Premature Capacitation of Frozen-Thawed Spermatozoa from Subfertile Japanese Black Cattle

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Abstract
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 CaCl2-lacking medium. In contrast, most of the sperm from the fertile bulls and other AI-subfertile bulls (S1-S4) remained uncapacitated. Addition of CaCl2 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.

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