Cryopreservation of strip spawned sperm using programmable freezing technique in the blue mussel Mytilus galloprovincialis

Abstract

In this study, a programmable freezing technique has been developed for strip spawned sperm in the blue mussel, Mytilus galloprovincialis. The optimized key parameters include cooling rate, endpoint temperature, thawing temperature, sugar addition and sperm to oocyte ratio. The sperm quality was assessed by the fertilization rate or the integrity of sperm component and organelle. The highest post-thaw sperm fertilization rate was 91%, which was produced with sperm cryopreserved in 8% dimethyl sulfoxide at the cooling rate of -4°C/min from 2°C to -30°C before being plunged into liquid nitrogen for at least 12 h, thawed in a 20°C seawater bath and fertilized at sperm to egg ratio of 50 000:1. The addition of glucose, sucrose or trehalose to 8% dimethyl sulfoxide could not further improve fertilization rates. The fluorescent assessments showed that the post-thaw sperm plasma membrane integrity and acrosome integrity were significantly damaged in comparison with fresh sperm.