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

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Endocrinology of the superovulated cow and subsequent prostaglandin Fâ‚‚[alpha] regression of the multiple corpora lutea
Creator:
Lopez Barbella, Sergio Rafael, 1945-
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English
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xiii, 105 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Beef cattle ( jstor )
Cattle ( jstor )
Estrogens ( jstor )
Estrus ( jstor )
Heifers ( jstor )
Hormones ( jstor )
Ovulation ( jstor )
Plasmas ( jstor )
Prostaglandins ( jstor )
Steroids ( jstor )
Animal Science thesis Ph. D
Beef cattle -- Breeding ( fast )
Dissertations, Academic -- Animal Science -- UF
Prostaglandins ( fast )
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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).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
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





84














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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
TABLE 17. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR LUTEINIZING HORMONE
Portion of curve
Source
under estimation
Estimated regression line
R2
PR>F
Group 1
from
1AM
to
5PM
y
_
5.97 Q.68x + 0.03x2 0.0002x3
0.5700
0.001
>3 CL
from
5PM
to
6AM
y
=
- 121.27+ 2.39x
0.3844
0.056
f rom
6AM
to
7AM
y
=
8232.59 248.28x + 1.86x2
0.4107
0.042
from
7AM
to
22PM
y
=
4.84 0.04x + O.OOOlx2
\
0.2591
0.001
Group 2
from
1AM
to
5PM
y
=
17.98 3 26x + 0.23x2 0.007x3 +
2 to 3 CL
O.OOOlx4
0.5419
0.001
from
5M to 6AM
y
=
- 75.11 + 1.51x
0.1960
0.272
from
6AM
to
7AM
y
=
1598.69 47.05x + 0.35x2
O
LO
OO
vx>
0.510
from
7AM
to
18PM
y
=
76.88 3.22x + 0.05x2 0.0005x3
0.5169
0.001
Group 3
from
1AM
to
6AM
y
=
16.92 + 3.55x 0.26x2 + 0.009x3
0.5894
0.052
0 to 1 CL
from
6AM
to
6PM
y
=
-210.4+3-5x
0.9421
0.094
from
6PM
to
7AM
y
=
33.69 0.44x
0.1189
0.118
from
7AM
to
1 8pm
y
=
46.25 1.79x + 0.03x2 0.0002x3
0.2458
0.001


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 over
night. 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 yl 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, ^H-203-P (oxidized to 3P) and 3H-E
facilitated quantification of these steroids. Aliquots of 200 yl and
500 y 1'of the buffered unknown was pipetted (Eppendorf, Brinkman Ins
truments) 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 yl aliquot was adjusted to
500 yl by the addition of 300 yl of gelatinized assay buffer. An ad
ditional 500 yl 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 mini
mum of 4 hr at 4C, followed by addition of 500 yl of charcoal suspen
sion to each tube, vortexed briefly and incubated 20 min at **C. All
assay tubes received this 500 yl 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 8. OVULATION DISTRIBUTION P0ST-PGF2ct INDUCED ESTRUS IN COWS IN EXPERIMENT 3
Behavioral estrus
after second PGF2a
(days)
Ovu1 a tion
in PMSG
treated <
g roups
Ovulat ion
in non-PMSG
trea ted
groups
Rt
ova ry
Lt
ova ry
Overa 11
Rt
ovary
Lt
ova ry
Overa 11
1
A.3
1.2
A. 0
+
1 .0
8.0
1 ,7a
0.0
0.0
1 .0
0.0
1 .0
+
0.0
2
3.7
2.0
3. A
3.1
7.1
A. 6
0.3
0.6
0.7
0.6
1 .0
+
0.0
3
3.5
1-9
2.3
+
1 .0
6.0
2.1
0.6
0.5
0.2
0. A
0.8
+
0.5
A
3.0
0.0
3.0
+
2.0
5.8
2.8
0.6
0.5
0. A
0.5
1 1
+
0.3b
Non respond i ng
0.00
+
0.00
0.60
0.55
Overa 11
6. A5
+
7.5A
0.91
0. A1
ax S.E.
^One cow had a double ovulation.
/
ON


100
Lopez-Barbella, S.R., A.C. Warnick, T.H. Wise and M.J. Fields. 1976.
Prostaglandin F201 to regress multiple CL in cows. J. Anim. Sci.
*3:273. (Abstr.).
Lopez-Barbella, S.R., A.C. Warnick, T.H. Wise, D.R. Hardin, T.R.
Thompson, and M.J. Fields. 1977. PGF201: 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 PGF2ct in cows. J. Anim. Sci. 35: 2^7 (Abstr.)7
Lou is, T.M., H.D.
Hafs,
and
D.A
. Morrow.
1972b.
Estrus and ovulation
after PGF2ct
in cows.
J.
Anim. Sci.
35:1121
. (Abstr.).
Lou is, T.M., H.D.
Hafs,
and
D.A
. Morrow.
1974a.
Intrauterine adminis
tration of prostaglandin F2a in cows: progesterone, LH, estrus
and ovulation. J. Anim. -Sci. 38:347.
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. 143:152.
Louis, T.M., J.N. Stellflug, H.A. Tucker, and H.D. Hafs. 1974b. 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 ef
fect 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 pro
gesterone 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 super
ovulation in cattle: A method for multiple embryo production.
69th Annual Meeting. Am. Soc. Anim. Sci. (Abstr.).
Mikhail, G., J. Zander, and W.M. Allen. 1963- Steroids in human ovarian
vein blood. J. Clin. Endocrinol. 23:1267.
Moody, E.L. and J.W. Lauderdale. 1977. Fertility of cattle following
PGF?r, controlled ovualtion. 69th Annual Meeting. Am. Soc. Anim.
Sci. (Abstr.).
Nett, T.M., D.W. Holtan, and V.L. Estergreen. 1973- Plasma 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 pros
taglandin F2a in the rabbit ovary. Am. J. Obstet. Gynecol.
117:381.


I0
Scanlon, P.F. 1972b. Ovarian response of cows following pregnant mare
serum gonadotrophin 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. Agrie. Sci. 70:179.
Schilling, E. and W. Holm. 1963- Investigation on induction of limited
multiple ovulation in cattle. J. Reprod. Frtil. 5:283-
Schwartz, F.L. and D.R. Shelby. 1969- Induced multiple ovu lat ion" i n
the bovine. J. Anim. Sci. 29:198. (Abstr.).
Shol1, S.A. and R.C. Wolf. 1974. Quantification of 20a- and 203-dihydro-
progesterone in plasma of the pregnant Rhesus Monkey. Steroids
23:269.
Short, R.V. 1962a. Steroid concentrations in normal follicular fluid
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Short, R.V. 1962b. Steroids present in the follicular fluid of the
cow. J. Endocrinol. 23:401.
Smith, J.G., J. Hawks, and R.V. Short. 1969- Biochemical observations
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J. Reprod. Frtil. 20:111.
Smith, L.E., G.D. Sitton, and C.K. Vincent. 1973- Limited injections
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Snook, R.B., M.A. Brunner, R.R. Saatman, and W. Hansel. 1969- The
effect of antisera to bovine LH in hysterectomized and intact
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Spears, L.L., A.B. Vercovitz, W.L. Reynolds, J.L. Kreider, and R.A.
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Luteinizing hormone levels in superovulated prepuberal and post-
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Sprague, E.A., M.L. Hopwood, G.D. Niswender, and J.N. Wiltbank. 1971
Progesterone and luteinizing hormone levels in peripheral blood
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Stabenfeldt, G.H., L.L. Ewing, and L.E. McDonald. 1969- Peripheral
plasma progesterone levels during the bovine oestrus cycle. J.
Reprod. Frtil. 19:433-


95
Chenault, J.R. 1973. Transitory changes in plasma progestins, estradiol
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Cupps, P.T. 1969. Steroid biosynthesis by the bovine ovary. J. Anim.
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Cupps, P.T., G.B. Anderson, M. Drost, B. Darien, and M.B. Horton. 1976.
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and adrenal after estrus and breeding of normal and abnormal cows.
J Da iry Sci. 44:888.


TABLE 22. ANALYSIS OF VARIANCE IN EXPERIMENT 3
Source
df
Sums of
squa res
F value
PR>F
Treatment (T)
3
182.36
PMSG (G)
1
176.65
55.54
0.0001**
Bred (B)
1
0.01
0.00
0.9644
P x B
1
5.70
,1.79
0.1812
Day (L)
1
476.36
149-77
0.0001**
Day (0)
1
240.33
75.56
0.0001**
Day (C)
1
12.26
3.86
0.0503*
Error
398
1265.93

T x Day
24
263.89
Cow (T) x Day
328
753.89
Cow (T)
41
240.91
Lack of fit
5
7.24
Corrected Total
404
2177.24
*(P<.05)
**(P<.01).
l


LU, PROGrSTERONC, 20l l-DIHYDROPROGt'.STERONE (ncj/m!)
Figure 7-
Concentration of progesterone
and estrogens ( ) in plasma
(*), 203-dihydroprogesterone ( ), luteinizing hormone
from PMSG treated cows with four or more corpora ltea.
( ),
-F


97
Gomez, W.R., V.L. Estergreen, Jr., O.L. Frost, and R.E. Erb. 1963-
Progestin levels in jugular and ovarian venous blood, corpora
ltea, and ovaries of the nonpregnant bovine. J. Dairy Sci.
46:553-
Gomez, W.R., O.L. Frost, and V.L. Estergreen, Jr. 1962. Progestins
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Gordon, I., G. Williams, and J., Edwards. 1962. The use of serum gonado
trophin (P.M.S.) in the induction of twin-pregnancy in the cow.
J. Agrie. Sci 59:143-
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gestins of the bovine ovary. Endocrinol. 62:234.
Gorski; J., R.E. Erb, W.M. Dickson, and H.C. Butter. 1958b. Sources
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Guthrie, H.D., D.M. Henricks, and D.L. Hand! in. 1974. Plasma hormone
levels and fertility in pigs induced to superovulate with PMSG.
J. Reprod. Frtil. 41:361.
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tility properties of prostaglandin F2ct. Biol. Reprod. 1:367-
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gesterone synthesis during the bovine estrous cycle. J. Anim.
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prostaglandin F2a in cattle. Vet. Rec. 96:134.
Hafs, H.D., J.G. Manns, and G.E. Lamming. 1975b. Fertility of cattle
from Al after PGF2a. J. Anim. Sci. 41:355. (Abstr.).
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Meyerhoeffer. 1975a. Reproductive response of the bovine to
PMSG. J. Anim. Sci. 40:187- (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.)


/ b
the introduction of bO mg PGF2a 13 days post-PMSG to superovulated 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 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.


TABLE 4. DISTRIBUTION OF ANIMALS SHOWING ESTRUS AFTER THE SECOND PGF2a ADMINISTRATION AND SUB
SEQUENT OVARIAN3 RESPONSE OF ANIMALS TREATED WITH PMSG IN EXPERIMENT 1
Behavioral estrus Estrus response to treatment Ovulation rate at laparotomy
PGF2a (hr)
Hou r1y
Accumu1 at ive
Rt
ova ry
Lt
ova ry
Overa 11
x S.E.
Range
0- 48
4/15
(26.7%)b
4/15
(26.7%)
3.50
1.73
2.00
\
1.15
5.50 1.29c
4-7
k3- 60
4/15
(26.7%)
8/15
(53.3%)
0.75
0.50
0.75
0.50
1.50 1.00*
0-2
61- 72
3/15
(20.0%)
11/15
(73-3%)
1.33
2.00
1.00
1.00
2.67 1.53*
0-4
73-120
3/15
(20.0%)
14/15
(93.3%)
0.67
1.15
0.67
1.58
0.67 0.82**
0-2
aBased upon number of CL at laparotomy.
^Number of animals responding total animals.
cDifferent superscripts are significantly different.
*(P<.05)
**(P<.01) .
I
-c-
-fc-


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 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 a 1 (1953), Hallford e_t^ aj_. (1975a,b), and
Turman et^ a_l_. (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 PGF2a was significantly (P< .01) affected by PMSG treat
ment (table 22, Appendix). The regress ion curves for individual treat
ments groups are depicted in figures 11 and 12. Plasma progestins con
centrations and predicted equations are summarized in tables 23 and 24
(Appendix), respectively.


ENDOCRINOLOGY OF THE SUPEROVULATED COW AND
SUBSEQUENT PROSTAGLANDIN F2a REGRESSION OF
THE MULTIPLE CORPORA LUTEA
By
SERGIO RAFAE4. 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 multipd-e
endeavors. This one is no exception. Since its very beginning, many
persons and institutions have provided enormous contributions. With
out 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 know
ledge 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 Agronoma, for providing economical support throughout my graduate
work.
I am also grateful to the Upjohn Company, especially to Dr. J.
Lauderdale, for providing PGF201 and reviewing my experimental pro
tocols. To Dr. J. L. Fleeger, Texas A&M University, and Dr. L. V.
Estergreen, Washington State University, my gratitude is extended for

their collaboration in providing the progesterone and estrogen antisera,
respectively. Special thanks are also due to Dr. J. H. Hentges, Univer
sity 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, 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. RossinaLFernandez for her typing of this manuscript.
i i i

TABLE OF CONTENTS
£age
ACKNOWLEDGEMENTS u
LIST OF TABLES *. vi
LIST OF APPENDIX TABLES vii
LIST OF FIGURES ix
ABSTRACT xi
CHAPTER I: INTRODUCTION 1
CHAPTER II: LITERATURE REVIEW 4
Prostaglandins and Reproduction 4
Prostaglandin F2a and Estrous Synchronization . 4
Luteolytic Effect of Prostaglandin F2a 6
Prostaglandin F2ct and Therapeutic Abortion. ... 8
Prostaglandin F2a and Superovulation 10
Gonadotropic Hormones and Limited Multiple Births ... 11
Endocrinology of the Superovu1ated Cow 14
20B~Dihydroprogesterone (4-Pregnen-208-o1-3_one). ... 16
CHAPTER III: MATERIAL AND METHODS 21
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE
(P), 2O3-DIHYDR0PR0GESTERONE (2O3-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
iv
27

TABLE OF CONTENTS Continued
Page
Conversion of Progesterone (^P) to 20B-Dihydro-
progesterone (3H-20f3-P). . 28
Use and Storage of Antibody 29
Serum Extraction and Chromatography 29
Conversion of 3H-20g-P to 3P . 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. ... ^0
Precision
Statistical Analysis
CHAPTER IV: RESULTS AND DISCUSSION 43
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE
(P) 20R-DI HYDROPROGESTERONE (200-P),
ESTROGENS (E), AND LUTEINIZING HORMONE
(LH) IN THE PMSG SUPEROVULATED COW SYN
CHRONIZED WITH PGF2a-THAM SALT 43
Reproductive Response 43
Endocrine Response 48
Progesterone 48
Estrogens 53
Luteinizing Hormone 54
20g-D ihydroprogesterone 55
EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN
THE SUPEROVULATED BEEF COW FOLLOWING
ADMINISTRATION OF 40 MG PGF2a~THAM SALT 57
Reproductive Response 57
Endocrine Response 58
EXPERIMENT 3- LUTEOLYTIC EFFECT OF PGF2crTHAM SALT IN
THE PMSG BRED \IS_ 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
1 EFFICIENCY OF 3H-P CONVERSION TO 3H-20(3-P AT
VARYING LEVELS OF COFACTORS. 31
2 STANDARD CURVE DILUTIONS 36
3 ADDITIONAL TUBES USED TO SUPPLEMENT RIA ANALYSIS . 37
A DISTRIBUTION OF ANIMALS SHOWING ESTRUS AFTER THE
SECOND PGF2a ADMINISTRATION AND SUBSEQUENT OVARIAN
RESPONSE OF ANIMALS TREATED WITH PMSG IN EXPERI
MENT 1 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 PGF2ra-THAM SALT AND
PMSG 39
7 DISTRIBUTION OF ESTRUS IN COWS AFTER A DUAL 33-5
MG SYNCHRONIZING INJECTION OF PGF2a-THAM SALT
AND ONE 40 MG ABORT I FAC I ENT DOSE OF PGF2a 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

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, 208-DI HYDRO
PROGESTERONE LUTEINIZING HORMONE, AND ESTROGENS
IN COWS HAVING MORE THAN THREE CORPORA LUTEA AT
LAPAROTOMY 80
13 PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DI HYDRO
PROGESTERONE LUTEINIZING HORMONE, AND ESTROGENS IN
COWS HAVING TWO OR THREE CORPORA LUTEA AT LAPAROTOMY 82
14 PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DI HYDRO
PROGESTERONE 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 208-DI HYDROPROGESTERONE (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
PGF2a IN COWS TREATED WITH OR WITHOUT PMSG 88
20 SPLIT-PLOT ON TIME ANALYSIS OF VARIANCE OF PROGES
TERONE 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
v i i

LIST OF APPENDIX TABLES Continued
TABLE Page
24 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTIN 93
v i i i

LIST OF FIGURES
FIGURE Page
1 Treatment protocol for experiment 1 involving
endocrine characterization following PMSG and
PGF2a ; 22
2 Treatment protocol for experiment 2 evaluating
the effectiveness of PGF2ct to regress multiple
corpora ltea in nonbred cows. . . 2k
3 Treatment protocol for experiment 3 comparing
the efficacy of PGF2ct to regress multiple
corpora ltea in the bred cow 26
k- LH-20 profile of 3H-1,2 progesterone (P) con
version to 3H-1 ,2-20(3-d i hydroproges terone
(208-P) using hexane:benzenermethanol (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 re
presents the mean of triplicates on seven
determinations.) 39
7 Concentration of progesterone ('') 208-dihydro-
progesterone ( ), luteinizing hormone ( ),
and estrogens ( ) in plasma from PMSG treated
cows with four or more corpora ltea kS
8 Concentration of progesterone ('*), 208~d ¡ hydro
progesterone ( ), luteinizing hormone ( ),
and estrogens ( ) in plasma from PMSG treated
cows with two or three corpora ltea 51
9 Concentration of progesterone ( ), 208-dihydro-
progesterone ( ), luteinizing hormone ( ),
and estrogens ( ) in plasma from PMSG treated
cows with zero or one corpora ltea 52
i x

LIST OF FIGURES Continued
FIGURE Page
10 Effectiveness of PGF2a to regress corpora ltea and
reduce plasma progesterone in PGF^ct synchronized
cows treated with PMSG ( ) and without PMSG ( ). 61
11 Plasfna progestin profile in PMSG-BRED ( ) and
PMSG-CYCL ING (---) cows after 40 mg PGF2a 68
12 Plasma progestin in BRED ( ) and CYCLING ( )
cows after **0 mg PGF2a 69
V
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 Barbel la
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 F2a (PGF2Ct) at a 12 day interval. Ovarian stimu
lation was achieved by injecting these cows with 2,000 i.u. PMSG 2k hr
prior to the second PGF2a administration. Cows were artificially in
seminated three times with Brahman semen at 0, 12, and 2k hr after
detection of the PMSG-PGF2a-induced estrus. Progesterone, 206-dihydro-
progesterone, 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 supra
vaginal 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

of the interval from PMSG-PGFpct to estrus and superovulation. This
possibility was supported by observed changes in the plasma endocrine
patterns.
When the endocrinology of the superovu1ated 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 post-
PMSG levels of progesterone, but estrogen concentrations were higher
in superovul ated cows (Pc.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 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 PGF20t. Although there was a significant (Pc.Ol) decline
in plasma progesterone levels and an absence of palpable CL during
the 7 days post-40 mg PGF201, 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 PGF2ct was indadequate for the in
creased CL mass. In an attempt to answer these questions, two experi
ments were conducted to test the efficacy of PGF2a to regress mu 11 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 2b 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

concentration by RIA. Forty milligrams of PGF2a was effective in re
gressing 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-AO 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 results from the
previous experiment. There were no observed differences between mated
and nonmated animals in response to kO mg PGF2a-
In conclus ionthese 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 PGF201 arid 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 preseht-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, fer
tility must assume an even greater importance in the economy of pro
duct 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 prof
itable alternative utilization. A primary means available for effi
ciently improving production in the beef cow is to wean a greater num
ber 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.

