Cryopreservation of Greenshell™ Mussel ( Perna canaliculus ) sperm. II. Effect of cryopreservation on fertility, motility, viability and chromatin integrity

The effect of cryopreservation on aspects of sperm function was investigated for sperm of the Greenshell™ mussel (Perna canaliculus). Fertility, DNA integrity, viability and motility were measured before and after cryopreservation. Cryopreservation had a significant effect on fertility with many cryopreserved samples failing to achieve 50% oocyte fertilisation at the highest sperm concentration measured. Sperm DNA integrity, measured using the Sperm Chromatin Structure Assay, showed a small but significant increase in DNA damage as a result of cryopreservation. Sperm viability, measured using propidium iodide/SYBR-14 dual staining, decreased significantly following cryopreservation (69.9±3.5% for fresh sperm versus 25.3±2.9% for cryopreserved sperm). Aspects of motility were similarly affected. The number of sperm tracked, curvilinear velocity (VCL), average path velocity (VAP), variation of straight line velocity (PFT), mean angular displacement (MAD), beat cross frequency (BCF), amplitude of lateral head displacement (ALH) and dance mean (DMN) all decreased following cryopreservation. Correlation analyses between the various aspects of sperm function were carried out. However, no single aspect of sperm function was significantly correlated with sperm fertility following cryopreservation. In addition, the technique for fertility measurement is relatively quicker and less dependent on high tech equipment than the other parameters studied. Fertility therefore remains the most important and relevant end point to measure. Further optimisation of the cryopreservation protocol for Greenshell™ mussel sperm is needed to improve post-thaw fertility.