‘bV1ESI_J RETURNING MATERIALS: Place in book drop to ”311511155 remove this checkout from w your record. FINES win be charged if book is returned after the date stamped be10w. -‘:,'.'1h":?' Esau-w gun-.1” a“ -. II; '1 I 7,1 k ? “ad? in § dog In FOLLICULAR OOCYTE MATURATION FOR USE IN BOVINE XENOGENOUS AND IN_VITRO FERTILIZATION By Vernon D. Dooiey A DISSERTATION Submitted to Michigan State University in partial fquiIIment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animai Science 1983 ABSTRACT FOLLICULAR OOCYTE MATURATION FOR USE IN BOVINE XENOGENOUS AND IN VITRO FERTILIZATION by Vernon D. Dooley A series of experiments was conducted to morphologically charac- terize follicular oocytes from bovine ovaries collected at slaughter, to define a culture system for consistent jg_vjt§9_maturation of these oocytes, and to assess the feasibility of using xenogenous and 13_ vitrg_fertilization to produce early embryos from jn_vjtrg_matured oocytes. There was no difference between oocytes from cull cows or heifers, or between oocytes ipsilateral and contralateral to the most recent corpus luteum. Follicles larger than 3 mm contained a higher propor- tion of degenerate oocytes than smaller follicles. 0f the 12.5 oocytes collected per ovary throughout the series of experiments, 20 to 30% were degenerate. Germinal vesicles were found in 76.9% of the remaining oocytes. The vital dye, trypan blue, indicated 83.3% of such oocytes were viable. Trypan blue exclusion and fluorescein diacetate uptake were correlated with each other, and with morphologi- cal classifications. No oocytes with severely degenerate ooplasm (score 3) and only 2% of oocytes devoid of surrounding cumulus cells Vernon D. Dooley matured during 27 to 30 hours of culture. The percentage of oocytes maturing was 25.6 and l3.8 for ovaries transported to the laboratory at 37°C and 0°C, respectively, despite similar oocyte appearance at collection. Oocyte maturation was not enhanced by lactate (50 or l00 mM) in medium containing glucose and pyruvate. Neither the proportion of oocytes remaining viable (survival), nor maturation, differed between a phosphate buffered saline and a TClQQ-based medium. Addition of FSH (l0 ug/ml) tended to reduce maturation. Reducing the atmospheric oxygen content from 20% to 5% did not alter maturation. Increasing culture temperature from 37° to 39° did not affect survival but tended to increase maturation. Estradiol (l ug/ml) or progesterone (l ug/ml) in the culture medium, alone or in combination with FSH (10 ug/ml), reduced survival and did not alter maturation. Fertilization (2 pro- nuclei or cleavage) was obtained in a pseudopregnant rabbit oviduct (xenogenous fertilization) in 5l. % of oocytes expected to have matured at 37°C under 20% 02. Percents fertilized were 70.8 and 52.7 following maturation under 5% 02 at 37°C and 39°C, respectively. After adjustment for the normal maturation rate, jg_vjtrg_fertiliza- tion (sperm penetration and one pronucleus) was achieved in 5l.0% of oocytes and 40.0% of fertilized ova formed two pronuclei. No differ- ences were found among media. ACKNOWLEDGEMENTS Sincere thanks and appreciation are expressed to the many people whose aid and encouragement contributed to this dissertation. I wish to extend special thanks to Dr. w. R. Dukelow for his advice, guidance and friendship. Gratitude is also due those who so willingly served as guiding committee members: Drs. D. Eversole, R. L. Fogwell, J. Gunther and D. Hawkins. Appreciation is extended to Bonnie Cleeves and my colleagues at the ERU. They have made this task immeasurably less difficult. I am indebted to Ms. Diane Hummel for the fast yet accurate typing of this manuscript, and to R. G. Rawlins for assistance in its final preparation. Appreciation and thanks are expressed to my wife, Susan, for moral and financial support throughout the many years of study. And finally, in return for trust, patience and encouragement, I thank my parents and assure them "...the roots of my raisin' run deep...”. 11 TABLE OF CONTENTS Page LIST OF TABLES --------------------------------------------------- iv LIST OF FIGURES -------------------------------------------------- vii INTRODUCTION ----------------------------------------------------- 1 LITERATURE REVIEW ------------------------------------------------ 3 Ig_Vitro Maturation ----------------------------------------- 3 In_VTTF6 Fertilization -------------------------------------- 2l Embryo Culture in a Foreign Tract --------------------------- 30 Xenogenous Fertilization ------------------------------------ 34 MATERIALS AND METHODS -------------------------------------------- 38 Oocyte Collection ------------------------------------------- 38 Oocyte Culture ---------------------------------------------- 44 Xenogenous Fertilization ------------------------------------ 45 In Vitro Fertilization -------------------------------------- 47 Statistical Analysis ---------------------------------------- 48 RESULTS ---------------------------------------------------------- 49 DISCUSSION ------------------------------------------------------- 79 SUMMARY AND CONCLUSIONS ------------------------------------------ 88 LITERATURE CITED ------------------------------------------------- 91 APPENDIX --------------------------------------------------------- l09 Publications by the Author ---------------------------------- lO9 Vita -------------------------------------------------------- llO Table 10 ll 12 I3 LIST OF TABLES Summary of in_vitro maturation studies with bovine oocytes ................................................ Summary of fertilization studies using bovine oocytes-- The culture of domestic animal embryos in the rabbit oviduct ................................................ Summary of xenogenous fertilization studies in the bovine ................................................. Changes in ovarian structures during the bovine estrous cycle -------------------------------------------------- The basic contents of TC199 medium for gamete handling and culture -------------------------------------------- The basic contents of PBS medium for gamete handling and culture -------------------------------------------- Classification system for oocyte evaluations ----------- Subclass frequencies of oocytes with various investment scores within follicle size on cow or heifer ovaries-~- Summary of follicle size effects on investment scores across age groups ...................................... Proportions of follicles in each size group and rela- tion to ovarian activity ------------------------------- Proportions of oocytes in each investment classifica- tion and relation to ovarian activity ------------------ Proportions of oocytes in each ooplasm classification and relation to ovarian activity ----------------------- iv Page 10 26 31 36 39 41 42 43 50 51 52 53 54 LIST OF TABLES (continued) Table l4 15 l6 l7 TB 19 20 21 22 23 24 25 26 27 28 Page Frequency of oocytes in each classification and rela- tion to follicle size ---------------------------------- 56 Effect of interval from slaughter to oocyte culture on proportion of oocytes in each classification ----------- 57 Meiotic stage of follicular oocytes at collection ------ 60 Effects of transport temperature on oocyte character- istics at follicle aspiration -------------------------- 6l Effects of transport temperature on oocyte character- istics after in_vitro culture -------------------------- 62 Proportion of follicular oocytes within an investment- ooplasm rating that mature jn_vitro -------------------- 64 Effect of preselecting oocytes by vital dyes on in_ vitro maturation rates --------------------------------- 65 Effect of lactate addition on oocyte characteristics after ig_vitro culture --------------------------------- 66 Effects of culture media and atmospheric environment on survival and maturation of oocytes during culture--- 67 Effect of two different incubation temperatures on sur- vival and maturation of oocytes in several media ------- 69 Comparisons between modified media in ability to support jg_vitro maturation under 5% 02 at 39° --------- 70 Effects of steroid addition to PBS medium on oocyte viability and maturation ------------------------------- 7l Effects of in vitro culture for varying lengths of time on subsequent xenogenous fertilization ----------------- 73 Xenogenous fertilization ratios of ova jg_vitro matured in various media at 37° under 5% C02 in air ------------ 74 Xenogenous fertilization ratios of ova in_vitro matured in various media at 37° under 5% 02:5% C02:90% N2 ------ 75 LIST OF TABLES (continued) Table Page 29 Xenogenous fertilization of ova in vitro matured in various media at 39° under 5% 02: 5%“ C02: .90% N2 --------- 76 30 Effect of in vitro maturation on subsequent fertiliza- tion in vitrow ------------------------------------------ 78 vi LIST OF FIGURES Figure Page l Oocyte viability assessments and relationship to ooplasm quality score at collection -------------------- 59 vii INTRODUCTION Mammalian ovaries harbor a large supply of genetic material which is not fully utilized. The number of ova ovulated spontaneously at estrus is a minute portion of the oocytes in the ovaries at birth. The remainder degenerate within the ovary through follicular atresia. Recovery of oocytes from bovine vesicular follicles could greatly increase the germ cell population available for fertilization and production of embryos. However, before fertilization can occur, quiescent follicular oocytes must resume meiosis to metaphase II, i.e. mature. The primary objective of the present study was to deter- mine the culture conditions necessary to preclude degeneration and induce maturation of bovine oocytes, so that oocytes liberated from developing follicles could be utilized for embryo production. Alternatives to natural fertilization can provide rapid, rela- tively inexpensive methods of producing embryos for morphological, biochemical, or teratological analyses. In addition, these have clinical and laboratory significance as a means of diagnosing some infertility problems, to extend usage of valuable semen, to assess the functional performance of male and female gametes, and to provide synchronously developing zygotes for research on such new technqiues as nuclear transfer and gene injection. 2 Ig_yjtrg_fertilization of laboratory animals has resulted in detailed morphological descriptions of fertilization as well as an increased understanding of biochemical events involved and environ- mental conditions required for fertilization (Stambaugh, 1978; Bavi- ster, l98l). Species differences exist and the optimal conditions for ig_yitrg_fertilization of domestic animal oocytes are not well de- fined. Xenogenous fertilization, the fertilization of ova by homo- logous sperm in the oviduct of a heterologous species, provides a natural fertilization environment and more closely controls the culture conditions for the researcher. Fertilization of ig_yjtrg_ matured ova was tested using both jg_yit§g and xenogenous systems in this study. The objective was to determine the effects of various environmental conditions on maturation and fertilization of bovine oocytes. This specifically included the atmospheric environment, temperature, and media. Additionally, the feasibility of using xeno- genous and jg_yjtrg_fertilization techniques to produce early bovine embryos was to be determined. II. LITERATURE REVIEW In Vitro Maturation Gametogenic cells of female mammals initiate meiosis during late stages of fetal development (Erickson, l966a; Thibault, l976). Meiotic progression is arrested, however, and at birth bovine ovaries contain several thousand immature oocytes with their nuclei in pro- phase of meiosis I. These nuclei are surrounded by a nuclear mem- brane, termed a germinal vesicle (GV) (Erickson, l966b; Baker and Franchi, l967). Follicular cells maintain the oocyte in this arrested state by producing a maturation inhibiting substance(s) or by with- holding essential nutrients. The oocyte in turn can prevent luteini- zation of granulosa and thecal cells (Mauleon and Mariana, l977). These primordial follicles therefore represent a resting pool of genetic material from which small numbers of oocytes will be recruited for further growth. Development into a vesicular follicle is charac- terized by formation of a fluid-filled antrum, thecal and granulosa cell hyperplasia, and differentiation of the granulosa cell layer into a zone surrounding the oocyte, the cumulus oophorus, and a zone lining the remainder of the follicle (Chang gt_al,, l978). A layer of more densely packed cells, the corona radiata, develops within the cumulus oophorus and in direct contact with the oocyte. Vesicular bovine follicles near ovulation are 10-20 mm in diameter (Staigmiller and 4 England, 1982). A few hours before ovulation an endogenous luteini- zing hormone (LH) surge induces follicular changes which allow resump- tion of meiosis (Donahue, 1972; Thibault gt_al,, 1975). The actual mechanism of this LH action on meiosis remains obscure. Once ini- tiated, meiosis proceeds through the germinal vesicle breakdown (GVBD) stage, metaphase I (met I), anaphase I, and telophase I (Fleming and Saacke, 1972). Oocytes in these stages are considered to be maturing in the present study. Oocytes that complete the first meiotic reduc- tional division extrude the first polar body into the perivitelline space between the cell membrane and zona pellucida and progress to metaphase II (Met II) (Donahue, 1972). These oocytes are meiotically mature and prepared for ovulation and penetration by spermatazoa. Hormonal cyclicity results in release of one such oocyte from the ovary of a mature, nonpregnant cow approximately every 21 days. A variety of techniques have been used in attempts to harvest the large number of follicular oocytes that would normally remain arrested in ovaries. Yet the optimum conditions for collection and maturation of these oocytes remains largely obscure. Early work showed explanta- tion of an oocyte from its follicular environment into a simple cul- ture medium is sufficient to stimulate resumption of meiosis through Met II (Pincus and Enzmann, 1935; Edwards, 1965; Foote and Thibault, 1969). These investigators and others further demonstrated the rate of maturation jg_vitro was similar to that obtained in_vivo following an endogenous LH surge. Ovulation of a mature bovine