32 Bovine serum albumin improves anuran sperm motility and mitochondrial vesicle stability during cold storage

Short-term cold storage of anuran sperm is a valuable tool for managing asynchronous gamete release for IVF procedures and for transfer of valuable gametes between institutions for genetic management of endangered species. Unfortunately, the motility of anuran sperm declines rapidly in the first couple days of cold storage, either from depletion of energy stores or breakdown of structural integrity of the spermatozoa from membrane-damaging compounds. In some species of anurans, motility of spermatozoa requires attachment of the mitochondrial vesicle (MV), a membrane enclosed structure loosely connected to the head, to power flagellar motion (Chen et al. 2022 Comp. Biochem. Physiol. Part A 268, 111191). Association of the MV with the sperm cell may be maintained by supplementing with bovine serum albumin (BSA), which is known to maintain mitochondrial membrane potential, prevent lipid peroxidation, increase ATP stores and improve GADPH activity in mammalian sperm. Here, we investigated the impact of BSA at three different concentrations (0.25%, 0.5%, and 0.75%) on cold storage of sperm from the Fowler’s toad (Anaxyrus fowleri). Males (n = 14) were administered 300 IU of human chorionic gonadotropin (hCG) intraperitoneally to stimulate spermiation, and spermic urine samples were collected at 3, 4, 5, and 6 h postinjection, pooled and divided into the three BSA treatments, or control, and stored at 4°C for 14 days. Sperm from each treatment were assessed for motility and MV presence on Days 1, 2, 4, 7, 11, and 14 and compared with the control. Forward motile (FM), stationary motile (SM), non-motile with MV intact (PNM), and non-motile without MV (NM) were counted until 100 sperm were assessed per sample per day. Total motility was calculated by summing FM and SM counts, while MV presence was calculated by adding FM, SM, and PNM together because any motile sperm requires MV to be intact (Chen et al. 2022). A generalized linear model was used to analyze the effect of treatment and time on motility parameters and presence of MV, while a Dunnett’s test was used to compare treatments with the control. Sperm samples co-incubated with BSA at 0.25%, 0.5%, and 0.75% maintained a significantly higher (P < 0.05) motility and MV presence over the 14-day period, compared with the control. At Day 7, TM for all three treatments were significantly higher than the control (0.25% M = 41.3% ± SE 5.35, 0.5% M = 34.25% ± SE 5, 0.75% M = 39.1% ± SE 5.5, 0% M = 3.6% ± SE 4). By Day 14, all treatments still had remaining TM where the control did not (0.25% M = 3.6% ± SE 1.5, 0.5% M = 4.1 ± SE 1.7, 0.75% M = 2.2 ± SE 0.74, 0% M = 0 ± SE 0). The addition of BSA to Fowler’s toad cold-stored sperm samples can significantly extend MV attachment and sperm motility for weeks, suggesting conservation breeding programs may be able to share genetics between populations. The One Plan approach to conservation is the sharing of genetics between and within ex-situ and in-situ populations for improved conservation management and requires the short-term cold storage of gametes over time and space.