Cryopreservation of masu salmon sperm using glucose-methanol extender and seminal plasma biomarkers related to post-thaw sperm motility

The aim of this study was to evaluate the cryopreservation of masu salmon (Oncorhynchus masou) semen, using 0.18 M glucose in 9% methanol as an extender, and to characterize semen parameters (sperm motility; sperm concentration; and seminal plasma parameters such as pH, electric conductivity, DNA concentration, and protein concentration), with respect to post-thaw sperm motility. Semen samples were collected from fishes in the middle of the spawning season, at Toya Lake station, Hokkaido University, and the end of the spawning season, at the Nanae freshwater station, Hokkaido University. The semen was diluted with the extender at a 1:5 ratio (semen:extender) and then equilibrated for 15 min. The final concentration was 0.15 M and 7.5% for glucose and methanol, respectively. Sperm motility was assessed for fresh, fresh-diluted, and post-thaw semen using computer-assisted sperm analysis. The post-thaw sperm showed lower motility, compared with those of fresh and fresh-diluted sperm, which ranged from 33.9% to 82.9% and 1.5% to 27.1% in the semen collected in the middle and end the spawning season, respectively. Sperm concentration, pH, and electric conductivity of the semen collected at the end of the spawning season were lower than those of semen collected in the middle of the spawning season. Moreover, remarkably high DNA concentrations were detected in the semen at the end of the spawning season, although no difference was observed in protein concentration. Finally, semen cryopreserved using the aforementioned method could be used for artificial fertilization to produce offspring. These results indicate that the glucose-methanol extender is useful for sperm cryopreservation in masu salmon and that the suitability of semen for cryopreservation is affected by the condition of the parental fish.