Vitrification of sperm from marine fish: effect on motility and membrane integrity

Abstract

Our goal was to develop a standardized approach for sperm vitrification of marine fishes that can be applied generally in aquatic species. The objectives were to: 1) estimate acute toxicity of cryoprotectants over a range of concentrations; 2) evaluate the properties of vitrification solutions (VS); 3) evaluate different thawing solutions, and 4) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10-µL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post-thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X-1000™ + 1% Z-1000™ had an average post-thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to high osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.

Vitrification as an Alternative Approach for Sperm Cryopreservation in Marine Fishes

“…A previous study in Summer Flounder that used EG for conventional cryopreservation at 20% reported postthaw motilities of 70% (Liu et al 2015). In a vitrification study that used 20% EG + 20% Gly to vitrify sperm from Red Snapper, the average postthaw motility was 23% (Cuevas-Uribe et al 2015), which was similar to the motility yielded of this study (26%). In a vitrification study with the freshwater Green Swordtail, the same vitrification solutions yielded postthaw motilities of ∼10% (Cuevas-Uribe et al 2011b).…”

“…Recently vitrification has been applied for sperm cryopreservation of marine fishes (Cuevas-Uribe et al 2015; Kasa et al, in press). Vitrified sperm samples from Spotted Seatrout, Red Snapper, and Red Drum yielded motility as high as 60% and membrane integrity as high as 23%.…”

“…In the present study, we used vitrification solutions developed from previous studies with marine fish (Cuevas-Uribe et al 2015) and the freshwater Green Swordtail (Cuevas-Uribe et al 2011b). Also, cryoprotectants were selected to formulate the vitrification solutions based on our previous study of high-throughput sperm cryopreservation in Southern Flounder (Hu et al 2016) and from other studies on paralichthid flounders.…”

“…In experiment 2, two vitrification solutions developed for use with marine fishes that yielded high motility (>20%) (Red Snapper, Spotted Seatrout, and Red Drum) were evaluated (Cuevas-Uribe et al 2015). To enhance glass formation, trehalose or the proprietary polymers X-1000 and Z-1000 were added to cryoprotectant mixtures.…”

“…Our recent studies evaluated sperm vitrification in three marine species, Spotted Seatrout Cynoscion nebulosus , Red Drum Sciaenops ocellatus , and Red Snapper Lutjanus campechanus , in which sperm motility and membrane integrity were general indicators of gamete quality (Cuevas-Uribe et al 2015). Most recently, sperm motility and sperm head morphometry were evaluated after vitrification of the marine European Eel Anguilla anguilla (Kasa et al, in press).…”