IN vmzo ovum MATURATION AND ANESTRUS ovuumon INDUCTION m SHEEP Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DAVID ANDREW SNYDER 1977 Date This is to certify that the thesis entitled In Slim Ovum Mnturction and Anestrus Ovulation Induction in Sheep presented by David Ardrow Smdor has been accepted towards fulfillment of the requirements for the Ph . D . Animal Husbandry degree in a]? 10 August 1977 0-7639 I Major professor IN VITRO OVUM MATURATION AND ANESTRUS OVULATION INDUCTION IN SHEEP By David Andrew Snyder A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1977 Ch. 9'- I": 1”,. rot!‘ P. D e P . \‘m . ' "fa .‘re‘ “*1;- ABSTRACT IN_VITR0 ovum MATURATION AND ANESTRUS OVULATION INDUCTION IN SHEEP By David Andrew Snyder Experiments were conducted to determine the effect of various endogenous, cyclic reproductive patterns on the ability of sheep follicular oocytes to mature in gi££g_and to determine the ability of FSH, HCG, testosterone, and estradiol to induce estrus and ovulation in the anestrous ewe. A total of 103 ova, which were recovered from ovaries collected at slaughter and stored for approximately three hours during transport to the laboratory, were used to determine the most efficient means of storage. Percents ova maturing were 16.7, 50.0, and 37.8 for ovaries transported at 30-3700, 21°C, and 1°C, respectivelyu Ova recovered from ovaries that were stored at 21°C were more normal in appearance than those from ovaries that were stored at 1°C. For subsequent studies, ovaries were stored at approximately 21°C. To determine the effect of 10 or 20% Fetal Calf Serum (FCS) added to Hams F10 culture media for ovum maturation, 162 ova were recovered from anestrous ewes at laparotomy and at slaughter. Very little differences were found among the culture media (57.6, 51.3, and 53.1% maturation for Hams F10, Hams F10 with 10% FCS, and Hams F10 with 20% FCS, respectively). For subsequent studies, Hams F10 alone was used David Andrew Snyder because of its highly defined nature. The time of ovum maturation in 31539 was determined using 77 ova recovered from anestrous ewes at slaughter. The time sequence of maturation beginning with removal of the ovum from its follicular environment was similar to that observed in vivg_following the endogenous LH surge. However, 25% of the ova were atretic upon recovery and an additional 20.5% became atretic during the first 12 hours of culture. An additional 319 ova were recovered at laparotomy and slaughter from cycling, anestrous, pregnant, and prepuberal ewes. No consistent differences in maturation rate were found among ova recovered from ewes under various endogenous hormonal influences or from CL bearing or opposite ovaries. There were also very little differences between large (> 2 mm) or small (< 2 mm) follicles of origin. Ovulation was induced in anestrous ewes with 5 mg FSH or 5 mg FSH and 100 in HCG. However, coincident estrus was not exhibited. Estradiol and testosterone (l mg/da each) were not effective in the induction of estrus without progesterone pretreatment. Following a five day progesterone pretreatment (20 mg/da), ovulation was inhibited with estradiol but not with testosterone. Bi VITRO OVUM MATURATION AND ANESTRUS OVULATION INDUCTION IN SHEEP By David Andrew Snyder A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1977 ACKNOWLEDGEMENTS Special acknowledgement is due to Dr. Robert H. Douglas, Dr. R. A. Merkel, and Dr. Gabel H. Conner for their advice and encouragement throughout my program. Appreciation is expressed to Dr. Ronald Nelson, Chairman of the Department of Animal Husbandry, for financial support during the course of this study. Appreciation is also expressed to Wolverine Packing Company, Detroit, Michigan, for allowing me to collect samples in their plant. Finally, greatest appreciation is expressed to my parents, Mr. and Mrs. Andrew J. Snyder, for their ongoing advice and encourage- ment and especially to my wife, Renate, for moral uplifting, technical assistance, and financial support. ii TABLE OF CONTENTS INTRODUCTION..................................... ....... .. IN XIIRQ_MATURATION 0F FOLLICULAR 00CYTES................. Literature Review.................................... Materials and Methods................................ Results......... ..... ................................ DiSCUSSionOOOOOOOO......OOOOOO0.000.000.0000. ..... ... INDUCTION 0F ESTRUS AND OVULATION IN ANESTROUS EWES ...... .. Literature Review.................................... Materials and Methods................................ Results.......... ...... ..... ...... ............. ...... Discussion............ ....... ............ ....... ..... SUMMARY AND CONCLUSIONS................................... APPENDIX ......... . ............ . ........... . ............ ... BIBLIOGRAPIH.........OOOOOOOOOOOCOOOO ........ 0.... ........ iii 15 22 33 38 38 40 42 43 46 48 51 58 TABLE 10 11 LIST OF TABLES Commonly used tissue culture media............. ....... Effects of storage temperature, culture media, and ovary of origin on subsequent in_vitro ovum maturation: experimental variables........... ...... .. Effects of various culture media on in vitro ovum maturation: experimental variables............. ...... Time sequence of i§_vitro ovum maturation: experimental variables................................ Effects of ovum collection on various days of the estrous cycle and ovary of origin on in vitro ovum maturation: experimental variables................... Effects of the luteal state and ovary of origin on in_vitro ovum maturation: experimental variables..... Effects of puberty, ovary of origin, and follicle size on in vitro ovum maturation: experimental variables.........OO‘I.........OOOOOOOOOOOOOI.0.0.0.... Effects of luteal state, ovary of origin, and follicle size on in_vitro ovum maturation: experimental variables................................ Effects of storage temperature, culture media, and ovary of origin on subsequent in_vitro ovum maturation.....OOOOOOO0.00.0000.........OOOOOOOOIOOOOO Summary of the main effect of ovary storage temperature on subsequent in vitro maturation of follicular oocyteSOOOOOOOOOOOCC....00...........IOOOIOOOOOOOOO... Summary of the main effect of culture media on in_ vitro ovum maturation.. ....... ............. ......... .. iv 16 18 20 20 21 21 21 23 23 24 TABLE 12 13 14 15 16 l7 l8 19 20 21 22 23 24 25 Summary of the main effect of ovary of origin on subsequent in_vitro maturation of follicular oocyteSOC......O....O...................O.....00...... Effects of various culture media on in vitro ovum maturation..............O.....O..........O0...... Summary of the main effects of culture media and follicle size of origin on in_vitro ovum maturation............................................ Time sequence of in_vitro ovum maturation............. l§_vitro maturation of ova collected at laparotomy on various days of the estrous cycle and from CL bearing or opposite ovaries........................... ln_vitro maturation of ova collected at slaughter from cycling and prepuberal lambs..................... In_vitro maturation of ova collected at slaughter from ewes in various luteal states.................... In vitro maturation of ova collected from cycling and anestrous ewes and lambs.......................... ln_vitro maturation of ova collected from pregnant, cycling, anestrous, and prepuberal ewes and lambs..... In vitro maturation of ova collected from pregnant, cycling, anestrous, and prepuberal ewes and lambs, arranged by ovary of origin........................... Ovulation induction with FSH and H00 ........... ....... Time of ovulation after FSH and HCG.. ............... .. Induction of estrus with testosterone and estradiOlOOO................................ 0000000000 Induction of estrus and ovulation with progesterone, estradiol, and testosterone. ....O... ... ......O. ....... 24 26 26 28 29 29 30 30 31 31 43 44 LIST OF FIGURES FIGURE 1 Sheep and swine surgery table......................... 49 vi INTRODUCTION The mammalian ovary is a seemingly endless storehouse of genetic material capable of being transmitted to the next generation. Most of the reproductive potential of the ovary is never realized due to wastage through various natural causes. Although the ovary contains many oocytes that have the ability to mature and ovulate at any partic- ular time, only a few will do so and only at specific times. Two methods of increasing the utilization of the ovaries reproductive potential would be to induce ovulation of viable oocytes at times other than those normally dictated by the animal or to place oocytes into an artificial environment that would preclude natural degeneration and allow maturation with continued development. The sheep ovary is in a dynamic state of follicle growth and atresia throughout its reproductive life. Oocytes within those follicles represent a ready source of genetic material that can be harvested with relative ease. One of the objectives of the present study was to determine the effects of the endogenous hormonal state of the ewe on subsequent in vitro develoPment of follicular oocytes. Through environmental influences the ewe undergoes a period of seasonal anestrus. This can be interrupted with appropriate hormonal treatments allowing the ewe to conceive at a time other than the normal breeding season. In this study, the efficiency of low doses of gonadotropins and steroids for the induction of estrus and ovulation l 2 was determined in anestrous ewes. IN_VITRO MATURATION OF FOLLICULAR OOCYTES Literature Review Oocytes within the ovaries of most mammals initiate meiosis in pre-natal or early post-natal life (Thibault, 1972). Development is arrested at the dictyate stage of prophase in meiosis I until a few hours before ovulation when an endogenous gonadotopic surge initiates the resumption of maturation to metaphase of meiosis II (Donahue, 1972 and Thibault, 1976). The following is a review of some of the factors that control oocyte maturation. When possible, species differences will be indicated but with the present state of knowledge these have not been fully elucidated. Oocyte maturational stages have generally been evaluated using the chromatin configuration. For the purposes of this review the following abbreviations (Baker and Neal, 1972) will be used: GV oocyte in the dictyate stage of Prophase I with a germinal vesicle present. GVBD germinal vesicle breakdown indicating resumption of meiosis beyond the dictyate stage. Met I oocyte in which maturation has progressed beyond the dictyate stage but not to the point of extrusion of the first polar body. 3 4 This will include oocytes in metaphase I, anaphase I and telophase I. Met II oocyte with chromatin in metaphase configuration with a polar body present. Early work has shown that explantation of the oocyte from its follicular environment into a simple culture medium is sufficient to cause the resumption of meiosis up to Met II (Pincus and Enzmann, 1935; Pincus and Saunders, 1939; Chang, 1955; Edwards, 1965a, b; and Foote and Thibault, 1969). It was further demonstrated by these investigators, and confirmed by others (Jagiello, Miller, Ducayen, and Lin, 1974; Shea, Latour, Bedirian, and Baker, 1976) that the rate of maturation in yitgg was very similar to the rate of intra-follicular maturation in givg_following the endogenous LH surge or adequate stimulation with an exogenous LH source. Experimental evidence has shown that cow and pig oocytes cultured in direct contact with granulosa cells do not resume meiosis in vi££g_(Foote and Thibault, 1969; Tsafriri and Channing, 1975). In this same study, follicular fluid was also found to be inhibitory to oocyte maturation, however, this inhibition was overcome by the addition of 5 ug/ml LH to the culture media (Tsafriri, Pomerantz, and Channing, 1976). A 50:50 mixture of Tyrodes solution plus 1 mg/ml BSA and homologous follicular fluid supported in gi££g_maturation of bovine follicular oocytes only to Met I (Hunter, Lawson, and Rowson, 1972). This was attributed to inadequacies in the medium. However, in light of the findings of Tsafriri, 25 31. (1976), there appears to be an inhibition of matura- tion by bovine follicular fluid. These studies indicate that a substance associated with the follicular environment is responsible 5 for the prolonged prophase arrest observed in mammals. Some of these early workers used a variety of crudely prepared media for oocyte culture: sterile rabbit blood plasma (Pincus and Enzmann, 1935); human serum (Pincus and Saunders, 1939); a 50:50 mixture of rabbit serum and Ringer-Lock's solution (Chang, 1955); or human follicular fluid (Edwards, Bravister, and Steptoe, 1969). Resumption of meiosis with maturation to Met II was achieved in each of these media. However, results were quite variable due to their undefined nature. The use of chemically defined media facilitated physiological studies with in vitro cultured oocytes. Some of the most common commercial media are shown in Table 1. Addition or deletion of various constituents to or from these media make comparisons between studies and, in some cases, within studies, difficult. One of the most common additions to a basic nutrient media is a source of large molecular weight protein. This is usually in the form