Cryoprotective Medium Research Articles

CRYOPRESERVATION OF REPRODUCTIVE CELLS OF MALE RUSSIAN STURGEON

Currently, cryopreservation of sperm is recognized as one of the promising ways to preserve the genetic diversity of fish, not only rare and endangered species, but also aquaculture objects. Scientific research was carried out in the scientific research center "Fisheries" of the S. Seifullin Kazakh Agro Technical University and the JSC "Republican Center for livestock breeding "Asyl Tulik" in 2022. The purpose of the research was to study methods of cryopreservation of Russian sturgeon sperm using cryoprotectors using dimethyl sulfide oxide with a concentration of 5% and 10% and methanol with a concentration of 3% and 8%. In the course of the research, generally accepted methods for assessing and freezing the sperm of the studied object were used. During the experimental work, the method of freezing the reproductive cells of the Russian sturgeon with a 15-minute exposure to -21°C in a box with subsequent immersion in a Dewar vessel was studied. The survival and lifetime of spermatozoa after defrosting were also studied. According to the results of the assessment of the quality of defrosted sperm of the Russian sturgeon, it was found that the most effective cryoprotective medium for cryopreservation is based on methanol with a concentration of 8%. The number of spermatozoa with translational movements in the three groups ranged from 23.8% to 31.2%. At the same time, the lifetime of spermatozoa in this cryoprotective medium was 118s. Methanol with a concentration of 8% provides the best resistance to oxidative stress experienced by cells during freezing. The results of the research make it possible to create a cryobank of the Russian sturgeon gene pool at fish hatcheries to preserve genetic diversity.

High sperm concentration during cryopreservation decreases post-thaw motility percentage without compromising in vitro fertilization outcomes in common carp

To avoid mechanical compression of spermatozoa during the cryopreservation, a high dilution ratio with the cryoprotective medium is applied. Due to this, the sample volume increases, which entails increased cryobank capacity. Moreover, the high volume of cryomedias does not allow to fertilize of large volumes of eggs in practical artificial reproduction of common carp aquaculture. The current study demonstrated that reasonable lowering of dilution rates might still be effective for carp sperm cryobanking —elevation of spermatozoa concentration up to 13 × 109 spz mL−1 resulted in a significant decrease in post-thaw sperm motility percentage to 20% compared with 39% in 0.5 × 109 spz mL−1. Nevertheless, no significant differences in sperm kinetic parameters (VCL and LIN) were found in this case. The fertilization outcome (embryo development and hatching rates) was similar after applying thawed sperm samples with “optimal” (2.2–2.4 × 109 spz mL−1) and “sub-optimal” (11.0–13.0 × 109 spz mL−1) concentrations (sperm/egg ratio at fertilization was in the range of 0.3–4.5 × 105 spz/egg). Thus, applying a low dilution rate such as one part of sperm to one of the cryoprotective mediums is favorable for decreasing cryo-storage space and the sperm volume needed to fertilize big egg numbers. The experiment also shows that the 4.5 × 105 spz/egg ratio is not sufficient for good fertilization, and it is necessary to use higher sperm concentrations per egg or improve the method of fertilization.

Use of Biological and Synthetic Polymers for Human Spermatozoa Cryopreservation

BACKGROUND: Spermatozoa cryopreservation is an integral part of the assisted reproductive technologies for treatment of infertility. It is also used to preserve the reproductive potential of men. However, using a standard freezing method with glycerol leads to a decrease in morphological and functional characteristics of spermatozoa in the case of oligoasthenoteratozoospermia (OAT). Therefore, it is relevant to develop effective methods of cryopreservation for such sperm. The use of various biopolymers can stabilize the membrane and bind excess water, which forms ice crystals in the medium that causes cell damage when temperature decreases. OBJECTIVE: To study the effectiveness of using cryoprotectant mixtures based on biological and synthetic polymers [serum albumin, polyvinylpyrrolidone (PVP) and insulin] for the cryopreservation of human spermatozoa with OAT. MATERIALS AND METHODS: Human spermatozoa with OAT were cryopreserved using different cryoprotectant media containing 10 % glycerol or 10 % PVP, 20 % albumin and 1 μg/m L human insulin. The viability, motility and mitochondrial membrane potential of spermatozoa were assessed after rewarming. RESULTS: A cryoprotectant solution containing 10 % PVP, 20 % human serum albumin and 1 μg/m L insulin enabled a similar level (%) of viable gametes compared with the standard method using glycerol, while the number of motile cells was significantly lower (p < 0.008). The membrane mitochondrial potential did not differ significantly from fresh sperm. CONCLUSION: The data obtained in this study show the effectiveness of a biopolymer mixture containing PVP, serum albumin and insulin for the cryopreservation of human OAT spermatozoa.

