Brito Leonardo F C, Barth Albert D, Bilodeau-Goeseels Sylvie, Panich Paul L, Kastelic John P
Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
Theriogenology. 2003 Nov;60(8):1539-51. doi: 10.1016/s0093-691x(03)00174-2.
The objectives of this study were to compare different methods of evaluating sperm plasmalemma and to determine their relationship with in vitro fertilization rate. A single batch of frozen semen from each of eight beef bulls was used for assessment of sperm viability and for in vitro fertilization. Conventional viability tests included sperm morphology, motility, acrosome integrity, and abnormal DNA condensation. Methods for evaluation of the sperm plasmalemma included eosin/nigrosin (EN) and trypan-blue (TB) vital stains, propidium iodide (PI) in combination with carboxyfluorescein diacetate (CFDA) or SYBR-14 (SYBR) fluorescent vital stains, and the hypoosmotic swelling test (HOST). A total of 133-150 oocytes were fertilized in vitro with sperm from each bull and cleavage rates were determined. There were high correlations between the results obtained with vital stains and good to excellent interclass correlation coefficients of agreement, indicating that these stains provide measures of the same sperm attribute, i.e. plasmalemma integrity. However, the proportions of membrane-intact sperm identified by EN or TB stains were greater (P<0.0001) than identified by CFDA/PI or SYBR/PI fluorescent stains. The results obtained with the HOST had moderate correlations but poor agreement with the results of the vital stains. The proportion of viable sperm identified by the HOST was lower (P<0.05) than the proportion identified by vital stains, indicating that response to the HOST did not depend only on the integrity of the plasmalemma. Although there were significant differences in fertilization rates and sperm viability among bulls, there was no sharp distinction for the results of sperm viability tests from bulls producing different in vitro fertilization rates. Proportions of normal, motile, acrosome-intact, and HOST-responsive sperm were identified as significant predictors of in vitro fertilizing potential; each of these endpoints explained 12-18% of the variation when evaluated separately (linear regression) and 48% when evaluated collectively (stepwise regression). In conclusion, EN and TB stains overestimated the proportion of plasmalemma-intact sperm compared to PI-based fluorescent stains. Vital stains evaluated the morphological integrity of the plasmalemma, whereas the HOST assessed plasmalemma function. In that regard, the HOST was the only plasmalemma evaluation method that significantly contributed to conventional sperm quality tests in predicting in vitro fertilization rate, indicating that the test could be incorporated to the routine of semen analysis.
本研究的目的是比较评估精子质膜的不同方法，并确定它们与体外受精率的关系。从八头肉用公牛中获取的每一批冷冻精液用于评估精子活力和体外受精。传统的活力测试包括精子形态、活力、顶体完整性和异常DNA凝聚。评估精子质膜的方法包括伊红/黑色素（EN）和台盼蓝（TB）活体染色、碘化丙啶（PI）与羧基荧光素二乙酸酯（CFDA）或SYBR-14（SYBR）荧光活体染色，以及低渗肿胀试验（HOST）。用每头公牛的精子对总共133 - 150个卵母细胞进行体外受精，并测定分裂率。活体染色获得的结果之间存在高度相关性，类间相关系数的一致性良好到优秀，表明这些染色提供了相同精子属性即质膜完整性的测量方法。然而，EN或TB染色鉴定出的膜完整精子比例高于CFDA/PI或SYBR/PI荧光染色鉴定出的比例（P<0.0001）。HOST获得的结果与活体染色的结果有中等相关性但一致性较差。HOST鉴定出的活精子比例低于活体染色鉴定出的比例（P<0.05），表明对HOST的反应不仅仅取决于质膜的完整性。尽管公牛之间的受精率和精子活力存在显著差异，但产生不同体外受精率的公牛的精子活力测试结果没有明显区别。正常、有活力、顶体完整和对HOST有反应的精子比例被确定为体外受精潜力的重要预测指标；当分别评估时（线性回归），这些终点中的每一个都解释了12 - 18%的变异，而当共同评估时（逐步回归）解释了48%的变异。总之，与基于PI的荧光染色相比，EN和TB染色高估了质膜完整精子的比例。活体染色评估质膜的形态完整性，而HOST评估质膜功能。在这方面，HOST是唯一一种在预测体外受精率方面对传统精子质量测试有显著贡献的质膜评估方法，表明该测试可以纳入精液分析常规流程。
