Reproduction technologies for the sustainable management of Caudata (salamander) and Gymnophiona (caecilian) biodiversity.

We review the use of reproduction technologies (RTs) to support the sustainable management of threatened Caudata (salamanders) and Gymnophiona (caecilian) biodiversity in conservation breeding programs (CBPs) or through biobanking alone. The Caudata include ∼760 species with ∼55% threatened, the Gymnophiona include ∼215 species with an undetermined but substantial number threatened, with 80% of Caudata and 65% of Gymnophiona habitat unprotected. Reproduction technologies include: (1) the exogenous hormonal induction of spermatozoa, eggs, or mating, (2) in vitro fertilisation, (3) intracytoplasmic sperm injection (ICSI), (4) the refrigerated storage of spermatozoa, (5) the cryopreservation of sperm, cell or tissues, (6) cloning, and (7) gonadal tissue or cell transplantation into living amphibians to eventually produce gametes and then individuals. Exogenous hormone regimens have been applied to 11 Caudata species to stimulate mating and to 14 species to enable the collection of spermatozoa or eggs. In vitro fertilisation has been successful in eight species, spermatozoa have been cryopreserved in seven species, and in two species in vitro fertilisation with cryopreserved spermatozoa has resulted in mature reproductive adults. However, the application of RTs to Caudata needs research and development over a broader range of species. Reproduction technologies are only now being developed for Gymnophiona, with many discoveries and pioneering achievement to be made. Species with the potential for repopulation are the focus of the few currently available amphibian CBPs. As Caudata and Gymnophiona eggs or larvae cannot be cryopreserved, and the capacity of CBPs is limited, the perpetuation of the biodiversity of an increasing number of species depends on the development of RTs to recover female individuals from cryopreserved and biobanked cells or tissues.

Cryopreservation of spermatozoa obtained postmortem from the European common frog Rana temporaria.

Cryopreserved spermatozoa offers a reliable, efficient and cost-effective means to perpetuate the genetic variation of endangered amphibian species in concert with conservation breeding programs. Here we describe successful cryopreservation of testicular spermatozoa of the common frog Rana temporaria, preliminarily stored in the carcasses of decapitated animals at +4°C for 0, 1 and 4 days. The motility, membrane integrity and fertilisation capability of fresh testicular spermatozoa treated with cryoprotective medium supplemented with 15% dimethylformamide (DMF) or 15% dimethylsulfoxide (DMSO) were examined. DMSO had a significantly greater toxic effect on fresh frog spermatozoa than DMF. Low levels of DNA fragmentation were seen in spermatozoa stored in the testis for different times and then treated with DMF (mean (±s.e.m.) 8.2±0.7% and 18.2±1.8% after 0 and 4 days storage respectively). After 1 day of storage in frog carcasses, the quality of spermatozoa cryopreserved with DMF was not significantly different from that of control spermatozoa (0 days of storage). After 4 days of storage, the quality of frozen-thawed spermatozoa was significantly lower in the DMF-treated than control group: 35% of the spermatozoa cryopreserved with DMF retained motility, 25% maintained the ability to fertilise fresh oocytes and 80% of fertilised oocytes survived to hatch.

Sperm collection and storage for the sustainable management of amphibian biodiversity.

Current rates of biodiversity loss pose an unprecedented challenge to the conservation community, particularly with amphibians and freshwater fish as the most threatened vertebrates. An increasing number of environmental challenges, including habitat loss, pathogens, and global warming, demand a global response toward the sustainable management of ecosystems and their biodiversity. Conservation Breeding Programs (CBPs) are needed for the sustainable management of amphibian species threatened with extinction. CBPs support species survival while increasing public awareness and political influence. Current CBPs only cater for 10% of the almost 500 amphibian species in need. However, the use of sperm storage to increase efficiency and reliability, along with an increased number of CBPs, offer the potential to significantly reduce species loss. The establishment and refinement of techniques over the last two decades, for the collection and storage of amphibian spermatozoa, gives confidence for their use in CBPs and other biotechnical applications. Cryopreserved spermatozoa has produced breeding pairs of frogs and salamanders and the stage is set for Lifecycle Proof of Concept Programs that use cryopreserved sperm in CBPs along with repopulation, supplementation, and translocation programs. The application of cryopreserved sperm in CBPs, is complimentary to but separate from archival gene banking and general cell and tissue storage. However, where appropriate amphibian sperm banking should be integrated into other global biobanking projects, especially those for fish, and those that include the use of cryopreserved material for genomics and other research. Research over a broader range of amphibian species, and more uniformity in experimental methodology, is needed to inform both theory and application. Genomics is revolutionising our understanding of biological processes and increasingly guiding species conservation through the identification of evolutionary significant units as the conservation focus, and through revealing the intimate relationship between evolutionary history and sperm physiology that ultimately affects the amenability of sperm to refrigerated or frozen storage. In the present review we provide a nascent phylogenetic framework for integration with other research lines to further the potential of amphibian sperm banking.

Refrigerated storage of European common frog Rana temporaria oocytes.

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1-5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1-5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days.