福岡県農業総合試験場研究報告

培養液交換および共培養がウシ体外受精胚の発生および耐凍性に及ぼす影響

[摘要]耐凍性の高いウシ体外受精胚を効率的に生産するため、修正ＣＲ１ａａからβメルカプトエタノール添加ＴＣＭ１９９への発生培養液の交換及び培養液中の卵丘細胞の有無（共培養または非共培養）が胚の発生と耐凍性に及ぼす影響について検討した。①受精後３日目に培養液を修正ＣＲ１ａａからβメルカプトエタノール添加ＴＣＭ１９９へ交換し卵丘細胞を除去する非共培養法及び受精後５日目に培養液を交換する共培養及び非共培養法により、修正ＣＲ１ａａ単独の培養法と同等またはそれ以外の高い胚盤胞発生率（29.4％ｖｓ28.3～35.5％）及び同等の卵巣当たり胚盤胞作出数（3.1胚／卵巣ｖｓ2.8～3.9胚／卵巣）が得られた。②凍結融解による胚の耐凍性試験では、受精後３日目に培養液を交換する非共培養法及び受精後５日目に交換する共培養法により、ＣＲ1ａａ単独培養と比較して高い耐凍性（生存率：50.8％ｖｓ72.9，90.0％、脱出率：30.0％ｖｓ62.5、86.7％）が得られた。特に、受精後３日日に培養液を交換し卵丘細胞を除去する非共培養法は、発生率が高く耐凍性にも優れているため、凍結移植可能な体外受精胚を高率に生産できる。

［キーワード：ウシ体外受精胚、培養液、卵丘細胞、発生率、耐凍性］

　Effect of a Changed Culture Medium with or without Cumulus Cells on the Development and the Frozen -thawed Viability of In Vitro Fertilized Bovine Embryos. KASA Shojiro, Shuji UEDA and Yoshihiro ISOZAKI (Fukuoka Aguricultural Research Center, Chikushino, Fukuoka 818-8549, Japan). Bull. Fukuoka Agric. Res. Cent., 18: 122 - 125 (1999)　　　

　 Investigation was made on the effect of a changed culture medium with or without cumulus cells on the development and the frozen -thawed viability of in vitro fertilized (IVF) bovine embryos. The culture medium CR1aa supplemented with 5%CS (m-CR1) was replaced with TCM199 supplemented with 20% FBS 0.1mMβ-mercaptatoethanol (β-199) on Day2, Day3 or Day5 (Day0=IVF) with or without cumulus cells. Control IVF embryos were designed to be nurtured as in an unchanged culture medium (m-CR1) with cumulus cells for all of the cultured period.

　 1　The changes of the culture medium (m-CR1 / β-199) on Day3 without cumulus cells and on Day5 with or without cumulus cells resulted in equal or high percentages (cont.:29.4% vs 28.3 - 35.5%) of blastocysts (blastocysts / oocytes) and a similar rate (cont.:3.1blastocysts / ovary vs 2.8 - 3.9 blastocysts / ovary) of blastocysts per ovary compared to the control .

　 2　Changes to the culture medium (m-CR1 / β-199) on Day3 without cumulus cells and on Day5 with cumulus cells resulted in high percentages (p<0.01) of survival (cont.:50.8% vs 72.9 , 90.0%), and on Day3 without cumulus cells and on Day5 with cumulus cells resulted in high percentages (p<0.05) of hatched blastocysts (cont.:30.0% vs 62.5, 86.7%) after cryopreservation compared to the control.

　 These results indicate that the IVF bovine embryos in the replaced culture medium (m-CR1 / β-199) and cumulus cells removed on Day3 have a high rate of blastocysts and high viability after cryopreservation.

　 It is expected that the IVF bovine embryos cultured by this method have a high pregnancy rate in embryo transfer.

[Key words: in vitro fertilized bovine embryos, culture medium, cumulus cells, development, frozen-thawed viability ]　　　　　　　　　　　　　　