of serum or bovine serum albumin (BSA). A lower rate of oocyte degeneration was found with the addition of 1% BSA to a Kreb's bicarbonate media which would indicate that the macromolecule provides either a protective or supportive function. Some discrepancy is found in the literature regarding the importance of the macromolecular source and the macromolecular source that yields the best results. Shea, Baker, and Latour (1976) found a greater number of rabbit oocytes undergoing GVBD and maturing to Met II in TCM 199 plus 10% rabbit serum (92% and 82%, respectively) than in TCM 199 plus 3% BSA (73% and 54%, respectively). In contrast, Cross and Brinster (1970) found no difference in mouse oocytes maturing to Met II in either TCM 199 plus 15% calf serum (86.7%) or TCM 199 plus 6 TABLE 1. Commonly used tissue culture media. b c d CH1 CH2 BMOC3 Hams F10 TCM 199 Balanced Salt Solution + + + + + Energy Sources Pyruvate Lactate Glucose +++ +++ +++ Macro Molecules BSA + + PVP + Amino Acids Alanine + Arginine + Asparagine Aspartic acid Cysteine + Cystine Glutamic acid Glutamine + + Glycine Histadine + Hydroxyproline Isoleucine + Leucine Lysine Methionine + Phenylalanine + Proline Serine Threonine Tryptophan Tyrosine Valine + ++ ++++ +++++ ++ ++++ +++++++++++++++++++ +++++++++++ :Haidri and Gwatkin, 1973. Grand Island Biological Company, 1976-1977 catalog, p. 123. Cl-lam, 1963. dMorgan, Morton, and Parker, 1950. 7 3% BSA (96.7%). No difference was found by Kennedy and Donahue (1969) in the ability of human oocytes to mature to Met II in either TCM 199 with serum (51%) or Hams F without serum (63.2%). Hams F 0 alone 10 l or with 0.4% or 3% BSA were equally effective in supporting 22% maturation of human follicular oocytes to Met II (Zamboni, Thompson, and Smith, 1972). McGaughey and Polge (1971) demonstrated greater maturation of pig follicular oocytes in medium 199 with 1-10 mg bovine plasma albumin (BPA) per m1, compared to Hams F10 with 1-10 mg BPA per ml. The maturation of bovine ova was no different in SOF medium with 1% BSA or with 3% BSA (Pope and Stephens, 1974). When polyvinylpyro- ladine (PVP) replaced BSA as a macromolecule source there was no decrease in maturation of mouse follicular oocytes (Haidri, Miller, and Gwatkin, 1971). However, in subsequent studies (Haidri and Gwatkin, 1973) involving maturation of oocytes from large hamster follicles, it was not possible to replace BSA with PVP. Energy requirements for maturation of oocytes must be supplied by the culture media. The addition of pyruvate, lactate, and glucose to Krebs-Ringer solution was as effective in supporting in 21532_ maturation of human oocytes as Hams F10 which contains glucose, pyruvate, and glutamine as energy sources (Kennedy and Donahue, 1969). Maturation of rat oocytes in balanced salt solution (31%) was shown by Zeilmaker and Verhamme (1974) to increase slightly with the addition of pyruvate (39%), and more so with the addition of lactate (47%). The addition of pyruvate, lactate, and insulin to TCM 199 (which contains glucose and glutamine) with 15% pig serum resulted in a higher incidence of maturation in pig oocytes (Tsafriri and Channing, 1975b). 8 Gwatkin and Haidri (1974) found that with the addition of several amino acids (CH-2 media, Table 1), BSA, pyruvate, lactate, and glucose could be eliminated from the culture media. This may reflect the ability of the ovum to metabolize amino acids for macromolecule and energy sources. The nutritional role of granulosa cells in oocyte maturation has been investigated. No difference was found between denuded or cumulus covered oocytes from rabbits in their ability to utilize glutamine (Bae and Foote, 1975) or from rats in their ability to utilize glucose, lactate, pyruvate or oxalacetate (Hillensjo, Hamberger, and Ahren, 1975) as energy sources. Intact cumulus cells were necessary for the incorporation of uridine into the nucleus of mouse follicular oocytes for RNA Synthesis (Wassarman and Letourneau, 1976). McGaughey (1977) showed approximately 50% lower maturation of pig oocytes without granulosa cells compared to those with the granulosa cells intact. The mammalian ovary is in a dynamic state of follicle growth r and atresia with changing luteal and other hormonal influences, all of which may effect the ability of follicular oocytes to undergo maturation. Early work with the rabbit (Chang, 1955) and mouse (Edwards, 1962) showed no differences between small and large follicles of origin or between pregnant and pseudopregnant animals in subsequent oocyte maturation in gitrg, When human oocytes were collected from polycystic ovaries, ovaries at various stages of the menstrual cycle, during pregnancy, or several years after hysterectomy, no correlation was found with maturation in_vi££9_(Edwards, 1965b). This was confirmed by Shea, Baker and Latour (1975) in the human and by Suzuki and Mastroianni (1966) in the Rhesus monkey. Oocytes from immature 9 rats were also shown to mature in xiE£g_(Niwa and Chang, 1975). In contrast, Szybek (1972) demonstrated an increase in the diameter of follicular oocytes from 11 to 17 days of age in mice with a concurrent increase in in_vi££2_maturability. Mouse oocytes within medium sized antral follicles respond to HCG both in vivg_and in zi££g_with the resumption of meiosis. However, maturation is arrested at Met I while those in large antral follicles mature to Met II (Neal and Baker, 1973). Lee (1976) demonstrated a four-fold increase in HCG binding sites in granulosa cells from small (1-2 mm) to medium (3-5 mm) pig ovarian follicles and a 26-fold increase in cells from small to large (6-12 mm) follicles. GH-l medium (Table l), with pyruvate, lactate, and glucose as energy sources, supported the maturation of hamster oocytes from small follicles, but the addition of BSA and eight more amino acids (CH—2, Table 1) was necessary to support maturation of oocytes from large follicles (Haidri and Gwatkin, 1973). A correlation between follicle size or oocyte diameter and the subsequent ability of the oocyte to mature in 21552 was also demonstrated in the hamster (Iwamatsu and Yanagimachi, 1975). Oocytes from follicles 126-166 um in diameter or oocytes of 70-78 um in diameter showed no in_gi££g_maturation while 70-93% of those from 202-710 um follicles or 79-91 um in diameter were in Met II after 12 hours. Prepubertal hamsters less than 22 days of age had only follicles less than 150 u in diameter and showed no in zi££g_oocyte maturation. Oocytes from small (1-2 mm) pig follicles had a lower total rate of GVBD and a lower rate of maturation to Met II than oocytes from medium (3-5 mm) or large (6-12 mm) follicles (Tsafriri and Channing, 1975b). Oocytes from medium sized follicles also had a lower rate of maturation to 10 Met II than oocytes from large follicles, but this difference was overcome by increasing the pig serum content of the culture medium from 15% to 50%. Although Thibault, Gerard, and Menezo (1976) found no difference in in yi££g_oocyte maturation between follicle sizes in the calf and rabbit, they demonstrated a definite correlation in the monkey. Oocytes from small follicles (less than 1 mm) failed to mature and those from medium sized follicles (1 mm to 1.4 mm) matured only to Met I while those from large follicles (greater than 1.5 mm) matured to Met II. The inhibitory effects of granulosa cells on oocyte maturation were found to be correlated with follicle size of origin (Tsafriri and Channing, 1975a). Granulosa cells from small follicles were ten times more potent than granulosa cells from large follicles. These studies clearly indicate an effect of follicle size of origin on subsequent in 22532 maturation of oocytes. However, they also show that more complete media can mask these endogenous effects. The factors respon- sible for this correlation between oocyte maturation and follicle size remain to be determined. Changes in intrafollicular protein composition, in relation to follicle development, have been demonstrated. Andersen, Kroll, Byskov, and Faber (1976) found a correlation between increases in follicle size and increases in the concentration of «xmacroglobulin in cow follicles. Slow migratingcx-globulin was shown to increase with early oocyte maturational stages in the pig (McGaughey, 1975). Intracellular protein synthesis is necessary for in gi££g_maturation of mouse oocytes (Nassarman, 1974; Wassarman, Josefowicz, and Letourneau, 1976). Proteins synthesized early in maturation are concentrated in the ll nucleus and, although not required for GV breakdown, are necessary for completion of meiosis I and progression to Met II (Wassarman and Letourneau, 1976a; Schultz and Wassarman, 1977). The necessity of 5-10% 02, in the culture atmosphere, was demon- strated for maximum in gi££g_maturation of oocytes from mice (Haidri, Miller, and Gwatkin, 1971) and from hamsters (Gwatkin and Haidri, 1974). This requirement, however, was found necessary only for initiation of GVBD in rat oocytes, which would continue maturation when transferred to an 02 free atmosphere (Zeilmaker and Verhamme, 1974). Pig oocytes matured best in an atmosphere with 20% 0 but 45% and 95% 02 atmos- 2» pheres were also capable of supporting maturation (Tsafriri and Channing, 1976b). The mechanism of gonadotropic stimulation of oocyte maturation in_vi!g_has been investigated using the in giggg system. Early workers added pituitary extracts to their culture media and demonstrated the lack of a direct stimulatory effect on the resumption of meiosis (Pincus and Enzmann, 1935; Chang, 1955). It has been postulated that gonadotropins exert their effect through intermediates such as steriods, c AMP, or prostaglandins. Meiotic division of rat oocytes within intact follicles in vitro is increased by the addition of LB or prostaglandin E to the culture medium or by microinjection of c AMP 2 (Tsafriri, Lindner, Zor, and Lamprecht, 1972). This effect was not seen with prolactin, progesterone, 20 a dihydr0progesterone, oestradiol -l73, linolenic acid or adenosine -5' - monophosphate. These findings suggest that a multistep process is involved in the stimulation of oocyte maturation with c AMP being an intrafollicular messenger. Weiss, Seamark, McIntosh, and Moor (1976) demonstrated a rapid increase 12 in c AMP production within sheep follicles after treatment with LH, HCG, PGE2 or noradrenalin. In the pig, increased c AMP in response to HCG was correlated with follicle size (Lee, 1976). Cyclic AMP showed a 1.5, 3.7, and 13-fold increase for small (1-2 mm), medium (3-5 mm), and large (6-12 mm) follicles, respectively, as compared to untreated controls. Hillensjo (1975), using follicle enclosed rat oocytes, found an increased GVBD response to LH when a phosphodiesterase inhibitor was also added to the medium. Jagiello, Ducayen, Miller, Graffeo, and Fang (1975) added c AMP to oocyte cultures and stimulated human and monkey oocyte maturation in.McCoy's medium, whereas maturation of cow and sheep oocytes was inhibited in Diploid medium. Differences in effect may be the result of dosage differences since Hillensjo (1975) demonstrated stimulation of GVBD in follicle enclosed rat oocytes in response to 1 mM dibutyryl c AMP, but no effect with 5-10 mM dibutyryl c AMP. In contrast to this work, Stern and Wassarman (1973), and Wassarman (1974) have demonstrated the ability of 0.4 mM dibutyryl c AMP to block maturation of mouse oocytes prior to GVBD. The enhance- ment of this inhibition by disulfide reducing agents led these investigators to conclude that c AMP acts to prevent dissolution of the nuclear (germinal vesicle) membrane (Wassarman and Turner, 1976). The suggestion has been made that fluctuating levels of intrafollicular c AMP may be active in controlling oocyte maturation in the mouse (Wassarman, Josefowicz, and Letourneau, 1976). Intrafollicular progesterone levels vary in a specific pattern throughout the menstrual cycle in humans (McNatty, Hunter, McNeilly, and Sawers, 1975). During the follicular phase, these investigators 13 demonstrated highest progesterone levels in the large follicle population, while during the luteal phase the small follicle population had the highest progesterone. Rush, Tibbits, and Foote (1973) found no effect of LH and FSH, progesterone, or LH, FSH, and progesterone on the in yiggg_maturation of oocytes from puberal heifers. Progesterone (10 ug/ml) added to Hams F or medium 199, each with 10 10% fetal calf serum, had no effect on germinal vesicle breakdown in human oocytes (Shea, 25 al., 1975). However, a significant increase in maturation of prepubertal monkey oocytes to Met II was achieved with the addition of 25 ug/ml progesterone with or without 8 ug/ml c AMP to Hams F10 with 15% fetal calf serum (Gould and Graham, 1976). No increase in maturation rate was found with c AMP alone. Progesterone (32 ug/ml) added to a basic salt solution containing pyruvate or glutamine as energy sources, decreased the time of matura— tion of rabbit oocytes from large follicles (Bae and Foote, 1975). However, no effect of progesterone was seen on oocytes from small follicles or with LH on oocytes from large or small follicles. No effect on in gi££g_maturation of cow oocytes resulted from the addition of progesterone or progesterone and estradiol to BMOC-3 media (Table 