed in ovarian response and conception to FSH treatment was due to the actual biological potency of the gonadotropin. Similarly, Schwartz and Shelby (1969), Laster et al. (1971a,b), and Scanlon (1972b) reported considerable differences in mean ovulation rates with similar treatment regimens of PMSG. A differential response in ovulation rate, doses of PMSG and/or FSH, and onset of the CAP synchronized estrus due to breed has been reported by Lamond (1972).

The infusion of FSH for 72 hr (Laster, 1972) did not decrease the variability in ovarian response that was achieved with twice daily injections for 5 days (Bellows et al., 1969). In contrast, when FSH was injected for 3 days, more cows ovulated from one FSH injection per day (5.16 4.78) than two injections per day (1.00 0.00) (Staigmiller et al., 1976). However, for 5 days of injections, fewer cows superovulated from one FSH injection per day (1.00 0.00) than from two (1.37 0.79).

Lamond (1972) and Laster (1973) reported that PMSG treatment resulted in a more desirable and less variable ovulation rate than treatment with FSH. It was of interest that abortions occurred in 20% of the animals treated with PMSG or FSH at 81 to 104 days after insemination. Similar observations of fetal wastage have been made by M. J. Fields, A. C. Warnick, and J. H. Hentges (unpublished data). Godke et al.(1977) reported cows treated with 1,6000 i.u. PMSG or 1-5 mg/day FSH-P resulted in pregnancy rates, cows pregnant with multiple CL and number of cows returning to estrus after 100 days gestation of




14





71.4% and 64.2%, 100% and 88.8%, 70.0% and 62.5%, respectively. At 220 days of gestation, there were no differences in pregnancy between cows treated with PMSG (16.6%) or FSH (17.7%).

Hill et al. (1973, 1976) reported that PMSG, given concomitantly

with PGF2a, resulted in a lower and less variable ovulation rate (4.33 3.60) than when PGF2a was injected at 24 hr post-PMSG (8.03 9.46). A high incidence of split estrus (55%), with a large number of unovulated follicles was observed when PMSG was given concomitantly with PGF2a as compared to an 8% incidence of split estrus when a 24 hr interval between hormonal treatments was allowed. These data suggest that the follicles had not had sufficient time to mature before estrous control was attempted with PGF2a. Absence of split estrus has been reported by Rajamahendran et al. (1976) and Lopez-Barbella et al. (1976) following a single injection of 2,000 i.u. PMSG 24 hr prior to a second injection of synchronizing PGF2a.


Endocrinology of the Superovulated Cow


Hallford et al. (1975a) using two PMSG injections, on days 5 and 17 of the estrous cycle, reported a significant (P<.005) increase in midluteal plasma progesterone levels over the nontreated controls or animals receiving a single injection of PMSG on day 17. Plasma LH levels, however, were similar in PMSG treated groups, and generally below one ng/ml (Hallford, 1975b). These two treatments were not compared against the non-PMSG treated cow.

According to Ford and Stormshak (1975) daily serum levels of LH

were elevated (P<.Ol) in heifers treated with PMSG vs nontreated heifers. In heifers treated with PMSG, GnRH injections produced lower plasma LH









levels when compared to non-PMSG treated heifers. Apparently, the differential LH response observed in the PMSG treated group could be due to a positive feedback of estradiol at the hypothalamic-pituitary level. Although Hallford et al. (1975b) reported a positive correlation between the number of CL and plasma estradiol levels (r = 0.64) on day 19 and increased plasma estradiol in PMSG-treated heifers,plasma LH levels did not differ between the PMSG and non-PMSG treated heifers (vide supra). Hill et al. (1972) and Dickey et al. (1973) reported plasma estradiol levels in PMSG superovulated heifers to be much higher prior to mating than in controls. These values, however, were for superovulated rather than cows of limited ovulations.

An increased ovulation rate with 2,000 i.u. PMSG resulted in an ovulatory surge of LH at 84 48 hr post-PMSG with a mean basal plasma LH level ranging from 0.6 to 2.5 ng/ml (Lopez-Barbella et al., 1976). A synchronizing injection of PGF2t 24 hr post-2,000 i.u. PMSG resulted in a decrease in plasma progesterone levels from 7.9 0.45 to 0.94

0.17 ng/ml within 72 hr (Fournier et al., 1976).

The stereotypic response to PMSG was clearly demonstrated by

Rajamahendran et al. (1976) who reported ovulation rates from I to 17 in heifers treated with 2,000 i.u. PMSG followed 48 hr later by 15 mg PGF2,. Two heifers, each with 17 CL, had peak progesterone levels of 38.4 and 27.8 ng/ml which were still high (9.6 and 26.5 ng/ml) by day 21. Progesterone levels of three heifers with 4-9 CL did not differ (P<.05) from those of three heifers with single CL. Six additional heifers had low progesterone levels (<] ng/ml) on days 8-14 post-PMSG suggesting premature CL regression or lack of ovulation.




16




206-Dihydroprogesterone (4-Preqnen-2O0-ol-3-one)


An interesting steroid, concerned with bovine steroidogenesis, that, to date, has been considered the major metabolite of progesterone (P) is 20-dihydroprogesterone (20B3-P) (Spilman et al., 1973).

Histologically, CL development has been described as a progressive arrangement and growth of the luteinizing granulosa cells in the cavity left at the ovulatory site (Harrison, 1946). The 14 day. CL has reached itsmaximum size filling the collapsed follicular cavity with an extensive vacuolation of luteal cells. Steroidogenically, this structural change is accompanied by a differential secretion of progesterone-derived hormones according to Short (1962a,b) in his "two cell theory." From the two tell theory of Short (1962a,b), the ovarian theca interna was proposed to covert P to estrogen in the absence of a 20-reductase system, i.e., 206-hydroxysteroid dehydrogenase (205-HSD). La Croix et al. (1974) have since shown that bovine theca cells synthesize androgens which serve as precursors for the granulosa cell to convert to estrogens.

The granulosa cell undergoing luteinization was proposed to contain the 20-reductase system and thus, 206-P secretion by the growing follicles was an indicator of luteinization. This was indirectly confirmed by other when P and 20B-P were isolated from luteal tissue (Savard and Teledgy, 1965). Lobel and Levy (1968), however, did find that 20-HSD activity resided in both the granulosa and theca cell layers of the rat follicle.

Hayano et al. (1975) first demonstrated the conversion of P to 206P by the bovine CL. Noticeable quantities of ovarian 20$-P levels in cycling cows at levels approximately 10 to 20% of that of P have been reported (Gorski et al., 1958a,b; Erb and Stormshak, 1961, Hafs and









Armstrong, 1968; Garverick et al., 1971). Brandau et al. (1972) and Brandau and Mutzke (1972) using homogenized bovine ovaries found that 20-HSD increased slowly to a maximum on the day 15 of the cycle. Levels of 200-P in the bovine are highest when the CL reaches maturity, with a delayed decrease in 208-P concentration following the P decline of the regressing CL (Erb and Stormshak, 1961; Staples and Hansel,-1961; Mares et al., 1962; Gomez et al., 1963; Gomez and Erb, 1965). Erb et al. (1968) reported that 201-P increases when synthesis br release of luteal P decreases late in the estrous cycle.

Sasser and Cupps (1969) incubated CL recovered at.various times during the bovine estrous cycle and found maximal P synthesis on days 10 to 12. These workers postulated that, prior to estrus, luteal regression could result in increased lysosomal activity which would result in reduced cellular pH and create optimal conditions for 205-P formation. When a similar study was conducted with porcine CL, only

0.6% of the P was converted to 206-P (Weiss et al., 1976).

In laboratory animals, in which the a-epimer (20a-P) of 208-P is the more predominant hydroxylated ovarian steroid, Barraclough et al. (1971) noted an increase in ovarian vein levels of 20a-P approximately 2.5 hr prior to the LH discharge in cycling female rats. This suggested the possibility that 20a-P has a positive feedback on the pituitary for discharge of LH. Ichikawa et al. (1971) found an increased secretion of both P and 20a-P in ovarian vein plasma after injection of LH into early proestrus rats. It is possible that sustained synthesis and release of 20a-P is caused by LH since LH will increase 20a-HSD activity (Kidwell et al., 1966).




18




Telegdy and Savard (1966) found the rabbit ovary to produce both 20a-P and 20-P, with 20-P being the more predominant hydroxylated steroid. Hilliard et al. (1967) in an elegant study showed, in the rabbit, that coitus triggers a transient release of LH sufficient to activate the synthesis and release of 20a-P which subsequently prolongs and heightens the LH discharge at a level and duration necessary f6r ovulation. Goodman and Neill (1976), however, were not able to confirm this role of 20a-P in a similar study.

The role of gonadotropins in bovine ovarian steroidogenesis has also been investigated. Romanoff (1966) perfused both the luteal and contralateral follicular ovaries from the same cow, each ovary serving as a control to the other. Perfusion of Follicle Stimulating Hormone (FSH) with acetate-1-1"C increased 14CP synthesis 3.3 fold and increased the P to 206-P ratio from 7.2 to 11.4 for the luteal ovary. Perfusion of FSH was not tested in the follicular ovaries. Perfusion of LH into the luteal ovary resulted in increased synthesis of both P and 20-P but did not affect the ratio between the two steroids. The perfusion of LH into follicular ovaries increased synthesis of P seven-fold with a 3.6 fold increase in 20$-P. Not unexpectedly, there was significantly less synthesis of P and 20$-P in follicular than luteal ovaries. Prolactin perfusion in this study, had no effect on the 20-HSD system.

Snook et al. (1969) working with hysterectomized heifers with sustained luteal function, however, showed that LH has a preferential effect on 20-HSD. They used LH-antisera to neutralize endogenous LH. This LH-antisera reportedly decreased total ovarian progestin concentration primarily due to a significant reduction in 20$-P while P concentrations remained unaltered. In the intact heifer, Spilman et al. (1973)









reported increased plasma 206-P levels after either PMSG or HCG treatment.

Kidwell et al. (1966) working with PMSG superovulated rats, found a three-fold increase in the 20-HSD activity 5 days post-treatment. When these primed rats were administered LH, the 20-HSD activity increased from 0.03 0.01 to 2.82 2.74 MU/mg 5 days after LH injection. The administration of HCG, however, resulted in a ten-fold increase in the 20-HSD activity. A significant increase in the glucose6-phosphate dehydrogenase activity was also observed after LH and HCG administration.

The possibility that 20B-P in the bovine might be playing a similar role as 20-P in other species is not apparent. The systemic patterns are different for P and 200-P which are inversely related while in the case of 208-P it follows a similar but slightly delayed pattern as P. Not only has 208-P been isolated from bovine ovarian vein plasma and tissue, but has been shown to be of ovarian origin in humans (Mikhail et al., 1963), Chinchilla (Tam, 1971) and the African elephant (Smith et al., 1969).

Staples and Hansel (1961) presented data to suggest that embryo

survival at day 15 may be influenced by circulating 206-P levels. Gomez et al. (1962) quantitated P and 20a-P levels in the utero-ovarian vein ipsilateral to the CL in cows between 250 and 282 days of gestation. Levels of 206-P remained near nondetectable levels until parturition, at which time there was a sharp rise which accompanied the decrease in P levels. At parturition, the P:206-P ratio was approximately one. Levels of 206-P in the cycling post-partum cow have been shown to be 10 to 15% of that of P (Tribble, 1973; Castenson et al., 1976).




20




To date, reports on 206-P are limited to plasma levels during the luteal phase of the cow's estrous cycle. Little or no information has been reported on levels of this hormone at or near estrus. In this study, plasma 206-P will be characterized in PMSG superovulated cows over a 21 day bleeding period.














CHAPTER III
MATERIAL AND METHODS



EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P), 7 206-DIHYDROPROGESTERONE (20B-P), TOTAL ESTROGENS (E), AND LUTEINIZING HORMONE (LH) IN PMSG SUPEROVULATED COWS SYNCHRONIZED WITH PGF2aTHAM SALT


The objectives of this experiment were (1) to characterize plasma P, 20-P, E (combined El and E2), and LH concentrations in the PMSG superovulated cow and (2) to determine if 40 mg PGF2U-Tham Salt was effective in regressing multiple corpora lutea (CL).

Fifteen exhibiting normal estrous cycles parous Angus cows were examined per rectum to verify that the reproductive tracts were normal and that a CL was present. All animals were injected twice intramuscularly with 33.5 mg PGF2a-Tham Salt (Upjohn) at a 12 day interval. Cows were administered 2,000 i.u. PMSG (Organon) subcutaneously 24 hr prior to the second PGF2a injection. Animals were observed twice daily for signs of estrous behavior throughout the experiment. Artificial ininsemination (Al) three times with Brahman semen was at 0, 12, and 24 hr after detection of the PMSG-PGF2G-induced estrus. Approximately 10 days following AI cows were exposed to an Angus bull for a 90 day breeding period (figure 1).

A 40 ml blood sample was collected via jugular vein venipuncture at 12 hr intervals from each animal for 21 days starting 48 hr prior to time of injection of PMSG. Blood samples were transported to the


21





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23




laboratory in an ice bath and immediately centrifuged at 2,000 g for 20min at 4C. Plasma was stored at -20C until analyzed for plasma P, 20B-P, E, and LH concentrations.

Ovulation rate was determined by supravaginal laparotomy 13 days post-PMSG. In an attempt to reduce fetal wastage from the superovulation effect, cows with more than three CL were injected with 40 mg'PGF2. to induce CL regression.


Surgical Description of Supravaginal Laparotomy


Prior to surgery animals were taken off feed overnight. Animals were restrained and tranquilized with 2 ml Acepromizine (Ayerst), an incision made in the upper anterior vagina, the ovaries were exteriorized through this incision,and the CL were counted. At time of surgery and 24 hr post-surgery cows were treated with two million i.u. penicilline (Combiotic, Pfizer).



EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN PMSG SUPEROVULATED BEEF CATTLE FOLLOWING ADMINISTRATION OF 40 MG PGF2,-THAM SALT


The objective of this trial was to determine in the nonbred beef cow if 40 mg PGF2a was effective in regressing multiple CL in response to PMSG treatment.

Thirteen parous Angus cows exhibiting normal estrous cycles were treated with two sequential injections of 33.5 mg PGF2a-Tham Salt to achieve synchronization of estrus (figure 2). A 2,000 i.u. PMSG injection was administered subcutaneously to 10 cows 24 hr prior to the second PGF2a injection. Animals were observed twice daily for signs of estrous behavior for 32 days starting the day of first PGF2. injection.




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Ovulation rate was determined by supravaginal laparotomy 13 days post-PMSG and, on the same day, all cows were treated with 40 mg PGF2ct.

A 10 ml blood sample was collected via jugular vein venipuncture daily for 10 days starting 2 days prior to laparotomy (vide supra).



EXPERIMENT 3. LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN BRED VS CYCLING BEEF COWS PREVIOUSLY TREATED WITH PMSG.


This experiment was conducted to determine the efficacy of PGF2a in regressing multiple CL in bred vs cycling cows treated with PMSG.

Forty-five parous Angus cows exhibiting normal estrous cycles were treated with a dual injection of 33.5 mg PGF2c-Tham Salt at a 12 day interval to achieve synchronization of estrus and randomly assigned to a 2x2 factorial with treatments of 2,000 i.u. PMSG and breeding (figure 3). Treatments were (1) 11 cows injected with 2,000 i.u. PMSG 24 hr prior to the second PGF2, injection and immediately exposed to natural breeding for 10 days; (2) 11 cows injected with 2,000 i.u. PMSG 24 hr prior to the second PGF2a injection and not mated; (3) 12 cows exposed to natural breeding for 10 days starting the day of the second PGF2a treatment; and (4) 11 cows were treated with PGF2a but received no PMSG and were not mated; thus, they served as a double control. Bleeding and laparotomy were as described in experiment 2.

Cows were checked for estrous behavior twice daily for 21 days prior to the beginning of the experiment, three times daily during the experimental period and twice daily for 70 days following laparotomy and injection of 40 mg PGF2a. Cows were checked for pregnancy 70 days post-laparotomy.


















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27




Radioimmunoassay of Steroid Hormones


Cleaning of Glassware


Excluding RIA disposable culture tubes (10 x 75 mm, Corning), all glassware was rinsed with ethanol, boiled in a soapy-distilled water for 1 min, and'rinsed withwarm tap water (3X) followed by deionizedwater (3X). Prior to use, the glassware was rinsed with ethanol, siliconized with 1% silicone solution (Siliclad, Clay Adams) for 20 sec and oven dried for 24 hr.


Organic Solvents


Analytical grades of benzene, ethanol, methanol, hexane, and ethyl acetate were distilled over boiling chips prior to use. Hexane was washed with sulfuric acid, distilled water, and dried over calcium chloride prior to distillation. Ether was used immediately following distillation over metallic sodium to remove peroxides and water. Preparation, Use, and Storage of Radioactive Steroids


Tritiated steroids, 1,2-H3-Progesterone (3p) (55.7 Ci/mM) and 2, 4,6,7-3H-Estradiol (3E2) (98.5 Ci/mM) were purchased from New England Nuclear Corporation. Upon receipt of tritiated steroids they were further purified on a 25 cm LH-20 column to eliminate undesired labelled impurities that could result in decreased accuracy and sensitivity of the assay. The center profile of the eluted tritiated steroid was pooled and stored in benzene:ethanol (9:1) at 4C. For assay purposes, a known amount of tritiated steroid was transferred to a 250 ml flask and dried under nitrogen gas (N2) at 37C. Gelatinized phosphate assay buffer (see




28




table 10, Appendix) was added to give a concentration of 200,000 dpm/ml. This mixture was vortexed for 5 min and equilibrated for I hr at room temperature. A volume 100 pl (20,000 + 1,000 dpm) was used in the assay. This solution was stored at 4C and used for no longer than I wk. P and E tritiated steroids were further diluted with benzene to 2,000 dpm/100 pl and stored'at 4C for determining recovery of extracted steroi.ds-.

Since tritiated 206-dihydroprogesterone was not commercially available it was synthesized from 3P as described by Tribble (1973). Conversion of Progesterone (3p)> to 208-Dihydroprogesterone (3H-206-P)


Forty thousand dpm of 3p was dried under N2 in a siliconized

conical centrifuge tube and subsequently dissolved in a drop of ethanol. To this dissolved 3p was added 0.5 ml of 0.15 M phosphate buffer [pH =

5.2, which contained 100 mg percent EDTA], 30 pl of NADH [(Sigma Chemical Company, Grade III) in 0.1 M Tris buffer (Eastman), pH = 8.1] (see table II, Appendix) and 30 pl of 208-hydroxysteroid dehydrogenase enzyme [(Calbiochem, Activity 4.95 i.u./ml at 30C) in 0.005 M Tris buffer (pH =

8.2) and vortex mixed. The reaction was incubated for 3 hr in a water bath at 37C. The reaction was stopped with the addition of I ml distilled water. The 3H-20-P was extracted with I ml ethyl acetate (3X), quantitatively transferred to a 13 x 100 mm culture tube and dried under N2 at 37C. The dried residue was quantitatively transferred to a LH-20 column (vide infra) and the 3H-20-P fraction was collected into a 13 x 100 mm culture tube. Elutions containing 3p were collected directly into scintillation vials and counted. From this fraction an estimate of the percent conversion of 3p to 3H-206-P was subsequently determined. Fractions containing 3H-20-P were stored in benzene:











ethanol (9:1) at 4C. A typical profile of 3p conversion to 3H-206-P and its LH-20 chromatography separation is illustrated in figure 4.

The above procedure for synthesis of 3H-20-P was adopted after a series of reactions to determine optimal- conditions for conversion were attempted. The efficiency of conversion as well as levels of cofactors tested are illustrated in table 1. Use and Storage of Antibody


The P and E antisera used- in this study were kindly supplied by Dr. Lee Fleeger, Texas A&M University (PR #24 and PR #281), and Dr. V. L. Estergreen, Washington State University, respectively. The lyophil.ized P antiserum was dissolved in 400p]I distilled H20 to form a 1:1 stock solution. Twenty microliter fractions of this 1:1 stock solution were stored in separate test tubes at -40C eliminating breakdown due to repeated freezing and thawing. A 1:7,000 dilution of the P antiserum resulted in 40 to 50% binding, while for estrogens, a 1:30,000 dilution of the antiserum resulted in 65 to 70% binding, using 3H-estradiol as a tracer, when a total incubating volume of 1.2 ml and 20,000 dpm were used in the assay.


Serum Extraction and Chromatography


For progesterone extraction 1 ml aliquots of plasma were pipetted into 20 x 150 mm test tubes. To each tube 100 ol 3P, 500 Pl of 0.05 M iThe addition of normal rabbit serum (1:400) improved the sensitivity of this antiserum.





30
























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TABLE 1. EFFICIENCY OF 3H-P CONVERSION TO 3H-20H-PAT VARYING LEVELS OF COFACTORS


Tritiated Efficiency of 3p progesterone NADHa 206-HSDb conversion to 3H-20OP
(dpm) n (m]) (ml) (%)

40,000 10 0.03 0.03 94,35 80,000 10 0.06 0.06 42.00 120;000 10 0.09 0.09 69.12 160,000 10 0.12 0.12 37.78 160,000 10 0.09 0.09 43.09 160,000 10 0.12 0.09 51.12 160,000 10 0.09 0.12 50.62

a5 mg 8-NADH/3 ml of 0.1 M Tris buffer.
b0.2 ml 208-HSD/0.8 ml of 0.005 M Tris buffer.









NaOH, and 15 ml of hexane was added and vortexed 1 min. The lower aqueous phase was quickly frozen in liquid nitrogen while the upper lipidcontaining organic phase was decanted into a 20 x 150 mm test tube and evaporated to dryness under N2 at 37C. The extract was resolubilized in 500 pl hexane:benzene:methanol (80:15:5), vortexed for 30 sec, and applied to a 7 cm LH-20 column. The progesterone fraction was eluted into a 13 x 100 mm tube, evaporated to dryness under N2 at 37C. To this dried progesterone 2 ml of gelatinized assay buffer was added, vortexed for 5 min and 500 Pl transferred to a scintillation vial to estimate recovery. The remainder was used for assay.

Quantification of 3P and 3H-206-P from the same plasma sample was accomplished using an ether rather than hexane extraction in the manner'described above.

The dried ether extract was subjected to chromatography on a 5 x .5 cm LH-20 column. These columns were disposable 5 ml glass pipettes (Corning) with 3 mm of glass wool packed in the bottom of the pipette. Then Sephadex LH-20 was allowed to swell overnight in hexane:benzene: methanol (80:15:5) and subsequently packed into each column to a height of 7 cm. The packed columnswere washed with 10 ml hexane:benzene: methanol (80:15:5) and the elution profile characterized with appropriate tritium-labelled steroids. The dried residue from the ether extract was quantitatively transferred to the column with two 500 Pl aliquots of hexane:benzene:methanol (80:15:5). Progesterone was eluted from the column using 5 ml of the above organic solvent while 20a-dihydroprogesterone was eluted 2 ml after 3p. The 3P and 3H-206-P eluted fractions were collected into 13 x 100 mm culture tubes and dried under N2 at 37C. Subsequent handling of these elutes was similar to that described for progesterone (vide supra).




33




Quantification of estrogens was accomplished as described by

Abraham et al. (1971) using the assay procedure of Nett et al. (1973). Quantification of LH was as described by Chenault (1973). The quantification of 208-dihydroprogesterone was accomplished by converting this progestogen to progesterone and subsequently assaying for progesterone. Conversion of 3H-20-P to 3p


The chemical conversion of 3H-20-P to 3p was similar to the procedure described by Sholl and Wolf (1974). To the dried column elute of 3H-208-P was added 2001Il of a 0.4% (w/v) solution of chromium trioxide in 90% acetic acid, vortexed for 1 min, and incubated in the dark at room temperature for 2 hr. The reaction was terminated with the addition of I ml of distilled water and the sample extracted with 3 ml of ethyl acetate (2X). This extract was washed with 500 Vl distilled water to remove any residual acetic or chromic acid. Removal of the aqueous wash was facilitated by freezing in liquid nitrogen and transferring the organic phase to a 13 x 100 mm culture tube. The organic phase was dried under N2 at 37C. The dried residue was subjected to chromatography on a LH-20 column (vide supra) and the 3p fraction was collected into a 13 x 100 mm culture tube for RIA. Preparation, Use, and Storage of Assay Buffer


In working with the phosphate assay buffer (Appendix), it was

observed that deionized water and a pH lower than 7.2 resulted in loss of sensitivity of the assay. The working gelatinized assay buffer was prepared by adding 0.1% Knox gelatine (Knox Gelatin Inc.) to the above stock solution. The gelatinized assay buffer was stored at 4C and used for no longer than 4 wk.




34




Preparation, Use, and Storage of Charcoal Suspension


To a 100 ml flask was added 0.625 g of Norit A (Matheson, Coleman, Coleman and Bell), 0.0625 g of Dextran T-70 (Mann Research Labs) and 100 ml of gelatinized assay buffer. Flasks were stoppered and shaken vigorously for 30 sec. The charcoal suspension was stored at 4C and used for no longer than 4 wk. A volume of 500 pl of this suspension was used to absorb and precipitate the free steroids after assay incubation. The charcoal absorption step of the assay was strictly carried out at 4C since higher temperatures resulted in stripping of the bound steroid. Twenty minuteswerefound to be sufficient time to absorb all free steroid at this temperature.


Preparation and Use of Counting Solution


To an amber bottle containing 800 ml of toluene was added 3.2 g Omni Scint I (98% PP0/2% BIS-MSB). A total of 3.5 ml of this counting fluid was used per scintillation vial. Cocktail and sample were equilibrated for 24 hr prior to counting to allow movement of the 3H steroid into the organic phase. No solubilizers were used. Preparation, Use, and Storage of Standard Steroids


Upon receipt of unlabelled steroids (Steraloids, Inc.) a series of crystallizations in ethanol, methanol, and acetone were performed until a constant melting point was achieved. From the final crystallization a stock solution of I g/ml unlabelled hormone in absolute ethanol was made. An aliquot of 20 il stock solution was transferred to a 20 x 150 mm test tube and dried under nitrogen gas at 37C. Then









10 ml of gelatinized phosphate buffer was added and subsequently vortexed for 5 min. The lack of solubility of progesterone in this concentration (20 ng/ml) in buffer necessitated the incubation of this Solution A overnight. Tables 2 and 3 represent the various dilutions used for the standard curve in the RIA analysis while figures 5 and 6 depict a typical standard curve.

A volume of 500 pl at each concentration was used in the assay. These solutions were good for up to 6 mo when kept at -40C. Radioimmunoassay


Isolation of recovered 3p, 3H-208-P (oxidized to 3P) and 3H-E

facilitated quantification of these steroids. Aliquots of 200 1l and 500 Pl'of the buffered unknown was pipetted (Eppendorf, Brinkman Instruments) into two 10 x 75 mm disposable culture tubes. These two dilutions were made in an effort to assure that one or both aliquots were located on the linear portion of the standard curve. The volume in the culture tubes containing the 200 Wl aliquot was adjusted to 500 Pl by the addition of 300 Pl of gelatinized assay buffer. An additional 500 l of sample was placed in a scintillation vial with cocktail and counted for recovery purposes (vide supra). Tritiated hormones and antibody were added to the assay tube as specified in table 3 and vortexed briefly. All assay tubes were incubated a minimum of 4 hr at 4C, followed by addition of 500 Pl of charcoal suspension to each tube, vortexed briefly and incubated 20 min at 4C. All assay tubes received this 500 Hl of charcoal suspension except the total count and background tubes which received an equivalent amount of assay buffer. Following charcoal incubation tubes were centrifuged






















TABLE 2. STANDARD CURVE DILUTIONSa


Mass in
500 l
Solution Preparation (pg)


A 20 pl of stock in 10 ml assay buffer 1,000 B 5 ml of solution A in 5 ml assay buffer 500 C 5 ml of solution B in 5 ml assay buffer 250 D 5 ml of solution C in 5 ml assay buffer 125 E 5 ml of solution D in 5 ml assay buffer 63 F 5 ml of solution E in 5 ml assay buffer 32 G 5 ml of solution F in 5 ml assay buffer 16

aUsed for both progesterone and estrogens.




3/
















TABLE 3. ADDITIONAL TUBES USED TO SUPPLEMENT RIA ANALYSISa


Amount of Amount of Amount of Solution assay buffer antibody 3H-Steroid Code added added added
(ml) (ml) (ml)


BGb 1.2

TCc 1.1 0.1

CHd o0.6 0.1

BTe 0.5 0.1 0.1

STf 0.1 0.1


aEach run in triplicate to a volume of 1.2 ml.
bTo determine the background counts.
cTotal count tube to determine cpm of 3H-steroid added to
each tube.
dTo determine residual counts left following addition of
charcoal.
eTo determine the percent of the 3H-steroid bound to the
antibody.
fAssay buffer was replaced by 0.5 ml of either standards
or unknown samples





36o


























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a

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00







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39












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u



















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44


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at 4C for 20 min at 2,000 g. Then, 500 pl of this incubate was withdrawn by a repipette and deposited in a scintillation vial along with 3.5 ml of scintillation cocktail. Counting for 5 min followed overnight equilibration at room temperature. Calculations


% Recovery = (recovered counts background counts) (4) x 100 original counts added background counts



BT tube counts
% Total Binding -BTubeoun x 100 TC tube counts

The standard curve and quantification of unknowns were calculated by logarithmic transformation of the data. For this, a Monroe 1860 computed the concentration of steroid in log scale and the percent dpm bound in linear scale.

