Modeling of oocyte maturation systems in farm animals is based on the available information on the composition of follicular fluid (FF), however, there are no data on the hormonal status of porcine follicular fluid in relation to the viability and functional state of the lipidome in granulosa cells (GCs) in the dynamics of folliculogenesis. We analyzed the hormonal profile (LH, FSH, T3, T4, cortisol) of porcine antral follicle fluid and obtained data on the effect of these hormones on the viability and lipid content (lipid droplets-LDs) of porcine GCs in follicles of different diameters (ø <3 mm, ø 3-5 mm and ø>5 mm). An inverse correlation was found between the proportion of GCs with high fluorescence intensity of the lipid droplet complex – NileRed (IFNileRed/LD - a marker of lipid droplets content) and the concentration of cortisol in the dynamics of folliculogenesis (-0.830, p<0.05), as well as an inverse correlation between the cortisol content and the proportion of viable cells (- 0.995, p<0.05). A direct correlation was found between the content of both T3 and T4 with IFNileRed/LD (0.901, 0.946, p<0.05) in GCs, which probably suggests accumulation of the energy substrate in the cytosol. There were no reliable correlation values in the content of LH in the FF and the proportion of GCs with high IFNileRed/LD intensity. The high level of correlation in the concentration of T3, T4 in the FF and cortisol with the lipid’s content in the GCs indicates a significant influence of these hormones on the energy reserve and viability of the GCs during folliculogenesis and allows us to consider them as potential candidates for an indepth study of the mechanisms of metabolic disorders that provoke abnormalities in the development of offspring obtained at in vitro maturation and fertilization of female gametes, as well as for modeling the composition of the media for the formation of a mature porcine ovum in vitro .

У клинически здоровых коров голштинской породы (n=20) изучена динамика гормонального обмена в период глубокой стельности и после отела. У них в сыворотке крови определяли концентрацию кисспептина, 17β-эстрадиола и прогестерона. За животными вели регулярное наблюдение с проведением клинических и акушерско-гинекологических исследований. В зависимости от концентрации глюкозы и бета-гидроксимасляной кислоты на 5-й день после отела коров разделили на 2 группы: в I группу вошли 15 особей с концентрацией в крови глюкозы больше 3 ммоль/л и уровнем бета-гидроксимасляной кислоты ниже 1 ммоль/л. Во IIгруппе у 5 коров зафиксированы нарушения в энергообмене (глюкоза меньше 3 ммоль/л, и уровень бета-гидроксимасляной кислоты выше 1 ммоль/л). На 15-й день после отела была отмечена нормализация энергетического гомеостаза. Концентрация кисспептина в предотельный период в обеих группах снижалась к отелу. Его количество в крови животных в группе с нарушениями энергообмена было выше на 9, 7-й и 5-й дни до отела в 2,4—3 раза (P<0,01—0,05), по сравнению с I группой. Концентрация 17β-эстрадиола в предотельный период снижалась в обеих группах. Более высокое количество перед отелом зафиксировано во IIгруппе — в 2—3,2 раза больше, по сравнению с I групой (P<0,05). Уровень прогестерона к моменту отела повышался в крови животных обеих групп. Концентрация трех гормонов после отела резко снизилась. В дальнейшем существенных различий в концентрации кисспептина не зафиксировано, кроме 5-го дня после отела (P<0,05). Уровень 17β-эстрадиола в послеотельный период между группами достоверно различался в 1, 3, 5-й и 7-й дни (P<0,05). Более высокая концентрация после отела зафиксирована во II группе — в 2—2,4 раза. Уровень прогестерона в послеотельный период начал повышаться в I группе с 17-го дня после отела, а во II— с 11-го дня. Достоверные различия зафиксированы на 15, 17, 19-й и 21-й дни после отела (P<0,05). Полученные экспериментальные данные показывают, что кисспептин играет важную роль в адаптации к нарушениям в энергетическом обмене, которые наиболее явно проявляются у высокопродуктивных молочных коров во вторую половину транзитного периода при появлении отрицательного энергетического баланса. Дальнейшее уточнение концентраций кисспептина при фиксации уровня стероидных гормонов позволит раскрыть генез многих нарушений репродуктивной функции при отрицательном энергетическом балансе и его негативных последствиях. The dynamics of hormonal metabolism during deep pregnancy and after calving was studied in clinically healthy Holstein cows (n=20). The concentrations of kisspeptin, 17β-estradiol and progesterone were determined in their blood serum. The animals were regularly monitored with clinical and obstetric-gynecological studies. Depending on the concentration of glucose and beta-hydroxybutyric, acid, on the 5th day after calving, cows were divided into 2 groups: Group I included 15 animals with a blood glucose concentration of more than 3 mmol/l and a beta-hydroxybutyric acid level below 1 mmol/l. In group II, 5 cows showed disturbances in energy metabolism (glucose less than 3 mmol/l, and the level of beta-hydroxybutyric acid above 1 mmol/l). On the 15th day after calving, normalization of energy homeostasis was noted. The concentration of kisspeptin in the pre-calving period in both groups decreased towards calving. Its amount in the blood of animals in the group with energy metabolism disorders was higher on the 9th, 7th and 5th days before calving by 2.4—3 times (P<0.01—0.05), compared with group I. The concentration of 17β-estradiol in the prepartum period decreased in both groups. A higher concentration before calving was recorded in group II — 2—3.2 times (P<0.05). The level of progesterone at the time of calving increased in the blood of animals of both groups. The concentrations of the three hormones decreased sharply after calving. Subsequently, no significant differences in kisspeptin concentration were recorded, except on the 5th day after calving (P<0.05). Postpartum 17β-estradiol levels were significantly different between groups on days 1, 3, 5, and 7 (P<0.05). A higher concentration after calving was recorded in group II — 2—2.4 times. The level of progesterone in the post-calving period began to increase in group I from the 17th day after calving, and in group II — from the 11th day. Significant differences were recorded on the 15th, 17th, 19th and 21st days after calving (P<0.05). The experimental data obtained show that kisspeptin plays an important role in adaptation to disturbances in energy metabolism, which are most clearly manifested in highly productive dairy cows in the second half of the transit period when a negative energy balance appears. Further clarification of kisspeptin concentrations when fixing the level of steroid hormones will reveal the genesis of many reproductive dysfunctions with a negative energy balance and its negative consequences.

