Cryopreservation of striped bass (Morone saxatilis) spermatozoa

Several experiments were conducted with striped bass (Morone saxatilis) spermatozoa, in order to (1) develop suitable extender(s) for refrigerated short-term storage and cryopreservation of striped bass spermatozoa, (2) determine the cryoprotectants, which could enable high post-thaw sperm motility and fertilization, (3) verify the ability of certain amino acids to improve post-thaw sperm motility and fertilization, and (4) identify the cryo-injuries to plasma membrane and mitochondria before freezing and post thawing. It was fast demonstrated that the initiation of sperm motility in striped bass was not regulated by osmolality or extracellular ions (K +, Ca2+), which are the two most common regulatory factor observed in other teleost species. Although the extender isotonic to seminal plasma failed to keep striped bass spermatozoa immotile, an isotonic extender was developed and yielded 70% sperm motility after 24 h refrigerated short-term storage. A hypertonic extender with an osmolality of 500 mmol/kg was also developed for cryopreservation, which resulted in significant higher post-thawed motility (P < 0.05) than the isotonic extender. In another study, cryoprotectants and equilibration time was evaluated. Dimethyl sulfoxide (DMSO) yielded significantly higher post-thaw motility (P < 0.05) compared to methanol and dimethylacetamide (DMA), and 5% DMSO yielded significantly higher (P < 0.05) post-thaw motility than 1.25, 2.5, 10 and 15% DMSO. In terms of adding amino acid to improve sperm motility, both glycine and alanine significantly improved (P < 0.05) sperm motility after short-term storage and cryopreservation. The best result based on post-thaw motility was obtained from cryopreserved striped bass sperm in Extender 2 with 5% DMSO and 50 mM glycine as the cryoprotectants at a freezing rate of -40°C/min and thawing the semen in a 35°C water bath for 8 sec. The effects of DMSO and glycine on plasma membrane integrity, mitochondrial function and ATP content were evaluated in another study. The data demonstrated that the higher DMSO concentrations tested (2.5--10%) provided better protection of the plasma membranes during the freeze-thaw process, however higher DMSO concentrations resulted in lower mitochondrial activity and ATP content. Adding glycine to cryomedia increased the percentage of spermatozoa with functional mitochondria and ATP content during cryopreservation. The highest percentage of spermatozoa with intact plasma membrane and functional mitochondria was achieved by 75 mM glycine with 7.5% DMSO. In the final study, the effects of DMSO and glycine on fertilization in vitro were studied. Spermatozoa cryopreserved with 75 mM glycine and 7.5% DMSO resulted in the highest fertilization rate of 54 +/- 5.6%, which was 90% of fresh sperm control.