Survival and activity of russian sturgeon spermatozoa using various cryoenvironments

The article studied the survival and activity of Russian sturgeon spermatozoa using various cryogenic media, assessing the effectiveness of using cryopreserved sperm in the artificial reproduction of Russian sturgeon.

Introduction At present, the survival and activity of Russian sturgeon spermatozoa are being studied using various cryogenic media. Possibility of increasing the low cryoresistance of sturgeon sperm due to the use of antioxidants in the composition of basic cryoprotective media. However, today, inbreeding of individuals is often carried out at fish hatcheries, which threatens with the loss of the natural genetic diversity of populations, inbreeding and, accordingly, a decrease in the adaptive potential of animals. While the preservation of cryopreserved sperm during artificial reproduction, at fish farms, will allow obtaining genetically heterogeneous offspring, reduce the area and cost of maintaining males, thereby allowing an increase in the production herd of females. Biotechnical development of cryopreservation of spermatozoa in Russian sturgeon for the purpose of their long-term storage at low and ultralow temperatures is an important area of research in the field of aquaculture and involves the creation of cryobanks of sturgeon genomes. It is known that aqueous plasma crystals and the cell itself often cause spermatolysis when frozen. Therefore, the search for substances with cryoprotective properties continues to this day. In this work, we studied various cryomedia in various proportions using sturgeon seminal fluid. The results obtained were compared with the base medium. In addition, the freezing and storage of seminal fluid was carried out in two versions: at low and ultra-low temperatures. The cold resistance of spermatozoa was assessed by spermatozoa motility and morphological analysis of cells by light microscopy. It is shown that this is due to the composition of cryoprotectants. The most severe violations were observed when adding osmotically active substances, including some inorganic compounds.

Оbject. The object of the study is the spermatozoa of the Russian sturgeon.

Materials and methods. Reproductive cells of Russian sturgeon males obtained in the scientific center "Breeding of valuable sturgeon breeds" were used for the study. The studies were carried out at the Scientific Center "Breeding of valuable sturgeon breeds" of the Volgograd State Agrarian University. For the study, Russian sturgeon sperm, obtained at the research center, was used. The studies were carried out in 2020-2022. sperm was obtained by the method of Tsvetkova et al. Collection, placement and storage of samples in a Dewar vessel equipped with canisters for placing biomaterial, which are suspended in a vessel on fiberglass handles to reduce heat gain and obtain maximum storage time at liquid nitrogen temperature, freezing was carried out in accordance with the “Regulations on the collection genetic low-temperature fish sperm bank” (1996) and "Procedure for collecting and laying in a low-temperature bank of genetic collections of fish sperm" (1996), approved by the Interdepartmental Ichthyological Commission. Also used polyethylene containers for freezing and storage with a volume of 1.5 and 0.6 ml. binocular microscope Micromed, Goryaev's camera, which was necessary to count the number of cells in a given volume of liquid. In studies on cryopreservation, semen with an activity of 4 and 5 points was used. Cryopreservation of reproductive cells was carried out according to a previously developed method using the most optimal cryoenvironment. The essence of the freezing technique is as follows. Freezing of a biological object in nitrogen vapor occurs from the initial temperature to the eutectic one. When the temperature reaches the cryoscopic value, the stage of ice crystal formation begins. When the crystal formation process ends, and the temperature throughout the volume of the test tube with the biological object reaches the eutectic temperature, the test tube with the biological object is immersed in liquid nitrogen, continuing its freezing to a final temperature of –196 C. After that, the test tube with the biological object is left in liquid nitrogen for long-term storage. Russian sturgeon sperm samples were kept in liquid nitrogen for 12 hours.

Results and conclusions. When freezing the seminal fluid of Russian sturgeon in cryo-medium № 1, the life time of spermatozoa during dilution averaged 228.1 seconds, with defrosting 134.6 seconds, while the activity of diluted sperm was 57.8%, and defrosted 43.8%. During cryopreservation in medium № 2, in comparison with cryo-medium № 1, the activity and lifetime of diluted sperm increased by 6.32% and 5.11%, defrosted sperm by 4.95% and 4.43%, respectively. The use of cryo-medium № 3 in comparison with cryo-medium № 1 did not have a significant effect on the increase in the activity of spermatozoa in dilute and defrosted seminal fluid (3.38 and 2.22%, respectively), however, an increase in the lifetime of spermatozoa by 8.84% was recorded. (diluted) and 8.21% (defrosted). When using medium № 4 containing soy lecithin, it was recorded that, in comparison with cryo-medium № 1, the activity of diluted sperm increased by 13.55, and defrosted by 10.6%, while the lifetime of spermatozoa increased by 2.79 and 2.05%. Thus, the implementation of research results will increase the demand for cryotechnologies for large volumes of sperm required for the formation of industrial cryobanks.

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