Sperm Cryopreservation as a Tool for Amphibian Conservation: Production of F2 Generation Offspring from Cryo-Produced F1 Progeny

Abstract

Sperm cryopreservation and biobanking are emerging as tools for supporting genetic management of small and threatened populations in amphibian conservation programs. However, there is little to no evidence demonstrating reproductive maturity and viability of offspring generated with cryopreserved sperm, potentially limiting widespread integration of these technologies. The purpose of this report is to demonstrate that amphibian sperm can be cryopreserved and thawed to successfully produce individuals of an F1 generation that can reach adulthood and reproductive maturity, to generating viable gametes and an F2 generation. Species-specific exogenous hormones were administered to both F0 and F1 adults to stimulate spermiation and oviposition in the eastern tiger salamander (Ambystoma tigrinum), dusky gopher frog (Lithobates sevosa), and Puerto Rican crested toad (Peltophryne lemur). Sperm cells collected non-lethally from F0 adults were cryopreserved, thawed, and used for in vitro fertilization (IVF) to produce F1 offspring. Individuals of the F1 generation are shown to reach adulthood, express viable gametes, and produce offspring through facilitated breeding, or IVF. The production of amphibian F2 generations shown here demonstrates that amphibian sperm collected non-lethally can be banked and used to generate reproductively viable animals of subsequent generations, thus maintaining valuable genetic linages and diversity in threatened amphibian species. The incredible value that cryopreservation of sperm has for long-term genetic management aids in the sustainability of both in situ and ex situ conservation efforts for this taxon.

The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species

“…The process of cryopreservation exerts stress on sperm, and can minimise both functionality and survival [ 153 ]. The freezing process exposes sperm cells to osmotic stress, intracellular ice-crystal formation, and cryoprotectant toxicity, which can damage cells and make them unviable [ 154 , 155 ]. Thus, the cryoprotectant components within the cryopreservation medium and the cooling rate must be carefully considered and tested to develop successful cryopreservation protocols [ 156 ].…”

“…Oocytes were collected by stripping (manual abdominal massage) following hormonal induction of ovulation [ 35 , 38 , 152 , 154 , 178 , 213 , 214 ], collected from amplectant females after first signs of oviposition [ 36 , 57 ] or collected directly from the oviducts following euthanasia [ 183 ]. Prior to AF, sperm were reactivated in either distilled water [ 36 , 57 , 213 , 214 ], sterilised embryo transfer water [ 38 , 145 , 152 , 154 ], Marc’s modified Ringer [ 178 ], or saline solution. In addition, Upton et al [ 57 ] centrifuged sperm prior to reactivation and AF to wash samples of CPAs.…”

“…In the amphibian studies that have compared hatching success between cryo-derived offspring and offspring generated from AF using fresh sperm, the use of frozen–thawed sperm for AF generally appears to result in lower fertilisation and hatching success ([ 34 , 35 , 38 , 145 , 152 , 154 , 183 ]; Table 4 ). However, the magnitude of this difference varies across species, with significant differences only demonstrated in four of the eight species studied ( Table 4 ).…”

“…The findings reported in these studies were variable, though encouraging, as cryo-derived offspring were used to successfully generate offspring in each case. In a recent report by Lampert et al [ 154 ], an F1 generation of three species of amphibians, the Eastern tiger salamander ( A. tigrinum ), the Puerto Rican crested toad ( P. lemur ), and the dusky gopher frog ( L. sevosus ), were produced via AF using frozen–thawed sperm and fresh oocytes. These offspring were then reared to sexual maturity and used to produce an F2 generation (using one cryo-derived F1 female with a control male for the Eastern tiger salamander and one cryo-derived F1 male with a control female for both the Puerto Rican crested toad and the dusky gopher frog).…”

Biobanks, offspring fitness and the influence of developmental plasticity in conservation biology

“…Unlike the situation with wild, but endangered, mammals and birds, a body of preliminary evidence about the development and survival of tadpoles and adults derived from frozen-thawed spermatozoa, is accumulating. In some cases, there is no suspicion that the use of cryopreserved amphibian spermatozoa might result in offspring with compromised survival or fertility ( Lampert et al, 2022 ). Indeed, the successful use of cryopreserved spermatozoa in breeding programs for threatened amphibian species, and for establishing biobanks and insurance populations as a hedge against extinction, is now widely regarded as an important aspect of amphibian conservation ( Kouba et al, 2013 ; Silla and Byrne, 2019 ; Upton et al, 2021 ).…”