Effect of seasonality on hormonally induced sperm in Epidalea calamita (Amphibia, Anura, Bufonidae) and its refrigerated and cryopreservated storage

Abstract

An understanding of how seasonality impacts amphibian's sperm quality is crucial for the development and implementation of assisted reproductive technologies for threatened species. We analysed sperm quality both within and outside the breeding season for the common natterjack toad (Epidalea calamita) in a population close to Madrid (Spain). Fresh hormonally induced sperm samples were assessed for sperm concentration, total activated sperm (TAS), forward motility (FM), quality of motility (QM) and DNA integrity (DNAi). Motility and DNAi were re-evaluated after cold-storage at 5 °C for 24 h. After freezing and thawing DNAi was re-analysed and a fertilization trial performed to test post-thaw sperm functionality. More males presented sperm after hormone administration during the breeding season compared to out of season (78.3 vs. 52.2%), although the difference was not significant. Fresh sperm motility and DNAi were not affected by season (p > 0.05). However, sperm concentration was higher (p = 0.037) during the breeding season (5.2 vs. 2.9 × 106 sperm/ml). After cold-storage sperm obtained in the breeding season presented higher FM (36.1 ± 4.6 vs. 22.0 ± 3.5%; p = 0.013) and QM (1.28 ± 0.2 vs. 0.68 ± 0.2; p = 0.017) than samples from out of season. DNAi decreased after freeze-thawing in both seasons (p < 0.01) but no differences were found between seasons. Fertilization experiment showed that neurulas were found in 72.7% (outside breeding season) and 68.8% (breeding season) sperm samples. Later, hatched embryos were observed in 18.2% (outside breeding season) and 56.3% (breeding season) sperm samples and the percentage of embryos was higher (p = 0.026) in sperm samples from males collected during the breeding season (38.4 ± 6.9 vs. 3.5 ± 0.2% embryos/sample). Overall, sperm obtained during the breeding season had a higher sperm concentration, was less sensitive to cold temperatures and maintained a better fertilization capacity after cryopreservation. Therefore, seasonality affects sperm quality and should be considered when planning sperm collection for assisted reproductive technologies.