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 hor
monally induced controlled ovulation rate.
Superovulation in beef cattle has been induced by various ex
tracts of the'anterior pituitary; however, pregnant mare serum gonado
tropin (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 ex
tracts require a series of injections; however, in either case ovula
tion rate is highly, variable.
Almost without exception, researchers in the field of super
ovulation 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 2a (PGf2a) may introduced into the
hormonal regimen to induce premature regression of multiple corpora
ltea (CL) resulting from an excessive ovulation rate in an attempt to
reduce fetal wastage. Thus, it was the objective of this study to de
termine 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 PGF2a and if this would minimize the necessity of determining the
exact stage of the estrous cycle for giving the injections of PMSG.

3
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 syn
chronizing es t rous wi th PGF2ct when used in conjunction with PMSG was
evaluated through studying the levels and interrelationships of the
reproductive hormones. "

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 PGF201 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 effec
tive 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 pseudo
pregnant rats (Pharris and Wyngarden, 19&9), others observed that treat
ment with a single injection of PGF2a (Rowson et al., 1972; Lauderdale,
1972; Inskeep, 1973; Oxender et_ aj_. 197^; Rodriguez, 197^+; Roche, 1974;
Fields et_ aj_. 1975; Thatcher and Chenault, 1976) or syn thet i c ana 1 ogues

5
of PGF2a (Tervit ej^ aj_. 1973; Cooper, 197^; Fields et_ ak 1977b) was
effective in causing luteolysis in heifers and cows except for the
first 5 days post-estrus (Rowson et al., 1972; Louis et a 1. 1973)
In cattle, a single injection of PGF2ct given during the responsive
stage of the estrous cycle is followed by an ovulation and normal fer
tility. There are, however, several factors associated with the ule
of a single injection of PGF2ct 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 a 1., 197*0- Others have reported similar results
following PGF2Ct treatment (Louis e_t_ a_l_. 1973; Louis ej^ aj_. 1974a;
Chenault ejt_ 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 10 to 12
day interval, as suggested by Inskeep (1973). Cooper (197*+) reported
that this dual injection technique, with an ICI analogue of PGF2a (ICI
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 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 PGF2ct in
jection. Several researchers have shown this synchronized estrus to
be fertile (inskeep, 1973; Lauderdale et al., 1974; Roche, 1974;

Rodriguez, 197**; Cooper and Jackson, 1975; Hafs et_ 1975b; Turman
et aj_. 1975; Ellicott and Thompson, 1976; Fields £t_ aj_. 1977b;
Moody and Lauderdale (1977)-
Luteolytic Effect of Prostaglandin F2ct
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 es-
trus. The induction of estrus is rapid and precise and the induced CL
has a normal life-span.
Louis et_ a_l_. (1972a) injected 5 mg PGF2aTham Salt into the uter
ine 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 in
jection of 30 mg PGF2a during diestrus resulted in a 60% decrease in
plasma progesterone within 12 hr with the initial progesterone concen
tration of 4 ng/ml declining to .8 ng/ml by 2b hr (Louis et a 1., 1972a,
1973). The interval to onset of estrus, LH peak and ovulation was 74,
77, and 104 hr post-PGF2a treatment, respectively. Similar results in
declining plasma progesterone were reported by Oxender et a 1. (1974)
even when PGF2a was given intramuscularly in varying numbers of injec
tions. The spaced double injection regimen produced precise synchro
nization of estrus in all the heifers responding to treatment (Dobson
et a 1. 1975). After both injections, the CL showed rapid morpholog
ical regression that was similar to that observed for a single injec
tion 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 98 hr after the first, and 48 to
55 hr after the second PGF2a treatment. A preovulatory surge of LH
occurred 62 to 103 hr after the first, and 48 to 62 hr after the sec
ond PGF2a treatment, and was followed by ovulation. Similar changes
have been reported by Stellflug et_ a_l_. (1973), Louis et_ aj_. (1973),
and Louis et^ aj_ (1974a,b).
Chenault et aj_. (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 in
creased and apparently stimulated an ovulatory surge of LH at 72 21
hr post-treatment. Ovulation occurred at 99-5 19 hr after PGF2ct ad
ministration. Gimenez et_ a_L (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 in
jection. In the same study, plasma estrogens concentrations ranged
from 25 to 100 pg/ml prior to treatment with no observed change in es
trogens levels occurring after giving PGF2ct when compared to the saline-
treated cows.
The mechanism by which PGF2a initiates CL regression is unknown.
Novy and Cook (1973) and Thornburn and Hales (1972) demonstrated that
PGF2ct 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 re
gression or physical destruction of the luteal cell.
Because structural regression was preceded by accumulation of
lipid droplets, Stacy e_t aj_. (1976) postulated functional CL regression

8
was most likely due to a blockage in one or more stages in steroido
genesis. 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 F2ct and Therapeutic Abortion
An effective means of inducing parturition in the cow could re
duce calving losses and labor costs by decreasing the calving period
to a shorter and predictable period. Parturition can be induced in
the cow by t reat ing >wi th estrogens (Spears e_t_ aj_. 197*0, corticoids
(Jochle, 1973), and PGF2a (Lauderdale, 1972). In addition, PGF2ct 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 PGF2ot (Gutknecht et a 1 1969). In humans,
PGF2a 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 e_t aj_. 197*0, and
bovine (Lauderdale, 1972, 197**; Fields et_ aj_. 1977a) species. Adminis
tration of PGF2ct either intramuscularly or systemically at early stages
of gestation in the bovine resulted in a dramatic decline in plasma pro
gesterone within 2*4 hr and behavioral estrus 2 to 16 days post-treatment
(Zerobin et_ aj_. 1973; Douglas et al., 197*4).

Louis et_ aj_. (1974a) injected 5 mg PGF2a into the uterine horn ipsi-
lateral for the CL In cows 11 days postmating and reported plasma pro
gesterone 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 concen
trations 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-PGF2oT
treatment. 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 PGF201 induced CL re
gression 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 post
abortion. In contrast, cows at l60 to l80 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 a 1. (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 con
centration declined to basal levels of 1.5 0.6 ng/ml within 48 hr
post-cloprostenol. No retained placentas were observed. Fields et a 1.
(1977a) reported 250 to 500 yg 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 e^ aj_. (1976) reported that- the administration of PGF2C1
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 a 1., 1976).
Dobson et a 1. (1975) suggested that the second PGF2ainduced luteolysis
may allow the wave of growing f-ollicles that originated after the first
PGF2C- i nduced estrus to progress and ovulate rather than become atretic.
i
Rajakoski (i960) reported such a wave of follicular growth approximately
b 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 PGF201 administration.
In line with this concept, Tervit £t_ ak (1973) reported that a
pros tag 1 anding F2ct analog (ICI 79,939) given to cattle with large PMSG-
induced follicles demonstrated a shorter interval to estrus. Archbald
(1976) reported excellent synchronization of estrus when a second in
jection 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 8l% of the cows responding behaviorally within 48 to 132 hr following
t reatmen t.

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 Hor
mone (FSH). Different gonadotropins, alone or in combination, have been
used by many researchers with consistent results. Casida et_ aj_. (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 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 injec
tion' of 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 e_t aj_. (1969, 1971) used this same hormonal protocol
and obtained a 109% weaned calf crop, from treated animals.
Kidder et a 1. (1952) and Dawson (1961) reported cows with double
ovulations gave birth to a very low number of twins. Gordon et a 1.
(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

12
with double ovulations from a single ovary (29%). The precise relation
ship between the number of ovulations per ovary and maintenance of preg
nancy to term remains to be established. The minimal intrauterine mi
gration (1:200) in the bovine (Perkins et_ a]_. 1954; Gordon e_t a]_. 1962;
Rowson et^ aj_. 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_ a_L (1969) reported 625 mg of FSH injected twice daily
for 5 days, in heifers synchronized by feeding 180 mg medroxyprogesterone
acetate (MAP) dail/ 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_ aj_. 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 e_t a_l_. (1969), but considerably lower than the calving rate
of 173% with PMSG treatment reported by Turman et a 1. (1971).
Reynolds et_ aj_. (1970), Vincent and Mills (1972), and Smith et a 1 .
(1973) attempted to prolong the action of FSH by using a 1% sodium
carboxymethy1 cellulose and polyviny1pirrolidone. 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 treat
ment was due to the actual biological potency of the gonadotropin.
Similarly, Schwartz and Shelby (1969), Laster et a 1. (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 syn
chronized 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 in
jections for 5 days (Bellows et_ aj_. 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
e^t aj_. 1976). However, for 5 days of injections, fewer cows super-
ovulated 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 8l to 104 days after inse
mination. Similar observations of fetal wastage have been made by
M. J. Fields, A. C. Warnick, and J. H. Hentges (unpublished data).
Godke e^ aj_. (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_ a_l_. (1973, 1976) reported that PMSG, given concomitantly
with PGF2a, resulted in a lower and less variable ovulation rate (4.33
3.60) than when PGF2ct was injected at 24 hr post-PMSG (8.03 9-46). A
high incidence of split estrus (55%), w.ith 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 be
tween hormonal treatments was allowed. These data suggest that the
follicles had not had sufficient time to mature before estrous control
was attempted with PGF2c(. Absence of split estrus has been reported
by Rajamahendran ajL (1976) and Lopez-Barbella et a 1. (1976)
following a single injection of 2,000 i.u. PMSG 24 hr prior to a second
injection of synchronizing PGF2c(.
Endocrinology of the Superovu1ated Cow
Hallford ej^ aj_. (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<.01) 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 dif
ferential LH response observed in the PMSG treated group could be due
to a positive feedback of estradiol at the hypothalamic-pituitary
level. Although Hallford e^ a_L (1975b) reported a positive correla
tion 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 e_t aj_. (1972) and Dickey et a 1 (1973) re
ported plasma estradiol levels in PMSG superovu1ated heifers to be
much higher prior to mating than in controls. These values, however,
were for superovu1ated 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-Barbel1 a et a 1., 1976).
A synchronizing injection of PGF2C1 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 a 1., 1976).
The stereotypic response to PMSG was clearly demonstrated by
Rajamahendran et a 1. (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
PGF2cr 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) f rom 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.

16
20g-D ihydroprogesterone (A-Pregnen-20g-ol-3~one)
An interesting steroid, concerned with bovine steroidogenesis, that,
to date, has been considered the major metabolite of progesterone (P) is
20g-dihydroprogesterone (20g-P) (Spilman 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 lA day- CL has reached
its maximum size filling the collapsed follicular cavity with an extensive
vacuolation of luteal cells. Stero idogenica11y, this structural change
is accompanied by a differential secretion of progesterone-derived hor
mones according to Short (1962a,b) in his "two cell theory." From the
two Cell theory of Short (1962a,b), the ovarian theca interna was pro
posed to covert P to estrogen in the absence of a 20-reductase system,
i.e., 20g-hydroxysteroid dehydrogenase (20g-HSD). LaCroix et_ 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 1uteinization was proposed to contain
the 20g-reductase system and thus, 20g-P secretion by the growing folli
cles was an indicator of 1uteinization. This was indirectly confirmed
by other when P and 20g-P were isolated from luteal tissue (Savard and
Teledgy, 1965). Lobel and Levy (1968), however, did find that 20g-HSD
activity resided in both the granulosa and theca cell layers of the rat
follicle.
Hayano et_ aj_. (1975) first demonstrated the conversion of P to 20g-
P by the bovine CL. Noticeable quantities of ovarian 20g-P levels in
cycling cows at levels approximately 10 to 20% of that of P have been
reported (Gorski et a 1., 1958a,b; Erb and Stormshak, 1961, Hafs and

17
Armstrong, 1968; Garverick et^ ajL 1971). Brandau e_t^ aj_. (1972) and
Brandau and Mutzke (1972) using homogenized bovine ovaries found that
206-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 203-P concentration following the P decline
of the regressing CL (Erb and Stormshak, 1961 ; Staples and Hanse1,-l961
Mares aj_- 1962; Gomez et aj_. 1963; Gomez and Erb, 1965)* Erb £t
al. (1968) reported that 20f3-P increases when synthesis or 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 re
gression could result in increased lysosomal activity which would
result in reduced cellular pH and create optimal conditions for 208"P
formation. When a similar study was conducted with porcine CL, only
0.6% of the P was converted to 20g-P (Weiss e_^ aj_. 1976).
In laboratory animals, in which the a-epimer (20a-P) of 208-P
is the more predominant hydroxylated ovarian steroid, Barraclough e^
a 1. (1971) noted an increase in ovarian vein levels of 20a-P approxi
mately 2.5 hr prior to the LH discharge in cycling female rats. This
suggested the possibility that 20ct_P has a positive feedback on the
pituitary for discharge of LH. Ichikawa et a 1. (1971) found an in
creased secretion of both P and 20a-P in ovarian vein plasma after in
jection 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 20g-P, with 20ct-P being the more predominant hydroxylated
steroid. Hilliard et^ (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 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 (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-ll*C increased 14CP synthesis 3-3 fold and increased
the P to 200-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 20g-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 200-P. Not unexpectedly, there was significantly
less synthesis of P and 200-P in follicular than luteal ovaries. Pro
lactin perfusion in this study, had no effect on the 20-HSD system.
Snook e_t aj_. (1969) working with hysterectomized heifers with sus
tained luteal function, however, showed that LH has a preferential ef
fect on 20-HSD. They used LH-antisera to neutralize endogenous LH.
This LH-antisera reportedly decreased total ovarian progestin concentra
tion primarily due to a significant reduction in 200-P while P concen
trations remained unaltered. In the intact heifer, Spilman et al. (1973)

reported increased plasma 208-P levels after either PMSG or HCG
treatment.
Kidwell et^ ak (1966) working with PMSG superovu 1 a ted 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 in
creased from 0.03 0.01 to 2.82 2.~]k MU/mg 5 days after LH injec
tion. The administration of HCG, however, resulted in a ten-fold in
crease in the 20-HSD activity. A significant increase in the glucose-
6-phosphate dehydrogenase activity was also observed after LH and HCG
administ rat ion.
The possibility that 20B-P in the bovine might be playing a simi
lar role as 20B-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^ a_l_. 1963), Chinchilla (Tam, 1971) and the African elephant (Smith e_t_
ah, 1969) -
Staples and Hansel (1961) presented data to suggest that embryo
survival at day 15 may be influenced by circulating 208-P levels. Gomez
et_ a_L (1962) quantitated P and 208-P levels in the utero-ovarian vein
ipsilateral to the CL in cows between 250 and 282 days of gestation.
Levels of 20BP 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:208_P ratio was approximately one.
Levels of 208-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 208-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 208-P will be characterized in PMSG superovu1ated cows
over a 21 day bleeding period.
V

CHAPTER I I I
MATERIAL AND METHODS
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P), '
20B-DI HYDROPROGESTERONE (206-P), TOTAL ESTRO
GENS (E), AND LUTEINIZING HORMONE (LH)lN PMSG
SUPEROVULATED COWS SYNCHRONIZED WITH PGF201-
THAM SALT
The objectives of this experiment were (l) to characterize plasma
P, 20g-P, E (combined Ei and E2) and LH concentrations in the PMSG
superovu1 a ted cow and (2) to determine if 40 mg PGF2ot-Tham Salt was ef
fective in regressing multiple corpora ltea (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 intra
muscularly with 33-5 mg PGF2ct-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 in
insemination (Al) three times with Brahman semen was at 0, 12, and 24 hr
after detection of the PMSG-PGF2ct-i nduced estrus. Approximately 10 days
following Al 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

33.5
PGF
MG
2 a
HEAT A I
AT 0,
LAPAROTOMY
OVARIAN
2 000 i. u.
33.5 MG
12, 24
40 MG PGFz a
RECTAL
PMSG
PG F2 a
HR
TO COW > 3 CL
PALPATION
1
7
DAY 1 DAY 10 DAY 12 DAY 13
DAY 2 5
DAY 32
BLEEDING TWICE DAILY
Figure 1.
Treatment protocol
following PMSG and
for experiment
PGF20C
involving endocrine characterization
N3
N3

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 superovula
tion effect, cows with more than three CL were injected with 40 mg~~PGF2a
to induce CL regression.
\
Surgical Description of Supravaginal Laparotomy
Prior to surgery animals were taken off feed overnight. Animals
were restrained andytranqu i 1ized 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. penicilline
(Combiotic, Pfizer).
EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN PMSG SUPER-
OVULATED BEEF CATTLE FOLLOWING ADMINISTRATION OF
40 MG PGF2a-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
ach ievesynchronization of estrus (figure 2). A 2,000 i.u. PMSG injec
tion was administered subcutaneously to 10. cows 24 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 PGF2a injection.

33.5 MG
PGF
2a
2,000 i.u
PMSG
33.5 MG
PGF, a
LAPAROTOMY
40 MG PGF.
2a
OVARIAN
RECTAL
PALPATION
DAY 1
DAY 12
DAY 13 DAY 23
DAY 2 5
DAY 3 2
Li DAILY BLEEDING
Figure 2.
Treatment protocol for experiment 2 evaluating the effec- 1
tiveness of PGF2a to regress multiple corpora ltea in
nonbred cows.
ro
-C-

23
Ovulation rate was determined by supravaginal laparotomy 13 days
post-PMSG and, on the same day, all cows were treated with 40 mg PGF2a-
/
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 PGF2orTHAM 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
L
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 24 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 2b 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-1aparotomy.

33.5 MG
PGF2a
GROUPS I S II
2, 000 i.u.
PMSG
33.5 MG
P G F 2 ct
LAPAROTOMY
4 0 MG PGF2 a
1
DAY
DAY 12
DAY 1 3
DAY 23 DAY 25
GROUPS I S III
NATURAL BREEDING
DAILY BLEEDING
DAY 3 3
Figure 3- Treatment protocol for experiment 3 comparing the efficacy of PGF2a to regress
multiple corpora ltea in the bred cow.

