| Summary: | The aim of this research was to investigate different factors, including
cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times,
freezing rates, and thawing methods to optimize cryopreservation protocols for
large yellow croaker (Larimichthys crocea). The parameters
evaluated were sperm motility, sperm activity index (SAI), survival rate, and
DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO),
propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested
for sperm preservation. The highest motility, SAI, and survival rate were
observed when EG was used. Different diluents such as Stein’s solution,
Hank’s balanced salt solution, marine fish Ringer’s solution,
artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution
were investigated. The highest post-thaw motility was observed upon using ASP as
the diluent. Different concentrations of EG were then mixed with ASP to identify
the optimal EG concentration. Experimental results showed that the motility
(70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of
post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and
diluent of cryopreservation, respectively. Post-thaw sperm motility was high at
equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm:
CPA + diluent) and was not significantly different compared with fresh sperm
motility. The freezing rate was found to be slow below −10°C/min.
The thawing temperature of 45°C was identified as ideal. The percentage
of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was
found to have more significant DNA damage than that in fresh sperm but
significantly lower damage than that in post-thaw sperm at EG concentrations of
5%, 15%, and 20% (p < 0.05). The cryopreservation
protocols obtained in this study will be useful in large yellow croaker
hatcheries.
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