Long-term fertility preservation of spin-dried ram spermatozoa stored at room temperature

Biobanking allows the conservation of somatic cells and gametes for biodiversity preservation and medical applications. Currently, biobanks are kept in liquid nitrogen (LN), a well mastered technology which requires though a great consumption of resources with a strong economic and environmental impact. Alternative methods, such as dry storage, may represent suitable green alternatives. Freeze-drying spermatozoa is the first method reported offspring production in mice in 1998. However, on- going experiments in our laboratory indicate that alternative methods, such as Spin-Dry (SD), might be less damaging. SD entails the rapid water subtraction in non-frozen samples in a controlled centrifuge kept under vacuum, a much smoother procedure comparing to lyophilization. Furthermore, dry samples are packaged into a laser sealed mini-capsules under an anhydrous atmosphere saturated with 98% helium and 2% argon to avoid long term oxidative damage of DNA. In this work, we tested fertility preservation of spin-dried ram spermatozoa stored at room temperature (RT) for 3 years. Additionally, we simulated a long-term storage by exposing spin-dried spermatozoa to a thermal stress, which is known to induce damage on DNA. Briefly, SD sperm were subjected to a thermal stress of 65 (65C) and 100°C (100C) for 10 min. The samples were heated in hot water inside their stainless steel/glass containers, rehydrated with bi-distilled water and then used for Intra-Cytoplasmic Sperm Injection (ICSI). In vitro matured sheep oocytes were fertilized by ICSI, 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% CO 2 , 7% O 2 in IVC-medium (ivf Bioscience, cat. 71005). ICSI was performed with spin-dried spermatozoa stored at RT for 2 and 3 years. Blastocyst percentage was 13,21% and 12, 37% respectively. After sperm exposure at high temperature, blastocyst percentage is 11,6% (65C) and 9% (100C). There is not a statistical difference between 2 and 3 years of storage, suggesting that spin-dried spermatozoa can be stable preserved for years. This is the first report of embryonic development following ICSI with spin-dried spermatozoa kept a RT for a relatively long time. We can conclude that storage at RT of ram spin-dried spermatozoa might be a realistic option.