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-disti 11ed water
for 1 min,and rinsed with warm tap water (3X) followed by deionized"
water (3X). Prior to use, the glassware was rinsed with ethanol, sili
conized with 1% silicone solution (Siliclad, Clay Adams) for 20 sec and
oven dried for 2b hr.
Organic Solvents
3-
Analytical grades of benzene, ethanol, methanol, hexane, and ethyl
acetat 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,
b, 6,7"3H-Estradiol (3 E 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 im
purities 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 bC. 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 1 hr at room
temperature. A volume 100 pi (20,000 1,000 dpm) was used in the assay.
This solution was stored at AC and used for no longer than 1 wk. P and
E tritiated steroids were further diluted with benzene to 2,000 dpm/100
pi and stored at AC for determining recovery of extracted steroi.dsT
Since tritiated 200-dihydroprogesterone was not commercially avail
able it was synthesized from 3P as described by Tribble (1973).
Conversion of Progesterone (3P)'to 203-Dihydroprogesterone (3H-20B~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 CpH =
5-2, which contained 100 mg percent EDTAH, 30 pi of NADH C(Sigma Chemical
Company, Grade III) in 0.1 M Tris buffer (Eastman), pH = 8.1 (see table
11, Appendix) and 30 pi of 208-hydroxysteroid dehydrogenase enzyme
C(Ca1biochem, Activity A.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 ml dis
tilled water. The 3H-20g-P was extracted with 1 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-20p-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-2O0-P was subsequently
determined. Fractions containing 3H-20S~P were stored in benzene:

29
ethanol (9:1) at 4C. A typical profile of 3P conversion to 3H-20g-P
an,d its LH-20 chromatography separation is illustrated in figure 4.
The above procedure for synthesis of 3H-20g-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 usecf in this study were kindly supplied by
Dr. Lee Fleeger, Texas A&M University (PR ft24 and PR #281), and Dr. V.
L. Estergreen, Washington State University, respectively. The lyo-
phil-ized P antiserum was dissolved in 400 pi 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 break
down 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 ul 3P, 500 yl of 0.05 M
3The addition of normal rabbit serum (1:400) improved the sen
sitivity of this antiserum.

C PM
ML ELUENT
I
Figure h. LH-20 profile of 3H 1 ,2 progesterone (P) conversion to 3H-l,2-20f3-
dfhydroprogesterone (203-P) using hexane: benzene :met Hanoi (80:15 ; 5)
o

31
TABLE 1. EFFICIENCY OF 3H-P CONVERSION TO 3H-206-PAT
VARYING LEVELS OF COFACTORS
Tri tiated
progesterone
(dpm)
n
NADHa
(ml )
20B-HSDb
(ml)
Efficiency of 3P
conversion to 3H-20BP
(%)
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-
b0.2 ml
-NADH/3 ml
20B-HSD/0.
of 0.1 M Tris
.8 ml of 0.005
buffer.
M Tris
buffer.

32
NaOH, and 15 ml of hexane was added and vortexed 1 min. The lower aque
ous phase was quickly frozen in liquid nitrogen while the upper lipid-
containing 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 pi 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 pi transferred to a scintillation vial to
estimate recovery. The remainder was used for assay.
Quantification of 3P and 3H-20g-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 appro
priate tritiurn-1 abe 1 led steroids. The dried residue from the ether ex
tract was quantitatively transferred to the column with two 500 pi ali
quots of hexane:benzene:methanol (80:15:5). Progesterone was eluted
from the column using 5 ml of the above organic solvent while 208-dihydro-
progesterone was eluted 2 ml after 3P. The 3P and 3H-208-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 des
cribed for progesterone (vide supra).

33
Quantification of estrogens was accomplished as described by
Abraham et aj_. (1971) using the assay procedure of Nett et_ aj_. (1973).
Quant i f ication of LH was as described by Chenault (1973). The quantifi
cation of 20g-dihydroprogesterone was accomplished by converting this
progestogen to progesterone and subsequently assaying for progesterone.
Conversion of 3H-20g-P to 3P
The chemical conversion of 3H-20g-P to 3P was similar to the
procedure described by Shol1 and Wolf (197*0- To the dried column
elute of 3H-20g-P was added 200pl 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 1 ml of distilled water and the sample extracted with
3 ml of ethyl acetate (2X). This extract was washed with 500 yl dis
tilled 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 sub
jected to chromatography on a LH-20 column (vide supra) and the 3P frac
t i on 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 A 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 vigor
ously for 30 sec. The charcoal suspension was stored at 4C and used for
no longer than 4 wk. A volume of 500 pi 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 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 I (98% PPO/2% BIS-MSB). A total of 3-5 ml of this counting
fluid was used per scintillation vial. Cocktail and sample were equi
librated for 2k 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 crystal
lization a stock solution of 1 g/ml unlabelled hormone in absolute
ethanol was made. An aliquot of 20 pi 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 over
night. 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 yl 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, ^H-203-P (oxidized to 3P) and 3H-E
facilitated quantification of these steroids. Aliquots of 200 yl and
500 y 1'of the buffered unknown was pipetted (Eppendorf, Brinkman Ins
truments) 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 yl aliquot was adjusted to
500 yl by the addition of 300 yl of gelatinized assay buffer. An ad
ditional 500 yl 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 mini
mum of 4 hr at 4C, followed by addition of 500 yl of charcoal suspen
sion to each tube, vortexed briefly and incubated 20 min at **C. All
assay tubes received this 500 yl 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 DILUTIONS3
Hass 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 &3
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.

37
TABLE 3-
ADDITIONAL TUBES USED TO
SUPPLEMENT RIA ANALYSI
ISa
Solution
Code
Amount of
assay buffer
added
(ml)
Amount of
antibody
added
(ml)
Amount of
3H-Steroid
added
(ml)
BGb
1 .2
-
-
TCc
1 1
-
0. 1
CHb '
0.6
-
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.
^To determine residual counts left following addition of
cha rcoa1 .
eTo determine the percent of the 3H-steroid bound to the
antibody.
^Assay buffer was replaced by 0.5 ml of either standards
or unknown samples

90
8 0
70
60
50
40
30
20
10
flM#
CONCENTRATION OF PROGESTERONE (DG/ML) /
Figure 5- Standard curve of progesterone, (Each point represents the mean of
triplicates on 20 determinations.)
O.

I C!D f NG
10 25 50 100 250 500 1000
CONCENTRATION OF ESTRADIOL (PG/ML)
I
Figure 6. Standard curve of estradiol. (Each point represents the mean of triplicates on seven
determinations.)
US

*4
at 4C for 20 min at 2,000 g. Then, 500 yl of this incubate was with
drawn by a repipette and deposited in a scintillation vial along with
3.5 ml of scintillation cocktail. Counting for 5 min followed over
night equilibration at room temperature.
Calculations
Recovery =
(recovered counts background counts) (4) x |qq
original counts added background counts
% Total Binding
BT tube counts
x 1 00
TC tube counts
The standard curve and quantification of unknowns were calculated
by logarithmic transformation of the data. For this, a Monroe i860 com
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 follows:
Steroid Concentration =
concentration of unknown
% 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 20g-d¡hydroprogesterone
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

41
progesterone was eluted within the first 3 ml and the more polar 208-P
was eluted between the fifth and seventh ml fractions.
Precision
The between and within assay coefficient of variation (c.v.) was
calculated from 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, 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 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
intraassay c.v. (14.4 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, i960) from which the following parameters
were obtained:
(1) Identification of classes (group, day, and group x day inter
action) with their respective least-squares means and standard errors
for progesterone, 206-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, 208-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 dis
cussion of the results;
(3) Test for day effect within hormone was computed using tire
appropriate error term obtained from Harvey's printout (Steel and
Torrie, i960). Reproductive responses were tested by LSD; and
{b) Additional computations were obtained according to the pro
cedures described in SAS 76 (Barr eT a_K 1976).
y

CHAPTER IV
RESULTS AND DISCUSSION
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P)
20g-DIHYDROPROGESTERONE (20g-P) ESTROGENS
(E), AND LUTEINIZING HORMONE (LH) IN THE
PMSG SUPEROVULATED COW SYNCHRONIZED WITH
PGF2ct-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-PGF2ra. The single animal not responding to this dual injection
of PGF2a was detected in estrus 36 hr prior to the second PGF2ct 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 48, 60, 72,
and 120 hr, respectively, following PGF2a injection. This is in agree
ment with Cupps e_^ £]_ (1976) who reported that the administration of
PGF?ct 24 hr post-PMSG, to previously synchronized heifers: (l) in
creased the number of animals exhibiting estrus; (2) shortened the in
terval 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% re
ported by Archbald (1976).
Estimates of ovulation rate, determined at laparotomy, are pre
sented in table 4. Significant differences were detected in total
43

TABLE 4. DISTRIBUTION OF ANIMALS SHOWING ESTRUS AFTER THE SECOND PGF2a ADMINISTRATION AND SUB
SEQUENT OVARIAN3 RESPONSE OF ANIMALS TREATED WITH PMSG IN EXPERIMENT 1
Behavioral estrus Estrus response to treatment Ovulation rate at laparotomy
PGF2a (hr)
Hou r1y
Accumu1 at ive
Rt
ova ry
Lt
ova ry
Overa 11
x S.E.
Range
0- 48
4/15
(26.7%)b
4/15
(26.7%)
3.50
1.73
2.00
\
1.15
5.50 1.29c
4-7
k3- 60
4/15
(26.7%)
8/15
(53.3%)
0.75
0.50
0.75
0.50
1.50 1.00*
0-2
61- 72
3/15
(20.0%)
11/15
(73-3%)
1.33
2.00
1.00
1.00
2.67 1.53*
0-4
73-120
3/15
(20.0%)
14/15
(93.3%)
0.67
1.15
0.67
1.58
0.67 0.82**
0-2
aBased upon number of CL at laparotomy.
^Number of animals responding total animals.
cDifferent superscripts are significantly different.
*(P<.05)
**(P<.01) .
I
-c-
-fc-

ovarian response when cows were partitioned according to when they ex
hibited behavioral estrus. A higher ovulation rate (P<.01) was ob
served in cows expressing estrus by 48 hr post-inject ion (5-50 1.29)
vs 0.67 0.82 for those expressing estrus from 73 to 120 hr. The dif
ference 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 (1949), Brock and Rowson
(1952), and Scanlon et aj_. (1968) in that a more desirable and res
tricted 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 e_t^ aj_. (1973) demonstrated in
cattle'with large PMSG-induced follicles that a PGF2a analog (ICI
79,939) shorten the interval to estrus. Dobson et_ a_l_. (1975) suggested
that the second PGF2ct-i nduced 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 ob
servation that the induction of estrus was more rapid and precise after
the second PGF2a 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
24 hr earlier following the second injection than the first injection.
Rajakoski's (I960) 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 et_ aj_. (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.

40
The present study suggests the possibility of a relationship be
tween 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 res
ponse of 0.67 0.82 ovulations per cow.
The percentage of animals conceiving to either the first post-
PMSG 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_ aj_. (1971), Laster e_t aj_. (1971b) and Hallford et a 1 .
(1975a,b) for superovulated cows. Multiple fetuses determined by calving
date were lower than those estimated earlier by palpation. These dis
crepancies 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 PGF2a,
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
X 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
includes one animal pregnant to treatment that died due to
Tympani tes.
^Post-mortem examination of reproductive tract revealed ovi
duct 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 PGF2ct. This treatment, in it
self, allowed for the subgrouping of animals according to their ovula
tion rate. Since it was determined that the time-trend in plasma hor
mones among 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 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 proges
terone curve (table 15, Appendix) was exhibited prior to PMSG treat
ment. 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), proges
terone decreased to a basal concentration of 0.72 0.14 ng/ml by day
6 (PM). These changes were best described by a significant (Pc.Ol)
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<.01) curvilinear (table 15,
Appendix) increase to 16.46 + 2.36 ng/ml on day 15 (AM) was observed.

LU, PROGrSTERONC, 20l l-DIHYDROPROGt'.STERONE (ncj/m!)
Figure 7-
Concentration of progesterone
and estrogens ( ) in plasma
(*), 203-dihydroprogesterone ( ), luteinizing hormone
from PMSG treated cows with four or more corpora ltea.
( ),
-F

50
As described initially, ovaries were examined by laparotomy and
those with more than three CL received kO mg PGF2a on day 15 (AM). A
significant (Pc.Ol) reduction in plasma progesterone was observed after
AO mg PGF2a 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
1A, 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
Henri cks £t ak (1973) Sp i Iman et^ a_l_. (1973), and Ha 1 1 ford e_t aj_.
(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 PGF2a 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<.01; table 18, Appendix) is in agreement
with Lamond and Gaddy (1972) and Spilman et al. (1973) and in disagree
ment with Rajamahendran ej^ al. (1976) who reported that a much larger
number of observations was needed to assess statistical difference be
tween superovu1ated and normal cycling cows.

LI I, l'HOGLS fSIKONE, 20{3-DIHYDR0PK0GLSTF.I\0NE (ng/ml)
Figure 8. Concentration of progesterone(), 20g-dihydroprogesterone ( ), lutei
nizing hormone ( ), and estrogens ( ) in plasma from PMSG treated cows
with two or three corpora ltea.

ESTROGENS (pg/rnl), LH, PROGESTERONE, AND
2O0-DIHYDROPROGESTERONE (qg/ml)
DAY OF BLEEDING
Figure 9- Concentration of progesterone (), 20f3-d ihydroprogesterone ( ),
luteinizing hormone ( ), and estrogens ( ) in plasma from PMSG
treated cows with zero or one corpora ltea.
vn

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<-01) increase to 13-62 2.06 pg/ml within
24 hr. In conjunction with the luteolytic dose of PGF2a on day b fAM),
estrogen levels continued to increase tc> a peak of 37-04 11.47 pg/ml
by 72 hr post-PMSG. These changes were best characterized by a third
order regression equation which accounted for b7% 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 PGF2a coupled with increasing levels of
estrogen led to the expression of estrus. As a consequence of the re
initiation of a PGF2a-induced estrous cycle, plasma estrogen declined
significantly (P<.01) 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 PGF?a 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 1 (r = 0.68, Pc.Ol; table 18, Appendix) is in

agreement with that reported by Hallford et a 1 (1975b). Henricks e_t
a 1 (1973), in cattle, and Guthrie et_ aj_. (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
fol1icles.
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_ aj_. (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_ aj_. (1973) that the
ovulatory surge of LH was earlier in PMSG treated heifers than in non-
treated controls. Although untreated PMSG-treated animals were not
i
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 1 (vide supra) Hallford e^ aj_. (1975b) failed to detect this
treatment effect; they only obtained plasma samples at 24 hr intervals.
Henricks et_ aj_. (1973) and Hallford et_ aj_. (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 rela
tionship between plasma LH and plasma estrogen or between plasma LH and
ovulation rate can be provided from this trial.
203-Pi hydroprogesterone
The individual plasma values and plot-ted levels of 20g-P are re
ported in tables 12 to 14 (Appendix) and figures 7 to 9, respectively.
There was a curvilinear relationship between plasma 203P and day of
bleeding (table 16, Appendix). A nonsignificant (P>.10) increase of
2O3-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. after PMSG treatment with 20g-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, 2O3-P levels to be 10 to 15% of that of P. In these
studies the maximum levels of plasma 2O3-P were 2.5 ng/ml which is com
parable 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-PGF201 induced estrous cycle. Although 2O3-P appeared
not to be influenced by gonadotropin administration, there was a 3-3
fold greater increase in P over 2O3-P synthesis at 24 hr post-PMSG treat
ment. Romanoff (1966) 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 lutei-
nization which is, in turn, maintaining 2O3-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 20g-P, it seems
likely that only luteal cells have an active 2O3-HSD. Thus in this

experiment, it may be that the origin of the 206-P was from reactiva
tion of luteal cells from regressing CL or 1uteinization of granulosa
cells from follicles. Hillard et_ ak (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 a-P.
Data accumulated in this study suggest that PMSG has profound ef
fects 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 208-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 40 mg PGF2a exhibited both
a functional and morphological regression of multiple CL with no abor
tion, it is of importance to test whether kO 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 ef
fect, 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.

5/
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 superovu1ated pregnant cow. The question then
arose as to whether 40 mg PGF?a 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 PGF^ dose, at 13
days post-PMSG, was effective in regressing multiple CL in the non
pregnant superovu1ated 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
importnce to review this concept and compare these results with the
endocrinological profile reported in experiment 1.
Reproductive Response
It is well established that PGF2ni 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
PGF201 when 10 of 13 animals exhibited estrus within 96 hr after treat
ment (table 6) followed by 84.6% at the time of the second injection.
Cooper (1974) and Chenau 11 e_t_ aj. (1976) clearly demonstrated that a single
injection of PGF2a 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 injec
tion 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_ aj_. (1976) and Archbald (1976) and support
the concept of a shortened interval and reduced variability to onset of
estrus after the administration of PGF2cx 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 shorter interva 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 proges
terone 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 pro
gesterone concentration in cows after the administration of 40 mg PGF2a
was significantly affected by treatment (P<.01) 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

TABLE 6. DISTRIBUTION OF ESTRUS AND OVULATION RATE IN COWS AFTER A DUAL INJECTION OF PGF2cT
THAM SALT AND PMSG
Time after PGF2a
First PGF2a
iniect ion
Second PGF2a
injection
ovulat ion
PMSG
rate at
1aparotomy
inj ection
(hr)
Number of
animals
% of
animals
Number of
animals
% of
animals
Rt
ovary
Lt
ovary
Overa 1 1
x S.E.
0-48
5
38.5
8
61.5
4.7 3-3
2.4 1
.9 4.3 3-5
49-72
4
30.8
3a
23- 1
3.0 1.4
1 .0 1
.4 4.0 2.8
73-96
1
7.7
0
0
97-120
0
0
0
0
121-144
2
15.4
0
0
1 45-more
0
0
2b
15-4
Tota 1
12/13
92.3
13/13
100
3-9 3.
1
a0ne animal
bTwo animals
did not receive
did not receive
2,000 i.u.
2,000 i.u.
PMSG.
. PMSG,

- 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 PGF2ct administered at laparotomy (figure 10)
plasma progesterone declined significantly (P<.01) 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 post
laparotomy 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 regres
sion, 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 PGF^ remains un
defined, data accumulated in this study clearly suggest that 40 mg
PGF2a *s effective in regressing multiple CL in the cycling nonpregnant
beef cow based upon the reduction in plasma progesterone and CL regres-
stion evidenced by the absence of palpable CL by 7 days post-treatment.
The question still remains as to whether or not 40 mg PGF?_a
is effective

PPOGES l'ERONE
(NG/ML)
4 0 MG
1 2 3 4 5 6 7 8 9 10
DAY OF BLEEDING
Figure 10. Effectiveness of PGF2a to regress corpora ltea and reduce plasma progesterone
in PGF2a synchronized cows treated with PMSG ( ) and without PMSG ( ).

6
in regressing multiple CL of PMSG-PREGNANT cows since in the first ex
periment, 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 1 indicated that ^0 mg PGF2a-Tham Salt
apparently induced only partial functional and structural CL regres
sion 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 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 PGF2a 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

TABLE 7. DISTRIBUTION OF ESTRUS IN COWS AFTER A DUAL 33-5 MG SYNCHRONIZING INJECTION OF PGF2a-
THAM SALT AND ONE 40 MG ABORT I FAC I ENT DOSE OF PGF2a
Time after
PGF2C1 injection
(days)
Estrus post-first PGF2a
synchronizing dose
Estrus post
synchron
-second PGF2ct
¡zing dose
Estrus post-
ABORTIFAC 1 ENT PGF2a
dose
PMSG Group
No. %
Non-PMSG Group
No. %
PMSG
No.
Group
%
Non-PMSG
No.
Group
*
PMSG
No.
Group Non-PMSG
No.
G roup
*
1
2 9
3 13
3
14
1
v 4
2
10 45
11 48
10
45
3
13
1
4
3
6 28
5 21
6
28
5
21

1
4
4
2 9
2
9
9
39
1 1
48
5-10
14
64 8
35
11-15
6
27
Non respondinga
2/22 3%
4/23 18*
1/22
4*
5/23
21*
2/22
3% 2/23
31
aCows were considered estrous synchronized if detected in estrus within 168 hr post 33-5 mg PGF2a
and within 15 days post 40 mg PGF2ct. Animals not expressing estrus within these periods were
considered nonresponding.
/

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 abort i fac ient dos'e
of PGF2d was longer in the PMSG treated animals when compared to the
nonsuperovulated group (15 ys_ 7 days, respectively, P<.01). Ninety-
one 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 behav
ioral ly between 11 and 15 days post-treatment with a mean interval of
13.50 1.05 days. Fifty days post-40 mg PGF2ct 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 ejt^ aj_. 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 1. (1974) in that cows aborted in
early gestation may return to estrus within 2 days after abortion.
Results from th i stri a 1 show the PMSG-BRED group to be comparable to
that of Zerobin et^ aj_. (1973) in that the administration of PGF2ct to
non-superovu1ated 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 1 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 a 1 (1953), Hallford e_t^ aj_. (1975a,b), and
Turman et^ a_l_. (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 PGF2a was significantly (P< .01) affected by PMSG treat
ment (table 22, Appendix). The regress ion curves for individual treat
ments groups are depicted in figures 11 and 12. Plasma progestins con
centrations and predicted equations are summarized in tables 23 and 24
(Appendix), respectively.