The amount of steroid present in one ml of serum was calculated as follows:


concentration of unknown
Steroid Concentration =recovery


x 2 (for 0.5 ml) or 5 (for 0.2 ml) Separation by LH-20 Column Chromatography


With the use of tritiated progesterone and 20B-dihydroprogesterone it was determined that these steroids were soluble in hexane:benzene: methanol (80:15:5) prior to chromatography.

Sephadex LH-20 and hexane:benzene:methanol (80:15:5) served as the partition system. When a 7 cm LH-20 column was used the less polar




4'




progesterone was eluted within the first 3 ml and the more polar 20B-P was eluted between the fifth and seventh ml fractions. Precision


The between and within assay coefficient of variation (c.v.) was calculated frbm two different duplicate determinations from a stock of standard plasma samples run with each assay. The c.v. from the means was estimated by the followed formula (Steel and Torrie, 1960): c.v.= 100 S/R, in which S is the standard deviation and R is the mean.

In a total of 58 duplicate observations, the intra- and interassay c.v. for plasma progesterone was 3.3 to 16.2% and 10.71%, respectively. For estrogens, with 24 duplicate observations more variability in the intraajsay c.v. (14.4 to 20.9%) and in the interassayc.v. (21.33%) was found than that for progesterone.



Statistical Analysis


The basic statistical method utilized was least-squares multiple regression analysis (Harvey, 1960) from which the following parameters were obtained:

(1) Identification of classes (group, day, and group x day interaction) with their respective least-squares means and standard errors for progesterone, 20B-dihydroprogesterone, estrogens, and luteinizing hormone;

(2) Least-squares analysis of variance and listing of polynomial regressions for each hormone. Since analysis of variance indicated that P, 20B-P, and E were statistically affected by day, group, and









day x group interaction, it was necessary to characterize these trends by regression analyses. Therefore, a set of regression equations for each hormone within source was computed and that which best characterized the hormonal response was considered the hypothetical model for discussion of the results;

(3) Test for day effect within hormone was computed using the appropriate error term obtained from Harvey's printout (Steel and Torrie, 1960). Reproductive responses were tested by LSD; and

(4) Additional computations were obtained according to the procedures described in SAS 76 (Barr et al., 1976).













CHAPTER IV
RESULTS AND DISCUSSION



EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P) 20B-DIHYDROPROGESTERONE (20B-P), ESTROGENS
(E), AND LUTEINIZING HORMONE (LH) IN THE PMSG SUPEROVULATED COW SYNCHRONIZED WITH PGF2a-THAM SALT


Reproductive Response


The efficacy of dual administration of PGF2a for synchronizing estrus in PMSG superovulated cowsis depicted in table 4. Behavioral estrus was observed in 14 of 15 (93.3%) treated animals by 120 hr post-PGF2,. The single animal not responding to this dual injection of PGF20 was detected in estrus 36 hr prior to the second PGF2a administration. Stratification of estrus at 12 hr-intervals resulted in 26.7, 53.3, 73.3, and 93.3% of the animals exhibiting estrus by 48, 60, 72, and 120 hr, respectively, following PGF2a injection. This is in agreement with Cupps et al. (1976) who reported that the administration of PGF2a 24 hr post-PMSG, to previously synchronized heifers: (1) increased the number of animals exhibiting estrus; (2) shortened the interval from treatment to estrus; and (3) reduced the variability in time to onset of estrus. The percentage of animals exhibiting estrus by 72 hr (73.3%) in this study, was slightly higher than the 67% reported by Archbald (1976).

Estimates of ovulation rate, determined at laparotomy, are presented in table 4. Significant differences were detected in total


43




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ovarian response when cows were partitioned according to when they exhibited behavioral estrus. A higher ovulation rate (P<.Ol) was observed in cows expressing estrus by 48 hr post-injection (5.50 1.29) vs 0.67 0.82 for those expressing estrus from 73 to 120 hr. The difference in ovulation rate in animals exhibiting estrus from 49 to 60 hr vs 61 to 72 hr was nonsianificant (P>.25). It was encouraging that only 5 of 15 (33.3%) of the cows had more than four ovulations (CL). These results agree with those of Hammond (1949), Brock and Rowson (1952), and Scanlon et al. (1968) in that a more desirable and restricted level of superovulation was obtained when the interval between PMSG or FSH injection and subsequent estrus was relatively short.

In line with this concept, Tervit et al. (1973) demonstrated in cattle'with large PMSG-induced follicles that a PGF2a analog (ICI 79,939) shorten the interval to estrus. Dobson et al. (1975) suggested that the second PGF2a-induced luteolysis may allow a wave of growing follicles originating after the first PGF2a-induced estrus to progress and ovulate rather than become atretic. This might account for the observation that the induction of estrus was more rapid and precise after the second PGF2. administration. R. Hardin, A. C. Warnick, S. R. Lopez Barbella, T. H. Wise, and M. J. Fields (unpublished) demonstrated that cows with dual injection of PGF2a analog (ICI 80,996) displayed estrus 24 hr earlier following the second injection than the first injection. Rajakoski's (1960) report that there are two waves of growing follicles during the estrouscycle of the cow tends to support the above findings. In addition, Goodman et al. (1977) reported that the largest follicle developed during the early phase of the estrous cycle retained its dominant position for a longer period of time than did the dominant follicles for other stages of the estrous cycle.









The present study suggests the possibility of a relationship between length of the interval from PMSG to estrus and superovulatory response as demonstrated with the longer interval from treatment estrus of 73 to 120 hr in conjunction with a lower superovulatory response of 0.67 0.82 ovulations per cow.

The percentage of animals conceiving to either the first postPMSG estrus and/or subsequent estrus as determined by rectal palpation 60, 100, and 145 days after treatment with PMSG or by subsequent calving dates is given in table 5. The conception rate of 100% determined by palpation at 60 days post-PMSG is higher than the 60 to 65% reported by Turman et al. (1971), Laster et al. (1971b), and Hallford et al. (1975a,b) for superovulated cows. Multiple fetuses determined by calving date were lower than those estimated earlier by palpation. These discrepancies were attributed to an inability to predict multiple fetuses by rectal palpation at earlier stages of gestation. In addition, none of the cows bearing two or three CL and only one of the five cows with more than four CL gave birth to multiple calves. When working with multiple fetuses, Gordon et al (1962) and Schwartz and Shelby (1969) suggested that both rectal palpation and laparotomy may be involved in increasing the incidence of embryonic mortality.

Although a reduction in embryonic mortality was achieved with 40 mg PGF2a, the presence of Brahman calves, a genetic marker indicating conception to breeding at the PMSG-induced estrus prior to 40 mg PGF2., raised the question of the fetus in some manner rescuing the CL, as suggested by Mapletoft et al. (1976). Alternatively, it could be a question of inadequate levels of PGF2a for the increased mass of CL tissue. These questions will be addressed in the next two experiments.





















TABLE 5. CONCEPTION RATES FOLLOWING ARTIFICIAL INSEMINATION
POST-TREATMENT WITH PMSG and PGF2a AND EXPOSURE TO
THE BULL FOR 90 DAYS IN EXPERIMENT 1


Time % pregnant to treatment Nonpregnant (days) Single Multiple Open to treatment


60 53.3 46.7 0.0 100 20.0 53.3 26.7 145a 93.3 0.0 6.7

Pregnant to term 26.6 6.7 6.7b 60.0c


alncludes one animal pregnant to treatment that died due to
Tympanites.
bpost-mortem examination of reproductive tract revealed oviduct blockage in the single open cow.
cDetermined by phenotypic appearance of the calves.









Endocrine Response


In an attempt to reduce fetal wastage due to the superovulation effect resulting from PMSG, five cows with ovulation rates greater than three CL were injected with 40 mg PGF2a. This treatment, in itself, allowed for the subgrouping of animals according to their ovulation rate. Since it was determined that the time-trend in plasma hormones among group I (cows with more than three CL), group 2 (cows with two or three CL), and group 3 (cows with zero or one CL) were similar, emphasis was placed on the magnitude of the response for subgrouping.

Concentrations of progesterone, 208-dihydroprogesterone, luteinizing hormone, and estrogens in plasma from each group are summarized in tables 12 to 14 of Appendix. Progesterone

Plasma progesterone concentrations in cows from group 1 plotted by days of bleeding is illustrated in figure 7. A near normal progesterone curve (table 15, Appendix) was exhibited prior to PMSG treatment. By 24 hr after gonadotropin administration, plasma progesterone concentrations had increased from 5.66 1.17 to 9.06 2.99 ng/ml (P<.05). After the luteolytic dose of PGF2a on day 4 (AM), progesterone decreased to a basal concentration of 0.72 0.14 ng/ml by day

6 (PM). These changes were best described by a significant (P<.0l) third order regression equation (table 15, Appendix) which accounted for 47% of the variation. Plasma progesterone remained at basal levels for 24 hr after which a significant (P<.Ol) curvilinear (table 15, Appendix) increase to 16.46 + 2.36 ng/ml on day 15 (AM) was observed.






















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(V)

















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LO
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As described initially, ovaries were examined by laparotomy and those with more than three CL received 40 mg PGF2. on day 15 (AM). A significant (P<.O1) reduction in plasma progesterone was observed after 40 mg PGF2. reaching a nadir of 1.78 0.77 ng/ml by day 20 (PM), followed by a low but steady increase characteristic of the pregnant animal. These changes were best characterized by a third order re gression equation (table 15, Appendix) which accounted for 61% of the variability. In the case of cows in groups 2 and 3 which did not receive 40 mg PGF2a (figures 8 and 9), a steady increase (tables 13 and 14, Appendix) in plasma progesterone was observed as expected in the nontreated cows. Characterization of these trends are shown in table 15 (Appendix).

The progesterone increase following PMSG has been reported by Henricks et al. (1973), Spilman et al. (1973), and Hallford et al. (1975b). Although the source of the increased progesterone could not be determined from this study, PMSG may have had a luteotrophic effect on luteal tissue or on the granulosa cells of mature follicles. The reduction in plasma progesterone after the second PGF2. injection, found in this study, closely paralleled that reported by Fournier et al. (1976). The relationship between progesterone secretion and number of CL in animals of group I (r = 0.73, P<.01l; table 18, Appendix) is in agreement with Lamond and Gaddy (1972) and Spilman et al. (1973) and in disagreement with Rajamahendran et al. (1976) who reported that a much larger number of observations was needed to assess statistical difference between superovulated and normal cycling cows.





















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53




Estrogens

Plasma estrogens for each group are depicted in figures 7, 8, and 9, respectively. Plasma estrogens in group 1 were generally below 5 pg/ml (table 12, Appendix) prior to PMSG administration after which there was a significant (P<.Ol) increase to 13.62 2.06 pg/ml within 24 hr. In conjunction with the luteolytic dose of PGF2a on day 4 fAM), estrogen levels continued to increase to a peak of 37.04 11.47 pg/mi by 72 hr post-PMSG. These changes were best characterized by a third order regression equation which accounted for 47% of the variability (table 16, Appendix). This increase probably resulted from increased follicular growth stimulated by PMSG; however, the experimental design did not allow a direct test of this assumption. Declining levels of progesterone in response to PGF2. coupled with increasing levels of estrogen led to the expression of estrus. As a consequence of the reinitiation of a PGF2,-induced estrous cycle, plasma estrogen declined significantly (P<.01l) from day 6 (AM) to day 11 (AM) (1.54 0.09 pg/ ml). Estrogen levels then remained at basal concentrations until an increase following the 40 mg dose of PGF2a given at laparotomy on day 15 (AM). These sequences of events were best described by a second order regression equation which accounted for 55% of the variability. Since cows in groups 2 and 3 (figures 8 and 9) were not administered 40 mg PGF2a, plasma estrogen levels remained at base line (table 13, Appendix), as might be expected, in the luteal phase of a normal cycling cow bled every 12 hr. Characterization of these trends are shown in table 16 (Appendix).

The relationship between estrogen secretion and number of CL in animals of group I (r = 0.68, P<.01; table 18, Appendix) is in









agreement with that reported by Hallford et al. (1975b). Henricks et al. (1973), in cattle, and Guthrie et al. (1974), in swine, concluded these types of comparisons have to be made with caution since the normal pro-estrus surge of estrogens could be confounded with an estrogen rise resulting from PMSG. As a result of this, they failed to report any'correlation between plasma estrogen and number of follicles.

Luteinizing hormone

The variability in plasma LH (tables 12 to 14, Appendix) was

most likely due to the infrequent collection of samples (figures 7 to 9). LH values prior to PMSG treatment were approximately 1.0 ng/ml. Elevations in LH to above 2 ng/ml were evident in each group between days 3"(AM) (day of PMSG administration) and day of the LH surge. This is in agreement with Spilman et al. (1973) who demonstrated that plasma LH rose sharply the day after PMSG injection and declined thereafter. Differential timing of the LH surge among groups in this study also support the observation of Henricks et al. (1973) that the ovulatory surge of LH was earlier in PMSG treated heifers than in nontreated controls. Although untreated PMSG-treated animals were not available for comparison in experiment 1, the LH surge in cows of group 1 occurred 12 hr earlier than that for animals in group 3. This is consistent with the earlier onset of behavioral estrus in cows in group I (vide supra). Hallford et al. (1975b) failed to detect this treatment effect; they only obtained plasma samples at 24 hr intervals. Henricks et al. (1973) and Hallford et al. (1975b) failed to detect a relationship between plasma LH and reproductive criteria after PMSG treatment. Since a 12 hr bleeding interval is not sufficient to fully








characterize the LH surge, no additional information regarding the relationship between plasma LH and plasma estrogen or between plasma LH and ovulation rate can be provided from this trial. 20B-Dihydroprogesterone

The individual plasma values and plotted levels of 208-P are reported in tables 12 to 14 (Appendix) and figures 7 to 9, respectively. There was a curvilinear relationship between plasma 208-P and day of bleeding (table 16, Appendix). A nonsignificant (P>.10) increase of 200-P levels was observed during the luteal phase (3.88 0.48 ng/ml) when plasma progesterone was at its maximum (18.44 4.59 ng/ml).

Spilman et al. (1973) reported an increase plasma 20B-P level

after PMSG treatment with 20B-P levels being 15 to 20% of that for P while Tribble (1973) and Castenson et al. (1976) showed, in the cycling post-partum cow, 20B-P levels to be 10 to 15% of that of P. In these studies the maximum levels of plasma 206-P were 2.5 ng/ml which is comparable to the concentrations found in this trial. In addition, similar ratios were observed in this study when cows were on day 5 of the luteal phase of the PMSG-PGF2a induced estrous cycle. Although 208-P appeared not to be influenced by gonadotropin administration, there was a 3.3 fold greater increase in P over 208-P synthesis at 24 hr post-PMSG treatment. Romanoff (1966) reported a similar ratio of P to 208-P when the ovary was perfused with FSH.

Finally, with the data available from this experiment, one may

speculate that the surge of LH at estrus is leading to follicular luteinization which is, in turn, maintaining 208-P levels. Since follicular fluid does not contain 20B-P (Short, 1962a,b) and CL (Savard and Teledgy, 1965) and luteal cysts (Short, 1962a,b) do contain 20B-P, it seems likely that only luteal cells have an active 20B-HSD. Thus in this









experiment, it may be that the origin of the 20-P was from reactivation of luteal cells from regressing CL or luteinization of granulosa cells from follicles. Hillard et al. (1967), in the rabbit, and Ichikawa et al. (1971), in the rat, demonstrated the release of LH at coitus and that exogenous LH injections activated the synthesis and release of 2C ceP.

Data accumulated in this study suggest that PMSG has profound effects on both the ovarian and endocrine response of the bovine female. Additional research aimed at understanding the change in progesterone and estrogen concentration and their role in follicular growth at the time of the post-PMSG estrus should provide further insight into the questions associated with the hormonal induction of multiple fetuses in cattle. Although the role of 20-P during the cow's estrous cycle remains obscure, experiments are needed to further characterize the endocrinology of this steroid.

Finally, although cows treated with 40 mg PGF2. exhibited both a functional and morphological regression of multiple CL with no abortion, it is of importance to test whether 40 mg PGF2a is effective in nonpregnant or pregnant cows in regressing multiple CL following PMSG treatment. If the introduction of PGF2a results in abortion of undesired multiple pregnancies associated with the superovulatory effect, then it could be used to minimize fetal wastage by regressing multiple CL and rebreeding the cow in a short breeding season. This could lead to an increase in the percent calf crop with restricted multiple fetuses. These alternatives are tested in the next experiment.









EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN THE SUPEROVULATED BEEF COW FOLLOWING ADMINISTRATION OF 40 MG PGF2a-THAM SALT


Results from experiment 1 indicated that PGF2a was ineffective in inducing abortion in the superovulated pregnant cow. The question then arose as to whether 40 mg PGF2c was inadequate to regress multiple'CL and subsequently allow the cow to be bred in a short breeding season. This experiment was designed to determine if this PGF2C dose, at 13 days post-PMSG, was effective in regressing multiple CL in the nonpregnant superovulated cow.

Although it was not the objective of this trial to evaluate the efficacy of a dual injection of PGF2a to synchronize estrus, it is of importance to review this concept and compare these results with the endocrinological profile reported in experiment 1. Reproductive Response


It is well established that PGF2a is not effective during the first 5 days of the estrous cycle. Therefore, in a large population of cycling cows 75% of the animals (based on a 21 day cycle) would be expected to be in a potentially responsive stage of the cycle (day 6 to 21) on any one day selected at random. For some unexplained reason these cows were somewhat synchronized prior to the first injection of PGF2a when 10 of 13 animals exhibited estrus within 96 hr after treatment (table 6) followed by 84.6% at the time of the second injection. Cooper (1974) and Chenault et al. (1976) clearly demonstrated that a single injection of PGF2 manipulated the estrous cycle so that cows were in a potentially more responsive stage of the cycle 12 days later. This observation is substantiated by the progesterone profiles of experiment 1.









The distribution of observed onset of estrus following an injection of PGF2a is shown in table 6. The percentage of animals exhibiting estrus 48 hr post-second PGF2a treatment (61.5%) was significantly (P< .01) greater than that after the first injection (38.5%). These results are in agreement with Cupps et al. (1976) and Archbald (1976) and support the concept of a shortened interval and reduced variability to onset of estrus after the administration of PGF2, 24 hr post-PMSG, to previously synchronized cows.

Ovarian estimates determined at laparotomy are presented in table

6. There was no significant difference in ovulation rate between cows that exhibited estrus by 48 hr (4.3 3.3) as compared to 72 hr (4.0 2.8). This does not agree with data from the first experiment in which cows with the shorterinterval to estrus had a higher ovulation rate. Endocrine Response


Ovarian stimulation by PMSG was monitored by measuring plasma progesterone on days 11, 12, and 13 post-PMSG at which time progesterone concentrations were 8.79 2.12, 10.34 2.22, and 10.22 1.89 ng/ml, respectively (table 19, Appendix). Bleeding was continued for

7 more days in an attempt to characterize the reduction in plasma progesterone following 40 mg PGF2a on day 13.

Analysis of variance indicated that the reduction in plasma progesterone concentration in cows after the administration of 40 mg PGF2. was significantly affected by treatment (P<.Ol) and day x treatment interaction (P<.05) (table 20, Appendix). Time trends of progesterone were characterized by regression analysis (table 21, Appendix). For the PMSG-treated group, a third order regression equation (y = 39.12





59








u- co




OIO +1 m 0
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0O IX I o -1S > +


0 e 01 -7
- m -0* H Le a + +1 L1 0 *







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F- > O ru










< c


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C 0 (D


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i H


















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- 13.99x + 1.73x2 0.07x3, R2 = 0.7323) best characterized this trend whereas a second order regression equation (y = 2.40 + 0.75x 0.26x2, R2 = 0.8697).best described the reduction in plasma progesterone in the non-PMSG treated group.

In response to 40 mg PGF2a administered at laparotomy (figure 10) plasma progesterone declined significantly (P<.Ol) to a basal level of 0.62 0.07 ng/ml in 96 hr. Plasma progesterone did not increase again (P>.10) until the luteal phase following the 40 mg PGF2a. A similar trend was observed in the non-PMSG treated group, although the magnitude of response was smaller. Ovarian palpation 7 days postlaparotomy confirmed multiple CL regression and subsequent estrus indicated the reinitiation of a new estrous cycle.

The change in slope of the progesterone decline following PGF2a possibly reflects functional CL regression. Functional CL regression is the termination of progesterone secretion while structural regression, i.e., the physical destruction of the luteal cell, is believed to be the result of changing intra-ovarian blood flow distribution post-PGF2a. Novy and Cook (1973) and Thornburn and Hales (1972) demonstrated that blood flow to the CL was reduced, whereas blood flow to the stroma and follicular component of the ovary was increased following PGF2a administration.

Although the mechanism of luteolysis after PGF2a remains undefined, data accumulated in this study clearly suggest that 40 mg PGF2a is effective in regressing multiple CL in the cycling nonpregnant beef cow based upon the reduction in plasma progesterone and CL regresstion evidenced by the absence of palpable CL by 7 days post-treatment. The question still remains as to whether or not 40 mg PGF2. is effective




















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in regressing multiple CL of PMSG-PREGNANT cows since in the first experiment, both functional and morphological CL regression was presumed. Unexpectedly, however, pregnancy was maintained. The next experiment deals with the effect of pregnancy on the regression of multiple CL in mated cows.



EXPERIMENT 3. LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN THE PMSG BRED VS NONBRED BEEF
COW


Data from experiment I indicated that 40 mg PGF2a-Tham Salt

apparently induced only partial functional and structural CL regression in PMSG-PREGNANT cows, since CL appeared to have regained their functional role and pregnancy was maintained to term. In experiment 2, however, 40 mg PGF2a was effective in regressing multiple CL in cycling cows when measured by a rapid decline in plasma progesterone to below I ng/ml and the absence of a palpable CL 7 days post-treatment. Experiment 3 was thus designed to test the difference in response to PMSG by bred vs cycling cows.


Reproductive Response


The distribution of estrus after a dual estrous synchronization scheme with PGF2. is summarized in table 7. As expected, a greater percentage of animals expressed estrus following the second injection of PGF2a than following the first injection in the PMSG treated group (96 vs 91%, respectively). Unexpectedly in the non-PMSG group,there was not an increase in the percentage of animals responding to the second injection of PGF2a (82 vs 79%, P>.10). The PMSG superovulated















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64




animals, by 72 hr post-PGF2a, expressed estrus to a greater degree (P< .01) than the non-PMSG animals (87 vs 39%, respectively). Whether the influence of PMSG on this reduction in interval from PGF2a to behavioral estrus results in differential fertility remains to be investigated.

The interval to estrus following the 40 mg abortifacient dose of PGF2a was longer in the PMSG treated animals when compared to the nonsuperovulated group (15 vs 7 days, respectively, P<.0O'). Ninetyone percent of the cows in the non-PMSG group responded within 7 days post-40 mg PGF2a with a mean interval to estrus of 4.48 1.12 days. Two types of estrous response were noted in the PMSG treated animals: one type, representing 64% of the animals, displayed estrus between 5 to 10 days post-treatment with a mean interval of 6.43 2.24 days and the second type, comprising 27% of the animals, responded behaviorally between 11 and 15 days post-treatment with a mean interval of 13.50 1.05 days. Fifty days post-40 mg PGF2. all animals cycled at least once and post-mortem examination of the reproductive tracts showed no evidence of pregnancy.

The abortifacient property of PGF2a in beef cattle has been widely demonstrated (Lauderdale, 1972, 1974; Fields et al., 1977a). There is only limited data available with regard to behavioral estrus post-abortion, in the PMSG-BRED cow. Results from the non-PMSG group substantiate that of Douglas et al. (1974) in that cows aborted in early gestation may return to estrus within 2 days after abortion. Results from this trial show the PMSG-BRED group to be comparable to that of Zerobin et al. (1973) in that the administration of PGF2a to non-superovulated pregnant cows at early stages of gestation resulted in behavioral estrus 2 to 16 days post-treatment.









Ovarian response estimates, determined at laparotomy, are presented in table 8. In experiment 1, a differential ovulation rate with respect to day of behavioral estrus was observed. In this trial, although there was a reduction in ovulation rate from day I to day 4 of 8.00 1.73 vs

5.88 2.83, respectively, this difference was nonsignificant (P>.10).

Six of the 22 superovulated cows had been treated with PMSG tn a previous trial. No refractoriness to the PMSG given in this trial was detected (table 9). The increased refractoriness to successive PMSG reported by Willett et al. (1953), Hallford et al. (1975a,b), and Turman et al. (1977) and the lack of refractoriness in this study might be attributed to the relative longer time elapsed between the two gonadotrophin injections.

Post-mortem examination of the reproductive tracts revealed no ovarian abnormalities, e.g., cystic follicles and indicated that the cows were cycling. It is important to mention, however, that there was observed a high incidence of ovarian adhesions in response to supravaginal laparotomy. This was particularly noticeable in the PMSG group.


Endocrine Response

Analysis of variance of the reduction of plasma progestin

after 40 mg PGF2. was significantly (P<.Ol) affected by PMSG treatment (table 22, Appendix). The regressioncurves for individual treatments groups are depicted in figures 11 and 12. Plasma progestins concentrations and predicted equations are summarized in tables 23 and 24 (Appendix), respectively.





66













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67















TABLE 9. OVULATION RATES IN COWS TREATED TWICE WITH
PMSG AT FOUR MONTH INTERVAL


First PMSG Second PMSG Cow Treatment (6/18/76) Treatment (10/29/76) number
RT ovary LT ovary RT ovary LT ovary

38 0 1 3 1

-42 5 2 3 3 44 4 2 5 1 45 1 2 1 1 46 3 4 4 4 47 5 4 5 0


Overall 3.0 + 2.1 2.5 3.0 3.5 1.5 1.7 1.9




68















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69















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Cows treated with PMSG exhibited higher mean plasma progestin concentration of 7.63 1.79 and 8.86 1.57 ng/ml for the bred and nonbred groups, respectively, when compared to those for non-superovulated cows of 3.07 0.15 and 2.64 0.21 ng/ml for the same respective groups at laparotomy. Similar results have been reported previously for cycling cattle (Plotka et al., 1967; Stabenfeldt et al-, 1969; Kazama and Hansel, 1970; Sprague et al., 1971; Wettemann et al., 1972; Glencross et al., 1973). AFter 40 mg PGF2a injection on day 3, plasma progestin in the PMSG group declined significantly (P<.0l) to a minimum of 1.37 0.23 and 1.25 0.22 ng/ml, on day 9, for the bred and nonbred groups, respectively. In the non-superovulated animals,minimum progestin levels of 1.06 0.19 and 0.96 0.16 ng/ml wereachieved by day 8. These trends were considered to be curvilinear (table 24, Appendix). On an individual animal basis, all cows achieved progestin levels below I ng/ml post 40 mg PGF2,.

Burrell and Wiltbank (1977) demonstrated that CL of a pregnant cows on day 17 and CL of a nonpregnant cow on day 6 differ in their response to exogenous hormones when 33.4% of the pregnant cows and 100% of the nonpregnant cows resumed cyclicity after a single dose of 4 mg norgestomet. With PGF2a, it only takes 24 hr to achieve a 60 to 75% reduction in plasma progesterone; however, for the subsequent expression of estrus it takes up to 16 days (Zerobin et al., 1973; Douglas et al., 1974).

In the PMSG superovulated cycling cow, PGF2a, was shown to be effective in regressing multiple CL when measured by a rapid decline in plasma progesterone, absence of a palpable CL, and resumption of cyclicity (Lopez-Barbella et al., 1976,1977). In the superovulated




71




pregnant cow, however, they indicated 40 mg PGF2a to have induced a significant (P<.Ol) decline in plasma progesterone but failed to induce abortion. The discrepancy in results between these reports and the data accumulated in this study might be explained by the animal variability, differences in time and/or other random sources of variability.

In conclusion, when induction of multiple fetuses is attempted with gonadotrophic hormones, abortions associated with the superovulation effect might be minimized by regressing the multiple CL with PGF2a and rebreeding the cow in a short breeding season. This, possibly could lead to a restricted number of cows with multiple calves which would increase the percent calf crop.















CHAPTER V
SUMMARY AND CONCLUSIONS



In an attempt to characterize the trends of plasma progesterone, 206-dihydroprogesterone, luteinizing hormone, and estrogens in response to the PMSG induction of superovulation, 15 cycling parous Angus cows were estrous synchronized with two sequential treatments of 33.5 mg PGF2,-Tham Salt (IM) at a 12-day interval. An injection of 2,000 i.u. PMSG (Organon) was administered 24 hr prior to the second PGF2a and cows were inseminated three times at 0, 12, and 24 hr post-PMSGPGF2a-induced estrus. Blood samples were collected twice daily for 21 days from all animals starting two days prior to the PMSG injection and plasma hormone levels analyzed by RIA (vide supra).

In response to the PMSG treatment cows exhibited a stereotypic ovulation rate (2.67 2.19). The highest ovulation rate (5.50 1.29) occurred in cows with the shortest interval from treatment to estrus (24 hr). The lowest ovulation rate (0.67 + 0.82) was in conjunction with the longer interval from treatment to estrus (48 to 96 hr) suggesting the possibility of a relationship between length of the interval from PMSG to estrus and superovulatory response.

