修正Earle平衡塩類液で培養したネコ体外受精卵の発育:添加蛋白ならびに培養皿の表面処理が受精率, 胚盤胞発生率に及ぼす影響 Development of in vitro Fertilized Feline Embryos in a Modified Earle's Balanced Salt Solution : Influence of Protein Supplements and Culture Dishes on Fertilization Success and Blastocyst Formation

新たに考案した修正Earle平衡塩類液;MK-1とMedium199(M-199)を用いて, 添加蛋白ならびに培養皿の表面処理がネコ体外受精卵子の発育に及ぼす影響を調べた.添加蛋白には非働化ヒト血清(HS), FCS, BSAを用い, 培養皿には組織培養用(TCD)と浮遊培養用(SCD)を用いた.TCDを用いた場合, 体外受精(IVF)後の卵割率はMK-1+10%HSが94.7%で最も高く, 以下MK-1+0.4%BSA(74.4%), MK-1+10%FCS(56.1%), M-199+10%FCS(51.4%)の順であった.IVFにより得られた卵割胚は何れの実験区においても培養96時間目の桑実胚までは良好な発育を示したが, 144時間目の胚盤胞発生率ではMK-1+HS(50.0%)と他の実験区(5.3〜17.2%)との間の有意差が認められた.次にMK-1+HSを用いて培養皿の検討を行ったところ, IVF後の卵割率ではTCDとSCDで差は認められなかった.TCDでは卵丘細胞が培養皿に付着増殖するため胚は培養皿の底面に固定された状態となったが, SCDでは同様の現象は認められなかった.胚盤胞発生率では, SCDが培養120時間目(47.2%), 144時間目(71.7%)の何れの時点でもTCD(22.2%, 55.6%)を上回った.MK-1+HS(SCD)で120時間培養した胚盤胞, ならびにMK-1+BSA(SCD)で120時間培養した後期桑実胚をrecipientの子宮角に1頭当たり6個移植し, 胎子への発生能を調べた.その結果, 胚盤胞を移植した10頭中8頭が, 桑実胚を移植した7頭中5頭が受胎し, このうち12頭が1〜5(平均2.6)頭の子ネコを正常分娩した.以上の成績から, ヒト血清はネコ体外受精卵子の卵割率ならびに胚盤胞発生率を向上させることが明らかとなった.

The effects of protein supplements and culture dish type on in vitro fertilization (IVF) and embryo development in culture were examined in the domestic cat. In experiment I, follicular oocytes were fertilized and cultured in either 1) modified Earle's balanced salt solution, designated MK-1, supplemented with one of the following : 10% human serum (HS), 10% FCS or 0.4% BSA, or 2) Medium 199 (M-199) supplemented with 10% FCS. Fertilization rates were lower (P<0.01) in MK-1+BSA (74.4%), MK-1+FCS (56.1%), and M-199+FCS (51.4%) than in MK-1+HS (94.7%). A greater (P<0.01) percentage of blastocysts was obtained in MK-1+HS (50.0%) than in other treatment groups (range, 4.3-17.2%). In Experiment II, the effect of dish type (tissue culture dish, TCD, versus suspension culture dish, SCD) on embryo development was evaluated in MK-1 supplemented with either HS or BSA. Significantly higher proportions of IVF-derived embryos developed to blastocysts at 120 and 144 hr post-insemination, respectively, when cultured in HS/SCD (47.2 and 71.7%) than in BSA/SCD (11.4 and 27.3%) or BSA/TCD (10.4 and 25.0%). At 120 hr post-insemination, there was a lower (P<0.01) percentage of blastocysts in HS/TCD (22.2%) than in HS/SCD. In Experiment III, six embryos per cat were transferred to the uterine horns of 17 recipients at 144 hr after hCG treatment. Five of 7 recipients which received late morulac cultured in MK-1+BSA (SCD) for 120 hr became pregnant (71.4%). Eight of 10 recipients which received early blastocysts cultured in MK-1+HS (SCD) for 120 hr became pregnant (80.0%). We conclude that MK-1 containing HS is highly beneficial for overcoming the in vitro developmental block of IVF-derived feline embryos and increasing the success rate of IVF/ET.