TABLE 8. OVULATION DISTRIBUTION P0ST-PGF2ct INDUCED ESTRUS IN COWS IN EXPERIMENT 3
Behavioral estrus
after second PGF2a
(days)
Ovu1 a tion
in PMSG
treated <
g roups
Ovulat ion
in non-PMSG
trea ted
groups
Rt
ova ry
Lt
ova ry
Overa 11
Rt
ovary
Lt
ova ry
Overa 11
1
A.3
1.2
A. 0
+
1 .0
8.0
1 ,7a
0.0
0.0
1 .0
0.0
1 .0
+
0.0
2
3.7
2.0
3. A
3.1
7.1
A. 6
0.3
0.6
0.7
0.6
1 .0
+
0.0
3
3.5
1-9
2.3
+
1 .0
6.0
2.1
0.6
0.5
0.2
0. A
0.8
+
0.5
A
3.0
0.0
3.0
+
2.0
5.8
2.8
0.6
0.5
0. A
0.5
1 1
+
0.3b
Non respond i ng
0.00
+
0.00
0.60
0.55
Overa 11
6. A5
+
7.5A
0.91
0. A1
ax S.E.
^One cow had a double ovulation.
/
ON

67
TABLE 9.
OVULATION RATES IN
PMSG AT FOUR MONTH
COWS TREATED TWICE
INTERVAL
WITH
Cow
number
Fi rst
T reatment
PMSG
(6/18/76)
Second
Treatment (
PMSG
10/29/76)
RT ovary
LT ova ry
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
Overa 11
3-0 2.1
2.5 3-0
3-5 1.5
1.7 1 -9

40 MG
PGF 2 a
\
3 4 5 6 7 8 9,1011
DAY OF BLEEDING
Plasma progestin profile in PMSG-BRED ( ) and PMSG-CYCLING ( ) cows after
40 mg PGFa
Figure 11.

PROGESTIN
(NG/MO
1 2 3 4 5. 6 7 8 9 in 11
DAY OF BLEEDING
Figure 12. Plasma progestin in BRED () and CYCLING ( ) cows after 40 mg PGF2a.
c
CO

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-super-
ovulated cows of 3-07 0.15 and 2.64 0.21 ng/ml for the same res
pective groups at laparotomy. Similar results have been reported pre
viously for cycling cattle (Plotka et a 1., 1987; Stabenfeldt et alT,
19&9; Kazama and Hansel, 1970; Sprague et_ aj_. 1971; Wettemann et al ,
1972; G1 encross et_ aj_. 1973). AFter 40 mg PGF2a injection on day 3,
plasma progestin in the PMSG group declined significantly (Pc.Ol) 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-superovu1ated
anima1s, min¡mum progestin levels of 1.06 0.19 and O.96 0.16 ng/ml
were achieved by day 8. These trends were considered to be curvilinear
(table 24, Appendix). On an individual animal basis, all cows achieved
progestin levels below 1 ng/ml post 40 mg PGF2ct.
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 ex
pression of estrus it takes up to 16 days (Zerobin et a 1., 1973;
Douglas et_ al_. 1974).
In the PMSG superovu1ated 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-Barbel la et al., 1976,1977). In the superovu1ated

71
pregnant cow, however, they indicated kO mg PGF2ct 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 vari
ability, 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 superovula
tion 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,
208-dihydroprogesterone, luteinizing hormone, and estrogens in res
ponse to the PMSG induction of superovulation, 15 cycling parous Angus
cows were estrous synchronized with two sequential treatments of 33.5
mg PGF2a-Tham Salt (lM) at a 12-day interval. An injection of 2,000
i.u. PMSG (Organon)>was administered 2b hr prior to the second PGF2a
and cows were inseminated three times at 0, 12, and 2b hr post-PMSG-
PGF2a-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 (5-501.29)
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) sug
gesting the possibility of a relationship between length of the inter
val 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 treat
ment after which a 50% increase was observed by 2b hr post-gonadotropin
administration. Simultaneously, a substantial increase in plasma
72

73
estrogens and LH was also recorded with no significant change in 208"
dihydroprogesterone levels. The hormonal trends after the luteolytic
dose of PGF2oi 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 PHSG. 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-PGF2a-induced cycle was characterized
by a rather significant (Pc.Ol) increase in plasma progesterone which
reached a maximum of 18.44 4.59 ng/ml on the day of injection of 40
mg PGF2cx. A dramatic decline in plasma LH and estrogen levels was ob
served 'post-estrus and remained at base line thereafter.
The plasma 208-dihydroprogesterone trend did not change signif
icantly (P>.10) during the course of this study. When progesterone and
208-dihydroprogesterone ratios were computed, however, a significant
(P<.01) trend was detected suggesting a relationship between P and 208P.
The lowest ratios of 20Q-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
1uteinization of the granulosa cells or from luteal cells of the re
gressing 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 endocri
nology of the PMSG superovu1ated cow differs from that of the normal
cycling cow. Not only the magnitude of the hormonal secretion is dif
ferent, but the length of the interval from PMSG to estrus and sup^r-
ovulatory response are affected. In addition, the early LH surge and
early increase in LH post-PMSG treatment might be partially due to dif
ferential follicular growth and estrogen secretion in response to
gonadotrophin administration. This experiment, however, was not
designed to test the hypothesis of differential follicular growth post
PMSG. Research designed to test the hypothesis of differential follic
ular-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 thishormonal 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 PGF2 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 PGF2a. 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 PGF2ct, 13 days post-PMSG, evoked a dramatic decline in plasma

P in both PMSG and non-PMSG treated cows. Plasma P reached basal con
centrations by 96 hr post-AO mg PGF2a- Ovarian palpation 7 days post
laparotomy confirmed multiple CL regression. Shortly thereafter cows
expressed estrus.
In conclusion, this experiment demonstrated that administration
of bO 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 b$ 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 PGF2a. The reduction in
plasma progestin following bO mg PGF2a was significantly (P<.01) af
fected 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 post
treatment vs^ 6b% 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 sug
gest that profound effects at both the ovarian and endocrine levels in
the bovine female in response to PMSG could be modulated by PGF2a. Thu

/ b
the introduction of bO mg PGF2a 13 days post-PMSG to superovulated 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 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 disti 11ed-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/100 ml) to the above stock solution. The assay buffer should be
stored at 4C for no longer than A weeks.
, aDo not breathe or contaminate skin. Extremely poisonous. Re
acts 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 amino-
methane in 500 ml disti 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 HC1. Then add disti 11ed-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 pM) and 3 ml of 0.1 M
Tris buffer pH 8.1.

80
TABLE 12. PLASMA CONCENTRATIONS OF PROGESTERONE, 206-DI HYDROPROGESTERONE,
LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING MORE THAN
THREE CORPORA LUTEA AT LAPAROTOMY
Day of
Progesterone
2 0 0-Dihydro-
Prog'esterone
Lute in¡zing
hormone
Estrogens
b1eeding
x S.E.
x S.E.
x S.E.
x S.E.
ng/ml
ng/ml
ng/ml
pg/ml
1AM
3.
72
+
0
34
2
60
+
0
65
0
97
+
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

0
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
1 1
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
10AM
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

0
1 1
11AM
6
30
+
1
37
1
72

0
50
1
82

0
1 1
1
54
+
0
09
PM
7
98
f
1
54
1
96

0
37
1
94

0
22
12AM
10
80

1
42
2
22

0
27
2
04

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

0
32
1
40
+
0
28
2
20
+
0
15
14AM
15
24
+
1
23
2
32

0
19
1
56

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

1
46
2
.82

0
35
1
40

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

TABLE 12.
(Cont¡nued)
Day of
b1eeding
Progesterone
x S.E.
ng/ml
203-Dihydro-
Progesterone
x S.E.
- ng/ml
Luteinizing
hormone
x S.E.
ng/ml
Estrogens
x S.E.
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, 208-DI HYDROPROGES
TERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING
TWO OR THREE CORPORA LUTEA AT LAPAROTOMY
Day of
Progesterone
20B-Dihydro-
Progesterone
Lute iniz¡ng
hormone
Estrogens
b1eed i ng
x S.E.
x S.E.
x S.E.
x S.T.
ng/ml
ng/ml
ng/m!
pg/ml
1AM
3-
.85
+
0.3 b
2,
.50

0.
37
1.
.23

0.
46
1
.73
+
0.09
PM
3.
95
+
0.38
2,
.55

0.
40
1.
.28

0.
13
2AM
3.
98

0.46
2,
.10

0.
37
0.
.73

0.
09
1
.65
4;
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)
b.
.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,
.90
0.
31
7
.28
+
1.97
PM
1 .
.18

0.45
1 ,
.58

0.
33
2,
.08

0.
36
9AM
1 .
.**3

0.49
1
.83

0.
.33
2.
.20

0.
, 12
5
.13
+
1.59
PM
1 .
b7

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
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 .
.95

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

0.
27
1
.60

0.40
i4am
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
b.
30
+
0.68
1
.83

0.
.39
1 .
.15

0.
.25
1
.67

0.18
PM
b.
.20
+
0.41
2.
.00

0.
,62
1 ,
.33

0.
30
16AM
b.
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
1 7AM
5.
.60
+
0.56
2,
.48

0.
.45
0,
.98

0.
,10
1
.69

0.17
PM
b.
.88
+
0.36
2
.95

0.
.49
1 .
. 10

0.
19
18am
b.
.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, 20B-01 HYDROPROGES
TERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS WITH
ZERO OR ONE CORPORA LUTEA AT LAPAROTOMY
20 3-Dihydro -
Lute¡nizing
Day of
Progesterone
Progesterone
hormone
Est rogens
b1eed ing
x S.E.
x S.E.
x S.E.
x S7E.
ng/ml
ng/ml
ng/ml
pg/ml
1AM
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
1 1AM
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
1 4AM
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

TABLE 15. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR PROGESTERONE
Portion of curve
Source
under
' estimation
Estimated regression line
R2
PR>F
Group 1
from
1AM
to
4AM
y
_
5.48 0.26x + O.Olx2
0.3285
0.002
>3 CL
from
4AM
to
6PM
y
=
1431.79 83.68x + 1.62x2 O.Olx3
0.4708
0.001
from
6pm
to
15 PM
y
=
- 3.73 0.02x + O.OOlx2
0.7563
0.001
from
1 5PM
1 tc
> 22PM
y
=
1056.72 15.48x + 0.07x2 O.OOOlx3
O.6O86
0.001
Group 2
from
1AM
to
3PM
y
=
5.62 0.24x + 0.008x2
0.2686
0.037
2 to 3 CL
from
3PM
to
7PM
y
=
21.97 0.65x + 0.005x2
0.7213
0.001
from
7PM
to
18pm
y
=
4.51 + 0.08x
0.6842
0.001
Group 3
from
1AM
to
1 8pm
y
=
0.77 + 0.26x 0.009x2 + 0.0001x3
0.2764
0.001
0 to 1 CL

TABLE 16. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES 'FOR ESTROGENS AND 20 3-DI HYDROPROGESTERONE (20g-P)
Hormone
Sou rce
Port
under
on of curve
estimation
Estimated regression line
v-
R2
PR>F
Estrogens
Group
1
from
1AM
to
6AM
y =
= 18.46 2.20x + 0.07x2 0.0005x3
0.4771
0.010
>3 CL
from
6AM
to
22PM
y =
= 212.18 4.lOx + 0.02x2
0.5529
0.011
G roup
2
f rom
1AM
to
6AM
y =
= 1493.00 67.75x + 1.T4x2 0.008x3
0.2791
0.163
2 to
3
CL
f rom
6AM
to
1 8pm
y =
= 28.39 4.36x + 0.22x2 0.004x3
0.3250
0.097
Group
3
from
1AM
to
18pm
y =
= 2.10 + 0.02x + 0.005x2
0.1181
0.050
0 to
1
CL
20g- P
G roup
1
f rom
1AM
to
22PM
y =
= 2.31 + O.Olx O.OOlx2
0.2059
0.054
Group
2
f rom
1AM
to
1 8pm
y =
= 3-84 0.I4x + 0.003x3
0.1762
0.066
G roup
3
from
1AM
to
1 8pm
y =
= 2.18 0.08x + 0.003x3
0.5121
0.051
I
CD
\-n

TABLE 17. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR LUTEINIZING HORMONE
Portion of curve
Source
under estimation
Estimated regression line
R2
PR>F
Group 1
from
1AM
to
5PM
y
_
5.97 Q.68x + 0.03x2 0.0002x3
0.5700
0.001
>3 CL
from
5PM
to
6AM
y
=
- 121.27+ 2.39x
0.3844
0.056
f rom
6AM
to
7AM
y
=
8232.59 248.28x + 1.86x2
0.4107
0.042
from
7AM
to
22PM
y
=
4.84 0.04x + O.OOOlx2
\
0.2591
0.001
Group 2
from
1AM
to
5PM
y
=
17.98 3 26x + 0.23x2 0.007x3 +
2 to 3 CL
O.OOOlx4
0.5419
0.001
from
5M to 6AM
y
=
- 75.11 + 1.51x
0.1960
0.272
from
6AM
to
7AM
y
=
1598.69 47.05x + 0.35x2
O
LO
OO
vx>
0.510
from
7AM
to
18PM
y
=
76.88 3.22x + 0.05x2 0.0005x3
0.5169
0.001
Group 3
from
1AM
to
6AM
y
=
16.92 + 3.55x 0.26x2 + 0.009x3
0.5894
0.052
0 to 1 CL
from
6AM
to
6PM
y
=
-210.4+3-5x
0.9421
0.094
from
6PM
to
7AM
y
=
33.69 0.44x
0.1189
0.118
from
7AM
to
1 8pm
y
=
46.25 1.79x + 0.03x2 0.0002x3
0.2458
0.001

87
TABLE 18. COEFFICIENTS OF CORRELATION BETWEEN PLASMA HORMONAL LEVELS
AND NUMBER OF CORPORA LUTEA3
Source
Progesterone
20B-Dihydro -
progesterone
Luteinizing
hormone
Estrogens
Group 1
0.73b
0.16
0.26
0.68
> 3 C L
(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. 1 1
0.35
0.42
0 to 1 CL
(0.1822)
(0.4846)
(0.0837)
(0.0053)
Expressed upon grouping criteria (see text).
^Correlation coefficient.
cProbabi 1ity.

88
TABLE 19. PLASMA CONCENTRATIONS OF PROGESTERONE AFTER 40 MG PGF2a~
IN COWS TREATED WITH OR WITHOUT PMSG
Proges terone
Day of bleeding (ng/ml)
PMSG group Non-PMSG group
1
8.79
2.12a
2.6 0.06
2
10.3*4
2.22
2.3 0.06
3 (PGF)
10.02
1.89
3.0 0.06
4
*4.59
0.67
2.1 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
0.47 0.15
8
0.61
0. 10
0.47 0.22
9
0.71
0.06
0.87 0.15
10
0.93
0.04
1.07 0.12
ax S.E.

89
TABLE 20. SPLIT-PLOT ON TIME ANALYSIS- OF VARIANCE OF PROGESTERONE IN
EXPERIMENT 2
Source
df
Sums of Squares
Mean Square
F value
TotaJ
103
1185.73
T reatment
1
26.02
26.02
3.29**
Cow (treatment)
1 1
87.09
7.92
Day
7
647.97
96.42
25.19**
T reatmentxday
7
102.96
14.71
3.84*
Cow (treatment)xday
77
294.69
3.83
* (P<05)
**(P<.01).

