MOLECULAR REPRODUCTION AND DEVELOPMENT 31:72-77 (1992)

Effect of Oocyte Maturation Medium on In Vitro Development of In Vitro Fertilized Bovine Embryos TERESA A. ROSE AND BARRY D. BAVISTER Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin Leibfried and Bavister, 1983; Shalgi, 1984; Fleming et al., 1985; Bavister, 1987). In many cases, by supplementing the culture medium with gonadotropins and/or steroid hormones, spontaneously matured oocytes could be fertilized and could undergo cleavage. These observations suggested that oocyte maturation consists of events occurring in both the nucleus and the cytoplasm. Additionally, because nuclear maturation (GVBD, metaphase 11, or extrusion of the first polar body) can occur in oocytes that may not have undergone cytoplasmic maturation, it is necessary to use end points other than nuclear morphology for assessing oocyte maturation. The developmental capacity of embryos derived from in vitro matured (IVM)/in vitro fertilized (IVF) oocytes is a much more definitive way to assess the normality of maturation. Embryo development can be used as a n end point to study oocyte maturation in cattle, in that IVMIIVF ova are able to develop to morulae and blastocysts in vitro. Although in early studies such ova exhibited a n in vitro block to development a t the eight- to 16-cell stage (Thibault, 1966; Camous et al., 1984; Eyestone and First, 1989; Fukui, 1990), recent advances in culture techniques have largely eliminated this problem. By coculturing embryos with oviductal epithelial cells, or certain other somatic cells, or using media conditioned by these cells, 20-50% of IVMIIVF ova can develop into morulae and blastocysts (see, e.g., Goto et al., 1988; Aoyagi e t al., 1990; Ellington et al., 1990b; Fukui, 1990; Kim et al., 1990; Nakao and Nakatsuji, 1990; Saeki et al., 1990). Recently, bovine IVM/IVF embryos were shown to be capable of developing into morulae and blastocysts in serum-free medium without somatic cell conditioning (Pinyopummintr, 1990; PinyopumKey Words: Oocyte maturation, Bovine, Cattle, Culmintr and Bavister, 1991). ture media, Embryo IVF At least some IVMiIVF morulae and blastocysts are viable, because subsets transferred to surrogate females have produced term offspring (Lu et al., 1988; INTRODUCTION Aoyagi et al., 1990; Fukuda et al., 1990; Kajihara et al., To study oocyte maturation in vitro, meaningful 1990; Goto et al., 1991; Jiang et al., 1991; Xu et al., experimental end points must be used. It was first 1991). Preimplantation development in vitro therefore demonstrated in 1935 (Pincus and Enzmann, 1935) appears to be a meaningful criterion for studying oocyte that rabbit oocytes removed from antral follicles, in the maturation and embryogenesis and is far less costly absence of gonadotropins, undergo spontaneous nuclear maturation a s determined by germinal vesicle breakdown (GVBD). It was later shown that many of Received March 27, 1991; accepted August 26, 1991. these spontaneously matured oocytes failed to be fer- Address reprint requests to Teresa A. Rose, DVM, Department of tilized or to produce viable embryos (Thibault, 1977; Veterinary Science, 1655 Linden Drive, Madison, WI 53706. The objective of this study was to ABSTRACT examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 m m follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 pg/ml luteinizing hormone (LH), 0.5 p.g/ml follicle-stimulating hormone (FSH),and 1 pg/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEMa, TCM199, MEMa/+, RPMI:MEMa) better supported normal oocyte maturation as determined by embryo development to the two-cell (7&82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/1 or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52%and 37%,respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave t o the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts. This study demonstrates that 1) the culture medium used for bovine oocyte maturation can markedly affect subsequent embryo development at several stages, including blastocysts, and 2) bovine oocytes can be matured and fertilized and develop to blastocysts in serum-free medium.

0 1992 WILEY-LISS, INC.

