THE SELECTION OF A TEMPERATURE MODE OF FREEZING DURING CRYOPRESERVATION OF INCONNU OOCYTES

At present, the use of methods of low temperature preservation remains an attractive and rapidly developing direction of conservation of rare and endangered species. Deep freezing of ova and embryos of fish is one of the central problems in the field of cryobiology of this class of animals. The rate of freezing during cryopreservation of cells is important. It has been ascertained that when the cooling is slow enough, the cells can rapidly lose water by osmosis. On the other hand, if the cooling is too fast, the extracellular solution decreases more rapidly than the intracellular one, and, as the result, the latter will eventually form intracellular ice crystals that proved to be fatal to cells.

In connection with the above the goal of the research was to determine the most suitable method of cryopreservation of inconnu eggs - either an ultra-fast one or a slow one.

The study was carried out at Alexandrovsky Fish Hatchery in Astrakhan Region in November 2016. Eggs from two inconnu females were used as research object. Obtaining of inconnu eggs was performed according to the method of draught. The mixture of triglycerides was used as a cryoprotectant. Ova were mixed with the cryoprotectant and placed in Eppendorf vials with their further placement in the Dewar glasses. Slow freezing was carried out at the rate of 2°/min to t= -70°C with subsequent immersion in liquid nitrogen (t= -196°C). Cryopreservation of inconnu eggs by the method of ultra-fast freezing was performed by quick immersion of glass samples in liquid nitrogen (1,500 °/min).

The thawing of eggs, cryopreserved by the method of ultra-fast freezing, indicated that 1/3 of the oocytes after thawing was with a damaged shell. It shows that there was an internal restructuring in the oocytes - fat and yolk vacuoles have merged, in contrast to the native eggs, which vacuoles fill the cell uniformly and evenly.

When thawing the eggs, stored by the method of slow freezing, it was ascertained that the oocytes were externally non-damaged/whole, with only a few having a damaged shell. When zooming one can see that all eggs have internal adjustments, but only single eggs were with the restructuring at the initial stages.

Thus, the implemented experiment indicated that cryopreservation of inconnu eggs by the method of slow freezing allows obtaining a greater number of (visually) undamaged oocytes after thawing.

It is likely that to get the expected result, the cryopreservation of the native inconnu eggs is recommended according to the slow freezing method. Due to the fact that the experiment was conducted using only two females, it is necessary to conduct further studies in this direction.

The study is implemented within Agreement No. 14.607.21.0163 of 03.10.2016 (the Unique Identifier RFMEFI60716X0163).