Cryopreservation of Giraffe Epidydimal Spermatozoa Using Different Extenders and Cryoprotectants

SIMPLE SUMMARY: Giraffe numbers have been plummeting over the last 30 years by 30–40%. As such, efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques such as sperm cryopreservation can help preserve the genetic diversity of giraffe subspecies or enhance genetic exchange between populations. However, to date, the post-thaw motility of recovered sperm has been variable. In this study, spermatozoa were collected from the epididymides of seven giraffes to investigate whether an alternative cryoprotectant could improve sperm motility following conventional cryopreservation. For this, we compared the motility and viability of sperm prior to and after freezing in three different extenders: a commercial equine extender (BotuCrio(®); Nidacon, Moedal, Schweden), a commercial bovine extender (Steridyl, Minitube, Tiefenbach, Germany), and an in-house “made” bovine egg yolk extender (TEY). Each was further supplemented with either glycerol or a mix of glycerol and methylformamide cryoprotectants. The results show that spermatozoa frozen with a mix of two cryoprotectants had significantly higher post-thaw motility compared to glycerol alone. Specifically, spermatozoa frozen in TEY and a mix of cryoprotectants achieved post-thaw sperm motility of 57 ± 3%. These results might serve as a blueprint for an improved protocol for giraffe sperm cryopreservation. ABSTRACT: Giraffe numbers have plummeted over the last 30 years by 30–40%. Thus, their conservation status has been raised from least concern to vulnerable. Efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques (ART) such as sperm cryopreservation could help preserve the genetic diversity of giraffe subspecies and, when used for artificial inseminations, enhance genetic exchange between isolated populations. However, to date, the post-thaw motility of recovered sperm has been low and inconsistent. In this study, epididymal sperm collected from the testes of giraffes (n = 7) was frozen in three different extenders, namely, BotuCrio, Steridyl, and test egg yolk (TEY), each supplemented with one of two different cryoprotectants (5% glycerol or a mix of 1% glycerol and 4% methylformamide) and frozen over liquid nitrogen vapor. Across all three extenders, sperm showed significantly better post-thaw results when frozen with a mix of glycerol and methylformamide compared with glycerol alone. Sperm frozen with TEY and a mix of glycerol and methylformamide achieved superior post-thaw total and progressive sperm motility of 57 ± 3% and 45 ± 3%, respectively. These results show the benefit of using alternative cryoprotectants for freezing giraffe spermatozoa and could aid in the application of ARTs for giraffe subspecies or the closely related endangered Okapi.