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A Bell & Howell Information Com pany 300Nortn Zeeb Road. Ann Arbor.Ml 48106-1346USA 313/761-4700 800/521-0600 EFFECTS OF TIME OF MATING AND EXOGENOUS ESTRADIOL AND TESTOSTERONE ON DEVELOPMENT AND SURVIVAL OF SWINE BLASTOCYSTS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of

The Ohio State University

By

Horacio Cardenas Seijas, B.S., M.S.

*****

The Ohio State University

1995

Dissertation Committee Approved by W .F . Pope D.C. Mahan F.L. Schanbacher jij-tild ,tvW W " c -o-c,______S.K. St. Martin Adviser ' D.L. Stetson Department of Animal Science UMI Number: 9526005

UMI Microform 9526005 Copyright 1995, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 To my son, wife and parents

ii ACKNOWLEDGMENTS

I express sincere appreciation to my adviser, Dr. Bill

Pope, for his guidance, insight and constant support throughout the research. I would like to thank the other members of my graduate committee, Drs. David Stetson, Don

Mahan, Floyd Schanbacher and Steve St. Martin, for their assistance during the course of my studies. I would like to thank The Ohio State University and the OARDC for providing the animals and other materials used in my experiments. The help of the swine farm crew, who took excellent care of the animals, is greatly appreciated. Sincere thanks to my wife, Luz, and son Daniel, for their love and understanding of my constant absences.

iii VITA

April 2, 1956...... Born, Saposoa, Peru 1980 ...... B.S., National Agrarian University La Molina, Lima, Peru 198 1...... Farm manager, Huaral, Peru

1982-1985...... Teaching and Research Assistant, National Agrarian University, La Molina, Lima, Peru 1986-1989...... Assistant Professor, Nacional Agrarian University, La Molina, Lima, Peru 1990-Presen t ...... Associate Professor, National Agrarian University, La Molina, Lima, Peru

1990...... M.S., Montana State University, USA. 1991-Presen t ...... Graduate Research Associate, The Ohio State University

PUBLICATIONS

Refereed Journal Articles

Cardenas, H., and W. F. Pope. 1994. Administration of testosterone during the of the estrous cycle increased number of corpora lutea in gilts. J. Anim. Sci. 72:2930.

iv Cardenas, H., J. G. Berardinelli, P. J. Burfening, and R. Adair. 1994. Histomorphology, oLH and hCG receptors, and testosterone secretion in vitro in Rambouillet rams from lines in which females had been selected for high or low reproductive rate. J. Reprod. Fertil. 102:201. Cardenas, H., and W. F. Pope. 1993. Effect of time of mating relative to ovulation on morphological diversity of swine blastocysts. Biol. Reprod. 49:1015. Cdrdenas, H., K. E. McClure, and W. F. Pope. 1993. Luteal function and blastocyst development in ewes following treatment with PGF2a and GnRH. Theriogenology 40:865. Cdrdenas, H., A. Padilla, E. Alvarado, W. Vivanco, and J. G. Berardinelli. 1991. Natural and prostaglandin F (PG)- synchronized estrous cycle in Brown Swiss and Simmental heifers in the highland of Peru. Anim. Reprod. Sci. 26:211. Cardenas Seijas, H., and J. Ch&vez Cossio. 1985. Consanguinidad y heterosis en caracteristicas productivas hasta el destete en cerdos en la granja de la Universidad Nacional Agraria La Molina. [Inbreeding and heterosis in pre-weaning productivity traits at the National Agrarian University La Molina swine farm]. Revista Anales Cientificos (in press). Pope, W. F. , H. Cardenas, T. M. Wiley, and K. E. McClure. 1994. Dose-response relationships of exogenous progesterone shortly after ovulation on estrous cycle length and blastocyst development in sheep. Anim. Reprod. Sci. (in press).

Nephew K. P., H. Cardenas, and W. F. Pope. 1994. Effects of progesterone pretreatment on fertility of gilts mated at an induced pubertal estrus. Theriogenology 42:99.

Pope W. F., H. Cardenas, and K. E. McClure. 1994. Influence of short term fasting on blastocyst morphology and survival in ewes. SID Sheep Res. J. 10:16. Nephew, K. P., H. Cardenas, K. E. McClure, T. L. Ott, F. W. Bazer and W. F. Pope. 1993. Effect of administration of human chorionic gonadotrophin or progesterone before maternal recognition of pregnancy on blastocyst development and pregnancy in sheep. J. Anim. Sci. 72:453.

Sviatko, M. B., H. Cardenas, K. E. McClure, and W. F. Pope. 1993. The effect of small doses of progesterone on blastocyst morphology in sheep. SID Sheep Res. J. 9:119.

v Lane, M. A., J. G. Berardinelli, H. Cardenas and R. B. Staigmiller. 1993. Sperm transport and distribution during the puberal transition in ewe lambs. J. Anim. Sci. 71:707. Berardinelli, J. G., R. W. Godfrey, R. Adair, D. D. Lunstra, D. J. Byerley, H. Cardenas, and R. D. Randel. 1992. Cortisol and prolactin concentrations during three different seasons in relocated Brahman and Hereford bulls. Theriogenology 37:641.

Abstracts Cardenas, H. and W. F. Pope. 1993. Effect of time of mating relative to ovulation on embryonic diversity in pigs. J. Anim. Sci. 71 (Suppl. 1):70. Cdrdenas, H., J. G. Berardinelli, P. J. Burfening and R. Adair. 1990. In vitro stimulation of testosterone secretion and testicular characteristics in rams from lines selected for high or low reproductive rate. J. Anim. Sci. 68 (Suppl. 1):179. C&rdenas, H., A. Padilla, E. Alvarado, W. Vivanco and J. G. Berardinelli. 1989. Comparison of natural and prostaglandin F (PG)-synchronized estrous cycles in Brown Swiss and Simmental heifers in the highland of Peru. Proc. West. Sect. Amer. Soc. of Anim. Sci. 40:284. Berardinelli, J. G, H. Cdrdenas, P. J. Burfening, and R. Adair. 1991. Testicular hCG/oLH receptors and in vitro gonadotropin-stimulated testosterone (T) secretion in Rambouillet rams from lines selected for low or high reproductive rate. Biol. Reprod. 44 (Suppl. 1):124.

Berardinelli, J. G, H. Cardenas, P. J. Burfening, and R. Adair. 1991. Testicular histomorphology and seminal characteristics of Rambouillet rams from lines selected for low or high reproductive rate. J. Anim. Sci. 69 (Suppl. 1):460. Lane, M. A., J. G. Berardinelli, H. Cardenas, and R. Staigmiller. 1990. Sperm transport during the puberal transition in ewe lambs. J. Anim. Sci. 68 (Suppl. 1):150. Berardinelli, J. G., P. J. Burfening, H. Cardenas, and R. Adair. 1989. In vitro technique for evaluating sensitivity of testicular tissue to human chorionic gonadotrophin (hCG) in rams. Proc. West. Sect. Amer. Soc. of Anim. Sci. 40:280.

vi Vivanco, H. W. , J. C. Donaire, and H. Cardenas. 1988. Evaluation of some factors influencing post freezing- thawing sperm motility of Peruvian Paso Horse frozen semen. 11th International Congress on Animal Reproduction and Artificial Insemination: 307. Vivanco, H. W., H. Cdrdenas, and R. Cox. 1987. Immunization against steroid hormones in female alpacas (Lama_pacos) to increase ovulatory response. J. Anim. Sci. 65 (Suppl. 1):416.

Other publications C&rdenas, H., W. Vivanco, E. Alvarado, A. Padilla, and A. Flores. 1990. Comparative studies of estrous cycles and ovulation characteristics of cattle kept at different altitudes in Peru. In: International Atomic Energy Agency, Livestock Reproduction in Latin America, p 55. Vienna. Vivanco, H. W., E. A. Nelson, W. C. Foote, G. Sides, C. Novoa, H. Cdrdenas, J. Camacho, and G. S. Riera. 1986. Scrotal circumference and semen characteristics of Criollo, Corriedale, and Junin rams in the High Central Sierra of Peru. Small Ruminant Collaborative Research Support Program. Technical Report Series 70. Utah State University. Vivanco, H. W . , E. A. Nelson, W. C. Foote, G. Sides, C. Novoa, H. Cardenas, and J. Camacho. 1986. Puberty in Corriedale, Criollo and Junin male sheep in the Central Sierra of Peru. Small Ruminant Collaborative Research Support Program. Technical Report Series 69. Utah State University. Vivanco, H. W., W. C. Foote, G. S. Riera, H. Cardenas, and J. Camacho. 1986. Hormone therapy to induce estrus and ovulation in sheep during the anestrous season. Small Ruminant Collaborative Research Support Program. Technical Report Series 68. Utah State University. Vivanco, H. W., W. C. Foote, G. S. Riera, C. Novoa, J. Camacho, H. Cardenas, and V. Alarcon. 1986. Postpartum interval (PPI) in sheep lambing during the breeding and anestrous seasons in the High Central Sierra of Peru. Small Ruminant Collaborative Research Support Program. Technical Report Series 67. Utah State University. Vivanco, H. W., W. C. Foote, C. Novoa, G. Sides, G. S. Riera, H. Cardenas, and J. Camacho. 1986. Fertility of criollo and and Junin female sheep in the Central Sierra of Peru. Small Ruminant Collaborative Research Support Program. Technical Report Series 66. Utah State University.

vii Vivanco H. W . , W. C. Foote, C. Novoa, G. Sides, G. S. Riera, H. Cardenas, and J. Camacho. 1986. Estrus and ovulation in Criollo, Corriedale and Junin sheep in the Central Sierra of Peru. Small Ruminant Collaborative Research Support Program. Technical Report Series 65. Utah State University.

FIELDS OF STUDY Major Field: Animal Science Studies in reproduction of pigs (blastocyst development and survival and follicular development), sheep (estrous cycle, embryonic survival, testicular physiology, artificial insemination), cattle (estrous cycle), horses (artificial insemination) and alpacas (ovulation, puberty, artificial insemination).

viii TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... iii VITA...... iv LIST OF TABLES...... xi LIST OF FIGURES...... xiii CHAPTER PAGE

I. INTRODUCTION...... 1 II. LITERATURE REVIEW...... 3 Follicular Growth...... 3 Control of Follicular Growth...... 7 Follicular Dynamics; Recruitment and Selection. 9 Follicular Atresia...... 13 Follicular Endocrinology During Final Maturation...... 17 ...... 23 Fertilization...... 27 Embryonic Development...... 30 Embryonic Secretions...... 33 Embryonic mortality...... 38

III. EFFECTS OF TIME OF MATING RELATIVE TO OVULATION ON MORPHOLOGICAL DIVERSITY OF SWINE BLASTOCYSTS..... 49 Introduction...... 50 Materials and Methods...... 51 Results...... 54 Discussion...... 59

IV. INHIBITORY AND STIMULATORY EFFECTS OF EXOGENOUS ESTRADIOL ON BLASTOCYST ELONGATION IN SWINE...... 63

Introduction...... 64 Materials and Methods...... 66 Results...... 70 Discussion...... 77

ix V. ADMINISTRATION OF TESTOSTERONE DURING THE FOLLICULAR PHASE OF THE ESTROUS CYCLE INCREASED THE NUMBER OF CORPORA LUTEA GILTS...... 83

Introduction...... 84 Materials and Methods...... 85 Results...... 90 Discussion...... 95 VI. ADMINISTRATION OF TESTOSTERONE FROM DAY 13 OF THE ESTROUS CYCLE TO ESTRUS INCREASED THE NUMBER OF CORPORA LUTEA AND BLASTOCYST SURVIVAL IN GILTS 100

Introduction...... 101 Materials and Methods...... 102 Results...... 106 Discussion...... 110

VII. SUMMARY...... 118 BIBLIOGRAPH Y ...... 123

X LIST OF TABLES

TABLES PAGE

1. Means of characteristics of blastocysts of gilts mated at different times after an injection of hCG to induce ovulation...... 55 2. Means of blastocyst size and total amounts of various components of uterine flushings in normal- and delay-mated gilts treated with estradiol-17B (E^) at 278 h and evaluated at 290 h post-hCG given to induce ovulation...... 72 3. Amounts of components of uterine flushings per mg of protein in uterine flushings in normal- and delay-mated gilts treated with estradiol-17B (E^) at 278 h and evaluated at 290 h post-hCG given to induce ovulation...... 75 4. Correlation coefficients among blastocyst size and total amounts of various components of uterine flushings around the time of blastocyst elongation in normal-mated (before ovulation) gilts...... 76 5. Correlation coefficients among blastocyst size and total amounts of various components of uterine flushings around the time of blastocyst elongation in delay-mated (after ovulation had begun) gilts...... 76 6. Mean number of large (> 6 mm) follicles on d 19 of the estrous cycle in gilts treated with testosterone...... 91 7. Mean number of medium (3 to 5 mm) follicles on d 19 of the estrous cycle in gilts treated with testosterone...... 91

8. Concentrations (ng/mL) of estradiol and testosterone in of large follicles on d 19 of the estrous cycle in gilts treated with various doses of testosterone...... 93

xi 9. Reproductive characteristics of gilts treated with vehicle or testosterone...... 93 10. Plasma concentrations of testosterone and estradiol in gilts treated with testosterone on d 17 of the estrous cycle...... 94 11. Characteristics of corpora lutea (CL) in gilts administered daily injections of 1 mg of testosterone from d 13 or d 16 of the estrous cycle to estrus...... 108 12. Characteristics of d 11.5 blastocysts of gilts administered daily injections of 1 mg of testosterone from d 13 or d 16 of the estrous cycle to estrus...... 109

13. Mean number of embryos at three stages of development in gilts administered daily injections of 1 mg of testosterone or vehicle from d 13 of the estrous cycle to estrus of mating...... Ill 14. Mean percentage of embryonic recovery at three stages of development in gilts administered daily injections of 1 mg of testosterone or vehicle from d 13 of the estrous cycle to estrus of mating...... 112

xii LIST OF FIGURES

FIGURES PAGE 1. Range and mean size of blastocysts per litter (A) and distribution of blastocysts among morphological classifications (B) in gilts mated at different times after an injection of hCG given to induce ovulation and evaluated 288 h later. Blastocysts of gilts mated 43 h post-hCG were evaluated at either 288 or 295 h post-hCG...... 57 2. Mean size (A) and estradiol secretion in vitro (B) by spherical blastocysts of gilts mated at different times after an injection of hCG given to induce ovulation and evaluated 288 h later. Blastocysts of gilts mated 43 h post-hCG were evaluated at either 288 or 295 h post-hCG. Bars in A represent means and those in B represent the antilogarithm of least squares means. Bars without a common letter character differ (P < .05)...... 58 3. Experimental design and treatment schedule (NM-278 = normal-mated gilts evaluated at 278 h post-hCG, DM-278 = delay-mated gilts evaluated at 278 h, NM-VE = normal-mated gilts treated with vehicle, NM-E2 = normal-mated gilts treated with E2, DM-VE = delay-mated gilts treated with vehicle, DM-E2 = delay-mated gilts treated with E2, M = mating, OVU = estimated time of ovulation, E2 = estradiol-176, HYS = ovariohysterectomy)...... 68

4. Distribution of blastocysts among morphological classifications in normal- (before ovulation, 24 to 32 h post hCG) and delay-mated (after ovulation has begun, 43 h post-hCG) gilts at 278 h post-hCG and in normal- and delay-mated

xiii gilts treated with vehicle or E2 at 278 h and evaluated at 290 h post-hCG given to induce ovulation. Litters and individual blastocysts are represented by rows and black dots, respectively......

xiv CHAPTER I

INTRODUCTION

Embryonic and fetal losses in pigs has been estimated at approximately 40%. Despite the increase in knowledge and understanding of conceptus mortality over the last 50 years, losses of concepti in pigs have basically remained the same. Early experiments were performed to quantify conceptus mortality before or after d 25 to 30 of pregnancy. From these experiments it was estimated that most losses (about 75% of the total) occurred during the first month of pregnancy. Current knowledge indicates that embryonic mortality is not uniformly distributed during the first month of pregnancy, but instead, there are critical periods when the proportion of losses is high. Although not completely clear, it appears that mortality is minimal from fertilization to the morula stage, increases by the time of blastocyst formation, and then increases again during or after blastocyst elongation.

Embryonic mortality associated with the period of blastocyst elongation might be caused by uterine asynchrony generated by blastocysts that elongate and secrete estradiol sooner than others (morphological and functional diversity). How uterine asynchrony might cause the demise of some embryos

1 around this time of development is not known. Experimental results have been accumulating to indicate that some proportion of embryonic death might be related to events that occur during follicular and oocyte development. For example, the state of follicular and oocyte maturation at the time of the LH surge may be of primary importance for subseguent physiological processes, including embryonic development.

The experiments presented here will increase our insight into the causes of embryonic diversity, the process of blastocyst elongation, and the roles of embryonic estrogen in blastocyst elongation. Perhaps most importantly, information will be presented to support the concept of a "follice-oocyte- embryo" relationship as related ultimately to embryonic mortality. CHAPTER II

LITERATURE REVIEW

Follicular Growth Primary follicles form the stock from which all follicles emerge and constitute the majority of follicles in the at all ages (Peters, 1978). In pigs, primary follicles are first observed 75 d postcoitum (Oxender et al., 1979) and the number increases to approximately 500,000 by 10 d after birth

(Black and Erickson, 1968). The size of primary follicles varies among species. For instance, primary follicles of pigs measure approximately 40 fim in diameter and those of rats 24 fxm (Peters, 1978) . Primary follicles contain a centrally- located oocyte encircled by a single layer of flattened epithelial cells (Zamboni, 1974; Peters, 1978). These encircling cells constitute the . Each follicle is separated from the surrounding tissue by a basement membrane. Neighboring granulosa cells are frequently associated by desmosomes, otherwise they are separated, resulting in direct exposure of portions of the oolemma to the extracellular matrix of the ovarian stroma (Zamboni, 1974). Primary follicles show little evidence of biosynthetic activity (Moore et al ., 1974; Guraya, 1985) and appear to grow

3 4 at a very slow rate (Hirshfield, 1989; Morbeck et al., 1992). Initiation of growth of primary follicles is marked by the appearance of mitotic activity in granulosa cells and deposition of components of the . Proteins that constitute the zona pellucida appear to be secreted by the oocyte in pigs (Tagaki et al., 1989). Granulosa cells change from flattened to cuboidal or columnar, forming a continuous and thicker layer around the oocyte. Localized deposition of more zona pellucida material brings about a noticeable separation of the oocyte from the follicle cells. Cytoplasmic processes begin to traverse from the granulosa cells through the zona pellucida and make contact with the oolemma by means of desmosomes. The zona pellucida then becomes a continuous structure around the oocyte by further deposition (Zamboni, 1974) . By this time, abundant microvilli and micropapillae are present and project out from the oocyte surface (Pedersen and Seidel, Jr.; 1972, Zamboni, 1974).

Soon after the initiation of follicular growth, another type of follicular cell appears on the surface of the follicle. These cells constitute the thecal layer which differentiate into and later during follicular growth. All pig follicles that reach 250 jum in diameter have already developed a thecal layer (Morbeck et al., 1992).

Granulosa cells continue to divide and gradually become arranged into layers. At this stage, granulosa cells begin to separate from each other by accumulating a fluid, called liquor folliculi, into small lacunae located around the cells. Accumulation of more liquor folliculi induces coalescence of the lacunae resulting in formation of a common cavity called the antrum (Zamboni, 1974). In pigs, formation of the antrum occurs when follicles reach approximately 400 nm in diameter

(Morbeck et al., 1992). The antrum compresses the follicular cells into two groups, one associated with the follicular wall and the other with the oocyte. Granulosa cells immediately circumscribing the oocyte form the . Growth rate of follicles increases as follicles increase in size, and consequently, the rate of growth is lower in preantral than antral follicles. Growth rates of antral follicles of pigs, cows, sheep and rats follow a exponential curve produced by a dramatic increase in proliferation of granulosa cells and increase in size of the antrum

(Hirshfield, 1991; Morbeck et al., 1992). Activated primary follicles of pigs require approximately 84 d for growing to the antral stage and an additional 19 d to grow to the preovulatory size of 10 mm (Morbeck et al., 1992). The rate of growth of porcine follicles from 3 to 10 mm in diameter has been estimated to be 1.14 mm per d (Dailey et al., 1976).

