First successful artificial insemination of a reptile using frozen-thawed semen

The use of assisted reproductive technologies (ARTs) has considerable value for conservation. However, their development has been limited to relatively few species of charismatic megafauna. Non-avian reptiles are incredibly speciose and up to a third are threatened with extinction, but progress developing and implementing ARTs into conservation programs has lagged other taxonomic groups. Here, we describe successful artificial insemination of an endangered snake using frozen-thawed semen within the context of a species recovery program. Six female Louisiana pinesnakes (Pituophis ruthveni) were inseminated with frozen-thawed semen from six donor males resulting in oviposition of two fertile cutches producing three viable hatchlings. Paternity of hatchlings was confirmed using genetic analyses. This is the first documented case of successful artificial insemination using frozen-thawed semen in a reptile and is a significant step forward in the conservation of reptiles. The methods described here hold the potential for zoos and conservation organizations to diversify reptile gene pools in captivity and the transfer of genetic material between wild populations and captive breeding programs. In addition, the ability to use frozen-thawed semen for AI emphasizes the conservation value of establishing cryobanks for reptile semen. The development and implementation of ARTs in recovery programs for reptiles has great conservation potential and warrants exploration across a broader range of species.