1) compared to BMOC-B media without steroids (McGaughey, 1977). Addition of estradiol -17B also had no effect on pig oocytes with granulosa cells intact. However, maturation of denuded oocytes was inhibited. LH or PGE2 caused an increase in both GVBD and glycolysis in rat Graafian follicles in gi££g_(Hillensjo, 1975). LH was shown to over- come the inhibitory action of follicular fluid on oocyte maturation (Tsafriri,_g£_al., 1976). Addition of LH to a balanced salt media, 14 containing pyruvate or oxalacetate as energy sources, decreased the oxygen uptake of rat oocytes (Hillensjo, g£_§l,, 1975). LR and prostaglandins of the E series were shown to increase sheep and monkey oocyte maturation in_vi££g (Jagiello, Ducayen, Miller, Graffeo, and Fang, 1975). However, this study was complicated by high concentrations of steroids in the serum used as a supplement and by differences in the basic culture media, making it difficult to reach conclusions on the mechanisms of maturation. Most studies of in;!}££2_ovum maturation involve ova that are collected at laparotomy or immediately after ovariectomy or death of the donor. However, in some studies, especially with ova from domestic animals, ovaries are collected in a commercial abattoir and must be transported to a laboratory for ovum recovery and culture. Jagiello, §£_§l,, (1975) collected sheep and cow ovaries at slaughter, washed them with sterile saline, and transported them in dry containers for about 30 minutes. They achieved maturation rates up to 100% using this system. With pig ovaries transported in sterile saline with penicillin and streptomycin at room temperature for up to five hours, Tsafriri and Channing (1975b) achieved up to 82% subsequent ova maturation. Shea, Latour, Bedirian, and Baker (1976) transported cow ovaries with no precautions to maintain body temperature and found no difference in GVBD between one and two hour storage times (88% vs 86%, respectively). In another study (Shea, Baker, and Latour, 1976) a significant decrease in GVBD in rabbit ova occurred between 0.5 hour and two hour storage times (88% vs 12%, respectively). 15 Materials and Methods Sheep ovaries were to be collected for much of this research along the slaughter line at WOlverine Packing Company, Detroit, Michigan. Storage time for transport to the laboratory was two to four hours depending on conditions. Because most studies in the literature involve only 30 or 40 minute storage times, little information was available on the transport and storage of ovaries. A.preliminary study was therefore undertaken to determine the optimum storage temperature for the purposes of in gi££g_ova maturation studies. Sixteen pairs of ovaries were collected along the slaughter line. Upon removal from the carcass, each pair of ovaries was sutured together, rinsed with sterile saline containing 100 iu penicillin/ml, and placed in one of three flasks containing sterile saline and penicillin. Flask l was in a waterbath at 370 and was stored in a styrofoam box with warm water bottles to maintain the temperature during transport to the laboratory. Flask 2 was a thermos bottle and was transported with no attempt to regulate the external temperature. Flask. 3 was transported in a styrofoam box filled with ice. Approximately 30 minutes were required to collect the ovaries and storage time was three hours. Upon arrival at the laboratory, the temperature of the storage saline was 30°C, 21°C, and 1°C in flasks 1, 2 and 3, respectively. Ovaries were removed from the storage solution in a warm room and all visible follicles were aspirated with a 25 ga needle and a 1 ml syringe. Aspirates were placed in the chambers of Lab—Tech tissue culture chamber slides in 0.25 ml of Hams F with 100 in penicillin/ml, 10 6.7 iu heparin/m1, and either 10% fetal calf serum (FCS), 20% FCS, 16 10% FCS plus 10 in human chorionic gonadotropin (HCG)/ml or 20% FCS plus 10 in HCG/m1. These were cultured in an atmosphere of 5% 002 in air at 37°C for 24 to 48 hours. Following culture, cumulus cells were removed by treating the ova with 10% hyaluronidase for five minutes, followed by repeated passage through a drawn pipette. Ova were mounted on glass slides, fixed in 10% neutral formalin for 20 minutes, stained with 1% orcein in 45% acetic acid, and examined by light microscopy. Maturational stages were determined by chromosomal configuration as described by Donahue (1972). Experimental variables involved in this study are shown in Table 2. TABLE 2. Effects of storage temperature, culture media, and ovary of origin on subsequent in_vitro ovum maturation: expertmental variables. Storage Temperature Culture Media 923$! 300 - 37°C Hams Floa + 10% FCSb Corpus Luteum Bearing 21°C Hams F10 + 20% FCS Opposite 1°C Hams F10 + 10% FCS + 10 iu/ml HCGC Hams F10 + 20% FCS + 10 iu/ml HCG aAlso contains 100 iu penicillin/m1 and 6.7 iu heparin/ml. Heat inactivated Fetal Calf Serum, (GIBCO), % by volume. uman Chorionic Gonadotropin (Ayerst). Effects of Various Media The purpose of this experiment was to compare the ability of Hams F10, alone or with two levels of fetal calf serum added, to support in_vitro maturation of sheep follicular oocytes. To reduce the variables involved, only ova from anestrous ewes were used in 17 this study. In the first portion of this study ova were collected from anestrous ewes at laparatomy. To detect estrus a vasectomized ram, with oil containing colored dye applied to his brisket, was penned continuously with a flock of 25 ewes. Ewes were examined daily for fresh breeding marks. Only ewes which had not shown estrus for 25 days and that did not have evidence of luteal tissue on their ovaries were used for this study. Ova were collected from 15 ewes. Mid-ventral laparotomy was performed and the ovaries exposed for follicle aspiration with a 1 ml syringe and a 25 ga needle. Aspirates were grouped by follicle size (greater than 5 mm, 2-5 mm, or less than 2 mm) and ovary of origin, and placed into the chambers of an 8 chamber tissue culture chamber slide (Lab-Tech) in 0.25 ml of media (Hams F 10’ Hams F + 10% FCS, or Hams F + 20% FCS randomized separately for 10 10 left and right ovaries). Chamber slides were placed immediately in a desicator jar maintained at 37°C in a waterbath until completion of surgery, at which time they were transferred to an atmosphere controlled incubator. Incubation was for 36 hours, at 37°C in a saturated 5% 002 in air atmosphere. After culture ova were recovered from the culture chambers under a dissecting microscope at 10-15X. Those ova with portions of the cumulus oophorus intact were placed in depression slides in a fresh 1% hyaluronidase