The aim of this study was to create balanced media for the cryopreservation of rooster semen in pellets to maintain the functional state of the sperm after thawing. Fructose was replaced by trehalose in experimental media in proportions of 10% (LCM-T10) and 20% (LCM-T20), while LCM was used as a control. After artificial insemination of the hens, the eggs were incubated (n = 400). To determine the functional safety of spermatozoa in the genital tract of hens after 5, 10, and 15 days from the last insemination, we used a method for assessing the interaction of sperm with the perivitelline membrane. Significantly higher rates of egg fertilization (82–86%) were obtained when using LCM-T10 and LCM-T20 compared to control (79%, p < 0.05). Egg fertility on the 5th day from the last insemination with the LCM-T20 diluent reached 100% versus 86% in the control; on the 10th day, the fertility rates were 55% versus 20%, respectively. The best results for fertility duration were obtained by freezing spermatozoa with LCM-T20 medium. The numbers of interaction points of spermatozoa with the perivitelline membrane were as follows: on the 5th day from the last insemination with LCM-T20—461.5 ± 11.5 holes/cm² (LCM-control—13.7 ± 2.7 holes/cm²), p < 0.01; on the 10th day with LCM-T20—319.3 ± 12.9 holes/cm² (LCM-control—14.9 ± 3.5 holes/cm²); and on the 15th day with LCM-T20—345.2 ± 11.1 holes/cm² (LCM-control—0 holes/cm²). In conclusion, the use of trehalose in LCM diluent medium can increase the fertility of frozen/thawed sperm and the duration of their fertility in the genital tract of hens.

Relevance. Identification and analysis of the reasons for reducing egg productivity is an urgent problem in quail breeding. The aim of this study is to assess the destructive processes in the somatic cells in ovarian follicles (the level of apoptosis and pycnosis) of mature quail layers of different productivity in connection with egg production and quality indicators of eggs.Methods. In experiments, the level of apoptosis and pyknosis in granulosa cells of ovarian follicles of 3–8 mm females of Estonian, Manchurian, English white, tuxedo, English black and Pharaoh breeds was evaluated. The quails were kept in individual cages in monopreed pairs with a muted 14-hour light day. The age of the females was 160–180 days. The level of pycnotic cells was assessed by the Tarkowski cytological method, apoptotic processes were analyzed by staining with fluorochromes, propidium iodide and acridine orange (10 mcg/mlPI/AO).Results. chromatin. The results of the study of chromatin destruction using PI/AO showed a high average percentage of apoptotic cells in almost all breeds except thep < 0.05). The maximum proportion of dead granulosa cells was tested in Tuxedo and English Black layers (52% and 43% respectively). At the same time, the English white breed of quails at the age of 183 days has one of the best indicators of egg production and the proportion of yolk in relation to egg weight, which indicates the possibility of prolonging the period of their use as laying hens.

Reproductive technologies are some of the key directions in the context of the need to preserve and select highly productive farmed animals in terms of economically useful traits. Improvements of the existing models of the in vitro oocyte maturation system help to solve the problem of low yield of porcine embryos at the final stages of preimplantation development. In the present study, a model of culture medium for gametes (NCSU-23 with 10 % homologous follicular fluid, 10 IU hCG and 10 IU eCG) modernized by the addition of 1·10⁶ granulosa cells (GCs) per ml and 0.001 % of highly dispersed silica nanoparticles (HDSn) is proposed for use in the IVM and IVF technology of donor porcine oocytes. Analysis of the oocyte chromatin status by the Tarkowsky method and assessment of the level of destructive changes in chromatin (apoptosis, pyknosis) revealed a significant percentage increase in matured oocytes and a decrease in the proportion of granulosa cells with degenerated chromatin when using the original culture system. The positive effects of a joint addition of GCs and HDSn to the maturation system have made it possible to increase the indicators of the meiotic maturation and fertilization of oocytes. Optimal results of developmental competence of oocytes were achieved with the joint use of GCs and HDSn in the maturation system (the proportion of matured cells reached 89 %, the level of oocytes with chromosome degeneration was 12 %, 39 % of embryos reached the final stage of preimplantation development). The positive effect of HDSn on oocyte fertilization was accompanied by an abrupt decrease in destructive processes in GCs during culture in the presence of HDSn. The level of somatic cells with pyknotic nuclei was 32 % and the level of apoptosis (TUNEL-test), 21 %, compared with the control (43 and 31 %, p <0.01, respectively). Thus, a high efficiency of the porcine oocyte maturation system in the joint culture of gametes with GCs and HDSn was revealed. It makes it possible to recommend a model of this culture medium at the IVM of female gametes of Sus scrofa domesticus for improving the quality of donor oocytes used in cell and genetic engineering.

