Sperm activation and effects of cryopreservation on motility, ultrastructure and DNA integrity in Grey mullet Mugil cephalus

Abstract

The effects of osmolality and pH of the seawater and non-ionic media (glucose) on sperm activation in Mugil cephalus was evaluated. The effect of cryopreservation was documented by cryopreserving the sperm diluted with a cryomedium (V2 extender + 10% dimethylsulfoxide) in a programmable freezer. The highest motility grade (4) or (3) was recorded when sperm were activated with seawater with osmolality above 600 (mOsmol/kg) and pH 6 to 9. Significant difference (P < 0.05) was found in the duration of sperm motility with pH 7 (316 ± 8 s) followed by pH 6 and pH 8.2. In non-ionic media, the highest motility grade (4) and maximum duration of sperm motility (152 ± 23 s) was recorded when sperm activation carried out with 800 mM of glucose. The frozen-thawed sperm registered a motility grade of 2.44 ± 0.72. Frozen- thawed spermatozoa revealed changes in ultrastructure like damage of plasma membrane around the sperm head, shrinkage and swelling of the mid-piece region, partial fragmentation, and complete loss of flagellum when observed under electron microscope. However, comet assay indicated a non-significant (P > 0.05) DNA damage in frozen-thawed spermatozoa (3.68 ± 2.69% of tail DNA) compared to fresh spermatozoa (3.01 ± 2.13% of tail DNA). Over-all, the sperm activation experiments indicated that sperm of M. cephalus can be activated by a media having osmolality above 600 (mOsmol/kg) and pH ranging from 6 to 9. Although DNA damage is minimal in frozen- thawed spermatozoa, the ultrastructural changes are prominent. Therefore, further experiments are required for the modifications of the composition of cryomedium to minimize the cryodamage.