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

Abstract

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 LNfree 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/10 9 cells vs. 72.9 nM/10 9 cells) and hatching rate (86.3% vs. 45.7%), accompanied with an increasing lipid peroxidation level (MDA content: 11.9 nM/10 9 cells vs. 4.9 nM/10 9 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.

Evaluation of different sugar extenders and large volume cryotube for sperm cryopreservation of brown‐marbled grouper ( Epinephelus fuscoguttatus )

“…Motility parameters were examined with an Integrated Semen Analysis Systems (ISAS 2.0, Spain) as described earlier (Yang et al, 2021). Briefly, the semen sample was activated with filtered seawater (supplemented with 0.1% bovine serum albumin, w/v) in a 1.8-mL Eppendorf tube at a dilution of 1:200.…”

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study

“…The benefits of fish sperm cryopreservation include: (i) storing high-quality sperm for fertilization of oocytes at any time of the year, avoiding the problems of gonadal asynchrony; (ii) establishing databases to carry out a germplasm bank; (iii) optimizing the exchange of semen between reproduction centers; (iv) improving species by allowing the introduction of new genetic lines; and (v) keeping a permanent supply of gametes for optimal use in hatcheries/breeding grounds, or for research [11,12]. However, despite the increasing successes of cryopreservation, this technique may cause serious damage to spermatozoa [13,14]. This is due to the osmotic stress produced by the process, combined with the formation of ice crystals and the toxicity of some cryoprotectants [15][16][17], causing rupturing of the plasma membrane, changes in the mitochondrial membrane potential, and fragmentation of nuclear DNA [18].…”