214 Impact of bovine serum albumin concentrations on morphology of Fowler’s toad (Anaxyrus fowleri) sperm during short-term 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 temperature is 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 effects on toad sperm structure, males (n = 14) were administered 300 IU intraperitoneally of human chorionic gonadotrophin (hCG), and spermic urine samples were collected at 3, 4, 5, and 6 h 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, on the proportion of normal sperm, abnormal head, abnormal tail, and abnormal head + tail. Normal amphibian sperm should appear filiform, with an elongated head, well-developed mid piece containing a mitochondrial vesicle, and a ribbon-like membrane that runs down the length of the double-axoneme tail. 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. Initial descriptive statistics revealed that all three concentrations of BSA had a greater proportion of normal sperm over time compared with the control. Across all time points, there was about twice the percentage of normal sperm found in the BSA treatments versus the control. As an illustration, on Day 11 postcollection, all three BSA treatments had 49.3% (low), 44.9% (medium), and 45.9% (high) normal sperm, respectively, whereas the control only had 18.7% normal sperm. Overall, these findings offer important insights on the beneficial addition of BSA for short-term cold storage. Enhancing the normal morphological structure of toad sperm during chilled storage should provide higher fertilization rates and overcome poor sperm quality issues resulting from asynchronous gamete release.