主要探讨精子活动促进剂对水牛Bubalus bubalis卵母细胞体外受精及随后胚胎发育的影响. 来自屠宰场水牛卵巢的卵母细胞和卵丘细胞复合体,在体积分数为5%CO2 的培养箱中培养24～26 h,然后通过体外受精测定其受精和胚胎发育能力. 试验1:解冻的精子用50 g/mL的胰蛋白酶处理30 min,然后进行受精;试验2:受精在含不同浓度咖啡因 (0, 2.5, 5.0 和 10.0 mmol/L)的受精液中进行;试验3:在含不同浓度的钙离子载体A23187 (0, 0.1, 1.0 和 2.5 μmol/L)的受精液中进行体外受精;试验4:在PHE、咖啡因和A23187不同组合(空白, PHE, PHE+2.5 mmol/L咖啡因+2.5 μmol/L A23187和PHE+2.5 μmol/L A23187) 的受精液中进行体外受精. 试验结果表明,精子用胰蛋白酶处理后的受精卵囊胚发育率显著下降 (12.5%和2.6%,P<0.05),而用咖啡因和钙离子载体A23187处理,对卵母细胞的卵裂率和囊胚发育率没有明显影响. 在体外受精中PHE和钙离子载体的组合使用能提高受精卵的胚胎发育率,而高浓度的咖啡因则降低卵母细胞的卵裂率和胚胎发育率.%Effects of sperm-motility enhancing agents on the in vitro fertilization of buffalo oocytes and their subsequent development in vitro were examined in this study. Buffalo oocytes from ovaries taken at slaughter were matured in vitro for 24-26 h at 38.5 ℃ under a humidified 5% CO2 in air, and then fertilized in vitro using buffalo spermatozoa washed with 50 μg/mL trypsin in experiment 1, treated with different concentration of caffeine (0, 2.5, 5.0, and 10.0 mmol/L) in experiment 2, calcium-ionophore A23187 (0, 0.1, 1.0 and 2.5 μmol/L) in experiment 3 or PHE only, PHE+2.5 mmol/L caffeine+2.5 μmol/L A23187, and PHE+2.5 μmol/L A23187 in experiment 4. At 24 to 26 h post insemination, the oocytes were co-cultured with granulosa cell monolayer in droplets containing culture medium to evaluate their embryonic development. There was a significant decrease (12.5% vs 2.6%, P<0.05) in the proportion of cleaved embryos that developed to blastocyst after sperm treatment with trypsin. The treatment of spermatozoa with caffeine and calcium-ionophore A23187 had no significant effect on cleavage and blastocyst yield. There was a minor improvement in blastocyst yield when PHE combined with calcium-ionophore was employed during IVF. However, high concentrations of caffeine seem to be detrimental to cleavage rate and blastocyst development.
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