OOCYTE MATURATION MEDIUM and time consuming than embryo transfer and pregnancy determinations. Although a wide variety of different protocols has been described for the production of IVMiIVF bovine embryos, no one embryo culture procedure results in consistently better embryo development. A common factor may be that the pool of oocytes used in all such studies are suboptimally matured, even though a proportion are capable of morphologically normal development following IVM and IVF. Inadequate oocyte maturation could underlie failure to cleave or abnormal embryo development. The concept that events occurring during oocyte maturation affect subsequent embryo development has recently been addressed (van de Sandt et al., 1990). Mouse oocytes matured in different culture media had varying capacities to undergo preimplantation development in vitro and in vivo, clearly showing the importance of the culture milieu on oocyte maturation. To date, this kind of analysis has not been carried out for any other species. The present study was designed to determine the influence of the culture medium used for IVM of bovine oocytes on subsequent embryo development in vitro. For this purpose, the suitability of seven commercially available media was examined, with the goals of determining 1)to what extent using different culture media for maturation of oocytes affects their developmental capability and 2) which media are most suitable for supporting normal oocyte maturation.

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Oocyte Recovery, Evaluation, and Culture Ovaries were collected at a local abattoir and transported in a thermos flask at 23-27°C in 0.9% NaCl supplemented with antibioticsiantimycotic (PSA). Time from ovary collection to arrival at the laboratory was from 1 to 3.5 h. In the laboratory, ovaries were immediately washed in 23-27°C tap water and then transferred into the same temperature 0.9% NaCl supplemented with PSA. Oocytes were aspirated with a n 18-gauge needle from follicles 2-5 mm in diameter and placed into a 50 ml conical tube. After processing ovaries, the oocytes were allowed to settle by gravity for approximately 15 min. The sediment was aliquoted into 60 x 15 mm petri dishes (Falcon Plastics No. 10071, and oocyte-cumulus complexes (OCCs) were removed. Only oocytes with a n intact, compact cumulus oophorus and evenly granulated cytoplasm (Leibfried and First, 1979) were cultured. Selected OCCs were washed twice in one of the seven C02-equilibrated maturation media prior to final IVM culture, which consisted of 10-12 oocytes in a 50 p1 drop of oocyte maturation medium covered with 10 ml paraffin oil (extracted three times and preequilibrated with sterile 0.9% NaC1) in a 60 x 15 mm petri dish. Oocytes were cultured in one of these seven media a t 39°C in 5%CO, in air and 100% humidity for 23-25 h. In Vitro Fertilization