Hormonal characterization in cows bearing more than three CL

post-PMSG at laparotomy (see figure 7 for experiment 1) showed plasma progesterone exhibited an expected secretion curve prior to PMSG treatment after which a 50% increase was observed by 24 hr post-gonadotropin administration. Simultaneously, a substantial increase in plasma

72









estrogens and LH was also recorded with no significant change in 208dihydroprogesterone levels. The hormonal trends after the luteolytic dose of PGF2a 24 hr post-PMSG were similar to those observed during CL regression in the normal cycling cow with an increase in plasma estrogens being correlated to the apparent increase in follicular growth stimulated by the PMSG. Although plasma LH was variable, tha LH surge was 12 hr earlier in cows with more than one CL compared to those with only a single CL following PMSG treatment.

The initiation of the PMSG-PGF2a-induced cycle was characterized by a rather significant (P<.01) increase in plasma progesterone which reached a maximum of 18.44 4.59 ng/ml on the day of injection of 40 mg PGF2,. A dramatic decline in plasma LH and estrogen levels was observed'post-estrus and remained at base line thereafter.

The plasma 208-dihydroprogesterone trend did not change significantly (P>.lO0) during the course of this study. When progesterone and 20B-dihydroprogesterone ratios were computed, however, a significant (P<.Ol) trend was detected suggesting a relationship between P and 208-P. The lowest ratios of 208-P/P were 13 to 15% during the luteal phase and increased to 80 to 100% when cows were approaching estrus. At estrus, however, 208-P levels were always higher than P. The experimental design did not reveal whether this 208-P was a result of PMSG induced luteinization of the granulosa cells or from luteal cells of the regressing CL.

The administration of 40 mg PGF2a to cows with greater than three CL resulted in a dramatic decline in plasma progesterone concentrations and the absence of palpable CL. This dose, however, was not adequate to induce abortion and to sustain plasma progesterone at basal









concentrations for an adequate time to permit the animal to express estrus.

In conclusion, these data clearly demonstrated that the endocrinology of the PMSG superovulated cow differs from that of the normal cycling cow. Not only the magnitude of the hormonal secretion is different, but the length of the interval from PMSG to estrus and superovulatory response are affected. In addition, the early LH surge and early increase in LH post-PMSG treatment might be partially due to differential follicular growth and estrogen secretion in response to gonadotrophin administration. This experiment, however, was not designed to test the hypothesis of differential follicular growth postPMSG. Research designed to test the hypothesis of differential follicular-growth post-PMSG may explain the etiology of these differential hormonal patterns associated with inducing multiple fetuses in cattle. The introduction of 40 mg PGF2a into this hormonal treatment regime first appeared to be an ineffective avenue to induce abortion in the PMSG-bred cow. The efficiency of 40 mg PGF2a to induce CL regression and subsequent abortion in cows, with multiple CL was tested in two additional experiments.

In the second experiment 13 animals received two sequential

treatments of 33.5 mg PGF2a 12 days apart. Ten cows were administered 2,000 i.u. PMSG 24 hr prior to the second PGF2a injection. Ovulation rate was determined by supravaginal laparotomy 13 days post-PMSG and cows were given 40 mg PGF2,. Regression of CL was monitored by bleeding all animals daily for 11 days starting 2 days prior to laparotomy and analyzing for plasma P concentrations. Administration of 40 mg PGF2,, 13 days post-PMSG, evoked a dramatic decline in plasma









P in both PMSG and non-PMSG treated cows. Plasma P reached basal concentrations by 96 hr post-40 mg PGF2a. Ovarian palpation 7 days postlaparotomy confirmed multiple CL regression. Shortly thereafter cows expressed estrus.

In conclusion, this experiment demonstrated that administration

of 40 mg PGF2., 13 days post-PMSG, was effective in regressing multiple CL, in superovulated cycling cows, when measured by a dramatic decline in plasma P, the absence of palpable CL 7 days post-treatment, and reinitiation of a new estrous period.

In the third experiment 45 cows were hormonally treated as in experiment 3. In addition, cows were divided into four factorial groups involving PMSG and MATING as main factors. Cows assigned to breeding were exposed to bulls for a 10 day breeding period starting the day of the second synchronizing dose of PGF2,. The reduction in plasma progestin following 40 mg PGF2a was significantly (P<.Ol) affected by PMSG treatment, with P declining at a slower rate. Although plasma P reached a basal concentration by 96 to 120 hr post-PMSG, behavioral estrus was observed in a cascade type fashion with 91% of the non-PMSG cows, responding within the first 7 days posttreatment vs 64% of the PMSG-treated animals, responding within 5 to 10 days post-treatment while 27% responded 11 to 15 days post-treatment. Post-mortem examination of reproductive tracts 50 days post-treatment confirmed multiple CL regression, reinitiation of cyclicity, and no evidence of pregnancies.

In conclusion, data accumulated in this experiment clearly suggest that profound effects at both the ovarian and endocrine levels in the bovine female in response to PMSG could be modulated by PGF2,. Thus









the introduction of 40 mg PGF2a 13 days post-PMSG to superovulated pregnant cows might result in a means to prevent fetal wastage from the superovulation effect by bringing cows with more than three CL to a premature abortion and rebreeding them to a bull in the normal breeding season. Trials need to be established as to whether this may be a means by which a herd of cows could be successfully induced to have a 100 to 110% calf crop through the induction of limited multiple births.







































APPENDIX






















TABLE 10. PREPARATION AND STORAGE OF ASSAY BUFFER


To a 2 1 volumetric flask add:

32.7 g of sodium phosphate dibasic heptahydrate (MW = 268)

10.8 g of sodium phosphate monobasic monohydrate (MW = 138)

18.0 g of sodium chloride (MW = 58)

2.0 g of sodium azidea (MW = 65)


Then add 2X distilled-tap water (pH = 7.0 0.1) to a total volume of

2 1. Adjust to pH = 7.20 0.01 adding concentrated solution of sodium hydroxide. This stock solution may be stored at 4C as long as there is no evidence of mold or bacterial growth. The working assay buffer is prepared by adding 0.1% Knox gelatin (100 mg/lOO ml) to the above stock solution. The assay buffer should be stored at 4C for no longer than 4 weeks.


,aDo not breathe or contaminate skin. Extremely poisonous. Reacts with drain pipe, resulting in residue build up over time.
Extremely explosive.










78




79

















TABLE 11. PREPARATION OF NADH IN 0.1 M TRIS BUFFER


A 0.1 M Tris buffer is obtained by mixing 6.057 g hydroxymethyl aminomethane in 500 ml distilled-tap water (2X). To a 100 ml volumetric flask add 50 ml of this stock solution and 26 ml of 0.1 N HCI. Then add distilled-tap water (2X) to a final volume of 100 ml. Adjust to pH = 8.10 0.01. To a small vial cylinder add 5 mg NADH (6.40 Mll) and 3 ml of 0.1 M Tris buffer pH 8.1.




80







TABLE 12. PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DIHYDROPROGESTERONE,
LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING MORE THAN
THREE CORPORA LUTEA AT LAPAROTOMY


20-Dihydro- Luteinizing
Day of Progesterone Prog'esterone hormone Estrogens bleeding + S.E. R S.E. A + S.E. R S.E.
ng/ml ng/ml ng/ml pg/ml


IAM 3.72 + 0.34 2.60 0.65 0.97 1 0.11 1.52 0.16 PM 3.72 0.27 2.34 0.48 1.58 0.27
2AM 3.70 0.32 1.96 0.56 1.04 0.22 2.48 + 0.30 PM 3.88 0.39 -1.78 0.30 0.80 0.08
3AM (PMSG) 5.66 1.17 1.68 0.45 0.72 0.08 3.28 0.86 PM 6.18 1.27 2.12 0.36 3.36 0.39
4AM (PGF) 9.06 2.99 1.88 0.18 3.34 0.17 13.62 + 2.06 PM 3.86 1.34 1.60 0.36 3.34 O0.11
5AM 2.02 0.64 1.36 0.30 2.42 0.36 30.08 13.32 PM. 1.40 + 0.29 1.44 2.32 2.84 0.31
6AM 0.90 0.19 1.40 0.27 24.32 + 9.60 37.04 11.47 PM 0.72 0.14 1.48 0.28 5.34 3.13
7AM 0.82 + 0.09 1.50 0.47 2.30 0.29 19.60 6.42 PM 1.00 0.23 1.42 0.36 2.42 0.35
8AM 1.34 + 0.39 1.52 0.34 2.60 + 0.38 11.80 3.91 PM 1.88 + 0.45 1.62 + 0.45 2.42 + 0.27
9AM 2.28 0.55 1.58 0.28 2.36 + 0.19 2.92 0.71 PM 2.94 + 0.64 1.46 0.33 2.18 0.22
IOAM 4.06 0.87 1.54 0.25 1.56 + 0.26 2.92 0.57
PM 5.68 1.63 1.40 0.27 1.54 + O.11
I1AM 6.30 1.37 1.72 0.50 1.82 0.11 1.54 0.09
PM 7.98 1.54 1.96 0.37 1.94 0.22
12AM 10.80 + 1.42 2.22 0.27 2.04 1 0.12 2.20 0.20
PM 14.04 1.63 1.88 0.11 1.72 0.09
13AM 15.34 1.40 2.08 i 0.32 1.40 0.28 2.20 0.15 14AM 15.24 1.23 2.32 0.19 1.56 i 0.10 2.18 0.21
PM 16.30 + 1.52 2.64 0.35 1.46 0.17
15AM 16.46 2.36 2.52 0.41 1.36 0.08 2.05 0.18
PM 18.44 4.'59 3.38 0.48 1.40 0.15
16AM (PGF) 14.22 2.94 3.10 0.31 1.50 + 0.19 5.85 0.34
PM 8.46 I 1.46 2.82 0.35 1.40 4 0.21
17AM 5.88 1.25 2.26 0.45 1.74 0.27 4.71 0.40
PM 3.98 1.36 1.86 0.53 1.48 0.26
18AM 2.90 + 0.83 1.88 0.46 1.30 0.18 5.23 0.41
PM 2.96 + 0.88 2.22 0.49 1.98 0.34
19AM 2.70 1.05 2.46 0.28 1.28 0.29 5.00 0.50




81















TABLE 12. (Continued)


20-Dihydro- Luteinizing
Day of Progesterone Progesterone hormone Estrogens bleeding S.E. 2 S.E. i + S.E. + S.E.
ng/ml ng/ml ng/ml pg/ml


19PM 2.90 1.15 2.84 0.27 1.32 0.29 20AM 2.12 0.85 2.14 0.23 1.14 0.15 3.30 0.33
PM 1.78 0.77 1.86 0.29 1.26 0.17
21AM 1.96 0.98 1.90 0.23 1.20 0.23 2.45 0.25
PM 2.22 1.20 1.94 0.32 1.15 + 0.34
22AM 2.62 + 1.59 2.38 0.37 2.38 0.37 2.12 0.71
PM 2.70 + 1.63 2.24 0.42 2.24 0.42




82







TABLE 13. PLASMA CONCENTRATIONS OF PROGESTERONE, 20B-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING
TWO OR THREE CORPORA LUTEA AT LAPAROTOMY


20-Dihydro- Luteinizing
Day of Progesterone Progesterone hormone Estrogens bleeding R R S.E. R S.E. R S.E. R S.E.
ng/ml ng/ml ng/ml pg/ml


1AM 3.85 0.34 2.50 0.37 1.23 0.46 1.73 0.09 PM 3.95 0.38 2.55 0.40 1.281 0.13
2AM 3.98 0.46 2.10 0.37 0.73 0.09 1.65 0.26 PM 4.28 0.62 -1.80 0.17 1.30 0.47
3AM (PMSG) 5.43 + 1.46 1.23 0.15 1.45+ 0.43 4.03 1.42 PM 6.30 + 1.16 1.43 0.25 3.23 0.52
4AM (PGF) 4.03 1.11 1.48 + 0.45 2.68 0.68 11.75 4.67 PM 2.70 0.64 1.65 + 0.59 4.23 0.55
5AM 1.85 0.48 1.28 0.31 3.68 0.74 9.83 2.68 PM. 1.00 0.11 1.23 0.29 3.18 0.14
6AM 1.08 0.25 1.55 0.32 16.73 11.19 14.30 7.89 PM 0.88 0.09 1.63 0.16 12.25 8.53
7AM 0.68 0.09 1.60 + 0.07 3.13 0.49 5.00 0.89 PM 0.65 0.06 1.43 + 0.13 2.70+ 0.37
8AM 0.85 0.21 1.50 0.29 2.904 0.31 7.28 1.97 PM 1.18 + 0.45 1.58 0.33 2.08 0.36
9AM 1.43 0.49 1.83 0.33 2.20 0.12 5.13 1.59 PM 1.47 0.82 1.83 + 0.44 2.87 0.77
IOAM 2.50 0.37 2.15 0.34 2.38+ 0.31 4.98 1.16
PM 2.35 0.69 2.05 0.48 2.18+ 0.15
11AM 2.78 + 0.70 1.65 0.43 2.30+ 0.20 1.95 0.37
PM 3.03 0.71 1.73 0.57 2.03 0.32
12AM 3.13 0.52 1.80 0.51 1.95f 0.26 1.54 0.35
PM 3.45 + 0.72 2.03 + 0.84 1.93+ 0.09
13AM 3.08 + 0.43 2.85 0.97 1.68 t 0.27 1.60 0.40 14AM 3.88 0.68 2.00 + 0.18 1.58. 0.10 1.58 0.38
PM 3.63 0.45 1.85 0.38 1.50 + 0.29
15AM 4.30 0.68 1.83 0.39 1.15+ 0.25 1.67 0.18
PM 4.20 + 0.41 2.00 + 0.62 1.33+ 0.30
16AM 4.30 0.33 2.05 0.55 1.50 0.34 1.70 0.09
PM 5.45 0.74 2.15 0.73 1.85 0.24
17AM 5.60 0.56 2.48 0.45 0.98 0.10 1.69 0.17
PM 4.88 0.36 2.95 0.49 1.10 0.19
18AM 4.88 + 0.46 2.65 0.60 0.50 0.00 1.73 0.17
PM 5.00 0.27 2.38 + 0.34 1.05 0.22




83







TABLE 14. PLASMA CONCENTRATIONS OF PROGESTERONE, 203-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS WITH
ZERO OR ONE CORPORA LUTEA AT LAPAROTOMY


20 B-Dihydro- Luteinizing
Day of Progesterone Progesterone hormone Estrogens bleeding + S.E. R S.E. + S.E. x S.
ng/ml ng/ml ng/ml pg/ml


IAM 2.88 + 0.71 1.47 0.27 0.54 0.09 2.68 1.39 PM 2.60 0.58 1.65 0.29 0.86 0.24
2AM 2.75 0.55 1.71 0.27 0.70 0.09 3.91 1.02 PM 3.08 + 0.64 -1.55 0.22 1.26 0.28
3AM (PMSG) 3.01 0.54 1.72 0.12 0.70 0.12 3.68 0.81 PM 3.65 0.51 1.88 0.25 2.40 0.29
4AM (PGF) 3.23 0.53 1.80 0.28 2.51 0.21 5.00 1.79 PM 2.10 + 0.53 1.71 0.29 3.73 + 0.64
5AM 1.47 0.44 1.67 0.32 2.80 0.28 4.03 1.38 PM. 1.37 0.40 1.95 + 0.59 3.00 0.19
6AM 1.07 0.32 2.12 0.74 3.10 0.59 4.38 1.24 PM 0.88 + 0.49 2.28 0.79 6.60 3.38
7AM 0.80 + 0.32 2.20 + 0.60 2.67 0.19 3.05 0.71 PM 1.21 + 0.59 2.40 0.59 2.61 0.13
8AM 1.45 0.78 2.33 0.78 2.46 0.21 3.20 0.66 PM 1.13 0.47 2.20 0.85 2.17 0.16
9AM 0.83 0.42 2.05 0.65 2.22 + 0.22 2.60 0.70 PM 0.80 0.17 1.63 0.29 2.40 0.15
10AM 1.45 0.58 1.68 0.27 1.68 + 0.12 2.35 0.52
PM 1.37 0.48 1.70 0.20 2.03 0.14
IIAM 1.80 + 0.52 1.95 + 0.30 2.43 0.29 1.75 + 0.17
PM 1.95 + 0.49 1.53 0.22 2.28 0.21
12AM 1.88 0.47 1.63 0.27 2.27 0.14 1.09 0.10
PM 1.75 + 0.31 1.38 0.14 1.95 0.26
13AM 1.90 0.47 1.20 0.20 1.57 0.17 1.10 0.08 14AM 2.15 0.43 1.58 0.30 1.72 0.30 1.00 + 0.08
PM 2.22 0.41 1.83 0.30 1.60 0.19
15AM 2.53 0.50 1.65 + 0.27 1.33 0.16 1.37 0.13
PM 2.52 0.46 1.52 0.31 2.03 0.69
16AM 2.20 + 0.32 1.63 + 0.26 1.65 + 0.41 1.40 0.08
PM 2.31 + 0.28 1.96 0.31 2.36 0.79
17AM 2.70 0.19 1.67 0.19 1.18 + 0.24 1.43 0.23
PM 2.53 + 0.19 1.67 0.29 1.02 0.19
18AM 2.52 + 0.26 1.62 0.32 0.96 + 0.27 1.40 0.14
PM 2.43 0.25 1.78 0.32 1.40 0.27





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TABLE 18. COEFFICIENTS OF CORRELATION BETWEEN PLASMA HORMONAL LEVELS
AND NUMBER OF CORPORA LUTEAa


20O-Dihydro- Luteinizing
Source Progesterone progesterone hormone Estrogens


Group 1 0.73b 0.16 0.26 0.68 >3CL (0.0001)c (0.3092) (0.3017) (0.0001)

Group 2 0.42 0.14 0.29 0.49
2 to 3 CL (0.0062) (0.4115) (0.2280) (0.0008)

Group 3 0.21 0.11 0.35 0.42
0 to 1 CL (0.1822) (0.4846) (0.0837) (0.0053)


aExpressed upon grouping criteria (see text).
bCorrelation coefficient.
CProbability.




Full Text

PAGE 1

ENDOCRINOLOGY OF THE SUPEROVULATED COW AND SUBSEQUENT PROSTAGLANDIN F2a REGRESSION OF THE MULTIPLE CORPORA LUTEA By SERGIO RAFABL LOPEZ BARBELLA A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1977

PAGE 2

ACKNOWLEDGEMENTS In most cases, every research product is the result of multip+e endeavors. This one is no exception. Since its very beginning, many persons and institutions have provided enormous contributions. Without them this dissertation would never have been possible. The list is long as deep is my indebtedness. Special thanks, go to every member of my Supervisory Committee: to Dr. Michael J. Fields, my chairman, for his help, patience, and encouragement not only during every phase of this research but throughout these years of graduate work; to Drs. Fuller W. Bazer and William W. Thatcher for always being available to share their knowledge and for their valuable comments; to Dr. Alvin C. Warnick, for providing his experience in the area through sound remarks; to Dr. Pejaver V. Rao, for statistical assistance; and to Drs. Daniel C. Sharp III and Robert J. Collier for reading and correcting the initials manuscripts of this study. My appreciation to Universidad Central de Venezuela, Facultad de Agronomia, for providing economical support throughout my graduate work I am also grateful to the Upjohn Company, especially to Dr. J. Lauderdale, for providing PGF2a and reviewing my experimental protocols. To Dr. J. L. Fleeger, Texas AsM University, and Dr. L. V. Estergreen, Washington State University, my gratitude is extended for

PAGE 3

their collaboration in providing the progesterone and estrogen antisera, respectively. Special thanks are also due to Dr. J. H. Hentges, University of Florida, and Mr. Ardeen Wiggins, University of Florida Foundation for providing the Angus cows used in these experiments. To my fellows graduate students Tomas H. Wise, Jorge Beltran, Daniel Hardin, and Thomas Thompson my indebtedness for their help at different phases of this study. Finally, and above all, the author wishes to express gratitude and appreciation to his mothe r, Reg i na to his wife, Lourdes, and to his children, Sergio and Ana, for their love in both good and difficult times and to Mrs. Ross i nav Fe rnandez for her typing of this manuscript.

PAGE 4

H TABLE OF CONTENTS ACKNOWLEDGEMENTS ii LIST OF TABLES •. vi LIST OF APPENDIX TABLES vii LIST OF FIGURES Ix ABSTRACT xi CHAPTER I: INTRODUCTION 1 CHAPTER II: LITERATURE REVIEW k Prostaglandins and Reproduction h Prostaglandin F2a and Estrous Synchronization k Luteolytic Effect of Prostaglandin F2a 6 Prostaglandin Fja and Therapeutic Abortion. ... 8 Prostaglandin F2ct and Superovu lat ion 10 Gonadotropic Hormones and Limited Multiple Births ... 11 Endocrinology of the Superovu lated Cow \k 203-Dihydroprogesterone (4-Pregnen-20B-o 1 -3-one) ... 16 CHAPTER III: MATERIAL AND METHODS 21 EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P), 20e-DIHYDR0PR0GESTER0NE (2O3-P) TOTAL ESTROGENS (E) AND LUTEINIZING HORMONE (LH) IN PMSG SUPEROVULATED COWS SYNCHRONIZED WITH PGFga-THAM SALT. ... 21 Surgical Description of Supravaginal Laparotomy 23 EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN PMSG SUPEROVULATED BEEF CATTLE FOLLOWING ADMINISTRATION OF 40 MG PGF2a-THAM SALT. 23 EXPERIMENT 3LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN BRED y£ CYCLING BEEF COWS PREVIOUSLY TREATED WITH PMSG 25 Radioimmunoassay of Steroid Hormones 27 Cleaning of Glassware 27 Organic Solvents 27 Preparation, Use, and Storage of Radioactive Steroids 27 fv

PAGE 5

TABLE OF CONTENTS Continued Page Conversion of Progesterone ( P) to 206-Dihydroprogesterone (3H-203-P) •• 28 Use and Storage of Antibody 29 Serum Extraction and Chromatography 29 Conversion of 3H-20B-P to 33 Preparation, Use, and Storage of Assay Buffer. 33 Preparation, Use, and Storage of Charcoal Suspension 3^ Preparation and Use of Counting Solution .... 3'* Preparation, Use, and Storage of Standard Steroids 3^ Radioimmunoassay 35 Calculations ^0 Separation by LH-20 Column Chromatography. ... hO Precision Statistical Analysis ^1 CHAPTER IV: RESULTS AND DISCUSSION A3 EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P) 20B-DIHYDR0PR0GESTER0NE (203-P) ESTROGENS (E) AND LUTEINIZING HORMONE (LH) IN THE PMSG 5UPER0VULATED COW SYNCHRONIZED WITH PGF2a-THAM SALT Reproductive Response ^3 Endocrine Response 'tS Progesterone ^8 Estrogens 53 Luteinizing Hormone 5^ 20g-D ihydroprogesterone 55 EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN THE SUPEROVULATED BEEF COW FOLLOWING ADMINISTRATION OF hO MG PGF2a-THAM SALT 57 Reproductive Response 57 Endocrine Response 58 EXPERIMENT 3LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN THE PMSG BRED VS NONBRED BEEF COW 62 Reproductive Response 62 Endocrine Response 65 CHAPTER V: SUMMARY AND CONCLUSIONS 72 APPENDIX 78 LITERATURE CITED 9^ BIOGRAPHICAL SKETCH 105 v

PAGE 6

LIST OF TABLES TABLE Page 1 EFFICIENCY OF ^H-P CONVERSION TO ^H-ZOB-P AT VARYING LEVELS OF COFACTORS. 31 2 STANDARD CURVE DILUTIONS 56 3 ADDITIONAL TUBES USED TO SUPPLEMENT RIA ANALYSIS 37 k DISTRIBUTION OF ANIMALS SHOWING ESTRUS AFTER THE SECOND PGF2a ADMINISTRATION AND SUBSEQUENT OVARIAN RESPONSE OF ANIMALS TREATED WITH PMSG IN EXPERIMENT 1 ..... 5. CONCEPTION RATES FOLLOWING ARTIFICIAL INSEMINATION POST-TREATMENT WITH PMSG AND PGF2a AND EXPOSURE TO THE BULL FOR 90 DAYS IN EXPERIMENT ] 4? 6 DISTRIBUTION OF ESTRUS AND OVULATION RATE IN COWS AFTER A DUAL INJECTION OF PGF2a-THAM SALT AND PMSG 59 7 DISTRIBUTION OF ESTRUS IN COWS AFTER A DUAL 33-5 MG SYNCHRONIZING INJECTION OF PGF2a-THAM SALT AND ONE kO MG ABORT I FAC I ENT DOSE OF PGF2a 63 8 OVULATION DISTRIBUTION P0ST-PGF2a INDUCED ESTRUS IN COWS IN EXPERIMENT 3 66 9 OVULATION RATES IN COWS TREATED TWICE WITH PMSG AT FOUR MONTH INTERVAL 67

PAGE 7

LIST OF APPENDIX TABLES TABLE Page 10 PREPARATION AND STORAGE OF ASSAY BUFFER 78 11 PREPARATION OF NADH IN 0.1 M TRIS BUFFER ...... 79 12 PLASMA CONCENTRATIONS OF PROGESTERONE, 20e-DIHYDR0PROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING MORE THAN THREE CORPORA LUTEA AT LAPAROTOMY 80 13 PLASMA CONCENTRATIONS OF PROGESTERONE, 206-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING TWO OR THREE CORPORA LUTEA AT LAPAROTOMY 82 ]h PLASMA CONCENTRATIONS OF PROGESTERONE, 206-DIHYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS WITH ZERO OR ONE CORPORA LUTEA AT LAPAROTOMY. 83 15 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR PROGESTERONE 8'* 16 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR ESTROGENS AND 2O3-D I HYDROPROGESTERONE (20B-P) ... 85 17 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR LUTEINIZING HORMONE 86 18 COEFFICIENTS OF CORRELATION BETWEEN PLASMA HORMONAL LEVELS AND NUMBER OF CORPORA LUTEA 87 19 PLASMA CONCENTRATIONS OF PROGESTERONE AFTER kO MG PGF2a IN COWS TREATED WITH OR WITHOUT PMSG 88 20 SPLIT-PLOT ON TIME ANALYSIS OF VARIANCE OF PROGESTERONE IN EXPERIMENT 2 89 21 LEAST SQUARES REGRESSION EQUATIONS AND R^ VALUES FOR PROGESTERONE 90 22 ANALYSIS OF VARIANCE IN EXPERIMENT 3 91 23. PLASMA CONCENTRATIONS OF PROGESTIN AFTER hO MG PGF2a IN COWS SUPEROVULATED WITH PMSG AND EXPOSED TO BREEDING 92 vl i

PAGE 8

LIST OF APPENDIX TABLES Continued TABLE P^gg 2i LEAST SQUARES REGRESSION EQUATIONS AND R^ VALUES FOR PROGESTIN 93 V i if

PAGE 9

LIST OF FIGURES FIGURE 1 Treatment protocol for experiment 1 involving endocrine characterization following PMSG and • PGFsa • • 2 Treatment protocol for experiment 2 evaluating the effectiveness of PGFga to regress multiple corpora lutea in nonbred cows '3 Treatment protocol for experiment 3 comparing the efficacy of PGFga to regress multiple corpora lutea in the bred cow iLH-20 profile of 3h-1,2 progesterone (P) conversion to 3h-1 ,2-203-dihydroprogesterone (206-P) using hexane : benzene :methano I (80:15:5)• 5 Standard curve of progesterone. (Each point represents the mean of triplicates on 20 determinat ions. ) 6 Standard curve of estradiol. (Each point represents the mean of triplicates on seven determinations.) 7 Concentration of progesterone (•*•), 203-dihydroprogesterone ( ), luteinizing hormone ( ), and estrogens ( ) in plasma from PMSG treated cows with four or more corpora lutea 8 Concentration of progesterone (•"•), 20g-dihydroprogesterone ( ), luteinizing hormone ( ), and estrogens ( ) in plasma from PMSG treated cows with two or three corpora lutea 9 Concentration of progesterone (• • • ), 20B-dihydroprogesterone ( ), luteinizing hormone ( ), and estrogens ( ) in plasma from PMSG treated cows with zero or one corpora lutea ix

PAGE 10

LIST OF FIGURES Continued FIGURE Page 10 Effectiveness of PGF2a to regress corpora lutea and reduce plasma progesterone in PGF-2a synchronized cows treated with PMSG ( ) and without PMSG ( ). 61 11 Plashia progestin profile in PMSG-BRED ( ) and ~ PMSG-CYCLING (---) cows after ^0 mg PGF2a 68 12 Plasma progestin in BRED ( )'and CYCLING (---) cows after kO mg PGF2a ^9 V X

PAGE 11

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENDOCRINOLOGY OF THE SUPEROVULATED COW AND SUBSEQUENT PROSTAGLANDIN F2a REGRESSION OF THE MULTIPLE CORPORA LUTEA By Sergio Rafael Lopez Barbel la December 1977 Chairman: M. J. Fields Major Department: Animal Science The endocrinology of the Pregnant Mare Serum Gonadotropin (PMSG) superovu 1 ated beef cow was studied in one experiment in which estrous synchronization of 15 Angus cows was achieved with a dual injection of 33.5 mg prostaglandin (PGF2a) at a 12 day interval. Ovarian stimulation was achieved by injecting these cows with 2,000 i.u. PMSG 2k hr prior to the second PGF2a administration. Cows were artificially inseminated three times with Brahman semen at 0, 12, and 2k hr after detection of the PMSG-PGF2ai nduced estrus. Progesterone, 203-dihydroprogesterone luteinizing hormone and estrogen levels were monitored by RIA of plasma from cows bled twice daily for 21 days starting 2 days prior to PMSG administration. Ovulation rate, determined by supravaginal laparotomy 13 days after PMSG administration, indicated that 66Z (10/15) of the animals had more than one corpus luteum (CL) Cows with the longest interval from treatment to estrus displayed the lowest superovulatory response. This suggested a relationship between length X i

