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

Abstract

Brown-marbled grouper (Epinephelus fuscoguttatus) is a large-bodied coral reef fish with high ecological and economic value in South-East Asia. Previous studies have developed cryopreservation protocols in 0.25- or 0.5-ml straws, but this small volume may limit the utilization of cryopreserved sperm for large-scale artificial fertilization, considering the high fecundity of brown-marbled grouper. To aid its commercial application, the main objective of the present study was to further elaborate cryopreservation protocol in this species, by evaluating the cryoprotective efficacy of different sugar extenders (glucose, sucrose or trehalose) and the feasibility of a 5-ml cryotube for cryopreservation. After frozen in a simple media containing 10% DMSO and 0.3-M sugar extender using a 0.5-ml straw, there was a deterioration in sperm motility, viability, mitochondrial membrane potential (MMP) and ATP content compared with fresh semen, but fertilization success of cryopreserved samples was not compromised at the ratio of 10,000 spermatozoa per egg. Besides, the type of sugar extender had no obvious effect on post-thaw sperm quality and fertility, thus it is suggested that glucose, sucrose or trehalose are fully interchangeable for the cryopreservation of brown-marbled grouper semen. By employing a faster freezing rate (freezing height: 2 cm vs. 3 cm) and longer thawing time (40°C, 90 s vs. 40°C, 7 s), samples cryopreserved in a 5-ml cryotube exhibited similar post-thaw motility parameters and fertility with those in 0.5-ml straw. Overall, our results demonstrate the effectiveness of new sugar extenders and the applicability of a 5-ml cryotube for sperm cryopreservation in brown-marbled grouper, which would allow for better flexibility for large-scale artificial fertilization in hatchery practice.