Effects of cooling and freezing on sperm motility of the endangered fish piracanjuba Brycon orbignyanus (Characiformes, Characidae)

The effects of extenders and cryoprotectants on sperm motility of the endangered fish piracanjuba (Brycon orbignyanus, Characiformes, Characidae) after storage at 4-6oC and at -196oC were evaluated. In Experiment 1, 20 extender-cryoprotectant combinations (5 extenders x 3 cryoprotectants, plus 5 solutions containing only extender without cryoprotectant) were tested. The extenders tested were: 154 mM NaCl, 200 mM NaCl, Saad (mM: 200 NaCl, 30 Tris), coconut water, and Kurokura (mM: 128.4 NaCl; 2.7 KCl; 1.4 CaCl2; 2.4 NaHCO3). The cryoprotectants (dimethyl sulphoxide DMSO, methanol, and methylglycol) were added at 10% of the total volume. One aliquot of semen was kept undiluted and served as a control. Motility was subjectively estimated at 0, 1, and 2 days after cold storage. In Experiment 2, the extender-cryoprotectant combinations that produced motility above 70% on Day 0 (Experiment 1) were selected as freezing media. The amount of egg yolk and semen included in each medium was 5 and 10% of the total volume, respectively. Three 0.5-mL straws for each freezing medium were frozen in nitrogen vapor container (Taylor-Wharton, CP 300) at -170°C and then stored in liquid nitrogen. Straws were thawed in a water bath at 60oC for 8 seconds. Sperm motility one day after cooling was higher in samples diluted in Saad solution (82%), 200 mM NaCl (67%), and 154 mM NaCl DMSO (77%). Undiluted samples yielded 53% motility. Higher post-thaw sperm motility (66%) was observed in semen cryopreserved in 154 mM NaCl egg yolk methylglycol compared to all the other samples. Piracanjuba semen diluted in Saad solution or 200 mM NaCl and stored at 4-6oC for one day or frozen in 154 mM NaCl yolk methylglycol maintained most of its sperm motility.