90
TABLE 21. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTERONE
Source Estimated regression line R2 PR>F
PMS Group y = 39.12 13-99x + 1.73x2 0.07x3 0.7323 0.001
Non-PMS Group y = 2.4 + 0.75x 0.26x2 0.8697 0.001

TABLE 22. ANALYSIS OF VARIANCE IN EXPERIMENT 3
Source
df
Sums of
squa res
F value
PR>F
Treatment (T)
3
182.36
PMSG (G)
1
176.65
55.54
0.0001**
Bred (B)
1
0.01
0.00
0.9644
P x B
1
5.70
,1.79
0.1812
Day (L)
1
476.36
149-77
0.0001**
Day (0)
1
240.33
75.56
0.0001**
Day (C)
1
12.26
3.86
0.0503*
Error
398
1265.93

T x Day
24
263.89
Cow (T) x Day
328
753.89
Cow (T)
41
240.91
Lack of fit
5
7.24
Corrected Total
404
2177.24
*(P<.05)
**(P<.01).
l

TABLE 23. PLASMA CONCENTRATIONS OF PROGESTIN .AFTER 40 MG PGF2ct IN COWS SUPER-
OVULATED WITH PMSG AND EXPOSED TO BREEDING
Progest in
ng/m 1
Day of bleeding Group 1 Group 2 Group 3 Group 4
(PMSG + bred) (PMSG-Nonbred) (Non-PMSG-bred) (control)
1
7.25
+
1 ,65a
9-65
1.35
2.68

0. 18
2.97
+
0.32
2
8.63
+
2.01
10.71
1.69
3.22
+
\
0.15
2.75
+
0.25
3 (PGF)
7.63

1.79
8.86
1.57
3.07
+
0.15
2.64
0.21
4
4.49

0.79
5. 11
0.92
2.67
+
0. 11
2.27
+
0.21
5
3.09

0.49
2.70
0.48
1.81

0. 1 8
1.65
+
0.14
6
2.30

0.31
2.83
0.49
1.49

0.17
1.38
0.09
7
1.49
+
0. 19
2.22
0.41
1 .22

0.13
1.14
+
0.12
8
1.45
+
0.27
1.60
0.32
1 .06

0. 19
0.96
0.15
9
1.37
+
0.23
1.25
0.22
1.14

0.17
0.84
+
0.09
10
1.55
+
0.23
1.31
0.19
1 .40

0. 19
1.11
+
0.09
11
1.88
+
0. 19
1.65
0.18
1 .62

0.12
1 ./46
+
0.09
ax S.E.
V£>
NJ

93
TABLE 24. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTIN
Source
Estimated regression line
R2
PR>F
PMSG + breeding
y = 32.22
- 10.90x + 1,29x2- 0.05x3
0.6642
0.001
PMSG + no
b reeding
y = 37.22
- l4.39x + 2.OOx2- 0.09x3
0.5781
0.001
No PMSG +
breeding
y = 6.3^ -
1.28x + 0.08x2
0.8301
0.001
No PMSG +
no breeding
y = 5.26 -
0.96x + 0.05x2
0.7693
0.001

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and
D.A
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after PGF2ct
in cows.
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101
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I0
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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 1963, he was ac
cepted in the Facultad de Agronoma, Universidad Central de Venezuela,
where he received the degree of Ingeniero Agronomo in May, 1969. In
October 1969, he entered in the Facultad de Agronoma as Instructor
of Beef Cattle Production and research assistant.
Since September 1973, he has been enrolled in the Graduate
School-of the University of Florida, with a scholarship granted by the
Facultad de Agronoma, 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

I certify that I have read this study and that in my opinion it
cpnforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
-
- yf .
Michael J. Fields, Chairman
Assistant Profssor 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.
lu t'M/i' c
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.
Fuller W.
Associate
Bazer
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.
William W. Thatcher
Associate Professor of Dairy 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.
Pejaver V. Rao
Professor of Statistics
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December, 1977
Dean, Graduate School



67
TABLE 9.
OVULATION RATES IN
PMSG AT FOUR MONTH
COWS TREATED TWICE
INTERVAL
WITH
Cow
number
Fi rst
T reatment
PMSG
(6/18/76)
Second
Treatment (
PMSG
10/29/76)
RT ovary
LT ova ry
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
Overa 11
3-0 2.1
2.5 3-0
3-5 1.5
1.7 1 -9


CHAPTER V
SUMMARY AND CONCLUSIONS
In an attempt to characterize the trends of plasma progesterone,
208-dihydroprogesterone, luteinizing hormone, and estrogens in res
ponse to the PMSG induction of superovulation, 15 cycling parous Angus
cows were estrous synchronized with two sequential treatments of 33.5
mg PGF2a-Tham Salt (lM) at a 12-day interval. An injection of 2,000
i.u. PMSG (Organon)>was administered 2b hr prior to the second PGF2a
and cows were inseminated three times at 0, 12, and 2b hr post-PMSG-
PGF2a-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 (5-501.29)
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) sug
gesting the possibility of a relationship between length of the inter
val 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 treat
ment after which a 50% increase was observed by 2b hr post-gonadotropin
administration. Simultaneously, a substantial increase in plasma
72


reported increased plasma 208-P levels after either PMSG or HCG
treatment.
Kidwell et^ ak (1966) working with PMSG superovu 1 a ted 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 in
creased from 0.03 0.01 to 2.82 2.~]k MU/mg 5 days after LH injec
tion. The administration of HCG, however, resulted in a ten-fold in
crease in the 20-HSD activity. A significant increase in the glucose-
6-phosphate dehydrogenase activity was also observed after LH and HCG
administ rat ion.
The possibility that 20B-P in the bovine might be playing a simi
lar role as 20B-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^ a_l_. 1963), Chinchilla (Tam, 1971) and the African elephant (Smith e_t_
ah, 1969) -
Staples and Hansel (1961) presented data to suggest that embryo
survival at day 15 may be influenced by circulating 208-P levels. Gomez
et_ a_L (1962) quantitated P and 208-P levels in the utero-ovarian vein
ipsilateral to the CL in cows between 250 and 282 days of gestation.
Levels of 20BP 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:208_P ratio was approximately one.
Levels of 208-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).


LIST OF FIGURES Continued
FIGURE Page
10 Effectiveness of PGF2a to regress corpora ltea and
reduce plasma progesterone in PGF^ct synchronized
cows treated with PMSG ( ) and without PMSG ( ). 61
11 Plasfna progestin profile in PMSG-BRED ( ) and
PMSG-CYCL ING (---) cows after 40 mg PGF2a 68
12 Plasma progestin in BRED ( ) and CYCLING ( )
cows after **0 mg PGF2a 69
V
x


41
progesterone was eluted within the first 3 ml and the more polar 208-P
was eluted between the fifth and seventh ml fractions.
Precision
The between and within assay coefficient of variation (c.v.) was
calculated from 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, 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 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
intraassay c.v. (14.4 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, i960) from which the following parameters
were obtained:
(1) Identification of classes (group, day, and group x day inter
action) with their respective least-squares means and standard errors
for progesterone, 206-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, 208-P, and E were statistically affected by day, group, and


7
ensued with a return to estrus 48 to 98 hr after the first, and 48 to
55 hr after the second PGF2a treatment. A preovulatory surge of LH
occurred 62 to 103 hr after the first, and 48 to 62 hr after the sec
ond PGF2a treatment, and was followed by ovulation. Similar changes
have been reported by Stellflug et_ a_l_. (1973), Louis et_ aj_. (1973),
and Louis et^ aj_ (1974a,b).
Chenault et aj_. (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 in
creased and apparently stimulated an ovulatory surge of LH at 72 21
hr post-treatment. Ovulation occurred at 99-5 19 hr after PGF2ct ad
ministration. Gimenez et_ a_L (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 in
jection. In the same study, plasma estrogens concentrations ranged
from 25 to 100 pg/ml prior to treatment with no observed change in es
trogens levels occurring after giving PGF2ct when compared to the saline-
treated cows.
The mechanism by which PGF2a initiates CL regression is unknown.
Novy and Cook (1973) and Thornburn and Hales (1972) demonstrated that
PGF2ct 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 re
gression or physical destruction of the luteal cell.
Because structural regression was preceded by accumulation of
lipid droplets, Stacy e_t aj_. (1976) postulated functional CL regression


83
TABLE 14. PLASMA CONCENTRATIONS OF PROGESTERONE, 20B-01 HYDROPROGES
TERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS WITH
ZERO OR ONE CORPORA LUTEA AT LAPAROTOMY
20 3-Dihydro -
Lute¡nizing
Day of
Progesterone
Progesterone
hormone
Est rogens
b1eed ing
x S.E.
x S.E.
x S.E.
x S7E.
ng/ml
ng/ml
ng/ml
pg/ml
1AM
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
1 1AM
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
1 4AM
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


32
NaOH, and 15 ml of hexane was added and vortexed 1 min. The lower aque
ous phase was quickly frozen in liquid nitrogen while the upper lipid-
containing 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 pi 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 pi transferred to a scintillation vial to
estimate recovery. The remainder was used for assay.
Quantification of 3P and 3H-20g-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 appro
priate tritiurn-1 abe 1 led steroids. The dried residue from the ether ex
tract was quantitatively transferred to the column with two 500 pi ali
quots of hexane:benzene:methanol (80:15:5). Progesterone was eluted
from the column using 5 ml of the above organic solvent while 208-dihydro-
progesterone was eluted 2 ml after 3P. The 3P and 3H-208-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 des
cribed for progesterone (vide supra).


APPENDIX


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. 3^:77-
Weiss, J.R., H.J. Brinkley, and E.P. Young-. 1976. In vitro steroido-
genesis in porcine corpora ltea. 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. 3^:1020.
Willett, E.L., P.J. Buckner, and W.H. McShan. 1953. Refractoriness
of cows repeatedly superovu1ated with gonadotrophins. J. Dairy
Sci. 36:1083.
Zerobin, K., W. Jockle, and Ch. Steingruber. 1973- Termination of
pregnancy with prostaglandins (E2 (PGE2) and F201 (PGF201) in
cattle. Prostaglandins A:891.


ENDOCRINOLOGY OF THE SUPEROVULATED COW AND
SUBSEQUENT PROSTAGLANDIN F2a REGRESSION OF
THE MULTIPLE CORPORA LUTEA
By
SERGIO RAFAE4. 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


their collaboration in providing the progesterone and estrogen antisera,
respectively. Special thanks are also due to Dr. J. H. Hentges, Univer
sity 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, 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. RossinaLFernandez for her typing of this manuscript.
i i i


TABLE 15. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR PROGESTERONE
Portion of curve
Source
under
' estimation
Estimated regression line
R2
PR>F
Group 1
from
1AM
to
4AM
y
_
5.48 0.26x + O.Olx2
0.3285
0.002
>3 CL
from
4AM
to
6PM
y
=
1431.79 83.68x + 1.62x2 O.Olx3
0.4708
0.001
from
6pm
to
15 PM
y
=
- 3.73 0.02x + O.OOlx2
0.7563
0.001
from
1 5PM
1 tc
> 22PM
y
=
1056.72 15.48x + 0.07x2 O.OOOlx3
O.6O86
0.001
Group 2
from
1AM
to
3PM
y
=
5.62 0.24x + 0.008x2
0.2686
0.037
2 to 3 CL
from
3PM
to
7PM
y
=
21.97 0.65x + 0.005x2
0.7213
0.001
from
7PM
to
18pm
y
=
4.51 + 0.08x
0.6842
0.001
Group 3
from
1AM
to
1 8pm
y
=
0.77 + 0.26x 0.009x2 + 0.0001x3
0.2764
0.001
0 to 1 CL


90
8 0
70
60
50
40
30
20
10
flM#
CONCENTRATION OF PROGESTERONE (DG/ML) /
Figure 5- Standard curve of progesterone, (Each point represents the mean of
triplicates on 20 determinations.)
O.


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 dis
cussion of the results;
(3) Test for day effect within hormone was computed using tire
appropriate error term obtained from Harvey's printout (Steel and
Torrie, i960). Reproductive responses were tested by LSD; and
{b) Additional computations were obtained according to the pro
cedures described in SAS 76 (Barr eT a_K 1976).
y


33
Quantification of estrogens was accomplished as described by
Abraham et aj_. (1971) using the assay procedure of Nett et_ aj_. (1973).
Quant i f ication of LH was as described by Chenault (1973). The quantifi
cation of 20g-dihydroprogesterone was accomplished by converting this
progestogen to progesterone and subsequently assaying for progesterone.
Conversion of 3H-20g-P to 3P
The chemical conversion of 3H-20g-P to 3P was similar to the
procedure described by Shol1 and Wolf (197*0- To the dried column
elute of 3H-20g-P was added 200pl 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 1 ml of distilled water and the sample extracted with
3 ml of ethyl acetate (2X). This extract was washed with 500 yl dis
tilled 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 sub
jected to chromatography on a LH-20 column (vide supra) and the 3P frac
t i on 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 A wk.


89
TABLE 20. SPLIT-PLOT ON TIME ANALYSIS- OF VARIANCE OF PROGESTERONE IN
EXPERIMENT 2
Source
df
Sums of Squares
Mean Square
F value
TotaJ
103
1185.73
T reatment
1
26.02
26.02
3.29**
Cow (treatment)
1 1
87.09
7.92
Day
7
647.97
96.42
25.19**
T reatmentxday
7
102.96
14.71
3.84*
Cow (treatment)xday
77
294.69
3.83
* (P<05)
**(P<.01).


I certify that I have read this study and that in my opinion it
cpnforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
-
- yf .
Michael J. Fields, Chairman
Assistant Profssor 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.
lu t'M/i' c
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.
Fuller W.
Associate
Bazer
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.
William W. Thatcher
Associate Professor of Dairy Science


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 PGF2ct. This treatment, in it
self, allowed for the subgrouping of animals according to their ovula
tion rate. Since it was determined that the time-trend in plasma hor
mones among 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 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 proges
terone curve (table 15, Appendix) was exhibited prior to PMSG treat
ment. 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), proges
terone decreased to a basal concentration of 0.72 0.14 ng/ml by day
6 (PM). These changes were best described by a significant (Pc.Ol)
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<.01) curvilinear (table 15,
Appendix) increase to 16.46 + 2.36 ng/ml on day 15 (AM) was observed.


Stacy, B.D., R.T. Gremmell, and G.D. Thorburn. 1976. Morphology of
the corpus luteum in the sheep during regression induced by prosta
glandin F2a- Biol. Reprod. 1 4 :2 80.
Staigmiller, R.B., R.A. Bellows, R.E. Short, and J.B. Carr. 1976.
Ovarian response to FSH injections in beef cows, J. Anim. Sci.
43(1) :306. (Abstr.).
Staples, R.E. and W. Hansel. 1961. Luteal function and embryo survival
in the bovine J. Dairy Sci. 44:240.
Steel, R.G.D. and J.H. Torrie. I960. 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. Luteo-
lysis after 30 or 60 mg PGF2a in heifers. J. Anim. Sci. 37:330.
(Abstr.).
Tam, W.H. 1971. The production of hormonal steroids by ovarian tissues
of the chinchilla (Ch i nch illa 1 aniger) J. Endocrinol. 50:267.
Telegdy, G. and K. Savard. 1966. Steroid formation in vitro in rabbit
ovary. Steroids 8:685.
Tervif, H.R., L.E.A. Rowson, and A. Brand. 1973- Synchronization of
oestrus in cattle using a prostaglandin F2aanalogue (ICI -79,939)
J. Reprod. Frtil. 34:179-
Thatcher, W.W. and J.R. Chenault. 1976. Reproductive physiological
responses of cattle to exogenous prostaglandin F2ct. J. 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 PGF201 into a
uterine vein. Proc. Austral. Phys. & Phar. Soc. 3:145. (Abstr.).
Tribble, R.L. 1973- Peripheral serum progesterone, 17~hydroxyproges-
terone and 203-hydroxyprogesterone levels in suckled and nonsuckled
primiparous Hereford heifers. Ph.D. Dissertation. Texas A&M
University.
Turman, E.J., D.B. Laster, R.E. Renbarger, D.F. Stephens, and R.H.
Edwards. 1969- The experimental production of multiple births
in beef cows by hormone injections. Mise. Pub. Agrie. Exp. Sta.
Okla. State University. 82:5.
Turman, 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 PGF2ct. J. Anim. Sci. 41:382.
(Abstr.).


BIOGRAPHICAL SKETCH
Sergio R. Lopez Barbel la was born November 1, 19^5, in Caracas,
Venezuela. After graduation from high school in 1963, he was ac
cepted in the Facultad de Agronoma, Universidad Central de Venezuela,
where he received the degree of Ingeniero Agronomo in May, 1969. In
October 1969, he entered in the Facultad de Agronoma as Instructor
of Beef Cattle Production and research assistant.
Since September 1973, he has been enrolled in the Graduate
School-of the University of Florida, with a scholarship granted by the
Facultad de Agronoma, 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


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, 208-DI HYDRO
PROGESTERONE LUTEINIZING HORMONE, AND ESTROGENS
IN COWS HAVING MORE THAN THREE CORPORA LUTEA AT
LAPAROTOMY 80
13 PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DI HYDRO
PROGESTERONE LUTEINIZING HORMONE, AND ESTROGENS IN
COWS HAVING TWO OR THREE CORPORA LUTEA AT LAPAROTOMY 82
14 PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DI HYDRO
PROGESTERONE 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 208-DI HYDROPROGESTERONE (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
PGF2a IN COWS TREATED WITH OR WITHOUT PMSG 88
20 SPLIT-PLOT ON TIME ANALYSIS OF VARIANCE OF PROGES
TERONE 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
v i i


9B
Hammond, J. 19^9. Induced tw in ovu1 at ion and multiple pregnancy in
cattle. J. Agrie. Sci. 39:322.
Harrison, R.J. 1946. The early development of the corpus luteum in
the mare. J. Anat. 80:l60.
Harvey, W.R. I960. Least squares analysis of data with unequal sub
class numbers. U.S.D.A. ARS-20-8. .
Hayano, M. M.C. Lindberg, M. Wiener, H. Rosenkrantz, and R.l. Dorfman.
1975- Steroid transformat ions by corpus luteum tissue. Endo
crinol. 55:326.
Henderson, K.M. and K.P. McNatty. 1975. A biochemical hypothesis to
explain the mechanism of luteal regression. Prostaglandin 9:779-
Henricks, C.H. 1972. Prostaglandins and therapeutic abortion =
Summary of present status,. J. Reprod. Med. 9:465.
Henricks, D.M., J.R. Hill, Jr., J.F. Dickey, and D.R. Lamond. 1973-
Plasma hormone levels in beef cows with induced multiple ovula
tions. J. Reprod. Frtil. 35:225.
Henricks, D.M., N.C. Rawlings, A.R. Ellicott, J.F. Dickey, and J.R.
Hill. 1977- Use of prostaglandin F201 to induce parturition in
beef heifers. J. Anim. Sci. 44:438.
Hill, J.R. Jr., J.F. Dickey, and D.M. Henricks. 1973- Gonadal hor
mones in PGF?a-PMS treated heifers. J. Anim. Sci. 37:307.
Hill, J.R. Jr., T. Gimnez, A.R. Ellicott, W.R. Boone, and D.M. Henricks
1976. Ovulation in cows after PGF2ot 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. 35:245. (Abstr.).
Hilliard, J., R. Penardi, and C.H. Sawyer. 1967. A functional role
for 20-hydroxypreg-4-en-3-one in the rabbit. Endocrinol. 80:901.
Ichikawa, S., H. Morioka, and T. Savada. 1971. Identification of the
neutral steroids in the ovarian venous plasma of LH-stimulated
rats. Endocrinol. 88:372.
Inskeep, E.K. 1973- Potential uses of prostaglandins in control of
reproductive cycles in domestic animals. J. Anim. Sci. 36:1149.
Jackson, P.S. and M.J. Cooper. 1977. The use of cloprostenol for the
termination of pregnancy and the expulsion of mummified fetus in
cattle. Vet. Rec. 100:361.
Jdchle, W. 1973- Corticoid-induced parturition in domestic animals.
Annu. Review Pharmacol. 13:33-


88
TABLE 19. PLASMA CONCENTRATIONS OF PROGESTERONE AFTER 40 MG PGF2a~
IN COWS TREATED WITH OR WITHOUT PMSG
Proges terone
Day of bleeding (ng/ml)
PMSG group Non-PMSG group
1
8.79
2.12a
2.6 0.06
2
10.3*4
2.22
2.3 0.06
3 (PGF)
10.02
1.89
3.0 0.06
4
*4.59
0.67
2.1 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
0.47 0.15
8
0.61
0. 10
0.47 0.22
9
0.71
0.06
0.87 0.15
10
0.93
0.04
1.07 0.12
ax S.E.


P in both PMSG and non-PMSG treated cows. Plasma P reached basal con
centrations by 96 hr post-AO mg PGF2a- Ovarian palpation 7 days post
laparotomy confirmed multiple CL regression. Shortly thereafter cows
expressed estrus.
In conclusion, this experiment demonstrated that administration
of bO 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 b$ 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 PGF2a. The reduction in
plasma progestin following bO mg PGF2a was significantly (P<.01) af
fected 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 post
treatment vs^ 6b% 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 sug
gest that profound effects at both the ovarian and endocrine levels in
the bovine female in response to PMSG could be modulated by PGF2a. Thu


40
The present study suggests the possibility of a relationship be
tween 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 res
ponse of 0.67 0.82 ovulations per cow.
The percentage of animals conceiving to either the first post-
PMSG 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_ aj_. (1971), Laster e_t aj_. (1971b) and Hallford et a 1 .
(1975a,b) for superovulated cows. Multiple fetuses determined by calving
date were lower than those estimated earlier by palpation. These dis
crepancies 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 PGF2a,
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.