PAGE 12

of the interval from PMSG-PGFja to estrus and superovu 1 a t i on This possibility was supported by observed changes in the plasma endocrine patterns. When the endocrinology of the superovu 1 ated cow was studied by partitioning animals according to number of CL and progesterone levels, it was found that hormonal trends were similar with respect to postPMSG levels of progesterone, but estrogen concentrations were higher in superovulated cows (P<.Ol). Although no significant relationship was detected between plasma LH concentrations and ovulation rate, the surge of LH was 12 hr earlier in cows having more than four CL when compared to t reated,_ cows having only one CL. In an attempt to reduce fetal wastage from the superovu 1 atory response, cows having more than three CL, at laparotomy, were treated with kO mg PGF2a. Although there was a significant (P<.01) decline in plasma progesterone levels and an absence of palpable CL during the 7 days post-^0 mg PGF2a, cows continued to carry the fetuses throughout gestation. The question thus arose as to whether the fetus was rescueing the CL or the dose of PGF2a was indadequate for the increased CL mass. In an attempt to answer these questions, two experiments were conducted to test the efficacy of PGF2a to regress mu 1 1 i p 1 e CL in bred vs nonbred cows. in the second experiment estrous was synchronized in 13 Angus cows with a dual injection of 33-5 mg PGFpa at a 12 day interval. Ten cows received 2,000 i.u. PMSG 2k hr prior to the second PGFaa injection. Cows were not bred. Determination of ovulation rate and treatment was as previously described. Cows were bled daily for 11 days, starting 2 days prior to laparotomy and plasma was analyzed for progesterone

PAGE 13

concentration by RIA. Forty milligrams of PGF2a was effective in regressing multiple CL in these PMSG-treated nonpregnant cows based on a precipitous decline in plasma progesterone levels, the absence of palpable CL on day 7 post-^0 mg PGF2a and the resumption of recurring estrous cycles. In experiment 3, the inclusion of mating post-treatment resulted in a 2x2 factorial with PMSG and MATING as treatments for comparison. The accumulated data from this trial supported the resul'ts from the previous experiment. There were no observed differences between mated and nonmated animals in response to kO mg PGF2aIn concl us ion these series of experiments confirmed that the ovarian and endocrine response of the bovine female is affected by PMSG. -When PMSG is used in an attempt to attain multiple fetuses, the high abortion rate .assoc iated with the superovu 1 at ion effect might be minimized by regressing the multiple CL with PGF2a and rebreeding the cow in a short breeding season. This possibly could lead to a number of cows with multiple fetuses at birth which would increase the percent calf crop. X i i i

PAGE 14

5^. CHAPTER I INTRODUCTION In presertt-day beef production in the United States the trend^is towards a reduction in the life cycle by finishing cattle at an earlier age. Steady progress can be expected towards eliminating the long holding periods enabling a faster turnover of capital and a finished beef animal that will be more acceptable to both butcher and consumer. As age of s 1 aughter i s reduced by advances in breeding and feeding, fertility must assume an even greater importance in the economy of product ion. Unfortunately, the beef cow is one of the least efficient of all the meat animals, i.e., the animal is maintained year round to produce only one useful product, a calf. Failure of the cow to wean a calf leaves the maintenance costs to be borne by the productive members of the cow herd. On the other hand, one-cow-one-calf per year is often a tenuous economical position when applied to land which offers profitable alternative utilization. A primary means available for efficiently improving production in the beef cow is to wean a greater number of calves each year. With little or no increase in cow numbers and only a limited rise in production costs, a substantial increase in net income could possibly be achieved. One way in which this could be accomplished would be to increase the rate of twinning in the beef cow. 1

PAGE 15

Unfortunately, the heritability and incidence of natural twin births is low. Therefore, some alternative means for increasing the twinning rate needs to be developed, i.e., embryo transfer or hormonal ly induced controlled ovulation rate. Superovulat ion in beef cattle has been induced by various extracts of the'anterior pituitary; however, pregnant mare serum gonadotropin (PMSG) has the advantage of being more readily available. In addition, PMSG is not as readily destroyed by the body which allows the use of a single injection for ovarian stimulation. Pituitary extracts require a series of injections; however, in either case ovulation rate is h i gh 1 y; var i ab 1 e Almost without exception, researchers in the field of superovulatfon have reported high death losses among triplet and larger litters, with a lower mortality rate among twins. Therefore, in the case of the superovu 1 at ion effect from exogenous gonadotropins, it is proposed that Prostaglandin Fza (PGFaa) may be introduced into the hormonal regimen to induce premature regression of multiple corpora lutea (CL) resulting from an excessive ovulation rate in an attempt to reduce fetal wastage. Thus, it was the objective of this study to determine if PGF2ct can effectively regress multiple CL in the pregnant cow. it was important that the gonadotropin injections be timed rather precisely with reference to the occurrence of the previous estrus. Therefore, it was of interest to determine if synchronization of the estrous cycle could be accomplished with a series of two injections of PGF2ct and if this would minimize the necessity of determining the exact stage of the estrous cycle for giving the injections of PMSG.

PAGE 16

Since several reports indicate that ovulation, egg transport, embryo transport, and fertilization in the superovu 1 ated cow might be affected by an imbalance in the hormonal pattern, the effect of synchronizing es t rous wi th PGF2„ when used in conjunction with PMSG was evaluated through studying the levels and interrelationships of the reproductive hormones. ~~

PAGE 17

CHAPTER I I LITERATURE REVIEW Prostaglandins and Reproduction ^ Prostaglandins have been intimately linked with reproduction since their first extraction from human semen and sheep vesicular glands by von Euler in 193^. Studies in basic reproductive endocrinology with prostaglandins were stimulated by the report of Pharris and Wyngarden (1969) who demonstrated the luteolytic action of PGF2a in the rat. To datQ, prostaglandins have been shown to be involved in nearly all phases of the endocrine system regulating reproductive function. The following review attempts to highlight the effect of PGF2a in synchronizing estrus and as an abort i fac i en t agent in the bovine. Prostaglandin F2a and Estrous Synchronization The successful agent for synchronization of estrus should allow effective synchronization of estrus and normal fertility of the synchronized estrus. In addition, the method has to be practical enough to be used under range conditions. After the initial report of PGF2a induced luteolysis in pseudopregnant rats (Pharris and Wyngarden, 1969), others observed that treatment with a single injection of PGF2a (Rowson et al., 1972; Lauderdale, 1972; Inskeep, 1973; Oxender et^ aj_. 197^*; Rodriguez, 197'*; Roche, 197^; Fields e_^ aj_. 1975; Thatcher and Chenault, 1976) or synthet ic ana logues

PAGE 18

of PGF2a (Tervit et aj_. 1973; Cooper, \3lk; Fields etai-, 1977b) was effective in causing luteolysis in heifers and cows except for the first 5 days post-estrus (Rowson e£ aj_. 1972; Louis £t aj_. 1973). In cattle, a single injection of PGF2a given during the responsive stage of the estrous cycle is followed by an ovulation and normal fertility. There are, however, several factors associated with the use of a single injection of PGF2a which limits its practical usefulness for ovulation control. It is well documented that PGF2a is effective only after day 5 of the estrous cycle when a mature corpus luteum (CL) is present (Lauderdale, 1972; Cooper, 197^*). Administration of PGF2a to animals with a palpable CL resulted in (>5% of the treated animals displaying visual signs of estrus within 7 days pos ti n j ect i on (Lauderdale et^ a_l_. 197'*). Others have reported similar results following PGF2a treatment (Louis e_t aj_. 1973; Louis e^ aj_. 197Aa; Chenault e_t aj_. 1976) In an attempt to by-pass the unresponsive days 1 to 5 of the estrous cycle, animals can be injected twice with PGF2a at a '0 to 12 day interval, as suggested by Inskeep (1973). Cooper (197'*) reported that this dual injection technique, with an ICI analogue of PGF2a ('CI 80,996), resulted in only 2 of 175 animals failing to respond to the second PGF2a treatment. Furthermore, 90^ of the animals were in estrus between k% and 72 hr after the second treatment and fertility of this second estrus was normal. Using this technique, in a large field study with heifers and cows, Hafs (1975) found 68% of the heifers and 62? of the cows were in estrus hi to 8^* hr after the second PGF2a injection. Several researchers have shown this synchronized estrus to be fertile (inskeep, 1973; Lauderda 1 e e^ all_. 197^*; Roche, 197^*;

PAGE 19

6 Rodriguez, IS?'*; Cooper and Jackson 1 975 ; Hafs e^ aj_. 1975b; Turman et a1., 1975; Ellicott and Thompson, 1976; Fields et^ aj_. 1977b; Moody and Lauderdale (1977)Luteolytic Effect of Prostaglandin Fzg The sequence of changes in the reproductive tract and plasma Jevels of gonadotropic and ovarian hormones that occur during synchronization of estrus with PGF2ct are similar to those occurring around natural estrus. The induction of estrus is rapid and precise and the induced CL has a normal life-span. Louis et al. (1972a) injected 5 mg PGF2a-Tham Salt into the uterine horn ipsi lateral to the CL and reported the interval to estrus was 72 hr, to LH peak 71 hr, and to ovulation 96 hr. An intramuscular injection of 30 mg PGF2a during diestrus resulted in a (yO% decrease in plasma progesterone within 12 hr with the initial progesterone concentration of k ng/ml declining to .8 ng/ml by ih hr (Louis aj^, 1972a, 1973). The interval to onset of estrus, LH peak and ovulation was 7^, 77, and lOA hr post-PGF2a treatment, respectively. Similar results in declining plasma progesterone were reported by Oxender aj_(^97'*) even when PGF2a was given intramuscularly in varying numbers of injections. The spaced double injection regimen produced precise synchronization of estrus in all the heifers responding to treatment (Dobson et al., 1975). After both injections, the CL showed rapid morphological regression that was similar to that observed for a single injection and was associated with a significant fall in plasma progesterone concentration 6 hr post-treatment with basal values being reached within 2^4 hr. Rapid follicular growth and secretion of estradiol

PAGE 20

7 ensued with a return to estrus ^48 to 96 hr after the first, and h8 to 55 hr after the second PGF2a treatment. A preovulatory surge of LH occurred 62 to 103 hr after the first, and k8 to 62 hr after the second PGF2a treatment, and was followed by ovulation. Similar changes have been reported by Stellflug al_. (1973), Louis et aj_. (1973), and Louis aj_ (197^*3, b). Chenault et aj^. (1976) reported thvat a single injection of PGF2a produced a rapid decline in plasma progestin to estrus concentrations by 2i hr post-treatment, whereas, estradiol concentrations slowly increased and apparently stimulated an ovulatory surge of LH at 72 21 hr post-treatment. 'Ovulation occurred at 99-5 19 hr after PGF2a administration. Gimenez et^ aj_. (1976) reported plasma progesterone in the uterine vein decreased from 1,000 ng/ml to 2 ng/ml within 5 days after PGFo„ intrauterine treatment and 2h hr after intramuscular injection. In the same study, plasma estrogens concentrations ranged from 25 to 100 pg/ml prior to treatment with no observed change in estrogens levels occurring after giving PGF2a when compared to the salinetreated cows. The mechanism by which PGF2c( initiates CL regression is unknown. Novy and Cook (1973) and Thornburn and Hales (1972) demonstrated that PGF2a may redistribute intraovarian blood flow, reducing the amount of blood flowing to the CL and increasing that to the stroma and follicular component of the ovary. Morphologically, this will result in both a functional termination in progesterone secretion and a structural regression or physical destruction of the luteal cell. Because structural regression was preceded by accumulation of lipid droplets, Stacy et aj[. (1976) postulated functional CL regression

PAGE 21

8 was most likely due to a blockage in one or more stages in steroidogenesis. Finally, Henderson and McNatty (1975) presented a biochemical hypothesis by which PGFpa may initiate CL regression through a direct or indirect action at the adenylate cyclase catalytic site at the cellular membrane level. Prostaglandin ^^id Therapeutic Abortion An effective means of inducing parturition in the cow could reduce calving losses and labor costs by decreasing the calving period to a shorter and predictable period. Parturition can be induced in the cow by treating with estrogens (Spears aj_. 197^), corticoids (Jbchle, 1973), and PGFja (Lauderdale, 1972). In addition, PGF2a can be used to terminate unwanted pregnancies. The abort ifacient property of PGF2a was initially demonstrated in laboratory species when pregnancy was terminated in 100^ of rats given three daily injections of PGF2a (Gutknecht ei_ a]_. 1969)In humans, PGF2ct has been used, with varying degrees of success, to terminate pregnancy at different stages of gestation (Henricks, 1972). In farm animals, vascular infusions or systemic injections of PGF2a have been reported to terminate pregnancy in porcine (Diehl and Day, 1973), caprine (Currie and Thorburn, 1973), equine (Douglas £t aj_. 197^), and bovine (Lauderdale, 1972, 197^*; F i e 1 ds e^ a_l_. 1977a) species. Administration of PGF2a either intramuscularly or systemically at early stages of gestation in the bovine resulted in a dramatic decline in plasma progesterone within 2k hr and behavioral estrus 2 to 16 days post-treatment (Zerobin et a1., 1973; Douglas et al., 197^).

PAGE 22

9 Louis et_a]_. (l97'+a) injected 5 mg PGFga into the uterine horn ipsilateral for the CL In cows 11 days postmating and reported plasma progesterone at 0, 2k, and hr declined from 3-6 0.3 to 1.7 0.2 ng/ ml and then to 1.0 0.1 ng/ml respectively. Plasma estradiol concentrations increased from 5-0 1.0 pg/ml to 6.1 O.i* pg/ml to 11.3 0.7 pg/ml, and 12.7 1-3 pg/ml at 0, 12, 2k, and 48 hr post-PGFza"treatment. The LH peak was detected at. 71 k post-treatment followed by estrus at 72 5 hr and ovulation at 95 5 hr post-treatment. Although hormonal patterns resulting from PGF2a induced CL regression of pregnant vs_ nonpregnant cows were similar, the expression of estrus and ovulation appear to be dependent on stage of gestation. Douglas (1974) reported cows at BO to 90 days of pregnancy to be in estrus 2 days following abortion and ovulating 6.5 days postabortion. In contrast, cows at l60 to iBO days of gestation showed estrus and ovulated 15.9 and 29-3 days post-abortion. In addition the late pregnant cow had a high incidence of retained placenta. Similar observations have been made by Henricks ej^ a_l_. (1977). With the use of c 1 op ros tenol a PGF2a analog, for the termination of pregnancy, Jackson and Cooper (1977) reported that cows in the first, second, and third trimester of gestation aborted with 58^ abortions in less than 7 days, 25'-^ between 7 and 14 days, and 13^ in more than 14 days post-treatment, even though the composite plasma progesterone concentration declined to basal levels of 1.5 i 0.6 ng/ml within 48 hr post-cl oprostenol No retained placentas were observed. Fields et a 1 (1977a) reported 250 to 500 pg cloprostenol aborted 100 to 107 heifers between 60 and 120 days of pregnancy, with no complications.

PAGE 23

10 Prostaglandin F2a and Superovu ] at i on Prostaglandin F2a has been used in conjunction with Pregnant Mare Serum Gonadotropin (PMSG) for the induction of superovu lat ion in beef cattle. Cupps et_ al. (1976) reported that' the administration of PGF2a 2h hr post-PMSG, to previously synchronized heifers, shortened the interval and reduced the variability to onset of estrus and increased the number of animals responding behavioral ly (Cupps e^.al., 1976). Dobson e_^ aj_. (1975) suggested that the second PGFpa" nduced luteolysis may allow the wave of growing follicles that originated after the first PGF20(i nduced estrus to progress and ovulate rather than become atretic. Rajakoski (i960) reported such a wave of follicular growth approximately h days after estrus culminating in a single follicle growing to day 10 or 12 of the cycle. He suggested the presence of the CL and thus the absence of an ovulatory surge of LH resulted in the follicle becoming atretic. This might account for the observation that the induction of estrus was more rapid and precise after the second PGF2a administration. In line with this concept, Tervit e_t^ a_l_. (1973) reported that a prostaglanding F2a analog (ICI 79,939) given to cattle with large PMSGinduced follicles demonstrated a shorter interval to estrus. Archbald (1976) reported excellent synchronization of estrus when a second injection of PGF2a was given ^8 hr post-PMSG with 67^ of the cows in estrus within k8 to 72 hr. Menino and Wright (1977) reported the administration of 2,000 i.u. PMSG 2k hr prior to the hormonal i nduced-estrus resulted in 8]t of the cows responding behavioral ly within k8 to 132 hr following treatment.

PAGE 24

n Go nadotropic Hormones and Limited Multiple Births Among the several methods available to increase ovulation rate in the bovine, the most promi s i ng are hormonal treatments with Pregnant Mare Serum Gonadotropin (PMSG) and pituitary Follicle Stimulating Hormone (FSH). Different gonadotropins, alone or in combination, have been used by many researchers with consistent results. Casida a_l_. (19^3) and Dowling (19^9), in early trials, reported successful, induction of multiple ovulations in mature animals from injecting FSH and PMSG during the follicular phase of the est'rous cycle. Schilling and Holm (1963) injected 1,000 to 1,500 i.u. PMSG to 11 cows on day 5 following estrus.On day 16 to 18, the CL was enucleated, followed by an injection' of 2,000 i.u. PMSG. In an attempt to synchronize ovulation, an intravenous dose of k ,000 i.u. of LH was given at estrus. In these trials, more than 701 of the cows ovulated the desired number of 2 to 3 eggs. Turman e_t aj_. (1969, 1971) used this same hormonal protocol and obtained a 109% weaned calf crop, from treated animals. .Kidder et al. (1952) and Dawson (1961) reported cows with double ovulations gave birth to a very low number of twins. Gordon et al (1962) in an extensive study of induced multiple births, used various levels of PMSG in one injection on day I6 or 17 of the estrous cycle. Six weeks following artificial insemination of the ^416 treated cows, 76Z were pregnant. In one trial, 33% of the cattle treated with 1,600 i.u. of PMSG carried multiple fetuses while 32 of 67 double ovulating cows for all treatments possessed twins. There was further noted an increase in fetal survival, from 29 to 62%, when the eggs were shed by both ovaries rather than one. Cows with three ovulations had a greater ability to sustain twins in a single horn {h(>%) than those

PAGE 25

\2 with double ovulations from a single ovary {13%). The precise relationship between the number of ovulations per ovary and maintenance of pregnancy to term remains to be established. The minimal intrauterine migration (1:200) in the bovine (Perkins aJL, 195^; Gordon et_ al, 1962; Rowson et al_., 1971; Scanlon, 1972a). does pose some serious problems to limited multiple births through the use of exogenous administration of gonadotropin hormones as an avenue to increase calf crop. The mechanism of intrauterine migration in the bovine remains undetermined. Bellows e^a]_. (1969) reported 6.25 mg of FSH injected twice daily for 5 days, in heifers synchronized by feeding 1 80 mg medroxyprogesterone acetate (MAP) daily for 9 to 1 1 days and injected with 5 mg estradiol valerate on day 2, resulted in a controlled ovulation rate. When the FSH injections were begun on day 8 of MAP treatment, it resulted in 8 cows with 17 ovulations ( Bel lows et a^. 1970). When this treatment was combined with breeding by natural service, twins were produced by 5 of 43 heifers. Vincent and Mills (1972) in a similar study reported 6.3 to 12.5 mg FSH, given with norethandrolone injections for preventing simultaneous estrus, resulted in kSl of all treated cows with multiple ovulations. Only 5 of cows were estimated to have more than three CL. There were, however, no significant differences in ovulation or pregnancy rates between levels of FSH. Furthermore, calving rates for cows responding to PMSG treatment were similar (121?) to those reported by Bellows et aj_. (I969), but considerably lower than the calving rate of 173? with PMSG treatment reported by Turman e^ aj_. (1971). Reynolds et_ aj_. (1970), Vincent and Mills (1972), and Smith et_ a\_. (1973) attempted to prolong the action of FSH by using a \% sodium carboxymethyl cellulose and pol yv i ny 1 p i r rol i done It was concluded that

PAGE 26

13 these diluents, used for one or two injections of FSH, were not as satisfactory as a series of injections. According to Laster (1972, 1973) and Smith e^ a]_. (1973), the discrepancy observed in ovarian response and conception to FSH treatment was due to the actual biological potency of the gonadotropin. Similarly, Schwartz and Shelby (1969), Laster et aj_. (1971a, b), an'd Scanlon (1972b) reported considerable differences in mean ovulation rates with similar treatment regimens of PMSG. A differentia] response in ovulation rate, doses of PMSG and/or FSH, and onset of the CAP synchronized estrus due to breed has been reported by Lamond (1972). The infusion of FSH for 72 hr (Laster, 1972) did not decrease the variability in ovarian response that was achieved with twice daily injections for 5 days (Bellows e^ a_l_. 1969)In contrast, when FSH was injected for 3 days, more cows ovulated from one FSH injection per day (5.16 '4.78) than two injections per day (l.OO O.OO) (Staigmiller et al., 1976). However, for 5 days of injections, fewer cows superovulated from one FSH injection per day (l.OO O.OO) than from two (1.37 0.79). Lamond (1972) and Laster (1973) reported that PMSG treatment resulted in a more desirable and less variable ovulation rate than treatment with FSH. It was of interest that abortions occurred in 20^ of the animals treated with PMSG or FSH at 8I to lOA days after insemination. Similar observations of fetal wastage have been made by M. J. Fields, A. C. Warnick, and J. H. Hentges (unpublished data). Godke e_t a_I_. ( 1977) reported cows treated with I ,6000 i.u. PMSG or 1-5 mg/day FSH-P resulted in pregnancy rates, cows pregnant with multiple CL and number of cows returning to estrus after 100 days gestation of

PAGE 27

7].k% and ]00% and 88.81, 70.0% and SZ.51, respectively. At 220 days of gestation, there were no differences in pregnancy between cows treated with PMSG (l6.6^) or FSH (17.7^). Hill et al. (1973, 1976) reported that PMSG, given concomitantly with PGF2a, resulted in a lower and less variable ovulation rate (4.33 3.60) than when PGF2a was injected at 2k hr post-PMSG (8.03 3 k^) A high incidence of split estrus (55%), wj th a large number of unovulated follicles was observed when PMSG was given concomitantly with PGF2a as compared to an 8% incidence of split estrus when a 2h hr interval between hormonal treatments was allowed. These data suggest that the follicles had not had sufficient time to mature before estrous control was attempted with PGFj^. Absence of split estrus has been reported by Rajamahendran et ajl_. (1976) and Lopez-Barbel 1 a e£ aj_. (1976) following a single injection of 2,000 i.u. PMSG 2k hr prior to a second injection of synchronizing PCFjaEndocrinology of the Superovul ated Cow Hallford et^ aj_. (I975a) using two PMSG injections, on days 5 and 17 of the estrous cycle, reported a significant (P<.005) increase in midluteal plasma progesterone levels over the nontreated controls or animals receiving a single injection of PMSG on day 17. Plasma LH levels, however, were similar in PMSG treated groups, and generally below one ng/ml (Hallford, 1975b). These two treatments were not compared against the non-PMSG treated cow. • According to Ford and Stormshak (1975) daily serum levels of LH were elevated (P<.01) in heifers treated with PMSG vs^ nontreated heifers, in heifers treated with PMSG, GnRH injections produced lower plasma LH

PAGE 28

levels when compared to non-PMSG treated heifers. Apparently, the differential LH response observed in the PMSG treated group could be due to a positive feedback of estradiol at the hypotha 1 ami c-p i tu i ta ry level. Although Hallford a_1_. (1975b) reported a positive correlation between the number of CL and plasma estradiol levels (r = O.Sh) on day 19 and increased plasma estradiol in PMSG-treated heifers, plasma LH levels did not differ between the PMSG and non-PMSG treated heifers ( vide supra ) Hill a]_. (1972) and D i ckey e_t^ ajl_. (1973) reported plasma estradiol levels in PMSG superovu 1 ated heifers to be much higher prior to mating than in controls. These values, however, were for superovu 1 ated rather than cows of limited ovulations. An increased ovulation rate with 2,000 i.u. PMSG resulted in an ovulatory surge of LH at 8^* 48 hr post-PMSG with a mean basal plasma LH level ranging from 0.6 to 2.5 ng/ml (Lopez-Barbe 1 1 a e_t aj_. 1976). A synchronizing injection of PGF2a 24 hr post-2,000 i.u. PMSG resulted in a decrease in plasma progesterone levels from 7.9 0.45 to 0.94 0.17 ng/ml within 72 hr (Fournier e^ aj_. 1976). The stereotypic response to PMSG was clearly demonstrated by Rajamahendran e_t^ a_l_. (1976) who reported ovulation rates from 1 to 17 in heifers treated with 2,000 i.u. PMSG followed 48 hr later by 15 mg PGF2ct. Two heifers, each with 17 CL, had peak progesterone levels of 38.4 and 27.8 ng/ml which were still high (9-6 and 26.5 ng/ml) by day 21. Progesterone levels of three heifers with 4-9 CL did not differ (P<.05) from those of three heifers with single CL. Six additional heifers had low progesterone levels (<1 ng/ml) on days 8-14 post-PMSG suggesting premature CL regression or lack of ovulation.

PAGE 29

16 20B-D ihydroprogesterone (A-Preqnen-20p-ol-3-one) An interesting steroid, concerned with bovine steroidogenesis, that, to date, has been considered the major metabolite of progesterone (P) is 20B-dihydroprogesterone (206-P) (Sp i 1 man et_ a_l_. 1973). Histologically, CL development has been described as a progressive arrangement and growth of the luteinizing granulosa cells in the cavity left at the ovulatory site (Harrison, 19^*6). The 14 dayCL has reached its maximum size filling the collapsed follicular cavity with an extensive vacuolation of luteal cells. S'tero i dogen ica 1 1 y this structural change is accompanied by a differential secretion of progesterone-derived hormones according to Short (1962a, b) in his "two cell theory." From the two cell theory of Short (1962a, b), the ovarian theca interna was proposed to covert P to estrogen in the absence of a 20-reductase system, i.e., 206-hydroxysteroid dehydrogenase (20B-HSD). La Croix e_^ a_l_. (197^) have since shown that bovine theca cells synthesize androgens which serve as precursors for the granulosa cell to convert to estrogens. The granulosa cell undergoing 1 u te i n i zat ion was proposed to contain the 203-reductase system and thus 20B-P secretion by the growing follicles was an indicator of I ute i n i zat i on This was indirectly confirmed by other when P and 20g-P were isolated from luteal tissue (Savard and Teledgy, I965). Lobel and Levy (I968), however, did find that 20g-HSD activity resided in both the granulosa and theca cell layers of the rat f o I 1 i c 1 e Hayano e_^ aj_. (1975) first demonstrated the conversion of P to 2O3P by the bovine CL. Noticeable quantities of ovarian 2O3-P levels in cycling cows at levels approximately 10 to 20% of that of P have been reported (Gorski e^t al., 1958a, b; Erb and Stormshak, I96I, Hafs and

PAGE 30

17 Armstrong, 1968; Garver i ck aj_. 1971)Brandau aj_. (1972) and Brandau and Mutzke (1972) using homogenized bovine ovaries found that 2O3-HSD increased slowly to a maximum on the day 15 of the cycle. Levels of 20g-P in the bovine are highest when the CL reaches maturity, with a delayed decrease in 2O3-P concentration following the P decline of the regressing CL (Erb and Stormshak, I96I; Staples and Hansel, -I96I Mares et aj_. 19^2; Gomez et^ aj_. 1963; Gomez and Erb, I965). Erb et_ al. (1968) reported that 2O3-P increases when synthesis or release of luteal P decreases late in the estrous cycle. Sasser and Cupps (I969) incubated CL recovered at various times during the bovine estrous cycle and found maximal P synthesis on days 10 to 12. These workers postulated that, prior to estrus, luteal regression could result in increased lysosomal activity which would result in reduced cellular pH and create optimal conditions for 2O3-P formation. When a similar study was conducted with porcine CL, only 0.6^ of the P was converted to 20B-P (Weiss e^ a_1_. 1976). In laboratory animals, in which the a-epimer (20a-P) of 206-P is the more predominant hydroxylated ovarian steroid, Barraclough e_t a 1 (1971) noted an increase in ovarian vein levels of 20a-P approximately 2.5 hr prior to the LH discharge in cycling female rats. This suggested the possibility that 20a-P has a positive feedback on the pituitary for discharge of LH. Ichikawa e_t^ aj^(1971) found an increased secretion of both P and 20a-P in ovarian vein plasma after injection of LH into early proestrus rats. It is possible that sustained synthesis and release of 20a-P is caused by LH since LH will increase 20a-HSD activity (Kidwell et al., I966).