During the process of follicular growth, oocytes also grow and undergo changes in the structural organization of cellular organelles. Mitochondria, endoplasmic reticulum, Golgi apparatus, and cortical granules become more abundant at the 6 periphery of the ooplasm, leaving the central region of the cell almost free of organelles. Pig oocytes reach their maximum diameter of approximately 110 when follicles have a diameter of about 250 /im (Morbeck et al., 1992). The number of intercellular bridges between the cumulus oophorus cells and the oolemma increases and penetrate to different degrees into the ooplasm (Zamboni, 1974; de Loos et al., 1991). These processes, which also contain organelles such as mitochondria,

lipid droplets and lysosome-like bodies, make association with the oolemma through desmosomes and gap junctions (Amsterdan et al., 1976; Anderson and Albertini, 1976; Gilula et al., 1978). Gap junctions become more numerous than desmosomes as follicular growth progresses (Anderson and Albertini, 1976). The oocyte and cumulus cells stay in contact through gap junctions for most part of the period of follicular growth and maturation. Mammalian gap junctions are permeable to small molecules ranging up to 1000D molecular weight (Loewenstein,

1974) . Oocytes are therefore considered to depend on the surrounding cumulus cells for uptake of aminoacids (Colonna and Mangia, 1983) and other small molecules such as uridine

(Moor et al., 1980; Heller et al., 1981; Eppig, 1982), choline

(de Loos et al., 1991) and growth factors (Coskun and Lin, 1993). In co-cultures of free oocytes and follicle cells in which cell contact did not occur, no growth or minimal growth was observed in oocytes (Eppig, 1979; Bachvarova et al.,

1980). Transfer of substances through gap junctions has been 7 postulated to be a mechanism to compensate for limitations in uptake of certain compounds by the oocyte due to the small

surface to volume ratio (Brower and Schultz, 1982). Near the time of ovulation, when the follicle is undergoing final maturation, the cytoplasmic processes of the cumulus oophorus cells are retracted from the oocyte. This causes an interruption of morphological relationships between the oocyte and the surrounding follicle cells (Gilula et al.,

1978; Eppig, 1982; Hyttel, 1987; Phillips and Dekel, 1991).

Control of Follicular Growth Controlling the initiation of primary follicle growth is complex and appears to involve endocrine actions and regulatory effects of local factors from the somatic cells of the follicle (Toneta and diZerega, 1986; Hirshfield, 1991; Lobb and Dorrington, 1992) and probably from the growing oocyte (Hirshfield, 1991; Vanderhyden et al., 1992). Initiation of primary follicle growth is a pivotal question that has not been answered clearly primarily because it is technically difficult to distinguish follicles that have just begun to grow from those that are still quiescent (Hirshfield, 1991). The involvement of gonadotropins LH and FSH, in the initiation of follicular growth is controversial. Some results that support this assumption are; 1) coincidental changes during the estrous cycle in the number of very small follicles 8

showing biosynthetic activity and the concentrations of gonadotropins (Pedersen, 1970), 2) decrease in the number of growing follicles in rats administered a GnRH antagonist (Van

Capellen et al., 1989), and 3) effects of FSH on the number of follicles that begin growth and on the rate of hyperplasia of granulosa cells in vitro (Ryle, 1972) . Furthermore, receptors for FSH are present in granulosa cells of rats at all stages of development (Richards et al., 1976; Hirshfield, 1991).

Greenwald (1974) postulated that selective growth of primary follicles is controlled by the access of the primary follicles to FSH. For instance, in infantile rodent , the primary follicles near the more vascularized regions are the first to growth (Byskov, 1975). Total withdrawal of gonadotropins as a result of hypophysectomy does not produce complete blockage of granulosa cell division but results in inhibition of formation of the (Dufour et al., 1979; Hirshfield, 1991).

Therefore, gonadotropins appear not to be absolutely necessary for initiation and maintenance of preantral follicular growth.

However, the completion of antrum formation and follicular maturation requires continuous gonadotropin support. These concepts agree with results of chronic immunoneutralization of GnRH in ewes (McNeilly et al., 1986) and treatment of rats with FSH (Zelesnick et al., 1974). Moreover, sheep follicles do not respond to exogenous gonadotropins until the layers of granulosa and theca are well developed, which occurs at approximately two to four weeks of age (Mauleon, 1969; Kennedy et al., 1974) . Ovulation and formation of corpora lutea can be induced by gonadotropin treatment in lambs not earlier than five to six months of age (Worthington and Kennedy, 1979) . In pigs, ovulation following exogenous gonadotropins occurs after 60 d of age which coincides approximately with the time when some antral follicles are present in the ovaries (Casida, 1934; Oxender et al. , 1979). Based on these different effects of gonadotropins, follicular development is sometimes divided into gonadotropin-independent and gonadotropin-dependent stages (Lobb and Dorrington, 1992). It has been hypothesized that onset and continuation of follicular growth during the gonadotropin-independent stage might be controlled by growth factors produced by individual follicles (Beldell and Dorrington, 1990; Hirshfield, 1991).

The influence of growth factors can be extended to the stage of antral development since it is known that gonadotropins can not directly promote granulosa cell growth (Lobb and Dorrington, 1992).

Follicle dynamics; Recruitment and Selection Follicles are not stimulated to grow at the same time. In mammals, this characteristic increases the possibilities of having progeny throughout a rather long reproductive life. Little is known about the patterns (dynamics) of development of preantral or antral follicles smaller than 1 mm in 10 diameter. However, some knowledge has been gained on development of larger follicles which can be monitored using ultrasound techniques, particularly in large domestic species such as cattle.

Follicular recruitment refers to the formation of a pool of antral follicles from which the ovulatory follicle(s) is subsequently selected (Fortune et al., 1991). There are some differences among species in these processes. In dairy cows, two or three groups (waves) of growing follicles (depending on the duration of the estrous cycle) can be identified on d 0 (day of estrus) and 10 or d 0, 9 and 16 of the estrous cycle, respectively (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989b). The largest follicle of each wave, the so called dominant follicle, is considered to suppress the growth of smaller (subordinate) follicles and the initiation of the next follicular wave (Ginther et al., 1989a; Ko et al.,

1991). Therefore, either atresia or ovulation of the dominant follicle would allow development of the next group or wave of follicles. Follicular waves and resulting development of dominant follicles occur during the prepuberal period (Adams et al., 1994). Waves of follicular development in cattle appear to be induced by small elevations in basal FSH; however, maintenance of the dominant follicle requires pulses of LH (Adams et al., 1992). A different mechanism of follicular dominance has been proposed in primates such that the largest follicle develops a greater sensitivity to FSH and 11 keeps growing. As the largest follicle grows estradiol secretion inhibits the basal concentrations of FSH and results in atresia of smaller follicles (Zeleznik and Kubik, 1986). Results regarding follicular dynamics in sheep are controversial. Some investigators observed follicular growth occurring in waves (Smeaton and Robertson, 1971; Brand and de Long, 1973; Noel et al., 1993). Others did not observe follicular waves during the estrous cycle or pregnancy, and observed follicular dominance only just before ovulation (Al- Gubory et al., 1992; Ravindra et al., 1994).

In pigs, large follicles are found only during the follicular phase of the estrous cycle. Fortune (1994) speculated that the absence of follicular waves in pigs may be related to sensitivity to, and(or) high concentrations of, feed-back regulators such as estradiol and inhibin. Unlike cattle, in which a dominant follicle appears to inhibit development of subordinate follicles, large follicles in pigs may promote steroidogenesis of smaller follicles by serving as a source of steroids (Foxcroft and Hunter, 1985; Foxcroft et al., 1987). Follicular recruitment in pigs occurs between d 14 and d

16 of the estrous cycle (Clark et al., 1975; Clark et al.,

1982; Foxcroft and Hunter, 1985) . On d 16, approximately 40 to

50 follicles 2 to 6 mm in diameter are present in both ovaries (Grant et al., 1989). A small proportion (about 25%) of these follicles are selected to complete final maturation and 12 ovulate while the remaining recruited follicles become atretic, most of them before reaching 6 mm in diameter (Grant et al., 1989; Dailey et al. , 1975). Ovulatory follicles are not readily identifiable until d 20 (Grant et al., 1989).

Selection of these follicles might be a continuous process that occurs during the follicular phase (Foxcroft et al., 1987). Selection could, therefore, be modulated by the rate of atresia among recruited follicles (Dailey et al., 1975).

Follicular recruitment appears to be controlled, at least partially, by FSH (Foxcroft and Hunter, 1985). There are several relationships that support this notion. Plasma concentrations of FSH increase just before recruitment (d 12 and 13) in gilts (Guthrie and Bolt, 1990; Guthrie et al., 1993) and in weaned sows (Shaw and Foxcroft, 1985; Foxcroft et al., 1987). Treatment with FSH or pregnant mare serum gonadotropin (PMSG) on d 15 and 16 of the estrous cycle increase the number of ovulations in gilts but has no effect when given after d 16. Partial suppression of FSH by administration of porcine follicular fluid to gilts during the estimated time of follicular recruitment and the early follicular phase reduces the subsequent number of small, medium or large follicles. However, populations of medium size follicles are partially restored when FSH is given following treatment with porcine follicular fluid (Guthrie et al., 1988;

Knox and Zimmerman, 1993). 13

Follicular Atresia Most follicles, and the oocytes they contain, degenerate and disappear from the ovaries through a process that seems to be the normal fate and not the exception. This loss is called follicular atresia. Although atresia may occur any time during development of antral follicles, most follicles, for a given species, are lost during the transition from the small to large size (Carson et al., 1979; Fortune, 1994). The biological significance and the mechanisms that induce follicular atresia are not known. At present, atretic follicles do not appear to play a clear role in ovarian function. However, it seems possible that progesterone and androgens synthesized by atretic follicles may be used by nonatretic follicles as substrates during steroidogenesis (Westhof et al., 1991). Perhaps, follicular atresia has evolved as a mechanism for eliminating poor developing follicles or oocytes and for maintaining number of ovulations within a narrow range for each mammalian species. Degeneration of granulosa cells is the first morphological alteration observed in atretic follicles (Hay et al., 1976; Tsafriri and Braw, 1984). Degeneration results in pyknosis and reduction in the number of granulosa cells due to cell lysis and phagocytosis. Due to cell lysis, accumulation of cellular debris occurs in the follicular fluid (Hay et al., 1976; Peluso et al., 1980; Maxson et al., 1985). Degeneration of cumulus cells and oocytes appears to begin after these 14 changes in granulosa cells are observed (Hay et al., 1976; Tsafriri and Braw, 1984). When measuring total blood flow to sheep follicles, Bruce and Moor (1976) found no difference between healthy and atretic follicles. However, subsequent studies determined that reorganization of the follicular capillary bed and reduction of blood flow to the thecal layer may account for some of the changes observed during atresia (Hay et al., 1976; Maxson et al., 1985). Antral follicles undergoing atresia have lower concentrations of estradiol and contain similar or greater concentrations of progesterone and androgens in the follicular fluid than nonatretic follicles (Moor et al., 1978; Carson et al., 1981; Maxson et al., 1985; Spicer et al., 1987; Guthrie et al., 1993). Estradiol stimulates granulosa cell proliferation and, therefore, is required for follicular growth (Peters and McNatty, 1980). Given the functions of estradiol in follicular development, it was logical to suppose that decreasing concentrations of estradiol could play a major role in follicular atresia. Small amounts of estradiol might be the result of decreased aromatase activity, decreased availability of aromatizable androgens, or both. Loss of aromatase activity has been considered the cause of decreased amounts of estradiol in atretic follicles of pigs (Maxson et al., 1985).

However, the involvement of gonadotropin binding in decreasing the aromatase activity is not clear since the specific binding 15 to gonadotropins was maintained in both atretic and healthy follicles of sheep (Carson et al., 1979) and cows (Spicer et al., 1987; Grimes et al., 1987) but decreased relative to healthy follicles in pigs (Maxson et al., 1985). Consistent with the findings of Maxson et al. (1985) in pigs, Guthrie et al. (1993) found that granulosa cells of atretic follicles obtained during the follicular phase exhibited loss of gonadotropin-sensitive adenylate cyclase activity simultaneously with a decrease in plasma concentrations of FSH. These investigators considered the loss of the ability of follicles to bind gonadotropins as a mechanism of atresia in gilts.

Although androgens have been considered inducers of follicular atresia in rats (Hillier and Ross, 1979; Bagnell et al. , 1982) , these experiments were performed using prepuberal, hypophysectomized animals and, therefore, do not reflect physiological situations as there was no gonadotropin support for follicular growth. When more physiological experimental conditions were applied to rats or other species, the role of testosterone in inducing atresia was not confirmed.

Examination of follicles obtained from pigs at known physiological stages allowed for the determination that the supply of aromatizable androgens may be more important than aromatase activity for the synthesis of estradiol during the follicular phase (Foxcroft and Hunter, 1985; Foxcroft et al.,

1987; Grant et al., 1989). 16

The importance of testosterone for estradiol synthesis and viability of maturing follicles found in pigs agrees with results in other species. For example, one of the early signs of atresia of preovulatory follicles of rats treated with sodium pentobarbital to block ovulation, was a decrease in the capacity to synthesize testosterone. Before morphological signs of atresia were detected, follicles showed low concentrations of estradiol but maintained aromatase activity and their capacity to synthesize progesterone under LH stimulation in vitro (Uilenbroek et al., 1980). Greenwald (1993), proposed that the superovulatory effect of hCG in hamsters was due to increase in androgen and estradiol production which, supposedly, resulted in the rescue of some follicles from atresia. Similarly, estrogenic follicles present in anestrous ewes do not complete maturation and ovulate probably because of inadequate LH-stimulated testosterone synthesis (McNatty et al., 1984). It is also possible, as suggested by Tsafriri and Braw (1984), that androgens may be atretogenic for immature follicles but necessary for rescue from atresia of maturing follicles. Programmed cell death or apoptosis, a process characterized by internucleosomal DNA fragmentation, has been recently implicated in follicular atresia in several species, including pigs. Atresia of pig follicles was associated with apoptosis of granulosa and theca cells and with a decrease in expression of mRNAs for aromatase and gonadotropin receptors 17

(Tilly et al., 1992). The loss of mRNA for aromatase is

consistent with loss of aromatase activity in atretic follicles of pigs (Maxson et al., 1985). Recently, a flow cytometric procedure based on amount of degraded DNA in granulosa cells was used to differentiate atretic and nonatretic follicles in pigs (Guthrie et al., 1994). Estrogen treatment decreased apoptosis in granulosa cells of rats while testosterone antagonized the effects of estrogens (Billing et al., 1993) suggesting a role for steroids in apoptosis.

Follicular Endocrinology During Final Maturation Pig follicles grow most rapidly during the follicular phase of the estrous cycle (Clark et al., 1975; Grant et al.,

1989) . During final maturation, cells of the theca and granulosa layers synthesize and secrete significant amounts of steroids, peptide hormones, prostaglandins and other substances. These hormones are important because they participate locally in follicular development (Sanyal et al., 1974; McNatty et al., 1979) and, some of them, convey signals that coordinate the function of the ovarian-hypothalamic- hypophyseal axis. Furthermore, some steroids may only serve as substrates in steroidogenesis, while others, such as estradiol and perhaps testosterone (Hild-Pepito et al., 1991), produce receptor-mediated actions in the ovary. The endocrine activity of follicles is primarily reflected in hormonal concentrations within the follicular fluid. In pigs, follicular concentrations of progesterone, testosterone, androstenedione, estradiol and other steroids remain low during the luteal phase, increase during follicular growth to the preovulatory stage and then decrease rapidly as follicles approach ovulation (Hunter et al., 1976; Babalola and Shapiro, 1988; Grant et al., 1989; Xie et al., 1990c; Guthrie et al., 1993; Conley et al., 1994). Before ovulation the low concentrations of androgens and estrogens may result from inhibitory effects of the LH surge on aromatase and cytochrome P450 17a-hydroxylase /17-20 lyase, the enzyme that catalyzes the conversion of 17a-OH progesterone into androstenedione (Tsang et al., 1985; Conley et al., 1994). Changes in concentrations of follicular estradiol are correlated to changes in plasma concentrations of this hormone

(Guthrie et al., 1972; Guthrie and Bolt, 1990). Considering the changes in gonadotropin hormones during final maturation of the follicle, plasma concentrations of FSH in gilts are elevated during luteolysis and then decrease gradually until estrus (Van de Wiel, 1981; Guthrie and Bolt,

1990; Guthrie et al., 1993). These decreasing concentrations of FSH occur while follicles are growing and increasing in size during the follicular phase. Similarly, FSH binding and FSH-stimulated adenylate cyclase activity by granulosa cells decrease as follicular size increases (Nakano et al., 1977;

Lindsey and Channing, 1979). Unlike FSH, LH does not change much during the follicular phase, except near the onset of estrus when the preovulatory LH surge occurs (Brinkley, 1981; Guthrie and Bolt, 1990; Guthrie et al., 1993). Directly associated with these changes in plasma LH, LH receptors in theca and granulosa cells are more abundant as follicles increase in size (Stouffer et al., 1976; Nakano et al., 1977; Foxcroft and Hunter, 1985). Secretion of gonadotropins during the follicular phase is highly influenced by feedback mechanisms. For example, increasing amounts of estradiol first suppress FSH and LH, and then they induce the preovulatory surge of LH (Guthrie et al., 1972; Brinkley, 1981). Follicular thecal and granulosa cells undergo steroidogenesis in a cooperative manner. According to the two- gonadotropin two-cell theory that applies to swine (Evans et al., 1981; Haney and Schomberg, 1981) and other species

(Short, 1962; Ryan, 1968; Armstrong and Papkoff, 1976; Fortune and Armstrong, 1977; Moor, 1977), aromatizable androgens

(testosterone and androstenedione) are synthesized by thecal cells upon stimulation by LH, which then diffuse to granulosa cells for subsequent conversion into estradiol. The conversion of androgens into estradiol requires stimulation of the aromatase enzyme system by FSH (Dorrington et al., 1975; Moon et al., 1975). Furthermore, results of in vitro experiments using rats indicate that progesterone produced by granulosa may be used by theca cells for the synthesis of androgens (Liu and Hsueh, 1986) . This may also occur in pigs because granulosa cells are the major sites of follicular synthesis of 20 progesterone (Evans et al., 1981). In pigs, thecal cells have the ability to synthesize estradiol but in lower amounts than those synthesized by granulosa cells (Haney and Schomberg, 1981; Tsang et al., 1985; Tonetta et al., 1986). Synthesis of estradiol by theca cells is stimulated by LH since FSH binds only to granulosa cells (Nakano et al., 1977; Tonetta et al., 1986). Testosterone and androstenedione are synthesized from pregnenolone by the delta 5 (pregnenolone -+ 17a-OH pregnenolone -+ dehydroepiandrosterone ->• androstenedione -*■ testosterone) or delta 4 (Pregnenolone -► progesterone -*■ 17a-OH progesterone -♦ androstenedione -*■ testosterone) pathways.

Androstenedione can be converted into testosterone in a reversible reaction catalyzed by 176-hydroxysteroid dehydrogenase or aromatized to estrone, which then can be converted to estradiol-176. Testosterone is directly aromatized into estradiol-176 (Peters and McNatty, 1980). In pigs, synthesis by theca cells and follicular fluid concentrations of androstenedione during the mid and late follicular phase are several fold greater than those of testosterone (Evans et al., 1981; Tsang et al., 1985; Babalola and Shapiro, 1988). Although, these results do not necessarily mean that granulosa cells use one androgen preferentially over the other, recent in vitro experiments indicated that pig granulosa cells from preantral follicles synthesized more estradiol in the presence of androstenedione than in the 21 presence of testosterone (LaBarbera et al., 1994). Furthermore, decreases in estradiol concentrations in follicular fluid of sows treated with dexamethasone during the follicular phase were related to decreases in the concentration of androstenedione (Liptrap and Cummings, 1991) . It seems to be clear from these studies, and those discussed in the section of follicular atresia, that estradiol synthesis by granulosa cells during the follicular phase in pigs depends mainly on availability of aromatizable androgens. Conversely, the ability of theca cells to synthesize estradiol appears to be limited by the level of aromatase activity (Ainsworth et al. 1990). It remains to be determined whether limiting factors for estradiol synthesis are the same in follicles present during other phases of the estrous cycle. Estradiol and testosterone are known to play local roles in the ovary. Estradiol stimulates granulosa cell proliferation and appears to be absolutely necessary for follicular growth and maturation (Peters and McNatty, 1980). The actions of FSH on aromatase activity are enhanced by estradiol and aromatizable androgens in rat granulosa cells in vitro (Daniel and Armstrong, 1980; Adashi and Hsueh, 1982). In addition, testosterone enhances and estrogen inhibits the synthesis of progesterone by pig granulosa cells of preovulatory follicles (Haney and Schomberg, 1978).

