C-1013: Cryopreservation of spermic milt in the model species Ambystoma tigrinum (Tiger salamander) for application in endangered salamanders

Abstract

In the face of the amphibian extinction crisis, captive assurance colonies have been established to preserve genetic diversity. Sadly, many captive assurance colonies fail due to reproductive cues which are not entirely understood. In addition, the majority of amphibian conservation and genome banking research has focused on anurans (frogs/toads), despite the fact that caudates (salamanders/newts) face a larger threat of extinction. Therefore, it is critical that assisted reproductive technologies, such as gamete cryopreservation and artificial fertilization, be developed as conservation tools for application in genetic management of threatened caudates. It was hypothesized that a modified anuran sperm freezing protocol could be successfully used in cryopreservation of Ambystoma tigrinum (tiger salamander) sperm as a caudate model. Spermiation in male tiger salamanders was hormonally induced via injection with hCG, LHRH, or a combination of these two hormones. Average spermic milt concentrations were 2.75 × 107(±7.2 × 106)/mL. Average total motility upon collection of fresh milt was 53.6% (±17.6%). After collection and initial analysis, fresh milt samples were diluted 1:1 in chilled cryoprotectant treatment solutions. They were then frozen in 0.25 mL French straws using a stepwise slow-rate freezing process. Upon thawing, milt suspensions were diluted 1:10 with a 10% Holtfreter’s solution in order to activate the sperm for post thaw analysis. Frozen-thawed sperm in the 5% Me2SO treatment group had an average total motility of 19.1% (±15.8%) and sperm in the 5% Me2SO with 0.5% BSA treatment group had an average total motility of 21.75% (±14.8%). Early trials showed that caudate sperm are not susceptible to toxicity from several cryoprotective media. Moreover, initial fertilization trials indicate that frozen-thawed caudate sperm are capable of producing early cleavage stage embryos. Optimizing cryopreservation protocols for salamander sperm is an important step in building a global genetic resource bank for endangered caudates.