solution for three to five minutes and then cleaned mechanically by repeated passage through a drawn pipet. Ova were then placed on the center of a glass slide and a cover slip with a spot of petroleum jelly on each corner was applied and depressed until the ovum was slightly squashed. Mounted ova were fixed 18 in 10% neutral formalin for at least 20 minutes, stained with 0.5% aceto orcein for 10 minutes, and examined by light microscopy. Ova were classified as atretic, germinal vesicle, metaphase I, or metaphase II based on general morphology and chromatin configuration (Donahue, 1972). Ovaries were also collected from ewes at slaughter at a local slaughter house. Forty pairs of ovaries were collected on two separate days. These were immediately placed in sterile saline with 100 in penicillin/m1 and maintained at room temperature for the duration of transport. The minimum transport time to the laboratory was two hours. At the laboratory follicles were aspirated and ova were cultured and examined as described above. Results of this experiment were analyzed by Chi square. Maturation stages were analyzed by two methods. Percent maturing indicates any ova which have resumed meiosis beyond the dictyate stage while metaphase II indicates only those ova classified as metaphase II. Experimental variables for this study are shown in Table 3. TABLE 3. Effects of various culture media on in_vitro ovum maturation: experimental variables. Culture Media Follicle Size Hams F10a greater than 5 mm Hams Flo + 10% FCSb 2-5 mm Hams P10 + 20% FCS less than 2 mm aAlso contains 100 iu penicillin/m1 and 6.7 iu heparin/m1. Heat inactivated Fetal Calf Serum (GIBCO) % by volume. 19 Time of Maturation For this study, sheep ovaries were collected along the slaughter line at a local slaughter house. Only sheep with no indication of cyclic activity on either ovary were used. Left and right ovaries were pooled, placed into sterile saline with 100 iu penicillin/m1, and transported to the laboratory in a styrofoam box at room temperature. Transport time was between two and three hours. Two replicates were carried out with 21 pairs of ovaries used in the first replicate and 10 pairs in the second. All visible follicles on each ovary were aspirated as previously described and the aspirates were grouped by follicle size (greater than 5 mm, 2-5 mm, and less than 2 mm) and placed in the chambers of an 8 chamber tissue culture chamber slide (Lab-Tech). Chamber slides were randomized for ovum examination at 0, 12, 24, 36, 48, or 60 hours from the beginning of culture. Culture was in an atmosphere of 5% CO2 in air at 37°C. Upon recovery ova were cleaned, fixed and examined as previously described. Experimental variables for time of maturation are given in Table 4. Estrous Cycle Effects In the first portion of this study, eleven cycling ewes were used. For the detection of estrus, a vasectomized ram was run continuously with the ewes. Fresh colored dye mixed with motor oil was applied to his brisket daily and ewes were observed for fresh breeding marks. The first day of estrus was designated day 0. Ewes were randomly assigned to ovum collection on day 5, 10, or 15. Ova were collected by follicle asPiration at mid-ventral laparotomy as previously described. Aspirates were grouped by follicle size and 20 TABLE 4. Time sequence of in_vitro ovum maturation: experimental variables. Replicate Culture Time (hr) Follicle Size 1 0 greater than 5 mm 2 12 2-5 mm 24 less than 2 mm 36 48 6O ovary of origin, and placed in the chambers of Lab-Tech tissue culture chamber slides in 0.25 ml of Hams F10 media. Culture was for 36 hours in a saturated atmosphere of 5% CO2 in air at 37°C. Upon recovery, ova were prepared and examined as previously described. Experimental variables for estrous cycle effects: I, are given in Table 5. TABLE 5. Effects of ovum collection on various days of the estrous cycle and ovary of origin on in_vitro ovum maturation: experimental variables. Day of the Estrous Cyclea Ovary 5 Corpus luteum bearing 10 Opposite 15 8Day 0 = first day of estrus. Other portions of this study involved ovaries collected at three separate times along the slaughter line at a local slaughter house. These were stored in sterile saline with 100 in penicillin/ml and transported to the laboratory at room temperature. Ovaries were collected from 17 old ewes in the second portion, from 6 cycling and 13 prepuberal lambs in the third portion, and from 4 prepuberal, 21 2 anestrous, 26 cycling, and 3 pregnant ewes and lambs in the fourth portion of this study. Follicle aspiration, ova culture, recovery and examination were performed by previously described methods. Ova were cultured in Hams F for 36 hours. Experimental variables for 10 these studies are given in Tables 6, 7, and 8, respectively. TABLE 6. Effects of the luteal state and ovary of origin on in_vitro ovum maturation: experimental variables. Luteal State Ovary Early luteal Corpus luteum bearing Mid luteal Opposite Late luteal Anestrus TABLE 7. Effects of puberty, ovary of origin, and follicle size on in_vitro ovum maturation: experimental variables. Luteal State Ovary Follicle Size Cycling lambs Corpus luteum bearing 2-5 mm Prepuberal lambs Opposite less than 2 mm TABLE 8. Effects of luteal state, ovary of origin, and follicle size on in_vitro ovum maturation: experimental variables. Luteal State Ovary Follicle Size Pregnant Corpus luteum bearing greater than 5 mm Early luteal Opposite 2—5 mm Mid luteal Late luteal Anestrous Prepuberal 22 Results Results of the first study are given in Table 9. A total of 103 ova were recovered from ovaries collected at slaughter. Hams F10 with 10% FCS supported a greater percent maturation of oocytes recovered from CL bearing ovaries that were stored at 21°C than from those CL bearing ovaries stored at 30-37°C (P< 0.1) or at 1°C (P< 0.05). No differences in percent maturation among storage temperatures were demonstrable among ova from opposite ovaries. Differences among media were only apparent among ova from ovaries that were transported at 1°C. Ova from CL bearing ovaries showed a greater percent maturation when 10 iu HCG was added to either Hams F10 with 10% FCS or Hams F with 20% FCS (P< 0.05). However, ova from 10 opposite ovaries had a higher percent maturation without the addition of HCG. Ova from CL bearing ovaries had a lower percent maturation compared to opposite ovaries (P< 0.05) when the ovaries were stored at 21°C and ova were cultured in Hams F10 with 20% FCS or when ovaries were stored at 1°C and ova were cultured in Hams F10 with either 10% or 20% FCS. However, when ovaries were stored at 1°C and ova