Osmotic Characteristics of Erythrocytes After Freezing with Composition-Modified Cryopreservatives

Here, we have studied the osmotic parameters of erythrocytes frozen in the composition-modified media with glycerol and 1,2-propanediol. A decreased glycerol concentration in a cryopreservative and an increased content of impermeable protective component sorbitol were established to affect the erythrocyte characteristics at different stages of freezing and hypothermic storage. Reduction of 1,2-propanediol and NaCl concentrations when augmenting the sucrose content in cryopreservative agent enabled changing a two-stage mode of erythrocyte freeze-thawing to the single-stage one. The presented here modification of cryoprotective medium allowed to diminish the level of damage to erythrocytes during freeze-thawing-washing out, that might prevent the inflammation development in a body when transfusing these cells.

Comparison of sperm quality after double slow freezing and double vitrification of stallion sperm

Intracytoplasmic sperm injection (ICSI) is a valuable tool to optimize the remaining frozen sperm of subfertile or dead stallions. The possibility of thawing, diluting, and re-freezing, conventionally frozen spermatozoa has been previously studied, but not for vitrified sperm. We compared the effect of thawing/warming and re-freezing/re-vitrifying conventionally frozen/vitrified stallion sperm. For this purpose, 10 ejaculates from 2 stallions were divided into two aliquots and submitted to i)slow freezing (SF), extending sperm at 100 × 10 6 sperm/ml in permeable cryoprotectant medium (Botucrio, Betlabs, Lexington, KY), slow cooling for 2 hours and frozen 4cm over liquid nitrogen (LN 2 ) for 10 minutes; ii) or vitrification (VIT), extending sperm in permeable-cryoprotectant free medium (INRA96 + Trehalose 100mM + BSA 1%), cooled for 1 h and plunged directly into LN 2 . Two SF straws were thawed at 37 °C for 30 s, whereas two VIT straws were warmed at 60 °C for 5s. One straw was used for sperm parameters assessment while the second straw was re-frozen (RE-SF) or re-vitrified (RE-VIT) andthawed for sperm analysis. Sperm were analyzed for total (TMOT) and progressive (PMOT) motility (CASA, SCA, Microptic, Barcelona, Spain) and percentage of viable, non-reacted sperm (VNR, PI/PNA staining using flow cytometry). TMOT (80.6 ± 2.2 % vs. 70.2 ± 2.9% and 79.4 ± 2.5% vs. 70.1 ± 3.0%), PMOT (46.9 ± 6.1% vs. 31.3 ± 3.4% and 47.8 ± 6.4% vs. 38.1 ± 3.9%) and VNR (81.4 ± 2.4% vs. 71.2 ± 3.1% and 79.1 ± 3.1 vs. 72.2 ± 3.9%) showed significantly (p<0.05) lower values after SF vs. RE-SF and VIT vs RE-VIT, respectively. RE-VIT showed significantly (p<0.05) higher PMOT values than RE-SF (38.1 ± 3.9% vs. 31.3 ± 3.4%). In conclusion, both re-freezing and re-vitrification decrease sperm parameters after warming. Interestingly, re-vitrified sperm obtained higher progressive motility values than conventionally re-frozen sperm. Although further studies are needed to evaluate the effect of using vitrified sperm for ICSI, re-vitrification seems an interesting alternative for the optimization of valuable stallion ejaculates.