8
was most likely due to a blockage in one or more stages in steroido
genesis. 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 F2ct and Therapeutic Abortion
An effective means of inducing parturition in the cow could re
duce calving losses and labor costs by decreasing the calving period
to a shorter and predictable period. Parturition can be induced in
the cow by t reat ing >wi th estrogens (Spears e_t_ aj_. 197*0, corticoids
(Jochle, 1973), and PGF2a (Lauderdale, 1972). In addition, PGF2ct 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 PGF2ot (Gutknecht et a 1 1969). In humans,
PGF2a 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 e_t aj_. 197*0, and
bovine (Lauderdale, 1972, 197**; Fields et_ aj_. 1977a) species. Adminis
tration of PGF2ct either intramuscularly or systemically at early stages
of gestation in the bovine resulted in a dramatic decline in plasma pro
gesterone within 2*4 hr and behavioral estrus 2 to 16 days post-treatment
(Zerobin et_ aj_. 1973; Douglas et al., 197*4).


PPOGES l'ERONE
(NG/ML)
4 0 MG
1 2 3 4 5 6 7 8 9 10
DAY OF BLEEDING
Figure 10. Effectiveness of PGF2a to regress corpora ltea and reduce plasma progesterone
in PGF2a synchronized cows treated with PMSG ( ) and without PMSG ( ).


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 superovula
tion effect, cows with more than three CL were injected with 40 mg~~PGF2a
to induce CL regression.
\
Surgical Description of Supravaginal Laparotomy
Prior to surgery animals were taken off feed overnight. Animals
were restrained andytranqu i 1ized 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. penicilline
(Combiotic, Pfizer).
EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN PMSG SUPER-
OVULATED BEEF CATTLE FOLLOWING ADMINISTRATION OF
40 MG PGF2a-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
ach ievesynchronization of estrus (figure 2). A 2,000 i.u. PMSG injec
tion was administered subcutaneously to 10. cows 24 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 PGF2a injection.


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 pi (20,000 1,000 dpm) was used in the assay.
This solution was stored at AC and used for no longer than 1 wk. P and
E tritiated steroids were further diluted with benzene to 2,000 dpm/100
pi and stored at AC for determining recovery of extracted steroi.dsT
Since tritiated 200-dihydroprogesterone was not commercially avail
able it was synthesized from 3P as described by Tribble (1973).
Conversion of Progesterone (3P)'to 203-Dihydroprogesterone (3H-20B~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 CpH =
5-2, which contained 100 mg percent EDTAH, 30 pi of NADH C(Sigma Chemical
Company, Grade III) in 0.1 M Tris buffer (Eastman), pH = 8.1 (see table
11, Appendix) and 30 pi of 208-hydroxysteroid dehydrogenase enzyme
C(Ca1biochem, Activity A.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 ml dis
tilled water. The 3H-20g-P was extracted with 1 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-20p-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-2O0-P was subsequently
determined. Fractions containing 3H-20S~P were stored in benzene:


9b
Erb, R.E., V.L. Estergreen Jr., E.R. Gomes, E.D. Plotka, and O.L.
Frost. 1968. Progestin levels in corpora ltea and progesterone
in ovarian venous and jugular blood plasma of the pregnant bovine.
J. Dairy Sci. 51:401.
/
von Euler, R.S. 1934. Zur kenntnis der pharmakologischem wirkungen
von nat ivsekreten and extrakten mannlicher accessorischer
gisch 1 echtsdrugen. Arck. Exp. Patho-1 Pharmak. 175:78.
Fields, M.J., R.H. Schultz, J. Bass, and A.C. Warnick. 1977a. Abortion
in beef'heifers using cloprostenol (a PGF2a analogue). Amerr Soc.
Anim. Sci. Southern Section, Atlanta, Georgia. (Abstr.).
Fields, M.J., R.H. Schultz, T.H. Wise, S.R. Lopez-Barbella, K.R.
Eubanks, J.A. Wiggins, and A.C. Warnick. 1977b. Effect of in
semination time on fertility in beef cattle synchronized with
cloprostenol. Amer. Soc. Anim. Sci. Southern Section, Atlanta,
Georgia. (Abstr.).
Fields, M.J., A.C. Warnick. T. Wise, J. Bass, and M. Koger. 1975- A
reevaluation of artificial insemination in beef cattle. J. Anim.
Sci.41:352.
Fordj S.P. and F. Stormshak. 1975- Effect of PMS and GnRH and serum
LH and P4 in heifers. J. Anim. Sci. 41:353- (Abstr.).
Fournier, M.P., E.J. Turman, R.P. Wettemann, and T.D. Rick. 1976.
Plasma progesterone in cows after PMSG and PGF2ct- J- Anim. Sci.
43:248. (Abstr.).
Garverick, H.A., R.E. Erb, R.D. Randel, and M.D. Cunningham. 1971-
Dietary urea for dairy cattle. I. Relationship to luteal func
tion. J. Dairy Sci. 54:1669-
Gimnez, T., R. Chang, J. Thacher, and D.M. Henricks. 1976. Steroid
levels in bovine U-0 vein after PGF2a. J. Anim. Sci. 43:286.
(Abstr.).
Glencross, R.G., I.B. Munro, B.E. Senior, and G.S. Pope. 1973- Concen
trations of oestradiol-170, oestrus, and progesterone in jugular
venous plasma of cows during the oestrous cycle and in early
pregnancy. Acta Endocrinol. 73:374.
Godke, R.A., R.G. Root, R.H. Ingraham, and J. L. Kreider. 1977- Syn
chronization of superovulation in multiple birth cows with FSH-
P, PMSG, and PGF2a- Proc. South. Sect., Amer. Soc. Anim. Sci.
P- 50.
Gomez, W.R. and R.E. Erb. 1965- Progesterone in bovine reproduction:
A review. J. Dairy Sci. 48:314.


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INGEST IEID E9IQPLYGZ_CB5YPY INGEST_TIME 2014-11-07T19:23:08Z PACKAGE AA00026362_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


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
X 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
includes one animal pregnant to treatment that died due to
Tympani tes.
^Post-mortem examination of reproductive tract revealed ovi
duct blockage in the single open cow.
cDetermined by phenotypic appearance of the calves.


87
TABLE 18. COEFFICIENTS OF CORRELATION BETWEEN PLASMA HORMONAL LEVELS
AND NUMBER OF CORPORA LUTEA3
Source
Progesterone
20B-Dihydro -
progesterone
Luteinizing
hormone
Estrogens
Group 1
0.73b
0.16
0.26
0.68
> 3 C L
(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. 1 1
0.35
0.42
0 to 1 CL
(0.1822)
(0.4846)
(0.0837)
(0.0053)
Expressed upon grouping criteria (see text).
^Correlation coefficient.
cProbabi 1ity.


90
TABLE 21. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTERONE
Source Estimated regression line R2 PR>F
PMS Group y = 39.12 13-99x + 1.73x2 0.07x3 0.7323 0.001
Non-PMS Group y = 2.4 + 0.75x 0.26x2 0.8697 0.001


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 disti 11ed-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/100 ml) to the above stock solution. The assay buffer should be
stored at 4C for no longer than A weeks.
, aDo not breathe or contaminate skin. Extremely poisonous. Re
acts with drain pipe, resulting in residue build up over time.
Extremely explosive.
78


levels when compared to non-PMSG treated heifers. Apparently, the dif
ferential LH response observed in the PMSG treated group could be due
to a positive feedback of estradiol at the hypothalamic-pituitary
level. Although Hallford e^ a_L (1975b) reported a positive correla
tion 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 e_t aj_. (1972) and Dickey et a 1 (1973) re
ported plasma estradiol levels in PMSG superovu1ated heifers to be
much higher prior to mating than in controls. These values, however,
were for superovu1ated 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-Barbel1 a et a 1., 1976).
A synchronizing injection of PGF2C1 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 a 1., 1976).
The stereotypic response to PMSG was clearly demonstrated by
Rajamahendran et a 1. (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
PGF2cr 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) f rom 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.


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. Superovulation in the cow using pregnant mare's
serum gonadotropin and prostaglandin F20U Vet. Med. Small Anim.
Clin. 71:953.
Barr, A.J., J.H. Goodnight, J. P~ Sail, and J.T. Helwig. 1976. A user's
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. 88:1437-
Bellows, R.A., D.C. Anderson, and R.E. Short. 1969- Dose-response
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. 69:369-
Brock, H. and L.E. Rowson. 1952. The production of viable bovine ova.
J. Agrie. Sci. 42:179.
Burrell, W.C. and J.N. Wiltbank. 1977. CL 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. 1943. Effects
of pituitary gonadotropins on the ovaries and the induction of
superfecundity in cattle. Am. J. Vet. Res. 4:76.
Castenson, P.E., A.M. Sorensen, Jr., C.R. Cobos,and J.L. Fleeger. 1976.
Source of postpartum P and 206-0HP preceding estrus in heifers.
J. Anim. Sci. 43:277. (Abstr.).
94


TABLE 12.
(Cont¡nued)
Day of
b1eeding
Progesterone
x S.E.
ng/ml
203-Dihydro-
Progesterone
x S.E.
- ng/ml
Luteinizing
hormone
x S.E.
ng/ml
Estrogens
x S.E.
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


The distribution of observed onset of estrus following an injec
tion 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_ aj_. (1976) and Archbald (1976) and support
the concept of a shortened interval and reduced variability to onset of
estrus after the administration of PGF2cx 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 shorter interva 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 proges
terone 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 pro
gesterone concentration in cows after the administration of 40 mg PGF2a
was significantly affected by treatment (P<.01) 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


ACKNOWLEDGEMENTS
In most cases, every research product is the result of multipd-e
endeavors. This one is no exception. Since its very beginning, many
persons and institutions have provided enormous contributions. With
out 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 know
ledge 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 Agronoma, for providing economical support throughout my graduate
work.
I am also grateful to the Upjohn Company, especially to Dr. J.
Lauderdale, for providing PGF201 and reviewing my experimental pro
tocols. To Dr. J. L. Fleeger, Texas A&M University, and Dr. L. V.
Estergreen, Washington State University, my gratitude is extended for


93
TABLE 24. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTIN
Source
Estimated regression line
R2
PR>F
PMSG + breeding
y = 32.22
- 10.90x + 1,29x2- 0.05x3
0.6642
0.001
PMSG + no
b reeding
y = 37.22
- l4.39x + 2.OOx2- 0.09x3
0.5781
0.001
No PMSG +
breeding
y = 6.3^ -
1.28x + 0.08x2
0.8301
0.001
No PMSG +
no breeding
y = 5.26 -
0.96x + 0.05x2
0.7693
0.001


101
Oxender, W.D., P.A. Noden, T.M. Louis, and H.D. Hafs. 197**. A review
of Prostaglandin Fa for ovulation control in cows and mares.
Am. J. Vet. Res. 35:997.
Perkins, J.R., D. Olds, and D.M. Seath. 195**- A study of 1,000 bovine
genitalia. J. Dairy Sci. 37:1158.
Pharris, B.B. and L.J. Wyngarden. 1969- The effect of prostaglandin
Fa 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. I960. The ovarian follicular system in sexually maturq
heifers with special reference to seasonal, cyclical and left-
right variations. Acta Endocrinol. Suppl. 52:3**-
Rajamahendran, R., P.C. Lague, and R.D. Baker. 1976. Plasma proges
terone levels in cycling, and gonadotrophin-Prostag1 andin-
treated heifers. Can. J. Anim. Sci. 56:37-
Reynolds, W.L., R.A. Bellows, T.M. De-Rouen, and D.C. Meyerhoeffer.
1970. Cow response to FSH treatment. J. Anim. Sci. 31:229-
(Abstr.).
Roche, J.F. 197**. Synchronization of oestrus and fertility following
artificial insemination in heifers given prostaglandin F2a- J-
Reprod. Frtil. 37:135-
Rodriguez, T.R. 197**- Fertility of the synchronized ovulation in the
bovine following PGF2a"Tham Salt and GnRH. Thesis. University
of Florida, Gainesville, FL.
Romanoff, E.B. 1966. Steroidogenesis in the perfused bovine ovary.
J. Reprod. Fert., Suppl. 1:89-
Rowson, L.E.A., R.A.S. Lawson and R.M.
twins in cattle by egg transfer.
Moor. 1971- Production of
J. Reprod. Frtil. 25:261.
Rowson, L.E.A., H.R. Tervit, and A. Brand. 1972. The use of prosta
glandins for synchronization of oestrus in cattle. J. Reprod.
Frtil. A6:lA5.
Sasser, R.C. and P.T., Cupps. 1969- Conversion of pregnenolone-A-14C
to progesterone-**-1 *C by bovine luteal tissue at selected stages
of the estrous cycle. J. Dairy Sci. 52:217-
Savard, K. and G. Telegdy. 1965- Steroid formation in the bovine
corpus luteum. Steroids Suppl. 2:205-
Scanlon, P.F. 1972a. Frequency of transuterine migration of embryos
in ewes and cows. J. Anim. Sci. 3**:79T -


Rodriguez, 197**; Cooper and Jackson, 1975; Hafs et_ 1975b; Turman
et aj_. 1975; Ellicott and Thompson, 1976; Fields £t_ aj_. 1977b;
Moody and Lauderdale (1977)-
Luteolytic Effect of Prostaglandin F2ct
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 es-
trus. The induction of estrus is rapid and precise and the induced CL
has a normal life-span.
Louis et_ a_l_. (1972a) injected 5 mg PGF2aTham Salt into the uter
ine 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 in
jection of 30 mg PGF2a during diestrus resulted in a 60% decrease in
plasma progesterone within 12 hr with the initial progesterone concen
tration of 4 ng/ml declining to .8 ng/ml by 2b hr (Louis et a 1., 1972a,
1973). The interval to onset of estrus, LH peak and ovulation was 74,
77, and 104 hr post-PGF2a treatment, respectively. Similar results in
declining plasma progesterone were reported by Oxender et a 1. (1974)
even when PGF2a was given intramuscularly in varying numbers of injec
tions. The spaced double injection regimen produced precise synchro
nization of estrus in all the heifers responding to treatment (Dobson
et a 1. 1975). After both injections, the CL showed rapid morpholog
ical regression that was similar to that observed for a single injec
tion 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


33.5 MG
PGF
2a
2,000 i.u
PMSG
33.5 MG
PGF, a
LAPAROTOMY
40 MG PGF.
2a
OVARIAN
RECTAL
PALPATION
DAY 1
DAY 12
DAY 13 DAY 23
DAY 2 5
DAY 3 2
Li DAILY BLEEDING
Figure 2.
Treatment protocol for experiment 2 evaluating the effec- 1
tiveness of PGF2a to regress multiple corpora ltea in
nonbred cows.
ro
-C-


concentrations for an adequate time to permit the animal to express
estrus.
< In conclusion, these data clearly demonstrated that the endocri
nology of the PMSG superovu1ated cow differs from that of the normal
cycling cow. Not only the magnitude of the hormonal secretion is dif
ferent, but the length of the interval from PMSG to estrus and sup^r-
ovulatory response are affected. In addition, the early LH surge and
early increase in LH post-PMSG treatment might be partially due to dif
ferential follicular growth and estrogen secretion in response to
gonadotrophin administration. This experiment, however, was not
designed to test the hypothesis of differential follicular growth post
PMSG. Research designed to test the hypothesis of differential follic
ular-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 thishormonal 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 PGF2 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 PGF2a. 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 PGF2ct, 13 days post-PMSG, evoked a dramatic decline in plasma


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.
Pejaver V. Rao
Professor of Statistics
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December, 1977
Dean, Graduate School


ovarian response when cows were partitioned according to when they ex
hibited behavioral estrus. A higher ovulation rate (P<.01) was ob
served in cows expressing estrus by 48 hr post-inject ion (5-50 1.29)
vs 0.67 0.82 for those expressing estrus from 73 to 120 hr. The dif
ference 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 (1949), Brock and Rowson
(1952), and Scanlon et aj_. (1968) in that a more desirable and res
tricted 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 e_t^ aj_. (1973) demonstrated in
cattle'with large PMSG-induced follicles that a PGF2a analog (ICI
79,939) shorten the interval to estrus. Dobson et_ a_l_. (1975) suggested
that the second PGF2ct-i nduced 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 ob
servation that the induction of estrus was more rapid and precise after
the second PGF2a 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
24 hr earlier following the second injection than the first injection.
Rajakoski's (I960) 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 et_ aj_. (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.


PROGESTIN
(NG/MO
1 2 3 4 5. 6 7 8 9 in 11
DAY OF BLEEDING
Figure 12. Plasma progestin in BRED () and CYCLING ( ) cows after 40 mg PGF2a.
c
CO


I C!D f NG
10 25 50 100 250 500 1000
CONCENTRATION OF ESTRADIOL (PG/ML)
I
Figure 6. Standard curve of estradiol. (Each point represents the mean of triplicates on seven
determinations.)
US


of the interval from PMSG-PGFpct to estrus and superovulation. This
possibility was supported by observed changes in the plasma endocrine
patterns.
When the endocrinology of the superovu1ated 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 post-
PMSG levels of progesterone, but estrogen concentrations were higher
in superovul ated cows (Pc.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 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 PGF20t. Although there was a significant (Pc.Ol) decline
in plasma progesterone levels and an absence of palpable CL during
the 7 days post-40 mg PGF201, 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 PGF2ct was indadequate for the in
creased CL mass. In an attempt to answer these questions, two experi
ments were conducted to test the efficacy of PGF2a to regress mu 11 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 2b 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


LI I, l'HOGLS fSIKONE, 20{3-DIHYDR0PK0GLSTF.I\0NE (ng/ml)
Figure 8. Concentration of progesterone(), 20g-dihydroprogesterone ( ), lutei
nizing hormone ( ), and estrogens ( ) in plasma from PMSG treated cows
with two or three corpora ltea.


CHAPTER I I I
MATERIAL AND METHODS
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P), '
20B-DI HYDROPROGESTERONE (206-P), TOTAL ESTRO
GENS (E), AND LUTEINIZING HORMONE (LH)lN PMSG
SUPEROVULATED COWS SYNCHRONIZED WITH PGF201-
THAM SALT
The objectives of this experiment were (l) to characterize plasma
P, 20g-P, E (combined Ei and E2) and LH concentrations in the PMSG
superovu1 a ted cow and (2) to determine if 40 mg PGF2ot-Tham Salt was ef
fective in regressing multiple corpora ltea (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 intra
muscularly with 33-5 mg PGF2ct-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 in
insemination (Al) three times with Brahman semen was at 0, 12, and 24 hr
after detection of the PMSG-PGF2ct-i nduced estrus. Approximately 10 days
following Al 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


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_ a_l_. (1973, 1976) reported that PMSG, given concomitantly
with PGF2a, resulted in a lower and less variable ovulation rate (4.33
3.60) than when PGF2ct was injected at 24 hr post-PMSG (8.03 9-46). A
high incidence of split estrus (55%), w.ith 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 be
tween hormonal treatments was allowed. These data suggest that the
follicles had not had sufficient time to mature before estrous control
was attempted with PGF2c(. Absence of split estrus has been reported
by Rajamahendran ajL (1976) and Lopez-Barbella et a 1. (1976)
following a single injection of 2,000 i.u. PMSG 24 hr prior to a second
injection of synchronizing PGF2c(.
Endocrinology of the Superovu1ated Cow
Hallford ej^ aj_. (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<.01) in heifers treated with PMSG vs_ nontreated heifers.
In heifers treated with PMSG, GnRH injections produced lower plasma LH


31
TABLE 1. EFFICIENCY OF 3H-P CONVERSION TO 3H-206-PAT
VARYING LEVELS OF COFACTORS
Tri tiated
progesterone
(dpm)
n
NADHa
(ml )
20B-HSDb
(ml)
Efficiency of 3P
conversion to 3H-20BP
(%)
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-
b0.2 ml
-NADH/3 ml
20B-HSD/0.
of 0.1 M Tris
.8 ml of 0.005
buffer.
M Tris
buffer.


