Cryopreservation of grey wolf (Canis lupus) testicular tissue

Development of genomic preservation technologies for canids, especially for seasonally breeding species like the grey wolf (Canis lupus), is needed in advance of growing species conservation concerns. Here, we evaluated the efficacy of two cryopreservation protocols - needle immersion vitrification (NIV) and slow freezing (SF) on grey wolf (n = 7) testicular tissue morphology. NIV samples were equilibrated in a 7.5% v/v dimethyl sulfoxide (DMSO or Me2SO) + 7.5% ethylene glycol (EG) solution in minimum essential medium with 20% FBS for 10 min at 4 °C, then exposed to 15% DMSO + 15% EG + 0.5 M sucrose for 10 min at 4 °C before plunging into liquid nitrogen. For slow freezing, we assessed two cryoprotectant (CPA) strategies, DMSO, 15% v/v alone (SF-D) or 7.5% EG + 7.5% DMSO (SF-ED). Following thawing, there were no significant differences in seminiferous tubule area among treatment groups, although all cryopreserved tissues displayed reduced tubule size compared with fresh controls and increased apoptosis, the latter reaching significance for SF-D treated tissues. Slow freezing improved maintenance of testis architecture, with minimal detachment of seminiferous tubule basement membranes post-thaw. Spermatogonia densities were reduced in NIV tissues compared with fresh, with no differences in spermatocyte, spermatid, or Sertoli cell counts, or germ cell marker DDX4⁺ cell densities among groups. In sum, we conclude that slow freezing better maintained morphology of cryopreserved testicular tissues compared with needle vitrification with 15% each DMSO and EG and 0.5 M sucrose, and that DMSO + EG combination SF supports cell viability. This represents a first step in the development of male gonadal tissue preservation strategies for the grey wolf.