The effect of titanium tetrapolyethylene glycolate dissolved in polyethylene glycol (ratio 1:10) (abbreviated TTPEG*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 % TTPEG*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 TTPEG*10PEG, revealed that the addition of 0.1 % TTPEG*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 % TTPEG*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 % TTPEG*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 TTPEG*10PEG (0.1 %) was identified. TTPEG*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 TTPEG*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.

In the clinically healthy cows of the Holstein breed (n = 20), a hormonal background was studied during theperiod of deepness and after the edema. For this purpose, the concentration of kisspeptin, testosterone and cortisol was determined in serum cows in the blood cows. The animals conducted regular observation with the conduct of clinical and obstetric and gynecological studies. Depending on the concentration of glucose and beta-hydroxybutyric acid on the 5th day after calving, the cows were divided into two groups. The first group included 15 heads with a glucose concentration greater than 3 mmol/l and a beta-hydroxybutyric acid level below 1 mmol/l. In the second group, 5 heads had violations in energy exchange (glucose levels less than 3 mmol/l, beta-hydroxybutyric acid above 1 mmol/l). On the 15th day after the hotel, normalization of energy homeostasis was recorded. The concentration of kisspeptin in the preventive period in both groups decreased to the hotel. At the same time, the concentration of kisspeptin in the blood of animals in the group with energy exchange disorders was 2.4–3 times higher on the 9th, 7th and 5th days before calving (p < 0.01–0.05) compared with the first group. In production experience, the concentration of all three hormones after the hotel decreased with significant intergroup differences. The level of kisspeptin was reliably higher in the preventive period in the experimental group and on the 5th day after the hotel (p < 0.05). The cortisole level was held at a high level of up to 7 days after the hotel (p < 0.05). The concentration of testosterone in the blood throughout the transit period was reliably high in the animals of the experimental group.