Inhibin, activin and follistatin are peptide hormones synthesized and secreted by pig granulosa cells (Van de Wiel 22 et al., 1983, Miller et al. , 1991). Activin stimulates secretion of FSH, whereas inhibin and follistatin inhibit FSH. LH-induced androgen production by theca cells is regulated positively by inhibin and negatively by activin (Hsueh et al., 1987). In addition, activin increases the number of FSH receptors in preantral follicles and prevents premature luteinization of large antral follicles. The effect of activin on FSH receptors could be of particular importance for initiation of follicular development (Findlay, 1993). Conversely, follistatin regulates progesterone synthesis and has the capacity to bind activin and attenuate its actions

(Findlay, 1993). Growth factors are produced by granulosa cells and possibly modulate final follicular maturation through paracrine/autocrine actions. Initial stimulation of ovarian steroidogenesis occurring 24 h after weaning in sows was not related to follicular concentrations of insulin-like growth factor-I (IGF-I; Killen et al., 1992). During the follicular phase in gilts, concentrations of IGF-I in follicular fluid of large follicles was higher than in small follicles (Mondschein et al., 1991). Similarly, expression of mRNA for IGF-I increased during the follicular phase and was correlated with size and estradiol concentrations. However, mRNA for insulin­ like growth factor-binding protein-2 (IGFBP-2) decreased during the same period (Samaras et al., 1993). A decrease in

IGFBP-2 might result in more free IGF-I available for cellular 23 functions. These results, together with others from in vitro experiments (Hsu and Hammond, 1987), indicate that IGF-I may be involved in follicular maturation. Transforming growth factor-B is secreted by thecal cells of porcine follicles less than 7 mm in diameter and exhibits stimulatory and inhibitory effects on progesterone production in vitro depending on its concentrations (Gangrade and May, 1990; Chang et al., 1993).

Oogenesis

Porcine oocytes enter meiosis during fetal life and become arrested soon after birth at the dictyate stage of prophase I for a variable period of time (Black and Erickson, 1968). Oocytes regain meiotic competence after reaching their full size, which occurs coincidentally with follicular antrum formation (Erickson and Sorensen, 1974; Schultz, 1986, Christmann et al., 1994; Hirao et al., 1994). At present, only oocytes from antral follicles have shown the ability to sustain normal embryonic development (Pavlok et al., 1992). Growth of oocytes is characterized by a significant increase in synthesis and accumulation of proteins and RNA (Crozet et al., 1981; Lazzari et al., 1994) Starting around puberty, with every ovarian cycle, one or more oocytes (depending on the species) resume meiosis and complete the first meiotic division in response to a surge of LH. Oocytes enter the second meiotic division and are usually in metaphase II at ovulation. The second meiotic division is 24

completed with the formation of the second polar body when a sperm cell penetrates the oolemma (Howlett and Bolton, 1985). Oocytes collected from antral follicles but separated from the cumulus cells when cultured in vitro, resumed meiosis spontaneously as determined by chromatin condensation and germinal vesicle breakdown (Edwards, 1965). During resumption of meiosis in vivo there is an uncoupling of the cytoplasmic processes of cumulus cells from the oolemma (Gilula et al., 1978; Phillips and Dekel, 1991). Artificial separation of the oocyte from the follicular cells also induces resumption of meiosis in vitro (Edwards, 1965). These findings suggest that cumulus cells may play a major role in oocyte meiotic arrest

and resumption. Cyclic AMP, the second messenger in the hormonal action of the gonadotropins LH and FSH, was among the first compounds to be tested as the oocyte meiotic inhibitor produced by cumulus

cells. Meiotic arrest was maintained in vitro by supplementing the media with cAMP analogues or phosphodiesterase inhibitors that indirectly increase cAMP in the cell (Cho et al., 1974; Dekel and Beers, 1978; Downs et al., 1988). After their findings, Dekel and Beers (1978) hypothesized that meiotic arrest was maintained in the follicle by transfer of cAMP from the follicle cells into the oocytes via gap junctions.

Termination of the gap junctional communication between the oocyte and follicle cell following the LH surge interrupted the flow of cAMP and resulted in meiotic resumption. 25

Similar actions to those found for cAMP on meiotic arrest were found for several purines such as hypoxanthine and guanosine (Downs et al., 1988). The maintenance of meiotic arrest by cAMP and purines has been shown to be reversible after treatment with LH and FSH (Downs et al., 1991), macromolecules such as polyvinylpyrrolidone, polivinylalcohol or crystallized BSA (Downs et al., 1991), Ca+2 (Racowsky, 1986)

and epidermal growth factor (Downs et al., 1988).

Resumption of meiosis appears to be more complex than implied by the action of a single inhibitor. For example, it has been shown in several species that resumption of meiosis induced by LH requires active RNA synthesis by cumulus cells (Lindner et al, 1974; Osborn and Moor, 1983; Motlick et al., 1989). In addition, denuded oocytes of pig, sheep and cattle require protein synthesis (Fulka et al, 1986; Moor and Crosby, 1986; Hunter and Moore, 1987), proteolysis (Jagiello et al.,1978) and protein phosphorylation (Fulka et al., 1990) to undergo resumption of meiosis. Calcium levels in intact rat oocytes increase concomitant with increases in serum luteinizing hormone (Batta and Knudsen, 1980). When free calcium was decreased in the oocyte by administering neomycin, an inhibitor of phosphoinositide hydrolysis, germinal vesicle breakdown (indicator of meiosis resumption) was inhibited irreversibly (Homa, 1991).

Involvement of calcium in meiotic resumption of mouse oocytes has been confirmed by De Felici et al. (1991) by using a 26

membrane permeable calcium chelator of high affinity. Motlik and Kubelka (1990) proposed that in mammals the pre-ovulatory LH surge induces a metabolic pathway in the

granulosa cells that ends in the activation of a cytoplasmic factor, such as maturation promoting factor (MPF), within the oocyte. This activation and subsequent completion of chromatin condensation and germinal vesicle breakdown requires RNA and protein synthesis, phosphorylation and protease activity. The importance of MPF and phosphorylation in arrest of meiosis and resumption has been confirmed by studies on the cell cycle, the set of events responsible for the duplication

of the cell (Murray and Kirschner, 1989). Three control points in the cell cycle have been named START, ENTRY and EXIT (Whitaker and Patel, 1990). Cells in interphase are in G1 (gap one) of the cell cycle. Activation of key proteins during

START commit the cell to go through the S phase (DNA

synthesis). Once S phase is completed cells enter G2, the

second gap of the cycle. Cells become committed to undergo M phase (division) by activation of MPF during ENTRY (Murray and

Kirschner, 1989). MPF is a complex of three different proteins: p34cdc2, cyclin B and cdc25. The first protein is the product of the cdc2 gene and remains inactive in nondividing cells by being phosphorylated. The protein cdc25 is a phosphatase that has the capacity to dephosphorylate p34cdc2 resulting in activation of MPF. Maturation promoting factor has kinase activity and will participate in chromosome 27 condensation, breakdown of the nuclear envelope and formation of the mitotic spindle. After the cell completes metaphase, MPF is inactivated by degradation of cyclin B and the cell will go to anaphase and then telophase. Vertebrate oocytes arrested at metaphase II do not progress to anaphase unless fertilization occurs. Therefore, oocytes are arrested because they can not go through EXIT, indicating that MPF remains active. The mechanisms that maintain MPF activity are not known. The presence of a cytostatic factor (CSF), which inhibits the degradation of cyclin B, has been proposed (Hunt, 1992). Fertilization blocks this inhibition apparently by inducing the synthesis of another protein through the action of calcium as a second messenger (Parrish et al., 1992).

Fertilization

Approximately 34 to 36 h after onset of estrus in swine oocytes are released from the matured follicles by the process of ovulation (Polge, 1978; Pope et al., 1988; Wilmut et al., 1992). Released oocytes are picked up by the oviducts and then transported down to the ampullar-isthmic junction by ciliary currents where fertilization takes place (Gaddum-Rose and

Blandau, 1970). Attachment of sperm to the zona pellucida takes place by the action of adhesion molecules. Although these molecules have been studied mostly in mice, it is likely that homologous 28 molecules play the same roles in other species (Wassarman,

1994) . Adhesion molecule involved in fertilization are species specific (Wassarman, 1992) except when interspecies

fertilization is possible (sheep and goat, mink and ferret and horse and donkey; Adams, 1973). In mice, the glycoprotein mZP3, located in the zona pellucida is the primary sperm receptor, whereas the protein SP56, located on plasma membrane associated with the intact acrosome seems to be the primary egg-binding molecule (Bleil and Wassarman, 1980; Bleil and

Wassarman, 1990). Presumably SP56 recognizes and binds mZP3 (Bleil and Wassarman, 1990; Wassarman, 1992). A secondary sperm receptor (mZP2) has been identified, which may help acrosome-reacted sperm to remain bound to the zona pellucida (Bleil et al., 1988). Recently, proteins called spermadhesins, which are secreted by male sexual accessory glands have been implicated in sperm binding to the zona pellucida in swine (Sanz et al., 1993). Attainment of the ability to bind the zona pellucida occurs during sperm capacitation, which in swine requires 3 to 6 h (Hunter, 1972,

1990). Post-ovulatory age of oocytes, from 2 to 15 h, did not influence the number of spermatozoa in the zona pellucida or the proportion of eggs fertilized (Hunter and Dziuk, 1968).

Upon attachment to the zona pellucida, sperm undergo an acrosome reaction which results in exocytosis of the acrosome contents and allows for penetration of the zona pellucida and fusion with the plasma membrane of the oocyte (egg activation, 29

Yanagamachi, 1988) . Important biochemical and morphological changes occur at the time of activation. The primary biochemical event is an increase in intracellular Ca+2 (Jaffe, 1990) which works as second messenger in the process of resumption of the second meiotic division and its completion with extrusion of the second polar body (Shiina et al., 1993) . Formation of the male pronucleus proceeds from the sperm head, whereas formation of the female pronucleus does not begin until meiosis is completed. The pronuclei move next to each other at the center of the oocyte and then the chromosome align at a stage equivalent to zyngamy in other species. From penetration to zyngamy, it takes approximately 3.5 h in pigs (Hunter, 1972). Normal embryonic development is not compatible with penetration of the oolemma by more than one sperm

(polyspermy) . The block to polyspermy occurs by changes in the adhesin molecules of the zona pellucida during the zona reaction (Bleil et al ., 1981), changes in the plasma membrane, or both, depending on the species (Wolf, 1981). The zona reaction is induced by enzymes released from the cortical granules of the oocyte once the first sperm had penetrated the oolemma (Wassarman, 1988). The plasma membrane block to polyspermy in the mouse results from inability of the sperm to adhere to the plasma membrane and appears to be sperm dependent and not related to cortical granules (Horvath et al., 1993). 30

Embryonic Development Fertilized oocytes undergo a series of mitotic divisions whereby daughter cells (blastomeres) are smaller than mother cells resulting in minimum changes in overall embryonic size

(Hunter, 1974). Extrusion of the second polar body marks the beginning of the first cell cycle (Howlett and Bolton, 1985). The first cell cycle is long but the second cycle is shorter than the first and consists basically of S and M phases with the G1 and G2 phases practically absent (Johnson, 1981).

Because there is hot DNA transcription, early cleaving embryos utilize mRNA previously synthesized during oocyte growth for their development. Therefore, this part of embryonic development is controlled by the maternal genome independent from the embryonic genome (Johnson, 1981). In pigs, the transition from the maternal to embryonic control of development occurs at the 4-cell stage (Jarrell, et al., 1989,

Schoenbeck et al., 1992) when the cell cycle lasts longer due to appearance of the G1 and G2 gaps. Pig embryos begin cleaving 17 ± 9 h after ovulation, they remain at the 2-cell stage for 6 to 8 h, at the 3 to 4 cell stage for 20-24 h and at the 5 to 8 cell stage for 46 to 52 h (Hunter, 1974) . Porcine embryos enter the mainly at the 4-cell stage, approximately 46 to 48 h after ovulation (Hunter, 1974) which is equivalent to 3 to 4 d after onset of estrus (Perry and

Rowlands, 1962, Oxenreider and Day, 1965, Broermann et al.,

1990). 31

Concepti having 8 to 16 blastomeres are referred to as morulas (Bazer et al., 1987). In pigs (Barents et al., 1989) and other species (Reeve, 1981; Betteridge and Fletchon, 1988), blastomeres form tight junctions during the process of morula compaction and subsequent blastocyst formation. Tight junctions are morphological structures that keep cells together and help to establish cell polarization and an internal environment different from the external one (Biggers et al., 1988; Staehelin and Hull, 1978; Dardik and Schultz, 1991). Approximately 5 to 6 d after ovulation in pigs (Perry and

Rowlands, 1962, Hunter, 1974), morulas transform into blastocysts by internal accumulation of fluid and formation of a central cavity, the blastocoele. The outer layer of cells surrounding the blastocoele is called trophoblast. Another group of cells localized beneath the trophoblast, at one pole, form the embryoblast or inner cell mass. By d 10 pig, embryoblasts move to the surface by displacing the overlaying trophoblast cells also known as Rauber's cells (Geisert et al., 1982b; Stroband et al., 1984; Barends et al., 1989). Six days after ovulation, pig blastocysts escape (hatch) from the zona pellucida. Embryonic and(or) uterine enzymes may

(Lindner and Wright, 1978) or may not (Broermann et al., 1989) be necessary for zona hatching. Expansion of the blastocyst which is induced by cell hyperplasia and fluid accumulation in the blastocoele, seem to be the most important factor involved 32

in zona hatching (Bergstrom, 1972; Bazer et al., 1987). From hatching to d 12 after estrus, blastocysts move freely in the uterine lumen and usually become intermixed with blastocysts from the other uterine horn (Dziuk et al., 1964; Dhindsa et al., 1967). At the end of the migratory process, blastocysts are approximately spaced in the uterine lumen and do not overlap one another (Anderson, 1978). Spacing of blastocysts is of primary importance for implantation, which is a gradual process that begins as early as d 10 and finishes with formation of interlocking microvilli by d 18 (Perry et al., 1973; Anderson, 1978). Male preimplantational blastocysts of pigs (Cassar et al., 1994b), mice (Tsunoda et al., 1985) and cattle (Avery et al., 1989), appear to grow faster than female blastocysts. On d 12 after estrus, porcine blastocysts that reach approximately 10 mm in diameter begin a process of rapid elongation (Perry and Rowlands, 1962; Anderson, 1978; Geisert et al., 1982b; Pusateri et al., 1990). Because not all blastocysts reach this size at the same time, spherical, ovoidal, tubular and filamentous blastocysts are usually present on d 12. By day 13 more than 95% of blastocysts are filamentous (10 to 60 cm in length Anderson, 1978; Pusateri et al., 1990). Geisert et al. (1982b) estimated a growth rate of 0.25 mm/h for d 11 spherical blastocysts (from 2.5 to 10 mm in diameter) and 4.5 mm/h for elongating blastocysts. 33

Determinations of mitotic indexes and DNA content demonstrated that blastocysts elongated by cellular rearrangement in the trophectoderm, trophectoderm-endoderm interactions and changes in the distribution of extracellular matrix proteins (Geisert et al., 1982b; Mattson et al., 1990; Pusateri et al., 1990). Cells of trophectoderm of pig blastocysts of different morphological shapes are distinctive in their form, size and organization of membrane-associated f-actin (Mattson et al., 1990). Blastocyst elongation appears to be important for implantation, however, the mechanisms that control elongation are still poorly understood.

Embryonic secretions

Embryos actively secrete a variety of substances which are involved in embryonic and uterine development, in establishing embryo-uterus interactions, and possibly in influencing effects among embryos in polytocous species such as pigs. The secretory activity of a mammalian embryo begins soon after fertilization. For example, mouse zygotes release platelet- activating factor which seems to play a role in metabolism of nutrients (Ryan et al., 1990a) and later on during implantation (Ryan et al., 1990b; Spinks et al., 1990). Embryos of several species secrete prostaglandins which may be involved in paracrine signals to the and maintenance of cell function (Gandolfi et al., 1992). The most important prostaglandin secreted by pig blastocysts is 34 prostaglandin E2. Pig blastocysts also secrete prostaglandins

F (Lewis and Waterman, 1983; Stone et al., 1986). In horses, prostaglandin E2 secreted by the embryo signals initiation of transport of the embryo through the oviduct (Weber et al., 1991). Perry et al. (1973) first observed that pig blastocysts had the capacity to synthesize and secrete estradiol. Amounts of estradiol secreted by pig blastocysts are higher than those of all species examined (Gadsby et al., 1980). Horse and camel blastocysts also secrete significant amounts of estradiol (Heap et al., 1982; Skidmore et al., 1994). These species, like the pig, have diffuse placentae. Aromatase activity and estradiol secretion or content increase as pig spherical blastocysts grow from three to ten mm in diameter and then elongate to the tubular stage (Gadsby et al., 1980; Geisert et al., 1982a; Mondschein et al., 1985; Pope, 1988; van der

Meulen et al., 1989; Pusateri et al., 1990). Secretion of estradiol by filamentous blastocysts decreases relative to amounts secreted by tubular blastocysts (Geisert et al.,

1982a; Fisher et al., 1985) apparently because of decrease in expression of l7a-hydroxylase cytochrome P450 (Conley et al., 1992). Consequently, low amounts of estradiol are present by d 13 to 14 in uterine flushings, which increase again during the following days (Stone and Seamark, 1985). Pig embryonic estrogen induces and(or) modulates the secretion and synthesis of prostaglandins E and F, calcium, 35

and proteins such as insulin-like growth factor-I, uteroferrin and retinol-binding protein by the endometrium (Geisert et

al., 1982a; Simmen et al., 1990; Simmen et al., 1991; Trout et al., 1992). Estradiol plays a modulatory role on secretion of uteroferrin, IGF-I and retinol-binding protein since progesterone is the main inducer of these proteins (Adams et al., 1981; Clawitter et al., 1990). Furthermore, it is known from studies in rats, that estradiol has potent actions on cell proliferation and induction of epidermal growth factor receptor and c-fos oncogene mRNA in the uterus (Kirkland et al., 1979; Lingham et al., 1988; Loose-Mitchell et al., 1988). These multiple actions of estradiol support the notion that estradiol secreted by blastocysts may be the signal for stimulation of blastocyst migration and spacing, maternal recognition of pregnancy, implantation and changes in blood flow in the uterus (Ford et al., 1982; Pope et alw 1982a;

Pope et al., 1986b).

Pig blastocysts secrete other steroids such as pregnenolone, progesterone, androstenedione, testosterone, estrone and catechol estrogen (Mondschein et al., 1985; Stone et al., 1986). Progesterone from blastocysts may cooperate with progesterone from corpora lutea to stimulate uterine secretions, perhaps in a more localized fashion (Stone et al., 1986). The function of catechol estrogen could be mainly related to its greater effectiveness in stimulating prostaglandin synthesis than phenolic estrogen (Mondschein et 36 al., 1985). Spherical and elongating blastocysts secrete different polypeptides before and during elongation (Godkin et al., 1982), among them retinol-binding protein (Harney et al., 1990), IGF-I (Letcher et al., 1989) and interferons (Cross and Roberts, 1989; LaBornnardiere et al., 1991). The first signals that extend the life of the corpora lutea (maternal recognition of pregnancy) appear at approximately d 11-12 of pregnancy in pigs (Dhindsa and Dziuk, 1968; Geisert et al. , 1990). It is thought that estradiol, interacting with prolactin and perhaps conceptus proteins (Geisert et al., 1990), protects the corpora lutea by redirecting the flow of prostaglandin F2a from going out of the uterus and reaching the corpora lutea, to the uterine lumen (Bazer and Thatcher, 1977). Maintenance of the corpora lutea apparently requires estrogen stimulation on d 11 and a prolonged stimulation after d 14 (Geisert et al., 1987), which is consistent with the patterns of blastocyst estradiol secretion. This mechanism of action of estradiol on function appears to have more support than those based on stimulation of progesterone secretion (Ball and Day, 1982;

Garverick et al., 1982; Conley et al., 1989). Prostaglandin E has also been implicated in luteal maintenance in the pig (Bazer et al., 1984; Kraeling et al., 1985). Prostaglandin E and F may also participate in the establishment of pregnancy through their actions on embryo spacing, migration and attachment, vascular permeability and blood flow (Davis and 37

Blair, 1993). The effects of estradiol on cell proliferation and secretion of growth factors suggest a possible role of embryonic estrogen in stimulation of embryonic elongation. Receptors for epidermal growth factor, transforming growth factor-a, and IGF-I have been found in pig blastocysts during elongation (Corps et al., 1990; Fisher et al., 1994). However, receptors for IGF-I were not present before elongation (Chastant et al., 1994). The concept that a uterine secretory product(s) or some other uterine actions are needed for blastocyst elongation is supported by the fact that elongation does not occur in vitro even though concepti continue secreting estradiol. Determination of the effects of estradiol on elongation is somewhat difficult because both processes occur simultaneously. For example, a reduction of 57% of estradiol secretion by pig blastocysts in vivo, using aromatase inhibitors, did not block blastocyst elongation and maintenance of pregnancy (O'Neill et al., 1991). IGF-I, which is secreted by the uterus during the estrous cycle and in higher amounts during the preimplatational stage

(Simmen et al., 1992; Ko et al., 1994), might function in regulating endometrial remodeling (Simmen et al., 1992) and blastocyst expression of aromatase enzymes (Hofig et al., 1990; Ko et al. , 1994) and growth. Retinol might also be involved in remodeling during blastocyst elongation. Uterine and embryonic retinol-binding proteins participate as 38 transporter of retinol in the uterine lumen. Concentrations of retinol in uterine luminal fluid increase several fold during blastocyst elongation (Trout et al., 1992). Retinol-binding proteins secreted by elongating blastocysts perhaps protect blastocysts from the toxic effects of high amounts of free retinol (Trout et al., 1992; Roberts et al., 1993). Although no functions have been proposed for uteroferrin during the preimplantation period, its induction by estradiol suggests some role that warrants further investigation.

