First cryopreservation of phyllostomid bat sperm

Bats, the second-largest mammalian order, are widely distributed and provide crucial ecosystem services. Their reproductive biology comprises noteworthy characteristics, such as long-term sperm storage, delayed implantation or even delayed fetal development. The understanding of these mechanisms remains limited. Research in reproductive biology may become crucial for the conservation of endangered bat species. Indeed, the IUCN Red List of Threatened Species currently lists 76 bat species as endangered or critically endangered, for which conservation measures should be taken. The development of assisted reproductive technologies (ART) in bats would thus open new perspectives.In this context we here describe the first successful cryopreservation of bat sperm in the Seba’s short-tailed bat (Carollia perspicillata). Epididymal sperm of 30 males was pooled in 6 sample sets and diluted using four different cryo-diluents: Canifreeze, modified Canifreeze, BotuCrio® and Test egg yolk extender. Glycerol and a mixture of glycerol and methylformamide were used as cryoprotectants. Best post-thaw results were achieved when sperm was diluted in Test egg yolk extender containing a mixture of glycerol and methylformamide as cryoprotectant. Post-thaw sperm motility, progressive motility and acrosome integrity of 42.0 ± 3.4%, 28.3 ± 6.3% and 87.7 ± 1.7%, respectively were superior to all other variants tested. It remains to be determined if the post-thaw quality of cryopreserved bat spermatozoa achieved in this study is sufficient for the use in artificial inseminations and produces acceptable pregnancy rates. Successful cryopreservation of spermatozoa in C. perspicillata may serve as the first model to establish gamete rescue programs and develop ART in other, critically endangered bat species.