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Lay Summary: The decreasing rate of successful pregnancies, both naturally and through assisted conception, has led to innovations in the way eggs, sperm, and embryos are stored. Despite these advances, the use of assisted reproductive techniques to preserve endangered or rare species remains unexplored. Since the location where samples are collected and facilities are often far apart, we aim to address part of this challenge by comparing different methods to store and handle ovarian tissue before freezing. This may pave the way for further research in preserving endangered species, despite the challenges posed by the distance between sample collection sites and suitable facilities.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species.