Bovine serum albumin and caffeine enhance activation of sperm from the American toad (Anaxyrus americanus)

The collection and cryopreservation of anuran spermic urine results in multiple osmolality shifts that inactivate and reactivate sperm motility prior to in-vitro fertilization. These shifts in osmolality reduce motility recovery and fertilization potential. Here, we analyzed the effects of metabolic supplements for reactivation of sperm motility and forward progression from the American toad (Anaxyrus americanus). Male toads (n = 30) were administrated hormones to stimulate spermiation and samples were mixed 1:1 with 2× simplified amphibian ringers solution for sperm inactivation. Inactivated sperm samples were randomly assigned to five treatment groups to be reactivated in distilled water alone (control) or supplemented with BSA (0.1 %, 0.2 %, 0.4 %), caffeine (1, 10, 25 mM), sodium pyruvate (0.5, 1, 2 mM), sodium lactate (10, 20, 40 mM), or sodium bicarbonate (10, 20, 40 mM). Sperm samples were assessed for total motility (TM) and quality of forward progressive movement (QFPM) at 0, 5, 15, 30, 45, 60, 90, and 120 min after reactivation. Results showed that sperm activated with BSA produced higher (P < 0.05) recovered motility than the control, regardless of BSA concentration. Moreover, the addition of caffeine (1 mM) resulted in higher (P < 0.05) sperm QFPM following reactivation compared to the control. In contrast, bicarbonate and lactate treatments resulted in lower recovery of TM and QFPM, while pyruvate had no effect (P ≥ 0.05). Our findings highlight the efficacy of BSA and caffeine on enhancing sperm motility and forward progression, respectively, following inactivation and reactivation of sperm undergoing short-term storage or cryopreservation procedures.