oocyte usually occurs 24-28 hr after the LH peak (Hafez gt_al,, 1963; Christenson et_ 5 al,, 1974). Progression to Met II occurs when bovine follicular oocytes have been cultured for 24-30 hr (Sreenan, 1970; Thibault, 1972; Shea gt_al., 1976). Most laboratory animal and primate jg_yjtrg_oocyte maturation studies have involved oocytes collected at laparotomy, laparoscopy, or immediately after ovariectomy. In domestic animals, ovaries have been collected at commercial abattoirs and transported to a laboratory for ovum recovery and culture. Comparable maturation rates have been ob- served for bovine ova when ovaries were transported at 30-37°C (Fukui and Sakuma, 1980), at ambient temperature (Newcomb gt_al,, 1978), or at 0°C (Kruip and Vernooy, l982). Shea gt_al, (1976) found no differ- ence in GVBD between oocytes from ovaries stored at ambient tempera- ture for one or two hours. Snyder (1977a) studied the effects of transport temperature on ovine oocyte maturation. No detrimental effect on maturation was reported following ovary storage for up to four hours at 22° or 1° compared to 30-37°C. This study further found similar maturation rates for oocytes placed in culture immediately following laparotomy and those from ovaries transported from an abattoir. Crosby gt_al, (1971) found no advantage by removing ovine ovaries in a room adjacent to the oocyte recovery laboratory to ensure a minimal time interval between collection and start of culture. The mammalian ovary is in a dynamic state of follicle growth and atresia under changing hormonal influences, which in turn affects the ability of oocytes to mature after liberation from the follicle. Oocytes from small (1-2 mm) porcine follicles had a lower rate of GVBD, as well as a lower rate of maturation to Met II, than oocytes 6 from medium (3-5 mm) or large (6-12 mm) follicles (lsafriri and Channing, 1975a). Oocytes from medium sized porcine follicles also had a lower rate of maturation than those from large follicles, but the difference was overcome by increasing the serum content of the culture medium. Snyder (l977b) reported no consistent differences between ovine oocytes from follicles greater than 2 mm diameter and those from smaller follicles. Moor and Trounson (1977) classified ovine follicles on the basis of their opacity, vascularization, and the integrity and uniformity of the granulosa cell layer. Following culture of oocytes within intact, nonatretic follicles, they recovered greater numbers of mature oocytes from 3-5 mm follicles than from smaller follicles. In contrast, culture of follicles considered atretic resulted in maturation rates similar to 3-5 mm nonatretic follicles regardless of size. Size of follicle from which an oocyte is recovered does not influence jn_yjtgg_maturation of bovine oocytes (Thibault gt_al,, l976a; Leibfried and First, 1979; Fukui and Sakuma, 1980). However, a higher proportion of apparently degenerating oocytes was obtained from follicles greater than 3-5 mm in diameter. This is consonant with studies of bovine folliculogenesis. Several investigators have reported faster turnover rates in large bovine follicles (Ireland gt_al,, 1979; Matton gt_a1,, 1981; Staigmiller and England, 1982). Thus, one could expect a higher proportion of large follicles to be in various stages of atresia as compared to their smaller counterparts. Choudary gt_al, (1968) reported the mean diame- ters of the largest healthy follicles and largest atretic follicles 7 were 4.1 mm and 9.5 mm, respectively, during the majority of the bovine estrous cycle. Marion §t_al, (1968) and Rajakoski (1960) utilized micromorpholo- gical characteristics observed in thin paraffin embedded ovary sec- tions to classify follicles as normal or in early or late atresia. The relative proportions of each follicle class did not differ between follicular and luteal phase ovaries. Several other reports, based on macromorphology of ovine and bovine ovaries, concluded that vesicular follicle development is largely continuous and independent of estrus and ovulation (Kammalade §t_§l,, 1952; Bane and Rajakoski, 1961; Smeaton and Robertson, 1971; Brand and de Jong, 1973). The percentage of ovine follicular oocytes that matured ig_vit§g was not affected by stage of estrous cycle (Snyder, l977b). Fukui and Sakuma (1980) investigated the relation of bovine oocyte maturation to stage of the cycle. There was no difference in jn_yitrg_maturation of oocytes from ovaries with a corpus luteum compared with those having a corpus albican (49.6% gs, 45.9%). Leibfried and First (1979) used characteristics of corpora lutea to divide ovary pairs into three classes: early luteal, luteal, and follicular. They then examined the appearance of recovered oocytes as well as ig_yitrg_maturation in relation to cycle phase. In one experiment they found a slightly higher proportion of degenerate chromatin configurations from luteal phase ovaries. In a second trial, however, oocyte morphology, in- cluding post-culture chromatin configuration, was more normal in luteal phase oocytes. Thus, the stage of the estrous cycle would not appear to influence the jg_yjtrg_maturation rate of the oocytes from a particular ovary pair. 8 Evaluation of oocytes upon liberation from follicles reveals considerable variation in amount of adhering cumulus. The presence of cumulus cells surrounding the oocyte appears to improve maturation in_ yitrg_in porcine (Tsafriri and Channing, 1975a; McGaughey, 1977), ovine (Crosby gt_al,, 1981), and bovine material (Bedirian and Baker, 1975; Fukui and Sakuma, 1980). Studies designed to investigate the requirement for cumulus cells for oocyte maturation has often involved mechanical removal of adhering cells by repeated aspiration and expul- sion through small-bore pipettes. Kennedy and Donahue (1969) provided three possible explanations for fewer oocytes reaching Met II when divested of cumulus cells: (1) removal of cumulus cells may damage the oocyte, (2) cumulus cells may be required to supply required sub- stances to the oocyte, and (3) cumulus cells may detoxify substances in the medium inhibitory to maturation of the denuded oocyte. The first reason can be eliminated by studies utilizing oocytes lacking cumulus when collected. However, the presence of cumulus may be more consistent with descriptions of jn_yiyg_matured ova (Dziuk, 1965; Lobel and Levy, 1968; Marion gt_al,, 1968). During the preovulatory period, cohesiveneSs of the cumulus cells is disrupted, but the corona radiata remains anchored in the zona pellucida (Lorton and First, 1979; Cran gt_al,, 1980). Nude oocytes, those lacking cumulus cell investment, were probably recovered from follicles in advanced stages of atresia (Henricson and Rajakoski, 1963; Hay gt_al,, 1976; Hay and Cran, 1978). ' 9 Numerous vesicles or globules within the ooplasm are character- istic of normal bovine (Fleming and Saake, 1972) and ovine oocytes (Russe, 1975; Cran gt_al,, 1980). This presents a granulated appear- ance. Uneven granulation or formation of large cellular inclusions are characteristic of degenerating oocytes from follicles in late or tertiary atresia (Peluso, 1978; Leibfried and Frist, 1979). Little information is available in the