Embryonic Mortality Embryonic mortality includes the loss of embryos beginning at fertilization and continuing until implantation is completed (Jainudeen and Hafez, 1987). In general, 25 to 40% of embryos are lost during the first month of pregnancy in domestic species (Hanly, 1961; Pope and First, 1985; Jainudeen and Hafez, 1987). Pope (1994) calculated an average of 27.1% conceptus mortality in gilts (37 studies) and 29% in sows (2 studies) by d 25 to 30 of pregnancy. Similar values of mortality were more recently found in gilts in other experiments (23.5%, Lambert et al., 1991; 14.9%, Dyck, 1991;

28.2%, Ashworth et al., 1992; 23%, Cassar et al., 1994a). Although it is generally agreed that most conceptuses die during the first month of pregnancy, critical times for conceptus survival within this period have not been clearly identified. 39 Estimations of embryonic mortality in gilts for d 10 to 18 of pregnancy, a period coincident with implantation, were high and extremely variable (28%, Spies et al., 1959; 28.4%, Perry and Rowlands, 1962; 52%, Scofield et al. , 1974; 17%, Anderson, 1978). Results of recent experiments (see below) suggest that most embryos not accounted for by d 18 in gilts may die before the time of blastocyst elongation (d 12). Perry and Rowlands (1962) found embryonic degeneration beginning at the early blastocyst stage (d 6), similar to the findings of Menino et al. (1989) using blastocysts of first-estrous gilts developed in vitro. Bazer et al., (1988) estimated an overall embryonic mortality of 27% by d 8 to 12 of gestation. Lambert et al.

(1991) evaluated conceptus mortality in first estrous gilts and found (in pregnant gilts only) 5.5, 24.5, 23.5 and 26.7% on d 3, 10, 30 and end of gestation, respectively. Similarly,

Cassar et al., (1994a) determined 23% embryonic loss by d 10 in second estrous gilts, which basically remained the same by d 25 of gestation. Similar mortality by d 10 was found in a subsequent experiment from that laboratory (20%, Cassar et al., 1994b). Embryonic survival in multiestrous (>3 periods of estrus) gilts by d 11 to 15 has been consistently higher than those found in first or second estrous gilts just described (97%, Archibong et al., 1987; 92%, Cardenas and Pope, 1993; 88%,

Cardenas and Pope, 1994) . Together, these results suggest that in European breeds of pigs, critical times of embryonic 40 mortality may differ depending on reproductive age. It seems that the period of blastocyst formation and expansion may be critical for embryonic survival in first and second estrous gilts, whereas the period from blastocyst elongation to the end of implantation may be critical in multiestrous gilts.

Differences in embryonic survival, along with lower number of ovulations of first-estrous gilts (Archibong et al., 1987), probably are the main factors responsible for the smaller litter sizes found in pubertal vs third-estrous gilts

(MacPherson et al., 1977; Young and King, 1981). Important periods of embryonic mortality in sows are unknown. The possibilities of applying what is known for multiestrous gilts to sows may not be possible since multiestrous gilts do not go through the physiological implications of pregnancy and lactation. Although causes of embryonic mortality in pigs are multiple, some of them appear to be more important than others and some of these causes have been more frequently examined in the last 20 years than others. Appropriate uterine space is important for fetal survival and is not considered a problem before d 25 (Dziuk, 1968; Wu et al., 1989). Protection of the embryo by the zona pellucida is important for embryonic survival in the oviduct but not in the uterus (Broermann et al., 1990). It has been noticed, in several experiments, that the majority of embryonic losses occurred in a minority of gilts (Perry and Rowlands, 1962; Dyck, 1991; Lambert et al., 41

1991). Therefore, the estimated means of embryonic mortality in those studies, were disproportionaly influenced by the high values of embryonic mortality observed in few gilts. Therefore, inclusion of these gilts in the estimations of embryonic mortality becomes questionable, particularly of those having no embryos at the time of evaluation. When, for example, no embryos are found on d 11 after estrus, it is not possible to know whether embryos died (embryonic mortality) or never formed (fertilization failure). On the other hand, identification and examination of females with low embryonic survival could provide additional insights into the causes of embryonic mortality. Oocytes are not equal regarding their stage of meiosis when ovulation begins (Xie et al., 1990b; Koenig and Stormshak, 1993). Since oocytes and sperm are the cells that become embryos, it is logical to assume that deficiencies in these cells might be manifested later during embryonic development. Although studies have concentrated on oocytes, this does not mean that sperm do not represent a potential source of factors detrimental to embryonic survival. For example, it has been shown that paternal genes are required for proper trophoblast development in mice (Surani et al.,

1987). Follicles greatly influence oocyte development. This influence begins as early as the preantral stage of follicular development and changes markedly during follicular and oocyte 42 growth (Colonna et a l ., 1989; Cecconi et al., 1991; Lazzari et al., 1994). Moreover, pig follicular fluid, granulosa cells or follicular shells promoted maturation, fertilization, and development of pig oocytes in vitro (Mattioli et al., 1988; Yoshida et al., 1992; Ding and Foxcroft, 1993, 1994). Therefore, histological and endocrinological variability observed among ovulatory follicles (Hunter et al., 1976.; Grant et al., 1989; Hunter et al., 1989; Wiesak et al., 1990; Xie et al., 1990c; Biggs et al., 1993) may result in variability among oocytes. It becomes conceivable that healthy, well developed follicles, would give rise to well developed (good quality) oocytes. Quality of oocytes has been implicated in lower embryonic survival rates found in first-estrus versus third-estrus gilts as first-estrous gilts ovulated greater proportions (33.1 vs

24.1%, respectively) of immature ova than third-estrous gilts

(Koenig and Stormshak, 1993). These findings might be associated with low recovery rates found among embryos from first-estrous gilts when transferred to first-or third-estrous recipients (Archibong et al., 1992). Bazer (1994) speculated that such an incidence of immature oocytes plus chromosomal abnormalities found in blastocysts (10% McFeely, 1967) could account for almost all embryonic mortality found in gilts up to d 30 of pregnancy. Although it is unlikely that embryonic mortality is caused by a single factor, more research is needed to assess the importance of oocyte immaturity at 43

ovulation on embryonic mortality. Synchrony (appropriate relationship) between the uterus and the embryo is required for normal embryo development in all domestic species (Ashworth, 1992) . In pigs, complete uterine-embryonic synchrony is more difficult to establish than in monotocous species because of the presence of multiple embryos that usually differ in their growth potential. Morphological diversity among pigembryos can be demonstrated almost at all stages of development, however the time when embryos vary the most is during elongation on d 12. This extreme variability has been associated with subsequent embryonic mortality based on the findings that most blastocysts begin the process of elongation and estradiol secretion sooner than a minority of less developed blastocysts. Estradiol induces uterine advancement which is thought to be favorable for the elongating blastocysts but asynchronous and detrimental for the less developed blastocysts (Pope et al., 1982b; Pope and First, 1985; Pope et al., 1986a; Pope, 1988; Pope et al., 1990). Advancement of uterine secretions has been demonstrated in nonmated gilts after the administration of exogenous estradiol at the estimated time of blastocyst elongation (Geisert et al., 1982c) or before (d 9 and 10, Morgan et al., 1987). Uterine secretions induced by exogenous estradiol in these experiments were similar to those found in the uterine lumen during blastocysts elongation. Furthermore, uterine secretions 44 indicative of more advanced development were found in containing more developed blastocysts than in those containing less developed blastocysts (Xie et al.,1990a). However, advancement of uterine secretions by administration of estradiol 2 to 3 d before blastocyst elongation caused blastocyst death (Morgan et al., 1987; Gries et al., 1989) and produced drastic changes in the uterine glycocalyx (Blair et al., 1991) but did not alter the luminal epithelium (Blair et al., 1993). The possibility that less-developed blastocysts might be lost because of inherent abnormal development and not due to uterine asynchrony has been examined using embryo transfer techniques. When less developed blastocysts from d-7 donors were transferred to d-6 recipients, the resulting survival rates to d 12 of pregnancy were not different to that of the more developed blastocysts of those d-7 donors transferred to d-7 recipients (Wilde et al., 1988). These results suggested that d-7 littermate blastocysts did not differ in their survival capacity up to d 12 provided that development occurred in synchronous uteri. Mortality of less developed blastocysts after d 12 and whether these blastocysts die because of failure to elongate, implant, or other factors, needs to be examined. Recently, Roberts et al. (1993) postulated that retinol, which increases dramatically in uterine luminal fluid upon stimulation by blastocyst estradiol, may become toxic for nonelongating blastocysts 45 resulting in their demise. The possible implications of the relationship between blastocyst diversity and survival has drawn attention to the factors that induce morphological diversity of swine blastocysts. The pattern of ovulation might contribute to blastocyst diversity. The majority of oocytes in pigs are ovulated at the beginning while the remainder are ovulated later (Pope et al., 1988). The importance of this pattern as related to blastocyst diversity was demonstrated in a series of experiments. Electrocautery of 2 to 4 nonovulated follicles of gilts examined during the process of ovulation decreased morphological diversity among d-11 blastocysts (Pope et al., 1988). Furthermore, oocytes that were obtained during ovulation from nonovulated follicles, stained and then returned to the oviduct of the same gilt, became the smaller embryos on d 4, and the smaller embryos on d 4 became the smaller on d 12 of gestation (Xie et al., 1990a). The pattern of ovulation contributes to blastocyst diversity because it allows for fertilization to occur at different times over a period similar to the duration of ovulation (2 to 9 h, Hunter, 1972; Signoret et al., 1972; Kaufman and Holtz, 1982; Pope et al., 1988; Brussow et al.,

1990; Soede et al., 1992; Soede and Kemp, 1993). Because oocyte activation begins at fertilization, it is expected that differences in time of fertilization would generate differences in the stage of embryonic development at a given 46 time of gestation. Results of recent experiments, which examined the duration of ovulation by using ultrasound techniques in sows, showed that durations of ovulation of 2.3 to 4.6 h were not related to diversity of blastocysts by d 7. (Soede et al., 1992; Soede and Kemp, 1993). However, monitoring of many follicles, such as those present in sows, using ultrasound techniques may not be accurate and would need verification, which was not performed in these studies. Moreover, d 7 is not a time when embryonic diversity is greatest in pigs. Another factor that may contribute to morphological diversity of blastocysts is an inherent difference among ovulated oocytes. In pigs, a majority of oocytes collected at different times during the interval from estrus to ovulation were more developed than the remaining minority. This distribution was similar to the distribution of zygotic development (Xie et al., 1990b). Interestingly, similar endocrinological variability was also found among preovulatory follicles (Xie et al., 1990c). These results demonstrate that oocyte and follicular development may affect embryonic development not only by inducing diversity but also by affecting oocyte quality (Hunter and Wiesak, 1990; Pope, 1992) . Mice oocytes fertilized in vitro over a short period of time were already different in the time they reached the 2- cell stage (Howlett and Bolton, 1985). High embryonic survival and a tendency for low variability among embryos were observed 47 in gilts that had close synchrony between the LH surge, estrus and the peak of estradiol (Blair et al., 1994). This type of synchrony may be the result of a more homogeneous development of follicles (Foxcroft and Hunter., 1985). Prolific breeds of pigs have been used recently to study factors responsible for high embryonic survival. Chinese Meishan pigs have a higher percentage of conceptus survival than European breeds for a given number of ovulations (Wilmut et al., 1992). In an early experiment, Meishan gilts had higher embryonic survival on d 8 to 12 of gestation and less variation in blastocyst diameter on d 8, 10 and 11 than Large

White gilts (Bazer et al., 1988). The possibility that more uniformity in blastocyst size is responsible for high embryonic survival in Meishan pigs was not confirmed in subsequent experiments (Anderson et al., 1993; Ashworth et al., 1992; Wilmut et al., 1992). Alternatively, slower development and more gradual estradiol secretion exhibited by

Meishan blastocysts relative to domestic pigs may induce less dramatic changes in the uterine environment resulting in high rates of embryonic survival (Anderson et al., 1993; Youngs et al., 1993; Youngs et al., 1994). High embryonic survival of Meishan embryos, however, was not related to amounts of growth factors in uterine luminal fluid (Simmen et al., 1989) or to significant changes in follicular heterogeneity (Biggs et al.,

1993) . However, it has been suggested that the higher survival of Meishan than Large-White embryos could be due in part to 48 differences in follicular aromatase activity (Hunter et al.,

1994) . Other factors that affect conceptus survival are those related to the environment and to anatomical abnormalities in the reproductive organs that interfere with embryonic transport (Jainudeen and Hafez, 1987). Level of nutrition of the mother affects maternal metabolism, which, indirectly, could alter embryonic development (Cassar et al., 1994a). The influence of external factors may interact with physiological causes of mortality, resulting in complex and sometimes unique situations. Perhaps, total embryonic and fetal survival will never be achieved but even a moderate increase in survival would be of great benefit to the swine industry. CHAPTER III

EFFECTS OF TIME OF MATING RELATIVE TO OVULATION ON

MORPHOLOGICAL DIVERSITY OF SWINE BLASTOCYSTS

The effect of time of mating relative to ovulation on the amount of embryonic diversity was investigated in 37 mature crossbred gilts. Ovulation was induced by injecting proestrus gilts with 1000 IU of hCG. Gilts were naturally mated once at 24 to 32 (control), 41 or 43 h after hCG injection (n = 10, 9 and 18, respectively). Blastocysts were collected surgically

288 h post-hCG from gilts mated 24 to 32 h, 41 h and from 8 gilts mated at 43 h post-hCG. Collection of blastocysts was performed at 295 hours in 10 gilts mated at 43 h post-hCG.

Blastocysts within the ranges of 1.0 - 4.0, 4.5 - 7.0, 7.5 -

10.0, 10.5 - 15.0, 15.5 - 40.0 and > 40.0 mm were classified morphologically as small, medium and large spheres, ovoidal, tubular and filamentous, respectively. Blastocysts were incubated for 6 h to quantify secretion of estradiol-176

(estradiol). Morphological diversity (standard deviation of size) did not differ among blastocysts of treatment groups. Delaying the time of mating reduced (P < .07) the subsequent size and estradiol secretion of blastocysts. Waiting until 295 h after hCG to evaluate blastocysts of gilts mated at 43 h

49 50

resulted in blastocyst size and estradiol secretion similar to concepti of control gilts. Estradiol secretion by medium and large spherical blastocysts of gilts mated at 43 h post-hCG and evaluated at 288 h was lower (P < .05) than blastocysts of the same classifications of control gilts. Delaying mating, relative to ovulation, decreased size and estradiol secretion of blastocysts but did not affect the extent of their morphological diversity.

Introduction Morphological and functional diversity is a prevalent feature of embryonic development in swine and it appears to be related to survival of concepti (Pope et al., 1982b, Pope and First, 1985, Morgan et al., 1987). The pattern of ovulation contributes to diversity among swine blastocysts (Pope et al., 1988). Ovulation in pigs is skewed whereby the majority of oocytes are released from the follicles at the beginning of this process, during a short period of time, and the remainder during the following 2 to 6 hours (Pope et al., 1988). Xie et al. (1990a) demonstrated that oocytes identified as those to be ovulated at the beginning of ovulation, became the largest concepti of the litter by days 4 and 12 of gestation, whereas oocytes from late ovulating follicles became the smallest embryos. Injection of hCG during late proestrus does not appear to affect the pattern of ovulation (Pope et al., 1988).

Ovulation induced by hCG begins by 40 to 42 h after 51

administration (Pope et al., 1988, Dziuk and Baker, 1962, Hunter, 1967) and is completed by 46 to 48 h (Pope et al., 1988) . Mating gilts early during estrus allows time for sperm capacitation to occur before ovulation begins. Thus, oocytes

are fertilized soon after they are released from follicles, as the time from ovulation to fertilization of each oocyte is

relatively constant (Xie et al., 1990b). However, the conditions associated with fertilization of oocytes of gilts mated during or after ovulation are different than those of gilts mated early in estrus. Oocytes of gilts mated after

ovulation has begun reach the oviduct before capacitation of sperm is completed and some aging of the oocytes can occur before sperm penetration. As the influence of these conditions

on the degree of morphological diversity of blastocysts is unknown, the objective of this experiment was to examine the diversity among blastocysts in gilts mated after ovulation had begun.

Materials and Methods

Eighty-three crossbred cyclic gilts (120 to 150 kg) were given 1000 IU of hCG during proestrus to induce ovulation

(Pope et al., 1988, Dziuk and Baker, 1962, Hunter, 1967). Subsequently, gilts were monitored every 8 h in the presence of intact boars for signs of behavioral estrus. Proestrus was determined by observation of swelling and redness of the 52 , and changes in behavior such as restlessness and preference to stay near the boars. Thirty-seven gilts (44.6% of originally injected gilts) exhibited estrus between 16 and 32 h after hCG treatment and were utilized in the experiment.

The remaining gilts exhibited estrus either before 16 or after 32 h post-hCG. These gilts were not utilized in the experiment because of the possibility of occurrence of the preovulatory LH surge before hCG injection or induction of ovulation of

follicles not fully developed. Gilts were allowed to mate at 24 to 32 h post-hCG, corresponding to a before ovulation group (control, n = 10), at 41 h, early during ovulation (n = 9) or at 43 h, later during ovulation (n = 18) . Bilateral ovariohysterectomies were performed 288 h after hCG administration in gilts mated at 24 to 32 h, 41 h and in 8 gilts mated at 43 h post-hCG. The remaining 10 gilts mated at 43 h were examined at 295 h post- hCG. Gilts were evaluated at 295 h to determine developmental changes of blastocysts after they were given seven additional hours of development (295 vs 288) . This 7 h period was the estimated delay of fertilization imposed on gilts mated at 43 h post-hCG. Seven hours was established based on observations that ovulation begins at 40 h post-hCG and a period of 4 h for sperm capacitation corresponding to the time to fertilize 50% of the oocytes (Hunter, 1972, Hunter and Hall, 1974, 3 plus 4 h for gilts mated at 43 h post-hCG, respectively). 53

Blastocysts were recovered by flushing the uterus with sterile

saline. Maximum diameter or length of each embryo was measured to the closest .5 mm with the aid of a dissecting microscope. Blastocysts were incubated for 6 h in Modified Tyrodes medium for the purpose of determining estradiol-17B (estradiol) secretion. Blastocysts were washed twice in the culture media before incubation and then placed into 5 ml of fresh media. Modified Tyrodes medium was supplemented with Hepes (10 mM)

and its concentration of NaHC03 was decreased from 25 mM to 2 mM (Hagen et al., 1991). Concentrations of estradiol in the

culture media at time 0 and at the end of incubation were determined by using RIA procedures (Nephew et al., 1989a). Mean concentrations of estradiol at time 0 were below the sensitivity (5 pg) of the assay. Intra- and inter-assay

coefficients of variation were 7.6 and 9.8%, respectively.

Data were analyzed by analysis of variance using the GLM procedure of SAS (SAS, 1988). Least squares means were

compared using the PDIFF option of SAS. Heterogenous data regarding embryonic size and estradiol secretion were transformed to natural logarithms and then analyzed using models for repeated observations (Steel and Torrie, 1980).

