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

Antibiotics for the refrigerated storage at 4 °C of hormonally induced European сommon frog (Rana temporaria) spermatozoa

abstract: A valuable reproduction technology for the sustainable management of amphibian biodiversity is the refrigerated storage of spermatozoa at 4 °C. Antibiotics offer the potential to increase the storage life of refrigerated spermatozoa by inhibiting bacterial growth. In the present study, the effect of antibiotics lincomycin, enrofloxacin and gentamicin, alone or in combination, on the retention of motility, and longevity of spermatozoa in the spermic urine of the European common frog (Rana temporaria) during a 36-day storing period in the refrigerator at 4 °C was investigated. The percentage of motile spermatozoa declined in each treatment group over time; however, in samples supplemented with three antibiotics in combination or gentamicin alone, the sperm motility was significantly higher (from 17 % to 52 %) on Day 30 after sperm collection than that in samples in control and other antibacterial groups. Lincomycin did not improve the spermatozoa longevity during refrigerated storage, and enrofloxacin had a less pronounced beneficial effect than gentamicin did. Also, the effect of gentamicin at concentrations of 15, 30, 60, 100, 200, 400 µg/mL, and 1, 2, 4 mg/mL for maintaining sperm motility during refrigerated storage was evaluated. Gentamicin at concentrations of 30–400 µg/mL had a positive effect on the retention of sperm motility, with the highest rates of motility during storage at a concentration of 100 µg/mL. Gentamicin at a concentration of 15 µg/mL had no effect on maintaining sperm motility. The sperm motility was partially inhibited by gentamicin at concentrations of above 400 µg/mL (1 and 2 mg/mL) and stopped completely at 4 mg/mL. Moreover, gentamicin effects on the cleavage of fertilized eggs and subsequently on the hatch rates were evaluated. Аfter 30 days of refrigerated storage with 30–120 μg/mL gentamicin, the motility of the spermatozoa in more than half of the samples was 50 % and above. The cleavage rate of eggs (55.0 ± 5.9 %) and the hatching rate of cleaved eggs (68.2 ± 2.9 %) did not differ significantly from those obtained for fresh spermatozoa (P < 0.05). Over all storage periods of spermatozoa in the presence of 30–120 μg/mL gentamicin, the cleavage rate and hatch rate of cleaved eggs were high and consistent, which may indirectly indicate the preservation of genetic material in the fertilizing spermatozoa of treated spermic urine samples