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Effects of culture system on development of in vitro-fertilized bovine ova into blastocysts
THERIOGENOLOGY
EFFECTS OF CULTURE SYSTEM ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE OVA NT0 BLASTOCYSTS Y. Aoyagi!Y.Fukui?:Y.Iwazumi! M.Urakawd Y.Minegishiaand H.On$ IHokkaido Livestock Improvement’ Association Inc., Kunneppu Tokorogun 099-14,Japan 20bihiro University QKitasorachi Agricultural Mutual Aid Association The effects of co-culture with oviductal epithelial cells,cumulus cells,trophoblastic vesicles or amniotic sac cells on the development of eight-cell embryos derived from in vitro.fertilization into blastocysts were examined. Ovaries were collected from Holstein cows at slaughter. The cumulus oocyte complexes were collected from 2- to 5-mm follicles. The medium for oocyte maturation was TCM199 t 10% fetal calf serum(FCS) supplemented with LH(lOpg/ml) and estradiol(E2:lpg/ml). Thirty to 40 oocytes were placed in medium (500~1) covered by liquid paraffin and cultured for 22-24 h in Co2 incubator(39@C,S%COZinair). Sperm capacitation was accomplished with caffeine(lOmM)tCa-ionophore(O.1pM) for 2.5 h. At 68 h after insemination, ova were removed from the cumulus cell mass and examined for cleavage. Eightcell embryos were cultured in vitro(treatment A to E) or transferred to rabbit oviducts(treatment F). Five culture systems were examined: (A)Control,(B)Co-culture with bovine oviduct epithelial cells(Vet.Rec.121.259). (C)Coculture with bovine cumulus cells(Jpn.J.Anim.Reprod.33, 173), (D)Co-culture with bovine trophoblastic vesicles (J.Reprod.Fert.70,533),(E)Co-culture with amniotic sac cells. Each amniotic sac(Day 60-120) was dissected into small fragments(approx.lxlmm)..Seven to 10 fragments were placed in the culture well. For in vitro culture, TCM199 tlO%FCS was used. The proportion of embryos that developed into blastocysts is shown below. Culture No.of ova No.of ova developing into blastocysts 6 7 8 9 system recovered Total(%) or examined (Days after insemination) A 54 1 l( 1.9)a B 5 23(39.Ojbc 59 8 10 1 82 3 11 1 16(19.5)bd C 35(50.7)bc 69 7 23 5 2 27 7 : 36(48.6)bc 74 F 9 8 17(29.3)bd 58 Values with different superscripts within a column are different(a.b:P
168
JANUARY
1989 VOL. 31 NO. 1
EFFECTS OF CULTURE SYSTEM ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE OVA NT0 BLASTOCYSTS Y. Aoyagi!Y.Fukui?:Y.Iwazumi! M.Urakawd Y.Minegishiaand H.On$ IHokkaido Livestock Improvement’ Association Inc., Kunneppu Tokorogun 099-14,Japan 20bihiro University QKitasorachi Agricultural Mutual Aid Association The effects of co-culture with oviductal epithelial cells,cumulus cells,trophoblastic vesicles or amniotic sac cells on the development of eight-cell embryos derived from in vitro.fertilization into blastocysts were examined. Ovaries were collected from Holstein cows at slaughter. The cumulus oocyte complexes were collected from 2- to 5-mm follicles. The medium for oocyte maturation was TCM199 t 10% fetal calf serum(FCS) supplemented with LH(lOpg/ml) and estradiol(E2:lpg/ml). Thirty to 40 oocytes were placed in medium (500~1) covered by liquid paraffin and cultured for 22-24 h in Co2 incubator(39@C,S%COZinair). Sperm capacitation was accomplished with caffeine(lOmM)tCa-ionophore(O.1pM) for 2.5 h. At 68 h after insemination, ova were removed from the cumulus cell mass and examined for cleavage. Eightcell embryos were cultured in vitro(treatment A to E) or transferred to rabbit oviducts(treatment F). Five culture systems were examined: (A)Control,(B)Co-culture with bovine oviduct epithelial cells(Vet.Rec.121.259). (C)Coculture with bovine cumulus cells(Jpn.J.Anim.Reprod.33, 173), (D)Co-culture with bovine trophoblastic vesicles (J.Reprod.Fert.70,533),(E)Co-culture with amniotic sac cells. Each amniotic sac(Day 60-120) was dissected into small fragments(approx.lxlmm)..Seven to 10 fragments were placed in the culture well. For in vitro culture, TCM199 tlO%FCS was used. The proportion of embryos that developed into blastocysts is shown below. Culture No.of ova No.of ova developing into blastocysts 6 7 8 9 system recovered Total(%) or examined (Days after insemination) A 54 1 l( 1.9)a B 5 23(39.Ojbc 59 8 10 1 82 3 11 1 16(19.5)bd C 35(50.7)bc 69 7 23 5 2 27 7 : 36(48.6)bc 74 F 9 8 17(29.3)bd 58 Values with different superscripts within a column are different(a.b:P
168
JANUARY
1989 VOL. 31 NO. 1