Spermatological characteristics and effects of cryopreservation in Lebranche mullet spermatozoa (Mugil liza Valenciennes, 1836): First report of ultra-rapid freezing

The present study investigated the spermatological characteristics of raw semen of Lebranche mullet (Mugil liza), namely pH, and sperm density, and motility; and subsequently evaluated the effects of different times of exposure to cryoprotectants, and the application of an ultra-rapid freezing protocol, on sperm motility and plasma membrane integrity. Semen samples were analyzed undiluted (control) and diluted 1:50 v/v in CF-HBSS + 10% Dimethyl sulfoxide + 30% Ethylene glycol + 94.58 gL−1 Trehalose dehydrate (n = 15). Two treatments – diluted semen samples in cryoprotective medium without ultra-rapid freezing (T1), and diluted semen in cryoprotective medium with ultra-rapid freezing (T2) – were evaluated at 0, 2, 4, 6 and 8 min. The frozen samples were thawed at 37ºC for 60 s. The spermatological characteristics recorded for the semen were: pH: 7.57 ± 0.21; sperm density: 30.4 ± 2.9 × 109 sperm mL−1; motility: 82 ± 4.9%. Sperm motility presented differences after 2 min exposure to cryoprotectants (70.0 ± 2.7%) and ultra-rapid freezing (66.5 ± 5.8%) compared to the control group (98.5 ± 1.9% and 98.5 ± 2.1%, respectively; p < 0.05). On the other hand, the plasma membrane integrity of the spermatozoa after 2 min exposure to cryoprotectants (64.0 ± 8.6%) and ultra-rapid freezing (62.5 ± 5.2%) presented no differences compared to the control group (69.5 ± 3.9% and 70.0 ± 3.5%, respectively p > 0.05); however, differences were observed in the parameters evaluated after longer exposure and cryopreservation times. This is the first report evaluating the effects of different times of exposure to cryoprotectants and direct ultra-rapid freezing in liquid nitrogen on Mugil liza sperm. Our results demonstrated the protocol of sperm ultra-freezing is safe within a time´s window of 2 min of exposure to cryoprotectants, after which a toxicity effect on sperm can be observed.

Comparison of cryoprotectants in hematopoietic cell infusion-related adverse events.

The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated. To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration. The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats. Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

Viable human brain microvessels for the study of aging and neurodegenerative diseases.

The brain microvasculature is altered in normal aging and in the presence of disease processes, such as neurodegeneration or ischemia, but there are few methods for studying living tissues. We now report that viable microvessels (MV) are readily isolated from brain tissue of subjects enrolled in studies of neurodegenerative diseases who undergo rapid autopsy (performed with <12h postmortem interval - PMI). We find that these MV retain their morphology and cellular components and are fairly uniform in size. Sufficient MV (~3-5000) are obtained from 3 to 4g of tissue to allow for studies of cellular composition as well as extracellular matrix (ECM). Using live/dead assays, these MV are viable for up to 5days in tissue culture media (2D) designed to support endothelial cells and up to 11days post-isolation in a 3-dimensional (3D) matrix (Low Growth Factor Matrigel™). Assays that measure the reducing potential of live cells \demonstrated that the majority of the MV maintain high levels of metabolic activity for a similar number of days as the live/dead assays. Functional cellular components (such as tight junctions and transporter proteins) and ECM of MV in tissue culture media, and to a lesser extent in 3D matrices, were readily visualized using immunofluorescence techniques. MV in tissue culture media are lysed and protein content analyzed, but MV in 3D matrix first require removal of the supporting matrix, which can confound the analysis of MV ECM. Finally, MV can be preserved in cryoprotective media, whereby over 50% retain their baseline viability upon thawing. In summary, we find that MV isolated from human brains undergoing rapid autopsy are viable in standard tissue culture for up to 5days and the timeframe for experiments can be extended up to 11days by use of a supportive 3D matrix. Viable human MV allow for temporal and spatial analysis of relevant cellular and ECM components that have implications for microvascular function in neurodegenerative diseases, vascular brain injury, and neurotrauma.