literature on the use of vital dyes to assess oocyte viability. In general, the procedures used for other cell cultures can be adapted to ova and embryos. Correlations with morphological criteria or ensuing development, as well as toxicity evaluations, have been performed with embryos for trypan blue (TpB) (Thadani §t_al,, 1982) and fluorescein diacetate (FDA) (Mohr and Trounson, 1980; Looney gt_al,, 1982). TpB is an indicator of membrane integrity (Tennant, 1964). Cells with a dis- rupted membrane permeability are unable to exclude the dye. Con- versely, normal cells are permeable to the nonfluorescent compound, FDA (Nhittingham, 1978). ,The acetate groups of FDA are cleaved by non-specific esterases inside the cell, releasing free fluorescein intracellularly. Intact-plasma membranes are impermeable to this polar compound. The resulting green fluorescence under high-energy blue or ultraviolet light has been correlated with embryo development (Church and Raines, 1980; Renard gt_gl,, l982). Oocytes selected for culture must be placed in a medium that will support development. A variety of commercial media have been tested for bovine oocyte maturation (Table 1). This variation, coupled with individual modifications of media or culture environment, makes 10 ....mwom 5pm: mmwusum cowumcaumz ocpm>.mm mo xcm553m F m4mom :OEOOPO Ezcmm 2.26mN o cowuzpom mpmconcmuwm cmmcwmumnmcx umOmOcoz .Om_-wmp .OO .~w\mupumw mopmumu muosuoca mczppzu OOOOOH Nwmp acmaeou Poopmo_owm ccOFOH ucmco sacmm wcwocoa Eacwm mpmu Foam; met-D" ANNON . Hm.mm NOOLONO O_O_O .OOOOOOOO OONOONOOO_ OO OOO NO + N_O O.EO= LOO ON NOO N ON NOON .NOOOLOO OOO OOOLN NO OOO NON + OONO NN NONE NO OOO NON + O-OO2O NF O-OO2O OO OOO NON + N_m O.OO: OO NNO O.OO= OO OOO NmN + OO_OO .1.... NO OOPON --- OO-NN NOO_ ..PO O OONOE OO OOO NO + OO_ON LOO ON NOO NO OO-ON ONO. .OOONOO OOO OONOO OO OO NOO + OONON OO OO NO_ + OO_OO ON ON NOO + ZOO _N WOO NOO + ZOO N ON OOE NO_ + ZOO LOO ON OO NO ON-ON OOOO_ .OOLOO OOO OOOLOOOOO mmmpcmocwm mcmcamoeu< Aggy mswp cowumcsumz EOOumz mcappsu cowamnaucH wucmcmemm ...AOOOOOOOOOV _ OOOON 12 comparisons between studies and, in some cases, within studies, difficult to interpret. Quirke and Gordon (1971) reported negative results in a study on jg_vjt£9_maturation of ovine oocytes in TC199 medium, TC199 supple- mented with 15% fetal calf serum (FCS) or sheep serum. Bovine folli- cular fluid supported minimal meiotic resumption while 50% resumed meiosis in ovine follicular fluid. This team of researchers reported markedly superior results (91% maturation after 40 hours) in a synthe- tic growth medium. Crosby and Gordon (1971) confirmed successful maturation in this growth medium. This culture medium also supported GVBD of bovine oocytes similar to that observed in bovine follicular fluid (Sreenan, 1970). However, Met II was reached twice as often in growth medium (59.3%) as in follicular fluid (27.6%). One of the most common additions to a basic culture medium is a source of large molecular weight protein, generally serum or bovine serum albumin (BSA). Some discrepancy is found in the literature re- garding the importance of the macromolecular source. Jagiello gt_al, (1975) increased ovine oocyte maturation from 60.0 to 77.7% in McCoys medium with the addition of 22% ovine serum. Replacement of ovine serum with FCS provided similar maturation (77.4%). These workers also reported 64.9% Met II in Diploid medium containing 12% ovine serum and 83.3% in Diploid medium with 22% FCS. Maturation of bovine ova was no different in synthetic oviductal fluid (Table l) with 1% BSA or with 3% BSA (Pope and Stephens, 1974). However, in another study, maturation was enhanced by adding 10% FCS (Pope and Turman, 1974). Leibfried and First (1980) found no differ- ences bewteen 10% FCS, 50% FCS, or 50% porcine serum addition to l3 Eagle's Basal Medium. Use of 3 mg/ml BSA in TC199 for bovine oocytes (Fukui and Sakuma, 1980) was inferior to serum addition in one report (Leibfried and First, 1979a) but not in a second (Leibfried and First, 1980). Fukui et_al, (1982) found significantly greater maturation in a modified Krebs-Ringer Bicarbonate solution (KRB) when 20% FCS was included (22% gs, 53%). However, an earlier study had obtained 71% in, yjtrg_maturation using a KRB medium without FCS (Iritani and Niwa, l977). FCS addition improved results significantly in Brinsters medium for ovum culture #3 (BMOC—3), but no improvement was observed with Ham's F12 medium (Fukui gt_al,, 1982). Other workers (Kruip and Vernooy, 1982) obtained better maturation in Ham's F12 with 1% BSA (65%) than Fukui gt_al, (1982) reported for Ham's F12 with 20% FCS (46%). Energy requirements for maturation of oocytes must be supplied by the culture media. Glucose, pyruvate and glutamine are included in most media. Few studies have examined the importance of individual culture components. The addition of pyruvate, lactate and insulin to TC199 (which contains glucose and glutamine) with 15% pig serum re- sulted in a higher incidence of pig oocyte maturation (Tsafriri and Channing, 1975a). In contrast, McGaughey (1976) reported porcine ovum maturation with glucose as the only energy source (58%) was not sta- tistically.different from controls (68%) with glucose, lactate and pyruvate. Hormones have been added to culture media in an effort to mimic 1 vivo oocyte maturation. Peripheral gonadotropins are known to surge shortly before ovulation (Schams gt_al,, 1977; Rahe gt_al,, 14 1980). Follicle stimulating hormone (FSH) has been measured in bovine follicular fluid and no correlation has been found between FSH concen- tration and follicle size (Henderson gt_al,, 1982). However, concen- trations of LH fell significantly as follicle size increased, perhaps indicating utilization of fluid LH by follicular cells. LH has been postulated as the key to jg_vivo resumption of meiosis by overcoming the action of an oocyte maturation inhibitor (OMI) (Tsafriri gt_al,, 1982). Spontaneous maturation of porcine oocytes has been inhibited, presumably through OMI action, when cultured with follicular hemisec- tions (Leibfried and First, 1980b), granulosa cells (Foote and Thi- bault, 1969; Tsafriri gt 31,, 1975b), medium in which granulosa cells had been cultured, or follicular fluid (Tsafriri gt_al,, 1976; Stone gt_al,, 1978). The inhibition could, however, be overcome by LH addition. In contrast, Jagiello gt_al, (1977) showed no effect on maturation of ovine, porcine and bovine oocytes cultured with granu- losa cell monolayers. Others have failed to demonstrate OMI activity in follicular fluid or granulosa cells on porcine (Racowsky and McGaughey, 1982) or bovine oocytes (Leibfried and First, 1980a). Addition of LH to culture media has resulted in increased in_ yjt[9_maturation rates of ovine oocytes from 60% to 72% (Jagiello gt_ al,, 1975). However, the stimulatory effect was not observed when LH was added to media containing FCS or ovine serum. Bovine oocyte maturation was inhibited by ovine LH in either McCoy's or Diploid medium containing 22% FCS. Fukui gt_al, (1982) reported no signifi- cant effect on bovine oocyte maturation when 10 ug/ml ovine LH or 2 i.u./ml human chorionic gonadotropin (HCG) was added to