Suzuki K, Geshi M, Yamauchi N, Nagai T
Tohoku National Agricultural Experiment Station, Morioka 020-0123, Japan.
Anim Reprod Sci. 2003 Jul 15;77(3-4):157-72. doi: 10.1016/s0378-4320(03)00035-6.
Spermatozoa from two Japanese Black bulls (Bull-ATF and Bull-KTG) were separated by centrifugation at 700 x g for 15min in modified TALP with or without 45-90% Percoll. Control washed spermatozoa and those collected from the bottom of 45 and 90% Percoll fractions were examined for viability and membrane integrity (using Hoechst bis-benzimide 33258 or propidium iodide and 6-carboxyfluorescein diacetate (PI-CFDA)), acrosomal status (using fluorescence isothiocyanate (FITC) conjugated Pisum Sativum agglutinin (PSA) and Peanut agglutinin (PNA), Naphthol Yellow S and Erythrosin B (NE) or triple staining (TS)), capacitation status (using chlortetracycline (CTC)), motility characteristics (using a computer-assisted sperm motion analysis system (CASA)) and for in vitro fertility. Percoll-separated spermatozoa showed greater viability and membrane integrity than controls, as determined by supravital staining. Differences were observed in the results regarding viability and acrosomal status of spermatozoa among sperm staining methods. Bull-ATF, which showed significantly greater in vitro fertility than Bull-KTG (P<0.05), showed a significantly higher rate of CTC-B-pattern (capacitated) spermatozoa (P<0.01) than Bull-KTG. The motility characteristics of control washed spermatozoa and those separated by 45-90% Percoll were analyzed by CASA. More motile and progressively motile spermatozoa were observed in the fraction at the bottom of the 90% Percoll solution than in the 45% Percoll fraction or in controls (P<0.01). Moreover, the spermatozoa of Bull-KTG, which showed lower in vitro fertility than Bull-ATF, did not show significant differences in motility from those of Bull-ATF. These results provided basic information about Japanese Black bull spermatozoa, and suggested that spermatozoa with greater motility and viability can be obtained by Percoll separation than without separation. However, Percoll separation did not enhance their in vitro fertility.
将来自两头日本黑牛（公牛 - ATF和公牛 - KTG）的精子，在含有或不含有45 - 90% Percoll的改良台氏液（TALP）中以700 x g离心15分钟进行分离。对对照洗涤后的精子以及从45%和90% Percoll组分底部收集的精子进行活力、膜完整性检测（使用双苯甲酰亚胺 Hoechst 33258或碘化丙啶和6 - 羧基荧光素二乙酸酯（PI - CFDA））、顶体状态检测（使用异硫氰酸荧光素（FITC）偶联的豌豆凝集素（PSA）和花生凝集素（PNA）、萘酚黄S和赤藓红B（NE）或三重染色（TS））、获能状态检测（使用金霉素（CTC））、运动特性检测（使用计算机辅助精子运动分析系统（CASA））以及体外受精能力检测。通过体外活体染色测定，Percoll分离的精子比对照精子显示出更高的活力和膜完整性。在精子染色方法之间，观察到精子活力和顶体状态结果存在差异。体外受精能力显著高于公牛 - KTG（P<0.05）的公牛 - ATF，其显示出CTC - B型（获能）精子的比例显著高于公牛 - KTG（P<0.01）。通过CASA分析对照洗涤后的精子以及经45 - 90% Percoll分离的精子的运动特性。在90% Percoll溶液底部的组分中观察到的有活力和进行性运动的精子比在45% Percoll组分或对照中更多（P<0.01）。此外，体外受精能力低于公牛 - ATF的公牛 - KTG的精子，其运动能力与公牛 - ATF的精子没有显著差异。这些结果提供了有关日本黑牛精子的基础信息，并表明通过Percoll分离比不分离可获得具有更高运动能力和活力的精子。然而，Percoll分离并未提高它们的体外受精能力。
