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Development of bovine in vitro matured and fertilized oocytes co-cultured with oviductal tissue in serum-free medium
THERIOGENOLOGY
DEVELOPMENT OF BOVINE IN VITRO MATURED AND FERTILIZED OOCYTES CO-CULTURED WITH OVIDUCTAL TISSUE IN SERUM-FREE MEDIUM Y. Nagao, K. Saeki and H. Kainuma Embryo Transplantation Laboratory Snow Brand Milk Products Co., Ltd. Tomakomai-shi, 059-13, Japan for in with somatic cells has been used Co-culture the vitro development of bovine embryos. In this study, serum, effect of macro-molecular supplement (fetal calf FCS) on co-culture of bovine oocytes matured and fertilized in vitro with bovine oviductal tissue was investigated. collected from cumulus oocyte complexes were Bovine ovaries and cultured for 22-24 h in serumslaughterhouse free medium (Saeki et al., 1990, Theriogenology, 33, 376). In vitro fertilization was carried out by a method previously described (Iritani et al., 1988, Ann. N.Y. Acad. from Sci., 541, 583) with some modifications. Frozen semen was used for all replicates. Five hours after two bulls insemination, the oocytes were denuded and cultured in medium with or without heat-treated FCS (10%) and/or bovine oviductal tissue (BOT, Eyestone and First, 1989, J. Reprod. Fert., 85, 715) at 39'C under 5% CO2 in air with high humidity. The basic medium was 25 mM HEPES buffered TCM-199 supplemented with 0.3% polyvinyl pyrrolidone (PVP), 0.5 mM Na-pyruvate and antibiotics. Cleavage and blastocyst development were examined at 45-50 h and 192 h (8 days) after insemination, respectively. Results are shown in the table below.
Treatment PVP FCS BOT + PVP BOT + FCS
No. oocytes cultured 121 119 136 131
No. (%) oocytes cleaved 92(76) 83(70) 93(68) 97(74)
No. (%)a blastocysts l( 1,; 6( 7) 39(42 \c 'C 50(52 )
Data were pooled from 6 replicates. : Percentages of cleaved oocytes. bye: Values differ significantly (PO.O5); however, blastocyst development was higher with BOT present than without (PcO.01). These results indicate that oviductal tissue enhances the blastocyst (I) development of bovine oocytes matured and fertilized in vitro and that FCS is not necessary in this co-culture system and (2) that PVP works as well.
JANUARY
1991 VOL. 35 NO. 1
249
DEVELOPMENT OF BOVINE IN VITRO MATURED AND FERTILIZED OOCYTES CO-CULTURED WITH OVIDUCTAL TISSUE IN SERUM-FREE MEDIUM Y. Nagao, K. Saeki and H. Kainuma Embryo Transplantation Laboratory Snow Brand Milk Products Co., Ltd. Tomakomai-shi, 059-13, Japan for in with somatic cells has been used Co-culture the vitro development of bovine embryos. In this study, serum, effect of macro-molecular supplement (fetal calf FCS) on co-culture of bovine oocytes matured and fertilized in vitro with bovine oviductal tissue was investigated. collected from cumulus oocyte complexes were Bovine ovaries and cultured for 22-24 h in serumslaughterhouse free medium (Saeki et al., 1990, Theriogenology, 33, 376). In vitro fertilization was carried out by a method previously described (Iritani et al., 1988, Ann. N.Y. Acad. from Sci., 541, 583) with some modifications. Frozen semen was used for all replicates. Five hours after two bulls insemination, the oocytes were denuded and cultured in medium with or without heat-treated FCS (10%) and/or bovine oviductal tissue (BOT, Eyestone and First, 1989, J. Reprod. Fert., 85, 715) at 39'C under 5% CO2 in air with high humidity. The basic medium was 25 mM HEPES buffered TCM-199 supplemented with 0.3% polyvinyl pyrrolidone (PVP), 0.5 mM Na-pyruvate and antibiotics. Cleavage and blastocyst development were examined at 45-50 h and 192 h (8 days) after insemination, respectively. Results are shown in the table below.
Treatment PVP FCS BOT + PVP BOT + FCS
No. oocytes cultured 121 119 136 131
No. (%) oocytes cleaved 92(76) 83(70) 93(68) 97(74)
No. (%)a blastocysts l( 1,; 6( 7) 39(42 \c 'C 50(52 )
Data were pooled from 6 replicates. : Percentages of cleaved oocytes. bye: Values differ significantly (P
JANUARY
1991 VOL. 35 NO. 1
249