Kuroda Ken, Fukushima Moriyuki, Harayama Hiroshi
Graduate School of Science and Technology, Kobe University, Kobe, Japan.
J Reprod Dev. 2007 Oct;53(5):1079-86. doi: 10.1262/jrd.19031. Epub 2007 Jul 6.
Artificial insemination (AI) subfertility is an indication of failure of AI with frozen-thawed sperm classified as normal by conventional semen examination. Recently, 8 AI-subfertile Japanese Black cattle (S1-S8) were identified using the routine AI test or in vivo fertilization test, which included AI with frozen-thawed sperm of superovulated females and subsequent non-surgical recovery of presumptive zygotes. In the present study, we assessed capacitation states and in vitro oocyte penetration of frozen-thawed sperm from these bulls to estimate causal factors of AI subfertility. Frozen-thawed sperm from 8 AI-subfertile (S1-S8) and 9 fertile (F1-F9, control) bulls were washed and then used for a chlortetracycline (CTC) staining assay and in vitro fertilization test. The CTC staining assay revealed that approximately 50% of the sperm from 4 of the AI-subfertile bulls (S5-S8) were prematurely progressing into the capacitation state immediately after washing and resuspension in a CaCl(2)-lacking medium. In contrast, most of the sperm from the fertile bulls and other AI-subfertile bulls (S1-S4) remained uncapacitated. Addition of CaCl(2) to the medium effectively promoted a spontaneous acrosome reaction in the sperm samples from the AI-subfertile bulls (S5-S8). Moreover, the in vitro fertilization test showed that rates of sperm penetration into oocytes were significantly lower in sperm samples from the AI-subfertile bulls (S5-S8) than in the control sperm samples from the fertile bulls (F2-F4 and F7-F9). It has previously been suggested that prematurely capacitated sperm undergo a spontaneous acrosome reaction possibly due to uncontrolled influx of calcium ion, and consequently they possess relatively lower in vitro fertilizing ability. It is therefore possible that premature capacitation of sperm used for AI is a causal factor of subfertility of male Japanese Black cattle and a potentially good marker for identification of subfertile bulls for removal from AI programs.
人工授精（AI）不育是指经常规精液检查分类为正常的冻融精子进行人工授精失败的情况。最近，通过常规人工授精试验或体内受精试验，鉴定出8头人工授精不育的日本黑牛（S1 - S8），其中包括用超排雌性的冻融精子进行人工授精以及随后非手术回收假定的受精卵。在本研究中，我们评估了这些公牛冻融精子的获能状态和体外卵母细胞穿透能力，以估计人工授精不育的因果因素。将8头人工授精不育（S1 - S8）和9头可育（F1 - F9，对照）公牛的冻融精子洗涤后，用于金霉素（CTC）染色试验和体外受精试验。CTC染色试验显示，4头人工授精不育公牛（S5 - S8）中约50%的精子在洗涤并重新悬浮于无氯化钙的培养基后立即过早进入获能状态。相比之下，可育公牛和其他人工授精不育公牛（S1 - S4）的大多数精子仍未获能。向培养基中添加氯化钙有效地促进了人工授精不育公牛（S5 - S8）精子样本中的自发顶体反应。此外，体外受精试验表明，人工授精不育公牛（S5 - S8）的精子样本中精子穿透卵母细胞的比率显著低于可育公牛（Fz - F4和F7 - F9）的对照精子样本。此前有人提出，过早获能的精子可能由于钙离子不受控制的流入而发生自发顶体反应，因此它们的体外受精能力相对较低。因此，用于人工授精的精子过早获能可能是日本黑牛雄性不育的一个因果因素，也是识别不育公牛以便从人工授精计划中淘汰的一个潜在良好标志物。