PAGE 31

18 Telegdy and Savard (I966) found the rabbit ovary to produce both 20a-P and 206-P, with 20a-P being the more predominant hydroxylated steroid. Hilliard aj_. (I967) in an elegant study showed, in the rabbit, that coitus triggers a transient release of LH sufficient to activate the synthesis and release of 20a-P which subsequently prolongs and heightens "the LH discharge at a level and duration necessary for ovulation. Goodman and Neill (1976), however, were not able to confirm this role of 20a-P in a similar study. The role of gonadotropins in bovine ovarian steroidogenesis has also been investigated. Romanoff (I966) perfused both the luteal and contralateral follicular ovaries from the same cow, each ovary serving as a control to the other. Perfusion of Follicle Stimulating Hormone (FSH) with acetate-1 -^'*C increased ^'^CP synthesis 3-3 fold and increased the P to 206-P ratio from 7-2 to 11.^ for the luteal ovary. Perfusion of FSH was not tested in the follicular ovaries. Perfusion of LH into the luteal ovary resulted in increased synthesis of both P and 2O3-P but did not affect the ratio between the two steroids. The perfusion of LH into follicular ovaries increased synthesis of P seven-fold with a 3-6 fold increase in 20B-P. Not unexpectedly, there was significantly less synthesis of P and 206-P in follicular than luteal ovaries. Prolactin perfusion in this study, had no effect on the 20-HSD system. Snook e_t^ a_l_. (I969) working with hysterectomized heifers with sustained luteal function, however, showed that LH has a preferential effect on 20-HSD. They used LH-antisera to neutralize endogenous LH. This LH-antisera reportedly decreased total ovarian progestin concentration primarily due to a significant reduction in 206-P while P concen, trations remained unaltered. In the intact heifer, Spilman e^t al. (1973)

PAGE 32

reported increased plasma 206-P levels after either PMSG or HCG treatment. Kidwell et aj_. (1966) working with PMSG superovu 1 ated rats, found a three-fold increase in the 20-HSD activity 5 days post-treatment. When these primed rats were administered LH, the 20-HSD activity increased from 0.03 0.01 to 2.82 + l.lh MU/mg 5 days after LH injeC-tion. The administration of HCG, however, resulted in a ten-fold increase in the 20-HSD activity. A significant increase in the glucose6-phosphate dehydrogenase activity was also observed after LH and HCG administration. The possibility that 206-P in the bovine might be playing a similar role as 20B-P in other species is not apparent. The systemic patterns are different for P and 203-P which are inversely related while in the case of 206-P it follows a similar but slightly delayed pattern as P. Not only has 206-P been isolated from bovine ovarian vein plasma and tissue, but has been shown to be of ovarian origin in humans (Mikhail e_^ a|^. 1963), Chinchilla (Tam, 1971) and the African elephant (Smith et aj_-. 1969). Staples and Hansel (I96I) presented data to suggest that embryo survival at day 15 may be influenced by circulating 206-P levels. Gomez et^ a_l_. (1962) quantitated P and 206-P levels in the utero-ova r i an vein ipsi lateral to the CL in cows between 250 and 282 days of gestation. Levels of 203-P remained near nondetectable levels until parturition, at which time there was a sharp rise which accompanied the decrease in P levels. At parturition, the P:206-P ratio was approximately one. Levels of 203-P in the cycling post-partum cow have been shown to be 10 to \5% of that of P (Tribble, 1973; Castenson et al., 1976).

PAGE 33

20 To date, reports on 206-P are limited to piasma levels during the luteal phase of the cow's estrous cycle. Little or no information has been reported on levels of this hormone at or near estrus. In this study, plasma 20B-P will be characterized in PMSG superovu 1 ated cows over a 21 day bleeding period.

PAGE 34

CHAPTER I I I MATERIAL AND METHODS EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P) "T 203-DIHYDROPROGESTERONE (206-P) TOTAL ESTROGENS (E) AND LUTEINIZING HORMONE (LH)IN PMSG SUPEROVULATED COWS SYNCHRONIZED WITH PGF2aTHAM SALT The objectives of this experiment were (l) to characterize plasma P, 20B-P, E (combined Ei and E2) and LH concentrations in the PMSG superovulated cow and (2) to determine if 40 mg PGF2a-Tham Salt was effective in regressing multiple corpora lutea (CL) Fifteen exhibiting normal estrous cycles parous Angus cows were examined per rectum to verify that the reproductive tracts were normal and that a CL was present. All animals were injected twice intramuscularly with 33.5 mg PGF2a-Tham Salt (Upjohn) at a 12 day interval. Cows were administered 2,000 i.u. PMSG (Organon) subcu taneous 1 y 2A hr prior to the second PGF^a injection. Animals were observed twice daily for signs of estrous behavior throughout the experiment. Artificial ininsemination (AI) three times with Brahman semen was at 0, 12, and 2h hr after detection of the PMSG-PGF2ctinduced estrus. Approximately 10 days following Al cows were exposed to an Angus bull for a 90 day breeding per iod (f igure 1 ) A kO ml blood sample was collected via jugular vein venipuncture at 12 hr intervals from each animal for 21 days starting h8 hr prior to time of injection of PMSG. Blood samples were transported to the 21

PAGE 35

22 ? -J < r: p Ia o < > r~ <^ ^ o e 1CL o (J < 5: n. < o -J ju A O u o >Q LPl CM < (0 N u V u 1_ (U c L. O o C ) o> c o > c IV a: < H CNJ X LU < --I 2: LPl LL Q. 3 •r-l C Q. CSi < Q < Q >< O < Q O \Q LU LU _l CD c 0) e 1(U CL X
PAGE 36

23 laboratory in an ice bath and immodiately centrifuged at 2,000 g for 20min at ^C. Plasma was stored at -20C until analyzed for plasma P, 203-P, E, and LH concentrations. Ovulation rate was determined by supravaginal laparotomy 13 days post-PMSG. In an attempt to reduce fetal wastage from the superovulation effect, cows with more than three CL were injected with hO mg'~PGF2ct to induce CL regression. Surgical Description of Supravaginal Laparotomy Prior to surgery animals were taken off feed overnight. Animals were restrained and, t ranqu i 1 i zed with 2 ml Acepromizine (Ayerst) an incision made in the upper anterior vagina, the ovaries were exteriorized through this incision, and the CL were counted. At time of surgery and 2k hr post-surgery cows were treated with two million i.u. penicillins (Combiotic, Pfizer). EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN PMSG SUPEROVULATED BEEF CATTLE FOLLOWING ADMINISTRATION OF 40 MG PGFja-THAM SALT The objective of this trial was to determine in the nonbred beef cow if kO mg PGF2c( was effective in regressing multiple CL in response to PMSG treatment. Thirteen parous Angus cows exhibiting normal estrous cycles were treated with two sequential injections of 33.5 mg PGF2^-Tham Salt to ach ieve synchron i zat ion of estrus (figure 2). A 2,000 i.u. PMSG injection was administered subcutaneous 1 y to 10 cows 2k hr prior to the second PGF2ct injection. Animals were observed twice daily for signs of estrous behavior for 32 days starting the day of first PGF injection.

PAGE 37

2k 25^ — c ^ < in ll O CD O LD O O 2^ Q. < Q CN < G CN >< >< Q >< Q >< Q IS (J o LU LU _J >_j 03 a. 4-> *j c 3 E E VU1 0) a (1) X L. (U (1) L. o O o a o Cvl o Ll. *-> CD o Q_ 1a. MO c l/l (U l/l E !U t-j C tJ 0) 0) > u 14.) CN 0) v_ D C71 Ll.

PAGE 38

25 Ovulation rate was determined by supravaginal laparotomy 13 days post-PMSfj and, on the same day, all cows were treated with kO mg PGF2a. A 10 ml blood sample was collected via jugular vein venipuncture daily for 10 days starting 2 days prior to laparotomy ( v ide supra ) EXPERIMEf^T 3. LUTEOLYTIC EFFECT OF PGF2a-THAM SALT IN "~ BRED \/S_ CYCLING BEEF COWS PREVIOUSLY TREATED WITH PMSG. This experiment was conducted to determine the efficacy of PGF2a in regressing multiple CL in bred vs^ cycling cows treated with PMSG. Forty-five parous Angus cows exhibiting normal estrous cycles were treated with a dual injection of 33-5 mg PGF2a-Tham Salt at a 12 day interval to achieve synchronization of estrus and randomly assigned to a 2x2 factorial with treatments of 2,000 i.u. PMSG and breeding (figure 3). Treatments were (1) 11 cows injected with 2,000 i.u. PMSG 2k hr prior to the second PGF2a injection and immediately exposed to natural breeding for 10 days; (2) 11 cows injected with 2,000 i.u. PMSG 2k hr prior to the second PGF2a injection and not mated; (3) 12 cows exposed to natural breeding for 10 days starting the day of the second PGF2a treatment; and (k) 11 cows were treated with PGF2a but received no PMSG and were not mated; thus, they served as a double control. Bleeding and laparotomy were as described in experiment 2. Cows were checked for estrous behavior twice daily for 21 days prior to the beginning of the experiment, three times daily during the experimental period and twice daily for 70 days following laparotomy and injection of kO mg PGF2aCows were checked for pregnancy 70 days post1 aparotomy

PAGE 39

26 >o ^ in U• O Qt — D • f-l t—l C! o LO LO o Q_ o O CM DC CD LL • (J CL >< < >< Q Q lU tu _l CQ >< ^ Q LU 1-1 00 (/I 2: Q >< Q in tn V 1. cn 4) u CM a. o o 10)
PAGE 40

27 Radioimmunoassay of Steroid Hormones Cle aning of Glassware Excluding RIA disposable culture tubes (10 x 75 mm, Corning), all glassware was rinsed with ethanol boiled in a soapy-distilled water for 1 min, and' rinsed withwarmtap water (3X) followed by deionized^ water (3X). Prior to use, the glassware was rinsed with ethanol, siliconized with 1'-?; silicone solution (Siliclad, Clay Adams) for 20 sec and oven dried for 2k hr. • Organic Solvents Analytical grades of benzene, ethanol, methanol, hexane, and ethyl acetate were distilled over boiling chips prior to use. Hexane was washed with sulfuric acid, distilled water, and dried over calcium chloride prior to distillation. Ether was used immediately following distillation over metallic sodium to remove peroxides and water. Prepar ation, Use, and Stora g e o f Ra dioactive Steroids Tritiated steroids. 1 ,2-H^-Progesterone (3p) (55-7 Ci/mM) and 2, ^,6,7-3H-Estradiol (^£2) (98.5 Ci/mM) were purchased from New England Nuclear Corporation. Upon receipt of tritiated steroids they were further purified on a 25 cm LH-20 column to eliminate undesired labelled impurities that could result in decreased accuracy and sensitivity of the assay. The center profile of the eluted tritiated steroid was pooled and stored in benzenerethanol (9:1) at ^C. For assay purposes, a known amount of tritiated steroid was transferred to a 250 ml flask and dried under nitrogen gas (N„) at 37C. Gelatinized phosphate assay buffer (see

PAGE 41

28 table 10, Appendix) was added to give a concentration of 200,000 dpm/ml. This mixture was vortexed for 5 min and equilibrated for 1 hr at room temperature. A volume 100 yl (20,000 + 1,000 dpm) was used in the assay. This solution was stored at hC and used for no longer than 1 wk. P and E tritiated steroids were further diluted with benzene to 2,000 dpm/100 yl and stored' at hC for determining recovery of extracted steroi.dsT Since tritiated 206-d i hyd roprogesterone was not commercially available it was synthesized from as described by Tribble (1973)Conversion of Progesterone (^P)to 20g-D i hyd roproges terone (^H-20B-P) Forty thousand dpm of ^P was dried under N2 in a siliconized conical centrifuge tube and subsequently dissolved in a drop of ethanol To this dissolved was added 0.5 ml of 0.15 M phosphate buffer [pH = 5.2, which contained 100 mg percent EDTA], 30 yl of NADH [(Sigma Chemical Company, Grade III) in 0.1 M Tris buffer (Eastman), pH = 8.1] (see table 11, Appendix) and 30 yl of 206-hydroxysteroid dehydrogenase enzyme [(Calbiochem, Activity '4.95 i.u./ml at 30C) in 0.005 M Tris buffer (pH = 8.2) and vortex mixed. The reaction was incubated for 3 hr in a water bath at 37C. The reaction was stopped with the addition of 1 m) distilled water. The 3H-206-P was extracted with 1 ml ethyl acetate (3X) quantitatively transferred to a 1 3 x 100 mm culture tube and dried under N2 at 37C. The dried residue was quantitatively transferred to a LH-20 column ( vide infra ) and the 3H-203-P fraction was collected into a 13 X 100 mm culture tube. Elutions containing ^P were collected directly into scintillation vials and counted. From this fraction an estimate of the percent conversion of 3p to 3H-203-P was subsequently determined. Fractions containing ^H-ZOB-P were stored in benzene:

PAGE 42

29 ethanol (9:1) at kC A typical profile of conversion to ^H-ZOB-P anjd its LH-20 chromatography separation is illustrated in figure h. The above procedure for synthesis of 3h-20|3-P was adopted after a series of reactions to determine opt ima 1 • cond i t ions for conversion were attempted. The efficiency of conversion as well as levels of^ cofactors tested are illustrated in table 1. Use and Storage of Antibody The P and E antisera used in this study were kindly supplied by Dr. Lee Fleeger, Texas A&M University (PR Iflh and PR §28^), and Dr. V. L. Estergreen, Washington State University, respectively. The lyophil-ized P antiserum was dissolved in ^400 pi distilled H2O to form a 1:1 stock solution. Twenty microliter fractions of this 1:1 stock solution were stored in separate test tubes at -hOC eliminating breakdown due to repeated freezing and thawing. A 1:7,000 dilution of the P antiserum resulted in hO to 50'^ binding, while for estrogens, a 1:30,000 dilution of the antiserum resulted in 65 to 70% binding, using ^H-estradiol as a tracer, when a total incubating volume of 1.2 ml and 20,000 dpm were used in the assay. Serum Extraction and Chromatography For progesterone extraction 1 ml aliquots of plasma were pipetted into 20 X 150 mm test tubes. To each tube 100 Ml ^P, 500 Ml of 0.05 M ^The addition of normal rabbit serum (1:^00) improved the sensitivity of this antiserum.

PAGE 43

30 CO. un o •• CM UA I — c o 0 J3 OJ Q. C -~(C X — (U — cn O i_ Q. o a o o -o CN > X — _J T3 0) 1. 3 cn Q_ O

PAGE 44

TABLE 1. EFFICIENCY OF 3H-P CONVERSION TO 3H-206-PAT VARYING LEVELS OF COFACTORS Tritiated Efficiency of P progesterone NADH^ 20B-HSD^ conversion to ''h-20Bp (dpm) n (ml) (ml) {%) ijO.OOO w 0 03 0 03 9^*. 35 80,000 10 0 06 0 06 /2 00 120; 000 10 0 09 0 09 69. 12 160,000 10 0 12 0 12 37. 78 160,000 10 0 09 0 09 43 09 160,000 10 0 12 0 09 51 12 160,000 10 0 09 0 12 50 62 ^5 mg 6-NADH/3 ml of 0.1 M Tris buffer. ^0.2 ml 203-HSD/0.8 ml of 0.005 M Tris buffer.

PAGE 45

32 NaOH, and 15 ml o1' hexane was added and vortexed 1 min. The lower aqua ous phase was quickly frozen in liquid nitrogen while the upper lipidcontaining organic phase was decanted into a 20 x 150 mm test tube and evaporated to dryness under N2 at 37C. The extract was resol ub i 1 i zed in 500 pi hexane : benzene :methanol (80:15:5), vortexed for 30 sec, and applied to a 7 cm LH-20 column. The progesterone fraction was elut^d into a 13 X 100 mm tube, evaporated to dryness under N2 at 37C. To this dried progesterone 2 ml of gelatinized assay buffer was added, vortexed for 5 min and 500 yl transferred to a scintillation vial to estimate recovery. The remainder was used for assay. Quantification of and 3H-20g-P from the same plasma sample was accomplished using an ether rather than hexane extraction in the manner descr i bed above. The dried ether extract was subjected to chromatography on a 5 x .5 cm LH-20 column. These columns were disposable 5 ml glass pipettes (Corning) with 3 mm of glass wool packed in the bottom of the pipette. Then Sephadex LH-20 was allowed to swell overnight in hexane : benzene : methanol (80:15:5) and subsequently packed into each column to a height of 7 cm. The packed columnswere washed with 10 ml hexane : benzene : methanol (80:15:5) and the elution profile characterized with appropriate t r i t i urn1 abe 1 led steroids. The dried residue from the ether extract was quantitatively transferred to the column with two 500 yl aliquots of hexane : benzene :methano 1 (80:15:5). Progesterone was eluted from the column using 5 ml of the above organic solvent while 203-dihyd progesterone was eluted 2 ml after ^P. The ^P and 3n-20g-P eluted frac tions were collected into 13 x 100 mm culture tubes and dried under N2 at 37c. Subsequent handling of these elutes was similar to that described for progesterone (vide supra).

PAGE 46

33 Quantification of estrogens was accomplished as described by Abraham et aj_. (1971) using the assay procedure of Nett aj_. (1973). Quantification of LH was as described by Chenault (1973). The quantification of 20B-d i hydroprogesterone was accomplished by converting this progestogen to progesterone and subsequently assaying for progesterone. Conversion of 3h-20b-P to The chemical conversion of 3H-203-P to 3p was similar to the procedure described by Sholl and Wolf (197'*). To the dried column elute of 3H-20B-P was added 200)jl of a 0.4^ (w/v) solution of chromium trioxide in SOZ acetic acid, vortexed for 1 min, and incubated in the dark at room temperature for 2 hr. The reaction was terminated with the addition of 1 ml of distilled water and the sample extracted with 3 ml of ethyl acetate (2X) This extract was washed with 500 pi distilled water to remove any residual acetic or chromic acid. Removal of the aqueous wash was facilitated by freezing in liquid nitrogen and transferring the organic phase to a 13 x 100 mm culture tube. The organic phase was dried under N2 at 37C. The dried residue was subjected to chromatography on a LH-20 column ( vide supra ) and the 3p fraction was collected into a 13 x 100 mm culture tube for RIA. Preparation, Use, and Storage of Assay Buffer In working with the phosphate assay buffer (Appendix), it was observed that deionized water and a pH lower than 7-2 resulted in loss of sensitivity of the assay. The working gelatinized assay buffer was prepared by adding 0.1^ Knox gelatine (Knox Gelatin inc.) to the above stock solution. The gelatinized assay buffer was stored at kC and used for no longer than h wk.

PAGE 47

3* Preparation, Use, and Storage of Charcoal Suspension To a 100 ml flask was added 0.625 g of Norit A (Matheson, Coleman, Coleman and Bell), 0.0625 g of Dextran T-70 (Mann Research Labs) and 100 ml of gelatinized assay buffer. Flasks were stoppered and shaken vigorously for 30 sec. The charcoal suspension was stored at kZ and used for no longer than 4 wk. A volume of 500 y 1 of this suspension was used to absorb and precipitate the free steroids after assay incubation. The charcoal absorption step of the assay was strictly carried out at hC since higher temperatures resulted in stripping of the bound steroid. Twenty minutes were found to be sufficient time to absorb all free steroid at this temperature. Preparation and Use of Counting Solution To an amber bottle containing 800 ml of toluene was added 3-2 g Omni Scint 1 (98'^ PPO/2'^ BIS-MSB). A total of 3-5 ml of this counting fluid was used per scintillation vial. Cocktail and sample were equilibrated for 2k hr prior to counting to allow movement of the -^H steroid into the organic phase. No solubilizers were used. Preparation, Use, and Storage of Standard Steroids Upon receipt of unlabelled steroids (Steraloids, Inc.) a series of crystallizations in ethanol methanol, and acetone were performed until a constant melting point was achieved. From the final crystallization a stock solution of 1 g/ml unlabelled hormone in absolute ethanol was made. An aliquot of 20 u 1 stock solution was transferred to a 20 X 150 mm test tube and dried under nitrogen gas at 37C. Then

PAGE 48

35 10 m1 of gelatinized phosphate buffer was added and subsequently vortexed for 5 min. The lack of solubility of progesterone in this concentration (20 ng/ml) in buffer necessitated the incubation of this Solution A overnight. Tables 2 and 3 represent the various dilutions used for the standard curve in the RIA analysis while figures 5 and 6 depict a typical standard curve. ~ A volume of 500 pi at each concentration was used in the assay. These solutions were good for up to 6 mo when kept at -kOC. Rad io immunoassay Isolation of recovered 3H-20g-P (oxidized to 3p) and 3h-E facilitated quantification of these steroids. Al iquots of 200 yl and 500 yl'of the buffered unknown was pipetted (Eppendorf, Brinkman Instruments) into two 10 X 75 mm disposable culture tubes. These two dilutions were made in an effort to assure that one or both al iquots were located on the linear portion of the standard curve. The volume in the culture tubes containing the 200 pi aliquot was adjusted to 500 pi by the addition of 300 yl of gelatinized assay buffer. An additional 500 yl of sample was placed in a scintillation vial with cocktail and counted for recovery purposes ( v ide supra ) Tritiated hormones and antibody were added to the assay tube as specified in table 3 and vortexed briefly. All assay tubes were incubated a minimum of k hr at he, followed by addition of 500 yl of charcoal suspension to each tube, vortexed briefly and incubated 20 min at ^C. All assay tubes received this 500 yl of charcoal suspensfon except the total count and background tubes which received an equivalent amount of assay buffer. Following charcoal incubation tubes were centrifuged

PAGE 49

TABLE 2. STANDARD CURVE DILUTIONS^ Mass in 500 yl Solution Preparation (pg) A 20 yl of stock in 10 ml assay buffer 1 ,000 B' 5 ml of solution A in 5 ml assay buffer 500 C 5 ml of solution B in 5 ml assay buffer 250 D 5 ml of solution C in 5 ml assay buffer 125 E 5 ml of solution D in 5 ml assay buffer 63 F 5 ml of solution E in 5 ml assay buffer 32 G 5 ml of so 1 ut i on F in 5 ml assay buffer 16 ^Used for both progesterone and estrogens.

PAGE 50

37 TABLE 3ADDITIONAL TUBES USED TO SUPPLEMENT RIA ANALYS 1 S'^ Amount of Amount of Amount of Sol ut ion assay buffer ant i body ^H-Steroid Code added added added (ml) (ml) (ml) BG*^ ^ 1.2 TC^ 1. 1 0.1 CH^ 0.6 0.1 BT^ 0.5 0. 1 0.1 0. 1 0.1 ^Each run in triplicate to a volume of 1.2 ml. ^To determine the background counts. ''Total count tube to determine cpm of ^H-steroid added to each tube. •^To determine residual counts left following addition of cha rcoa 1 ^To determine the percent of the ^H-steroid bound to the ant ibody ^Assay buffer was replaced by 0.5 ml of either standards or unknown samples

PAGE 51

o o o o oo o o U3 O LA O O O Ci) SNlGNia

PAGE 52

39 o o o o o LA CN o o o a < IC/) UJ u. c z o < Iz UJ o z o o c > (A C o M ) 4-1 o L. c (U E 0) JC c 0) tn a> u a 0) L. o a JC o (t) o o o 0~i o CO o o o o o o CN o ID 1_ 4-1 1/1 c o D O M -D c 1_ ro i a i_ c (1) fD 4J •l-J 0) tn Co) SNlUiH'tJ NO u D cn

PAGE 53

^0 at for 20 min at 2,000 g. Then, 500 pi of this incubate was withdrawn by a repipette and deposited in a scintillation vial along with 3.5 ml of scintillation cocktail. Counting for 5 min followed overnight equilibration at room temperature. Calculations o, n (recovered counts background counts) (4) „ % Recovery = ~ -r-r—i 1 — \ 3 — r— ^ '^0 original counts added background counts o -,, r,. .. BT tube counts % Total Binding ^ — ; x 100 TC tube counts The standard curve and quantification of unknowns were calculated by logarithmic transformation of the data. For this, a Monroe i860 corrr puted the concentration of steroid in log scale and the percent dpm bound in linear scale. The amount of steroid present in one ml of serum was calculated as fol 1 ows : • concentration of unknown Steroid Concentration = 4, recovery X 2 (for 0.5 ml) or 5 (for 0.2 ml) Separation by LH-20 Column Chromatography With the use of tritiated progesterone and 206-d i hyd roproges terone it was determined that these steroids were soluble in hexane : benzene: methanol (80:15:5) prior to chromatography. Sephadex LH-20 and hexane: benzene:methanol (80:15:5) served as the partition system. When a 7 cm LH-20 column was used the less polar

PAGE 54

41 progesterone was eluted within the first 3 ml and the more polar 206-P was eluted between the fifth and seventh ml fractions. P rec i s ion The between and within assay coefficient of variation (c.v.) was calculated from two different duplicate determinations from a stocTc of standard plasma samples run with each assay. The c.v. from the means was estimated by the followed formula (Steel and Torrie, I960): c.v.= 100 S/x, in which S is the standard deviation and x is the mean. In a total of 58 duplicate observations, the intraand interassay c.v. for plasma progesterone was 3-3 to 16.2^ and 10.71^, respectively. For estrogens, with 2h duplicate observations more variability in the intraassay c.v. to 20.9^) and in the interassay c.v. (21.33^) was found than that for progesterone. Statistical Analysis The basic statistical method utilized was least-squares multiple regression analysis (Harvey, I96O) from which the following parameters were obtained: (1) Identification of classes (group, day, and group x day interaction) with their respective least-squares means and standard errors for progesterone, 206-d ihydroprogesterone estrogens, and luteinizing hormone; (2) Least-squares analysis of variance and listing of polynomial regressions for each hormone. Since analysis of variance indicated that P, 203-P, and E were statistically affected by day, group, and

PAGE 55

f2 day X group interaction, it was necessary to characterize these trends by regression analyses. Therefore, a set of regression equations for each hormone within source was computed and that which best characterized the hormonal response was considered the hypothetical model for discussion of the results; (3) Test for day effect within hormone was computed using th^ appropriate error term obtained from Harvey's printout (Steel and Torrie, I96O). Reproductive responses were tested by LSD; and (k) Additional computations were obtained according to the procedures described in SAS 76 (Barr et al., 1976).

PAGE 56

CHAPTER IV RESULTS AND DISCUSSION EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P) 20B-DIHYDR0PR0GESTER0NE (2O3-P) ESTROGENS (E), AND LUTEINIZING HORMONE (LH) IN THE PMSG SUPEROVULATED COW SYNCHRONIZED WITH PGF2a-THAM SALT Reproductive Response The efficacy of dual administration of PGF2a ^or synchronizing estrus in PMSG superovulated cows is depicted in table k. Behavioral estrus was observed in ]k of 15 {3?>-3%) treated animals by 120 hr post-PGF2cf The single animal not responding to this dual injection of PGF2a was detected in estrus 36 hr prior to the second PGF2a adminis tration. Stratification of estrus at 12 hr-intervals resulted in 26.7, 53.3, 73.3, and 93.3% of the animals exhibiting estrus by ^8, 60, 72, and 120 hr, respectively, following PG^za injection. This is in agreement with Cupps et aj_. (1976) who reported that the administration of PGF2(^ 2h hr post-PMSG, to previously synchronized heifers: (l) increased the number of animals exhibiting estrus; (2) shortened the interval from treatment to estrus; and (3) reduced the variability in time to onset of estrus. The percentage of animals exhibiting estrus by 72 hr (73.3-^) in this study, was slightly higher than the G7% reported by Archbald (1976). Estimates of ovulation rate, determined at laparotomy, are presented in table Significant differences v/ere detected in total k3

PAGE 57

CD ZD CO Q < o — 1— < 1— t/1 LlJ 2: — < LU 8 CI. CM X Ll. LU CJ Q. 0 CO 2: LjJ QCO LlJ 3: 1 — zn 1— ct^ UJ LU 1— 1— Ll_ "< < LU D£ CO H ZD DC CO \— _l m < LU 2: — CD ~~ < Ll 0 0 n: CO LU CO I/) 2: 0 < Q_ CO LU a: < ro z 0 < 0 < > H 0 CO H z cc LU H ID CO O* LU Q CO 0) c LU CO + 1 X > o >Iro > O l-J > c (1) 4-1 E nj j-i 0) 0) E 1_ •l-J U 0 < t-l (U in c 0 Q. 1/1 0) 1_ (/) >1_ 4-1 1_ 1/1 :3 LU 0 l/l D 14-1 Ll/l -C 1 4-1 -C in 0) 0 m CL (•! -3CM 1 -d0 1 0 1 0 0 0 OA LA -;c CM 00 — 0 +1 +1 +1 + 1 0 LA 0 LTv r-^ vD LTl (N 0 LH 0 LA 0 0 00 LA 0 + 1 +1 +1 + 1 0 0 LA 0 0 CM, 0 0 r-. 0 LA 0 0 LA 0 CM +1 +1 +1 +1 0 LA LA OA OA vO CO 0 — 0 OA OA CNI OA LA OA 1^ OA CA LTl LA LA LA \ -3\ 00 \ \ -aXI 0^ r~~ 0 0 0 0 CM CM CM LA LA LA LA \ -3\ OA \ OA 00 0 CM 0 JvD r~CM CD 1 crv 1 1 OA vD 4-1 c x> E >• c (D 4-1 >c E ro 0 ro u 4-" 4-J 0 0 uV_ 4-1 (U c Q. •Icn (D cn 1/1 c: 4-J <40) Q. 1\1_ in U 01 in XI ro u E E (U Q. C c ro in C 0 u4-1 Q, 0 c 0) i_ ILA TD (U 0) 0 0) XI uin E uV (H 3 CL OQ Q TO XI U

PAGE 58

ovarian response when cows were partitioned according to when they exhibited behavioral estrus. A higher ovulation rate (P<.01) was observed in cows expressing estrus by hr pos ti n j ect ion (5-50 1.29) vs 0.67 0.82 for those expressing estrus from 73 to 120 hr. The difference in ovulation rate in animals exhibiting estrus from 49 to 60 hr vs 61 to 72 hr was nonsignificant (P>.25). It was encouraging that only 5 of 15 (33.3%) of the cows had more than four ovulations (CL) These results agree with those of Hammond (19'<9), Brock and Rowson (1952), and Scanlon e_^ aj_. (I968) in that a more desirable and restricted level of superovulat ion was obtained when the interval between PMSG or FSH injection and subsequent estrus was relatively short. In line with this concept, Tervit et^ a_l_(1973) demonstrated in cattle'with large PMSG-induced follicles that a PGF2a analog (ICI 79,939) shorten the interval to estrus. Dobson et^ aj_. (1975) suggested that the second PGF2a' nduced luteolysis may allow a wave of growing follicles originating after the first PGF2a~ i nduced estrus to progress and ovulate rather than become atretic. This might account for the observation that the induction of estrus was more rapid and precise after the second PGF2c( administration. R. Hardin, A. C. Warnick, S. R. Lopez Barbel la, T. H. Wise, and M. J. Fields (unpublished) demonstrated that cows with dual injection of PGF2a analog (ICI 80,996) displayed estrus 2k hr earlier following the second injection than the first injection. Rajakoski's (I96O) report that there are two waves of growing follicles during the estrous cycle of the cow tends to support the above findings. In addition, Goodman e_^ a_l_. (1977) reported that the largest follicle developed during the early phase of the estrous cycle retained its dominant position for a longer period of time than did the dominant follicles for other stages of the estrous cycle.

