142 Horse oocyte intracytoplasmic sperm injection with freeze-dried and frozen donkey spermatozoa: preliminary results

Most donkey breeds face the risk of extinction. Only limited information about assisted reproductive technologies in donkeys is available so far. This study aimed to assess the in vitro fertilising ability of freeze-dried and frozen sperm from the Martina Franca donkey following intracytoplasmic sperm injection (ICSI) into horse oocytes aspirated from abattoir-derived ovaries. Nowadays, freeze-drying is considered the ultimate method for long-term storage of biological material from endangered species and breeds because it allows for storage independent from liquid nitrogen. Donkey sperm were diluted and frozen in INRAFreeze or diluted in drying media (trehalose, KCl, glucose, EDTA, Trolox), electroporated, freeze-dried in 200-µL aliquots, and stored at room temperature for several months. After holding at room temperature overnight, the oocytes were in vitro matured for 28–30 h in DMEM-F12 supplemented with 10% FCS, 50 ng/mL EGF, 100 ng/mL IGF1, and 0.1 IU/mL pFSH-LH. Matured oocytes were denuded, and those presenting an extruded polar body were selected. Donkey freeze-dried sperm were rehydrated by adding 200 μL of bi-distilled water. Then, a 5-μL aliquot was suspended in 100 μL of SOF-IVF. Frozen sperm were thawed, washed by centrifugation, and suspended in SOF-IVF. Both sperm types were diluted 1:1 with 12% PVP. Piezo-ICSI was performed using: (1) freeze-dried sperm (FD); (2) frozen motile sperm (Fm); and (3) frozen nonmotile sperm (Fnm). Presumptive zygotes were cultured in SOF-IVC. Cleavage rate and early embryo development were recorded 40 h after fertilisation. Embryos and non-cleaved oocytes were stained with Hoechst 33342 to assess the nuclei and sperm status, respectively. Non-cleaved oocytes were classified as having an intact sperm in the cytoplasm (INT), a decondensed sperm (DEC), or a male pronucleus (PN). The experiment included three replicates. Data were analysed by chi-squared test (IBM SPSS Statistics). Statistical significance was set at P < 0.05. 60 matured oocytes were injected. No cleavage was observed in the FD (0/31) and Fnm (0/12) groups, while 9/17 oocytes (52.9%) cleaved in the Fm group (P < 0.05). Four of the nine embryos were at the 2-cell stage, three at the 4-cell stage, and two at the 6–8-cell stage. No differences were observed (P > 0.05) among the non-cleaved oocytes (FD group: 6 INT, 19 DEC, 2 PN; Fm group: 2 INT, 2 DEC, 1 PN; Fnm group: 5 INT, 7 DEC). In conclusion, these preliminary results show that frozen donkey spermatozoa can fertilise equine oocytes by ICSI, while the freeze-dried and non-motile spermatozoa could not progress through the fertilisation process. DNA fragmentation issues could be a possible explanation for this failure. Further studies are ongoing to clarify this aspect.