Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

Abstract

The spotted halibut is species that has a high potential market value in Korea, but the supply of seed is unstable because of the limited milt production of males. The objective of this research was to explore different aspects, such as CPAs, diluents, dilution ratio, and freezing rates, to develop an optimal sperm cryopreservation. The parameters assessed were movable sperm ratio, sperm activity index, survival rate, and DNA damage. The CPAs tested in this research were propylene glycol, dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and glycerol. Different diluents, including 300 mM sucrose, 300 mM glucose, Stain’s solution, and Ringer’s solution, were investigated. The previous experiment showed that the optimal CPA for cryopreservation was DMSO with a concentration of 15% with 300 mM as diluent. To determine the effect of the dilution ratio, sperm was diluted to 1:1, 1:2, 1:10, 1:100, and 1:1000 with 300 mM sucrose containing DMSO at a final concentration of 15%. Lastly, the optimal freezing rate of the sperm was evaluated with four different freezing rates (−1, −5, −10, and −20 °C/min). Post-thaw sperm motility was higher with a dilution ratio lower than 1:2, and the freezing rate was less than −5 °C/min. In conclusion, these findings represent the development of a cryopreservation protocol for spotted halibut.

Vital Analysis of Cryopreserved Sperm of Marbled Flounder, Pseudopleuronectes yokohamae

“…In the present study, a sucrose base solution was used as extender-1. It had a composition similar to the extender used for the cryopreservation of sperm of summer flounder, Paralichthys dentatus (Brown et al, 2012 ); Brazilian flounder, Paralichthys orbignyanus (Lanes et al, 2008 ); and spotted halibut, Verasper variegatus (Tian et al, 2008 ). Glucose solution was used as extender-2.…”

“…Cryopreservation with MeOH resulted in a decreased post-thaw sperm motility compared to that with Me 2 SO, GLY, EG, or PG. It also produced the lowest post-thaw motility for sperm of other flounder species such as Paralichthys olivaceus (Zhang et al, 2003 ), Paralichthys dentatus (Brown et al, 2012 ), and V. variegatus (Tian et al, 2008 ). Although it is associated with a low post-thaw motility of sperm of marine flounders, it has been reported as an effective CPA for cryopreserving sperm of freshwater fish (Lahnsteiner et al, 1997 ).…”

“…Two dilution ratios (1:2 and 1:4) were used to establish the optimal cryopreservation technique based on the published results for other flounder species (Zhang et al, 2003 ; Tian et al, 2008 ; Brown et al, 2012 ). Two concentrations (10 and 12%) of CPAs (Me 2 SO and GLY) with extender-1 and extender-2 were used to select the best dilution ratio for the effective cryopreservation.…”

“…Currently, sperm cryopreservation techniques exist for over 200 fish species (Nahiduzzaman et al, 2011 ). Cryopreservation of sperm of several flounder species has been reported (Zhang et al, 2003 ; Lanes et al, 2008 ; Tian et al, 2008 ; Brown et al, 2012 ; Liu et al, 2015 ). However, very little is known about the cryopreservation of marbled flounder sperm.…”

Antioxidant Activity and Oxidative Stress-Oriented Apoptosis Pathway in Saccharides Supplemented Cryopreserved Sperm of Pacific Abalone, Haliotis discus hannai

“…Further, sperm cryopreservation can be used to preserve genetic materials of this important species. Sperm cryopreservation biotechnology has several benefits including genetic materials preservation, maintenance costs reduction of male broodstock, constant supply, simplified transportation and to reduce disease transfer [ 5 , 6 , 7 ].…”