The effect of titanium tetrapolyethylene glycolate dissolved in polyethylene glycol (ratio 1:10) (abbreviated TTP_EG*10PEG) on granulosa cells (GC) of the antral ovarian follicles of Sus scrofa domesticus in the dynamics of in vitro culture was assessed. Granulosa cells aspirated from post mortem ovarian porcine follicles (ø 3-5 mm) obtained at the Tosnensky Meat Processing Plant, were cultured without and in the presence of 0.1, 0.01 and 0.001 % TTP_EG*10PEG. After 22 and 44 hours, the GC were analyzed by flow cytometry. A comprehensive analysis of viability indicators (mitochondrial activity, apoptosis), as well as the generation of reactive oxygen species (ROS) in the GC after exposure to TTP_EG*10PEG, revealed that the addition of 0.1 % TTP_EG*10PEG to the culture medium after 22 and 44 hours causes disturbances in the functional activity of the GC, accompanied by a decrease of mitochondrial membrane potential compared to control (by 8 and 9 %, respectively, p<0.05) and cell death (the proportion of cells in apoptosis was 45 and 41 %, respectively, p<0.001). In the groups cultured in the presence of 0.01 % and 0.001 % TTP_EG*10PEG, no significant differences in the level of GC in the state of apoptosis were detected when cultured for either 22 or 44 hours. At the same time, an increase in mitochondrial activity was shown in these groups in comparison with the control and the group containing 0.1 % TTP_EG*10PEG (by 11 and 13 %, respectively, after 22 hours, p<0.001 and by 15 and 27 % after 44 hours, p< 0.001). No significant differences were found between the study groups in the rate of ROS generation in cells. In general, a dose-dependent negative effect of TTP_EG*10PEG (0.1 %) was identified. TTP_EG*10PEG at concentrations of 0.01 and 0.001% did not have a destructive effect on the studied cell population, which indicates the possibility of using TTP_EG*10PEG at the above concentrations (0.01 and 0.001 %) to simulate the composition of media used in porcine granulosa cells culture systems.

Searching biomarkers, that have an early prognostic trait and determinate functional status of male gametes Gallus Gallus is a promising direction for improving ejaculate’s quality reproductive characteristics. In context of a decreasing reproductive performance due to breeding selection of birds aimed to improving economically useful traits, this approach to selecting males, based on their sperm quality production will make it possible to evaluate sperm in a short time and identify potentially the best producers at early stages. The study determined possibility of using exDNA as a potential biomarker that determines quality indicators of native sperm obtained from roosters. Significant correlations were revealed between exDNA and proportion of cells with damaged plasma membrane (r = 0.35, p<0.05), which indicates a possible connection of this biomarker with necrotic and apoptotic processes occurring in cells. Variation coefficient of exDNA indicator was 30.28 %, demonstrating the possibility of using the proposed biomarker (exDNA concentration in the seminal fluid of Gallus Gallus) as a predictive criterion for assessing quality of male gametes. Significant correlations (r = 0.51, p<0.05) were established between proportion of cells exposed to oxidative stress, proportion of cells with damaged plasma membrane and high mitochondrial activity, which indicates negative impact of reactive oxygen species on the plasma membrane integrity and relationship increased generation of reactive oxygen species with functional status of mitochondria. On the basis of the analysis of research results, the indicator “exDNA content in seminal fluid” should be considered as a preventive non-invasive biomarker of the functional state of male gamete Gallus Gallus.

One of the ways to improve efficiency of artificial insemination in poultry is early selection of males producing sperm with high cryoresistant properties. Initial ejaculate evaluation plays an important role in predicting viability and can serve as basis for developing quality criteria for decryopreserved semen. In our study, which involved roosters (n=139) from 20 breeds and populations, we identified prognostic indicators that allow us to draw conclusions about the functional activity of decryopreserved sperm based on native ejaculate parameters. At sperm concentration of 3.5 billion/ml in native ejaculate after cryopreservation, was observed the highest percentage of gametes with high kinetic parameters (38.12±1.68%). Maximum percentage of decryopreserved spermatozoa with high rates of total motility (37.05±1.03%) was obtained in the case of freezing native sperm samples with total motility above 85%.