At the end of the oocyte IVM culture period, each treatment group of 10-12 OCCs was moved as a group MATERIALS AND METHODS through three washes in TL-HEPES (Bavister et al., Oocyte Maturation Media 1983) without glucose (Parrish et al., 1986) suppleSeven culture media were purchased in powdered mented with 3 mgiml BSA (Fraction V; Sigma Chemform containing L-glutamine from Sigma Chemical Co. ical Co.; A9647, lot 26F022), 0.25 mM pyruvate, and 50 (St. Louis, MO) or Gibco Laboratories (Grand Island, pgiml gentamicin sulfate (Sigma Chemical Co.). The NY). Media obtained from Sigma were: SFRE 199-2 oocytes were then placed in 50 p1 drops of IVF medium medium (SFRE), Eagle’s minimum essential medium-a consisting of TALP (Bavister e t al., 1983) modified to (MEMa), Eagle’s minimum essential medium-a with contain no glucose, 0.25 mM pyruvate, and 6 mg/ml of deoxy- and ribonucleosides (MEMa/+), 1:l RPMI- bovine serum albumin (fatty acid free; Sigma Chemical 1640:MEMa (RPMI:MEMa), Waymouth’s medium MB Co.; A7511, lot 29F9315) and 2 pgiml heparin. The IVF 752i1 (Waymouth’s medium) and Ham’s nutrient mix- medium drops were overlaid with paraffin oil. Incubature F-12 (Ham’s F-12). Tissue culture medium 199 tion conditions for IVF were 39°C in 5% C 0 2 in air and (TCM199) was obtained from Gibco Laboratories. Fresh 100% humidity for 18-20 hr. All experiments used frozen semen obtained from the media were made up the day before each replicate experiment using water purified by reverse osmosis same ejaculate of one bull packaged in straws a t and Milli-Q (Millipore Corporation, New Bedford, MA) 50 x 106 sperm/ml (donated by American Breeders filtration; before use, the water was tested with the Service, De Forest, WI). For each replicate, one or two hamster sperm motility quality-control assay (Bavister straws were thawed at 35°C for 1 min. The sperm and Andrews, 1988).All media were sterilized using a washing procedure was based on a protocol by Parrish 0.22 pm Millipore filter and stored a t 4°C. On the day et al. (1986), modified by W.H. Eyestone (1988, perof experiment, all media were supplemented with 0.25 sonal communication) and is as follows: 250 ~1 of mM sodium pyruvate, 6 mg/ml bovine serum albumin frozen-thawed semen was layered under 1 ml of TALP (fraction V; Sigma Chemical Co.; A9647 lot 26F022), 1 medium [modified to contain no glucose, 21.6 mM pgiml estradiol (Sigma @-estradiol;made up in 1OOOx sodium lactate, 1 mM pyruvate, 10 mM HEPES (N-2stock solution in absolute ethanol), 10 pg/ml NIAMDD hydroxyethylpiperazine-N’-2-ethanesulfonicacid), 6 ovine luteinizing hormone (oLH), 0.5 pgiml NIAMDD mg/ml BSA albumin (fraction V; Sigma Chemical Co.; ovine follicle-stimulating hormone (oFSH), and anti- A9647, lot 26F022), and 50 pgiml gentamicin sulfate] bioticiantimycotic (100 unitsiml penicillin G [K salt], contained in 12 x 55 mm test tubes and incubated at 39°C for 1 hr. The upper 800 pl of medium from each 100 pgiml streptomycin, and 0.25 pg/ml amphotericin test tube was recovered and washed by resuspending B; PSA).

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T.A. ROSE AND B.D. BAVISTER

the sperm twice in 3 ml of modified TALP medium and centrifuging a t 1OOg. The concentration of the final sperm pellet was determined with a hemocytometer, and the fertilization drops were inseminated at a final concentration of 0.5-1.5 x lo6 sperm/ml.

In Vitro Embryo Development and Evaluation After the IVF culture period, all eggs within each treatment group were washed three times in 200 p1 of HECM-1 (Schini and Bavister, 1988), supplemented with 6 mgiml BSA (fraction V; Sigma Chemical Co.; A9647, lot 26F022). During the washing process, most of the cumulus cells were removed mechanically by pipetting using flame-drawn pipette tips with a n inner diameter slightly larger than the egg diameter. Each treatment group of 10 to 12 eggs were cultured for 8 days in 50 pl drops of HECM-l/BSA medium, which was overlaid with 10 ml paraffin oil, at 39°C in 5% CO, in air and 100% humidity. Embryos were evaluated between days 4 and 8 with a n inverted microscope using phase and Nomarski interference contrast at ~40-100. On day 4, embryos were scored for ability to cleave to two or more cells. Between days 6 and 8, embryos were scored for development to morulae and blastocysts. Embryos showing compaction were classified as morulae and those with a blastocoel cavity a s blastocysts. Upon completion of culture, morulae and blastocysts from two replicate experiments were fixed and Hoechst stained. Statistical Analysis A protected (Fisher’s) analysis of variance (ANOVA) was performed using arcsin-transformed data in a randomized block design where the block is equal to days. All treatments were replicated four times, except for Ham’s F-12, which was replicated three times. Each replicate represented oocytes collected on a particular day. Significance level was P 0.05. RESULTS After IVM in seven different media, oocytes in all treatments were subjected to IVF, then cultured for 8 days in the same embryo culture medium (HECM-1/ BSA). Table 1 summarizes embryo development results based on total oocytes placed into culture at the beginning of the experiment. Figure 1results are based on the number of two-cell embryos produced. Cleavage to the Two-Cell Stage: Indication of Incidence of Fertilization and Developmental Potential Responses with the seven different oocyte maturation media could be divided into three significantly different groups (Table 1). In the first group, consisting of SFRE, TCM199, and the three MEM-containing media, 7 6 4 2 % of all oocytes cleaved to the two-cell stage (no statistical differences). However, in the other two groups, consisting of Waymouth’s medium and Ham’s F-12, only 52% and 37%, respectively, of all

