Extender composition, osmolality and cryoprotectant effects on the motility of sperm in the Brazilian endangered species Brycon opalinus (Characiformes)

Abstract

Sperm preservation is an important tool for conservation of endangered fish species, such as the Brycon opalinus (Characiformes). An optimum medium should prevent the initiation of sperm motility during storage. The aim of this paper was to study the effects of extender composition, osmolality and cryoprotectant agent (CPA) on the motility of B. opalinus sperm after a 30-min equilibration time. Eight media were prepared by switching extender compositions (NaCl or glucose) and osmolalities (245, 285, 325 or 365mOsm/kg). These media were then divided in three aliquots and combined with two CPAs (dimethyl sulfoxide, DMSO, or methylglycol, MG) at 1.4M; the third aliquot remained without CPA (control). After dilution, all samples were observed under light microscope to confirm whether all extender-CPA combinations actually prevented the initiation of sperm motility. Then, sperm motility was triggered in NaCl 92mOsm/kg as activating agent after 0- and 30-min equilibration time at 4°C and the percentage of motile sperm and duration of motility were determined. All combinations of glucose or NaCl media at high osmolalities (325 and 365mOsm/kg), completely suppressed the initiation of sperm motility. Low extender osmolalities (245mOsm/kg), however, did not prevent the initiation of sperm motility and more than 50% of sperm diluted in all combinations of glucose media, NaCl-control and in NaCl-DMSO were motile. When motility was triggered after both 0- and 30-min equilibration times, more than 77% motile sperm were observed in all combinations of NaCl and glucose media, except for glucose 245-DMSO and glucose 285-DMSO. The duration of motility in sperm diluted in all media was above 40s, except for glucose 245-DMSO. Based on these findings, we can conclude that the initiation of sperm motility is triggered by low osmolality rather than the ionic composition of the surrounding medium in B. opalinus. Glucose or NaCl solutions at high osmolalities combined with either DMSO or MG prevent the initiation of sperm motility during storage. Sperm diluted in these media yields motility upon activation above 77% and that should last long enough to fertilize oocytes. These media are recommended as freezing media for future essays in cryopreservation of B. opalinus sperm.