Effects of glutathione treatments during sperm washing and in vitro fertilization on the in vitro early development of embryos of Japanese Black cattle.

This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.

Persistence of undifferentiated spermatogonia in aged Japanese Black cattle.

Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age-related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age-related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.

Glutathione treatment of Japanese Black bull sperm prior to intracytoplasmic sperm injection promotes embryo development.

Intracytoplasmic sperm injection (ICSI) was expected to enable more efficient use of sperm from sires with preferable genetic traits and result in a generation containing a larger number of offspring with superior genetic characteristics in livestock. However, the efficiency of the early development of embryos produced by ICSI is still far from satisfactory in cattle. The present study aimed to investigate the effects of the treatment of cryopreserved sperm with glutathione (GSH) on the early development of embryos produced by ICSI in Japanese Black cattle. Moreover, the disulfide bond state and mitochondrial function were investigated in the sperm treated with GSH to confirm the effectiveness of the abovementioned treatment. We also investigated the effect of 7% ethanol activation treatment on the developmental ability of ICSI embryos using GSH-treated sperm. There was no effect on the blastocyst rate from the activation treatment. When sperm-injected oocytes were cultured in vitro, the treatment with GSH significantly improved the early development of embryos. Specifically, the rates of embryos reaching the 4-8-cell stage and blastocyst stage were significantly higher in ICSI with GSH-treated sperm (71.4% and 31.0%, respectively) than that with the control sperm (36.6% and 7.0%, respectively). Moreover, the GSH-treated sperm treatment significantly decreased the number of disulfide bonds in the sperm head (as shown by monobromobimane staining) and enhanced the mitochondrial function in the sperm middle piece (as shown by Rhodamine 123 staining and the adenosine triphosphate-dependent bioluminescence assay). Based on these results, we suggest that the treatment of cryopreserved sperm with GSH might contribute to the improvement of ICSI techniques for the production of blastocysts in Japanese Black cattle.