Ham’s F-12, only 52% and 37%, respectively, of all oocytes cleaved to the two-cell stage. To determine if the failure to cleave was due to errors in the fertilization process, a subset of oocytes from each of the two media was observed under the light microscope. These observations (data not shown) revealed the presence of decondensed sperm and formation of male pronuclei, indicating that fertilization, a s determined by morphology, had proceeded normally.

Development to the Morula andlor Blastocyst Stages The incidence of embryo development to morulae and blastocysts (Table 1)is based on the total number of oocytes placed into culture a t the onset of the experiment. Responses with SFRE (32%), MEMa (30%), TCM199 (27%), MEMa/+ (25%), and RPM1:MEMa (26%) were not significantly different from one another. The least morula and blastocyst development took place in Ham’s F-12 (3%) and was significantly different from the response in Waymouth’s medium (14%). In addition, the response in Waymouth’s medium was significantly different from those in SFRE or MEMa, but not significantly different from those in TCM199, MEMa/+, or RPM1:MEMa. With respect to blastocyst development, the seven different maturation media could be placed into two significantly different groups. In the first group, the frequencies of blastocyst development reached were SFRE (19%), MEMa (18%), TCM199 (15%), MEMa/+ (12%) and RPM1:MEMa (15%). Within this group, there were no significant differences. The responses in the remaining two media (Waymouth’s medium and Ham’s F-12) were not significantly different (3% and 1%, respectively) but were significantly different from those in the other five media (P 0.05). Developmental Potential of Oocytes That Cleaved to the Two-Cell Stage To assess better the delayed effects of oocyte maturation on cell divisions after the first cleavage stage, the percentage of morulae and/or blastocysts obtained were calculated based on the number of two-cell embryos obtained (Fig. 1). Two-cell embryos derived from oocytes matured in SFRE produced significantly more morulaeiblastocysts and blastocysts (43% and 26%, respectively) than Waymouth’s medium (27% and 6%) and Ham’s F-12 (9% and 3%), but the SFRE response was not different from that in MEMa, TCM199, MEMa/+, or RPM1:MEMa. Two-cell embryos derived from oocytes matured in Ham’s F-12 produced the lowest percentage of morulae/blastocysts (significantly different than all other maturation media). Maturing bovine oocytes in SFRE, MEMa, TCM199, or RPM1:MEMa resulted in significantly more blastocysts than oocytes matured in Waymouth’s medium MB 752/1 or Ham’s F-12. To assess further the morulae and blastocysts produced in this culture system, the numbers of nuclei

OOCYTE MATURATION MEDIUM

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TABLE 1. Development of Embryos in Vitro After Oocyte Maturation in Different Culture Media* Percent Percent morulae and Percent No of two or more blastocysts blastocysts Medium oocytes cells f s.e. iz s.e. s.e. SFRE 131 76 f 5a 32 f 5a 19 f 3a MEMa 145 80 f 4a 30 zk 6a 18 3a 27 6a,b 15 4a TCM199 163 78 6a MEM a/+ 154 77 7a 25 6”pb 12 4a RPM 1:MEMa 126 82 f 5a 26 f 9a,b 15 f 6a Waymouth’s 124 52 4b 14 f gb,“ 3 f 3b Ham’s F-12 117 37 f 5c 3 f 1c 1 lb