In clinically healthy Holstein cows (n=25), the dynamics of hormonal metabolism in the first trimester of cow pregnancy was studied. For this purpose, the concentration of kisspeptin, 17β-estradiol, progesterone and cortisol was determined in the blood serum of cows. The animals were regularly monitored with clinical and obstetricgynecological studies. The level of kisspeptin in the first trimester of pregnancy increased almost throughout the entire period under consideration with a decrease in concentration to 194.23±23.61 pg/ml on day 98. The level of estradiol during the first trimester increased from 49.66±9.68 to 76.20±17.84 pg/ml, respectively, which is also explained by the inclusion of the fetoplacental complex in its synthesis and secretion. Progesterone and cortisol levels were on average at the same level. During the experiment, out of 25 animals, embryonic death was recorded in 14 on the 30th day after insemination. As a result, two groups were formed: the 1st group - animals with a normal course of pregnancy and the 2nd group with embryonic death. Analyzing the data of enzyme immunoassay in animals of the 1st group, one can note a decrease in the level of kisspeptin by 11% on the 28th day compared to the 14th day after insemination. At the same time, the concentration of estradiol, on the contrary, increased by 29%. In the second group, on the contrary, the level of kisspeptin and estradiol did not change and, on average, remained almost at the same level. On the 28th day after insemination in the first group, the cortisol concentration was significantly higher compared to the second group - 15.32±3.23 and 9.00±0.89 nmol/l, respectively. Data for kisspeptin and estradiol showed no significant differences. However, it is of interest that in the first group, with an increase in the level of estradiol, a decrease in the level of kisspeptin occurs. In particular, this is consistent with previous studies, according to which such an interaction of these hormones can be regulated by a high concentration of progesterone.

У клинически здоровых коров голштинской породы (n=10) изучена динамика гормонального обмена в лютеиновую фазу полового цикла. С этой целью у коров в сыворотке крови определяли концентрацию кисспептина, 17β-эстрадиола и прогестерона. За животными вели регулярное наблюдение с проведением клинических и акушерско-гинекологических исследований. В зависимости от индекса осеменения коров разделяли на две группы: 1-ая группа: с индексом осеменения более 1 и 2-ая группа — равным 1. Уровень прогестерона в ходе лютеиновой фазы увеличивался, и концентрация в исследуемых группах практически совпадала: в 1-й группе — 8,56±0,92, во 2-ой — 8,94±0,74 нг/мл. Уровень 17β-эстрадиола в группе животных с индексом осеменения более 1 был ниже в сравнении со 2-ой группой — на 10-ый день лютеиновой фазы зафиксировано достоверное различие (P<0,05). Уровень кисспептина показал противоположную динамику. В группе коров с индексом осеменения 1 уровень кисспептина был достоверно ниже на 12-ый и 16-ый дни лютеиновой фазы (P<0,05). Наши исследования показывают схожую динамику по кисспептину и 17β-эстрадиолу в каждой группе животных. При этом в определенные дни лютеиновой фазы наблюдалось расхождение — при повышении уровня 17β-эстрадиола понижалась концентрация кисспептина и наоборот. В группе животных с индексом осеменения более 1 это отмечено с 8 по 12 день лютеиновой фазы, во 2-ой группе — с 10 по 14 день. Затем динамика вновь становилась схожей. Причина такого разобщения пока неясна и требует дополнительных исследований. In the clinically healthy cows of the Holstein breed (n = 10), the dynamics of hormonal metabolism in the luteal phase of the estrous cycle have been studied. To this end, the concentration of kisspeptin, 17β-estradiol and progesterone was determined in serum cows in serum cows. The animals conducted regular observation with the conduct of clinical, obstetric, and gynecological studies. Depending on the insemination index, the cows were divided into two groups: the 1st group: with the insemination index of more than 1, and the 2nd group with the insemination index 1. The level of progesterone during the luteal phase increased, and concentration in the studied groups almost coincided: in the first group - 8.56 ± 0.92, in the second - 8.94 ± 0.74 ng/ml. The level of 17β-estradiol in the group of animals with an insemination index of more than 1 was lower in comparison with the second group-on the 10th day of the lutein phase, a reliable difference was recorded (p <0.05). The level of Kisspeptin showed the opposite dynamics. In a group of cows with an insemination index 1 level of kisspeptin was reliably lower for the 12th and 16th days of the luteal phase (p <0.05). Our studies show similar dynamics by kisspeptin and 17β-estradiol in each group of animals. At the same time, on certain days of the lutein phase, a discrepancy was observed-with an increase in the level of 17β-estradiol, the concentration of kisspeptin and vice versa. In a group of animals with an insemination index of more than 1, this is noted from the 8th to 12th day of the lutein phase, in the second group - from 10 to 14 days. Then the dynamics again became similar. The reason for such a division is still unclear and requires additional research.