various media containing FCS, although some variability was noted. Percent of 15 oocytes maturing was not increased by LH addition to TC199. Rush gt a1, (1973) also reported no effect of either LH or FSH on bovine oocyte maturation. Addition of HCG to TC199 increased the proportions of matured porcine oocytes in one study (Meinecke and Meinecke-Till- mann, 1979a), while others reported porcine oocytes were not affected by gonadotropin addition (Hillensjo and Channing, 1980). The effect of gonadotropins is possibly not exerted directly on the oocyte, but through the mediation of granulosa and cumulus oopho- rus cells. One intermediate associated with gonadotropin action is cyclic AMP (cAMP) (Lindner _t__l,, 1974; Moor gt_al,, 1980). Weiss gt 31, (1976) has demonstrated a rapid increase in cAMP within ovine follicles after LH treatment. ‘Increased follicular cAMP in response to HCG was correlated withporcine follicle size, with the greatest response in large (6-12 mm) follicles (Lee, 1976). Direct addition of cAMP to oocyte culture media has resulted in reduced maturation rates (Racowsky, 1983; Rice and McGaughey, 1981). Maturation of cumulus-enclosed porcine oocytes fell from 74% to 58% with the addition of 1 mM cAMP derivative, N6,02-dibutyry1 adenosine 3',5'-cyc1ic monophosphoric acid (dbcAMP) (Rice and McGaughey, 1981). This inhibition was reversible. Maturation levels of oocytes cultured in dbcAMP-containing medium for 24 hours, then transferred to control medium for 24 hours, was not different than that for oocytes in con- trol medium for 24 hours (54.4% and 69.0%, respectively). Inhibited meiosis was also reported for ovine oocytes with l or 2 mM cAMP, and bovine oocyts with 2 mM cAMP (Jagiello gt_al,, 1975). Therefore, cAMP at the level of the oocyte appears to inhibit meiosis resumption. Its 16 action within the follicle may, however, be mediated by other cells. Tsafriri gt_gl, (1982) theorized that cAMP, the physiological inhibi- tor of meiosis, is transmitted from cumulus cells through gap junc- tions and that gonadotropins induce maturation by disrupting oocyte- cumulus cell junctions. This hypothesis was not supported by the finding that GVBD precedes demonstrative disruption of oocyte-cumulus cell communication (Moor et_al,, 1980). Moor gt_al, (1981) demon- strated induced maturation without affecting oocyte-cumulus cell coupling by adding low levels of LH to the culture system. They further stated that low levels of FSH suppressed intracellular coupling without inducing meiosis in ovine oocytes. Stimulation of cumulus expansion with FSH has also been reported in porcine and bovine oocytes (Ax and Ryan, 1979; Lenz gt_gl,, 1982; Hensleigh and Hunter, 1983). Furthermore, the FSH response was mimicked with dbcAMP addition (Schweitzer gt_§l,, 1981; Bellin and Ax, 1981). The impor- tance of cumulus expansion is not clear but it is known to be tem- porally related to nuclear maturation (Thibault gt_al,, 1975; Baker and Polge, 1976; Ainsworth gt_al., 1980). The preovulatory gonadotropin surge induces marked changes in follicular steroidogenesis (Linder §t_al,, 1974; Eiler and Nalbandov, 1977). This suggested that steroids are involved in the regulation of meiosis resumption. Prior to the gonadotropin surge, developing follicles produce primarily estradiol (Moor, 1973; Edwards, 1974; Webb and England, 1982). Estrogen levels fall abruptly in the pre- ovulatory follicle at the first detection of elevated serum LH (Moor, 1974; England gt_al,, 1981; Murdoch and Dunn, 1982). Androgen 17 concentrations in follicular fluid remain stable (Ainsworth gt_al,, 1980) or decline shortly after the LH peak (Baird gt_al,, 1981; Murdoch and Dunn, 1982). Progesterone levels, increasing during follicle growth, tend to plateau concomitant with the gonadotropin surge, only to increase again before ovulation (Bjersing gt_al,, 1972; Wheeler gt_al,, 1975; Moor §t_al,, 1975). Thus, GVBD occurs in follicular fluid characterized by declining estrogen levels and rela- tively high and constant levels of progesterone and androgen. Gerard gt_al, (1979) studied oocyte maturation by culturing whole porcine follicles jg_yjtrg, Exposure of follicles (>6 mm diameter) to HCG, FSH, or LH resulted in the expected drop in estrogen and rise in progesterone levels in follicular fluid and resumption of oocyte meiosis. However, while gonadotropins were mildly effective in inducing maturation of oocytes enclosed in <6 mm follicles, the characteristic inversion of the estrogen-progesterone ratio did not occur in these small follicles jg_yjtrg, Direct addition of steroids to culture medium has also given variable results. Inhibition of steroidogenesis did not prevent the LH-induced resumption of meiosis in ovine follicle-enclosed oocytes, but did block meiosis at the Met I stage (Moor and Narnes, 1978). Addition of estradiol to such cultures, together with the gonadotro- pin, prevented this block and improved fertilization of the oocytes. Jagiello gt_al, (1975) was not able to demonstrate increased matura- tion rates in ovine oocytes with the addition of estradiol-178 (1000 pg/ml) followed 0 to 6 hours later with the addition of LH (30 ng/ml). Neither progesterone nor estradiol-178 had a significant effect on maturation of cumulus-clad porcine oocytes (McGaughey, 1977). He did 18 report inhibited maturation of denuded oocytes with estradiol at 1.0 or 10.0 ug/ml, but this inhibition was overcome by including pro- gesterone in the medium or removing oocytes to steroid-free medium. This reversible inhibition by estradiol led to speculation that this is the mechanism by which follicles maintain oocytes in arrested meiosis. Testosterone was shown to inhibit oocyte maturation (Richter and McGaughey, 1979; Rice and McGaughey, 1981), and since granulosa cells aromatize androgens to estrogen (porcine: Bjersing and Carten- sen, 1967; ovine: Moor, 1977; bovine: Lacroix gt_al,, 1974), estradiol from granulosa cells may mediate the inhibition in small follicles known to contain high androgen levels (Carsen et 31,, 1981; Henderson gt_al,, 1982). An increased incidence of chromosomal abnormalities was observed in oocytes cultured in progesterone or estradiol containing medium (McGaughey, 1977). However, oocytes cultured in media containing both steroids exhibited a higher incidence of normal telophase I and Met II chromosomes than did control oocytes. Fukui gt a1, (1982), studying bovine oocyte maturation, reported that the effect of added hormones differed slightly according to the media tested. No significant effect was found for hormonal treatments in TC199 or modified KRB but was evident in Hams-F-12 and BMOC-3. Addition of either estradiol alone or estradiol and LH in combination to the culture medium significantly increased maturation rates. Pro- portions of matured oocytes were 47.8%, 61.7%, 61.0% and 55.2% for no additions, estradiol, estradiol + LH, and progesterone, respectively. Estradiol has also been reported to reduce maturation of cumulus l9 enclosed bovine oocytes to 12% compared to 60% for controls (Robertson and Baker, 1969). These workers reported that adding progesterone to medium TC199 increased GVBD as well as first polar body formation in denuded