** *

**

* ** * *

* Oocyte maturation was carried out in one of seven culture media, followed by IVF and embryo culture using the same protocols and media for all oocytes. s.e. represents nontransformed data. a-CSignificance(P5 0.05) was determined using arcsin-transformed data in a protected (Fisher’s) ANOVA. Within a column, values with no common superscript are significantly different. cally for the serum-free maintenance of primary baboon kidney cells (Weiss e t al., 1980), also resulted in embryos with relatively high developmental capacity. However, SFRE appeared to be marginally better than TCM199 for IVM of bovine oocytes. Although there was no statistical difference between these two media, IVM DISCUSSION of oocytes in SFRE resulted in statistically different This study has emphasized the influence of oocyte responses from Waymouth’s medium and Ham’s F-12, maturation medium on the developmental capacity of at both morulaetblastocysts and blastocyst stages of IVF embryos in vitro. Maturing bovine oocytes in one of development. In contrast, oocytes matured in TCM199 seven commercially available serum-free maturation were similar in developmental capacity to Waymouth’s media resulted in zygotes with highly variable abilities medium when the end point was the percentage of to cleave to the two-cell stage. Furthermore, the poten- morulae/blastocysts (Fig. 1). MEM was included in this study because many tial of these two-cell embryos to develop to morulae and/or blastocysts was enhanced or attenuated depend- oocyte maturation studies have used various formulations of this medium. In view of the positive results ing on the medium used for oocyte maturation. Oocyte maturation in SFRE, MEMa, TCM199, obtained with IVM of mouse oocytes using a MEMa MEMa/+, or RPM1:MEMa resulted in similar embryo formulation (van de Sandt et al., 1990), we investigated development responses: two or more cells (76-82%), the maturation of bovine oocytes in one of two MEMa compact morulae and blastocysts (25-32%), and blas- formulations: with (MEMa/+) or without (MEMa) tocysts (12-19%). Oocyte maturation in Waymouth’s deoxy- and ribonucleosides. In addition, the combinamedium or Ham’s F-12 resulted in significantly less tion of RPM1:MEMa was also evaluated. This study embryo development: two or more cells (52%, 37%), showed that oocytes matured in any of the MEMa compact morulae and blastocysts (14%, 3%), and blas- media derivatives resulted in embryos with relatively tocysts (3%, l%), respectively. The decrease in the high developmental capacity; there were no significant incidence of development to two or more cells in Way- differences between responses with these media. Howmouth’s medium and Ham’s F-12 did not appear to be ever, as in the case of SFRE, results with oocytes due to failure of fertilization, a s subsets of oocytes from matured in MEMa (without deoxy- and ribonucleothese two media were observed for sperm penetration, sides) were always statistically different from Waydecondensation, and pronuclear formation (data not mouth’s medium and Ham’s F-12. In contrast, oocytes shown). Moreover, two-cell embryos derived from matured in MEMa/+ or in RPM1:MEMa (as with oocytes matured in these two media were significantly TCM199) were similar in developmental capacity to less competent to undergo further embryonic develop- Waymouth’s medium when end points were the percentages of morulae and blastocysts (Table 1, Fig. 1). ment in vitro (Fig. 1). Ham’s media have previously been used for bovine TCM199 is commonly used for IVM of bovine oocytes, albeit usually with serum supplementation. Even with- oocyte maturation, and Ham’s F-10 is frequently used out serum, as in this study, maturing oocytes in for final oocyte maturation, IVF, and embryo culture in TCM199 resulted in embryos with relatively high human clinical protocols. Using Ham’s F-12 for IVM of developmental capacity. Furthermore, oocytes matured bovine oocytes produced a significant decrease in emin SFRE, a medium formulated from TCM199 specifi- bryo developmental capacity compared with SFRE,

were counted. Embryos (morulae, n = 44; blastocysts, n = 52) from two replicate experiments were fixed and stained with Hoechst DNA stain. The mean nuclei number (+sample standard deviation) were 38 (k15) and 97 (t30)for morulae and blastocysts, respectively.

