The Influence Of Several Factors On Crucian Car P(Carassius Cuvieri)oocytes Vitrification

This paper studied the vitrified cryopreservation of crucian carp(Carassius cuvieri) oocytes which had grown to stage IV and analyzed the effects of two different buffer solutions(KCl buffer solution and D-Hank’s buffer solution),two different vitrification solutions(VS4 and VSd), two different pre-equilibrium ways(three-steps pre-equilibrium and five steps pre-equilibrium), different frozen days(1d, 2d, 3d, 4d, 6d, 8d) and two different re-warming methods. The study aimed at selecting suitable buffer solution, pre-equilibrium way and re-warming method for vitrified cryopreservation of crucian carp oocytes by examining the cell survival rate, SDH activity and MDA content, thus providing experimental proof and technical support for establishing and improving the technical route of vitrified cryopreservation of fish oocytes. The research had three parts. Firstly, we analyzed the effect of different pre-equilibrium methods on the vitrified cryopreservation of crucian carp oocytes. Secondly, we vitrified cryopreserved the crucian carp oocytes with the suitable pre-equilibrium way according to the first result and analyzed the interaction of different buffer solutions and different re-warming methods by double-factor wariance analysis. Lastly, we analyzed the effect of different buffer solutions and different re-warming methods on the vitrified cryopreservation of crucian carp oocytes by one-way analysis of variance.(1) Selection of different pre-equilibrium methods: Crucian carp oocytes were pre-equilibriated by three steps and five steps pre-equilibrium ways. Three steps pre-equilibrium methods of VS4-K group and VSd-K group were better than five steps pre-equilibrium methods of VS4-K group and VSd-K group. Three steps pre-equilibrium methods of VS4-D group and VSd-D group were better than five steps pre-equilibrium methods of VS4-D group and VSd-D group. Therefore when it was 0℃, three steps pre-equilibrium was suitable for vitrified cryopreservation of crucian carp oocytes with each step balancing time for 10 minutes.(2) Interaction of multi factors on vitrified cryopreservation of crucian carp oocytes: Two factors combined contributed to vitrified cryopreservation of crucian carp oocytes. The experimental results showed that there was no interaction on oocytes between different buffers and re-warming methods, which meant that different buffer solutions had no significant effects on crucian carp oocytes no matter which re-warming method the researchers took.(3) Selection of different buffer: Four kinds of vitrification solutions were generated namely VS4-K, VSd-K, VS4-D, VSd-D by combining two buffer solutions and two vitrification solutions. Through comparing KCl buffer solution with D-Hank’s buffer solution, the cell survival rate and SDH activity of VS4-K group were higher than VS4-D group, while the MDA content was lower. The cell survival rate and SDH activity of VSd-K group were higher than VSd-D group, while the MDA content was lower. Therefore, KCI buffer solution was better than D-HanK’s buffer solution.(4) Selection of different re-warming methods: crucian carp oocytes were re-warmed by water bath or microwave re-warming after vitrified cryopreservation. It showed that the survival rate and SDH activity of microwave re-warming group were significantly higher than water bath re-warming group while the MDA content was lower. The result indicated that the damage of microwave re-warming to crucian carp oocytes was less than that of water bath and it could be used in the vitrified cryopreservation of crucian carp oocytes.