Cathepsin L involved in the freezing resistance of murine normal hatching embryos and dormant embryos

The cryopreservation of mammalian embryos is an important technology in embryo engineering. The discovery and application of the embryo's own high freezing resistance factors are the main methods to improve the utilization of mammalian embryos in cryopreservation. Cathepsin L gene expression in the frozen and thawed dormant embryos displayed a significant difference from those normal hatched ones. The aim of the present study was to dig out the potential role of Cathepsin L in anti-freezing capacity of murine blastocysts by investigating the location and expression of Cathepsin L in frozen and thawed both activated and dormant hatching blastocysts. Different concentrations of Cathepsin L recombinant protein and E-64d were then respectively added into the embryo cryoprotectant and pre-cryo culture medium. Our results found that down-regulation of Cathepsin L improves the freezing resistance of murine normal hatching embryos by reducing apoptosis. Cathepsin L inhibitors can be used to improve the efficiency of cryopreservation and recovery of blastocysts in vitro. Our study provides a theoretical basis for the further development and application of Cathepsin L.

Cryoprotective effect of antifreeze protein III on the rabbit ovary.

Ovarian tissue cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, ischemia reduces the lifespan of grafts. Microvascular transplantation of cryopreserved whole ovary may allow immediate revascularisation, but the damage incurred during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function. This study was designed to investigate the beneficial effects of antifreeze protein III (AFP III) on rabbit ovary cryopreservation. Ovaries (n =25) obtained from 5-month-old female rabbits (n =13) were frozen by slow freezing and vitrification. Cryoprotectant media were supplemented with and without 1mg/mL of AFP III. The experiment was divided into five groups: fresh control group (F), slow freezing group (S), slow freezing group with AFP III (AFP III-S), vitrification group (V) and vitrification group with AFP III (AFP III-V). All groups of ovaries were examined by histological characteristics analysis, ultrastructural analysis, apoptosis detection and follicle viability test. With slow freezing, the normal rate of change in follicle morphology, density of stromal cells and the survival rate of follicles in the AFP III supplemented group were significantly higher than those in the non-supplemented group, and a lower oocyte apoptotic rate was shown in the AFP III supplemented group. In the vitrification groups, the normal rate of change in follicle morphology and density of stromal cells in the AFP III supplemented group were significantly higher than those in the non-supplemented group, and a lower oocyte apoptotic rate was found in the AFP III supplemented group. But there was no obvious difference in the survival rate of follicles between the two groups. There was also no significant difference in the normal rate of change in follicle morphology, the survival rate of follicles and the apoptotic rate of oocytes between the vitrification and slow freezing groups (P >0.05), but the density of stromal cells in the vitrification groups was statistically higher than that of the slow freezing group (P <0.05). The addition of AFP III in slow freezing and vitrification could improve the cryoprotective effect of ovaries, which was more evident in slow freezing. The findings of this study provide a foundation for further research on the effects of AFP III in human ovarian tissue.

Dynamics of Dimethyl Sulfoxide Penetration Into L929 Cells and L929-Based Spheroids

The study proposes an algorithm for calculating of appreciable permeability coefficients for multicellular structures in a cryoprotectant medium using physical and mathematical model of mass transfer. The values of surface-area-to-volume ratio for L929 cells at different temperatures were determined and the thermal expansion coefficient of the surface area of cell membranes was calculated (β = 2.7 × 10-3 /°C). The osmotically inactive volume for L929 cells and their spheroids was determined. Filtration and permeability coefficients to DMSO for L929 cells and in toto spheroids were found from the dynamic curves of relative volume change. The calculated parameters are the highest for individual cells and significantly (p &lt;0.05) decrease for cells in the spheroids with increasing depth of their location, this reduction may be stipulated by a decrease in the available surface of cells in the spheroids for the penetration of extracellular substances. Obtained in this research permeability characteristics of spheroids can be used to develop optimal cryopreservation regimens for them.

Effects of melatonin supplementation on the quality of cryopreserved sperm in the neotropical fish Prochilodus lineatus

