Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.
Theriogenology. 2012 Oct 15;78(7):1570-7. doi: 10.1016/j.theriogenology.2012.06.026. Epub 2012 Aug 25.
The objectives were to standardize some methodological and analytical aspects of a direct technique to detect sperm-bound antisperm antibodies (ASAs) in bovine semen using flow cytometry, including the effects of prefixation of sperm membranes with formalin buffer solution and inclusion of dead cells in the analysis. Fourteen Angus bulls, including ASA-positive (experimentally induced ASAs) and 10 reproductively normal ASA-negative bulls, were used. Fixation of sperm membranes had no significant effect on the percentage of IgG- or IgA-bound spermatozoa detected by flow cytometry. However, including dead cells in the analysis increased the percentage of IgG-bound spermatozoa in fixed (live and dead 18.6 ± 9.7% and live 1.3 ± 0.5%; median ± SEM) and nonfixed samples (live and dead 18.8 ± 9.2%, live 1.5 ± 0.6%; P = 0.0029), as well as IgA-bound spermatozoa in fixed (live and dead 16.3 ± 6.4%, live 0.3 ± 0.5%) and nonfixed samples (live and dead 21.4 ± 4.6%, live 1.0 ± 0.5%; P = 0.0041) in semen from ASA-negative bulls. Intrasample, intra-assay, and interassay coefficients of variation (CV) were 0.8%, 4.6%, and 5.3%, respectively, for determination of sperm-bound IgG, and were 2.8%, 8.4%, and 40.3% for determination of sperm-bound IgA. Despite the high interassay CV for IgA determination, all ASA-positive bulls consistently had high percentages of IgA-bound spermatozoa. Flow cytometry correctly identified ASA-positive bulls. Confocal laser microscopy confirmed binding of ASAs to sperm heads and cytoplasmic droplets, and less frequently to midpieces and principal piece. In conclusion, although fixation was not necessary, dead cells should be excluded from the analysis, because ejaculates with a large proportion of dead cells can yield false-positive results. Flow cytometry was accurate and reliable for detection of sperm-bound IgG and IgA and discrimination between ASA-positive and ASA-negative bulls.
目的是标准化一种使用流式细胞术直接检测牛精液中精子结合抗精子抗体（ASAs）的方法，包括用福尔马林缓冲液固定精子膜和分析中包含死细胞的影响。使用了 14 头安格斯公牛，包括 ASA 阳性（实验诱导的 ASAs）和 10 头生殖正常的 ASA 阴性公牛。精子膜的固定对流式细胞术检测到的 IgG 或 IgA 结合精子的百分比没有显著影响。然而，在分析中包含死细胞会增加固定（活和死 18.6 ± 9.7%和活 1.3 ± 0.5%；中位数±SEM）和非固定样本（活和死 18.8 ± 9.2%，活 1.5 ± 0.6%；P = 0.0029）中 IgG 结合精子的百分比，以及非固定样本中 IgA 结合精子的百分比（活和死 16.3 ± 6.4%，活 0.3 ± 0.5%）和非固定样本（活和死 21.4 ± 4.6%，活 1.0 ± 0.5%；P = 0.0041）中的 IgG 结合精子和 IgA 结合精子。对于确定精子结合 IgG，测定的样品内、测定内和测定间变异系数（CV）分别为 0.8%、4.6%和 5.3%，而对于确定精子结合 IgA，测定的 CV 分别为 2.8%、8.4%和 40.3%。尽管 IgA 测定的测定间 CV 很高，但所有 ASA 阳性公牛的 IgA 结合精子百分比始终很高。流式细胞术正确识别了 ASA 阳性公牛。共聚焦激光显微镜证实了 ASAs 与精子头部和细胞质小滴结合，并且不太常见与中段和主段结合。总之，虽然固定不是必需的，但应从分析中排除死细胞，因为含有大量死细胞的精液可能会产生假阳性结果。流式细胞术对于检测精子结合 IgG 和 IgA 以及区分 ASA 阳性和 ASA 阴性公牛是准确和可靠的。
