Bovine serum albumin (BSA) impacts morphology of Fowler’s toad (Anaxyrus fowleri) sperm during short-term cold storage

Short-term cold storage of amphibian sperm is a valuable technique for overcoming challenges associated with asynchronous gamete release during breeding events or for transferring sperm between institutions for genetic management. Unfortunately, sperm longevity declines over time even when kept cool. Maintaining sperm form and function is critical to achieving the highest possible fertilization rates. Thus, cold storage of amphibian sperm would benefit from the addition of compounds that could extend their quality, improve fertilization and result in more offspring. One such compound is bovine serum albumin (BSA), which provides membrane stabilization, prevents lipid peroxidation of cells, protects against free radical damage and may even be a source of ATP. Very little is known about the beneficial effects of co-incubation of BSA with anuran sperm over time and such studies would be valuable for threatened amphibian species in conservation breeding programs. The aim of this study was to evaluate the morphology of chilled Fowler’s toad sperm over time with increasing concentrations of bovine serum albumin (BSA). To examine BSA’s effects on toad sperm structure, males (n=10) were administered 300 IU of human Chorionic Gonadotropin (hCG) intraperitoneally and spermic urine samples were collected at 3, 4, 5, and 6 hours post-injection. Subsequently, samples were evaluated, pooled and mixed with three treatments of BSA (weight/volume): Low (0.25%), Medium (0.5%), and High (0.75%). Several morphological parameters were analyzed, prior to- and after addition of BSA, including the proportion of: normal sperm (N), abnormal head (AH), abnormal tail (AT) and abnormal head + tail (AHT). After the addition of BSA to each treatment group, samples were stored at 4°C and a subsample removed for morphology assessment on days 1,2,4,7,11, and 14. Results revealed that all three concentrations of BSA had a greater proportion of normal sperm over time when compared to the control. Across all timepoints, there was a greater percentage of normal sperm found in the BSA treatments vs. the control. Statistical analysis revealed BSA concentrations and experimental day significantly impacted the percentage of normal sperm (F(3, 194) = 4.31, p = 0.0057) and (F(5, 194) = 7.57, p < 0.0001), respectively. However, there was no significant interaction between the treatment and experimental day that affected sperm structure (F(15, 194) = 1.04, p = 0.4202). Using a Kruskal-Wallis test showed no statistical significance in normal morphology on days 7, 11, and 14 amongst treatment groups. Pairwise comparisons showed statistical significance between 0.5% BSA and