The development
of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes
americanus (Walbaum): evaluation of cryoprotectants and diluents
R.M. Rideout, M.K. Litvak, E.A.
Trippel-2003
Aquaculture
Research, 34(8): 653-659
Abstract:
Three cryoprotectants (dimethyl
sulphoxide, propylene glycol and glycerol) and two diluents (sucrose based
and saline based) were mixed (9 parts diluent-1 part cryoprotectant)
factorially to produce six extenders that were tested to develop an
effective sperm cryopreservation protocol for winter flounder Pseudopleuronectes
americanus (Walbaum). Sperm were diluted 1:3 with each extender and
frozen by flotation on liquid nitrogen before being submerged and stored for
30 days. Sperm left unfrozen in each extender for 20 min showed no toxic
effects on motility. Extenders containing propylene glycol (PG) as
cryoprotectant yielded higher post-thaw sperm motilities than those
containing dimethyl sulphoxide (DMSO) or glycerol. The sucrose-based diluent
performed better than the saline-based diluent when DMSO was used as
cryoprotectant, but there were no differences in post-thaw motility between
diluents for the other cryoprotectants. Activating sperm with ovarian fluid
and sea water instead of sea water alone had no effect on post-thaw
motility. In fertilization trials, no differences were observed between any
of the extenders and fresh milt when milt, eggs and sea water were left in
contact for 1 h. When sperm were forced to compete for eggs by reducing
contact time to 20 s, fertilization results followed those of sperm
motility rates. Percentage hatch and morphology of larvae at hatching did
not differ for eggs fertilized by cryopreserved and fresh sperm. This study
represents the first reported successful attempt at cryopreserving winter
flounder sperm and should improve gamete and broodstock management protocols
for this species.
Fisheries and Oceans Canada, Biological
Station, 531 Brandy Cove Road, St Andrews, NB, Canada, E5B 2L9. E-mail: rideoutr@mar.dfo-mpo.gc.ca