bovine oocytes. No effect of progesterone addition was noted by others (Rush gt_al,, 1973; Fukui §t_al,, 1982). There is some evidence that the hormones in follicular fluid, or culture media, play a role in maturation beyond the nuclear changes normally monitored. Cytoplasmic and membrane maturation are poorly understood and generally assessed by development following fertiliza- tion. Nevertheless, the rate of amino acid uptake, rate of amino acid incorporation into proteins, and resulting patterns of protein synthe- sis in oocytes maturing jn_yjtg9_have been induced by gonadotropins to approximate those of jn_yjyg_matured ovine oocytes (Narnes gt_al,, 1977; Moor and Smith, 1978, 1979; Crosby gt_al,, 1981; Moor gt_al,, 1981). Other important aspects of the culture environment include pH and osmolality of the medium, and culture atmosphere. Very little re- search has been done to define optimum values of these components for oocytes from domestic animals. Studies with mice (Brinster, 1965) and rabbits (Kane, 1974) indicated development can occur over a wide range of pH values (6.0 to 7.8). Shea gt_al, (1976) stated that bovine oocytes matured in media with pH values from 6.7 to 7.6, but the highest proportion of GVBD (80%) and Met II (69%) was obtained at a pH range from 7.0 to 7.3. This also appears to be the pH range of folli- cular fluid (Edwards, 1974; Knudsen gt_al,, l978). 20 Commonly used culture media range in osmolality from 270 to 316 m05m (Wright and Bondiol, 1981). Bae and Foote (1980) matured rabbit oocytes in medium ranging from 250 to 310 mOSm. Best results were obtained in 270 mOSm medium. Similarly, porcine oocytes matured in media with calculated osmolalities of 265 to 305 mOSm (McGaughey, l977b). However, poor maturation rates were obtained in the higher ranges, and 285 mOSm was considered optimal. This is approximately the osmolality of plasma, and similar to that of follicular fluid (Olds and VanDemark, 1957; Edwards, 1974). Two culture atmospheres, 5% C02 in air and 5% C02, 5% 02, 90% N2, have been routinely used for jn_yitrg_culture of mammalian oocytes and embryos. The reduced oxygen atmosphere of this second mixture was beneficial to development of early bovine (Tervit §t_gl,, 1972), ovine (Trounson and Moore, 1974; Wright gt_al,, 1976a) and porcine (Wright, 1977) embryos. Contradictory evidence was reported by Wright et_al, (1976b), who were unable to demonstrate a beneficial effect of lower oxygen concentrations on bovine embryos cultured in medium under paraffin oil. The oil cover may mask the difference by restricting oxygen exchange (Gwatkin, 1972). While granulosa cells may best be sustained by a gas phase rich in oxygen (Moor, 1973), mammalian oocytes appear to require an oxygen concentration of less than 20%. Survival and maturation were sharply improved by reducing oxygen concentration in hamster oocyte cultures (Gwatkin and Haidri, 1973; 1974). Porcine oocytes have been demonstrated to be less sensitive to the oxygen content during culture (Tsafriri and Channing, 1975a). The 21 percentages of oocytes with one polar body were 52.7, 43.6 and 37.5 in 20%, 45% and 95% oxygen, respectively. Nearly twice as many dege- nerated oocytes were found in 95% 02. Trounson gt_al, (1977) reported superior bovine oocyte maturation under a reduced oxygen atmosphere (Table 1). A variety of culture vessels have been used for bovine oocyte culture, including stoppered test tubes (Pope and Turman, 1974; Troun- son §t_al,, 1977; Fukui and Sakuma, 1980), media droplets under oil (Hunter gt_al,, 1972; Iritani and Niwa, 1977; Liebfried and First, 1979b), and covered petri dishes (Shea gt_al,, 1976; Newcomb gt 21,, 1978). Studies within which comparisons were made reported no differ- ences (Leibfried and First, 1980a; Enright, 1982). In Vitro Fertilization Mammalian fertilization has been thoroughly reviewed (Chang and Pincus, 1951; Bedford, 1970; Stambaugh, 1978) and no attempt will be made here to describe the physiological events involved. In yjt§9_ fertilization (IVF) has also been the subject of reviews, including recent books (Mastroianni and Biggers, 1981; Hafez and Semm, 1982). Following recognition of the need for sperm to be capacitated in a female reproductive tract prior to fertilization (Austin, 1951; Chang, 1951), several reports of IVF in laboratory animals appeared. Significant among these was documentation of live rabbit offspring resulting from IVF and transfer of a 4-cell embryo (Chang, 1959). Offspring resulting from IVF have since been reported for mice (Whit- tingham, 1968), rats (Toyada and Chang, 1974), humans (Steptoe and Edwards, 1978), and cattle (Brackett gt al,, 1982). 22 Attempts at IVF of large domestic animals have largely been dis- appointing when compared to successes in laboratory species. Baker and Polge (1976) observed no penetration by sperm in yjtrg_using por- cine oocytes recovered from follicles 1-3 hours before ovulation or ova recovered from oviducts 1-4 hours after ovulation. The sperm added to the cultures were washed ejaculated sperm, epididymal sperm or sperm recovered from the tract of inseminated estrous gilts. Sperm penetration did occur if the oocyte and sperm mixture was transferred after 4 hours of in_yjtrg_incubation to oviducts of unmated estrous recipients. They suggested that the failure of sperm penetration in yit§9_resulted from lack of sperm capacitation (Polge, 1977). Iritani gt_al, (1975) determined the developmental time-course following porcine IVF. Ovaries were obtained at slaughter and follicular oocytes aspirated and cultured for 26 to 30 hours to maturity. When combined with collected semen preincubated in modified KRB, the ovi- duct, or the uterus of an estrous sow, fertilization rates of 7.3%, 5.2% and 11.5%, respectively, resulted. Sperm penetration into the periviteline space was observed three to five hours after insemination with penetration into the vitellus after five to ten hours. The second meiotic division, with extrusion of a second polar body, was observed at 10 to 16 hours. Two pronuclei were seen at 16 to 24 hours and cleavage at 16 to 30 hours. Using similar oocyte and semen treat- ments, ejaculated and epididymal sperm were then compared (Iritani gt_ 31,, 1978). No fertilization was reported in this study following sperm preincubation in KRB. Penetration after capacitation of epidi- dymal sperm was better than that of ejaculated sperm (23% gs, 13%). 