Cryopreservation studies on Silver carp (Hypophthalmichthysmolitrix) embryos

Abstract

The experiment was conducted to investigate the effect of differentcryoprotectants and mixture solution on the hatching rate of different development stage in Silver carp (H.molitrix) embryos. This study also conducted to evaluate the relative efficiency of ultrasound to promote the incorporation ofcryoprotectantsg.Pa) in three different embryonic developmental stages (Morula, 6-somites and tail elongation) of silver carp. Embryos at three developmental stages were immersed in five single cryoprotectants (DMSO, MeOH, PG, DFA, EG), in concentrations of 10, 15, 20 and 25% for 20 min and then hatching rates were determined.Twelve mixture solution, prepared based on the toxicity of single CPA were used at different concentration to test the toxicity in embryos. In case of ultrasound, embryos were exposed to ultrasound for 2, 3, 4,5, 6, 7,8,9, and 10 min (1st trail), exposed to ultrasound for 1,2, and 3 min in the presence of CPA mixture solutions (2ndtrial), and pre-exposed to CPA mixture solution for 8 min (4 steps of 2 min) prior to ultrasound treatment in mixture solution for 2 min (3rd trail). Hatching rates decreased with increasing cryoprotectant concentrations and toxicity to embryos varied in the order of PG < DMSO < EG <MeOH<DF A. The tail elongation stage was more tolerant to cryoprotectant solutions than other stages. Hatching rates of embryos exposed to cryoprotectant mixture solutions in ultrasound varied with the type of mixture solution. Embryos up to 2 min resulted in rapid decline of survival rate (without any CPA). The mixture solution ofCS3, CS6, CS 12 at 2 min in ultrasound and 4 x 2 min + 2 min in ultrasound gave hatching rate of 8-11% and 6-8% respectively. For both 6-somite and tail elongation stage, Embryos that were subjected to ultrasound before cooling showed higher good embryos (%) than the embryos that were not subjected to ultrasound. Although cryopreservation trial after ultrasound-mediated impregnation of 6-somites and tail elongation embryos with a CPA solution did not yield live embryos after freeze-thawing but resulted in the increase of the proportion of morphologically good embryos (%) after freeze-thawing. These results suggest that ultrasound treatment might be useful for silver carp fish embryo cryopreservation.