Antilogarithms of least squares means are presented. Embryos were classified according to their size in small, medium or large spheres, ovoidal, tubular or filamentous if their sizes were within the following ranges: 1.0 to 4.0, 4.5 to 7.0, 7.5 to 10.0, 10.5 to 15.0, 15.5 to 40.0 or greater than 40.0 mm, 54

respectively. A test of independence for distribution of

embryos among these classifications was performed using Chi- square (Snedecor and Cochran, 1990). Morphological diversity of blastocysts was compared by analyzing within-litter standard deviation of size. Because filamentous blastocysts became entangled during uterine flushing and it was not possible to separate them from each other in time for

incubation, estradiol data for this classification was not included in the analyses. Blastocysts of one gilt mated at 43

h post-hCG and evaluated at 295 h were all filamentous and it was not possible to measure their size accurately. Therefore this gilt is not represented in part A of Figure 1 but is included in part B.

Results

Embryonic size and diversity

Blastocyst recovery rates were not different (P > .10) among groups (Table 1). The number (mean ± SEM) of blastocysts

recovered per gilt were 14.3 ± 0.7, 13.1 ± 0.7, 13.8 ± 1.1 and 13.5 ± 1.3 for gilts mated between 24 and 32, at 41, 43

(evaluated at 288) and 43 (evaluated at 295) h post-hCG, respectively. Pregnancy rates did not differ (P > .10) among groups and were 90, 100, 100 and 80% for gilts mated between 24 and 32, at 41, 43 (evaluated at 288) and 43 (evaluated at 295) h post-hCG, respectively. 55

Time of mating relative to ovulation influenced (P < .07) blastocyst size. Blastocysts of gilts mated 43 h post-hCG were

smaller (P < .05) compared to those mated before ovulation. Blastocysts of gilts mated 41 h post-hCG were numerically

intermediate to those of gilts mated before ovulation or 43 h

post-hCG and evaluated at 288 h (Table 1) . Mean size of blastocysts of gilts mated at 43 h post-hCG and evaluated at 295 h was greater (P < .05) than those evaluated at 288 h and was not different from blastocysts of control gilts (Table 1). A certain amount of morphological diversity of blastocysts, as

Table 1. Means of characteristics of blastocysts of gilts mated at different times after an injection of hCG to induce ovulation.

Time of Recovery Size Standard Estradiol mating (%)d (mm)e deviation secretion after hCG of size per (h)a (mm)d blastocyst (ng)e 24-32 (288) 93.1 ± 2.8 9. 9b 6.5 ± 2.2 30. 0b 41 (288) 89.0 ± 2.8 7. 2bc 2.6 ± 0.9 23.8^ 43 (288) 97.3 ± 3.0 6. 3C 1.5 ± 0.5 10. 2C 43 (295) 88.4 ± 3.0 10.0b 4.7 ± 1.8 44.3b

8Time (hours) of embryonic evaluation post-hCG is in parenthesis. “Means without a common superscript within each column differ (P < .05). “Values are least squares means ± SEM. •Values are antilogarithms of least squares means.

determined by the range of size and by the number of morphological classifications, was observed in all mating

groups (Figure 1 A and B). The distribution of blastocysts 56

among classifications was influenced (P < .001) by time of mating such that more spherical blastocysts were recovered with increasing delay of mating (Figure 1 B) . The degree of morphological diversity of blastocysts expressed as within-

litter standard deviation of size was not different among groups (Table 1).

Estradiol Secretion Estradiol secretion per blastocyst was influenced (P < .07) by time of mating relative to ovulation (Table 1) .

Greater (P < .05) amounts of estradiol were secreted by blastocysts of gilts mated before ovulation and those of gilts mated 43 h post-hCG and evaluated at 295 h, compared to blastocysts of gilts mated 43 h post hCG and evaluated at 288 h. Mean estradiol secretion by blastocysts of gilts mated 41 h post-hCG did not differ (P > .10) from the other treatment groups (Table 1).

Overall, tubular blastocysts secreted more (P < .01) estradiol than ovoidal, ovoidal more than large spheres and large spheres more than medium and small spherical blastocysts

(148 ± 34.3, 93.1 ± 14.5, 45.1 ± 4.7, 23.7 ± 2.3 and 1.6 ± .5 ng, mean ± SEM, respectively) . Mean size of blastocysts within the spherical morphologies was not influenced by the main effect of treatment (P > .38) or by the interaction of treatment and morphology (small, medium and large spheres; P

> .65, Figure 2 A). However, within the medium and large spherical classifications, blastocysts collected at 288 h from 57

i. Bo­ at at « «' at N 30- >»in o 20 - «-•o a t 4- a io- ■■ 1 111 +" m + w f *tt+v+ •ft-f

24-32 41 43 (288) 43 (285) Time of mating after hCQ (h)

B Morphological classifications Spherical Small Medium Large Ovoidal Tubular Filamentous I I (=□ V///.X C*X*M n-129 n -110 n-110 n-108 100

CO o>* 75 o 0) cd 50

c © o 25 © Q.

24-32 41 43 (288) 43 (295) Time of mating after hCG (h)

Figure 1. Range and mean size of blastocysts per litter (A) and distribution of blastocysts among morphological classifications (B) in gilts mated at different times after an injection of hCG given to induce ovulation and evaluated 288 h later. Blastocysts of gilts mated 43 h post-hCG were evaluated at either 288 or 295 h post-hCG. 58

Time of mating after hCQ (h)

24-32 n rrn 41 <2m> v //A 4 3 (2 s s> 43 (285)

10 SE 8 E 8 E 7 « N 6 'co *— 5 CO o>» 4 ♦—o «10 3 m 2 1 0 Small spheres Medium spheres Large spheres Morphological classifications

B Time of mating after hCG (h)

24-32 I t-H-tl 41 V///A 43 (288) I I 43 (295)

a> c (0 o>» «—o CO as

as a. •o as 10 LU

Small spheres Medium spheres Large spheres Morphological classifications

Figure 2. Mean size (A) and estradiol secretion in vitro (B) by spherical blastocysts of gilts mated at different times after an injection of hCG given to induce ovulation and evaluated 288 h later. Blastocysts of gilts mated 43 h post- hCG were evaluated at either 288 or 295 h post-hCG. Bars in A represent means and those in B represent the antilogarithm of least squares means. Bars without a common letter character differ (P < .05). 59

gilts mated at 43 h post-hCG secreted lower (P < .05) amounts of estradiol than either those of gilts mated at 43 h and evaluated at 295 h or those of control gilts (Figure 2 B).

Discussion In this experiment we utilized hCG when given to proestrus gilts to precisely time ovulation. Previous observations performed in similar gilts indicate that ovulation begins at 39 to 41 h post-hCG injection (Pope et al., 1988). By using this methodology it was determined that the size of blastocysts was decreased by mating gilts after ovulation had

begun. This finding, together with the observation that blastocysts of gilts mated 43 h post-hCG and evaluated at 295 h were of similar size to blastocysts of control gilts, suggest that time of fertilization was the most important

factor influencing the size of blastocysts. Porcine blastocysts enter a phase of rapid growth by days

11 and 12 of gestation and demonstrate appreciable morphological and functional diversity at this time (Anderson, 1978, Geisert et al., 1982b, Pope 1988). The time for transition of littermate blastocysts from all spherical to those ranging from spheres to filamentous was less than 7 h in this experiment. This period of transition is consistent with the 6 h reported by Geisert et al. (1982b). The possibility that mating during ovulation could have produced a decrease in the total time of fertilization is speculative at best. Under 60 delayed mating conditions, a pool of unfertilized oocytes could accumulate in each oviduct before fertilization begins.

Once capacitated sperm become available, fertilization of this pool of oocytes could perhaps be more synchronous. Although this experiment was not designed to document the time or duration of fertilization, Hunter (1972) observed that in gilts mated at the time of ovulation, fertilization of oocytes still lasted several hours, beginning approximately three hours after mating and reaching a maximum rate by 5 h after mating.

Results of estradiol secretion in vitro support previous findings that secretion of this hormone by elongating blastocysts increases as size of the blastocyst increases

(Fisher et al., 1985, Pope, 1988, Pusateri et al., 1990). Blastocysts of gilts mated 43 h post-hCG and evaluated at 288 h secreted less estradiol than those of control gilts. When blastocysts of these delay-mated gilts were allowed to advance seven more hours, they developed to equivalent morphological stages and secreted amounts of estradiol comparable to those obtained from embryos of gilts mated before ovulation. This demonstrates that blastocysts of delay-mated gilts maintained their capacity to secrete high levels of estradiol and to eventually elongate. The decreases in size and estradiol secretion observed in this study apparently were not due to a decrease in post­ ovulatory oocyte viability. The estimated delay in fertilization did not exceed the viable life of the porcine oocyte, which is approximately 8 h (Hunter 1972). Preliminary studies indicated that allowing gilts to mate at 45 h post-hCG reduced recovery rates of blastocysts. Furthermore, pregnancy rates and embryonic recovery rates were high in all groups which is an indication of good oocyte viability. Finally, the observations that blastocysts had equivalent sizes and amounts of estradiol secretion after waiting seven additional hours before collection compared to controls, further reduced the concerns about oocyte viability in this experiment. Full expression of the steroidogenic capacity of blastocysts appears to be more complicated than a simple interrelationship with stage of morphological development. Blastocysts of delay-mated gilts reached the size of medium or large spheres before estrogen secretion was fully established for blastocysts of that size. Perhaps delaying fertilization induced blastocyst growth before steroidogenesis. More probable is that the uterine environment of delay-mated gilts resulted in slightly advancing growth of embryos before, or independent of, an induction of embryonic synthesis of estrogen. Considerable evidence exists to support the concept of a uterine ability to advance conceptus development (Wilmut et al., 1985, Wilmut et al., 1988) however, this is the first report in swine of advancement of morphology before other embryonic functions. The importance of estrogen in blastocyst migration (Pope et al., 1986b), induction of uterine secretions (Geisert et al., 1982a, Morgan et al., 1987) and maternal recognition of pregnancy (Bazer and Thatcher, 1977, Geisert et al., 1990), might have been conservatively preserved among species by being controlled and preset at the time of fertilization. In conclusion, delaying mating in relation to ovulation did not affect the amount of embryonic diversity but did decrease mean embryonic size and estrogen secretion in vitro.

However, if blastocysts were compared at equivalent times after fertilization, then they did not differ in size and expressed similar steroidogenic capacities. CHAPTER IV

INHIBITORY AND STIMULATORY EFFECTS OF EXOGENOUS ESTRADIOL-

170 ON BLASTOCYST DEVELOPMENT DURING THE PERIOD OF

ELONGATION IN SWINE

Objectives were to examine the effects of a single dose (4 mg) of estradiol-170 (E2) on blastocyst elongation. Proestrus gilts (n = 32) were induced to ovulate with hCG and were mated before ovulation (24 to 32 h post-hCG, normal mating) or after ovulation had begun (43 h post-hCG, delayed mating). This difference in time of mating has been demonstrated to result in approximately a 7 h difference in time of elongation. Normal- and delay-mated gilts were ovariohysterectomized at

278 h post-hCG or injected (i.m.) with E2 or vehicle (corn oil) at 278 h and then ovariohysterectomized at 290 h post-hCG

(5 or 6 per group). Blastocyst size was measured and concentrations of retinol, E2, uteroferrin, insulin-like growth factor-I (IGF-I), and protein in uterine flushings were quantified. Blastocyst size and components of uterine flushings did not differ (P > .10) between normal- and delay- mated gilts at 278 h post-hCG. Normal-mated gilts receiving E2 had smaller (P < .001) blastocysts than those receiving vehicle. Similarly, normal-mated gilts receiving E2 had

63 64 smaller (P < .01) total amounts or amounts per mg of protein of retinol in uterine flushings than those receiving vehicle. Conversely, delay-mated gilts treated with E2 had larger (P < .05) blastocysts and greater (P < .10) amounts of uteroferrin at 290 h post-hCG than those treated with vehicle. As expected, blastocysts of normal-mated gilts at 290 h were larger (P < .001, small spheres to filamentous) than those of delay-mated gilts (small spheres to ovoidal). Blastocyst size (P < .05) and total amounts of components of uterine flushings (P < .10) increased from 278 h to 290 h post-hCG in normal or delay-mated gilts not treated with E2 (except for IGF-I in delay-mated gilts, P > 0.10). Among gilts not treated with E2, blastocyst size was positively correlated (P < .10) with total amounts of all components of uterine flushings in normal-mated gilts and with total amounts of E2, retinol, uteroferrin and protein in delay-mated gilts. Results indicated that a single dose of E2 given before elongation can reduce or stimulate blastocyst development depending on how close blastocysts were to onset of elongation at the time of E2 treatment. Results also suggested important associations of retinol and uteroferrin in the morphological changes that occur just before and during elongation.

Introduction Swine blastocysts undergo elongation, a sequence of transformations from spherical (approximately 10 mm) into 65 ovoidal, tubular and filamentous shapes, on d 12 of pregnancy (Perry and Rowlands, 1962; Anderson, 1978) . Elongation results primarily from cellular rearrangement in the trophectoderm (Geisert et al., 1982b; Pusateri et al., 1990; Mattson et al., 1990). Mattson et al. (1990) suggested that cellular repositioning occurring during blastocyst elongation may be produced by forces generated by the actin cytoskeleton. Estradiol secreted by swine blastocysts increases gradually as they approach their maximal spherical size and then more rapidly during elongation (Geisert et al., 1982a; Pope, 1988; Xie et al., 1990a). Estradiol stimulates secretion of many endometrial products. Some of these estradiol-induced substances, such as IGF-I (Murphy et al., 1987; Simmen et al., 1990), uteroferrin (Roberts and Bazer, 1988; Simmen et al., 1991), retinol-binding proteins (Trout et al., 1992) and protease inhibitors (Fasleabas et al., 1983), have been associated with growth and development of pig conceptuses.

Therefore, it is possible that estradiol secreted by pre­ elongating (spherical) blastocysts might play a role in their subsequent elongation. However, because changes in morphology and steroidogenesis occur almost simultaneously during elongation, cause and effect relationships have been difficult to examine. For example, partial inhibition of blastocyst estradiol using aromatase inhibitors or estrogen antagonists did not block elongation in gilts (O'Neill et al., 1991). 66

Altering the time of mating to before or during ovulation results in blastocysts at different stages of development at 288 h post-hCG (used to induce ovulation) and can generate a difference in onset of elongation of approximately 7 h (C&rdenas and Pope, 1993). In the present experiment, gilts mated before or during ovulation were used to examined the effects of exogenous estradiol, when administered before elongation, on blastocyst development during the next 12 h. Timing of injection of estradiol was considered critical because transformation of blastocysts from large spheres to filamentous takes place relatively fast (less than 6 h,

Geisert et al., 1982b; Cardenas and Pope, 1993) .

Materials and Methods

Animals and experimental design Crossbred (Yorkshire x Landrace x Duroc) gilts, weighing

120 to 150 kg, were observed for display of estrus and then injected with 750 IU of hCG during late proestrus to induce ovulation as previously described (Cardenas and Pope, 1993).

Gilts injected with hCG during late proestrus begin ovulating at approximately 40 h post-hCG (Dziuk and Baker, 1962, Hunter, 1967, Pope et al., 1988). Gilts were mated to a boar at 24 to 32 h (before ovulation, normal mating) or 4 3 h (after ovulation had begun, delayed mating) post-hCG. Normal and delay-mated gilts were ovariohysterectomized at 278 h post-hCG or were treated with 4 mg of estradiol-176 (E2, Sigma 67

Chemical) or vehicle (corn oil) at 278 h and then ovariohysterectomized at 290 h post-hCG (5 or 6 per group) . It has been shown that 5 mg of estradiol valerate on d 11 of the estrous cycle induced endometrial secretions similar to those present in uterine flushings of gilts at the time of blastocyst elongation (Geisert et al., 1982c). Furthermore, a dose of 4 mg of E2 did not alter embryonic survival and pregnancy rate by d 30 (Pope et al., 1986c). A summary of the experimental design is presented in Fig. 3.

Blastocyst development Immediately after ovariohysterectomy, blastocysts and uterine luminal fluid were recovered by flushing the uterus with 50 ml of physiological saline (0.9% NaCl). Blastocysts were examined using a dissecting microscope and then classified according to size (diameter or length to the nearest millimeter) and morphology into small, medium or large spheres, ovoidal, tubular or filamentous (1 to 4, 5 to 7, 8 to

10, 11 to 15, 16 to 40 and > 40 mm, respectively) . Filamentous blastocysts that became entangled during flushing were counted based on the number of ends and were given the minimum value of size (41 mm) for that classification. Uterine flushings were snap-frozen in liquid nitrogen and then kept at -70"C until concentrations of components of uterine flushings were determined. Corpora lutea (CL) were counted and values were used to estimate the percentages of blastocyst recovery

(number of blastocysts divided by number of CL times 100). 68

M HYS y NM-278 V — i M HYS y y DM-278 — i M OIL V y NM-VE M e 2 y y NM-E2 M OIL DM-VE y y M e 2 y y d m -e 2 t t hCG ovu HYS WH- -H~ 0 24 40 43 278 290 TIME (h)

Figure 3. Experimental design and treatment schedule (NM-278 = normal-mated gilts evaluated at 278 h post-hCG, DM-278 = delay-mated gilts evaluated at 278 h, NM-VE = normal-mated gilts treated with vehicle, NM-E, = normal-mated gilts treated with estradiol-176, DM-VE = delay-mated gilts treated with vehicle, DM-E2= delay-mated gilts treated with estradiol-176, M = mating, OVU = estimated time of ovulation, E2 = estradiol- 178, HYS = hysterectomy). 69

Components of uterine luminal fluid

Estradiol-17B was extracted (Nephew et al., 1989b) from 500 jLtl of uterine flushing and then quantified by RIA procedures (Nephew et al., 1989a). All samples were included in a single assay (intra-assay CV < 10%). Concentrations of total retinol (vitamin A) was determined by spectrophotometric procedures (Trout et al., 1992). Uteroferrin was quantified by RIA procedures as described by Simmen et al. (1988). IGF-I concentrations were measured in acid-ethanol extracts following protocols described in Letcher et al. (1989) and Simmen et al.(1990). Protein concentrations were measured by the Bradford assay using bovine serum albumin as standard.

Statistical analyses

Data (untransformed or transformed to natural logarithms) for mean size of blastocysts per litter, recovery rates of blastocysts and total amounts or concentrations of components of uterine flushings were analyzed by least-squares ANOVA for a completely randomized design (six treatment groups, Fig. 1) .

Comparisons of means were performed using contrasts.

Bartlett's test was used for determining homogeneity of variance. These analyses were performed using SYSTAT

(Wilkinson, 1990). Pearson correlation coefficients were estimated using the CORR procedures of SAS (SAS, 1988). 70

Results

Blastocyst development Distribution of littermate blastocysts among morphological classifications is presented in Fig. 4. Blastocysts of normal- and delay-mated gilts at 278 h post-hCG were small or medium spheres (Fig. 2) and did not differ (P > .10) in mean diameter (Table 2) . Normal-mated gilts administered vehicle at 278 h and evaluated at 290 h post-hCG had blastocysts ranging from small spheres to tubular or filamentous, however, blastocysts of normal-mated gilts treated with E2 were primarily spherical (Fig. 4) . Consequently, mean blastocyst size at 290 h was greater (P < .01) in normal-mated gilts administered vehicle than in those treated with Ez (Table 2). Blastocysts of delay- mated gilts receiving E2 ranged from spherical to filamentous

(Fig. 4) and their mean size was greater (P < .01, Table 2) at 290 h post-hCG than those of gilts receiving vehicle which ranged from spherical to ovoidal. Blastocysts of normal-mated gilts were larger (P < .001) at 290 h (mostly filamentous) than at 278 h (small or medium spheres) post-hCG (Table 2) . Though blastocysts of delay-mated gilts were greater (P < .05) in diameter at 290 h than at 278 h post-hCG (Table 2) , most of them remained within the spherical classifications (Fig. 4). At 290 h post-hCG blastocyst of normal-mated gilts administered vehicle were greater in size (P < .01, Table 2) than those of delay-mated gilts receiving vehicle. 71

Group Small Medium Large Ovoidal Tubular Filamentous sphere sphere sphere (11-15 (16-40 (> 40 mm) (1-4 mm) (5-7 mm) (8-10 mm) mm) mm) 27tf h, normal mating • • • •

• •• • • 278 h, delayed • mating • •••

" 290 h, normal • • •• • • • • mating, • vehicle •• • • " 2 9 0 X " • ••• normal • • • • mating, ••• Ea • • ••• 290 h, • delayed • • •• mating, vehicle • • • ••• “290'h,.. • • • • delayed • •• • •• mating, • • •• E> • • ••

Figure 4. Distribution of blastocysts among morphological classifications in normal- (before ovulation, 24 to 32 h post- hCG) and delay-mated (after ovulation has begun, 43 h post- hCG) gilts at 278 h post-hCG and in normal- and delay- mated gilts treated with vehicle or estradiol-176 (E,) at 278 h and evaluated at 290 h post-hCG given to induce ovulation. Litters and individual blastocysts are represented by rows and black dots, respectively. Table 2. Means of blastocyst size and total amounts of various components of uterine flushings in normal- and delay-mated gilts treated with estradiol-176 (E2) at 278 h and evaluated at 290 h post-hCG given to induce ovulation.