The cryopreservation process causes damage to sperm structures and supplementation of the cryoprotective medium is an alternative to reduce these damages. The aim of this study was to determine the effect of melatonin supplementation on post-thaw sperm quality in Prochilodus lineatus. The cryoprotective medium was supplemented with 2.00, 2.75, 3.50, and 4.25 mM melatonin, and the control group (without melatonin). Sperm motility parameters, membrane integrity, sperm morphology, oxidative stress (lipid peroxidation and enzymatic activity), and fertilization capacity were analyzed in the post-thaw sperm. Samples cryopreserved with 2.00 mM melatonin yielded higher sperm motility rate than other treatments with the addition of melatonin. Sperm curvilinear velocity (VCL) and average path velocity (VAP) were higher in samples containing 2.00 mM melatonin than in other treatments. Samples from control and with 2.00 mM melatonin presented higher membrane integrity and morphological normality than samples containing 4.25 and 2.75 mM melatonin, respectively. Regarding oxidative stress, lower lipid peroxidation (LPO) occurred in 2.75 and 3.50 mM melatonin, compared to control. While higher enzyme activity of catalase (CAT) occurred in the control than in other treatments, no differences were observed in the activity of superoxide dismutase (SOD). Higher fertilization and hatching rates occurred at 2.75 mM melatonin compared to 4.25 mM. Although no significant differences were observed in LPO between the control and samples supplemented with 2 mM melatonin, it was observed that this dosage of melatonin allowed higher VCL and VSL and reduced values of CAT. However, as there are no differences in motility and fertilization rates between the control and the 2 mM concentration, it is suggested that further studies be carried out with lower concentrations of melatonin in order to determine its effectiveness as an antioxidant in the sperm of this species.

Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70–75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

Natural Competence for Transformation in Lactobacili

Natural Competence for Transformation in Lactobacili

Enhanced Cellular Cryopreservation by Biopolymer-Associated Suppression of RhoA/ROCK Signaling Pathway.

With increasing demands on long-term storage of cells, cryopreservation of cells is gaining more importance in cell-based research and applications. Dimethyl sulfoxide (DMSO) is a commonly used chemical cryoprotectant, providing increased cell survival during the freezing process. However, its use is limited in clinical applications due to its low biocompatibility above cryogenic temperatures. Herein, we present a new approach for reducing the use of DMSO in cryopreservation by using biodegradable hyaluronic acids (HAs). By adding HAs into cryoprotectant media containing a low concentration of DMSO, higher cell viability and cell proliferation rate were observed upon thawing after cryopreservation. The HA-supplemented cryopreservation media did not reduce the size of the ice crystal, which significantly influenced cell viability during cell freezing, but decreased the Ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling pathway related to apoptosis. The cell-interactive cryoprotectants containing HA can be applied to the development of a new cryoprotectant that reduces the adverse effect of DMSO.

Role of Mono- and Disaccharide Combination in Cryoprotective Medium for Rooster Semen to Ensure Cryoresistance of Spermatozoa.

The combination of saccharides in the composition of a cryopreservation medium may represent a promising method for the preservation of the reproductive cells of male birds. In the current study, cryoprotective media with a combined composition of mono- and di-saccharides were developed. The degree of penetration of reducing saccharide molecules (maltose—Mal20 medium) and non-reducing disaccharide molecules (trehalose—Treh20 medium) from the cryoprotective medium into the cytosol of rooster spermatozoa was studied. LCM control media without disaccharides were used as the control. The number of maltose molecules penetrating from the outside into the cytosol of the spermatozoon was 1.06 × 104, and the number of trehalose molecules was 3.98 × 104. Using a combination of maltose and fructose, the progressive motility of frozen/thawed semen and the fertility rates of eggs were significantly higher ((p < 0.05) 40.2% and 68.5%, respectively) than when using a combination of trehalose and fructose in a cryoprotective diluent (33.4% and 62.4%, respectively). A higher rate of chromatin integrity at the level of 92.4% was obtained when using Treh20 versus 74.5% Mal20 (p < 0.05). Maltose positively affected the preservation of frozen/thawed sperm in the genital tract of hens. On the seventh day from the last insemination when using Mal20, the fertilization of eggs was 42.6% and only 27.3% when using Treh20. Despite the same molecular weight, maltose and trehalose have different physicochemical and biological properties that determine their function and effectiveness as components of cryoprotective media.

Ocular Delivery of Predatory Bacteria with Cryomicroneedles Against Eye Infection.

