Effect of melatonin supplementation to a cytoprotective medium on post-thawed Brycon orbignyanus sperm quality preserved during different freezing times.

The aim of the present study is to verify the viability of frozen B. orbignyanus sperm cells after freezing with dilution media containing different concentrations of the melatonin and after different freezing times. Semen from 15 males was collected and pooled as five pools from three random animals. Oocytes (100) from three females were separately used for fertilization. There were three treatments: (C) Control Medium: 90% of extender Beltsville thawing solution (5% concentration) + 10% methylglycol (MG); (M1) Control Medium + 1 mM melatonin; and (M2) Control Medium + 2 mM melatonin. Sperm samples were diluted in media at a final proportion of 1:4 [125 µl sperm (25% V/V) + 337.5 µl BTS (65% V/V) + 37.5 µl MG (10% V/V)]. Melatonin was added at final solution. Three Dry shipper freezing times were used: T1 (15 min), T2 (12 h) and T3 (24 h). The samples were transferred, stored in a cryobank and thawed in a water bath at 60 °C for 5 s and evaluated concerning viability, morphology and fertilization rate. B. orbignyanus semen frozen in M2 presented the highest fertilization rate (8.40 ± 2.54%). The highest vitality (85.2 ± 2.8%), motility (64.63 ± 8.3%), motility duration (84.22 ± 11.4 s) and progressive motility (17.01 ± 1.2%) rates were maintained for M2. The highest number of altered cells was observed in C (57.4 ± 5.9%). Melatonin at 2 mmol L-1 associated with the cryoprotectant methylglycol in cryopreservation could be used to improve a cryobank for endangered Brycon orbignyanus populations.