Fertility preservation of spin dried ram spermatozoa following 3 years storage at room T°

Biobanking allows the conservation of somatic and gametic cells for the preservation of biodiversity and medical applications. Currently, biobanks are maintained in liquid nitrogen (LN). Storage in LN or at -80 ° C requires a great consumption of resources and energy, with a strong economic and environmental impact, thus, alternative methods, such as dry storage, may represent suitable alternatives. Freeze drying has been the first method reported to dehydrate spermatozoa. However, on-going experiments in our laboratory indicate that alternative methods of water removal might be less damaging. One of these methods is spin-dry (SD). SD allows the drying of spermatozoa under vacuum, a gentler procedure comparing with lyophilization. In this study we set up to investigate the preservation of fertilizing capacity of dry ram spermatozoa following spin dry and room temperature (RT) storage, comparing with the standard procedures of freeze drying. Moreover, a longer storage has been simulated by heating the dry spermatozoa at 65C° for 10 minutes (65C) and 100°C for 10 minutes (100C). Samples dehydrated by SD and stored at RT for 3 years, were subjected to a thermal stress of 65 and 100°C for 10 min. The samples were heated in hot water inside their stainless steel/glass containers, rehydrated with double distilled water and then utilized for intra cytoplasmatic sperm injection (ICSI). In vitro matured sheep oocytes (a total of 280 oocytes – 180 for 65C and 100 for 100C) were fertilized using the ICSI procedures in use in our laboratory, then chemically activated by 5 min incubation in ionomycin followed by 4 hours incubation in cycloheximide. Presumptive zygotes were cultured in humidified atmosphere at 38.5°C, 5% CO2, 7% O2 in IVC medium (ivf Bioscience, cat. 71005). The highest percentage of cleavage was observed in 65C then 100C (51.38% vs 24%, p<0.0001) against 47.16% of the control (ICSI with not treated SD semen on 53 oocytes) while the percentage of blastocysts was 11.6% and 9% respectively against 13.2% of the control. The obtained blastocysts obtained were stored in part in liquid nitrogen for future molecular analysis, and in part were cultured in vitro to obtain embryonic cell lines (outgrowth) for point-mutation analysis. These results indicate, for the first time, that spin dried ram spermatozoa, maintain the fertilizing capacity even after 3 years of room temperature storage. Moreover, the preservation of the fertilizing potential even after an intense thermic stress, indicated that long-term room temperature storage of spin dry spermatozoa might be a realistic option.