WWW.WAS.ORG • WORLD AQUACULTURE • MARCH 2018 63 composition of cryoprotectant, which can prevent destruction and loss of germ cells during low-temperature preservation, is species-specific in relation to the different characteristics of a particular type of sperm. Providing electrical stimulation of germ cells (Tikhomirov and Ponomareva 2008) increases the rate of penetration of the cryoprotectant into reproductive cells of sturgeon. In 2010, the Southern Scientific Center, Russian Academy of Sciences, jointly with Astrakhan State Technical University, received a patent for a method of increasing the survival of germ cells in sturgeon cryopreservation. Developed with the Institute of Cell Biophysics, a method of reducing the low-temperature jump during crystallization of cryoprotectant solutions was developed, allowing an increase in the integrity of thawed cells after cryopreservation. In the method, freezing the suspension with the biological material and cryprotectant in liquid nitrogen is carried out with application of ultrasonic waves with a frequency of 0.50-10 MHz. These studies demonstrated the effectiveness of freezing sperm on mesh in a thin film (Krasilnikova and Tikhomirov 2014a). Reducing the volume of toxic substances in the composition of cryoprotective medium for sturgeon sperm, which in turn reduced the toxic effect of cryoprotectants, has led to increased lifespan of thawed cells. The results obtained allowed us to recommend adjustment of the concentration of penetrating cryoprotectant solutions as a function of the quantity of intracellular water to improve survival of sperm cells after the double temperature shock (Krasilnikova and Tikhomirov 2014b, Krasilnikova and Tikhomirov 2015). To increase the level of heterogeneity in the resulting offspring and eliminate the negative consequences of inbreeding, a scheme was proposed for the formation of broodstock sturgeon in industrial conditions with the use of cryopreserved reproductive sperm stored in a low-temperature sperm bank. In the formation and operation of broodfish, it is recommended to use 10 percent of the sperm stored in liquid nitrogen in the cryobank annually. This gives the possibility of using high-quality sperm at any time, eliminating the risk of delayed maturation of broodfish and using a greater number of females in spawning. (CONTINUED ON PAGE 64) In the context of current global environmental challenges, the problem of biodiversity conservation has become increasingly important. Maintaining and increasing stocks of fish fauna of the Southern Seas of Russia is possible while maintaining natural reproduction and mandatory development of breeding to the greatest extent possible. However, hatcheries currently take a simplified approach to the formation of female populations due to a lack of broodstock. The use of closely related pairs in mating is fraught with the loss of natural genetic polymorphisms, inbreeding, and consequently, a significant reduction in the adaptive potential of the population. At the moment, cryotechnologies are of strategic importance, including anti-crisis technologies to solve problems related to conservation of fish genetic biodiversity. The use of cryopreserved sperm in hatcheries that apply artificial reproduction and aquaculture enterprises will allow the creation of a genetically diverse population, and will reduce the area required and the cost of maintaining males, thereby allowing an increase in the production of a population of females. Application is possible at any time, without the risk of delayed maturation of broodfish or obtaining sexual products of inadequate quality. Since 2004, the staff of the Southern Scientific Center of the Russian Academy of Sciences, jointly with the Astrakhan State Technical University, have been conducting research on lowtemperature preservation and long-term storage of genetic material of valuable fish species. The aim of the research is development of technology for cryopreservation and storage of fish reproductive cells to ensure their structural and functional integrity and the development of techniques for using cryopreserved sperm in aquaculture. The two most important issues of low-temperature preservation of gametes are the freezing rate and the composition of cryoprotective mixtures. Laboratory staff has conducted research on optimization of freezing and selection of compounds of cryoprotective mixtures for different fish species, including Russian sturgeon, Beluga sturgeon, stellate sturgeon, sterlet, carp, silver carp, common carp, and whitefish. Earlier studies formed the basis for development of cryopreservation methods for sturgeon sperm. The Cryopreservation of Fish Reproductive Cells: Development of Research in Southern Russia Aleksandra A. Krasilnikova, Elena N. Ponomareva and Andrey M. Tikhomirov A method of reducing the low-temperature jump during crystallization of cryoprotectant solutions was developed that allows an increase in the integrity of thawed cells after cryopreservation. In the method, freezing the suspension with the biological material and cryprotectant in liquid nitrogen is carried out with application of ultrasonic waves with a frequency of 0.50-10 MHz. Studies demonstrated the effectiveness of freezing sperm on mesh in a thin film. Reducing the volume of toxic substances in the composition of cryoprotective medium for sturgeon sperm has led to increased lifespan of thawed cells.
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