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T.A. ROSE AND B.D. BAVISTER Morulae and Blastocysts

Blastocysts

a

T

ab

T

SFFIE

m a

ab

T

TCM~W

ab ab

T

mai+

T

FIPMIMM

b

T

WAYMOUTH

HAM-12

b y t e Maturation Media Fig. 1. Development in vitro to morula and/or blastocyst of those bovine IVMiIVF oocytes able to undergo the first cleavage division. a , b, and c signify statistical differences ( P S 0.05) in percentage of morula/blastocyst development; d, e, and f signify statistical differences ( P i 0.05) in percentage of blastocyst development. Columns with no common superscript are significantly different. The means and standard error are based on nontransformed data, and the statistical analysis is based on arcsintransformed data using Fisher’s ANOVA.

TCM199, and the three MEMa-containing media. Although the two media are similar, Ham’s F-10 is sufficiently different from F-12 to preclude predictions about the outcome of bovine IVM in the F-10 formulation. Possibly, serum supplementation could overcome the limitations of maturing bovine oocytes in Ham’s F-12 encountered in this study. Waymouth’s medium MB 752/1 was evaluated in the present study because i t was the medium of choice for IVM of mouse oocytes, assessed by IVF and embryo development (van de Sandt et al., 1990). However, we found that culturing bovine oocytes in Waymouth’s medium for 23-25 h significantly decreased their in vitro developmental capacity following IVF. Plausible explanations for these contrasting results are 1)different nutrient requirements of bovine oocytes compared with those from the particular mouse strain used by van de Sandt et al. (1990) [(C57BL/K x SJL/J)Fll; 2) mouse oocytes were collected from hormonally (pregnant mares’ serum gonadotropin) primed mice and cultured €or 14-15 h r prior to IVF, while bovine oocytes were collected from cattle a t all stages of the estrous cycle and cultured for a longer time period with gonadotropin supplemented medium; and 3) mouse oocytes were cultured in serum-containing medium, whereas the present study used BSA a s a serum substitute.

Oocytes used in this study were matured in serumfree complex media, fertilized in vitro in the absence of serum, and developed into morulae and blastocysts in serum-free medium without epithelial cell contributions. Serum constituents (and concentrations) would undoubtedly influence oocyte maturation, being stimulatory or inhibitory (depending on the serum sample), regardless of the medium employed. Therefore, we chose not to confound the outcome of experiments by including serum in the medium used for any phase of IVM/IVF and embryo culture. The developmental data show clearly that neither serum nor coculture with somatic cells other than the surrounding cumulus oophorus is necessary for the production of blastocysts from IVM/IVF bovine oocytes. It is also of interest that the embryo culture medium used in these experiments (HECM-11,originally formulated in our laboratory to support hamster two-cell embryo development, is glucose- and phosphate free (Schini and Bavister, 1988). This indicates that exogenous glucose and phosphate are not necessary for bovine embryos derived from IVM/IVF oocytes to develop into morulae and blastocysts in vitro. The ability of bovine embryos to develop in vitro in the absence of glucose, although in the presence of oviductal cells, has recently been reported by Ellington et al. (1990a).

OOCYTE MATURATION MEDIUM These authors found that the presence of glucose during the first 48 h of culture actually inhibited subsequent blastulation. The quality of the embryos produced in this study was assessed using two criteria: 1)embryo morphology using a dissecting microscope and 2) nuclear morphology of a subset of the compact morulae and blastocysts using a Hoechst DNA stain; embryos that had been classified as morulae or blastocysts using a dissecting microscope were found to contain mostly healthy looking nuclei. Although morphologically the embryos appear to be “normal,” their viability was not confirmed by embryo transfer, the only unequivocal test. In conclusion, the environment supplied for in vitro oocyte maturation is critical for subsequent embryo development, even for events occurring after fertilization. Selection of a suitable oocyte culture medium is imperative for optimal results in bovine IVM/IVF studies, in order to 1)improve the overall efficiency of bovine embryo production, 2) increase understanding of the control of oocyte maturation and embryo development, and 3) interpret properly experimental results obtained from oocyte maturation, fertilization, and embryo development.

ACKNOWLEDGMENTS This work was supported by USDA-CRGO grant 89-37240-4562. We are indebted to American Breeders Service (DeForest, Wisconsin) for donating bovine semen. Gonadotropins were generously supplied by the National Institute of Diabetes and Digestive and Kidney Diseases and the National Hormone and Pituitary Program (University of Maryland School of Medicine). Special thanks are due to Donna Evenson for technical assistance.

