Cryopreservation of sperm in brown-marbled grouper ( Epinephelus fuscoguttatus )

The brown-marbled grouper, Epinephelus fuscoguttatus, is a highly commercial fishery and mariculture species listed as Near Threatened by IUCN. Adequate supply of good quality seed, at present, has become a bottleneck for grouper farming due to the rapid expansion of aquaculture practices. As sperm cryopreservation is of great potential not only for fish farming but also for species conservation, a series of sequential experiments were carried out to determine optimum cryoprotectant, freezing condition, dilution ratio, equilibration time, and thawing temperature for freezing brown-marbled grouper sperm. Based on post-thaw motility, 10% DMSO combined with 0.3 M glucose shown to be an effective cryomedium among the cryoprotectants evaluated here, and sperm was better cryopreserved by freezing 1–5 cm above the liquid nitrogen (LN) surface and cooled below − 80 °C before plunging into LN. Significantly higher post-thaw motility was observed for dilution ratios ranging from 4:1 to 1:3 than at higher dilution ratios (1:5, 1:9, and 1:19). Besides, equilibrated and post-thaw sperm motility was not affected by a 2-h equilibration period, indicating a high level of toxicity tolerance for 10% DMSO. Further evaluation of thawing temperatures showed that 30 °C and 40 °C yielded significantly higher post-thaw motility than at 25 °C, 50 °C, or 60 °C. In addition, brown-marbled grouper sperm could be refrigerated up to 48 h without decreasing its suitability for cryopreservation. Compared with fresh sperm, a decrease in motility and curvilinear velocity (VCL) was detected in thawed sperm. However, the thawed sperm could be stored up to 2 h at 4 °C without losing its fertilizing capacity, which was equal to that of fresh sperm (fertilization rate, 92.7 ± 2.5%; hatching rate, 87.7 ± 2.1%). The cryopreservation protocol developed in this study is efficient and will facilitate seed production for both aquaculture and conservation purposes.