31 Compounds enhancing American toad (Anaxyrus americanus) sperm activation: beneficial effects of bovine serum albumin and caffeine

Short-term cold storage and long-term sperm cryopreservation are pivotal tools for genetic management of threatened amphibians in human care. Chilled or frozen amphibian sperm are primarily used for IVF procedures to produce new offspring for continued management or release into the wild as part of a conservation program. The process of storing or cryopreserving sperm often results in active sperm becoming inactivated due to the higher osmolality of storage solutions; before IVF, sperm motility must be reactivated by dropping the osmolality. These changes in sperm cell activity states lead to reduced motility and forward progression, which likely affects function. This study analyzed the effects of five commonly used biological compounds, each tested at three different concentrations, on reactivation of anuran sperm motility and quality of forward progressive movement (QFPM). Human chorionic gonadotrophin (hCG; 300 IU) was administered intraperitoneally to induce spermiation in 30 American toads (Anaxyrus americanus). The toads were randomly assigned to five groups (n = 6 males per biological component). Spermic urine samples from each male were collected and inactivated by a 1:1 dilution in 2× simplified amphibian ringer solution. Each inactivated sample was then split into four aliquots and reactivated by 1:5 dilution in a distilled water reactivation medium containing either bovine serum albumin (BSA), caffeine, sodium lactate, sodium pyruvate, or sodium bicarbonate. The BSA medium was tested at concentrations of 0.1%, 0.2%, and 0.4%; caffeine at 1, 10, and 25 mM; sodium pyruvate at 0.5, 1, and 2 mM; and sodium lactate and bicarbonate at 10, 20, and 40 mM. Distilled water alone served as the control. Total motility and QFPM were assessed at 0, 5, 15, 30, 45, 60, 90, and 120 min post-reactivation. QFPM was assessed using a 0–5 scale, where 0 indicated no forward movement and 5 indicated forceful, rapid forward movement. The effects of dose and time were analyzed using generalized linear mixed models in each biological component group. Results showed that sperm activated with 0.1%, 0.2%, and 0.4% BSA concentrations produced significantly higher (P < 0.05) recovered motility compared with the control (62.1% ± 2.2, 60.8% ± 1.9, and 58.4% ± 2.5 vs. 36.6% ± 1.4, respectively). Furthermore, sperm activated with 1 mM caffeine produced significantly higher (P < 0.05) QFPM recovery compared with the control (1.8 ± 0.1 vs. 0.9 ± 0.1, respectively). In contrast, bicarbonate and lactate treatments resulted in significantly lower recovery of motility, while pyruvate had no effect (P > 0.05). Notably, there were no significant declines in the total motility during the first 45 min for all BSA and caffeine doses. Overall, our findings highlight the efficacy of BSA and caffeine in enhancing activation of sperm motility following short-term cold storage or cryopreservation, which may result in higher fertilization rates and more animals produced for threatened species recovery programs.