The generally accepted method for sperm preservation is by freezing in liquid nitrogen at -196 EC. However, this requires a constant replacement of liquid nitrogen for long-term preservation. Development of preservation techniques for male gametes at room temperature would allow us to store them with a simple and cost-effective manner. It has been known that food can be preserved in salt for long periods at room temperature. Therefore, in this study, we have tried to develop a new sperm preservation method at room temperature using a powder of salt and sugars. Whole cauda epididymides of mature B6D2F1 mouse were placed directly in a powder of pure salt (NaCl), sugars (glucose, raffinose or trehalose) or without any additives (untreated control) for 1 day to 1 month, at room temperature. Epididymides were then rehydrated with Hepes-CZB or NIM medium for 15 min at 4EC, and the sperm were collected by disrupting the epididymis using tweezers. In some experiments, cauda epididymides were gradually dehydrated using different concentrations of sugars in water, to avoid the rapid water replacement damage, and rehydration time was extended to avoid insufficient rehydration. The quality of stored spermatozoa was evaluated by their oocyte activation capacity and developmental potential after intracytoplasmic sperm injection (ICSI). When the cauda epididymis was stored for 1 week, there were no significant differences in the oocyte activation capacity of spermatozoa when compared to the fresh control, irrespective of storage conditions (stored: n= 533, fresh: n= 328; 88 to 100% vs 98%). However, when the cauda epididymis was stored for 1 month, the rate of activated oocytes decreased significantly in the untreated control (n= 130; 5%), whereas the cauda epididymis stored in salt or sugar showed that most spermatozoa still retained oocyte activation capacity after ICSI (n= 251; 83 to 95%). Activated oocytes formed male and female pronuclei which appeared normal and extrudes a second polar body 6 h after ICSI. We also examined pronuclear morphology and methylation status of histone H3 lysine 9 (H3K9) in zygotes by immunofluorescence staining, but we did not observe any differences between zygotes fertilized with stored and fresh spermatozoa (stored: n= 71, fresh: n= 30). However, the developmental ability of zygotes generated by ICSI using stored sperm showed a significant decrease. When epididymides were stored in glucose or raffinose for 1 day at room temperature, only 2% and 17% of embryos developed to blastocyst (glucose: n= 203, raffinose: n= 132), and after transfer into recipient females, only 1% and 7 % of them developed to full term (glucose: n= 208, raffinose: n= 182), respectively. However, when epididymides were stored for 1 month in glucose or raffinose, or stored in salt or trehalose for 1 day, none of embryos developed to the blastocyst stage. This could not be improved even when the epididymis was dehydrated gradually (n= 162) and rehydration time was extended (n= 259). We examined the cause of developmental arrest in embryos injected with sperm stored for 1 day and found that 100% (NaCl) or 87.8% (glucose) embryos showed aberrant chromosome segregation at the 2-cell stage (fresh: 7%). In conclusion, although the activation factor(s) is maintained in sperm preserved with salt or sugar for at least 1 month at room temperature, their developmental capacity were lost due to the abnormal distribution of the chromosomes.
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