PAGE 59

The present study suggests the possibility of a relationship between length of the interval from PMSG to estrus and superovulatory response as demonstrated with the longer interval from treatment estrus of 73 tolZOhr in conjunction with a lower superovulatory response of 0.67 0.82 ovulations per cow. The percentage of animals conceiving to either the first postPMSG estrus and/or subsequent estrus as determined by rectal palpation 60, 100, and 145 days after treatment with PMSG or by subsequent calving dates is given in table 5The conception rate of 100!^ determined by palpation at 60 days post-PMSG is higher than the 60 to dSt reported by Turman et_ aj_. (1971), Laster e_t aj_. (I971b), and Hallford et al (1975a, b) for superovulated cows. Multiple fetuses determined by calving date were lower than those estimated earlier by palpation. These discrepancies were attributed to an inability to predict multiple fetuses by rectal palpation at earlier stages of gestation. In addition, none of the cows bearing two or three CL and only one of the five cows with more than four CL gave birth to multiple calves. When working with multiple fetuses, Gordon et^ a_I_ (I962) and Schwartz and Shelby (I969) suggested that both rectal palpation and laparotomy may be involved in increasing the incidence of embryonic mortality. Although a reduction in embryonic mortality was achieved with kO mg PGF2a, the presence of Brahman calves, a genetic marker indicating conception to breeding at the PMSG-induced estrus prior to ^40 mg ^^^^2a^ raised the question of the fetus in some manner rescuing the CL, as suggested by Mapletoft aj_. (1976). Alternatively, it could be a question of inadequate levels of PGF2a fof the increased mass of CL tissue. These questions will be addressed in the next two experiments.

PAGE 60

TABLE 5. CONCEPTION RATES FOLLOWING ARTIFICIAL INSEMINATION POST-TREATMENT WITH PMSG and PGFza AND EXPOSURE TO THE BULL FOR 90 DAYS I N "eXPER I MENT 1 Time % pregnant to treatment Nonpregnant (days) Single Multiple Open to treatment 60 53.3 ^6.7 0.0 100 20.0 53.3 26.7 93.3 0.0 6.7 Pregnant to term 26.6 6.7 6.7*^ 60.0^ ^Includes one animal pregnant to treatment that died due to Tympanites. '^Post-mortem examination of reproductive tract revealed oviduct blockage in the single open cow. ''Determined by phenotypic appearance of the calves.

PAGE 61

Endocrine Response In an attempt to reduce fetal wastage due to the superovu 1 at ion effect resulting from PMSG, five cows with ovulation rates greater than three CL were injected with ^0 mg PGF2aThis treatment, in itself, allowed for the subgrouping of animals according to their ovulation rate. Since it was determined that the time-trend in plasma hormones anrang group 1 (cows with more than three CL) group 2 (cows with two or three CL) and group 3 (cows with zero or one CL) were similar, emphasis was placed on the magrwitude of the response for subgrouping. Concentrations of progesterone, 203 -d i hydroproges te rone luteinizing hormone, and estrogens in plasma from each group are summarized in tables 12 to 1 of Appendix. Progesterone Plasma progesterone concentrations in cows from group 1 plotted by days of bleeding is illustrated in figure 7. A near normal progesterone curve (table 15, Appendix) was exhibited prior to PMSG treatment. By 2h hr after gonadotropin administration, plasma progesterone concentrations had increased from 5.66 1.17 to 9-06 2.99 ng/ml (P<.05). After the luteolytic dose of P(^f2a ^ (AM), progesterone decreased to a basal concentration of 0.72 Q.\k ng/ml by day 6 (PM) These changes were best described by a signfficant (P<.01) third order regression equation (table 15, Appendix) which accounted for h7Z of the variation. Plasma progesterone remained at basal levels for 2k hr after which a significant (P<.01) curvilinear (table 15, Appendix) increase to 16. ^46 + 2.36 ng/ml on day 15 (AM) was observed.

PAGE 62

0) c o E ro 1Q) O +-) :3 CD c TO •— 1_ N O a C i•— o (U o 4-1 — 1 1 1 O i_ 0) o c uo -C 0) 4_l *i TO 0) 3 10 in I' ) C\J CM i\J (\) (\l J._.l_l_L_J_.LJ_J_J_J_J._l._L.J._l_J_l..J_J. O (T) (0 tID m M" ''5 01 O O) CU 1^ (Ll 'O vjI') iM •: 0o't (iiu/ijij) ;!^Jo^J3ls:loo^Jdo.^JaAlllG-! loi' 'jf^'O.MJiir.j'jojJu 'in

PAGE 63

50 As described initially, ovaries were examined by laparotomy and those with more than three CL received kO mg PGFpa on day 15 (AM) A significant (P<.01) reduction in plasma progesterone was observed after ^0 mg PGF2„ reaching a nadir of 1.78 0.77 ng/ml by day 20 (PM) followed by a low but steady increase characteristic of the pregnant animal. These changes were best characterized by a third order re^ gression equation (table 15, Appendix) which accounted for 6]% of the variability. In the case of cows in groups 2 and 3 which did not receive ^0 mg PGF2a (figures 8 and 9), a steady increase (tables 13 and 1^, Appendix) in plasma progesterone was observed as expected in the nontreated cows. Characterization of these trends are shown in table 1 5 (Append ix) The progesterone increase following PMSG has been reported by Henr icks e^ aj_. (1973), Sp i Iman et^ aj_. ( 1 973) and Hal Iford et al (I975b). Although the source of the increased progesterone could not be determined from this study, PMSG may have had a luteotrophic effect on luteal tissue or on the granulosa cells of mature follicles. The reduction in plasma progesterone after the second PGF2c( injection, found in this study, closely paralleled that reported by Fournier et al. (1976). The relationship between progesterone secretion and number of CL in animals of group 1 (r = 0.73, P<.OI; table 18, Appendix) is in agreement with Lamond and Caddy (1972) and Spilman aj_. (1973) and in disagreement with Rajamahendran e_t^ a_I_. (1976) who reported that a much larger number of observations was needed to assess statistical difference between supe rovu 1 a ted and normal cycling cows.

PAGE 64

5i (iuj/Lnj)'j;>i I'jum.i.';;.-] in 2 O u o (U 4-1 (D (U 1_ to c o •w 4> 01 O 1Cl O I. T3 >o CM ^ 1_ C o c o o 00 CD (/I c (U O) • O nJ 10) •U 4-J 1/1 D OJ — -D TO C lfD O D. I— ^ O O 0) 0) x: cn 4-1 c — jd N 4-P c 'i !^]0^l:JiSDoo^ .^MIG-OO^ 'INO^GJ.SjDOHcI 'I I'

PAGE 65

52 o> 00 (IUI/6U) 3NOLI-^lS3908dOHaAHia-E)02 I to I 3: J a. 4) O C L. 0 ui_ 0) (D -t-" E 01 ;/i 0) (T! CD — O Q. u a. c o — D ^ — (U I +-> OQ (/I 3 O C — -01• U1 • a) O 1o a. 1o o (1) c o (/I (U O 0) I0) N Q. C O <4e 4-j o u — o 2 C JZ O i/i — O) 3 •u c o (D — N 4-1 — TJ C C (U 0) — U O 0) (T! C -W (U O 3 1(_) — 4-1 o CD

PAGE 66

53 Est rogens Plasma estrogens for each group are depicted in figures 7, 8, and 9, respectively. Plasma estrogens in group 1 were genera 1 1 y be low 5 pg/ml (table 12, Appendix) prior to PMSG administration after which there was a significant (P<.Ol) increase to 13-62 2.06 pg/ml within 2h hr. In conjunction with the luteolytic dose of PGF^^ on day ^4 tAM) estrogen levels continued to increase to a peak of 37 -Oh 1 1.^*7 pg/ml by 72 hr post-PMSG. These changes were best characterized by a third order regression equation which accounted for k7% of the variability (table 16, Appendix). This increase probably resulted from increased follicular growth stimulated by PMSG; however, the experimental design did not allow a direct test of this assumption. Declining levels of progesterone in response to PGFg^^ coupled with increasing levels of estrogen led to the expression of estrus. As a consequence of the reinitiation of a PGFpcfinduced estrous cycle, plasma estrogen declined significantly (P<.Ol) from day 6 (AM) to day 11 (AM) (1.5'* 0.09 pg/ ml). Estrogen levels then remained at basal concentrations until an increase following the hO mg dose of PGFj,^ given at laparotomy on day 15 (am). These sequences of events were best described by a second order regression equation which accounted for 55?^ of the variability. Since cows in groups 2 and 3 (figures 8 and 9) were not administered kO mg PGF2^, plasma estrogen levels remained at base line (table 13, Appendix), as might be expected, in the luteal phase of a normal cycling cow bled every 12 hr. Characterization of these trends are shown in table 16 (Appendix). The relationship between estrogen secretion and number of CL in animals of group 1 (r = 0.68, P<.01; table 18, Appendix) is in

PAGE 67

5^ agreement with that reported by Hall ford et aj_. {1975b). Henricks eX a\_. (1973), in cattle, and Guthrie et aj_(197'^), in swine, concluded these types of comparisons have to be made with caution since the normal pro-estrus surge of estrogens could be confounded with an estrogen rise resulting from PMSG. As a result of this, they failed to report any' correlat ion between plasma estrogen and number of "~ fol 1 icles. Luteinizing hormone • The variability in plasma LH (tables 12 to 1 Appendix) was most likely due to the infrequent collection of samples (figures 7 to 9). LH values prior to PMSG treatment were approximately 1.0 ng/ml Elevations in LH to above 2 ng/ml were evident in each group between days 3'(AM) (day of PMSG administration) and day of the LH surge. This is in agreement with Spilman et^ a_l_(1973) who demonstrated that plasma LH rose sharply the day after PMSG injection and declined thereafter. Differential timing of the LH surge among groups in this study also support the observation of Henricks e_^ aj^('973) that the ovulatory surge of LH was earlier in PMSG treated heifers than in nontreated controls. Although untreated PMSG-treated animals were not available for comparison in experiment 1, the LH surge in cows of group 1 occurred 12 hr earlier than that for animals in group 3This is consistent with the earlier onset of behavioral estrus in cows in group 1 ( vide supra ) Hallford aj_. (I975b) failed to detect this treatment effect; they only obtained plasma samples at 2k hr intervals. Henricks ei^ aj_. (1973) and Hallford eX a]_. (1975b) failed to detect a relationship between plasma LH and reproductive criteria after PMSG treatment. Since a 12 hr bleeding interval is not sufficient to fully

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characterize the LH surge, no additional information regarding the relationship between plasma LH and plnsma estrogen or between plasma LH and ovulation rate can be provided from this trial. 20g-Di hydroprogesterone The individual plasma values and plot-ted levels of 20B-P are reported in tables 12 to I'* (Appendix) and figures 7 to 9, respectively. There was a curvilinear relationship between plasma 20B-P and day of bleeding (table 16, Appendix). A nonsignificant (P>.10) increase of 20g-P levels was observed during the luteal phase (3-88 O.^iS ng/ml) when plasma progesterone was at, its maximum h.SS ng/ml). Spilman et aj_. (1973) reported an increase plasma 20B-P level after PMSG treatment with 20B-P levels being 15 to 20% of that for P while Tribble (1973) and Castenson e^ aj^. (1976) showed, in the cycling post-partum cow, 20B-P levels to be 10 to ]5% of that of P. In these studies the maximum levels of plasma 20g-P were 2.5 ng/ml which is comparable to the concentrations found in this trial. In addition, similar ratios were observed in this study when cows were on day 5 of the luteal phase of the PMSG-PGFaa induced estrous cycle. Although 206-P appeared not to be influenced by gonadotropin administration, there was a 3-3 fold greater increase in P over 2O3-P synthesis at 2h hr post-PMSG treatment. Romanoff (I966) reported a similar ratio of P to 2O3-P when the ovary was perfused with FSH. Finally, with the data available from this experiment, one may speculate that the surge of LH at estrus is leading to follicular luteinization which is, in turn, maintaining 20B-P levels. Since follicular fluid does not contain 2O3-P (Short, 1962a, b) and CL (Savard and Teledgy, 1965) and luteal cysts (Short, 1962a, b) do contain 20b-P, it seems likely that only luteal cells have an active 20B-HSD. Thus in this

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56 experiment, it may be that the origin of the 206-P was from reactivation of lutea] cells from regressing CL or 1 ute i n i za t i on of granulosa cells from follicles. Hi Hard e^ a_l_. (1967), in the rabbit, and Ichikawa et al. (1971), in the rat, demonstrated the release of LH at coitus and that exogenous LH injections activated the synthesis and release of • Data accumulated in this study suggest that PMSG has profound effects on both the ovarian and endocrine response of the bovine female. Additional research aimed at understanding the change in progesterone and estrogen concentration and their role in follicular growth at the time of the post-PMSG estrus should provide further insight into the questions associated with the hormonal induction of multiple fetuses in cattle. Although the role of 206-P during the cow's estrous cycle rema ins obscure experiments are needed to further characterize the endocrinology of this steroid. Finally, although cows treated with kO mg PGF2„ exhibited both a functional and morphological regression of multiple CL with no abortion, it is of importance to test whether 'O mg PGF2a is effective in nonpregnant or pregnant cows in regressing multiple CL following PMSG treatment. If the introduction of PGFj^ results in abortion of undesired multiple pregnancies associated with the superovu 1 atory effect, then it could be used to minimize fetal wastage by regressing multiple CL and rebreeding the cow in a short breeding season. This could lead to an increase in the percent calf crop with restricted multiple fetuses. These alternatives are tested in the next experiment.

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57 EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN THE SUPEROVULATED BEEF COW FOLLOWING ADMINISTRATION OF kO HG PGF2a-THAM SALT Results from experiment 1 indicated that PGFg^ was ineffective in inducing abortion in the superovu 1 ated pregnant cow. The question then arose as to whether kO mg PGFj^ was inadequate to regress multipIe^CL and subsequently allow the cow to be bred in a short breeding season. This experiment was designed to determine if this P^^2a days post-PMSG, was effective in regressing multiple CL in the nonpregnant superovu 1 ated cow. Although it was not the objective of this trial to evaluate the efficacy of a dual injection of PGFg^ to synchronize estrus, it is of importance to review this concept and compare these results with the endocrinological profile reported in experiment 1. Reproductive Response It is well established that PGF2p( is not effective during the first 5 days of the estrous cycle. Therefore, in a large population of cycling cows 75% of the animals (based on a 21 day cycle) would be expected to be in a potentially responsive stage of the cycle (day 6 to 21) on any one day selected at random. For some unexplained reason these cows were somewhat synchronized prior to the first injection of PGF2a when 10 of 13 animals exhibited estrus within 96 hr after treatment (table 6) followed by Sh.GX at the time of the second injection. Cooper (IS?'*) and Chenau 1 1 et^ a_L ( 1 976 ) cl ea r 1 y demonstrated that a single injection of PGF2^ manipulated the estrous cycle so that cows were in a potentially more responsive stage of the cycle 12 days later. This observation is substantiated by the progesterone profiles of experiment 1

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58 The distribution of observed onset of estrus following an injection of PGF2c( is shown in table 6. The percentage of animals exhibiting estrus k8 hr post-second PGF2a treatment (61.5^) was significantly (P< .01) greater than that after the first injection (38.5^)These results are in agreement with Cupps et aj_. (1976) and Archbald (1976) and support the concept of a shortened interval and reduced variability to onset of estrus after the administration of PGF2ct 2^* hr post-PMSG, to previously synchronized cows. Ovarian estimates determined at laparotomy are presented in table 6. There was no significant difference in ovulation rate between cows that exhibited estrus by ^8 hr (4.3 3-3) as compared to 72 hr (4.0 2.8). This does not agree with data from the first experiment in which cows with the shorter in te rva 1 to estrus had a higher ovulation rate. Endocrine Response Ovarian stimulation by PMSG was monitored by measuring plasma progesterone on days 11, 12, and 13 post-PMSG at which time progesterone concentrations were 8.79 + 2.12, 10.34 2.22, and 10.22 1.89 ng/ml respectively (table 19, Appendix). Bleeding was continued for 7 more days in an attempt to characterize the reduction in plasma progesterone following 40 mg PGF2n on day 13Analysis of variance indicated that the reduction in plasma progesterone concentration in cows after the administration of 40 mg PGF2a was significantly affected by treatment (P<.0l) and day x treatment interaction (P<.05) (table 20, Appendix). Time trends of progesterone, were characterized by regression analysis (table 21, Appendix). For the PMSG-treated group, a third order regression equation (y = 39.12

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59 UTN CO >. CO E UJ +1 O 0) +1 4- \_ CO O 0) + 1 o 1> (C o IX -cr -3CL IT) -3> 1-1 m fa +1 +1 > o > 4-J o o a: -cr o 3-P 8 CM U. O Quo "D C 1_ ro 0 0) E o JD 0) E c 3 c OlUo IL. CD QC O L. .— aj 4-1 4-1 O 1u — 1 — c (U — E \0 oo oo CO -3I o oo CM -3Ln ft) ro o o J3 (Nl o CO r-~. — o CM O CM vD Osl -3r~-CTi — I r I I cn CO — -3cTi csi o E I UA -3CO +1 CO o o CJ CO CO s: Q3 3 CO CNI O cn O O o o o CNI 0) > > (U 0) o CO u
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60 13.99X + 1.73x^ 0.07x^, = 0.7323) best characterized this trend whereas a second order regression equation (y = 2.^0 + 0.75x 0.26x = 0.8697). best described the reduction in plasma progesterone in the non-PMSG treated group. In response to hO mg PQif^2a administered at laparotomy (figure 10) plasma progesterone declined significantly (P<.Ol) to a basal leveT of 0.62 0.07 ng/ml in 96 hr. Plasma progesterone did not increase again (P>.10) until the luteal phase following the kO mg PGF2a • A similar trend was observed in the non-PMSG treated group, although the magnitude of response was smaller. Ovarian palpation 7 days postlaparotomy confirmed multiple CL regression and subsequent estrus indicated the reinitiation of a new estrous cycle. The change in slope of the progesterone decline following PGF2a possibly reflects functional CL regression. Functional CL regression is the termination of progesterone secretion while structural regression, i.e., the physical destruction of the luteal cell, is believed to be the result of changing i n tra-ova r ian blood flow distribution post-PGF2cf Novy and Cook (1973) and Thornburn and Hales (1972) demonstrated that blood flow to the CL was reduced, whereas blood flow to the stroma and follicular component of the ovary was increased following PGF2a administration. Although the mechanism of luteolysis after PGFj,^ remains undefined, data accumulated in this study clearly suggest that 'O mg PGF2a is effective in regressing multiple CL in the cycling nonpregnant beef cow based upon the reduction in plasma progesterone and CL regresstion evidenced by the absence of palpable CL by 7 days post-treatment. The question still remains as to whether or not kO mg PGF2^ is effective

PAGE 74

bl ClW/t)N) aNO'da.i S3t)0dd

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62 in regressing multiple CL of PMSG-PREGNANT cows since in the first experiment, both functional and morphological CL regression was presumed. Unexpectedly, however, pregnancy was maintained. The next experiment deals with the effect of pregnancy on the regression of multiple CL in mated cows. EXPERIMENT 3. LUTEOLYTIC EFFECT OF PGFpa-THAM SALT IN THE PMSG BRED VS NONBRED BEEF COW Data from experiment 1 indicated that kO mg PGF2c(-Tham Salt apparently induced only partial functional and structural CL regression in PMSG-PREGNANT cows, since CL appeared to have regained their functional role and pregnancy was maintained to term. In experiment 2, however, kO mg PGF2a ^^s effective in regressing multiple CL in cycling cows when measured by a rapid decline in plasma progesterone to below 1 ng/ml and the absence of a palpable CL 7 days post-treatment Experiment 3 was thus designed to test the difference in response to PMSG by bred vs_ cycling cows. Reproductive Response The distribution of estrus after a dual estrous synchronization scheme with PCFj^ is summarized in table 7As expected, a greater percentage of animals expressed estrus following the second injection of PGFjct than following the first injection in the PMSG treated group (96 VS 91^, respectively). Unexpectedly in the non-PMSG group, there was not an increase in the percentage of animals responding to the second injection of PGF2a (82 v^lSl, P>.10). The PMSG superovulated

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CL 0) -3(/) o o (J 1 a CD 8 1 CxI CO CM J-J Ll. 2: LiCL • — — O Q1 0 Q. O. c Z H 0 1/1 "Z. -p' O 3 UJ U — Do l/l ct 0 UJ U_ L. 1— 1LU —i o CO r\ X < Ql z. M Q. — a 3 -3• — z (M 0 • — CM o U. 1_ cc C3 (1) CJ O. 1/1 o O C3 "D X) CO >C 2: • LA cn O cn Q_ 0 — u c 1 z: C3 s (U — C 2: CM U1 N 0 Ll. 1 • — z LA C3 M C CL in O O 1CL ro U. Q. ^ 3 LA CO o O 0 — CM _J i/l C v. < UJ u :^ (S> to L. l/l Q o 4-J Q Ul CO /-\ CO 0 < LU 2: — 1QLU UJ — u. < < CL U3 ro 00 > — — a 0 ^— CNJ :2 fl_ o QC Ll (U t3 O C3 1/1 DQ CL O < CO — LA l/l cn CL 0 — 2: 1C 1 Z r3 C q: o MN 0 H -a1 •— z CO LJ C UJ LU l/l O Z O 1a Li. o Q. -C 3 CTl LTv CO O U 0 3^ CM Q l/l C i_ 3 > C3 o < u in 4— C3 1— hin CO 0 CNl 0 _J LU 2: — < Z CO 1— tn < Q 1— c o o LLl L 0) _i 0) — — ^ in •L" C in < 14• — > CNl ro\ t3 X> E u. — o 1D00 LA -3OA ^ ^ — CO vD CM I I LA — — dJ 1_ C o a CNl Ll C3 OJ O. L. CM UJ cn CM E in LA T3 0 rA o\ rA L. (U 4J Q. l/l 0 0) CM CL l/l CM 0) JZ CM *j 00 c — — 3 CM l/l 3 2 i_ PA 1/1 1/1 CM 3 OJ L. LA 4-J cn in c X) CL c CM l/l CM a 0) X) l/l c c LA 0 0 c u C X! 0) 0) nj L. L. cn (U 4-1 0) c 2 XI 2 XI in in c > X) c 0 0 c 0 CL CJ 0 l/l CD

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64 animals, by 72 hr post-PGF2c(, expressed estrus to a greater degree (P< .01) than the non-PMSG animals (87 vs_33%, respectively). Whether the influence of PMSG on this reduction in interval from PGF2ct to behavioral estrus results in differential fertility remains to be investigated. The intferval to estrus following the ^40 mg abort i fac ient dos^e of PGF2a was longer in the PMSG treated animals when compared to the nonsuperovulated group ( 1 5 ys^ 7 days, respectively, P<.01). Ninetyone percent of the cows in the non-PMSG group responded within 7 days post-'40 mg PGF2a with a mean interval to estrus of ^.'8 + 1.12 days. Two types of estrous response were noted in the PMSG treated animals: one type, representing 6^4^ of the animals, displayed estrus between 5 to 10 days post-treatment with a mean interval of 6.^3 2.2h days and the second type, comprising 27^ of the animals, responded behaviorally between 11 and 15 days post-treatment with a mean interval of 13.50 1.05 days. Fifty days post-'tO mg PGF2ct all animals cycled at least once and post-mortem examination of the reproductive tracts showed no evidence of pregnancy. The aborti facient property of PGF2a in beef cattle has been widely demonstrated (Lauderdale, 1972, ]37h; F i e 1 ds et^ a2_. 1977a). There is only limited data available with regard to behavioral estrus post-abortion, in the PMSG-BRED cow. Results from the non-PMSG group substantiate that of Douglas et_ a]_. (197^) in that cows aborted in early gestation may return to estrus within 2 days after abortion. Results from this trial show the PMSG-BRED group to be comparable to that of Zerobin et^ a]_. (1973) in that the administration of PGFjq to non-supe rovu 1 ated pregnant cows at early stages of gestation resulted in behavioral estrus 2 to 16 days post-treatment.

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b!> Ovarian response estimates, determined at laparotomy, are presented in table 8. In experiment 1, a differential ovulation rate with respect to day of behavioral estrus was observed. In this trial, although there was a reduction in ovulation rate from day 1 to day h of 8.00 + 1.73 vs_ 5.88 2.83, respectively, this difference was nonsignificant (P>.10). Six of the 22 supe rovu 1 a ted cows had been treated with PMSG In a previous trial. No refractoriness to the PMSG given in this trial was detected (table 9). The increased refractoriness to successive PMSG reported by Willett et_ a\_. (1953), Hallford et aj_. (I975a,b), and Turman et aj_. (1977) and the lack of refractoriness in this study might be attributed to the relative longer time elapsed between the two gonadotrophin injections. Post-mortem examination of the reproductive tracts revealed no ovarian abnormalities, e.g., cystic follicles and indicated that the cows were cycling. It is important to mention, however, that there was observed a high incidence of ovarian adhesions in response to supravaginal laparotomy. This was particularly noticeable in the PMSG group. Endocrine Response Analysis of variance of the reduction of plasma progestin after hO mg PGF2ct was significantly (P<.01) affected by PMSG treatment (table 22, Appendix). The regress ion curves for individual treat; ments groups are depicted in figures 11 and 12. Plasma progestins concentrations and predicted equations are summarized in tables 23 and 2k (Appendix), respectively.

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66 Q. o tjl D o 3 > o > o >1_ in > o > a o 1/1 3 Lu \i-> CL oo l/l (U -a UJ c 01 _1 o >00 13 u ro < V0) -a O l/> > 1_ (U -C j-j 0) CO o o LA o o O O +i +1 +1 + 1 o o OO Q o vD LTV o O o O +1 +1 +1 + 1 o -3o O O o LA LA o o O O +1 +1 '+I +1 Q vD o O O o nj vD oo -3CM +1 + 1 +1 +1 o — O oo CO vO LTi o — O O (N +1 + 1 +1 +1 o roi o (N (N O CTl O O XI O D IQ a LU 2 • o LO (J •tl 0) c I X o TO -O 0) > o

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TABLE 9. OVULATION RATES IN COWS TREATED TWICE WITH PMSG AT FOUR MONTH INTERVAL Cow Fi rst Treatment PMSG (6/18/76) Second Treatment PMSG (10/29/76) number RT ovary LT ovary RT ovary LT ovary 38 0 1 3 1 k2_ 5 2 3 3 kk k 2 5 1 h5 1 2 1 1 ii6 3 k if k hi 5 k 5 0 Overal 1 3.0 2.1 2.5 3.0 3.5 1 .5 1 .7 1 -9

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68

PAGE 82

Hiis::30udd

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70 Cows treated with PMSG exhibited higher mean plasma progestin concentration of 7.63 1.79 and 8.86 1.57 ng/ml for the bred and nonbred groups, respectively, when compared to those for non-superovulated cows of 3-07 0.15 and 2.Gk 0.21 ng/ml for the same respective groups at laparotomy. Similar results have been reported previously for cycling cattle (Plotka et^ aj_. 1967; Stabenfeldt et al~, 1969; Kazama and Hansel, 1970; Sprague et^ a_l_. 1971; Wettemann et aj^. 1972; Glencross et aj_. 1973). AFter kO mg PGF2a inject'ion on day 3, plasma progestin in the PMSG group declined significantly (P<.01) to a minimum of 1.37 0.23 and 1.25 0.22 ng/ml, on day 9, for the bred and nonbred groups, respectively. in the non-superovu 1 ated animals, minimum progestin levels of 1.06 + 0.19 and 0.96 0.l6 ng/ml were achieved by day 8. These trends were considered to be curvilinear (table 2^*, Appendix). On an individual animal basis, all cows achieved progestin levels below 1 ng/ml post ^0 mg P^^2aBurrell and Wiltbank (1977) demonstrated that CL of a pregnant cows on day 17 and CL of a nonpregnant cow on day 6 differ in their response to exogenous hormones when 33. '^ of the pregnant cows and ]00% of the nonpregnant cows resumed cyclicity after a single dose of k mg norgestomet. With PGF2a, it only takes 2h hr to achieve a 60 to 75% reduction in plasma progesterone; however, for the subsequent expression of estrus it takes up to 16 days (Zerobin e_^ aj_. 1973; Douglas et a_l_. 197^) • In the PMSG superovu 1 ated cycling cow, PGFj^ was shown to be effective in regressing multiple CL when measured by a rapid decline in plasma progesterone, absence of a palpable CL, and resumption of cyclicity (LopezBarbel la e_t aj_. 1976,1977)In the superovulated

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71 pregnant cow, however, they indicated ^0 mg PGF2a to have induced a significant (P<.01) decline in plasma progesterone but failed to induce abortion. The discrepancy in results between these reports and the data accumulated in this study might be explained by the animal variability, differences in time and/or other random sources of variability. In conclusion, when induction of multiple fetuses is attempted with gonadotrophic hormones, abortions associated with the superovulation effect might be minimized by regressing the multiple CL with PGF2a and rebreeding the cow in a short breeding season. This, possibly could lead to a restricted number of cows with multiple calves which would increase the percent calf crop.