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mental capacity of rat oocytes matured in vitro in defined medium. Gamete Res 12:255-263. Fukuda Y, Ichikawa M, Naito K, Toyoda Y (1990): Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage. Biol Reprod 42:114-119. Fukui Y (1990): Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro. Mol Reprod Dev 2 6 : 4 0 4 6 . Goto K, Kajihara Y, Kosaka S, Koba M, Nakanishi Y, Ogawa K (1988): Pregnancies after co-culture of cumulus cells with bovine embryos derived from in-vitro fertilization of in-vitro matured follicular oocytes. J Reprod Fertil 83:753-758. Goto K, Kinoshita A, Takuma Y, Ogawa K (1991):Birth ofcalves after the transfers of oocytes fertilized by sperm injection. Theriogenology 35:205. Jiang JY, Zhong S, Fan BQ (1991): Calf born after the transfer of frozen-thawed embryos from follicular oocytes matured, fertilized and developed in vitro. Theriogenology 35217. Kajihara Y, Kometani N, Kobayashi S, Shitanaka Y, Koshiba Y, Hishiyama K, Shiraiwa K, Goto K (1990): Pregnancy rates and births after co-culture of cumulus cells with bovine embryos derived from in vitro fertilization of matured follicular oocytes. Theriogenology 33:264. Kim CI, Ellington J E , Foote RH (1990):Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture. Theriogenology 33:433-440. Leibfried ML, Bavister BD (1983):Fertilizability of in vitro matured oocytes from golden hamsters. J Exp Zoo1 226:481485. Leibfried L, First NL (1979): Characterization of bovine follicular oocyte and their ability to mature in vitro. J Anim Sci 53:76-86. Lu KH, Gordon I, Chen HB, Gallagher M, McGovern H (1988): Birth of twins after transfer of cattle embryos produced by in vitro techniques. Vet Rec 122539-540. Nakao H, Nakatsuji N (1990): Effects of co-culture, medium components and gas phase on in vitro culture of in vitro matured and in vitro fertilized bovine embryos. Theriogenology 33591-600. Parrish J J , Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL (1986): Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 25:591-600. Pincus G, Enzmann EV (1935): The comparative behavior of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. J Exp Med 62:665-675. Pinyopummintr T (1990): Bovine embryos develop to the morulai blastocyst stage in vitro in a chemically defined protein-free medium. Biol Reprod 42[Suppl 11:61. Pinyopummintr T, Bavister BD (1991): In vitro-maturediin vitrofertilized bovine oocytes can develop into morulaeiblastocysts in chemically defined, protein-free culture media. Biol Reprod (in press). Saeki K, Hoshi M, Leibfried-Rutledge ML, First NL (1990):In vitro fertilization and development of bovine oocytes matured with commercially available follicle stimulating hormone. Theriogenology 34:1035-1039. Schini SA, Bavister BD (1988): Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol Reprod 39:1183-1192. Shalgi R (1984):Developmental capacity of rat embryos produced by in vivo or in vitro fertilization. Gamete Res 10:77-82. Thibault C (1966): La culture in vitro de l’oeuf de vache. Ann Biol Anim Biochim Biophys 6:159-164. Thibault C (1977): Are follicular maturation and oocyte maturation independent processes? J Reprod Fertil 51:1-15. van de Sandt JJM, Schroeder AC, Eppig JJ (1990): Culture media for mouse oocyte maturation affect subsequent embryonic development. Mol Reprod Dev 25:164-171. Weiss SA, Lester TL, Kalter SS, Heberling RL (1980): Chemically defined serum-free media for the cultivation of primary cells and their susceptibility to viruses. In Vitro 16:616-628. Xu KP, Loskutoff NM, Betteridge K J (1991): Pregnancies resulting from bovine embryos derived from in vitro maturation and fertilization of follicular oocytes. Theriogenology 35296.