Long-Term Preservation of Testicular Tissue Integrity and Viability Using Vitrification in the Endangered Black-Footed Ferret (Mustela nigripes)

SIMPLE SUMMARY: In addition to semen preservation, systematic cryo-banking of testicular tissues is critical to preserve the genetic value of recently deceased or neutered black-footed ferrets. Mature sperm cells produced from vitrified-warmed tissues could be used for in vitro fertilization over multiple generations, which would enhance the genetic management of this rare and endangered species. The objective of the study was to evaluate structural and functional properties of vitrified testicular tissues directly after warming or after warming plus a short period of in vitro culture. Fresh, vitrified/warmed, and warmed/cultured tissues from five adults were analyzed through histology, DNA fragmentation, cell survival, and germ cell composition. Percentages of intact seminiferous tubules decreased right after vitrification/warming and improved after culture (reaching same percentages as the fresh controls). While proportions of cells with intact DNA and viable cells were affected by vitrification/warming, they were then similar or better after additional culture than in the fresh tissue. Proportions of cells labeled with differentiation markers also increased during in vitro culture. We demonstrated for the first time that black-footed ferret testicular tissues can be vitrified and revived, which will potentially contribute to new strategies to enhance assisted reproduction as well as conservation efforts in that rare and endangered species. ABSTRACT: Systematic cryo-banking of semen and testicular tissues is critical to preserve the genetic value of recently deceased or neutered black-footed ferrets (BFFs). Specifically, recovering or producing mature sperm cells from vitrified-warmed issues offers additional options in assisted reproduction. This could, in turn, enhance the genetic management of this rare and endangered species over multiple generations. The objective of the study was to evaluate structural properties, DNA fragmentation, cell viability, and germ cell composition in vitrified testicular tissues from BFFs directly after warming or after warming plus a short in vitro culture period. Tissue biopsies from five adult BFFs were either kept fresh or vitrified with a standard protocol (using dimethylsulphoxide (DMSO) and glycerol) and warmed at 50 °C for 5 s. Some of the warmed samples were then cultured in vitro for 24 h. Fresh, warmed, and warmed/cultured tissues were analyzed using different indicators: histology of seminiferous tubules, intact Sertoli cells (vimentin labeling), DNA integrity, cell viability, germ cell composition (Oct4 and Boule labeling). Percentages of intact seminiferous tubules decreased after vitrification/warming and returned to the level of fresh samples after culture. While percentages of cells labeled with vimentin, with intact DNA integrity, or proportions of viable cells were affected by vitrification/warming, they all reached similar or better levels than the fresh tissue after culture. Proportions of cells labeled with Boule antibodies also improved during in vitro culture post-warming. We demonstrated for the first time that BFF testes subjected to vitrification, rapid warming, and short in vitro culture were viable and maintained the ability to resume germ cell progression. Cryopreserved testicular tissues could potentially contribute to new strategies to enhance BFF assisted reproduction as well as conservation efforts.