Motility, fertility and ultrastructural changes of ocean pout ( Macrozoarces americanus L.) sperm after cryopreservation

Abstract

The present study examined the possibility of long term storage, by cryopreservation in liquid N 2, of the sperm of ocean pout ( Macrozoarces americanus L.), an internally fertilizing marine fish, and the changes in motility, fertility and ultrastructure of the sperm after freeze and thaw. Four cryoprotectants, including dimethyl sulfoxide (DMSO), and three semen diluents (A, B and C) were tested for their influence on sperm motility. Since the fresh sperm displayed the highest motility in diluent C, which had the closest chemical composition to that of the seminal plasma of ocean pout, with DMSO, this solution (C-DMSO) was chosen as a diluent for the present study of ocean pout semen cryopreservation. Fresh semen was diluted in three volumes of C-DMSO and filled in 0.25- or 1.7-ml straws, then frozen in liquid N 2 vapor. When the internal temperature of the straws had dropped to −95°C, the straws were plunged into the liquid N 2. To recover the sperm motility, the frozen semen was thawed in 1 or 30°C water bath for 30 and 7 s, respectively. It was demonstrated that the presence of DMSO in semen extender was essential for protecting the sperm from dying during freeze and thaw, and 20% of DMSO (v/v) yielded the highest post-thawed sperm motility (20–25% of the total cells). A freezing rate of average 9°C/min during the initial freezing phase (in liquid N 2 vapor) produced a higher post-thawed sperm motility than that produced by faster (18°C/min) and slower (6°C/min) freezing rates. Thawing the frozen semen in 30 or 1°C water did not cause any difference in terms of sperm motility. The loss of sperm motility during freeze and thaw might be due to the ultrastructural changes of sperm, e.g., severe swelling of the mitochondria or dehydration of cytoplasm at the midpiece, revealed by scanning electron microscope. The motile sperm in the post-thawed semen possessed fertility because in vitro artificial insemination of fresh eggs using the post-thawed semen yielded a fertilization rate of 33% vs. 48–58% from fresh semen.