230 Cryopreservation for Canis lupus conservation: Evaluating protocols to freeze grey wolf testicular tissue

Because of the severe decline in grey wolf (Canis lupus) abundance, preservation of genetic diversity within the species is essential to prevent inbreeding depression. Cryopreservation is a valuable tool in endangered species conservation, because it allows genetic material to be rescued from rare individuals for future use. The ability to preserve gonadal tissue is particularly useful in seasonal breeders, such as grey wolves, when mature gametes are not available during the non-breeding season. Optimization of species-specific cryopreservation protocols is necessary due to the toxicity of cryoprotectants. Here, we aimed to investigate three protocols to cryopreserve grey wolf testicular tissue, as a model for endangered canid species. Testicular tissue samples from four adult grey wolves were obtained and cryopreserved using slow-freezing (SF) and needle vitrification (nVT) techniques. Slow-frozen samples were exposed to 15% dimethylsulfoxide (DMSO) or 7.5% DMSO + 7.5% ethylene glycol (EG) in minimum essential medium (MEM) at room temperature, placed in a Mr. Frosty Freezing Container (Thermo Fisher Scientific) and cooled to −80°C overnight, before storage in liquid nitrogen. Needle-vitrified samples were equilibrated in a 7.5% DMSO + 7.5% EG solution in MEM for 10min at 4°C, then treated with 15% DMSO + 15% EG + 0.5M sucrose for 10min at 4°C before plunging into liquid nitrogen. Each sample was thawed using serial dilutions of sucrose (1, 0.5, and 0.25) into MEM at 37°C for 5min each, fixed in 4% paraformaldehyde, and embedded in paraffin wax. Five-micron-thick cross-sections of tissue samples were stained using haematoxylin and eosin. Tubule areas and quantity of each cell type (Sertoli cells, spermatogonia, spermatocytes, spermatids, and abnormal cells) were recorded and compared with a fresh control group for each wolf. Cell populations and tubule areas were analysed using a nonparametric Wilcoxon test through JMP statistical software (SAS Institute Inc.), and are presented as means±standard deviations. There was no difference in tubule areas between the treatment groups (fresh=5200±2870, nVT=5655±2370, SF DMSO=3607±814, SF DMSO+EG=3905±1450µm2; P>0.05). Although there was an increase in abnormal cells/tubule area (µm2) for nVT, SF DMSO, and SF DMSO+EG compared with the fresh samples (0.0015±0.0014, 0.0014±0.0013, 0.0016±0.0011, and 0.0007±0.0004, respectively; P>0.05), differences were not statistically significant. Moreover, the number of Sertoli cells, spermatogonia, and spermatocytes were reduced in the cryopreserved groups compared with fresh tissues, but the differences were not statistically different (P>0.05). The data suggest that the three tested protocols preserved testicular structure and cell populations of cryopreserved testicular tissue and could be useful in the genetic preservation of grey wolves.