Variables8 278 h post-hCG 290 h post-hCG Normal Delayed Normal mating Delayed mating

mating mating Vehicle E2 Vehicle E2 (n=5)b (n=6)c (n=5)b,d (n=5) (n=5)c,d (n=6)

Blastocyst size, mm 4 . 7ns 4.7 30.1*** 7.9 7.8* 18.6

Protein, mg 13 . 0NS 14.4 35. 6ms 29.9 24 . 1MS 28.2

E2, ng 0.5ns 0.3 6.1s 3.2 3. 5ms 5.9

Retinol, fig 1 . 0 NS 1.0 12 . 6*** 2.3 3 . 6ns 5.1

Uteroferrin, mg 0. 4ns 0.5 3 . 0NS 2.9 2.0s 4.1

IGF-I, fig 1. 8NS 1.6 3. 6hs 3.1 l . g N S 2.4 aData (except for protein) were transformed to logarithms for ANOVA because of heterogeneity of variance. Arithmetic means are presented. bNormal, 278 h vs vehicle, normal, 290 h differ (P < .01) in all variables (IGF-I, P = .09) . cDelayed mating at 278 h vs vehicle, delayed at 290 h differ (P < .05) in all variables except for IGF-I. 'Vehicle, normal mating vs vehicle, delayed mating at 290 h differ (P < .01) in blastocyst size, protein and retinol. NS = Nonsignificant (P > .10), s = P < .10, * = P < .05, ** = P < .01 and *** = P < .001, for comparisons with adjacent right column. 73

Components of uterine flushings When expressed as total amounts in uterine flushings, E2, retinol, uteroferrin, IGF-I, and protein were influenced by treatment group (P < .01; IGF-I, P = .08). At 278 h post-hCG, the various components of uterine flushings did not differ (P > .10) between normal- and delay-mated gilts (Table 2). Amounts of protein and retinol were greater (P < .01) in normal- than in delay-mated gilts treated with vehicle at 278 h and evaluated at 290 h post-hCG. Normal- and delay-mated gilts treated with vehicle did not differ (P > .10) in total amounts of E2, uteroferrin and IGF-I (Table 2) . At 290 h, normal-mated gilts treated with vehicle had greater amounts of E2 (P = .10) and retinol (P < .001) than those treated with E2 and did not differ (P > .10) in amounts of uteroferrin, IGF-I and protein. Delay-mated gilts administered E2 had greater amounts of uteroferrin (P = .10) than those receiving vehicle and did not differ (P > .10) in amounts of protein, retinol and IGF-I (Table 2) . Amounts of components of uterine flushings increased from 278 h to 290 h-post-hCG (vehicle- treated gilts only) in normal- (P < .01; IGF-I, P = .09) and delay-mated gilts (P < .05; except for IGF-I, P > .10). When components of uterine flushings were expressed as amounts per mg of protein; E2, retinol and uteroferrin were influenced (P < .05) by treatment group, whereas IGF-I was not altered (P > .10, Table 3). Amounts of E2, retinol and uteroferrin per mg of protein did not differ (P > .10) between 74 normal- and delay-mated gilts at 278 h post-hCG. Similarly, amounts of E2, retinol and uteroferrin per mg of protein did not differ (P > .10) at 290 h post-hCG between delay-mated gilts administered vehicle or E2. Amounts of retinol per mg of protein at 290 h post-hCG decreased (P < .01) in normal-mated gilts receiving E2 than in those treated with vehicle. Amounts of E2, retinol and uteroferrin per mg of protein increased (P < .06) from 278 h to 290 h (vehicle-treated gilts only) in normal- and delay-mated gilts (Table 3). Normal-mated gilts treated with vehicle had greater (P = .06) amounts of retinol per mg of protein at 290 h post-hCG than similarly treated delay-mated gilts (Table 3). Total amounts of components of uterine flushings in normal-mated gilts not treated with E2 (278 and 290 h, vehicle pooled) were positively correlated (P < .05, IGF-I, P = .07) with mean blastocyst size per litter. Components of uterine flushings were positively correlated to one another (P < .10) with the exception of IGF-I with uteroferrin or total protein (Table 4). In delay-mated gilts not treated with E2 (278 and

290 h, vehicle pooled), mean blastocyst size was positively correlated (P < .10) with retinol, E2, uteroferrin and protein but it was not correlated (P > .10) with IGF-I. Similarly, IGF-I was not correlated (P > .10) with retinol, E2 and uteroferrin but it was positively correlated (P < .10) with protein. All the other components of uterine flushings were positively correlated (P < .10) to one another with the Table 3. Concentrations of components of uterine flushings per mg of protein in normal- and delay-mated gilts treated with estradiol-176 (E2) at 278 h and evaluated at 290 h post-hCG given to induce ovulation.

Variables8 278 h post-hCG 290 h post-hCG Normal Delayed Normal mating Delayed mating

mating mating Vehicle E 2 Vehicle E 2 (n=5)b (n=6)c (n=5)b,d (n=5) (n=5)c,d (n=6)

e 2, pg 46. 9ns 16.8 173. 8ns 107.4 142. 5ns 221.9 Retinol, ng 81.0ns 70.0 346.1** 79.8 143. 6ns 183.4

Uteroferrin, fig 29. 7ns 36.6 83 . 7ns 99.1 86. 1NS 147.9 IGF-I, ng 141.1 107.6 100.8 104.8 84.5 88.1 aData (except for E2) , were transformed to logarithms for ANOVA because of heterogeneity of variance. Arithmetic means are presented. IGF-I was not influenced (P > .10) by treatment group. formal mating at 278 h vs vehicle, normal mating at 290 h differ in retinol (P < .001), E2 and uteroferrin (P < .05). cDelayed mating at 278 h vs vehicle, delayed mating at 290 h differ in E2, retinol (P < .05) and uteroferrin (P = .06). "Vehicle, normal mating vs vehicle, delayed mating at 290 h differ (P = .06) in retinol. NS = Nonsignificant (P > .10), = P < .01 for comparisons with adjacent right column. 76

Table 4. Correlation coefficients among blastocyst size and total amounts of various components of uterine flushings around the time of blastocyst elongation in normal-mated (before ovulation) gilts8.

Blastocyst Retinol E2b Uteroferrin IGF-I size

Retinol .97 E2b . 88 h i t it . 80 _ _ *** Uteroferrin . 89 .96 .69 IGF-IC .56§ • 52§ .72 _ .*** Protein .93 . 88« « *** .84 aIncludes normal-mated gilts evaluated at 278 h and normal- mated gilts treated with vehicle at 278 h and evaluated at 290 h post-hCG. Range of mean blastocyst size: 3.5 to 37.4 mm. hEstradiol-^B. NS = Nonsignificant (P > .10), § = p < .10, * = P < .05, ** = P < .01 and ** = p < .001.

Table 5. Correlation coefficients among blastocyst size and total amounts of various components of uterine flushings around the time of blastocyst elongation in delay-mated (after ovulation had begun) gilts8.

Blastocyst Retinol E2b Uteroferrin IGF-I size _ Retinol .78 O . w

M C .79 .81** Uteroferrin .79** .80** .68*

IGF-IC . 07ns . 02ns -. 09ns . 15ns

Protein . 59§ .63* .61§ .45ns . 56§ aIncludes delay-mated gilts evaluated at 278 h and delay-mated gilts treated with vehicle at 278 h and evaluated at 290 h post-hCG. Range of mean blastocyst size: 3.7 to 9.2 mm. “Estradiol-l?!!. Ns = Nonsignificant (P > .10), § = p < .1 0 , * = P < .05, ** = P < .01 and = P < .001. 77 exception of uteroferrin with protein (Table 5).

Discussion Mean size of blastocysts and amounts of E2, retinol, uteroferrin, IGF-I and protein in uterine flushings were not different between normal- and delay-mated gilts when evaluated at 278 h post-hCG. However, important differences were found at 290 h. Normal-mated gilts treated with vehicle at 278 h had blastocysts at more advanced stages of development and their uterine flushings contained greater amounts of protein and retinol than delay-mated gilts receiving vehicle. Although blastocysts of normal- and delay-mated gilts were apparently at similar stages of development at 278 h, blastocysts of normal-mated gilts elongated sooner than those of delay-mated gilts. Therefore, E2 treatment at 278 h post-hCG was given to gilts predisposed to having blastocysts with different developmental capabilities over the next 12 h period.

Experiments from our laboratory noted that blastocyst growth and elongation are related to post-fertilization age.

Most recently, Cardenas and Pope (1993) observed litters of normal-mated gilts that were in transition from spherical to filamentous at 288 h post-hCG, while those of delay-mated gilts were spherical. When delay-mated gilts were examined at 295 h, their blastocysts were elongating. It was estimated that the difference in age post-fertilization between blastocysts from these normal- and delay-mated gilts was approximately 7 h, which appears to be similar to the 78 difference in time of elongation (C&rdenas and Pope, 1993). The smaller blastocyst size in normal-mated gilts that received E2 than those of gilts receiving vehicle occurred simultaneously with decreases in retinol and E2 and no significant changes in uteroferrin, IGF-I or protein.

Furthermore, the amounts of retinol per mg of protein also decreased in these E2-treated normal-mated gilts. The association of less developed blastocysts with less E2 in flushings suggests that the contribution of exogenous E2 to uterine E2 by 290 h was minimal as the exposure of the uterus to exogenous E2 probably lasted less than 12 h. Although E2 decreased development of blastocysts of normal-mated gilts, it is possible that they maintained their ability to elongate. This notion is supported by the findings that blastocysts transferred to gilts, which were subsequently administered 5 mg of estradiol valerate on d 11, were filamentous on d 14

(Morgan et al., 1987). Furthermore, administration of up to 8 mg of E2 on d 12 and 13 was not detrimental to pregnancy rate and embryonic survival by d 30 (Pope et al., 1986c). Most retinol in uterine luminal fluid of pigs is bound to retinol-binding proteins (Trout et al., 1992). These proteins are secreted by the endometrium under the influence of progesterone (Adams et al., 1981, Clawitter et al., 1990] and by conceptuses before elongation and during the peri- implantation period (Harney et al., 1990). Estradiol stimulates secretion of endometrial retinol-binding proteins in the presence of progesterone (Trout et al., 1992). It is important to note that, until examined further, changes in components of uterine flushings 12 h post-E2 do not necessarily reflect more immediate changes in uterine luminal fluid components due to E2 treatment. Therefore, whether the smaller blastocyst size of normal-mated gilts treated with E2 was a cause of decreased retinol or a consequence of it, is not known. However, changes in retinol 12 h post- E2 and the high positive correlation (.97) between mean blastocyst size and retinol in normal- and delay-mated gilts suggest that this compound may be particularly important for the morphological transformations just before and during the transition of blastocysts from spherical to filamentous as suggested recently (Trout et al., 1992, Harney et al., 1990, Roberts et al., 1993). Retinoids are potent mprphogens and have profound effects on organ and fetal development in part due to their actions on gene transcription and cell integrity and proliferation (reviewed by Roberts et al., 1993 and Blomhoff et al., 1990).

A single injection of E2 to ovariectomized rats produced reduction of approximately 50% of total cellular estrogen receptors within 1 h of treatment (Copland et al., 1987) probably due to accelerated receptor processing and degradation (Rosser et al., 1993). Estrogen receptors were replenished gradually beginning at five h and returned to pre­ treatment values by 15 to 18 h (Cidlowski and Muldoon, 1978). 80

If the same phenomenon occurs in swine, then the reduction in estrogen receptors would coincide with the beginning of elongation in normal-mated gilts. This may be an indication of the importance of endometrium sensitivity to blastocyst estradiol at a critical time for elongation. The detrimental effects of Ez on blastocyst development in normal-mated gilts and stimulatory effects in delay-mated gilts represents an interesting finding. Perhaps, the opposite effects of E2 on blastocyst development were related to how close blastocysts were to onset of elongation. In delay-mated gilts treated with E2, increase in blastocyst size was not accompanied by significant changes in components of uterine flushings other than increase in amounts of uteroferrin. A single injectionof estradiol valerate on d 11 of the estrous cycle increased uteroferrin within 12 h (Geisert et al., 1982c) or 24 h (Simmen et al., 1991). The increase in uteroferrin found in delay-mated gilts together with high correlations of blastocyst size with amounts of uteroferrin in normal- or delay-mated gilts nontreated with E2, indicate that this compound might be involved in development of elongating blastocyst perhaps through its acid phosphatase activity for which no specific function to date has been suggested.

Uteroferrin participates in iron metabolism and as a growth factor in hematopoiesis during later gestation in pigs (Roberts et al., 1993). It is also possible that stimulation of blastocyst development found in delay-mated gilts resulted 81 in part from rapid increase of IGF-I (i.e. within 1 h, Simmen et al., 1990) and (or) other growth factors induced by exogenous E2. Insulin-like growth factor-I was positively correlated to blastocyst size and E2 and increased in normal-mated gilts from 278 h to 290 h post-hCG when blastocysts developed from spherical to filamentous. In delay-mated gilts, blastocysts stayed spherical and mean size increased from 4.7 mm at 278 h to 7.8 mm at 290 h. Insulin-like growth factor-I was not correlated with size nor did the total amounts or amounts per mg of protein of IGF-I in uterine flushings change from 278 h to 290 h post-hCG, suggesting that IGF-I may be more important for elongation than for growth of spherical blastocysts. This is consistent with the stimulatory effects of IGF-I on aromatase cytochrome P450 activity of d 12 elongating blastocysts but not d 10 spherical blastocysts (Hofig et al., 1991) and with the positive associations between amounts of

IGF-I, cytochrome P450 protein and estradiol secretion by elongating pig blastocysts (Ko et al., 1994).

In summary, effects of a single dose of E2 on blastocyst development depended upon how close the blastocysts were to time of elongation. A single injection of E2 decreased development of blastocysts that normally begin elongation within the next 12 h but was stimulatory for development of blastocysts that normally grow to the large spherical stage during that time period. It is probably more than coincidentally related the high positive associations of retinol and uteroferrin with changes in morphology occurring during blastocysts elongation. CHAPTER V

ADMINISTRATION OF TESTOSTERONE DURING THE FOLLICULAR PHASE

INCREASED THE NUMBER OF CORPORA LUTEA IN GILTS

The effects of 0 (vehicle), 1, 10, or 100 mg of testosterone, administered on d 17 and 18 of the estrous cycle (d 0 = 1st d of estrus) , on the number of preovulatory follicles on d 19 or the number of corpora lutea (CL) and blastocysts on d 11 of the subsequent cycle were examined in 82 gilts. The mean number of preovulatory follicles increased (P < .01) in a dose-dependent manner. Likewise, gilts that received 1, 10, or 100 mg of testosterone had more (P < .05) CL than gilts treated with vehicle. The mean number of blastocysts increased (P = .06) in gilts receiving 1 mg of testosterone but decreased (P < .05) in gilts treated with 10 mg of testosterone compared with gilts receiving vehicle.

Similarly, percentage of blastocyst recovery decreased (P < .05) in gilts treated with 10 or 100 mg of testosterone relative to gilts administered 0 or 1 mg of testosterone.

Plasma concentrations of testosterone and estradiol increased

(P < .05) 2 h following administration of 1 mg of testosterone on d 17 of the estrous cycle. These results indicate that although the 10- and 100-mg dosages of testosterone were

83 84 detrimental to blastocyst survival, the 1-mg dosage increased synthesis of estradiol, the number of CL and d-11 blastocysts.

Introduction Testosterone is used as a substrate for follicular synthesis of estradiol in swine (Evans et al., 1981; Ainsworth et al., 1990). Estradiol in turn is required by follicles to remain healthy (nonatretic) and grow (Richards et al., 1976;

McNatty, 1978). Although cause and effect relationships remain unknown, low amounts of antral estradiol may result from either decreased aromatase activity, low availability of substrate (Ainsworth et al., 1990), or both. Recent evidence indicates that in swine the decrease in estradiol concentrations in atretic follicles is due to a loss of aromatase activity (Maxson et al., 1985; Westhof et al., 1991;

Tilly et al., 1992). However, a primary role of androgen availability for estradiol synthesis during the follicular phase has been suggested by Grant et al. (1989). Androgens have been implicated in inducing follicular atresia in rats (reviewed by Tsafriri and Braw, 1984), and immunization against testosterone increased the number of ovulations in sheep (Scaramuzzi, 1979). In contrast, Greenwald (1993) concluded that the superovulatory response of hamsters to small doses of hCG was due to stimulation of the synthesis of follicular androgens. To our knowledge, no evidence exists to demonstrate that testosterone treatment affects ovulation 85 rate in any species. The objectives of the present experiments were to determine whether administration of testosterone during the follicular phase of the estrous cycle alters the number of preovulatory follicles, number of corpora lutea (CL) and d-11 blastocysts in gilts.

Materials and Methods

General A homogeneous group of Landrace (1/4) x Yorkshire (1/4) x

Duroc (1/2) gilts, weighing from 140 to 160 kg, were used in this study. Estrus was detected twice daily (0830 and 2000) in the presence of intact boars. The 1st d of estrus was considered d 0 of the estrous cycle. Only gilts that previously exhibited estrous cycles of 19.0 to 21.5 d were included in the experiments.

Experiment 1

Gilts (n = 5 per group) were administered i.m. injections of 0 (vehicle) , 1, 10, or 100 mg of testosterone (Sigma Chemical, St Louis, Mo) on d 17 and 18 of the estrous cycle. The volume of vehicle used (7.2 mL of corn oil) was the minimum that allowed 100 mg of testosterone to dissolve. Gilts were ovariectomized on d 19, and the numbers of nonatretic and atretic follicles > 3 mm in diameter were determined.

Follicular diameter was measured to the nearest millimeter at the surface of the ovary by use of a caliper. Follicles were 86 classified as medium (3 to 5 mm) or large (> 6 mm). Each follicle was considered nonatretic or atretic on the basis of macroscopic inspection of its color and integrity of the follicular wall (capillary density and transparency; Carson et al., 1979; Maxson et al., 1985). Nonatretic follicles > 6 mm in diameter were considered preovulatory. Samples of nontretic- and atretic-appearing follicles (> 3 of each class) were dissected from each pair of ovaries for purposes of microscopically assessing the accuracy of the macroscopic classification of atresia. Follicles were fixed in Bouin's solution for 24 h and then embedded in Paraplast

(Oxford Labware, St Louis, Mo) using standard techniques. Five to ten sections (5 /urn thickness) obtained from the middle part of each follicle were stained using Masson's trichrome procedure (Humason, 1972) . Microscopic assessment of atresia was performed by examining one representative section of each follicle, independent of the earlier macroscopic evaluation.

Follicles with dispersed or less than four layers of granulosa cells were considered atretic (Maxson et al., 1985). In addition, a high incidence of pyknotic nuclei among granulosa cells was an indication of atresia. Histological interpretations were then compared with those of the macroscopic examination; the accuracies of macroscopic assessment of nonatretic and atretic follicles were 92.6 and

84.6%, respectively. 87

The follicular fluid content of estradiol 17-6 (estradiol) and testosterone was determined in an additional subsample of large follicles to compare with the macroscopic classification of atresia. Follicular fluid (minimum of four follicles per gilt) was aspirated into a syringe containing .5 mL of PBS. Concentrations of testosterone in follicular fluid were quantified using a direct double-antibody RIA kit (Pentax, Santa Monica, CA). An inhibition curve of serially diluted samples of follicular fluid was parallel to the standard curve. Sensitivity of the assay was 0.1 ng/mL, and a test for additivity demonstrated 97.3% recovery of known amounts of hormone. Antiserum cross-reactivities (provided by the manufacturer) were 100% for testosterone, 6.9% for 5a- dihydrotestosterone, .52% for androsterone, and < .1% for other steroids. All concentrations of testosterone were estimated in a single assay (intraassay CV = 12.7%). Estradiol was extracted (Nephew et al., 1989b) from 100 jxL of diluted (20 to 200 fold) follicular fluid. Estradiol concentrations were quantified with a single-antibody RIA procedure (Nephew et al., 1989a). Standards ranged from 7.5 to

800 pg/mL, and the standard curve was parallel to an inhibition curve of serially diluted samples of follicular fluid. The least detectable concentration of estradiol was 5 pg/mL. Assay of samples to which known amounts of hormone were added demonstrated 98.9% recovery of hormone. Intra- and interassay CV were 4.1 and 9.6%, respectively. Follicles 88 having concentrations of estradiol > 100 ng/mL (estrogenically active; Foxcroft et al., 1987) and a ratio of estradiol:testosterone > 5 (estimated from Grant et al., 1989) were classified as nonatretic. These endocrine criteria of atresia and the macroscopic classifications of nonatretic and atretic follicles agreed in 94.6 and 88.4%, respectively, of the cases.