The development of potent antibiotic alternatives with rapid bactericidal properties is of great importance in addressing the current antibiotic crisis. One representative example is the topical delivery of predatory bacteria to treat ocular bacterial infections. However, there is a lack of suitable methods for the delivery of predatory bacteria into ocular tissue. This work introduces cryomicroneedles (cryoMN) for the ocular delivery of predatory Bdellovibrio bacteriovorus (B. bacteriovorus) bacteria. The cryoMN patches are prepared by freezing B. bacteriovorus containing a cryoprotectant medium in a microneedle template. The viability of B. bacteriovorus in cryoMNs remains above 80% as found in long‐term storage studies, and they successfully impede the growth of gram‐negative bacteria in vitro or in a rodent eye infection model. The infection is significantly relieved by nearly six times through 2.5 days of treatment without substantial effects on the cornea thickness and morphology. This approach represents the safe and efficient delivery of new class of antimicrobial armamentarium to otherwise impermeable ocular surface and opens up new avenues for the treatment of ocular surface disorders.

Influence of Xenon on Survival of Sperm of Common Frog Rana temporaria during Slow Freezing.

We studied the effect of xenon on the survival rate of the spermatozoa of the common frog Rana temporaria during slow freezing with saturation of the suspension with xenon at a pressure of up to 1.2 bar. The cryoprotective properties of xenon were analyzed in comparison with nitrogen. No specific cryoprotective effect of xenon was revealed. Viability of spermatozoa pretreated with xenon at atmospheric pressure (0 bar) or under excess pressure of 0.6 bar and frozen in a cryoprotective medium with dimethylformamide, sucrose, and BSA did not differ significantly. The use of overpressure of xenon of 1.0 or 1.2 bar in the pretreatment and freezing process significantly impaired viability of the biomaterial.

CRYOPRESERVATION OF MALE CARP REPRODUCTIVE CELLS

Currently, cryopreservation of fish reproductive cells is an urgent direction in the strategy of preserving genetic biodiversity, as well as the development of fisheries and aquaculture, scientific research was carried out in the research center "Fisheries" of the «S.Seifullin Kazakh Agro Technical University» and in the JSC "Republican Center for Breeding in Animal Husbandry "Assyl Tulik" in 2021. During the research, the method of cryopreservation of male carp reproductive cells using two types of cryoprotector based on 10% ethylene glycol and 10% dimethyl sulfide oxide was studied. During the experimental work, the generally accepted methods for conducting fish bonitization and freezing carp sperm were used. The assessment of the quality of carp sperm was carried out on a 5-point Persov scale. Also, when assessing the quality, the volume, concentration of sperm, the time of activity and mobility of carp sperm were taken into account. As a result of scientific research, it was found that the use of a cryoprotective medium consisting of 10% ethylene glycol has a positive effect in comparison with DMSO, since it reduces the osmotic and oxidative stress experienced by cells during freezing. The obtained results allow us to recommend adjusting the concentration of penetrating protectors in the cryoprotective solution depending on the amount of intracellular water to increase the survival rate of male fish reproductive cells after a double temperature shock. The results of the research make it possible to create a cryobank of the gene pool of carp breeding material at fish hatcheries to preserve the genetic diversity of these commercial objects.

Study of Interaction of Glycerol Cryoprotectant and Its Derivatives with Dimethylacetamide in Aqueous Solution Using Fluorescent Probes

In this paper we have studied the interaction of the mixtures of glycerol (GL) and its oxyethylated derivatives (OEG) with polymerization degree n = 3, 25 and 30 with dimethylacetamide (DMAc) in aqueous solution using 3-hydroxy-4´-(N, N dimethylaminoflavone) fluorescent probe. The combination of GL and its oxyethylated derivatives with DMAc was found to reduce the membranotropy of certain cryoprotective agents, forming a mixture. The combination of both GL and its low molecular weight derivative (OEGn=3) with DMAc reduced the membranotropy of the latter. At the same time, combining GL derivatives of high molecular weight (OEGn=25 and OEGn=30) with DMAc diminished the membranotropy of OEG. The OEGn=30 at concentrations above 1 wt.% was shown to form the micellar-type structures or micellar associates in aqueous solution. This enabled suggesting the membranotropic ability of high molecular weight OEG associates to be stipulated by possible interaction of their nonpolar segments with nonpolar sites on biomembrane surface. Structural rearrangements of molecular associates in aqueous solutions of low and high molecular weight cryoprotectant mixtures were designated as the experimentally established mechanism of cytotoxicity reduction in combined cryoprotective media.