Successful short term sperm cryopreservation in brown‐marbled grouper ( Epinephelus fuscoguttatus ) with the utility of ultra‐freezer (‐80 ℃)

Production of cryopreserved semen in fish generally requires liquid nitrogen (LN), which is not always easily available in remote areas. To reduce reliance on LN, the aim of the present study was to evaluate whether electric freezer could be a feasible LN-free alternative to cryopreserve brown-marbled grouper sperm. After loading, semen straws were put directly into freezers (-30 or -80°C) for freezing and then transferred to LN for storage. Compared with the conventional LN vapour freezing (straws were put horizontally 3 cm above the surface of LN), there was a significant reduction in all tested post-thaw sperm quality parameters in samples frozen at -30°C for 10 min, including kinetic parameters (total motility: 85.0% vs. 48.6%), viability (84.7% versus 51.7%), high mitochondrial membrane potential (86.4% vs. 63.7%), ATP content (106.9 nM/109 cells vs. 72.9 nM/109 cells) and hatching rate (86.3% vs. 45.7%), accompanied with an increasing lipid peroxidation level (MDA content: 11.9 nM/109 cells vs. 4.9 nM/109 cells). In contrast, frozen with -80 °C ultra-freezer (10 min or 12 hr) produced similar sperm quality parameters to those using LN, except that temporary storage (12 hr) at -80°C yielded lower average path velocity. In conclusion, this study confirmed that -80°C ultra-freezer is an effective alternative to LN for sperm freezing in brown-marbled grouper.