This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post-thaw sperm motility was achieved with the sperm being frozen at a cooling rate of -5°C min-1 to -30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post-thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L-ascorbic acid, on the other hand, significantly improved the post-thaw sperm motility. However, only the addition of 0.6% glycine improved the post-thaw sperm fertilization rate, which was further confirmed by the improvement of the post-thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.
- Haliotis laevigata
- Amino acids
- Greenlip abalone
- Programmable freezing technique