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CHAPTER V SUMMARY AND CONCLUSIONS !n an attempt to characterize the trends of plasma progesterone, 206-dihydroprogesterone, luteinizing hormone, and estrogens in response to the PMSG induction of superovulat ion 15 cycling parous Angus cows were estrous synchronized with two sequential treatments of 33-5 mg PGF2ct-Tham Salt (IM) at a 12-day interval. An injection of 2,000 i.u. PMSG (Organon) -was administered lU hr prior to the second PGF2a and cows were inseminated three times at 0, 12, and Ik hr post-PMSGPGF2a-"nduced estrus. Blood samples were collected twice daily for 21 days from all animals starting two days prior to the PMSG injection and plasma hormone levels analyzed by RIA ( vide supra ) In response to the PMSG treatment cows exhibited a stereotypic ovulation rate (2.67 + 2.19)The highest ovulation rate (5-5011.29) occurred in cows with the shortest interval from treatment to estrus {2k hr). The lowest ovulation rate (0.67 0.82) was in conjunction with the longer interval from treatment to estrus (48 to 96 hr) suggesting the possibility of a relationship between length of the interval from PMSG to estrus and superovu 1 a tory response. Hormonal characterization in cows bearing more than three CL post-PMSG at laparotomy (see figure 7 for experiment 1) showed plasma progesterone exhibited an expected secretion curve prior to PMSG treat ment after which a 50^ increase was observed by 2k hr post-gonadotrop i administration. Simultaneously, a substantial increase in plasma 72

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73 estrogens and LH was also recorded with no significant change in 206" dihydroprogesterone levels. The hormonal trends after the luteolytic dose of PGFgni 24 hr post-PMSG were similar to those observed during CL regression in the normal cycling cow with an increase in plasma estrogens being correlated to the apparent increase in follicular growth stimulated by the PMSG. Although plasma LH was variable, the LH surge was 12 hr earlier in cows with more than one CL compared to those with only a single CL following PMSG treatment. The initiation of the PMSG-PGF2ct' nduced cycle was characterized by a rather significant (P<.01) increase in plasma progesterone which reached a maximum of ]8.kk A. 59 ng/ml on the day of injection of kO mg PGF2(j. A dramatic decline in plasma LH and estrogen levels was observed pos t-es t rus and remained at base line thereafter. The plasma 20B-d i hyd rop roges te rone trend did not change significantly (P>.10) during the course of this study. When progesterone and 206-dihydroprogesterone ratios were computed, hov/ever, a significant (P<.01) trend was detected suggesting a relationship between P and 2O3-P. The lowest ratios of 2O3-P/P were 13 to 15^ during the luteal phase and increased to 80 to 100^ when cows were approaching estrus. At estrus, however, 20B-P levels were always higher than P. The experimental design did not reveal whether this 2O3-P was a result of PMSG induced 1 ute i n i zat ion of the granulosa cells or from luteal cells of the regressing CL. The administration of ^40 mg PGF2ct to cows with greater than three CL resulted in a dramatic decline in plasma progesterone concentrations and the absence of palpable CL. This dose, however, was not adequate to induce abortion and to sustain plasma progesterone at basal

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concentrations for an adequate time to permit the animal to express estrus. In conclusion, these data clearly demonstrated that the endocrinology of the PMSG superovulated cow differs from that of the normal cycling cow. Not only the magnitude of the hormonal secretion is different, but the length of the interval from PMSG to estrus and supgrovulatory response are affected. In addition, the early LH surge and early increase in LH post-PMSG treatment might be partia-lly due to differential follicular growth and estrogen secretion in response to gonadotroph in administration. This experiment, however, was not designed to test the hypothesis of differential follicular growth postPMSG. Research designed to test the hypothesis of differential follicular-growth post-PMSG may explain the etiology of these differential hormonal patterns associated with inducing multiple fetuses in cattle. The introduction of ^0 mg PGF2a into this hormonal treatment regime first appeared to be an ineffective avenue to induce abortion in the PMSG-bred cow. The efficiency of hO mg PGFgrx to induce CL regression and subsequent abortion in cows, with multiple CL was tested in two additional experiments. In the second experiment 13 animals received two sequential treatments of 33-5 mg PCFg^^ 12 days apart. Ten cows were administered 2,000 i.u. PMSG 2k hr prior to the second PGFg^ injection. Ovulation rate was determined by supravaginal laparotomy 13 days post-PMSG and cows were given kO mg PGF2p(. Regression of CL was monitored by bleeding all animals daily for 11 days starting 2 days prior to laparotomy and analyzing for plasma P concentrations. Administration of kO mg PGF2ct, 13 days post-PMSG, evoked a dramatic decline in plasma

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P in both PMSG and non-PMSG treated cows. Plasma P reached basal concentrations by 96 hr post-'^O mg PGF2ciOvarian palpation 7 days postlaparotomy confirmed multiple CL regression. Shortly thereafter cows expressed estrus. In conclusion, this experiment demonstrated that administration of mg PGF2a, 13 days post-PMSG, was effective in regressing multiple CL, in superovulated cycling cows, when measured by a dramatic decline in plasma P, the absence of palpable CL 7 days post-treatment, and reinitiation of a new estrous period. In the third experiment ^45 cows were hormonally treated as in experiment 3. In addition, cows were divided into four factorial groups involving PMSG and MATING as main factors. Cows assigned to breeding were exposed to bulls for a 10 day breeding period starting the day of the second synchronizing dose of ^^^2a'^^^ reduction in plasma progestin following kO mg P^^2a significantly (P<.01) affected by PMSG treatment, with P declining at a slower rate. Although plasma P reached a basal concentration by 96 to 120 hr post-PMSG, behavioral estrus was observed in a cascade type fashion with 91^ of the non-PMSG cows, responding within the first 7 days posttreatment vs of the PMSG-treated animals, responding within 5 to 10 days post-treatment while 27% responded 11 to 15 days post-treatment. Post-mortem examination of reproductive tracts 50 days post-treatment confirmed multiple CL regression, reinitiation of cyclicity, and no evidence of pregnancies. In conclusion, data accumulated in this experiment clearly suggest that profound effects at both the ovarian and endocrine levels in the bovine female in response to PMSG could be modulated by PGF2aThus

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/6 the introduction of kO mg PGF2ct 13 days post-PMSG to superovul ated preg nant cows might result in a means to prevent fetal wastage from the superovulation effect by bringing cows with more than three CL to a pre mature abortion and rebreeding them to a bull in the norma! breeding season. Trials need to be established as to whether this may be a means by which a herd of cows could be successfully induced to have a 100 to 110^ calf crop through the induction of limited multiple births.

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APPENDIX

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TABLE 10. PREPARATION AND STORAGE OF ASSAY BUFFER To a 2 1 volumetric flask add: 32.7 g of sodium phosphat-e dibasic heptahydrate (MW = 268) 10.8 g of sodium phosphate monobasic mono hydrate (MW = I38) 18.0 g of sodium chloride (MW = 58) 2.0 g of sodium azide^ (MW = 65) Then add 2X d ist i 1 led-tap water (pH = 7-0 + 0.1) to a total volume of 2 1. Adjust to pH = 7.20 0.01 adding concentrated solution of sodium hydroxide. This stock solution may be stored at kC as long as there is no evidence of mold or bacterial growth. The working assay buffer is prepared by adding O.R Knox gelatin (lOO mg/100 ml) to the above stock solution. The assay buffer should be stored at hC for no longer than k weeks. ^Do not breathe or contaminate skin. Extremely poisonous. Reacts with drain pipe, resulting in residue build up over time. Extremely explosive. 78

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TABLE 11. PREPARATION OF NADH IN 0.1 M TRIS BUFFER A 0.1 M Tris buffer is obtained by mixing 6.057 g hydroxymethy 1 aminomethane in 500 ml d i st i 1 led-tap water (2X) To a 100 ml volumetric flask add 50 ml of this stock solution and 26 ml of 0.1 N HCl. Then add d i s t i 1 1 edtap water (2X) to a final volume of 100 ml Adjust to pH = 8 1 0 0 0 1 To a small vial cylinder add 5 mg NADH (6.'40 iiM) and 3 ml of 0.1 M Tris buffer pH 8. 1

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80 TABLE 12. PLASMA CONCENTRATIONS OF PROGESTERONE, 206-D I HYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING MORE THAN THREE CORPORA LUTEA AT LAPAROTOMY 203-DihydroLute i n i z i ng Day of Progesterone Progesterone hormone Estrogens b 1 eed i ng X S.E. X S.E. X S.E. X S.E. ng/ml ng/ml ng/ml pg/ml 1AM 3. 72 + 0.3h 2. 60 0. 65 0 97 + 0. 1 1 1 52 0. 16 PM 3. 72 + 0.27 2. 3h 0. h8 1 58 + 0. 27 h8 2AM 3. 70 + 0.32 I 96 0, 56 1 Oh + 0. 22 2. 0. 30 PM 3. 88 + 0.39 '1 78 0. 30 0 80 + 0. 08 28 86 3AM (PMSG) 5. 66 + 1.17 1 68 0. h5 0 72 + 0. 08 3. 0. PM 6. 18 + 1.27 2 12 0. 36 3 36 + 0. 39 06 4am (PGF) 9. 06 + 2.99 1 88 0. 18 3 3h + 0. 17 13 62 2. PM 3. 86 + 1.3^* 1 60 0. 36 3 3h + 0. 1 1 5AM 2. 02 + o.eh 1 36 0. 30 2 h2 + 0. 36 30 08 13. 32 PM1 ho + 0.29 1 hh 2. 32 2 8h + 0 31 6am 0. 90 + 0.19 1 ho 0. 27 2h .32 + 9 60 37 04 1 1 hi PM 0. 72 + 0. l^t 1 h8 0 28 5 3h 1 3 13 h2 7AM 0 82 0.09 1 50 0 hi 2 .30 + 0 29 19 60 6. PM 1 00 0.23 1 hi 0 36 2 .'2 + 0 35 8AM 1 3A 0.39 1 52 0 3h 2 .60 + 0 38 1 1 80 3. 91 PM 1 88 O.hS 1 62 0 hS 2 .h2 4 0 27 9AM 2 28 0.55 1 58 0 28 2 .36 + 0 19 2 92 0. 71 PM 2 9'* O.eh 1 he 0 33 2 .18 + 0 22 10AM k 06 0.87 1 5h 0 25 1 .56 + 0 26 2 92 0. 57 PM 5 68 + 1.63 1 ho 0 27 1 .54 + 0 1 1 1 1AM 6 30 1 .37 1 72 0 50 1 .82 + 0 1 1 1 5h 0 09 PM 7.98 1.5'* 1 96 0 37 1 .Sh + 0 22 12AM 10 80 1.^42 2 22 + 0 27 2 .Oh 0 12 2 20 0 20 PM 1^ Oh 1.63 1 88 0 1 1 1 .72 + 0 09 13AM 15 3h ].hO 2 .08 0 32 1 .ho + 0 28 2 20 + 0 15 I'tAM 15 2h 1 .23 2 32 0 19 1 .56 0 10 2 18 0 21 PM 16 30 1.52 2 .eh + 0 35 1 .'6 + 0 17 15AM 16 he 2.36 2 .52 0 h] 1 .36 + 0 08 2 .05 0 18 PM 18 hh h:S3 3 .38 0 h8 1 .ho + 0 15 16AM (PGF) ]h 22 + 2.Sh 3 .10 0 31 1 .50 + 0 19 5 85 0 3h PM 8 he t ].he 2 .82 + 0 35 1 .ho + 0 21 17AM 5 88 + 1.25 2 .26 + 0 hS 1 .7h + 0 .27 h .71 0 ho PM 3 98 + 1.36 1 .86 + 0 53 1 .h8 + 0 .26 18AM 2 90 + 0.83 1 .88 + 0 he 1 .30 + 0 .18 5 .23 0 h] PM 2 96 + 0.88 2 .22 + 0 h3 1 .98 + 0 3h 19AM 2 70 + 1 .05 2 .he + 0 28 1 .28 + 0 29 5 .00 0 50

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81 TABLE 12. (Continued) 20P-D i hydroLuteinizing Day of Progesterone Progesterone hormone Estrogens bleeding x S.E. xS,E. xSE. xSE. )9PM 2.90 1.15 2.84 0.27 1.32 0.29 20AM 2.12 + 0.85 2.1'*0.23 1.1^4 0.15 3-30 0.33 PM 1 .78 0.77 1 .86 + 0.29 1-26 0.17 21AM 1.96 + 0.98 1.90 0.23 1.20 0.23 2.'*5 0.25 PM • 2.22 + 1.20 ].3k 0.32 1.15 0.3'* 22AM 2.62 1.59 2.38 + 0.37 2.38 0.37 2.12 0.71 PM 2.70 1.63 2.2h O.hl 2.1k 0.^42

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82 TABLE 13. PLASMA CONCENTRATIONS OF PROGESTERONE, 208-D I HYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING TWO OR THREE CORPORA LUTEA AT LAPAROTOMY 20B-DihydroLuteinizing Day of Progesterone Progesterone hormone Est rogens b 1 eed i ng X S.E. X S.E. X + S.E. X S.T. ng/ml ng/ml ng/ml pg/ml 1AM 3.85 + 0. 34 2 50 + 0 37 1 23 + 0 1 "7 0 15 + r\ no PM 3.95 + 0.38 2 55 + 0 40 1 1 2o + 0 1 3 0 25 2AM 3.98 + 0. 46 2. 1 0 + 0. 37 0. 73 + 0 09 1 65 + PM A. 28 + 0. 62 '1 80 + 0. 1 7 1 30 0 47 3AM (PMSG) 5. '3 + 1 46 1 23 + 0. 1 5 1 ,45 0. 43 1, 03 + 1 /i o 1 PM 6. 30 + 1 16 1 43 + 0 25 3. 23 + 0 52 hM (PGF) 4.03 + 1 1 1 1 48 + 0. 45 2 DO 0 L Q DO 1 1 1 1 75 + 4.0/ PM 2. 70 + 0. 64 1 65 + 0 59 4. ,23 -t 0 55 Z DO 5AM 1 Op1 .85 + 0. 48 1 0 0 2o + 0 31 3. DO + 0 9 03 + PM. 1 .00 + 0. 1 1 1 23 + 0. 29 3. 1 Q 1 1 0 0 1 /i 1 4 1 • 09 6am 1 .08 + 0. 25 1 .55 + 0 32 16. .73 + 1 1 1 1 1 9 1 ii 50 4PM 0.88 + 0. 09 1 63 + 0. 1 6 1 2 ,25 + 0 0 53 7AM 0.68 + 0. 09 1 ,60 + 0. .07 3. ,13 + 0. 49 5 00 + 0 09 PM 0.65 + 0. ,06 1 ,43 + 0. .13 2 70 + 0. 37 8am 0.85 + 0. ,21 1 .50 + 0. .29 2. ,90 1 0. .31 7 2o + 1 .97 PM 1.18 + 0. ,45 1 .58 0. .33 2 08 1 0. .36 9AM l.'*3 + 0. ,49 1 .83 + 0. .33 2. ,20 i 0, .12 5 .13 + 1.59 PM l.i7 + 0. ,82 1 .83 + 0. ,44 2, .87 0, .77 10AM 2.50 + 0. .37 2, .15 + 0. .34 2, .38 0, .31 4 .98 + 1.16 PM 2.35 + 0.69 2, .05 + 0. .48 2. 18 + 0, .15 1 lAM 2.78 + 0. .70 1 .65 + 0. .43 2, .30 + 0. ,20 1 .95 + 0.37 PM 3.03 + 0, .71 1 .73 + 0. .57 2 .03 + 0. .32 12AM 3.13 + 0, .52 1 ,80 + 0, .51 1 .95 0. .26 1 .54 + 0.35 PM 3.^*5 + 0. .72 2, .03 + 0, ,84 1 .93 + 0, .09 13AM 3.08 0. .43 2, .85 + 0. .97 1 .68 t 0. .27 1 .60 + 0.40 14am 3.88 + 0. ,68 2. ,00 0, ,18 1 .58 0. .10 1 .58 + 0.38 PM 3.63 + 0, .45 1 .85 + 0, .38 1 .50 0, .29 15AM '4.30 + 0. .68 1 .83 + 0, .39 1 .15 + 0, .25 1 .67 + 0.18 PM 4.20 + 0, ,41 2, .00 + 0, ,62 1 .33 0, .30 16am 4.30 + 0. .33 2, .05 + 0, .55 1 .50 0, .34 1 .70 + 0.09 PM 5.45 + 0. .74 2 .15 + 0, .73 1 .85 i 0 .24 17AM 5.60 + 0. .56 2, .48 + 0, .45 0 .98 0 .10 1 .69 + 0.17 PM 4.88 + 0, .36 2 .95 + 0, .49 1 .10 0 .19 18am 4.88 + 0, ,46 2, .65 + 0, .60 0 .50 0 .00 1 .73 + 0.17 PM 5.00 + 0, .27 2, .38 + 0, .34 1 .05 0 .22

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83 TABLE ]h. PLASMA CONCENTRATIONS OF PROGESTERONE, 206-D I HYDROPROGESTERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS WITH ZERO OR ONE CORPORA LUTEA AT LAPAROTOMY 20 e-Di hydro Luteinizing Day of Progesterone Progesterone hormone Est rogens bleeding X + S.E. X S.E. X S.E. X STE. ng/ml ng/ml ng/ml pg/ml 1AM 2 00 + 0 71 1 ] h7 40 O "7 2 / U n nQ o Z DO 1 1Q J J PM 2.60 + 0 r O 58 1 65 0 29 r\ U ob + n 0 /i U 2AM 2.75 + 0. 55 1 71 + 0. 27 0 70 4n no 5 Q 1 y 1 1 uz PM 3.08 + 0. 64 -1 55 + 0 22 1 26 + n oft DO 81 3AM (PMSG) 3.01 + 0. 54 1 72 + 0 1 2 0 /o n 1 0 U 1 Z o i 4n u PM 3.65 + 0. 51 1 Q Q 00 + 0 25 Z /in n 9 Q u zy km (PGF) 3.23 + 0. 53 1 Q n oO + 0 2o n z 5 1 U Z 1 r nn 41 1 /y PM 2.10 + 0. 53 1 .71 + 0. 29 3 73 + n L)i 0 04 jO 5AM 1 .47 + 0. 1. 1. 44 1 .67 0 32 Z oU n ofi U zo h H n'j u j -j1 1 PM. 1 .37 + 0. 40 1 .95 + 0 59 3 00 0.19 Z 4 6am 1 .07 + 0. 32 2 1 2 + 0 74 3 1 0 4r\ CO 0. by 4 7.9. 1 1 PM 0.88 + 0. 49 2 n O 2o + 0. 79 D dO 3 3o 7AM 0.8o + 0. 32 2 20 + 0 oO Z 0/ + C\ 1 Q i n c: Ub n u 7 1 PM 1 .21 + 0. 59 2 40 + 0 59 2 D 1 + 0.13 DO 8am 1 .45 + 0. 78 2 • 33 + 0 -70 7o 2 ) C 40 40. z 1 i 0 A zu n U PM 1.13 + 0. 47 2 20 0 o5 2 1 / U 1 o 9AM 0.83 + 0. 42 2 .05 + 0 65 2 22 + 0.22 2 .60 + 0 70 PM 0.80 + 0. 17 .63 + 0 29 2 40 + 0.15 lOAM 1.45 + 0. 58 .68 + 0 27 1 68 + 0. 12 2 .35 + 0 52 PM 1.37 + 0. 48 .70 + 0 20 2 03 + 0.14 1 1AM 1 .80 + 0 52 .95 + 0 30 2 43 + 0.29 1 .75 + 0 17 PM 1.95 + 0 49 .53 :h 0 22 2 28 + 0.21 12AM 1.88 + 0 47 .63 + 0 27 2 27 + 0.14 1 .09 + 0 10 PM 1.75 + 0 31 .38 + 0 14 1 95 + 0.26 08 13AM 1 .90 + 0 47 .20 + 0 20 1 57 + 0.17 1 .10 + 0 14AM 2. 15 + 0 43 .58 + 0 30 1 .72 + 0.30 1 .00 + 0 .08 PM 2.22 + 0 41 .83 + 0 30 1 .60 + 0.19 15AM 2.53 + 0 50 .65 + 0 .27 I .33 + 0.16 1 .37 + 0 .13 PM 2.52 + 0 46 .52 + 0 .31 2 .03 + 0.69 16AM 2.20 + 0 32 .63 + 0 .26 1 .65 + 0.41 1 .40 + 0 .08 PM 2.31 + 0 28 .96 0 .31 2 .36 + 0.79 17AM 2.70 + 0 19 .67 + 0 .19 1 .18 + 0.24 1 .43 + 0 .23 PM 2.53 + 0 19 .67 + 0 .29 1 .02 + 0. 19 18am 2.52 + 0 26 .62 + 0 .32 0 .96 + 0.27 1 .40 + 0 .14 PM 2.43 + 0 25 .78 + 0 .32 1 .40 + 0.27

PAGE 97

o DC to LU C3 O CC a. CC o to LU Z3 _l < > CM OC O < to o 1< o' 1x1 to I/) LU £ C3 LU CC 1/1 LU < cy in I< LU DO < Ll. A CC CM DC 0) c c o 1/1 in (U i_ cn -o 4-1 TO E 0) c > O 3 4-1 U 03 O l/l 4-J 0) L. 13 o c Q3 o L3 o to o o o o o o o o o o o o o o o o o o o o o o Ln oo vD CM -T CO o oo CO -T vO CNI LTv o \D CNI oo -3CM CM o o o o O o O o CD X o o I o o o o I Q. O 3 O 1A CJ3 O CM Q. O 3 +-> o 1CM C3 O O O Csl CM X Csl X + CM X (M vD o X CM CM o X LTV X X o o OO O cn o O o o + o + o O o o X + X o o CO CO + + vD X -3+ 1 CM X X o UTv X LA X X vD CO -dvD OO CM o CM o CM 1 O O 1 o o O cri CM 1 1 r~ro + + 1 — CO CM cn r-i Ln v£) LA r-^ o LTV 1 un CNI -cr o II II 11 II II II II II <>~ <> <><> <><> <> <> s: 2: QQ2: z: CM 2: 2: < LA CM Qa. 00 00 vD rA 0 o O 0 4-1 0 0 0 0 *-> -M 4-1 4-1 w 4-1 4-) 2: s: 2: Q_ 2; 2; 2: z: < < LA < DD< -aVD PA 1 — E E E E E E E E O 0 0 0 0 0 0 0 i_ i_ u u \L. 1i_ unMuu14y<+rA — D. O 3 4-1 O 1O

PAGE 98

CQ O O CC LU H LO LU CJ o cc: Cl. o o: Q >z: CQ O Q < CD O cc: o 00 LU q; Q Z < 1/1 z o o' LU C/) (/) LU D£ LU £ l/l LU < o" to LO < A CC CM DC 0) C c o Ul 0) i_ 0) E 1) c > O i_ — O (TJ E 14_ ._ O J-i C 0) o .— L. +-> a) o c Q. D o v_ o CO OQ < C o E i_ O 3: o o o X LA o o o o I rsl X o o + X o < O O — O — cn r-^ csi 1^ ITS X CM O o + X o oo CSI (N II < >< < — v£) E E O O 1L. CL O o <^ 1A CD in c cn o CM O X CO o o o I rvl X -3X LTV O O cn E o LA CM 0^ O X -3O o o I tN X CM CM O + X sD (v^ -dI cn OO CM Q. 2: DCM < 00 CM MD — 2: Z < < — ^ o o CM OA Q. O 4-) o 1CM C3 0 vD LTV LA \0 LA 0 0 Q 0 cn (N CO LA vD CM 0 r-~CM LA 0 0 0 0 CM cn CO X X X X LA rA OA 0 0 0 0 0 0 0 0 0 0 0 0 + 1 + + X X X X CM — 00 0 0 • — • 0 < 0 0 0 0 + + 1 0 -3C rA CO CM CN rA CSI II M II II < >< > < > < >~ s; CL Q. Q. Q. C (N 00 CO CM 0 0 0 0 4-1 4-1 4-> 4-J SI < < < < E E E E 0 0 0 0 i_ i_ 1MuMu_l <_) rA CNI rA Q. 0 Q. CL Ol 4-1 n D 0 0 0 0 1_ 0 11_ u CD CD CD CD a. I CO. o

PAGE 99

A Q. CM 0) o 1/1 t/1 (D s_ CD (1) 0) E (U o 1_ o I/) CNI o LA -do o o o o o o o o o 1^ c o cn oo LTV LA tNI o o o o X CNI o O O o I CM X rr\ O O + OO vD O I CTv un II < >X CPi ro CM + CM CM I II < >CM X vO OO X CO CM CO -cr tNI cn LA CM rA CM CO II < >CM o CNJ c o O LA cri o o CM LA O o o o o O O O o o o o en O CTv cn -Jen OO OO vD CO CA OO • O r-. r-~ • LA VD vD o 1 1^ II II II II II II < > < >< >< > < >~ < >c > o z: Qs: Q< < CNI Q. 3 4-1 LA CM LA U E O o o o 0 U4- 4-1 4J 4-1 4-1 o 4-1 in s: 2: c CD < Q< < < o LA vO u lU E E E E E T3 O o O 0 0 o C i_ u i_ 11QD uuMQ. O O CA LA CD O CM CA Q. O 4-1 o 1CM CJ3 X LA CA + O CM I X -4-Jo I CA nD CA CA II < > X CNI O o o o I CM X CA O O + X CA LA CNJ II 2: z: Q. 2: 2: 2: Q2: < 00 < Q< 00 < \X5 0 0 0 0 0 0 i-i 4-1 4-1 4-1 4-1 4-1 4-1 2: 2; 2: z: z: 2: < < < < a. < LA vD E E E E E E E 0 0 0 0 0 0 0 1u u i_ LL. "4uu"4141414Q. O n 4-1 o 1o C3

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87 TABLE 18. COEFFICIENTS OF CORRELATION BETWEEN PLASMA HORMONAL LEVELS AND NUMBER OF CORPORA LUTEA^ Source Progesterone 203-Dihydroprogesterone Luteinizing hormone Est rogens Group 1 >3CL 0.73'^ (0.0001 )c 0.16 (0.3092) 0.26 (0.3017) 0.68 (0.0001 ) Group 2 2 to 3'CL Q.kl (0.0062) {0.k]]5) 0.29 (0.2280) O.hS (0.0008) Group 3 0 to 1 CL 0.21 (0. 1822) 0. 1 1 (O.A8A6) 0.35 (0.0837) 0.42 (0.0053) ^Expressed upon grouping criteria (see text). ^Correlation coefficient. '^Probabi 1 i ty.

PAGE 101

88 TABLE 19. PLASMA CONCENTRATIONS OF PROGESTERONE AFTER kO MG PGFaa'^ IN COWS TREATED WITH OR WITHOUT PMSG Progesterone Day of bleeding (ng/m 1 ) PMSG group Non-PMSG group 1 8 79 2 12^ 2 6 0 06 2 10 3^4 2 22 2 3 0 06 3 (PGF) 10 02 1 89 3 0 0 06 i 59 0 67 2 I 0 03 5 2 55 0 28 1 9 0 03 6 1 77 + 0 18 1 5 +0 17 7 0 62 0 07 0 k7 0 15 8 0 61 0 10 0 47 0 22 9 0 71 0 06 0 87 0. 15 10 0 93 0 Ok 1 07 0. 12 S.E.