Experiment 2 Gilts (n = 13) were treated with testosterone as before and observed for the subsequent display of estrus. Gilts were bred twice to different boars during the 1st 24 h of estrus and then ovariohysterectomized on d 11. Blastocysts were recovered by flushing the uterus with physiological saline. Blastocysts were counted, and their maximum diameter and stage of morphological development recorded. Percentage of blastocyst recovery was calculated by dividing the number of blastocysts by the number of CL and multiplying by 100.

Experiment 3

Plasma and follicular concentrations of testosterone and(or) estradiol were determined following administration of vehicle or 1 mg of testosterone (n = 5 per group) on d 17 of the estrous cycle. The 1-mg dose was chosen because it increased the number of CL and embryos in the previous experiments. Jugular blood samples were obtained at 0, 2, and

4 h following testosterone administration. Immediately after the 4 h blood sample was obtained, gilts were ovariectomized, 89 and follicular fluid of eight nonatretic and four atretic follicles was aspirated. Mean follicular diameter of nonatretic or atretic follicles did not differ (P > .2) between treatment groups. Concentrations of estradiol in individual follicles and testosterone and estradiol in serum were quantified with RIA procedures described in Exp. 1.

Intra- and interassay CV for follicular fluid estradiol were 9.2 and 2.3%, respectively. The RIA procedures described in Exp. 1 were validated for their application to swine serum. Estradiol was extracted from 200 jiL of serum. Displacement curves of serially diluted samples of serum were parallel to the respective standard curve of estradiol or testosterone. Recoveries of known amounts of hormone, sensitivities, and intra- and interassay CV of the RIA for estradiol and testosterone were 101 and 100%, 5 pg and .1 ng/mL, 10.1 and 8.9%, and 10.0 and 12.1%, respectively.

Statistical analyses

Data for follicles, CL, blastocysts, and estrous cycle length were analyzed with ANOVA for a completely randomized design (Steel and Torrie, 1980). Means were compared to one another by using contrasts. These analyses were performed using SYSTAT (Wilkinson, 1990). Pregnancy rates (percentage of gilts from which blastocysts were recovered) were analyzed with chi-square tests of independence (FREQ procedure; SAS, 1988). Hormonal data and diameters of follicles and blastocysts were analyzed using the GLM procedures of SAS 90

(1988) . Concentrations of estradiol and testosterone in follicular fluid and diameters of blastocysts were evaluated with one-way ANOVA with subsamples (Steel and Torrie, 1980). The model included the effects of dose and animal within dose. Plasma concentrations of estradiol and testosterone were analyzed with ANOVA for repeated measures using a model with the effects of dose, animal within dose, hour, and dose by hour. Tests of hypothesis for comparing means of interaction effects were performed using the predicted difference (PDIFF) option of SAS (1988).

Results

Testosterone treatment influenced (P c.Ol) the number of preovulatory follicles on d 19. Gilts treated with 10 or 100 mg of testosterone had more (P < .05) nonatretic preovulatory follicles than did those treated with vehicle; however, the number of preovulatory follicles in gilts that received 1 mg of testosterone or vehicle did not differ (Table 6).

Testosterone treatment did not influence the total number

(nonatretic plus atretic) of follicles or the number and percentage of atretic follicles within the large (Table 6) and medium (Table 7) classifications. Concentrations of estradiol or testosterone in follicular fluid of large nonatretic or atretic follicles did not differ (P > .10) among treatment groups (Table 8). 91

Table 6. Mean number of large (> 6 mm) follicles on d 19 of the estrous cycle in gilts treated with testosterone

Group8 Total Nonatretic Atretic % Atretic (Preovulatory) 0 20.2 14.0 b 6.2 30.1 1 20.2 15. S1* 4.4 21.4 10 23.4 18. 0cd 5.4 22.2 100 25.0 20. 6d 4.4 16.2 SEMe 1.9 1.3 1.4 3.2 8Dosages of testosterone (milligrams/day). h^eans lacking common superscript letters differ (P < .05) en = 5 gilts/group.

Table 7. Mean number of medium (3 to 5 mm) follicles on d 1 of the estrous cycle in gilts treated with testosterone

Group8 Total Nonatretic Atretic % Atretic 0 14.0 1.2 12.8 92.7 1 16.4 4.4 12.0 80.9 10 15.8 1.8 14.0 91.3 100 14.8 1.8 13.0 89.8 SEM6 4.8 1.9 3 . 6 6.8 aDosages of testosterone (milligrams/day). bn = 5 gilts/group. Means are not different. 92

Gilts treated with 1, 10, or 100 mg of testosterone had more (P < .05) CL than gilts receiving vehicle (Table 9). The average increase in CL per gilt was 2.3 (1-mg dose), 2.7 (10- mg dose), and 3.5 (100-mg dose) relative to the gilts treated with vehicle. The number of blastocysts was greater (P = .06) in gilts that received 1 mg of testosterone, but it was less (P < .05) in gilts treated with 10 mg of testosterone than in gilts receiving vehicle (Table 9) . The number of blastocyst for gilts that received 100 mg of testosterone was not different (P > .10) from that for gilts receiving vehicle or 10 mg of testosterone (Table 9). Recovery rates of blastocysts from gilts treated with vehicle or 1 mg of testosterone did not differ (P > .10), but they were greater (P < .05) than those for gilts administered 10 or 100 mg of testosterone

(Table 9) . Blastocyst diameter was not altered by testosterone treatment (Table 9) , and all blastocysts were spherical in morphology. Pregnancy rates of gilts receiving vehicle, 1, or

10 mg of testosterone were 92.3, 100, and 92.3%, respectively, and were greater (P = .007) than that of gilts receiving 100 mg of testosterone (53.8%). The duration of the estrous cycle of gilts treated with 10 mg of testosterone was shorter (P < .05) than that of gilts treated with vehicle or 1 mg of testosterone and was not different from that of gilts treated with 100 mg of testosterone (Table 9). 93

Table 8. Concentrations (ng/mL) of estradiol and testosterone in follicular fluid of large follicles on d 19 of the estrous cycle in gilts treated with various doses of testosterone®

Group6 Nonatretic0 Atreticd

Estradiol Testosterone Estradiol Testosterone M C C"

0 402.2 ± 98.3 28.9 ± 24.6 6.2 + • 3.2 ± 6.8 1 347.4 + 76.5 53.6 + 18.9 5.0 + 2.9 14.4 + 7.4 10 192.5 ± 99.5 22.1 + 24.5 7.5 ± 3.0 14.6 + 6.8 100 325.5 + 65.8 59.6 + 17.1 10.7 + 4.0 17.6 + 9.1

“Values are least squares means ± standard errors of the least squares means. bDosages of testosterone (milligrams/day). cn = 10, 14, 10, and 18 for groups 0, 1, 10, and 100, respectively. dn = 10, 9, 10, and 6 for groups 0, l, 10, and 100, respectively.

Table 9. Reproductive characteristics of gilts treated with vehicle or testosterone® Group6 Number Number of Recovery, Blastocyst Estrous of CLc blastocystsd %d size, mmde cycle’ > 4 CO - 0 14. 4f 12.3 + 1.0f 84.7 + • 5.4 + .8 20.2f 1 16.79 15.0 + 1.0h 89.9 + 4. 6f 5.4 + .7 20. lf 10 17.19 8.9 + 1 . 09 52.4 + 4. 89 4.4 + 1.1 19.4s 100 17.9s 9.6 + 1.4fg 49.6 + 6. 39 3.2 + 1.4 19.79f “Values are means ± SEM. bDosages of testosterone (milligrams/day). CSEM = .7, n = 13. dn = 12, 13, 12, and 7 for the number of pregnant gilts per group receiving 0, 1, 10, and 100 mg of testosterone, respectively. “Least squares means ± standard error of the least squares mean (n=147,195, 96 and 67 for groups 0, 1, 10 and 100, respectively). f9hMeans within a column lacking a common superscript letter differ (P < .05). For number of blastocysts P < .06. ’Length (d) of the estrous cycle during which testosterone was injected on d 17 and 18, SEM = .2, n = 13. 94

Table 10. Plasma concentrations of testosterone and estradiol in gilts treated with testosterone on d 17 of the estrous cycle

Group8 Sampling time, Testosterone, Estradiol, h6 ng/mL pg/mL

Vehicle 0 . 3C 21.7C 2 .2° 18. 6ce 4 .2° 19. 7ce

Testosterone 0 .2c 12. 6d 2 1. 2d 18. 5ce 4 1. 2d 15. 6de SEMf .1 1.6

"Gilts were administered vehicle or 1 mg of testosterone on d 17. bAfter injection of vehicle or testosterone. cdeLeast sguares means within a column lacking a common superscript letter differ (P < .05). fStandard error of the least squares means, n = 5 gilts/group.

Plasma concentrations of testosterone increased at 2 and

4 h following administration of 1 mg of testosterone on d 17 and did not change in gilts receiving vehicle (dose by hour interaction, P < .001, Table 10). Plasma estradiol increased by 2 h and then decreased by 4 h in gilts treated with testosterone, while concentrations of estradiol at 0, 2, and

4 h were not different in gilts receiving vehicle (dose by hour interaction, P < .05, Table 10). Estradiol concentrations in follicular fluid were not different (P > .40) between gilts treated with testosterone and those administered vehicle (means ± SEM, 420.6 ± 105.7 and 296.2 ± 109.2 for nonatretic 95 follicles, 9 ± 12.4 and 12.3 ± 23.2 for atretic follicles, respectively).

Discussion The results of these experiments indicated that administration of testosterone during the follicular phase increased the number of preovulatory follicles and resulting CL in swine. Testosterone induced these effects without altering the total number of medium and large surface follicles on d 19 of the estrous cycle. Because only nonatretic and follicles undergoing atresia would be present at a given time, any significant increase in the number of nonatretic follicles should occur simultaneously with a decrease in atretic follicles. In the present study, however, only a numerical reduction in the percentage of large atretic follicles was detected (statistically nonsignificant, P =

.22). It is possible that the small number of gilts or the timing of follicular collection did not allow documentation of this effect. Regardless, the observation that the number of CL and plasma concentrations of estradiol increased confirmed that testosterone had indeed affected follicular maturation.

A heterogeneous group of 40 to 50 follicles, 2 to 8 mm in diameter (Grant et al., 1989), is present by the end of recruitment (d 16 of the estrous cycle, Clark et al., 1982; Foxcroft and Hunter. , 1985) in swine. The fate of most of these follicles is atresia (Grant et al., 1989) and only a 96 small proportion are selected to proceed to ovulation by still unknown mechanisms. Testosterone in swine is used as substrate for the synthesis of estradiol by ovarian follicles (Evans et al., 1981; Ainsworth et al., 1990). The synthesis of estradiol increases steadily as follicles grow and mature to the preovulatory stage (Guthrie et al., 1972; Guthrie and Bolt, 1990) . Administration of testosterone on d 17 and 18 in the present study was coincidental with this period of a rapid increase in estradiol synthesis. Although this experiment was not designed to examine this phenomenon, it is possible that during this period of increased steroidogenic activity and demand for estrogens, the supply of androgens may become critically deficient for some follicles. In a small trial in which five gilts were ovariectomized two weeks before treatment with 1 mg of testosterone, the concentrations of estradiol were undetectable, indicating that the contribution of extraovarian aromatization to plasma estradiol was not important. Therefore, the 68% increase in plasma concentrations of estradiol by 2 h following testosterone administration on d 17 indicates that follicles may have the capacity to respond to exogenous testosterone during the follicular phase by secreting additional amounts of estradiol. This response, however, seems to be short lasting because concentrations of follicular or plasma estradiol 24 and(or) 4 h after testosterone administration were not altered. The difference in plasma estradiol at 0 h between 97 gilts administered 1 mg of testosterone and those treated with vehicle, was probably due to the high variability among gilts in follicular maturation within each day of the follicular phase (Guthrie et al., 1972). However, the patterns of plasma estrogen within each group provided valid information on the response to testosterone. Other actions of testosterone may include modulation of follicular progesterone synthesis (Armstrong and Dorrington, 1976; Leung et al., 1979) and aromatase activity (Daniel and Armstrong, 1980; Hillier and deZwart, 1981). Androgen receptors present in the ovaries of rats (Schreiber et al., 1976) and primates (Hild-Pepito et al., 1991) may also be mediators of some of the paracrine actions of androgens. None of these endocrine actions can be excluded at this time and warrant further study. Androgens, however, do not seem to play a direct role in gonadotropin secretion in females (Schaison and Couzinet, 1991). Treatment of gilts with 50, 100, or 200 mg of testosterone for 10 d beginning on d 15 of the estrous cycle did not synchronize estrus or alter the number of CL; however, when testosterone treatment was initiated earlier than d 15 it blocked ovulation (Torres and First, 1974). Certainly, these doses of testosterone were considerably larger and exposure was for a longer period than the regimen used in our study. It is possible that massive treatment with testosterone may not have the same effects as a short-term treatment with smaller 98 amounts given during the follicular phase. For example, ovulations were induced by injecting 1 to 25 mg of testosterone propionate in anestrous ewes, but greater stimulation occurred with 1 and 5 mg than with larger doses (Radford and Wallace, 1971). Unlike the number of CL, embryonic survival and pregnancy rates were affected negatively by the larger doses of testosterone. These effects of testosterone have not been reported elsewhere. It is unknown whether the decrease in the percentage of blastocyst recovery was due to a decrease in the rate of fertilization, embryonic mortality before d 11, or both. Large doses of testosterone propionate produced degenerative changes in oocytes and preantral follicles in estrogen-stimulated hypophysectomized rats. These effects were not evident at the smaller dosages of testosterone (Payne et al., 1956; Hillier and Ross, 1979). It has also been reported that testosterone may interfere with the actions of estradiol in the ovary by decreasing the number of available estrogen receptors (Saiduddin and Zassenhaus, 1978). Changes in estrous and mating behavior were not detected in the present study; however, gilts that received 10 mg of testosterone had estrous cycles that were approximately 1 d shorter than those of control gilts but still within the range of normal duration.

In conclusion, treatment of gilts with 1 mg of testosterone during 2 d of the follicular phase increased the number of CL and number of recovered blastocysts. Exogenous 99 testosterone increased plasma concentrations of estradiol and dosages of testosterone > 1 mg decreased fertility. CHAPTER VI

ADMINISTRATION OF TESTOSTERONE FROM D 13 OF THE ESTROUS

CYCLE TO ESTRUS INCREASED NUMBER OF CORPORA LUTEA AND

BLASTOCYST SURVIVAL IN GILTS

Eighty nine crossbred gilts were used to examine the effects of exogenous testosterone on number of corpora lutea

(CL) and conceptus survival. In Experiment 1, gilts received daily i.m. injections of 1 mg of testosterone from d 13 (d 0 = 1st d of estrus, n = 18) or d 16 (n = 19) until the next estrus. Control gilts (n = 20) received vehicle (corn oil) from d 13 to estrus while those receiving testosterone beginning on d 16 received vehicle on d 13 to 15. Gilts were mated and then evaluated on d 11.5. In Experiment 2, embryonic survival was evaluated at the 4 to 8-cell, early blastocyst or hatching blastocyst stages in gilts treated with testosterone

(1 mg/d) or vehicle from d 13 to estrus. The number of CL was greater (P < .05) in gilts treated with testosterone from d 13 to estrus than in those receiving vehicle. The number of blastocysts and the percentage of blastocyst recovery were greater (P < .05) in gilts treated with testosterone from d 13 to estrus than in those treated with testosterone beginning on d 16 or those receiving vehicle. Likewise, the number of CL

100 101 and total number of concepti (normal plus degenerating) was greater (P < .01) and the percentage of embryonic recovery tended to be greater (P < .12) in gilts treated with testosterone versus those receiving vehicle in Experiment 2. Gilts treated with testosterone or vehicle did not differ (P > .05) in number of normal concepti at the 4 to 8-cell and hatching stages. However, prior treatment with testosterone delayed conceptus death as gilts treated with testosterone contained more (P < .05) concepti at the intermediate stage (early blastocyst) than those treated with vehicle. Treatment with testosterone during the follicular phase increased number of ovulations and embryonic survival through the early blastocyst stage of development.

Introduction Administration of 1 mg of testosterone to gilts on d 17 and 18 of the estrous cycle increased number of CL and d 11 blastocysts (Cardenas and Pope, 1994). Increases in plasma concentrations of estradiol-176 after testosterone administration on d 17 suggested that the effects of testosterone may be mediated through the actions of estradiol on follicular development resulting in a decrease in atresia (Cardenas and Pope, 1994). Stimulation of follicular function by exogenous testosterone may also indirectly affect oocyte maturation and subsequent embryonic development as relationships between the degree of follicular maturation and 102

oocyte development have been implicated in embryonic survival

in pigs (Hunter and Wiesak, 1990, Pope, 1992). Relationships between dose of testosterone and number of CL or blastocyst survival in pigs have been described (Cardenas and Pope, 1994). Because the response to testosterone may also be influenced by duration of treatment, the present experiments were performed to determine the effects of two different periods of testosterone administration on number of CL and survival of pre- implantational blastocysts.

Materials and Methods

Experiment 1

Gilts (1/2 Duroc X 1/4 Landrace X 1/4 Yorkshire, 7 to 8 months of age, 120 to 130 kg of body weight initially were observed at approximately 12 h interval for display of estrus in the presence of intact boars. Gilts that exhibited estrous cycles of 18.0 to 22.0 days were allocated to receive daily i.m. injections of the following treatments during their next cycle; l) Vehicle (corn oil) from d 13 of the estrous cycle (d 0 = first d of estrus) until the first d of the next estrus (n = 20) , 2) One mg of testosterone from d 13 of the estrous cycle until the first d of the next estrus (n = 18), or 3) vehicle on d 13, 14 and 15 and then 1 mg of testosterone from d 16 until the first d of the next estrus (n = 20). Gilts that displayed estrus 12 h after the last injection of testosterone 103 or vehicle, did not receive additional injections. Gilts within each group were assigned to receive 1 or 7 mL of vehicle. Seven mL was used in a previous experiment because it was the minimum volume of vehicle that allowed the largest dose of testosterone to dissolve. It was of interest in the present experiment to determine whether the volume of vehicle would alter the responses to testosterone administration. Characteristics evaluated were not altered by volume of vehicle, therefore, data were pooled across volume of vehicle within each treatment for the final statistical analyses. Similarly, in a small trial in which 1 mg of testosterone dissolved in 1 or 7 ml of vehicle was injected (i.m.) into ovariectomized gilts, plasma concentrations of testosterone at 0, 1, 2, 4, 6, 8, 12, 16 and 24 h after testosterone administration were not different between groups. In order to decrease variability due to duration of the estrous cycle when treatments were given, 7 out of 64 gilts were excluded because their estrous cycles did not fall within the range of 18.0 to 22.0 days. Duration of the estrous cycle of gilts that were included in the experiment were not different (P > 0.10) among groups and were 19.4 ± .2, 19.3 ± .2 and 19.0 ± .2 in gilts treated with testosterone beginning on d 13, d 16, and those treated with vehicle, respectively.

Gilts were mated to different boars at 12, 24 and 36 h after onset of estrus and then ovariohysterectomized on d 11.5. Corpora lutea were counted and then removed from the 104 ovaries and weighed. Blastocysts were recovered by flushing the uterus with physiological saline (.9% NaCl) within 10 to

15 minutes after surgery. Before flushing, the was separated from the uterus and the tip of each uterine horn was cut approximately .5 cm from the uterine-tubal junction. The uterus was flushed twice by infusing 50 ml (total of 100 ml) of saline into the tip of one horn, and recovering the flushing through the tip of the opposite uterine horn. Blastocysts were examined using a stereomicroscope and then measured to the nearest millimeter at their largest diameter. Number of filamentous blastocysts that became entangled during flushing were estimated based on the number of trophoblastic ends. Morphological diversity among littermate blastocysts was estimated by calculating the standard deviation of blastocyst diameter. Percentage of blastocyst recovery was calculated by dividing the number of blastocysts by the number of CL and multiplying by 100. Because it was not possible to know whether gilts from which no blastocysts were recovered lost their concepti or had total fertilization failure, they (one gilt per group) were not included in the calculations of percentage of blastocyst recovery.