6
in regressing multiple CL of PMSG-PREGNANT cows since in the first ex
periment, 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 1 indicated that ^0 mg PGF2a-Tham Salt
apparently induced only partial functional and structural CL regres
sion 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 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 PGF2a 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


50
As described initially, ovaries were examined by laparotomy and
those with more than three CL received kO mg PGF2a on day 15 (AM). A
significant (Pc.Ol) reduction in plasma progesterone was observed after
AO mg PGF2a 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
1A, 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
Henri cks £t ak (1973) Sp i Iman et^ a_l_. (1973), and Ha 1 1 ford e_t aj_.
(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 PGF2a 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<.01; table 18, Appendix) is in agreement
with Lamond and Gaddy (1972) and Spilman et al. (1973) and in disagree
ment with Rajamahendran ej^ al. (1976) who reported that a much larger
number of observations was needed to assess statistical difference be
tween superovu1ated and normal cycling cows.


TABLE OF CONTENTS
£age
ACKNOWLEDGEMENTS u
LIST OF TABLES *. vi
LIST OF APPENDIX TABLES vii
LIST OF FIGURES ix
ABSTRACT xi
CHAPTER I: INTRODUCTION 1
CHAPTER II: LITERATURE REVIEW 4
Prostaglandins and Reproduction 4
Prostaglandin F2a and Estrous Synchronization . 4
Luteolytic Effect of Prostaglandin F2a 6
Prostaglandin F2ct and Therapeutic Abortion. ... 8
Prostaglandin F2a and Superovulation 10
Gonadotropic Hormones and Limited Multiple Births ... 11
Endocrinology of the Superovu1ated Cow 14
20B~Dihydroprogesterone (4-Pregnen-208-o1-3_one). ... 16
CHAPTER III: MATERIAL AND METHODS 21
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE
(P), 2O3-DIHYDR0PR0GESTERONE (2O3-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
iv
27


40 MG
PGF 2 a
\
3 4 5 6 7 8 9,1011
DAY OF BLEEDING
Plasma progestin profile in PMSG-BRED ( ) and PMSG-CYCLING ( ) cows after
40 mg PGFa
Figure 11.


33.5 MG
PGF2a
GROUPS I S II
2, 000 i.u.
PMSG
33.5 MG
P G F 2 ct
LAPAROTOMY
4 0 MG PGF2 a
1
DAY
DAY 12
DAY 1 3
DAY 23 DAY 25
GROUPS I S III
NATURAL BREEDING
DAILY BLEEDING
DAY 3 3
Figure 3- Treatment protocol for experiment 3 comparing the efficacy of PGF2a to regress
multiple corpora ltea in the bred cow.


33.5
PGF
MG
2 a
HEAT A I
AT 0,
LAPAROTOMY
OVARIAN
2 000 i. u.
33.5 MG
12, 24
40 MG PGFz a
RECTAL
PMSG
PG F2 a
HR
TO COW > 3 CL
PALPATION
1
7
DAY 1 DAY 10 DAY 12 DAY 13
DAY 2 5
DAY 32
BLEEDING TWICE DAILY
Figure 1.
Treatment protocol
following PMSG and
for experiment
PGF20C
involving endocrine characterization
N3
N3


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 vigor
ously for 30 sec. The charcoal suspension was stored at 4C and used for
no longer than 4 wk. A volume of 500 pi 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 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 I (98% PPO/2% BIS-MSB). A total of 3-5 ml of this counting
fluid was used per scintillation vial. Cocktail and sample were equi
librated for 2k 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 crystal
lization a stock solution of 1 g/ml unlabelled hormone in absolute
ethanol was made. An aliquot of 20 pi stock solution was transferred
to a 20 x 150 mm test tube and dried under nitrogen gas at 37C. Then


37
TABLE 3-
ADDITIONAL TUBES USED TO
SUPPLEMENT RIA ANALYSI
ISa
Solution
Code
Amount of
assay buffer
added
(ml)
Amount of
antibody
added
(ml)
Amount of
3H-Steroid
added
(ml)
BGb
1 .2
-
-
TCc
1 1
-
0. 1
CHb '
0.6
-
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.
^To determine residual counts left following addition of
cha rcoa1 .
eTo determine the percent of the 3H-steroid bound to the
antibody.
^Assay buffer was replaced by 0.5 ml of either standards
or unknown samples


20
To date, reports on 208-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 208-P will be characterized in PMSG superovu1ated cows
over a 21 day bleeding period.
V


- 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 PGF2ct administered at laparotomy (figure 10)
plasma progesterone declined significantly (P<.01) 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 post
laparotomy 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 regres
sion, 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 PGF^ remains un
defined, data accumulated in this study clearly suggest that 40 mg
PGF2a *s effective in regressing multiple CL in the cycling nonpregnant
beef cow based upon the reduction in plasma progesterone and CL regres-
stion evidenced by the absence of palpable CL by 7 days post-treatment.
The question still remains as to whether or not 40 mg PGF?_a
is effective


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 treat
ment was due to the actual biological potency of the gonadotropin.
Similarly, Schwartz and Shelby (1969), Laster et a 1. (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 syn
chronized 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 in
jections for 5 days (Bellows et_ aj_. 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
e^t aj_. 1976). However, for 5 days of injections, fewer cows super-
ovulated 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 8l to 104 days after inse
mination. Similar observations of fetal wastage have been made by
M. J. Fields, A. C. Warnick, and J. H. Hentges (unpublished data).
Godke e^ aj_. (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


18
Telegdy and Savard (1966) found the rabbit ovary to produce both
20a-P and 20g-P, with 20ct-P being the more predominant hydroxylated
steroid. Hilliard et^ (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 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 (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-ll*C increased 14CP synthesis 3-3 fold and increased
the P to 200-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 20g-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 200-P. Not unexpectedly, there was significantly
less synthesis of P and 200-P in follicular than luteal ovaries. Pro
lactin perfusion in this study, had no effect on the 20-HSD system.
Snook e_t aj_. (1969) working with hysterectomized heifers with sus
tained luteal function, however, showed that LH has a preferential ef
fect on 20-HSD. They used LH-antisera to neutralize endogenous LH.
This LH-antisera reportedly decreased total ovarian progestin concentra
tion primarily due to a significant reduction in 200-P while P concen
trations remained unaltered. In the intact heifer, Spilman et al. (1973)


Louis et_ aj_. (1974a) injected 5 mg PGF2a into the uterine horn ipsi-
lateral for the CL In cows 11 days postmating and reported plasma pro
gesterone 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 concen
trations 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-PGF2oT
treatment. 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 PGF201 induced CL re
gression 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 post
abortion. In contrast, cows at l60 to l80 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 a 1. (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 con
centration declined to basal levels of 1.5 0.6 ng/ml within 48 hr
post-cloprostenol. No retained placentas were observed. Fields et a 1.
(1977a) reported 250 to 500 yg cloprostenol aborted 100 to 107 heifers
between 60 and 120 days of pregnancy, with no complications.


73
estrogens and LH was also recorded with no significant change in 208"
dihydroprogesterone levels. The hormonal trends after the luteolytic
dose of PGF2oi 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 PHSG. 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-PGF2a-induced cycle was characterized
by a rather significant (Pc.Ol) increase in plasma progesterone which
reached a maximum of 18.44 4.59 ng/ml on the day of injection of 40
mg PGF2cx. A dramatic decline in plasma LH and estrogen levels was ob
served 'post-estrus and remained at base line thereafter.
The plasma 208-dihydroprogesterone trend did not change signif
icantly (P>.10) during the course of this study. When progesterone and
208-dihydroprogesterone ratios were computed, however, a significant
(P<.01) trend was detected suggesting a relationship between P and 208P.
The lowest ratios of 20Q-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
1uteinization of the granulosa cells or from luteal cells of the re
gressing 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


5
of PGF2a (Tervit ej^ aj_. 1973; Cooper, 197^; Fields et_ ak 1977b) was
effective in causing luteolysis in heifers and cows except for the
first 5 days post-estrus (Rowson et al., 1972; Louis et a 1. 1973)
In cattle, a single injection of PGF2ct given during the responsive
stage of the estrous cycle is followed by an ovulation and normal fer
tility. There are, however, several factors associated with the ule
of a single injection of PGF2ct 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 a 1., 197*0- Others have reported similar results
following PGF2Ct treatment (Louis e_t_ a_l_. 1973; Louis ej^ aj_. 1974a;
Chenault ejt_ 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 10 to 12
day interval, as suggested by Inskeep (1973). Cooper (197*+) reported
that this dual injection technique, with an ICI analogue of PGF2a (ICI
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 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 PGF2ct in
jection. Several researchers have shown this synchronized estrus to
be fertile (inskeep, 1973; Lauderdale et al., 1974; Roche, 1974;


12
with double ovulations from a single ovary (29%). The precise relation
ship between the number of ovulations per ovary and maintenance of preg
nancy to term remains to be established. The minimal intrauterine mi
gration (1:200) in the bovine (Perkins et_ a]_. 1954; Gordon e_t a]_. 1962;
Rowson et^ aj_. 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_ a_L (1969) reported 625 mg of FSH injected twice daily
for 5 days, in heifers synchronized by feeding 180 mg medroxyprogesterone
acetate (MAP) dail/ 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_ aj_. 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 e_t a_l_. (1969), but considerably lower than the calving rate
of 173% with PMSG treatment reported by Turman et a 1. (1971).
Reynolds et_ aj_. (1970), Vincent and Mills (1972), and Smith et a 1 .
(1973) attempted to prolong the action of FSH by using a 1% sodium
carboxymethy1 cellulose and polyviny1pirrolidone. It was concluded that


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<-01) increase to 13-62 2.06 pg/ml within
24 hr. In conjunction with the luteolytic dose of PGF2a on day b fAM),
estrogen levels continued to increase tc> a peak of 37-04 11.47 pg/ml
by 72 hr post-PMSG. These changes were best characterized by a third
order regression equation which accounted for b7% 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 PGF2a coupled with increasing levels of
estrogen led to the expression of estrus. As a consequence of the re
initiation of a PGF2a-induced estrous cycle, plasma estrogen declined
significantly (P<.01) 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 PGF?a 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 1 (r = 0.68, Pc.Ol; table 18, Appendix) is in


TABLE OF CONTENTS Continued
Page
Conversion of Progesterone (^P) to 20B-Dihydro-
progesterone (3H-20f3-P). . 28
Use and Storage of Antibody 29
Serum Extraction and Chromatography 29
Conversion of 3H-20g-P to 3P . 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. ... ^0
Precision
Statistical Analysis
CHAPTER IV: RESULTS AND DISCUSSION 43
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE
(P) 20R-DI HYDROPROGESTERONE (200-P),
ESTROGENS (E), AND LUTEINIZING HORMONE
(LH) IN THE PMSG SUPEROVULATED COW SYN
CHRONIZED WITH PGF2a-THAM SALT 43
Reproductive Response 43
Endocrine Response 48
Progesterone 48
Estrogens 53
Luteinizing Hormone 54
20g-D ihydroprogesterone 55
EXPERIMENT 2. RESPONSE OF MULTIPLE CORPORA LUTEA IN
THE SUPEROVULATED BEEF COW FOLLOWING
ADMINISTRATION OF 40 MG PGF2a~THAM SALT 57
Reproductive Response 57
Endocrine Response 58
EXPERIMENT 3- LUTEOLYTIC EFFECT OF PGF2crTHAM SALT IN
THE PMSG BRED \IS_ 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


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 Hor
mone (FSH). Different gonadotropins, alone or in combination, have been
used by many researchers with consistent results. Casida et_ aj_. (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 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 injec
tion' of 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 e_t aj_. (1969, 1971) used this same hormonal protocol
and obtained a 109% weaned calf crop, from treated animals.
Kidder et a 1. (1952) and Dawson (1961) reported cows with double
ovulations gave birth to a very low number of twins. Gordon et a 1.
(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


agreement with that reported by Hallford et a 1 (1975b). Henricks e_t
a 1 (1973), in cattle, and Guthrie et_ aj_. (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
fol1icles.
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_ aj_. (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_ aj_. (1973) that the
ovulatory surge of LH was earlier in PMSG treated heifers than in non-
treated controls. Although untreated PMSG-treated animals were not
i
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 1 (vide supra) Hallford e^ aj_. (1975b) failed to detect this
treatment effect; they only obtained plasma samples at 24 hr intervals.
Henricks et_ aj_. (1973) and Hallford et_ aj_. (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


TABLE 7. DISTRIBUTION OF ESTRUS IN COWS AFTER A DUAL 33-5 MG SYNCHRONIZING INJECTION OF PGF2a-
THAM SALT AND ONE 40 MG ABORT I FAC I ENT DOSE OF PGF2a
Time after
PGF2C1 injection
(days)
Estrus post-first PGF2a
synchronizing dose
Estrus post
synchron
-second PGF2ct
¡zing dose
Estrus post-
ABORTIFAC 1 ENT PGF2a
dose
PMSG Group
No. %
Non-PMSG Group
No. %
PMSG
No.
Group
%
Non-PMSG
No.
Group
*
PMSG
No.
Group Non-PMSG
No.
G roup
*
1
2 9
3 13
3
14
1
v 4
2
10 45
11 48
10
45
3
13
1
4
3
6 28
5 21
6
28
5
21

1
4
4
2 9
2
9
9
39
1 1
48
5-10
14
64 8
35
11-15
6
27
Non respondinga
2/22 3%
4/23 18*
1/22
4*
5/23
21*
2/22
3% 2/23
31
aCows were considered estrous synchronized if detected in estrus within 168 hr post 33-5 mg PGF2a
and within 15 days post 40 mg PGF2ct. Animals not expressing estrus within these periods were
considered nonresponding.
/


3
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 syn
chronizing es t rous wi th PGF2ct when used in conjunction with PMSG was
evaluated through studying the levels and interrelationships of the
reproductive hormones. "


LIST OF FIGURES
FIGURE Page
1 Treatment protocol for experiment 1 involving
endocrine characterization following PMSG and
PGF2a ; 22
2 Treatment protocol for experiment 2 evaluating
the effectiveness of PGF2ct to regress multiple
corpora ltea in nonbred cows. . . 2k
3 Treatment protocol for experiment 3 comparing
the efficacy of PGF2ct to regress multiple
corpora ltea in the bred cow 26
k- LH-20 profile of 3H-1,2 progesterone (P) con
version to 3H-1 ,2-20(3-d i hydroproges terone
(208-P) using hexane:benzenermethanol (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 re
presents the mean of triplicates on seven
determinations.) 39
7 Concentration of progesterone ('') 208-dihydro-
progesterone ( ), luteinizing hormone ( ),
and estrogens ( ) in plasma from PMSG treated
cows with four or more corpora ltea kS
8 Concentration of progesterone ('*), 208~d ¡ hydro
progesterone ( ), luteinizing hormone ( ),
and estrogens ( ) in plasma from PMSG treated
cows with two or three corpora ltea 51
9 Concentration of progesterone ( ), 208-dihydro-
progesterone ( ), luteinizing hormone ( ),
and estrogens ( ) in plasma from PMSG treated
cows with zero or one corpora ltea 52
i x


experiment, it may be that the origin of the 206-P was from reactiva
tion of luteal cells from regressing CL or 1uteinization of granulosa
cells from follicles. Hillard et_ ak (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 a-P.
Data accumulated in this study suggest that PMSG has profound ef
fects 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 208-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 40 mg PGF2a exhibited both
a functional and morphological regression of multiple CL with no abor
tion, it is of importance to test whether kO 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 ef
fect, 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.


ESTROGENS (pg/rnl), LH, PROGESTERONE, AND
2O0-DIHYDROPROGESTERONE (qg/ml)
DAY OF BLEEDING
Figure 9- Concentration of progesterone (), 20f3-d ihydroprogesterone ( ),
luteinizing hormone ( ), and estrogens ( ) in plasma from PMSG
treated cows with zero or one corpora ltea.
vn


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 amino-
methane in 500 ml disti 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 HC1. Then add disti 11ed-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 pM) and 3 ml of 0.1 M
Tris buffer pH 8.1.


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 PGF201 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 effec
tive 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 pseudo
pregnant rats (Pharris and Wyngarden, 19&9), others observed that treat
ment with a single injection of PGF2a (Rowson et al., 1972; Lauderdale,
1972; Inskeep, 1973; Oxender et_ aj_. 197^; Rodriguez, 197^+; Roche, 1974;
Fields et_ aj_. 1975; Thatcher and Chenault, 1976) or syn thet i c ana 1 ogues


16
20g-D ihydroprogesterone (A-Pregnen-20g-ol-3~one)
An interesting steroid, concerned with bovine steroidogenesis, that,
to date, has been considered the major metabolite of progesterone (P) is
20g-dihydroprogesterone (20g-P) (Spilman 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 lA day- CL has reached
its maximum size filling the collapsed follicular cavity with an extensive
vacuolation of luteal cells. Stero idogenica11y, this structural change
is accompanied by a differential secretion of progesterone-derived hor
mones according to Short (1962a,b) in his "two cell theory." From the
two Cell theory of Short (1962a,b), the ovarian theca interna was pro
posed to covert P to estrogen in the absence of a 20-reductase system,
i.e., 20g-hydroxysteroid dehydrogenase (20g-HSD). LaCroix et_ 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 1uteinization was proposed to contain
the 20g-reductase system and thus, 20g-P secretion by the growing folli
cles was an indicator of 1uteinization. This was indirectly confirmed
by other when P and 20g-P were isolated from luteal tissue (Savard and
Teledgy, 1965). Lobel and Levy (1968), however, did find that 20g-HSD
activity resided in both the granulosa and theca cell layers of the rat
follicle.
Hayano et_ aj_. (1975) first demonstrated the conversion of P to 20g-
P by the bovine CL. Noticeable quantities of ovarian 20g-P levels in
cycling cows at levels approximately 10 to 20% of that of P have been
reported (Gorski et a 1., 1958a,b; Erb and Stormshak, 1961, Hafs and


CHAPTER I
INTRODUCTION
In preseht-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, fer
tility must assume an even greater importance in the economy of pro
duct 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 prof
itable alternative utilization. A primary means available for effi
ciently improving production in the beef cow is to wean a greater num
ber 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.


71
pregnant cow, however, they indicated kO mg PGF2ct 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 vari
ability, 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 superovula
tion 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.


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 e^ aj_. (1976) reported that- the administration of PGF2C1
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 a 1., 1976).
Dobson et a 1. (1975) suggested that the second PGF2ainduced luteolysis
may allow the wave of growing f-ollicles that originated after the first
PGF2C- i nduced estrus to progress and ovulate rather than become atretic.
i
Rajakoski (i960) reported such a wave of follicular growth approximately
b 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 PGF201 administration.
In line with this concept, Tervit £t_ ak (1973) reported that a
pros tag 1 anding F2ct analog (ICI 79,939) given to cattle with large PMSG-
induced follicles demonstrated a shorter interval to estrus. Archbald
(1976) reported excellent synchronization of estrus when a second in
jection 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 8l% of the cows responding behaviorally within 48 to 132 hr following
t reatmen t.


