Cryopreservation of hormonally induced sperm of the common toad (Bufo bufo) with a cushion centrifugation step: a preliminary study

One of the most important methods for preserving amphibian biodiversity is sperm cryobanking, in particular cryopreservation of spermic urine obtained by hormonal induction of spermiation. However, for bufonids, cryopreserved sperm obtained by this method has low fertilizing ability, which hampers its use in cryobanking and artificial breeding programs for endangered species. We hypothesized that the key cause of this problem is the low concentration of sperm in the spermic urine of toads. It is possible to increase sperm concentration by centrifugation. The purpose of this work is to study the resistance of common toad (Bufo bufo) sperm to centrifugation and the effect of centrifugation on sperm cryoresistance. The resulting samples were subjected to noncushioned ( × 200 g, 5 min and × 500 g, 5 min) and cushioned ( × 500 g, 5 min, with underlayering of a cushioned solution: Niu-Twitty medium + 10% bovine serum albumin (BSA) + 5% sucrose). A comparison was also made of programmable freezing (2 °C/min), freezing in nitrogen vapor (7-9 °C/min), and three thawing methods (ice bath, air at 18 °C, and water bath at 40 °C). It was found that noncushioned centrifugation did not reduce sperm motility but reduced its fertilizing ability. Cushioned centrifugation did not have a negative effect on the fertilizing ability of common toad sperm. The fertilizing ability of sperm frozen after cushioned centrifugation was 39 ± 10%, which significantly exceeded the fertilizing ability of sperm frozen without centrifugation (<1%) and after noncushioned centrifugation (4%). Programmable freezing followed by slow thawing in air resulted in better sperm survival compared with more rapid freezing and thawing.