were cultured in Hams F10 with 10% FCS and 10 iu HCG/m1, ova from CL bearing ovaries had a greater (P <0.05) percent maturation than Opposite ovaries. In Tables 10, 11, and 12, the data from Table 9 are grouped by main effects. Although accurate analysis of the main effects is not possible because of interactions, some inferences can be made. Maturation was inhibited in oocytes from ovaries that were stored at 30—37OC. There was no difference in maturation among oocytes from ovaries stored at 22°C or at 1°C. However, disruption of the cumulus 23 TABLE 9. Effects of storage temperature, culture media, and ovary of origin on subsequent ip_vitro ovum maturation. % Maturation Storage 1 Corpus Luteum Temperature Media Hams F10__with Bearinngvary Opposite Ovagy 3o-37°C 10% FCS2 0 (2)4 42.9 (7) 20% FCS 3 0 (4) 33.3 (3) 10% FCS + 10 iu HCG o (7) 0 (2) 20% FCS + 10 iu HCG 14.3 (7) 25.0 (4) 22°C 10% FCS 71.4 (7)8 33.3 (3) 20% FCS o (2) 100.0 (2) 10% FCS + 10 iu HCG 0 (0) 16.7 (6) 20% FCS + 10 iu HCG 50 (6) 75.0 (4) 1°C 10% FCS o (5): 60.0 (5)8 20% FCS 14.3 (7) s 100.0 (3): 10% FCS + 10 in HCG 100 (1)3b 0 (S) 20% res + 10 iu HCG 75 (4)8 42.9 (7) Values with different superscripts within columns are different at P< 0.05. Also contains 100 iu penicillin/ml and 6.7 iu heparin/m1. Heat inactivated Fetal Calf Serum (GIBCO), by volume. Human Chorionic Gonadotropin (Ayerst). Number in parenthesis = number of ova. L‘WNH TABLE 10. Summary of the main effect of ovary storage temperature on subsequent ip_vitro maturation of follicular oocytes. Storage Temperature % Maturation 3o-37°C 16.7 (36)a 22°C 50.0 (30)b 1°C 37.8 (37)b Values with different superscripts are different at P< 0.05. Number in parenthesis = number of ova. 24 TABLE 11. Summary of the main effect of culture media on ip_vitro ovum maturation. Media F10 With % Maturation 10% FCS 41.4 (29)a 20% FCS 33.3 (21)b 10% FCS + HCG 9.5 (21) 20% FCS + HCG 43.7 (32)8 Values with different superscripts are different at P< 0.05. Number in parenthesis = number of ova. TABLE 12. Summary of the main effect of ovary of origin on subsequent .ip_vitro maturation of follicular oocytes. Ovary % Maturation CL Bearing 26.9 (52) Opposite 41.2 (51) Number in parenthesis = number of ova. cell layer was apparent in oocytes from ovaries that were stored at 1°C. Ovum maturation was inhibited in Hams F10 with 10% FCS and HCG with 10% FCS, Hams F10 with 20% FCS and HCG with 20% FCS (P approaching 0.05). There was compared to Hams F10 (P< 0.05) and Hams Flo no difference in maturation between CL bearing and opposite ovaries. Effects of Various Media For purposes of analysis, it was found necessary to combine ova from greater than 5 mm follicles and those from 2-5 mm follicles into one category (large follicles) for comparison with ova from small follicles (less than 2 mm). A total of 39 ova were recovered from ewes at laparotomy and 123 ova from ovaries collected at slaughter 25 on two separate days. Using the Chi-square test for independence, a time effect was found among the three portions of this study: ova collected at laparotomy, ova collected at slaughter the first day, and ova collected at slaughter the second day. This made separate analysis of ova cultured from each collection time necessary. Results of this study are given in Table 13. In the first portion of this study, ova from small follicles initiated maturation and matured to Met II more readily in Hams F 10 with 20% FCS than in Hams F10 alone. There was a tendency (P<<0.l) for ova from large follicles to mature more readily in Hams F 0 alone 1 than in Hams F10 with 10% FCS. When Hams F10 was the culture media, the percent of ova initiating maturation was greater for ova from large: follicles compared to those from small follicles. No differences were found among culture media for ova collected at slaughter. For ova collected at the first slaughter time and cultured in Hams F10 with 10% FCS, there was a tendency (P< 0.1) for ova from small follicles to have a greater percent maturation than those from large follicles. However, at the second slaughter time, Hams F with 10 20% FCS supported greater (P< 0.05) percent maturation of ova from large follicles compared to small follicles. In Table 14 the main effects of media and follicle size from the three ova collection times are summarized from Table 13. Although statistical analysis cannot be performed on the data in this table, very little differences can be seen. 26 TABLE 13. Effects of various culture media on ip_vitro ovum maturation. Ova Follicle Total % 4 % 5 Collection Media Size3 Ova Maturing Met II 1 Hams F101 large 8 87.5: 62.58 2 small 8 37.5 0 with 10% FCS large 3 33.3 0 small 4 50.0 0 with 20% FCS large 6 83.3 50.0b small 10 90.08 50.0 2 Hams F10 large 6 66.7 16.7 small 12 75.0 25.0 with 10% FCS large 4 25.0 0 small 11 72.7 36.4 with 20% FCS large 2 100.0 50.0 small 16 43.75 31.3 3 Hams F10 large 9 55.6 22.2 small 16 37.5 18.8 with 10% FCS large 7 42.9 14.3 small 10 50.0 10.0 with 20% FCS large 8 75.0: 25.0 small 22 22.7 0.1 Values with different superscripts within ova collection times and within columns are different at P< 0.05. Also contains 100 iu penicillin/m1 and 6.7 in heparin/ml. Heat inactivated Fetal Calf Serum (GICBO). Large = greater than 2 mm, small = less than 2 mm. Includes ova in Met 1, Ana 1, Tel 1, or Met 11. Includes only ova with Metaphase chromosomes and a polar body. UlbLAJNH TABLE 14. Summary of the main effects of culture media and follicle size of origin on ip_vitro ovum maturation. Total Ova % Maturing Hams Flo alone 59 57.6 with 10% FCS 39 51.3 with 20% FCS 64 53.1 Follicle size large 53 64.2 small 109 49.5 27 Time of Maturation Results of this study are given in Table 15. The two replicates were found to be independent and were combined for further analysis. Also, for purposes of analysis, ova from greater than 5 mm follicles and ova from 2—5 mm follicles were combined into one category (large follicles) for comparison with less than 2 mm follicles (small follicles). A total of 77 ova were used in this study. A greater (P‘<0.05) percent of ova contained GVs at 0 hours compared to those ova cultured for 12 or more hours. No difference in percent of ova initiating maturation were found between ova from large or small follicles at 0, 12, 24, 36, 48, or 60 hours. At 36 hours, however, there was a greater number of ova from large follicles matured to Met 11 compared to ova from small follicles. There was a progressive increase in ova initiating maturation from 0 hours to 24 hours in culture. However, only at 0 hours were there significantly fewer ova initiating maturation. If ova from all follicle sizes were combined, the percent of ova maturing to Met II was constant among ova cultured for 24, 36, 48, or 60 hours with a significant increase between 12 and 60 hours. Estrous Cyele Effects Results of this study are given in Tables 16 through 21. In the first portion, no differences were found among ova collected by laparotomy on day 5, 10, or 15 of the estrus cycle or among ova from CL bearing or opposite ovaries for percent maturing or percent Met II. Ova from cycling lambs showed no differences in percent maturing among CL bearing or opposite ovaries of origin. There was a tendency 28 .hvon umaoa m mam mmsomoeounu mmM£QMumz suHB m>o haze movsaocH .uouoamav :w as N amnu mama moHoHHHom .uouoemfiv ea EB N amnu Houmouw moaoaaaom .HH no: no H H09 .H mc< .H no: aw m>o moenaoaH '4qu .mo.ouvm um ucmuomwfic mum msou :Hnufi3 mumfiuumuomsm udmuomwaw zufia wm=Hm> .m>o mo museum I mwmonuaoume ca nonaaz p0.0m heav 0.0a AOHv m.mH Andy 0.0H any we Amav 00.0 ANHV mocfinaoo m.