23 Formation of morphologically normal male and female pronuclei was low (27.8%). A delay in the transformation of the sperm head into a pronucleus was frequently observed. Similar observations in the rabbit lead to the suggestion that jfl_yitrg_oocyte maturation was incomplete. Synthesis of a male pronucleus growth factor (MPGF) in the egg cytoplasm during jn_yjy9_maturation may not accompany in_yit§9_ maturation (Thibault and Gerard, 1973; Thibault gt 21., 1975b, l976b). A role of ovarian steroids in this cytoplasmic maturation has been postulated (Thibault, 1977; Moor, 1978). Failure of normal fertili- zation and development in swine induced to ovulate with HCG while follicular estrogens were low supports this theory (Hunter gt 21,, 1976). Leman and Dziuk (1971), however, reported some normal em- bryonic development following transfer of oocytes collected from the ovaries of HCG-treated gilts 12 hours before the expected time Of ovulation. Normal fertilization has also been obtained following transfer of jn_yjtrg matured porcine oocytes to oviducts of insemi- nated sows (Motlik and Fulka, 1974). Thibault and Dauzer (1961) found that none of 41 ovine ova from seven ewes were fertilized following jg_yjtrg_insemination with ejacu- lated ram semen. These studies used ovulated ova. Four ova developed to the second polar body or 2-pronuclei stage following IVF of 78 ova from 38 ewes with sperm recovered from the uterus 12 to 24 hours after coitus. Using similar conditions (naturally ovulated ova and oviduc- tal-incubated semen), Kraemer (1966) reported that four of 23 ova formed a second polar body, but only one developed two pronuclei. Tubal ova, collected by flushing ewe oviducts, were successfully 24 fertilized in synthetic oviductal fluid (SOF) and Minimal Essential Medium (MEM) by jn_yjtrg capacitated sperm (Bondioli and Wright, 1980). Sperm treatment involved a 3 hour preincubation of ejaculated semen in the culture medium at 37°C before addition of ova. Sperm penetration was achieved in 9.5% of 294 ova, and cleavage in 53.6% of fertilized ova. No difference was found in the ability of SOF or MEM with no protein, 3% BSA, or 20% lamb serum to support fertilization. A high incidence of fragmentation (30 to 60%) was reported in all media. Four of six follicular oocytes from gonadotropin treated lambs extruded a second polar body in a preliminary trial of IVF following ig_yitrg_maturation of ovine oocytes (Dahlhausen gt_al,, 1980). Ram sperm capacitated in a rabbit uterus was capable of fertilizing in. vitrg_matured oocytes, as well as ovulated ova jn_yitrg (Bondioli and Wright, 1981). Penetration was observed in 22% of the oocytes and cleavage in 10% following IVF. Other groups have had limited success in obtaining fertilization following transfer of jg_vit:9_matured ovine oocytes to the oviduct of previously inseminated ewes (Crosby gt_al,, 1971: 5% 2-pronuclei; Quirke and Gordon, 1971: 25% 2-pronuclei). Woody et_al, (1961) trans- ferred follicular oocytes to mated recipient ewes without ig_yjt§9_ culture. While 25% of ova recovered 24 to 48 hours later had formed two pronuclei, none had cleaved. Conversely, 67% of ovulated ova cleaved in recipient ewes, and one pregnancy resulted following re- transfer (Woody and Ulberg, 1963). Moor and Trounson (1977) reported encouraging results after oOcyte maturation through follicular organ 25 culture. Normal blastocysts developed from less than 5% of ovine oocytes matured in follicles and then transferred to recipient ewes for 7 days when either no hormone or only FSH and LH were included in the culture medium. However, follicle-enclosed oocytes cultured 24 hours in medium containing 1 ug/ml estradiol-17B, 2 ug/ml FSH and l ug/ml LH before transfer resulted in 40% development to blastocysts. Only one of 61 ova cultured outside the follicle before transfer developed into a blastocyst, and four developed to the morula stage. Further studies confirmed adding estradiol to the medium during 3n vitrg_maturation within the follicle reduced fertilization and cleav- age abnormalities in the ovine (Moor, l978). Crosby gt_al, (1981) also reported fertilization of ig_yjtrg_matured ovine oocytes in ewe oviducts following transfer. Normal blastocysts developed from 4% of cumulus-enclosed oocytes matured outside the follicle in the absence of gonadotropins. The proportion of blastocysts increased to 30% with LH addition (10 ug/ml) during maturation. Cumulus cells were essen- tial for development. Only 3% of denuded oocytes cleaved during the 10 to 12 days in recipients. Removing cumulus cells after the first eight hours of the 24 hour maturation period before transfer resulted in 31% cleavage and 5.4% blastocyst development. IVF of bovine oocytes has received considerable attention in the last decade (Table 2). In several studies follicular oocytes have been recovered and transferred to oviducts of previously inseminated recipients. Hunter gt 21, (1972) reported 51.9% fertilization of fin yitrg_matured bovine oocytes by this method. Formation of two pro- nuclei was complete in 68.8% of the fertilized ova recovered 22 hours 26 cu m .umpmcpmcma Ne\op Amy an o .OONOONOOOO OO\__ ANO Omumcumcmn ~m\o A_v NO NON NOon 2 cum ....mwN NOOOO_OOOOO OE OOO OOOOOLO OOOOONOOLN OLNN>.mm .l.:1.AONO_O umumcumcma mp\m LOO cw mou Nm >O msowcm> Oumumpzumwm mom .PO um oncmz OLNO>.mm LO AONOFO OO_OO cowamcpmcwa o NO + Owums Ozowcm> >O macmp: Ovmumpsumnm mom was cmxmm OOONOO NO NON NOoNO OOOOOOLOOO OOOLONO .1.... 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F.Lw.llo OcaocF FpmxuomeFn cu Ocmoe OOOO «1N Fqu mnF FONOFV ..h|.w1cosz4 FFOu oFum OOOO N FFOU NuF FFNva.lwfilwm FOOFOm Fqu Ooqu ; mmnce Fqu oFuN FNOOFV .FO um :Ocomcm umxoouOOFn OOOO O Fqu mFum Fmova :oFcOum.mmAImO:OOcm pmxuouOOFO on OFacoe OOOO elm FFOu Nmum Fmova .FO um OEOO< Fqu mFuN OOOO N FF8 NuF Fmova OFmsm OOO NOFO: OOF>om FpmxuoumOFO ou Ocmoe OOOO Num FFOu OFuN FONNmFV .FO pm IsoszA FOOOOONOOFO OOOO Nue FFOu Nmnm Fwova..|FO1uO OEOO< FONOOOONOOFO o» Ocmos ; woFuNoF FFOU NuN FNmva FO pm lgmucaz OumauoumOFO on OFsgos OAOO mue FFOo qu FFOmF~1.FO um OEOO< ONOOOONOOFO o» OFOEoE OOOO One FFmo NFuN meva .FO pm FFFLO>< m=F>o mumxoouOOFO on FFOu m OOOO muF OFacos op FFOO F FNNOFV .11 OmFoa OcFucom umxooumOFO 11. OmzouO; on mumxuomeFn OFOOO : melee umauopOOFO op Fqu Nm FmNva :mFF< OOFOON OmOpm FOOOOEOOFO>OQ OFOOON LOOOOOOF NO Oco : OOFOOOm mcFuFOOmm :F OEFF OmOpm FOucmanFm>Oo 5» < ongEm NOOOO>O NOOOOO OON OF OOOOOOO FOOOO< OOOOOOOO OO OLONFOO OOF m m4mOOFO m .LOmFuzcogO N -- mnm\mmF Ozm Ozocpmm OONFFFNLON o -- -- NFOOOL OOOONOO FONOFV OOOOmgm ONFOO-N N O NN-O_ OONON NNOOOO OOOOFO OOOOOOO ONFOOON :oFNONFFFNLOO OEFF Ogm>oomm zcwwmwmm NOOOF>o NOOFOFOON OLOON=< OcF>om O m4mFNOOOOOL OON LON mmsoum NOOENOO>OF cmms ONO OFOOONOONOO :F ONOOEOZ F NOO.NO ON NON.NO ON NNOF.NO OFON OOO OOONOO NO.ON ONO O NN OOO O NO.OO OOO ON OO OOO N ON.ON ONN N O ONN N OOOONOO NOO.NO O NOO.NO NN .NO_.NO OOO OOO OOONOO NNO.ONO NON O O OO. O NON.OOV NON O ON ONN N NOO.ONV OO O N NO N OOOO Eam NA N-O mv Ogoum FNV zom NOOENOO>OH Nagy OONOOONO OFONFFOO OOFLO>o LONFO: co zou co ONFm OFOFFFoO :FONFz Omcoum Ncmspmw>cH maoFLO> ONFz mmuxooo Fo mmFucmacmLu mmOFonzm m m4m3 n (%) n (%) 1 222 (11.17) 6 (4.88) 2 1179 (59.34) 82 (66.67) 3 586 (29.49) 35 (28.45) 2 x = 5.331, p<.1 52 0%.: ammmwo N .Ncwmmgg chuFOFO maacoo u F u NOV NONON NOV NOWNOOHOO NOON ONNO ONONFNOO ONF>FNO< :OFLO>o ON :oFNOFOm OOO Ozone ONFm OOON OF OOFOFFFOO No OOOFNNOOONO FF m4mF u OOO NONON NOV _ “ONOW>M mgoom NOOENOO>ON Oquoo ONF>FNo< cOFcO>o ON :oFNOme OOO :oFNOuFNFOOOFu NOOENOO>ON ONON OF OONzuoo No mcoFNcoOoca NF mNmFNo< NOOEOZ F v F OOFOO>o Ocoum EOOFOoo ONF>FNO< :OFNO>o ON :oFNOFOm OOO coFNOuFNFOOOFu EOOFOoo OOON OF OOquoo No OcoFNNoaogm mF m4m3 Cumulus l 257 (10.88) 9 (3.64) Investment 2 1407 (59.57) 106 (66.06) Score2 3 698 (29.55) 50 (30.30) Oocyte 1 330 (35.99 17 (10.76) Cytoplasm 2 377 (41.11 89 (56.33) Score 3 210 (22.90) 52 (32.91) 1 criteria and follicle size. 2 2 X 3 2 X 