Production of live tiger salamander offspring using cryopreserved sperm

Amphibian conservation breeding programs often rely on assisted reproductive technologies (ART) to produce offspring due to the difficulty of replicating natural reproductive cues under human care. Reproductive management using in-vitro fertilization (IVF) with cryopreserved sperm has been successfully applied to a variety of anurans, yet the approach has seen limited use for internally-fertilizing caudates. This study aimed to test two cryoprotectants, dimethyl formamide (DMFA) and dimethyl sulfoxide (DMSO), in combination with three different sperm extenders, HAM's F-10, diluted HAM's F-10, and 10% Holtfreter's, for the production of salamander larvae from cryopreserved sperm. Fresh sperm was collected from tiger salamanders (n = 12) and diluted with one of three extenders, then subsequently mixed 1:1 with cryoprotectant. Samples were frozen using liquid nitrogen and later thawed to be applied in IVF. The diluted HAM's and DMFA treatment resulted in the highest (p < 0.05) fertilization rate, with over 40% of eggs cleaving. The DMFA treatments resulted in 87 hatched offspring, while DMSO treatments resulted in only one offspring. This research highlights the potential of biobanking to produce internally-fertilizing salamander larvae, with DMFA being the likely preferred sperm cryoprotectant. These findings can inform conservation breeding programs by increasing caudate population sustainability through genetic management, facilitated by ART.