Optimization of a sperm cryopreservation protocol for giant grouper (Epinephelus lanceolatus)

Characterized by the extremely fast growth performance, giant grouper (Epinephelus lanceolatus) is one of the most promising marine culture species in South East Asia. Motivated by the rapid expansion in grouper aqua culture, giant grouper semen remains in increasing demand for massive seed production, but which is often constrained by the shortage of broodstock and asynchronous availability of gametes for the bulk of farmers. Sperm cryopreservation has a great potential for increased flexibility and efficiency for artificial seed production in aquaculture. To develop an efficient and standardized cryopreservation protocol for giant grouper semen, some key factors were investigated in this study, including the effects of different extenders (MPRS, E2, ELRS3), freezing heights (3, 5, 7 and 9 cm) above the liquid nitrogen surface corresponding to different freezing rates, final sperm concentration in the straw (1-10 x 10(9) spermatozoa/mL), equilibration (0-180 min) and pre freezing storage time (0-60 h). Moreover, the fertilizing ability of cryopreserved semen was tested at sperm: egg ratios ranging from 100:1 to 1x 10(6):1. With 10% DMSO as cryoprotectant, the glucose-based extender ELRS3 at freezing height 5-7 cm produced higher post-thaw sperm motility. The optimal sperm concentrations in straws ranged from 2 to 8 x 10(9) spermatozoa/mL. Besides, sperm motility after thawing was not affected by a 120-min equilibration, but a significant decrease was detected after 180 min of equilibration. Concerning pre-freezing storage, semen stored within 24 h after collection could be successfully cryopreserved, while extended storage time would compromise the cryopreserved sperm motility. Similar fertilization results were obtained at the range of 1000 to 1 x 10(6) spermatozoa per egg for cryopreserved semen. Compared with fresh semen, the fertilization capacity of thawed sperm was not affected by 120 min of post-thaw storage, although with reduced motility parameters. This standardized procedure will be useful for the implementation of cryopreserved giant grouper semen in hatchery practice.

Automated summary

This study aimed to develop an efficient and standardized cryopreservation protocol for giant grouper semen. The results showed that using 10% DMSO as cryoprotectant and glucose-based extender ELRS3 at freezing height 5-7 cm produced higher post-thaw sperm motility. The optimal sperm concentration in straws ranged from 2 to 8 x 10(9) spermatozoa/mL.