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 the p < 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.

Functioning of the granulosa cells of animal ovarian follicles (OF) determines the growth and development of the OF and the oocyte formed in it, which determines their use in modeling the composition of media for oocyte maturation in vitro in cell reproductive technologies. The purpose of the study is to determine the peculiarities of functional activity of granulosa cells in follicles of different diameters (< 3, 3–5, 5–8 mm) using complex testing of granulosa cell viability parameters (apoptosis level, production of reactive oxygen species, functional activity of mitochondria and lipidome) and hormonal status of follicle fluid (estradiol, testosterone, anti-Müllerian hormone level) in order to optimize the composition of the culture media for in vitro maturation of pig oocytes. The experiments found that the proportion of apoptotic cells in the OF increased as the diameter of the OF increased (by 11% in OFs 5–8 mm in diameter, by 4% in OFs < 3 mm in diameter). There was an increase in the number of high reactive oxygen species cells (by 10% in 5–8 mm diameter OF compared with < 3 mm diameter OF) and high mitochondrial activity (by 12% in 5–8 mm diameter OF compared with < 3 mm diameter OF). Correlations were found between the group of cells with high generation of reactive oxygen species and the level of apoptosis (0.500, p < 0.05), as well as with high levels of mitochondrial activity (0.500, p < 0.05). Estradiol concentration in follicular fluid increased as the OF grew (1.50 ng/ml in OF < 3 mm diameter and 2.09 ng/ml in OF 3–5 mm diameter, p < 0.01), being almost unchanged in OF 5–8 mm diameter (2.11 ng/ml), as was the testosterone concentration. Correlations were found between the proportion of the cells with high NileRed/LD FI (a marker of lipid droplet functional activity) and the levels of estradiol (0.998, p < 0.05), testosterone (0.500, p < 0.05), and anti-Müllerian hormone (–0.500, p < 0.05) in the dynamics of folliculogenesis, as well as between estradiol concentration and the proportion of cells with high mitochondrial activity (0.746, p < 0.05). Correlations were detected between the concentrations of estradiol and anti-Müllerian hormone, testosterone and anti-Müllerian hormone (–1.000, p < 0.05), between the level of apoptotic cells and the concentration of anti-Müllerian hormone in follicular fluid (–1.000, p < 0.05). The results of the monitoring of the viability and functional activity of porcine OF granulosa cells supplement the available data on the peculiarities of their functioning in the dynamics of folliculogenesis and can be used in modeling the systems of in vitro maturation of donor oocytes of Sus Scrofa Domesticus .

The features of the functioning of native and devitrified porcine granulosa cells at prolonged culture have characterized. Ultra-low temperatures cause an increase in the level of generation of reactive oxygen species and the proportion of apoptotic granulosa cells. The addition of 0.001% nanoparticles of highly dispersed silica in the composition of cryoprotective and culture media reduces the level of apoptotic cells.

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 use of the indicator of membrane-bound calcium content (fluorescence intensity of the complex calcium + chlortetracycline + membrane) as a biomarker of the functional state (growing or fully grown oocytes) of donor oocytes in the technology of extracorporeal maturation of female gametes Sus scrofa domesticus is substantiated. The addition of highly dispersed silica nanoparticles with granulosa cells to culture media at in vitro maturation promotes the mobilization of calcium ions from intracellular stores of oocytes that have not finished the growth phase in vivo.