The effect of combination of methanol with several concentration of honey solution on sperm quality of botia Chromobotia macracanthus Bleeker 1852 after short-term storage at −80 ℃

Background: Clown loach or botia, Chromobotia macracanthus Bleeker 1852, is a popular freshwater ornamental fish. Lack of high quality broodstock and asynchronous gonad maturation are the main problem in breeding of this species. Cryopreservation is one of the methods to overcome this problem, and cryoprotectant plays a vital role in this process. Herein, we tested two types of cryoprotectants, namely methanol as a permeating cryoprotectant and honey solution as a natural non-permeating cryoprotectant. Hence, the objective of the present study was to determine best combination of honey solution and methanol for short-term storage of C. macracanthus sperm. Methods: One level of methanol (10%) combined with six levels of honey solution (0%, 0.1%, 0.3%, 0.5%, 0.7% and 0.9%) were tested in this study. Ringer’s solution was used as an extender. The diluted sperms were equilibrated for 25 min at 4 °C and then kept at −80 ℃ for 48 h. Sperms was thawed for 13 s at 40 °C. Spermatozoa viability, motility, and fertilization rates were then observed. Results: The one-way ANOVA showed that combination of methanol with several concentrations of honey had a significant effect on sperm viability and fertilization rates (P<0.05), but did have a significant effect on sperm motility (P>0.05). The study revealed that the 0.1% honey solution combined with 10% methanol resulted in the highest rates of motility (89.4 ± 5.45%), viability (85.75 ± 4.79 %), and fertilization (98.55 ± 1.69 %). Conclusion: A 0.1% honey solution combined with 10% methanol in Ringer’s was the best cryoprotectant for C. macracanthus spermatozoa preserved at -80 ℃.