8.890, p<.05 39.3141, p<.01, C = .1878 Numbers within parenthesis are percentages within a 57 TABLE 15 Effect of Interval from Slaughter to Oocyte Culture on Proportion of Oocytes in Each Classification Slaughter to Culture Time Score <4 hours >4 hours Cumulus Investment 1 11.56 10.95 2 60.26 60.10 3 28.18 28.95 mean 2.17 2.18 (n) (692) (1172) Ooplasm l 33.54 36.78 2 45.83 36.36 3 20.62 26.86 mean 1 87 1 90 (n) 430 726 58 processed at either time interval were classified with ooplasm with a score of l, and 71% had some cumulus cell investment. A relationship between ooplasm classification and viability, as assessed by vital dyes, is shown in Figure 1. A total of 891 oocytes were evaluated in 12 trials. TpB exclusion and FDA uptake were highly correlated (r = .987). Fewer oocytes were FDA+ than were TpB- in all categories. Oocytes fixed immediately after collection were predominantly in the germinal vesicle stage (66.67%, Table 16). The percentage of oocytes with an intact GV appeared to decline with higher scores, although the number of oocytes examined was small and differences were not significant. The number of oocytes in each investment and ooplasm category are shown in Table 17 for collections from ovaries tranSported to the laboratory at two temperatures. There was a tendency for more de- generate ooplasm in oocytes collected after transport on ice. No difference was found between temperatures for investment scores. Subsequent culture of the oocytes revealed transport temperature effects (Table 18). Survival (proportion which remain TpB-) during fin .11139_cu1ture was superior (p<.01) when ovaries had been kept close to 37°C. There was also a trend for more oocytes to mature to Met II after warm transport (p<.10). Changes in mean investment and ooplasm scores during culture were small (warm transport; -0.33, -0.34; cold transport, -0.29 and -0.37 for investment and ooplasm scores, re- spectively). 59 100 80 I ‘0 DD 60'“ :3 3 m 2 r- -I Z “J 'Tl U :2 35 40> CL + 20 l 2 3 OOCYTE MORPHOLOGY CLASSIFICATION *fi differs from preceding ooplasm classification (P<.01) Figure l. Oocyte viability assessments and relationship to ooplasm quality score at collection. 60 TABLE 16 Meiotic stage of Follicular Oocytes at Collection Ooplasm Score Cumulus Enclosed l 2 l 2 Number with GV1 5 18 5 8 % 83.33 75.00 66.67 53.33 Maturing2 1 2 3 5 % 16.67 11.11 33.33 33.33 Unidentifiable --- 4 1 2 Total 6 24 9 15 x2 = 3.200, n.s. x2 = 4.69, n.s. 61 TABLE 17 Effects of Transport Temperature on Oocyte Characteristics at Follicle Aspiration 1 Investment Score2 Transport 1 (%) 2 (%) 3 (%) Warm 11 (6.92) 102 (64.15) 46 (28.93) Cold 14 (8.19) 97 (56.72) 60 (35.09) 1 Ooplasm Score3 Transport 1 (%) 2 ' (%) 3 (%) Warm 97 (61.01) 57 (35.85) 5 (3.14) Cold 105 (61.40) 54 (31.58) 12 (7.02) 1 1.901 2.848, p<.1 warm = transport in an incubated container at 32-37°C; cold = transport on ice near 0°C. ’1 62 TABLE 18 Effects of Transport Temperature on Oocyte Characteristics After 13 Vitro Culture TransportTFv n TpB-2 (%) Mature3 (%) Warm 9O 79 (87.78) 23 (25.56) Cold 80 55 (68.75) 11 (13.75) 1warm = 32-37°C; cold = 0°C. 2 x2 = 9.19, p<.01 3 x2 = 3.69, p<.10 63 Maturation ratios (number mature/number cultured) for investment- ooplasm combinations are shown in Table 19. Oocytes devoid of cumulus cells (score 3) matured less often than those with surrounding cells (score 1 or 2). No maturation occurred in oocytes with score 3 ooplasm. Table 20 shows the maturation ratios for oocytes that were tested with TpB and FDA prior to culture. Significantly poorer maturation was again detected in oocytes with the poorest ooplasm rating. This difference was not found, however, among oocytes selected on the basis of TpB or FDA tests. Addition of lactate to TC199 medium was not beneficial for oocyte culture (Table 21). A significant interaction was found between medium and atmos- pheric environment during oocyte culture (Table 22). Addition of FSH tended to benefit survival when the basic medium was T0199 and the oocytes were incubated in 5% C02 in air (p<.10). No benefit was seen with PBS. No differences between media were detected in a 5% 02 environment. Survival was higher in the lower 02 environment in all cases, though statistical differences were found only for TC199 and PBS + FSH. Effects of these culture conditions on maturation can be seen in Table 22. The percentage of 02 in the atmosphere did not alter maturation ratios. The only media effect found was with FSH addition to PBS which resulted in fewer mature ova (p<.05). 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Survival x2 = 9.84, p<.05. 3 2 Maturation x = 2.79, n.s. abcRatios lacking a common superscript differ (P<-05)- 72 Xenogenous fertilization was attempted using the information gained through jg_vitrg_maturation experiments. The first trial was an attempt to demonstrate the benefit of a culture period prior to attempted fertilization in the rabbit oviduct (Table 26). No differ- ences were detected, using four to five rabbits for each culture group, in the number fertilized. Higher recovery rates of oocytes were experienced with longer culture times. Having determined the maturation percentage in various media, information was sought on fertilization rates following culture. Two to three replicates were performed for each of the media in Table 27. No statistical differences were detected among media in recovery or fertilization of ova. Failure to obtain fertilization in oocytes after 24-hour culture in FSH-containing medium seems noteworthy. Three media were tested in an atmosphere of 5% 02 at 37°C (Table 28). Again, there were no differences among media. Fertilization (l0%) was achieved after in vitrg_maturation in an FSH-containing medium. A comparison of data in Table 30 with that in Table 29 indicates a benefit to fertilization following culture in 5% 02 rather than 18%. In vitro maturation at 39°C rather than 37°C appeared to improve -subsequent xenogenous fertilization percentages (Table 29). All tested media supported fertilization equally well. Oocyte recovery was complete from the two rabbits used to test fertilization after culture in mPBS with FCS. Parthenogenesis was not induced in the XF system following in_ vitro maturation at 37° or 39°C. One oocyte (3.0%) inseminated with dead sperm formed two polar bodies but lacked pronuclei or evidence of 73 TABLE 26 Effects of In Vitro Culture for Varying Lengths of ~ Time on—Subsequent Xenogenous Fertilization Hours in Recovery (7) Fertilization (%) Culture Ratio ° Ratio 0 48/114a (42.10) 3/48 (6.25) 24 128/237b (54.01) 22/128 (17.19) 36 64/85c (75.29) 10/64 (15.62) x2=21.90, p<.01 x2 = 3.43, n.s. abc Ratios with different superscripts differ (p<.05). 74 e-=md _e\a: O.N .mcowwvucou mamm mnu gmvca vmumnaucw mmuxuoo mo mpasmm menu on moan; cowuogaums umcpEmemv apmaop>mga acmpmpoamgpxmp .m.= .me.e u Nx .m.= .mm.F u Nx FN\o Ao.msv m~\_m szd + mma e.me AP.FFV m_\N Am.mev m~\mp mad mF\o Am.eev mm\mp mime + mm_ue m.mm Am.o_v mop\F. Ap.mov ~m_\mop mmpue eremmmfiw.... :5 8mm”... s 5%. .82 P L.< cw Nopuxm twee: 0.5m be 6.58: 636.26) cw umcsumz ogpw> :H m>o mo momumm :oPHMNP_Pmem msocwmocmx mm MAm

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