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This research was carried out to examine the effect of various volumes (0.5, 0.75, 1.5 and 2 mL) of the frozen sample on cryopreservation of sturgeon sperm and also the possibility of using the method of vitrification of sperm under deep low-temperature cooling in the form of thin films on nets. The object of the study was the spermatozoa of the Russian sturgeon (Acipenser gueldenstaedtii Brandt, 1833) and the Siberian sturgeon of the Lena population (Acipenser baerii Brandt, 1869). There is a direct relationship between the volume of frozen material and the survival rate of defrosted sperm. With the increase in freeze sample preservation frozen-melted cells is falling, as is the range of cooling rate to freeze the sample, in which the majority of cells are frozen at a speed different from the optimal values. When cryopreservation of a sperm smear in the form of a thin film, the analysis of cell movement activity after defrosting showed the suitability of such sperm for use in the fish-breeding process. The highest life time of the sperm as it was observed during the freezing of the films on the plastic samples.

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The research aimed to determine the effect of different taurine concentrations on the duration of sperm fertility preservation and the results of further use during cryopreservation. The taurine was injected into the sperm of the Siberian sturgeon by various methods under conditions of low positive temperatures. Immediately after preparation, the taurine was injected into native sperm at concentrations of 0.01, 0.05, and 0.1 mmol/ml. Two methods of introducing taurine into native sperm were tested: 1 (dry method) - adding taurine powder directly to native sperm and 2 (wet method) - adding taurine dissolved in saline to native sperm. The studies have shown that injections of taurine in an amount of 0.05 - 0.1 mmol/ml both dry and wet can be recommended as the method of long-term preservation of the native sperm of Siberian sturgeon viability. While storing Siberian sturgeon sperm for further cryopreservation the optimal taurine concentration is 0.01 mmol/ml.

The article describes the methods of cryopreservation which provides the reliable protection of cell organelles integrity after freezing-defrosting processes, as well as the needed supply of organic substances generating metabolic process in cells and tissues after a double temperature shock, and helps to achieve a significant progress in the cell long-term storage. There are considered the aspects of low temperature preservation of sturgeon sperm. Reproductive cells of Russian sturgeon ( Acipenser gueldenstaedtii Brandt & Ratzeburg, 1833) and sterlet ( Acipenser ruthenus Linnaeus, 1758) obtained at sturgeon hatcheries of the Astrakhan region and the research-expeditionary base “Kagalnik” in the Rostov region during spawning campaign served as the material for research. The purpose of the work was to establish optimal freezing rates during sturgeon sperm cryopreservation process that could ensure saving structural components of reproductive cells. It has been found that the freezing rate is species-specific. The best freezing speed for Russian sturgeon sperm proved to be 3°C/min. When experimenting with sterlet sperm there was registered less damage after freezing and defrosting at 10°C/min. Freezing speed 3°C/min was found less effective for sterlet sperm. Staged freezing process showed worse results in both cases. However, the quality of defrosted sperm didn’t get lower the fish breeding standards in all three studied speeds, which justifies sturgeon sperm freezing at all three rates subject to different conditions of preservation.

Резюме. Цель. Повышение выживаемости репродуктивных клеток осетровых видов рыб при криоконсервации и получение надежной технологии, пригодной для использования в промышленных масштабах. Методы. В работе применяли стандартные методы замораживания, оттаивания репродуктивных клеток, оплодотворения и инкубации икры и подращивания личинок осетровых видов рыб. Принципиально новым был криопротективный состав: для сперматозоидов скорректирован состав криозащитной среды (для белуги 3% диметилсульфоксида, для русского осетра 4% диметилсульфоксида); для замораживания яйцеклеток применялась криопротекторная смесь из растительного нерафинированного и животного масел. Результаты. Увеличилась выживаемость дефростированных сперматозоидов осетровых видов рыб: у белуги на 20%, у русского осетра-на 47%. При осуществлении оплодотворения криоконсервированной икры русского осетра нативной спермой процент оплодотворения составил 41%. Выводы. Показана эффективность снижения токсического действия веществ в составе криозащитной среды, что привело к повышению выживаемости репродуктивных клеток осетровых видов рыб. При проведении оплодотворения икры, хранившейся в жидком азоте, полученное потомство было жизнеспособным и по реактивности центральной нервной системы и рецепторного комплекса не отличалось от молоди, полученной по традиционной технологии. Ключевые слова: криоконсервация, сперма рыб, яйцеклетки, криопротектор, осетровые рыбы, выживаемость, сохранение генофонда, искусственное воспроизводство.

The research on the sterlet roe artificial insemination using cryopreserved sperm was carried out in the research base of the RAS Southern Scientific Centre (the Rostov region). Reproductive cells (including cryopreserved cells), larvae, sterlet fry ( Acipenser ruthenus Linnaeus, 1758) were taken as an object of research. A half of the roe (1.7 kg) taken from female starlet was inseminated by native sperm (control group); another half was inseminated by defrosted sperm of two males, which was stored in liquid nitrogen at -196ºC during 3 years (pilot group). Incubation lasted 5 days at water temperature 14.5-18.2ºC, with daily fluctuations of temperature 1.9ºC. Roe insemination in the control group made 90%, in the pilot group - 70%. Roe embryonic growth in the control group was faster, but embryogenesis duration in the pilot group met the standard time limits. Hatching prolarvae in the control group started one hour earlier, than in the pilot group; it made 75% and 60% of all incubated roe, correspondingly. Waste during the period of larvae maturing before they pass to mixed feeding was negligible - 2% in the control group and 3.4% in the pilot group. According to the test results, "open field" of reactivity of the central nervous system in the pilot group fry didn’t change from the control group fry, but more active response to stimuli was noted in the pilot group, which is very important for fry adaptation to the conditions in natural water basins. It was established that sterlet offspring obtained with use of defrosted sexual cells does not differ from the offspring obtained using native sperm and has higher morphometric characteristics. The test results prove the possibility and practicability of using sexual cells stored in liquid nitrogen for artificial restoration and formation of sturgeon fish broodstocks.

To improve the quality of the frozen material during cryopreservation, scientists apply various effects on cells: mechanical, chemical or physical. In this work we use acoustic-mechanical effects on cells before cryopreservation. As a result of the studies, the optimal parameters of the impact of the piezoactuator were selected to improve the quality of defrosted reproductive cells of male sturgeons. The object of research was the sperm of the Russian sturgeon. The progressive motility time of native spermatozoa posure time (0.5 min; 1 min, 1.5 min) and frequency (300 Hz, 500 Hz, 550 Hz) were used. Analysis of the motility of thawed sperm showed that the best result in terms of the percentage of sperm motility was obtained when using a frequency of 500 Hz for 1 minute (27%). At the same time, the best indicator of sperm motility time was given by using a frequency of 300 Hz for 1 minute (390 s).