~H A00 H.m~ Amav ho Aoav 5.0a a0v 0 A00 0.NH Amy moHofiHHow Hanan £0.00 a0v 0 Amy xo.o< A00 0 AHV mo A00 0 A00 moauaaaom mwuma m 0 QHH u z a o0.00 AcHV 00.00 a0Hv oo.o0 Amav na.~m Amy n~.0q Amav mmm.m ANHV vmcwaoo m.nm A00 00.H0 Amav 0.00 Aoav 05.00 A00 0.00 A00 mm.NH A00 mmoauaaaom Hanan om.00 A00 m.mm Amv 00.00 Amy 0 Adv o.om A00 mo A00 mmoHoaaaom owuma stfiu5umz N no AQHV nmm.0 AOHV no AmHv a.o Anv om.m Amav m5.00 Amav >0 N do. Mm mm «IN. INA m 35 as: 3316 .:0fiumusuma ad>o ouuH>.mW mo mosmauom mafia .ma mqm 2 mm) or small (<12 mm) follicles of origin. Ovulation was induced in anestrous ewes with 5 mg FSH and 100 in HCG. However, coincident estrus was not exhibited. Estradiol and testosterone (1 mg/da each) were not effective in the induction of estrus without progesterone pretreatment. Following a five day progesterone pre- treatment (20 mg/da), ovulation was inhibited with estradiol but not with testosterone. APPENDIX APPENDIX Sheep and Swine Surgery Table Ovarian examination in the sheep and pig is generally accomplished by means of laparotomy or laparoscopy. To facilitate examination by either procedure, it is advantageous for the animal to be in a dorsal recumbent position with the posterior portion elevated at a 30-40 degree angle. Lamond and Urquhart (1961) designed a stationary laparotomy cradle for use in sheep. Hulet and Foote (1968) modified this cradle, making it possible to secure the ewe in a supine position, then increase the angle to that desired for examination. With some minor modifica- tions, this table was used for laparoscopic ovarian examination in the goat (Dukelow, Jarosz, Jewett, and Harrison, 1971), the pig (Wildt, Fujimoto, Spencer, and Dukelow, 1973), and the sheep (Snyder and Dukelow, 1974). This table was stationary and had no adjustment in the angle at which the animal was positioned necessitating much physical movement of anesthetized animals. Examination of larger pigs and sheep required the assistance of several people. This communication describes an examination table that was designed to facilitate loading of large pigs and sheep at the place of initial anesthesia, movement to the surgery area, and easy adjustment of the angle used for examination. A schematic diagram is presented in Figure l. 48 49, ...NN 3m :wH :0 .mumn mmouu 0am edema Houaou ufispsou :q\m .oaaoo oowo ooao =H .Hmafism wsfiusoom How mafia Odom .mwOH Houaafinmum mean :H .mHoons :0 .ouooaoa on ooao =n x =~\HIH .vsmum Hooam Opamafi maavaam ha pushed .omaa :H .mwoa manmumsflw< .oafie :N\HIH Suez moms .msmum uooam .Oanmu weaned“ Mom umaoa mawsomuum you wanna :0\H ‘< macacan5e4L5:514 .oanmu muowuom mafiam 0am nomnm. .H mmsuHm .qw / 7/ m ...VN m I .... / m Krill. o 7/ no 77 4: u a :«N . / . \ If :bN .% oi. ..m 50 Basic construction is of galvanized tubing to allow for strength and ease of cleaning. In the loading-unloading position the table is low to the floor to facilitate handling of large animals and rests on five-inch wheels so that large animals can easily be moved from holding pens into the surgery area while under general anesthesia. A hoist attached to the ceiling of the surgery room is used to raise the surgery table to the height desired for surgery. Removable and adjustable legs are attached to the posterior end of the table for stability. This surgery table has been used for fast and efficient laparotomies, laparoscopies, and other surgical procedures in sheep and pigs of various sizes. References Dukelow, W. R., S. J. Jarosz, D. A. Jewett, and R. M. Harrison. 1971. Laparoscopic examination of the ovaries in goats and primates. Lab Anim Sci 21:594-597. Hulet, C. V. and W. C. Foote. 1968. A rapid technique for observing the reproductive tract of living ewes. 'J_Anim Sci 27:142-145. Lamond, D. R. and E. J. Urquhart. 1961. Sheep laparotomy cradle. Austral Vet Journ 37:430-431. Snyder, D. A. and W. R. Dukelow. 1974. Laparoscopic studies of ovulation, pregnancy diagnosis, and follicle aspiration in sheep. Theriogen 2:143-148. Wildt, D. E., S. Fujimoto, J. L. Spencer, and W. R. Dukelow. 1973. Direct ovarian Observation in the pig by means Of laparoscopy. g_Reprod Fert 35:541-543. BIBLIOGRAPHY BIBLIOGRAPHY Andersen, M. M., J. Kroll, A. 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The porcine ovarian follicle: III. development of chorionic gonadotropin receptors associated with increase in adenyl cyclase activity during follicle maturation. Endocrin 99:42-48. 54 Martensz, N. D., D. T. Baird, R. J. Scaramuzzi, and P. F. A. VanLook. 1976. Androstenedione and the control of luteinizing hormone in the ewe during anoestrus. J_Endocr 69:227-237. McGaughey, R. W. 1975. A comparison of the fluids from small and large ovarian follicles of the pig. Biol Reprod 13:147-153. McGaughey, R. W. 1977. The culture of pig oocytes in minimal medium, and the influence of progesterone and estradiol -17B on meiotic maturation. Endocrin 100:39-45. McGaughey, R. W. and C. Polge. 1971. Cytogenetic analysis of pig oocytes matured ip_vitro. J_Exp Zol 176:383-396. McGovern, P. T., H. L. Williams, and J. L. Hancock. 1969. The time of ovulation in the ewe following treatment with human chorionic gonadotropin. _J_Reprod Fert 20:537-540. McNatty, K. P., W. M. Hunter, A. S. McNeilly, and R. S. 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Name: Born: Birthplace: Formal Education: Degrees Received: VITA David Andrew Snyder May 18, 1950 Beatrice, Nebraska Andrew's University Academy Berrien Springs, Michigan Andrew's University Berrien Springs, Michigan Michigan State University East Lansing, Michigan Bachelor of Science Michigan State University, 1972 Master Of Science Michigan State University, 1974 58