The subfertility derived from male factors is a problem of concern in domestic animals, because it could cause a disintegration of the breeding system and large economic losses, particularly when the subfertility affects genetically superior male animals. Therefore, it is urgent that causal factors of male subfertility be determined. Recently, an increasing number of subfertile bulls have been found among Japanese Black cattle, which is a representative breed of Japanese beef cattle. The purpose of the present study was to elucidate causal factors of male subfertility in Japanese Black cattle. Frozen–thawed spermatozoa from 8 subfertile (S1-S8) and 7 fertile (F1–F7, control) bulls were used for the assessment of fertilization-related parameters. The data obtained from each subfertile bull in the following experiments were individually compared with the mean values of the fertile bull group. In Experiment 1, sperm motility was observed in samples that were frozen-thawed and subsequently washed in PBS. Many spermatozoa (higher than 65%) exhibited flagellar movement in all samples from fertile and subfertile bulls. However, the percentages of progressively motile spermatozoa from 2 subfertile bulls were significantly lower (S2: 6%; S7: 7%; P < 0.05, ANOVA and Tukey's multiple range tests) than those from fertile bulls (average: 37%). Moreover, rapidly progressive movement was not observed in the spermatozoa from 4 subfertile bulls (S1, S2, S6, and S7). These data suggest abnormality in the motility system of sperm flagella in these 4 subfertile bulls. In Experiment 2, the capacitation–acrosome reaction state of frozen–thawed spermatozoa was examined by the CTC-staining assay. More than 50% of the frozen–thawed spermatozoa from 4 subfertile bulls (S5–S8) were prematurely progressing in the capacitation state immediately after washing and resuspension in the medium lacking CaCl2. Moreover, the addition of CaCl2 to the medium induced acrosomal loss in these sperm samples (percentages of spermatozoa without the acrosome: 36–49%). These findings indicate the occurrence of premature capacitation and a spontaneous acrosome reaction in spermatozoa from these 4 subfertile bulls. In Experiment 3, the in vitro fertilizing ability of frozen–thawed spermatozoa was evaluated by the IVF test. The percentages of fertilized eggs with both male and female pronuclei or developmental rates of fertilized eggs to the 2-cell or 4-cell stage were significantly lower in the spermatozoa from S6 to S8 bulls than in those from fertile bulls (P < 0.05, chi-squared tests). This may suggest that spermatozoa from these 3 subfertile bulls hardly accomplish the normal fertilization process. In summary, low progressive motility and low in vitro fertilizing ability because of premature capacitation were found in the spermatozoa from subfertile bulls. It is therefore possible that these are causal factors for the subfertility of male Japanese Black cattle.
Potassium ions modulate expression of mouse sperm fertilizing ability, acrosome reaction and hyperactivated motility in vitro