学会発表Factors affecting DNA damage in bovine spermatozoa after freeze drying
Recently, freeze-drying has been proposed as a novel method to preserve mammalian spermatozoa. If bovine spermatozoa are commonly stored by freeze-drying, it becomes an effective preservation method against great disasters. However, no calves have been obtained by FD spermatozoa, although blastocysts were produced (Keskintepe et al., 2002, Martins et al., 2007, Hara et al., 2014). In this study, factors affecting DNA damage in bovine freeze-dried (FD) spermatozoa were examined. Frozen spermatozoa were thawed and washed by Percoll density gradient centrifugation. Washed spermatozoa were incubated in BO supplemented with 5 mM dithiothreitol (DTT) or 8 mM glutathione (GSH) for 10 min at 38.5℃. Then, spermatozoa were suspended in two freeze drying solution, m-EGTA (50 mM EGTA, 100 mM Tris HCl, pH 8.2) and Na-EGTA (50 mM NaCl, 50 mM EGTA, 10 mM Tris HCl, pH 8.2), respectively. After freeze-drying, alkaline comet assay was used to assess DNA damage in spermatozoa. The FD spermatozoa were observed by Transmission Electron Microscopy (TEM). FD spermatozoa were also injected into matured oocytes. The rate of DNA-damaged spermatozoa was 0% in Na-EGTA and 2% in m-EGTA. The rate of DNA-damaged spermatozoa in GSH treatment was significantly lower than that in DTT treatment (0% vs. 25%, P<0.05). After freeze-drying, the nuclear membranes treated by DTT were destroyed, however, the nuclear membranes treated by GSH were intact according to TEM observation. The blastocyst formation rates in GSH treatment and DTT treatment were 8.5% and 0%, respectively. In conclusion, the DNA damage in bovine spermatozoa are suppressed after freeze-drying, when they are washed by Percoll density gradient centrifugation, incubated with GSH, and suspended in Na-EGTA. And GSH treatment for FD spermatozoa is effective to produce blastocysts.