Experiment 2 Gilts at approximately 6 to 7 months of age, weighing 100- 110 kg initially and of the same genetic background as those of Experiment 1 were used to examine embryonic survival at earlier stages of development. Gilts were administered 1 mg of 105 testosterone/d (n = 24) or vehicle (7 mL, n = 24) from d 13 of their first estrous cycle until estrus and then mated as described in Experiment 1. This treatment was selected because it resulted in the highest percentage of blastocyst recovery in Experiment 1. Corpora lutea and concepti were evaluated after ovariohysterectomies on d 4.0, 5.5 or 7.0. Embryos were recovered by flushing each oviduct and uterine horn three times with 25 ml of modified Tyrode's medium (TLH, Hagen et al., 1991). This medium was used instead of saline because the search for the concepti took longer than on d 11.5. Morphological development of concepti was determined by microscopic examination and then classified as normal or degenerating depending primarily on stage of development. Embryos that were more than 24 h behind (Perry and Rowlands, 1962) relative to the most developed embryo within a litter were considered degenerating (i.e. two-cell embryos on d 4 or cleaving embryos on d 7.0). Typically, concepti were at the 4 to 8-cell, early blastocyst and hatching blastocyst stages on d 4.0, 5.5, and 7.0, respectively. The early blastocyst stage included the period of blastocoele formation whereas the hatching stage comprised expanding and hatched blastocysts.

Statistical Analyses Observations from Experiment 1 were analyzed by one-way ANOVA using models that included the effect of treatment, except for CL weight and blastocyst diameter which were analyzed using models that included the effects of treatment 106 and animal within treatment. Data from seven gilts, which had unmeasurable filamentous blastocysts, were not included in the analyses of blastocysts diameter. Percentage of pregnant gilts and proportion of gilts having 100 % recovery rates were compared using chi-square test of independence (Steel and Torrie, 1980). Experiment 2 was a factorial (two doses of testosterone by three stages of embryonic development) and data were analyzed accordingly by ANOVA procedures. As expected, number of CL was not influenced by stage of development and was therefore pooled across stages and then analyzed by one-way ANOVA. Analyses of variance were performed using general linear models of the Statistical Analysis System [SAS, 1988]. Preplanned comparisons of means were performed using LSD tests (Steel and Torrie, 1980).

Results

Experiment 1 The number of CL was greater (P < .05) in gilts treated with testosterone from d 13 to estrus than in those treated with vehicle (Table 11). Gilts treated with testosterone from d 16 to estrus and those treated with vehicle were not different (P > .05). Weight of CL was not influenced by treatment (Table 11) . The number and recovery rates of blastocysts were greater (P < .05) in gilts treated with testosterone from d 13 to estrus than in those treated with testosterone from d 16 to estrus or vehicle-treated gilts 107

(Table 12) . Gilts treated with testosterone from d 16 to estrus and vehicle-treated gilts were not different (P > .05) in these characteristics. The proportion of gilts having 100% recovery rates on d 11.5 tended (P = .09) to be greater in gilts treated with testosterone from d 13 to estrus than in the other groups. Blastocyst diameter and standard deviation of blastocyst diameter were not influenced (P > .10) by treatment (Table 12). Standard deviations of blastocyst diameter were 1.9 ± 0.3, 1.3 ± 0.3, and 1.6 ± 0.2 mm (mean ± standard error of the mean) for gilts treated with testosterone beginning on d 13, d 16 and those receiving vehicle, respectively. The proportion of gilts that were pregnant was not influenced by treatment and were 94.4, 94.7 and 95.0% for gilts receiving testosterone beginning on d 13, d 16 or those receiving vehicle, respectively.

Experiment 2

The number of CL was greater (P < .01) in gilts treated with testosterone from d 13 to estrus than in those receiving vehicle (Table 11). The total number of concepti was influenced (P < .01) by testosterone treatment and stage of embryonic development (Table 13). Mean number of total concepti was greater (P < .01) in gilts treated with testosterone than in those treated with vehicle (13.6 vs 12.0, respectively). Mean number of total concepti at the 4 to 8- cell and early blastocyst stages did not differ (P > .05; 13.8 versus 13.6, respectively) but were greater (P < .01) than at 108

Table 11. Characteristics of corpora lutea (CL) in gilts administered daily injections of 1 mg of testosterone from d 13 or d 16 of the estrous cycle to estrus8.

Group Number of CL, Number of CL, CL weight, Exp. lb Exp. 2C mgd

D 13 16.2 ± . 5e 14.9 ± .4** 457.4 ± 21.9

D 16 15.0 ± . 5ef - 448.3 ± 22.1 • H U) Vehicle • 09 1+ 13.3 ± .4 487.6 ± 21.2

°Values are least squares means ± standard error of the least squares mean. bn = 18, 19 and 20 for D 13, D 16 and Control, respectively. cn = 24 per group. D 16 was not included in Experiment 2. dn = 278, 271 and 295 for D 13, D 16 and Control, respectively. CL from two gilts were accidentally lost. efMeans without a common superscript differ (P < .05). **D 13 different from Vehicle (P < .01). the hatching stage (11.2, Table 13). The number of normal concepti was influenced (P < .05) by the interaction of testosterone treatment by stage of embryonic development (Table 13). The mean number of early blastocysts was greater (P < .001) in gilts treated with testosterone than in those treated with vehicle (14.9 vs 10.8, respectively); however, the mean number of 4 to 8-cell embryos and hatching blastocysts were not different (P > .05) between these groups.

Although not statistically significant at the hatching stage, numerically 11.5 hatched blastocysts were recovered from testosterone treated gilts versus 9.9 from gilts treated with 109

Table 12. Characteristics of d 11.5 blastocysts of gilts administered daily injections of 1 mg of testosterone from d 13 or d 16 of the estrous cycle to estrus8.

Group Number of Recovery, %b Gilts Diameter blastocysts15 having mmd 100% recovery, %c

D 13 15.3 ± .6f 94.8 ± 2.8f 47.1 7.2 ± .7

D 16 12.8 ± .69 85.8 ± 2.79 16.7 5.1 ± .8

Vehicle 12.8 ± . 59 86.6 ± 2.79 21.1 6.4 ± .7

°Values are least squares means ± standard error of the least squares means. bn = 17, 18 and 19 for D 13, D 16 and Control, respectively. cRelative to gilts from which blastocysts were recovered. Effect of treatment group (P = .09) dn = 242, 184 and 226 for D 13, D 16 and Control, respectively (number of gilts = 16, 14, and 17 for D 13, D 16, and Control, respectively. Gilts having unmeasurable filamentous blastocysts were not included). f9Means without a common superscript differ (P < .05). vehicle. Number of normal concepti of gilts treated with testosterone did not change (P > .05) from the 4 to 8-cell to the early blastocyst stage (13.8 vs 14.9, respectively) and then decreased (P < .05) by the hatching stage (Table 13). Conversely, number of normal embryos in gilts treated with vehicle decreased (P < .05) from the 4 to 8-cell stage to the early blastocyst stage (13.4 vs 10.8) and numerically, but nonsignificantly, decreased by the hatching stage (9.9; Table 13) . 110

Recovery rates of total embryos were not influenced (P > .05) by the interaction or the main effect of treatment, however, this variable was highly influenced (P < .001) by stage of embryonic development (Table 14). Recovery rates of normal concepti only tended to be influenced (P = .12) by testosterone treatment (89.6 vs 85.0) but was influenced (P < .05) by stage of embryonic development (Table 14). Although the interaction effect was not significant (P = .25), comparisons of means showed that the difference of approximately 11% in percentage of embryonic recovery between gilts treated with testosterone and those treated with vehicle at the early blastocyst stage (95.5% vs 84.7%, respectively) were statistically different (P < .05). Recovery rates of total or normal embryos at the 4 to 8-cell and early blastocyst stages were not different (P > .05); however, percentage of embryonic recovery at these stages were greater

(P < .01) than at the hatching blastocyst stage (Table 14).

Discussion

It was previously observed in our laboratory that administration of 1 mg of testosterone/d on days 17 and 18 of the estrous cycle increased number of CL and the number of d 11 blastocysts (Cardenas and Pope, 1994). Mean percentage of blastocyst recovery was numerically, but nonsignificantly, higher (89.9% vs 84.7%) in gilts treated with testosterone than in those receiving vehicle in that previous experiment. Ill

Table 13. Mean number of embryos at three stages of development in gilts administered daily injections of 1 mg of testosterone or vehicle from d 13 of the estrous cycle to estrus of mating®.

Embryo stage Number of embryos/gilt

Testosterone Vehicle Total

Total embryos6

4 to 8-cell 13.9 13.6 13.8C

Early blastocyst 15.1 12.0 13. 6C

Hatching blastocyst 11.9 10.5 11.2d

Total 13.6** 12.0

Normal embryose

4 to 8-cell 13. 8f 13 .4fh 13.6

Early blastocyst 14. 9f 10.8s 12.8

Hatching blastocyst 11.59h 9.9s 10.7

Total 13.4 11. 3 aValues are least-sguares means. bSEM = .7 (n = 8) , .5 (n = 16) and .4 (n = 24) for embryo stage by treatment combinations, totals for stage and totals for treatment, respectively. cdMeans without a common superscript differ (P < .01). eSEM = .8 (n = 8) , .5 (n = 16), and .4 (n = 24) for embryo stage by treatment combinations, totals for stage and totals for treatment, respectively. fghMeans without a common superscript differ (P < .05) within the normal embryos group. **13.6 different from 12.0 vehicle (P = .01). 112

Table 14. Mean percentage of embryonic recovery at three stages of development in gilts administered daily injections of 1 mg of testosterone or vehicle from d 13 of the estrous cycle to estrus of mating®.

Embryonic stage Embryonic recovery/gilt, %

Testosterone Vehicle Total

Total embryos6

4 to 8-cell 95.0 96.8 95.9C

Early blastocyst 97.7 94.3 96.0C a • Hatching blastocyst 81.8 79.7 00 o

Total 91.5 90.3

Normal embryose o CO 4 to 8-cell 94.1 95.4 CTi ^1*

Early blastocyst 95.5 84.7 90. lc

Hatching blastocyst 79.2 74.9 77. ld

Total 89.6 85. 0

“Values are least-squares means. bSEM = 3.2 (n = 8), 2.3 (n = 16), and 1.9 (n = 24) for embryo stage by treatment combinations, totals for stage and totals for treatment, respectively. cdMeans, within total or normal embryo groups, without a common superscript differ (P < .001). eSEM = 3.5 (n = 8), 2.5 (n = 16), and 2.0 (n = 24) for embryo stage by treatment combinations, totals for stage and totals for treatment, respectively. 113

In the present experiments, a longer treatment, from d 13 to estrus, increased the number of CL and blastocysts and increased the percentage of blastocyst recovery. Although these effects of testosterone on blastocyst survival were clear in Experiment 1, the main effect of treatment in Experiment 2 only approached significance. The combined effects of testosterone on increasing the number of CL and delaying early blastocyst death accounted for increasing the number of normal blastocysts on day 5.5 (Exp. 2) and probably on d 11.5 (Exp. 1) . We previously hypothesized that the effects of testosterone on increasing the number of CL might be related to its capacity to increase concentrations of estradiol, resulting in stimulation of follicular development and decrease in percentage of large atretic follicles (Cardenas and Pope, 1994) . Although it is possible that the same hypothesis applies to the present results, it remains to be determined whether testosterone treatment from d 13 to estrus increased number of CL by altering follicular recruitment with or without additional changes in follicular atresia. If the effects of exogenous testosterone are mediated by estradiol, then it would be logical to expect similar effects of exogenous estradiol to those found for testosterone.

Interestingly, administration of estradiol benzoate to sows at the time of weaning (end of their lactation) or shortly after weaning decreased number of ovulation and altered estrous 114 behavior (Edwards and Foxcroft, 1983) . These negative effects were not observed in our studies, probably because the increases in estradiol induced by testosterone were small and transient (few hours, Cardenas and Pope, 1994). The lack of effects of testosterone when administration was initiated on d 16 suggests that exogenous testosterone during follicular recruitment (d 14 to 16, Foxcroft and Hunter, 1985) was important for the effects found in the present experiments. However, in a previous study, testosterone treatment on d 17 and 18, a period included in the treatment from d 16 to estrus, increased number of ovulations (C&rdenas and Pope, 1994). It appears that responses to testosterone might depend on complex and still unknown interactions between duration and initiation of testosterone treatment. A new and important finding in the present experiments was the positive effect of exogenous testosterone, when administered from d 13 to estrus, on the proportion of blastocyst survival. It seems logical to consider that effects which reduce follicular atresia also augment development of follicles that do not undergo atresia. For example, the effects of exogenous testosterone on follicular synthesis of estradiol (Cardenas and Pope, 1994), which might have been involved in increasing the number of CL, may have also improved oocyte quality resulting in enhanced blastocyst survival. Increasing concentrations of estradiol and other 115 steroids seems to be necessary for proper response of oocytes to the LH surge (Hunter et al., 1976, Ainsworth et al., 1980). Perhaps related is the observation that less developed oocytes were found in a minority of late developing preovulatory follicles, which also had a lower content of steroids in follicular fluid (less developed) than the remaining majority (Xie et al., 1990c). Furthermore, relationships between follicular and oocyte maturation have been implicated in embryonic survival in pigs (see Hunter and Wiesak, 1990 and

Pope, 1992 for review). For example, recent findings of high estrogen content of Meishan follicles were related to the high embryonic survival of this breed of pigs (Hunter et al., 1994) . In the present experiments, the effects of testosterone treatment on number of CL, did not induce changes in CL weight. Similarly, increases in mean percentage of blastocyst recovery were not accompanied by significant changes in morphological diversity of d 11.5 littermate blastocysts. Lower embryonic survival at the hatching than at the early blastocyst stage found in Experiment 2 is consistent with degeneration of blastocysts beginning on d 6 (Perry and

Rowlands, 1962) and with abnormalities noted during blastocyst formation and expansion in cultured embryos obtained from gilts at first estrus (Menino et al., 1989). This is one of few studies that monitored embryonic survival in a sequential manner from the end of cleavage to elongation. Mean percentage of embryonic recovery in vehicle- treated gilts at the elongation stage (Experiment 1) was approximately 12% higher than those of control gilts at the hatching stage (Exp. 2) . Estimates of embryonic recovery including gilts from which no blastocysts were recovered would still be 5 to 8% higher for elongating blastocyst than hatching blastocyst. Since the only difference between gilts of Experiments 1 and 2 was the unintentional difference in the number of estrous cycles before treatments were given (greater than three cycles versus 2nd estrous cycle in Exp. 1 versus Exp. 2, respectively), it seems possible to consider the role of reproductive age on the time when high amounts of embryonic death occurs. Perhaps a significant proportion of embryos were lost sooner in younger gilts (Exp.l) than in older gilts (Exp.2). A high incidence of immature oocytes was associated with low embryonic survival of prepuberal gilts induced to ovulate with PMSG/hCG (Wiesak et al., 1990) and gilts at their first estrus (Koenig and Stormshak). Furthermore, in vitro development and survival rates after transfer of embryos from gilts at first estrus was diminished relative to those obtained at third estrus (Menino et al., 1989, Archibong et al., 1992). Similarly, a significant amount of embryonic mortality (more than 25%) was found before blastocyst elongation (d 10) in first and second estrus gilts (Lambert et al., 1994, Cassar et al., 1994a). This large amount of mortality was consistent with our estimates of embryonic 117 mortality found at the hatching blastocyst stage in Experiment 2 (25% in gilts treated with vehicle). However, embryonic mortality by d 11 to 15 in multiestrous gilts has been significantly lower than those of gilts at first or second estrus (overall 3 to 15%, Archibong et al., 1987, Cardenas and Pope, 1993, 1994, present study). As has been proposed by others, perhaps the critical periods of embryonic mortality differ between gilts exhibiting several estrous cycles versus those at their first or second estrous. In conclusion, significant losses of embryos were found during the transition from the 4 to 8-cell stage to the hatching blastocyst stage in second estrous gilts. Testosterone treatment from d 13 to estrus was effective in increasing number of ovulations and survival of early blastocyst in both second and multiestrous gilts. Perhaps prior treatment with testosterone improved some aspects of oocyte quality and resulting embryonic development up to the elongation stage of blastocyst development. CHAPTER VII

SUMMARY

Approximately 60% of porcine concepti develop to term, 30% die by the first month of pregnancy and the remaining 10% die after the first month. These proportions indicate that conceptus mortality in swine is high and accounts for approximately 40% of the total number of concepti. Most conceptus mortality appears to occur from fertilization to the end of implantation (d 18) . This period of conceptus mortality is called embryonic mortality. Many factors cause embryonic mortality, such as suboptimal environmental conditions, management, nutrition and diseases.

However, even when these conditions are optimal, embryonic mortality still occurs. In these situations, embryonic-uterine asynchrony and factors related to the oocytes (i.e. chromosomal defects, stage of maturity at ovulation) are perhaps the most important causes of embryonic mortality.

Embryonic-uterine asynchrony is the disruption of the relationship between uterine advancement during pregnancy and embryonic development. The main cause of embryonic-uterine asynchrony is the morphological diversity among littermate blastocysts. This

118 119 diversity among littermate embryos occurs because morphological changes during embryonic development do not occur at the same time. Although diversity is present at all times during the first two weeks of gestation, it is greatest during blastocyst elongation, a period of rapid transformations from spherical into ovoidal, tubular and then filamentous forms. Porcine blastocysts at the time of elongation do not only differ in morphology but also in their capacity to secrete estradiol and other substances such as prostaglandins, growth factors, retinol binding protein and interferons. Estradiol secretion increases significantly as blastocysts develop from spherical, to ovoidal and then to tubular shapes. Estradiol regulates the synthesis and (or) secretion of uteroferrin, growth factors and binding proteins from the uterus which in turn stimulate embryonic development. Therefore, estradiol plays a primary role in the establishment of the physiological relationship between the embryos and the uterus.

Because of the significant changes in morphology and secretory activity that occur during blastocyst elongation, it is clear that blastocyst elongation is a critical period during development, however little is known about the factors that control this process. Results presented here indicate that estradiol plays a role(s) in the control of blastocyst elongation. Exogenous estradiol stimulated elongation of blastocysts that were approximately 12 h from onset of 120 elongation but was inhibitory for blastocysts that normally begin elongating within 12 h from the time of treatment. These inhibitory and stimulatory effects of exogenous estradiol on blastocyst elongation are difficult to interpret. However, the dual effects of estradiol neatly demonstrate how small differences in time (hours) during blastocyst elongation can produce completely different responses to changes in the uterine environment. Furthermore, results of this experiment supported previous findings of a significant increase in concentrations of retinol, uteroferrin, IGF-I and total proteins in uterine luminal fluid during blastocyst elongation. These substances, alone or together, might be the mediators of the observed effects of estradiol in this experiment. Pope et al. (1990) proposed that uterine advancement induced by estradiol secreted by elongating blastocysts is beneficial for their own development but becomes detrimental

(asynchronous) for the survival of less developed blastocysts.

Because of the possible association between blastocyst diversity and survival it has been of interest to determine the causes of embryonic diversity. Blastocyst diversity seems to be related to the pattern of ovulation. In pigs ovulation lasts 2 to 9 h. Most oocytes are ovulated at the beginning and few of them later during ovulation. The oocytes that are ovulated first are fertilized first and generate embryos that are more developed up to d 12 of gestation. Therefore, it 121 appears to be logical to predict that if oocytes are fertilized at approximately the same time blastocyst diversity would decrease. This possibility was investigated by presumably decreasing the duration of fertilization by mating gilts during ovulation (delay mating). Although delay mating decreased blastocyst size (by delaying fertilization), delay mating did not alter blastocyst morphological diversity.

Heterogeneity among ovulatory follicles and ovulated oocytes is present in pigs and has been related to blastocysts diversity. The same pattern of a majority being more developed than the remaining minority was found to be present among preovulatory follicles, maturing oocytes and zygotes. These associations appeared to be more than coincidental and reflected a possible relationship between follicular, oocyte and embryonic development. The presence of this relationship in addition to the well known associations between follicular and oocyte development and the fact that the oocyte participate in the formation of the embryo, suggested that embryonic development could be influenced by altering follicular growth during the follicular phase of the estrous cycle.

A hormone necessary for follicular development is estrogen, which is synthesized from testosterone and androstenedione mainly by the granulosa cells of the follicle.

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