PAGE 102

89 TABLE 20. SPLIT-PLOT ON TIME ANALYSISOF VARIANCE OF PROGESTERONE IN EXPERIMENT 2 Source df Sums of Squares Mean Square F value TotaJ 103 1 185.73 Treatmen t 1 26.02 26 02 3.29>v>'c Cow (treatment) 1 1 87.09 7 92 Day 7 6't7.97 96 k2 25. 19-;.--;.Treatmentxday 7 102.96 lit 71 3. Bit--Cow ( t reatmen t ) xday 77 29^4.69 3 83 ^--(P<.05) ••(?<. 01 )

PAGE 103

90 TABLE 21. LEAST SQUARES REGRESSION EQUATIONS AND VALUES FOR PROGESTERONE Source Estimated regression line r2 PR>F PMS Group y = 39. 12 13.99X + 1 .73x^ 0.07x^ 0.7323 0.001 Non-PMS Group y = 2.h + 0.75x 0.26x^ 0.8697 0.001

PAGE 104

91 a. X o < or < > O CO 00 > < < CM CO < A 0) > (U i_ 03 D D" (/I E D CO u(U u 1a o I/) 3CM — — <^ O -3— O O O O vD OO O O O CTi — O O O O O O O O O -3" O CTl vD VD Ln o 1^ un oo en o LA cn en -aLA — o \£> o v£) O LA rA CM CO \£) OA vD OA rA CM v£) O CNI r~--3— -3CM I— C5 CD CO 1-J C3 "D C OO 0) X E Q. CQ CL 4-1 03 (1) -J cy o >->->fo ta fD Q Q Q cn cr\ 3c OO cn CM rA OA o VD LA CM r-~CM OA cn LA vO CM OO — LA CM CM -3OA 00 PA rtl Q 03 tH O X 3 3 u o o oj H<->(_) _J -:3CM o -3O a o o o LA — o o V V

PAGE 105

92 CM LA — — -3CM LA CTl cn 00 CM CM CM — 0 — — 0 0 0 -a— • • • 0 0 0 0 0 0 0 0 0 0 0 0 1 Q. i+ 1 + 1 + 1 +1 +1 +1 + 1 + 1 +1 +1 +1 oc 0 C LLl 10 LA ^ 1^ LA CO vD -3' — vD Q. CJ u 1^ CM vD rA • — D0 01 C Li. Cs! 1X1 eed (PG —I DO CM rA LA 00 CA 0 < 1Day of

PAGE 106

93 TABLE 2h. LEAST SQUARES REGRESSION EQUATIONS AND VALUES FOR PROGESTIN Source Estimated regression line R PR>F PMSG + breeding y = 32.22 1 0 90x + 1 29x2 0.05x^ 0. 66^12 0.001 PMSG +'no breeding y = 37.22 ]h.33x + 2.00x2o.09x^ 0.5781 0.001 No PMSG + breeding y = 6.3^ 1.28x + O.OSx^ O.83OI 0.001 No PMSG + no breeding y = 5-26 0.96x + O.OSx^ 0.7693 0.001

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LITERATURE CITED Abraham, G.E., R. Swerdloff, D. Tulchinsky, and W.D. Odell. 1971. Rad ioimrtiunoassay of plasma progesterone. J. Clin. Endocr. 32:619. Archbald, L.F. 1976. Superovulat ion in the cow using pregnant mare's serum gonadotropin and prostaglandin F2a. Vet. Med. Small Anim. Clin. 71:953. Barr, A.J., J.H. Goodnight, J Sail, and J.T. Helwig. 1976. A user' guide to SAS 76. Sparks Press. Raleigh, North Carolina. Barraclough, C.A., R. Colla, R. Massa, and L. Martini. 1971. Temporal interrelationships between plasma LH, ovarian secretion rates and peripheral plasma progestin in concentrations in the rat; Effect of nembutal and exogenous gonadotropins. Endocr. 88:1^37. Bellows, R.A., D.C. Anderson, and R.E. Short. 1969Doseresponse relationships in synchronized beef heifers treated with follicle stimulating hormone. J. Anim. Sci 28:638. Bellows, R.A., R.E. Short, N.H. Wiltbank, and O.F. Pahnish. 1970. Mul tiple births and artificial rearing of calves. J. Anim. Sci. 30: 1030. (Abstr.). Brandau, H. and G. Mutzke. 1972. Activity patterns of hydroxysteroid dehydrogenases in the corpus luteum of the cow. Acta Endocr. Supp. 159:16. (Abstr.). Brandau, H., L. Brandau, and Mutzke. 1972. Cyclische schwankunger der aktivitaten von hydroxysteroid-dehydrogenase in corpus luteum. Des Rindus. Acta Endocr. 69:369Brock, H. and I.E. Rowson. 1952. The production of viable bovine ova. J. Agr ic. Sci ^42: I 79Burrell, W.C. and J.N. Wiltbank. 1977CL regression in pregnant and cycling cows. 69th Annual Meeting. Am. Soc. Anim. Sci. (Abstr.) Casida, L.E., R.K. Meyer, W.H. McShan and W. Wisnicky. ]3k3. Effects of pituitary gonadotropins on the ovaries and the induction of superfecund i ty in cattle. Am. J. Vet. Res. A : 76 Castenson, P.E., A.M. Sorensen, Jr., C.R. Cobos, and J.L. Fleeger. 1976 Source of postpartum P and 20B-0HP preceding estrus in heifers. J. Anim. Sci. ^3:277(Abstr.). 94

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95 Chenault, J.R. 1973. Transitory changes in plasma progestins, estradiol and LH approaching ovulation and after prostaglandin F2a injection in the bovine. M.S. Thesis. University of Florida. Chenault, J.R., W.W. Thatcher, P.S. Kalra, R.M. Abrams, and C.J. Wilcox. 1976. Plasma progestins, estradiol and luteinizing hormone following prostaglandin F^a injection. J. Dairy Sci. 59:13'*2. Cooper, M.J. 197^*. Control of oestrus cycle of heifers with a synthetic prostaglandin analogue. Vet. Rec. 95:200. Cooper, M.J. and P. Jackson. 1975. Control of the bovine oestrus cycle with a synthetic prostaglandin analogue I C I -80996-prel iminary field experiences in lactating beef cattle. Proc. Br. Soc. Anim. Prod. ^:115. Cupps, P.T. 1969. Steroid biosynthesis by the bovine ovary. J. Anim. Sci 29: 187. (Abstr. ) Cupps, P.T., G.B. Anderson, M. Drost, B. Darien, and M.B. Norton. 1976. Estrus synchronization of heifers with PGF2cc JAnim. Sci. 43: 280. (Abstr.). Curr.ie, W.B. and G.D. Thorburn. 1973Induction of premature parturition in goats by prostaglandin Fga administered into the uterine vein. Prostaglandins. ^4:201. Dawson, F.L.M. I96I. Corpus luteum enucleation in the cow = Therapeutic and traumatic effects. Vet. Rec. 73:661. Dickey, J.F., D.M. Henricks, and J.R. Hill. 1973. Gonadal hormones in PGFja/PMS treated heifers. J. Anim. Sci. 37:307. (Abstr.). Diehl, J.R. and B.N. Day. 1973. Effect of prostaglanin F2a on luteal function in swine. J. Anim. Sci. 37:307. (Abstr.). Dobson, H., M.J. Cooper, and B.J. A. Furr. 1975. Synchronization of estrus with ICI 79,939, and analogue of PGF2a, and associated changes in plasma progesterone, oestradiol 176 and LH in heifers. J. Reprod. Fertil. k2:]k]. Douglas, R.H., E.L. Squires, and O.J. Ginther. 197^*. Induction of abortion in mares with prostaglandin F2. J. Anim. Sci. 39:^*04. Do.wling, D.F. ]3k3. Problems of the transplantation of fertilized ova. J. Agric. Sci. 39:37^+. Ellicott, A.R. and C.E. Thompson. 1976. Estrous control using progesterone implants. J. Anim. Sci. ^3:282. (Abstr.). Erb, R.E. and F. Stormshak. I96I Progestins in corpora lutea, ovaries, and adrenal after estrus and breeding of normal and abnormal cows. J. Dai ry Sci A^t.-SSS.

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9b Erb, R.E., V.L. Estergreen Jr., E.R. Gomes, E.D. Plotka, and O.L. Frost. 1968. Progestin levels in corpora lutea and progesterone in ovarian venous and jugular blood plasma of tine pregnant bovine. J. Dairy Sci 51 :'*01 von Euler, R.S. 193^4. Zur l
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97 Gomez, W.R., V.L. Estergreen, Jr., O.L. Frost, and R.E. Erb. 1963Progestin levels in jugular and ovarian venous blood, corpora lutea, and ovaries of the nonpregnant bovine. J. Dairy Sci. '*6:553. Gomez, W.R., O.L. Frost, and V.L. Estergreen, Jr. 1962. Progestins in ovarian and peripheral blood of cows during late pregnancy. J. Dai ry Sci ^(5:670. (Abstr. ) Goodman, A.L. and J.D. Neill. 1976. Ovarian regulation of postcoital gonadotropin release in the rabbit: Reexamination of a functional role for 20a-Dihydroprogesterone. Endocrinol. 99:852. Goodman, A.L., W.E. Nixon, D.K. Johnson, and G.D. Hodgen. 1977. Regulation of fol 1 iculogenos is in the cycling Rhesus Monkey: Selection of the dominant follicle. Endocrinol. 100:155. Gordon, I., G. Williams, and J., Edwards. 1962. The use of serum gonadotrophin (P.M.S.) in the induction of twin-pregnancy in the cow. J. Agric. Sci. 59:1^*3. Gorski, J., O.V. Dominguez, L.T. Samuels, and R.E. Erb. 1958a. Progestins of the bovine ovary. Endocrinol. 62:23'*. Gorski; J., R.E. Erb, W.M. Dickson, and H.C. Butter. 1958b. Sources of progestins in the pregnant cow. J. Dairy Sci. ^1:1380. Guthrie, H.D., D.M. Henricks, and D.L. Handlin. 197^Plasma hormone levels and fertility in pigs induced to superovulate with PMSG. J. Reprod. Fertil. 41:361. Gutknecht, G.D., J.C. Cornette, and B.B. Pharriss. 1969. Anti-fertility properties of prostaglandin F2a. Biol. Reprod. 1:367. Hafs, H.D. 1975Onset of estrus and fertility of dairy heifers and suckled beef cows treated with prostaglandin F2aAnim. Prod. 21:13. Hafs, H.D. and D.T. Armstrong. I968. Corpus luteum growth and progesterone synthesis during the bovine estrous cycle. J. Anim. Sci. 27:13'*. Hafs, H.D., J.G. Manns, and B. Drew. 1975a. Onset of estrus after prostaglandin F2a in cattle. Vet. Rec. 96:13'*. Hafs, H.D., J.G. Manns, and G.E. Lamming. 1975b. Fertility of cattle from A! after PGFjaJAnim. Sci. 41:355(Abstr.). Hallford, D.M., E.J. Turman, R.P. Wettemann, C.E. Pope, and D.C. Meyerhoef fer 1975a. Reproductive response of the bovine to PMSG. J. Anim. Sci. 40 : 1 87 (Abstr.). Hallford, D.M., E.J. Turman, R.P. Wettemann, and C.E. Pope. 1975b. Plasma LH and estradiol in the bovine after PMSG. J. Anim. Sci. 41-356 (Abstr.)

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98 Hammond, J. ^ShS. I nduced tw in ovu 1 at i on and multiple pregnancy in cattle. J. Agric. Sci. 39:322. Harrison, R.J. 19^6. The early development of the corpus luteum in the mare. J. Anat. 80: 160. Harvey, W.R. I96O. Least squares analysis of data with unequal subclass numbers. U.S.D.A. ARS-20-8. Hayano, M. M.C. Lindberg, M. Wiener, H. Rosenkrantz, and R.I. Dorfman. 1975. Steroid transformat ions by corpus luteum tissue. Endo— cr inol 55: 326. Henderson, K.M. and K.P. McNatty. 1975'. A biochemical hypothesis to explain the mechanism of luteal regression. Prostaglandin 9:779Henricks, C.H. 1972. Prostaglandins and therapeutic abortion = Summary of present status,. J. Reprod. Med. 9:^65. Henricks, D.M. J.R. Hill, Jr., J.F. Dickey, and D.R. Lamond. 1973Plasma hormone levels in beef cows with induced multiple ovulations. J. Reprod. Fertil. 35:225. Henricks, D.M., N.C. Rawlings, A.R. Ellicott, J.F. Dickey, and J.R. Hill. 1977. Use of prostaglandin fza to induce parturition in beef heifers. J. Anim. Sci. kk:h3S. Hill, J.R. Jr., J.F. Dickey, and D.M. Henricks. 1973Gonadal hormones in PGFgfj-PMS treated heifers. J. Anim. Sci. 37:307. Hill, J.R. Jr., T. Gimenez, A.R. Ellicott, W.R. Boone, and D.M. Henricks 1976. Ovulation in cows after PGF2a and PMSG treatment. J. Anim. Sci ^43:289. (Abstr. ) Hill, J.R. Jr., D.M. Henricks, J.F. Dickey, and D.R. Lamond. 1972. Pro gesterone and estrogen profiles in multiple ovulated heifers. J. Anim. Sci. 35:2^45. (Abstr.). Milliard, J., R. Penardi, and C.H. Sawyer. 1967. A functional role for 20a-hydroxypreg-'4-en-3-one in the rabbit. Endocrinol. 80:90I. Ichikawa, S., H. Morioka, and T. Savada. 1971Identification of the, neutral steroids in the ovarian venous plasma of LH-st imulated rats. Endocrinol. 88:372. Inskeep, E.K. 1973Potential uses of prostaglandins in control of reproductive cycles in domestic animals. J. Anim. Sci. 36:11^9. Jackson, P.S. and M.J. Cooper. 1977The use of cloprostenol for the termination of pregnancy and the expulsion of mummified fetus in cattle. Vet. Rec. 100:361. Jdchle, W. 1973. Cort icoidinduced parturition in domestic animals. Annu. Review Pharmacol. 13:33-

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99 Kazama, N. and W. Hansel. 1970. Preovulatory changes in the progesterone level of bovine peripheral plasma. Endocrinol. 86:1252. Kidder, H.E., G.R. Barrett, and L.E. Casida. 1952. A study of ovulation in six familes of Hoi stein-Fr ies ians J. Dairy Sci. 35:'<36. Kidwell, W.R., K. Balogh, Jr., and W.G. Wiest. 1966. Effects of luteinizing hormones on g 1 ucose-6-phosphate and •20a-hydroxysteroid dehydrogenase activities in superovu 1 a ted rate ovaries. Endocrinol. 79:352. La Croix, E.,'W. Elchaute, and 1. Leusen. 197^. The b iosynthes i s^^of estrogens by cow follicles. Steroids 23:337Lamond, D.R. 1972. Hormonal induction of multiple ovulation in the bovine. J. Anim. Sci. 3':901. (Abstr.). Lamond, D.R. and R.G. Gaddy. 1972. Plasma progesterone in cows with multiple ovulations. J. -Reprod. Fertil. 29:307. Lamond, D.R., R.V. Tombinson, M. Drost, D.M. Henricks, and W. Jochle. 1973Studies of Prostaglandin F 2a the cow. Prostaglandin i*:269. Laster, D.B. 1972. Folicular development in heifers infused with follicle-stimulating hormone. J. Reprod. Fert. 28:285. Laster, D.B. 1973Ovulation, fertility, and prenatal mortality in heifers treated with PMSG (pregnant mare serum gonadotropin) or porcine FSH. J. Reprod. Fertil. 33:275Laster, D.B., E.J. Turman, D.F. Stephens, and R.E. Renbarger. 1971a. Superovulat ion of beef cows and heifers by injection of pregnant mare serum (PMS) following estrus synchronization by means of an oral progestogen. Misc. Pub. Agric. Exp. Sta. Okla. State Univ. 85:24. Laster, D.B., E.J. Turman, D.F. Stephens, and R.E. Renbarger. 1971b. Ovulation rates of beef cows and heifers treated with equine gonadotropin (PMS) and chorionic gonadotropin (HCG) J. Anim. Sci. 33:4'*3Lauderdale, J.W. 1972. Effect of PGFj^^ on pregnancy and estrous cyle of cattle. J. Anim. Sci. 35:2^6^ Lauderdale, J.W. 197'^Distribution and biological effects of prostaglandin. J. Anim. Sci. 38:22 (Suppl. l). Lauderdale, J.W., B.E. Seguin, J.N. Stellflug, J.R. Chenault, W.W. Thatcher, C.K. Vincent, and A.F. Layancano. 197'*Fertility of cattle following PGEj^ injection. J. Anim. Sci. 38:96'*. Lobel B.L. and E. Levy. 1968. Enzymatic correlates of development, secretory function and regression of follicles and corpora lutea in the bovine ovary. Acta Endocr. Suppl. 132:7-

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100 Lopez-Barbel la, S.R., A.C. Warnick, T.H. Wise and M.J. Fields. 1976. Prostaglandin to regress multiple CL in cov/s. J. Anim. Sci. ii3:273. (Abstr.). Lopez-Barbel la, S.R., A.C. Warnick, T.H. Wise, D.R. Hardin, T.R. Thompson, and M.J Fields. 1977PGF2a: regression of multiple CL in bred vs cycling cows. 69th Annual Meeting. Am. Soc Anim. Sci. (AbstrTT. Louis, T.M., H.D. Hafs, and D.A. Morrow. 1972a. Estrus and ovulation 'after uterine PGF2a in cows. J. Anim. Sci. 35:2^)7(Abstr. Louis, T.M., H.D. Hafs, and D.A. Morrow. 1972b. Estrus and ovulation after PGFja in cows. J. Anim. Sci. 35:1121. (Abstr.). Louis, T.M., H.D. Hafs, and D.A. Morrow. 197'a. Intrauterine administration of prostaglandin F2a in cows: progesterone, LH, estrus and ovulation. J. Anim. -Sci. 38:3'*7. Louis, T.M., H.D. Hafs, and B.E. Seguin. 1973. Progesterone, LH, estrus and ovulation after prostaglandin F2a in heifers. Proc. Soc. Exp. Biol. Med. li*3:152. Louis, T.M., J.N. Stellflug, H.A. Tucker, and H.D. Hafs. I97'*b. Plasma prolactin, growth hormone, luteinizing hormone and glucocorticoids after prostaglandin F2a in heifers. Proc. Soc. Exp. Biol. Med. 147:128. Mapletoft, R.J., D.R. Lapin, and O.J. Ginther. 1976. Luteotropic effect at ovarian level in pregnant ewes. J. Anim. Sci. ^43:296. (Abstr.). Mares, S.E., R.G. Zimbelman, and L.E. Casida. 1962. Variation in progesterone content of the bovine corpus luteum of the estrual cycle. J. Anim. Sci. 21:266. Menino, A.R. Jr., and R.W. Wright, Jr. 1977. Synchronization and superovulation in cattle: A method for multiple embryo production. 69th Annual Meeting. Am. Soc. Anim. Sci. (Abstr.). Mikhail, G., J. Zander, and W.M. Allen. 1963Steroids in human ovarian vein blood. J. Clin. Endocrinol. 23:1267Moody, E.L. and J.W. Lauderdale. 1977Fertility of cattle following PGF2a controlled ovualtion. 69th Annual Meeting. Am. Soc. Anim. Sci. (Abstr.). Nett, T.M., D.W. Holtan, and V.L. Estergreen. 1973Plasma estrogens in pregnant and post-partum mares. J. Anim. Sci. 37:962. Novy, M.J. and M.J. Cook. 1973. Redistribution of blood flow by prostaglandin F2a in the rabbit ovary. Am. J. Obstet. Gynecol. 1 17:381

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lot Oxender, W.D., P. A. Noden T.M. Louis, and H.D. Hafs. 197^. A review of Prostagland in f 2a for ovulation control in cows and mares. Am. J. Vet. Res. 35:997. Perkins, J.R., D. Olds, and D.M. Seath. 195'4A study of 1,000 bovine genitalia. J. Dairy Sci. 37:1158. Pharris, B.B. and L.J. Wyngarden. 1969The effect of prostaglandin F2a on the progesterone content of ovaries from pseudopregnant rats. Proc. Soc Exp. Biol. Med. 130:92. Plotka, E.D., R.E. Erb, C.J. Callahan, and W.R. Gomes. 1967. Levels of progesterone in peripheral blood plasma during the estrous cycle of the bovine. J. Dairy Sci. 50:1158. Rajakoski, E. I96O. The ovarian follicular system in sexually matur^ heifers with special reference to seasonal, cyclical and leftright variations. Acta Ejidocrinol. Suppl 52:3'*Rajamahendran, R., P.C. Lague, and R.D. Baker. 1976. Plasma progesterone levels in cycling, and gonadot roph i n-Pros tag 1 and i ntreated heifers. Can. J. Anim. Sci. 56:37. Reynolds, W.L., R.A. Bellows, T.M. De-Rouen, and D.C. Meye rhoef f er 1970. Cow response to FSH treatment. J. Anim. Sci. 31:229. (Abstr.). Roche, J.F. 1974. Synchronization of oestrus and fertility following artificial insemination in heifers given prostaglandin F2a^" Reprod. Fertil. 37:135. Rodriguez, T.R. 197't. Fertility of the synchronized ovulation in the bovine following PGF2a"Tham Salt and GnRH. Thesis. University of Florida, Gainesville, FL. Romanoff, E.B. I966. Steroidogenesis in the perfused bovine ovary. J. Reprod. Fert. Suppl. 1:89. Rowson, L.E.A., R.A.S. Lawson and R.M. Moor. 1971Production of twins in cattle by egg transfer. J. Reprod. Fertil. 25:261. Rowson, I.E. A., H.R. Tervit, and A. Brand. 1972. The use of prostaglandins for synchronization of oestrus in cattle. J. Reprod. Fertil. ^46:1^5. Sasser, R.C. and P.T., Cupps. 1969Conversion of pregnenol one-'-^'*C to progesterone-^-^^C by bovine luteal tissue at selected stages of the estrous cycle. J. Dairy Sci. 52:217Savard, K. and G. Telegdy. I965. Steroid formation in the bovine corpus luteum. Steroids Suppl. 2:205Scanlon, P.F. 1972a. Frequency of transuterine migration of embryos in ewes and cows. J. Anim. Sci. 3^:791-

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102 Scanlon, P.P. 1972b. Ovarian response of cows following pregnant mare serum gonadotroph in treatment during two successive estrous cycle. J. Dairy Sci 55:527. Scanlon, P., J. Sreenan, and I. Gordon. 1968. Hormonal induction of superovulation in cattle. J. Agric. Sci. 70:179. Schilling, E. and W. Holm. 1963Investigation on induction of limited multiple ovulation in cattle. J. Reprod. Fertil. 5:283. Schwartz, F.L; and D.R. Shelby. 1969Induced multiple ovulation-in the bovine. J. Anim. Sci. 29:198. (Abstr.). Sholl, S.A. and R.C. Wolf. 197^. Quantification of 20aand 20e-dihydro progesterone in plasma of the pregnant Rhesus Monkey. Steroids 23:269. Short, R.V. 1962a. Steroid concentrations in normal follicular fluid and ovarian cyst fluid from cows. J. Reprod. Pert. ':27. Short, R.V. 1962b. Steroids present in the follicular fluid of the cow J. Endocrinol. 23:'*01. Smith, J.G., J. Hawks, and R.V. Short. I969. Biochemical observations on the corpora lutea of the African elephant, Loxodonta africana J. Reprod. Perti 1 20: 1 11 Smith, L.E., G.D. Sitton, and C.K. Vincent. 1973Limited injections of follicle stimulating hormone for multiple births in beef cattle. J. Anim. Sci. 37:523. Snook, R.B., M.A. Brunner, R.R. Saatman, and W. Hansel. 1969The effect of antisera to bovine LH in hysterectomized and intact heifers. Biol, of Reprod. 1:'*9Spears, L.L., A.B. Vercovitz, W.L. Reynolds, J.L. Kreider, and R.A. Godke. 197^. Induction of parturition in beef cattle and estradiol and PGFpaJAnim. Sci. 39:227. (Abstr.). Spilman, C.H., G.E. Seidel, Jr., L.L. Larson, G.R. Vukman, and R.H. Poote. 1973Progesterone, 20p-Hyd roxypreg-'-en-3 one, and Luteinizing hormone levels in superovul ated prepuberal and postpuberal cattle. Biol, of Reprod. 9:116. Sprague, E.A., M.L. Hopwood, G.D. Niswender, and J.N. Wiltbank. 1971 Progesterone and luteinizing hormone levels in peripheral blood of cycling beef cows. J. Anim. Sci. 33:99Stabenfeldt, G.H., L.L. Ewing, and L.E. McDonald. 1969Peripheral plasma progesterone levels during the bovine oestrus cycle. J. Reprod. Fertil. 19:'33.

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10j> Stacy, B.D., R.T. Gremmell, and G D. Thorburn. 1976. Morphology of the corpus luteum in the shrep during regression induced by prostaglandin F2aBiol. Reprod. 1^:280. Staigmi 1 ler R.B., R.A. Bellows, R.E. Short, and J.B. Carr. 1976. Ovarian response to FSH injections in beef cows, J. Anim. Sci. A3(l):306. (Abstr.). Staples, R.E. and W. Hansel. I96I. Luteal function and embryo survival in the bovine J. Dairy Sci. kk:2kO. Steel, R.G.D. and J.H. Torrie. I96O. Principles and procedures of statistics. McGraw-Hill Book Company, Inc., New York, NY. Stellflug, J.N., T.M. Louis, B.E. Seguin, and H.D. Hafs. 1973. Luteolysis after 30 or 6O mg PGF2a heifers. J. Anim. Sci. 37:330. (Abstr.). Tam, W.H. 1971. The production of hormonal steroids by ovarian tissues of the chinchilla ( Chinchilla laniger ). J. Endocrinol. 50:267. Telegdy, G. and K. Savard. I966. Steroid formation in vi tro in rabbit ovary. Steroids 8:685. Tervit; H.R., L.E.A. Rowson, and A. Brand. 1973Synchronization of oestrus in cattle using a prostaglandin F2aanalogue ( I C I -79 939) • J. Reprod. Fertil. 3't:179. Thatcher, W.W. and J.R. Chenault. 1976. Reproductive physiological responses of cattle to exogenous prostaglandin F2a^Dairy Sci. 59:1366. Thornburn, G.D. and J.R.S. Hales. 1972. Selective reduction in blood flow to the ovine corpus luteum after infusion of PGF2a into a uterine vein. Proc. Austral. Phys.&Phar. Soc. 3:1'*5(Abstr.). Tribble, R.L. 1973Peripheral serum progesterone, 1 7-hyd roxyprogesterone and 20g-hydroxyprogesterone levels in suckled and nonsuckled primiparous Hereford heifers. Ph.D. Dissertation. Texas A&M Un i vers i ty Turman, E.J., D.B. Laster, R.E. Renbarger, D.F. Stephens, and R.H. Edwards. 1969The experimental production of multiple births in beef cows by hormone injections. Misc. Pub. Agric. Exp. Sta. Okla. State University. 82:5Turman, E.J., D.B. Laster, R.E. Renbarger, and D.F. Stephens. 1971. Multiple births in beef cows treated with equine gonadotropin (PMS) and chrionic gonadotropin (HCG) J. Anim. Sci. 32:962. Turman, E.J., R.P. Wettemann, T.D. Rich, and R. Totusek. 1975. Estrous synchronization of range cows with PGF2aJAnim. Sci. ^1:382. (Abstr.).

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104 Turman, E.J., R.P. Wettemann, and M.P. Fournier. 1977. Ovarian response of beef cows treated with PMSG during two consecutive breeding seasons. 69th Annual Meeting. Am. Soc. Anim. Sci. (Abstr.). Vincent, C.K. and A.C. Mills. 1972. Gonadotropin levels for multiple births in beef cattle. J. Anim. Sci. Z^-.ll Weiss, J.R., H.J. Brinkley, and E.P. Young-. 1976. jn^ vi_tro steroidogenesis in porcine corpora lutea. J. Anim. Sci. 42:121. Wettemann, R.P., H.D. Hafs, L.A. Edgerton, and L.V. Swanson. 1972: Estradiol and progesterone in blood serum during the bovine estrous cycle. J. Anim. Sci. Z^-AOIO. Willett, E.L., P.J. Buckner, andW.H. McShan. 1953. Refractoriness of cows repeatedly superovu 1 ated with gonadotroph i ns J. Dairy Sci. 36:1083. Zerobin, K. W. Jockle, and Ch. Steingruber. 1973. Termination of pregnancy with prostaglandins (E2 (PGE2) and V20. (PGFza) in cattle. Prostaglandins 't:891.

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BIOGRAPHICAL SKETCH Sergio R. Lopez Barbel la was born November 1, 19'5, in Caracas, Venezuela. After graduation from high school in I963, he was accepted in the Facultad de Agronomia, Universidad Central' de Venezuela, where he received the degree of Ingeniero Agronomo in May, 1969In October I969, he entered in the Facultad de Agronomia as instructor of Beef Cattle Production and research assistant. Since September 1973, he has been enrolled in the Graduate Schoolof the University of Florida, with a scholarship granted by the Facultad de Agronomia, Universidad Central de Venezuela. In August 1975 he received the degree of Master of Science in Agriculture from the University of Florida. The author is a candidate for the degree of Doctor of Philosophy. The author married Lourdes P. De Pina in June 1970 and they have two children, Sergio Antonio and Ana Sofia. At the end of his graduate training he will return to Venezuela where he will continue with teaching and research responsibilities in the area of physiology of reproduction in beef cattle. 105

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I certify that I have read this study and that in my opinion it cqnforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Michael J. Fields, Chairman Assistant Professor of Animal Science I certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and qifality, as a dissertation for the degree of Doctor of Philosophy. Alvin C. Warnick Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Fu 1 1 er W. Bazer Associate Professor of Animal Science I certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Wi 1 1 iam W. Thatcher Associate Professor of Dairy Science

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I certify that I have read tiiis study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Pejaver V. Rao Professor of Statistics This dissertation was submitted to the Graduate Faculty of the College of Agr-iculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December, 1977 ture Dean, Graduate School