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-disti 11ed water
for 1 min,and rinsed with warm tap water (3X) followed by deionized"
water (3X). Prior to use, the glassware was rinsed with ethanol, sili
conized with 1% silicone solution (Siliclad, Clay Adams) for 20 sec and
oven dried for 2b hr.
Organic Solvents
3-
Analytical grades of benzene, ethanol, methanol, hexane, and ethyl
acetat 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,
b, 6,7"3H-Estradiol (3 E 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 im
purities 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 bC. 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


CHAPTER IV
RESULTS AND DISCUSSION
EXPERIMENT 1. CHARACTERIZATION OF PLASMA PROGESTERONE (P)
20g-DIHYDROPROGESTERONE (20g-P) ESTROGENS
(E), AND LUTEINIZING HORMONE (LH) IN THE
PMSG SUPEROVULATED COW SYNCHRONIZED WITH
PGF2ct-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-PGF2ra. The single animal not responding to this dual injection
of PGF2a was detected in estrus 36 hr prior to the second PGF2ct 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 48, 60, 72,
and 120 hr, respectively, following PGF2a injection. This is in agree
ment with Cupps e_^ £]_ (1976) who reported that the administration of
PGF?ct 24 hr post-PMSG, to previously synchronized heifers: (l) in
creased the number of animals exhibiting estrus; (2) shortened the in
terval 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% re
ported by Archbald (1976).
Estimates of ovulation rate, determined at laparotomy, are pre
sented in table 4. Significant differences were detected in total
43


82
TABLE 13. PLASMA CONCENTRATIONS OF PROGESTERONE, 208-DI HYDROPROGES
TERONE, LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING
TWO OR THREE CORPORA LUTEA AT LAPAROTOMY
Day of
Progesterone
20B-Dihydro-
Progesterone
Lute iniz¡ng
hormone
Estrogens
b1eed i ng
x S.E.
x S.E.
x S.E.
x S.T.
ng/ml
ng/ml
ng/m!
pg/ml
1AM
3-
.85
+
0.3 b
2,
.50

0.
37
1.
.23

0.
46
1
.73
+
0.09
PM
3.
95
+
0.38
2,
.55

0.
40
1.
.28

0.
13
2AM
3.
98

0.46
2,
.10

0.
37
0.
.73

0.
09
1
.65
4;
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)
b.
.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,
.90
0.
31
7
.28
+
1.97
PM
1 .
.18

0.45
1 ,
.58

0.
33
2,
.08

0.
36
9AM
1 .
.**3

0.49
1
.83

0.
.33
2.
.20

0.
, 12
5
.13
+
1.59
PM
1 .
b7

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
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 .
.95

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

0.
27
1
.60

0.40
i4am
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
b.
30
+
0.68
1
.83

0.
.39
1 .
.15

0.
.25
1
.67

0.18
PM
b.
.20
+
0.41
2.
.00

0.
,62
1 ,
.33

0.
30
16AM
b.
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
1 7AM
5.
.60
+
0.56
2,
.48

0.
.45
0,
.98

0.
,10
1
.69

0.17
PM
b.
.88
+
0.36
2
.95

0.
.49
1 .
. 10

0.
19
18am
b.
.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


23
Ovulation rate was determined by supravaginal laparotomy 13 days
post-PMSG and, on the same day, all cows were treated with 40 mg PGF2a-
/
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 PGF2orTHAM 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
L
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 24 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 2b 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-1aparotomy.


concentration by RIA. Forty milligrams of PGF2a was effective in re
gressing 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-AO 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 results from the
previous experiment. There were no observed differences between mated
and nonmated animals in response to kO mg PGF2a-
In conclus ionthese 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 PGF201 arid 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


TABLE 2. STANDARD CURVE DILUTIONS3
Hass 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 &3
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.


17
Armstrong, 1968; Garverick et^ ajL 1971). Brandau e_t^ aj_. (1972) and
Brandau and Mutzke (1972) using homogenized bovine ovaries found that
206-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 203-P concentration following the P decline
of the regressing CL (Erb and Stormshak, 1961 ; Staples and Hanse1,-l961
Mares aj_- 1962; Gomez et aj_. 1963; Gomez and Erb, 1965)* Erb £t
al. (1968) reported that 20f3-P increases when synthesis or 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 re
gression could result in increased lysosomal activity which would
result in reduced cellular pH and create optimal conditions for 208"P
formation. When a similar study was conducted with porcine CL, only
0.6% of the P was converted to 20g-P (Weiss e_^ aj_. 1976).
In laboratory animals, in which the a-epimer (20a-P) of 208-P
is the more predominant hydroxylated ovarian steroid, Barraclough e^
a 1. (1971) noted an increase in ovarian vein levels of 20a-P approxi
mately 2.5 hr prior to the LH discharge in cycling female rats. This
suggested the possibility that 20ct_P has a positive feedback on the
pituitary for discharge of LH. Ichikawa et a 1. (1971) found an in
creased secretion of both P and 20a-P in ovarian vein plasma after in
jection 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).


C PM
ML ELUENT
I
Figure h. LH-20 profile of 3H 1 ,2 progesterone (P) conversion to 3H-l,2-20f3-
dfhydroprogesterone (203-P) using hexane: benzene :met Hanoi (80:15 ; 5)
o


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)
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 F2a (PGF2Ct) at a 12 day interval. Ovarian stimu
lation was achieved by injecting these cows with 2,000 i.u. PMSG 2k hr
prior to the second PGF2a administration. Cows were artificially in
seminated three times with Brahman semen at 0, 12, and 2k hr after
detection of the PMSG-PGF2a-induced estrus. Progesterone, 206-dihydro-
progesterone, 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 supra
vaginal 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


characterize the LH surge, no additional information regarding the rela
tionship between plasma LH and plasma estrogen or between plasma LH and
ovulation rate can be provided from this trial.
203-Pi hydroprogesterone
The individual plasma values and plot-ted levels of 20g-P are re
ported in tables 12 to 14 (Appendix) and figures 7 to 9, respectively.
There was a curvilinear relationship between plasma 203P and day of
bleeding (table 16, Appendix). A nonsignificant (P>.10) increase of
2O3-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. after PMSG treatment with 20g-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, 2O3-P levels to be 10 to 15% of that of P. In these
studies the maximum levels of plasma 2O3-P were 2.5 ng/ml which is com
parable 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-PGF201 induced estrous cycle. Although 2O3-P appeared
not to be influenced by gonadotropin administration, there was a 3-3
fold greater increase in P over 2O3-P synthesis at 24 hr post-PMSG treat
ment. Romanoff (1966) 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 lutei-
nization which is, in turn, maintaining 2O3-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 20g-P, it seems
likely that only luteal cells have an active 2O3-HSD. Thus in this


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 abort i fac ient dos'e
of PGF2d was longer in the PMSG treated animals when compared to the
nonsuperovulated group (15 ys_ 7 days, respectively, P<.01). Ninety-
one 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 behav
ioral ly between 11 and 15 days post-treatment with a mean interval of
13.50 1.05 days. Fifty days post-40 mg PGF2ct 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 ejt^ aj_. 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 1. (1974) in that cows aborted in
early gestation may return to estrus within 2 days after abortion.
Results from th i stri a 1 show the PMSG-BRED group to be comparable to
that of Zerobin et^ aj_. (1973) in that the administration of PGF2ct to
non-superovu1ated pregnant cows at early stages of gestation resulted
in behavioral estrus 2 to 16 days post-treatment.



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

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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

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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

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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

PAGE 86

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

PAGE 87

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

PAGE 88

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.

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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 )

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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
PAGE 110

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.)

PAGE 111

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-

PAGE 113

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-

PAGE 115

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.).

PAGE 117

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.

PAGE 118

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

PAGE 119

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

PAGE 120

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


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 Ho 1ste in-Fri es ians. J. Dairy Sci. 35:^36.
Kidwell, W.R., K. Balogh, Jr., and W.G. Wiest. 1966. Effects of luteinizing
hormones on g1ucose-6-phosphate and -20a-hydroxysteroid dehydrogenase
actiyities in superovulated rate ovaries. Endocrinol. 79:352.
La Croix, E.,'W. Elchaute, and I. Leusen. 197^. The biosynthesis"of
estrogens by cow follicles. Steroids 23:337-
Lamond, D.R. 1972. Hormonal induction of multiple ovulation in the
bovine. J. Anim. Sci. 34:901. (Abstr.).
Lamond, D.R. and R.G. Gaddy. 1972. Plasma progesterone in cows with
multiple ovulations. J. -Reprod. Frtil. 29:307-
Lamond, D.R., R.V. Tombinson, M. Drost, D.M. Henricks, and W. Jochle.
1973- Studies of Prostaglandin F2a in the cow. Prostaglandin
4:269-
Lastpr, D.B. 1972. Folicular development in heifers infused with
follicle-stimulating hormone. J. Reprod. Fert. 28:285.
Laster, D.B. 1973- Ovulation, fertility, and prenatal mortality in
heifers treated with PMSG (pregnant mare serum gonadotropin) or
porcine FSH. J. Reprod. Frtil. 33:275-
Laster, D.B., E.J. Turman, D.F. Stephens, and R.E. Renbarger. 1971a.
Superovulation of beef cows and heifers by injection of pregnant
mare serum (PMS) following estrus synchronization by means of
an oral progestogen. Mise. Pub. Agrie. 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. 33:443.
Lauderdale, J.W. 1972. Effect of PGF2a on pregnancy and estrous cyle
of cattle. J. Anim. Sci. 35:246.
Lauderdale, J.W. 1974. Distribution and biological effects of prosta
glandin. 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. 1974. Fertility
of cattle following PGF2a injection. J. Anim. Sci. 38:964.
Lobel, B.L. and E. Levy. 1968. Enzymatic correlates of development,
secretory function and regression of follicles and corpora ltea
in the bovine ovary. Acta Endocr. Suppl. 132:7.


LIST OF TABLES
TABLE Page
1 EFFICIENCY OF 3H-P CONVERSION TO 3H-20(3-P AT
VARYING LEVELS OF COFACTORS. 31
2 STANDARD CURVE DILUTIONS 36
3 ADDITIONAL TUBES USED TO SUPPLEMENT RIA ANALYSIS . 37
A DISTRIBUTION OF ANIMALS SHOWING ESTRUS AFTER THE
SECOND PGF2a ADMINISTRATION AND SUBSEQUENT OVARIAN
RESPONSE OF ANIMALS TREATED WITH PMSG IN EXPERI
MENT 1 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 PGF2ra-THAM SALT AND
PMSG 39
7 DISTRIBUTION OF ESTRUS IN COWS AFTER A DUAL 33-5
MG SYNCHRONIZING INJECTION OF PGF2a-THAM SALT
AND ONE 40 MG ABORT I FAC I ENT DOSE OF PGF2a 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


TABLE 23. PLASMA CONCENTRATIONS OF PROGESTIN .AFTER 40 MG PGF2ct IN COWS SUPER-
OVULATED WITH PMSG AND EXPOSED TO BREEDING
Progest in
ng/m 1
Day of bleeding Group 1 Group 2 Group 3 Group 4
(PMSG + bred) (PMSG-Nonbred) (Non-PMSG-bred) (control)
1
7.25
+
1 ,65a
9-65
1.35
2.68

0. 18
2.97
+
0.32
2
8.63
+
2.01
10.71
1.69
3.22
+
\
0.15
2.75
+
0.25
3 (PGF)
7.63

1.79
8.86
1.57
3.07
+
0.15
2.64
0.21
4
4.49

0.79
5. 11
0.92
2.67
+
0. 11
2.27
+
0.21
5
3.09

0.49
2.70
0.48
1.81

0. 1 8
1.65
+
0.14
6
2.30

0.31
2.83
0.49
1.49

0.17
1.38
0.09
7
1.49
+
0. 19
2.22
0.41
1 .22

0.13
1.14
+
0.12
8
1.45
+
0.27
1.60
0.32
1 .06

0. 19
0.96
0.15
9
1.37
+
0.23
1.25
0.22
1.14

0.17
0.84
+
0.09
10
1.55
+
0.23
1.31
0.19
1 .40

0. 19
1.11
+
0.09
11
1.88
+
0. 19
1.65
0.18
1 .62

0.12
1 ./46
+
0.09
ax S.E.
V£>
NJ


LIST OF APPENDIX TABLES Continued
TABLE Page
24 LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES FOR
PROGESTIN 93
v i i i


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-super-
ovulated cows of 3-07 0.15 and 2.64 0.21 ng/ml for the same res
pective groups at laparotomy. Similar results have been reported pre
viously for cycling cattle (Plotka et a 1., 1987; Stabenfeldt et alT,
19&9; Kazama and Hansel, 1970; Sprague et_ aj_. 1971; Wettemann et al ,
1972; G1 encross et_ aj_. 1973). AFter 40 mg PGF2a injection on day 3,
plasma progestin in the PMSG group declined significantly (Pc.Ol) 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-superovu1ated
anima1s, min¡mum progestin levels of 1.06 0.19 and O.96 0.16 ng/ml
were achieved by day 8. These trends were considered to be curvilinear
(table 24, Appendix). On an individual animal basis, all cows achieved
progestin levels below 1 ng/ml post 40 mg PGF2ct.
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 ex
pression of estrus it takes up to 16 days (Zerobin et a 1., 1973;
Douglas et_ al_. 1974).
In the PMSG superovu1ated 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-Barbel la et al., 1976,1977). In the superovu1ated


TABLE 16. LEAST SQUARES REGRESSION EQUATIONS AND R2 VALUES 'FOR ESTROGENS AND 20 3-DI HYDROPROGESTERONE (20g-P)
Hormone
Sou rce
Port
under
on of curve
estimation
Estimated regression line
v-
R2
PR>F
Estrogens
Group
1
from
1AM
to
6AM
y =
= 18.46 2.20x + 0.07x2 0.0005x3
0.4771
0.010
>3 CL
from
6AM
to
22PM
y =
= 212.18 4.lOx + 0.02x2
0.5529
0.011
G roup
2
f rom
1AM
to
6AM
y =
= 1493.00 67.75x + 1.T4x2 0.008x3
0.2791
0.163
2 to
3
CL
f rom
6AM
to
1 8pm
y =
= 28.39 4.36x + 0.22x2 0.004x3
0.3250
0.097
Group
3
from
1AM
to
18pm
y =
= 2.10 + 0.02x + 0.005x2
0.1181
0.050
0 to
1
CL
20g- P
G roup
1
f rom
1AM
to
22PM
y =
= 2.31 + O.Olx O.OOlx2
0.2059
0.054
Group
2
f rom
1AM
to
1 8pm
y =
= 3-84 0.I4x + 0.003x3
0.1762
0.066
G roup
3
from
1AM
to
1 8pm
y =
= 2.18 0.08x + 0.003x3
0.5121
0.051
I
CD
\-n


*4
at 4C for 20 min at 2,000 g. Then, 500 yl of this incubate was with
drawn by a repipette and deposited in a scintillation vial along with
3.5 ml of scintillation cocktail. Counting for 5 min followed over
night equilibration at room temperature.
Calculations
Recovery =
(recovered counts background counts) (4) x |qq
original counts added background counts
% Total Binding
BT tube counts
x 1 00
TC tube counts
The standard curve and quantification of unknowns were calculated
by logarithmic transformation of the data. For this, a Monroe i860 com
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 follows:
Steroid Concentration =
concentration of unknown
% 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 20g-d¡hydroprogesterone
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


5/
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 superovu1ated pregnant cow. The question then
arose as to whether 40 mg PGF?a 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 PGF^ dose, at 13
days post-PMSG, was effective in regressing multiple CL in the non
pregnant superovu1ated 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
importnce to review this concept and compare these results with the
endocrinological profile reported in experiment 1.
Reproductive Response
It is well established that PGF2ni 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
PGF201 when 10 of 13 animals exhibited estrus within 96 hr after treat
ment (table 6) followed by 84.6% at the time of the second injection.
Cooper (1974) and Chenau 11 e_t_ aj. (1976) clearly demonstrated that a single
injection of PGF2a 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.


TABLE 6. DISTRIBUTION OF ESTRUS AND OVULATION RATE IN COWS AFTER A DUAL INJECTION OF PGF2cT
THAM SALT AND PMSG
Time after PGF2a
First PGF2a
iniect ion
Second PGF2a
injection
ovulat ion
PMSG
rate at
1aparotomy
inj ection
(hr)
Number of
animals
% of
animals
Number of
animals
% of
animals
Rt
ovary
Lt
ovary
Overa 1 1
x S.E.
0-48
5
38.5
8
61.5
4.7 3-3
2.4 1
.9 4.3 3-5
49-72
4
30.8
3a
23- 1
3.0 1.4
1 .0 1
.4 4.0 2.8
73-96
1
7.7
0
0
97-120
0
0
0
0
121-144
2
15.4
0
0
1 45-more
0
0
2b
15-4
Tota 1
12/13
92.3
13/13
100
3-9 3.
1
a0ne animal
bTwo animals
did not receive
did not receive
2,000 i.u.
2,000 i.u.
PMSG.
. PMSG,


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 hor
monally induced controlled ovulation rate.
Superovulation in beef cattle has been induced by various ex
tracts of the'anterior pituitary; however, pregnant mare serum gonado
tropin (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 ex
tracts require a series of injections; however, in either case ovula
tion rate is highly, variable.
Almost without exception, researchers in the field of super
ovulation 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 2a (PGf2a) may introduced into the
hormonal regimen to induce premature regression of multiple corpora
ltea (CL) resulting from an excessive ovulation rate in an attempt to
reduce fetal wastage. Thus, it was the objective of this study to de
termine 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 PGF2a and if this would minimize the necessity of determining the
exact stage of the estrous cycle for giving the injections of PMSG.


29
ethanol (9:1) at 4C. A typical profile of 3P conversion to 3H-20g-P
an,d its LH-20 chromatography separation is illustrated in figure 4.
The above procedure for synthesis of 3H-20g-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 usecf in this study were kindly supplied by
Dr. Lee Fleeger, Texas A&M University (PR ft24 and PR #281), and Dr. V.
L. Estergreen, Washington State University, respectively. The lyo-
phil-ized P antiserum was dissolved in 400 pi 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 break
down 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 ul 3P, 500 yl of 0.05 M
3The addition of normal rabbit serum (1:400) improved the sen
sitivity of this antiserum.


80
TABLE 12. PLASMA CONCENTRATIONS OF PROGESTERONE, 206-DI HYDROPROGESTERONE,
LUTEINIZING HORMONE, AND ESTROGENS IN COWS HAVING MORE THAN
THREE CORPORA LUTEA AT LAPAROTOMY
Day of
Progesterone
2 0 0-Dihydro-
Prog'esterone
Lute in¡zing
hormone
Estrogens
b1eeding
x S.E.
x S.E.
x S.E.
x S.E.
ng/ml
ng/ml
ng/ml
pg/ml
1AM
3.
72
+
0
34
2
60
+
0
65
0
97
+
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

0
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
1 1
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
10AM
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

0
1 1
11AM
6
30
+
1
37
1
72

0
50
1
82

0
1 1
1
54
+
0
09
PM
7
98
f
1
54
1
96

0
37
1
94

0
22
12AM
10
80

1
42
2
22

0
27
2
04

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

0
32
1
40
+
0
28
2
20
+
0
15
14AM
15
24
+
1
23
2
32

0
19
1
56

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

1
46
2
.82

0
35
1
40

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