Cryopreservation of rainbow trout semen using a glucose-methanol extender

• Glucose concentration is important for the cryopreservation of rainbow trout semen.
• Concentrated extenders are not effective for freezing of rainbow trout milt.
• Post-thaw fertility of semen was similar to fresh one at a sperm/egg ratio 600,000:1.

The goal of this study was to improve post-thaw quality of cryopreserved rainbow trout semen. Quality was assessed by measuring sperm motility and fertilization rates at the eyed, hatching, and swim-up larvae stages. We first tested how varying glucose concentrations in a methanol extender (0.1, 0.2, and 0.3 M, n = 9 different males per concentration) and different semen-extender ratios (1:3, 1:1, and 3:1, n = 3 males and in a separate experiment 1:3 and 1:5, n = 6) affected post-thaw motility. Sperm motility and fertilization rates of samples with 0.18 M glucose in 9% methanol as an extender, a 1:5 sperm-to-extender ratio, and a 15 min equilibration period then were measured at sperm-to-egg ratios of 100,000, 300,000, and 600,000:1. The optimal cryopreservation conditions were found to be 0.15 M glucose in the extender, a 15 min equilibration period, and a 1:5 semen-to-extender ratio. This combination resulted in high post-thaw motility (49.9%) and hatching rates (67.1 ± 18.7, 78.6 ± 17.0, and 84.4 ± 10.0% for sperm-to-egg ratios of 100,000, 300,000 and 600,000:1, respectively). To our knowledge, this is the first report to demonstrate that the post-thaw fertilization ability of rainbow trout semen can be similar to that of fresh semen at a sperm-to-egg ratio as low as 600,000:1.