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.

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.

The plasma membrane of spermatozoa plays an important role in the formation and maintenance of many functions of spermatozoa, including during cryopreservation. As a result of chromatographic analysis, the content of lipids and fatty acids in the membranes of spermatozoa of roosters of two breeds was determined under the influence of cryoprotective media containing trehalose LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). The use of the cryoprotective diluent Treh20 made it possible to maintain a dynamic balance between the synthesis and degradation of phospholipids and sterols in the plasma membranes of frozen/thawed spermatozoa, close to that of native spermatozoa. This contributed to an increase in the preservation of frozen/thawed spermatozoa membranes from 48.3% to 52.2% in the egg breed and from 30.0% to 35.1% in the meat- and-egg breed. It was also noted that their kinetic apparatus (mobility indicators) remained at the level of 45.6% (egg breed) and 52.4% (meat-and-egg breed). An increase in the concentration of trehalose to 13.4 mM in a cryoprotective diluent for rooster sperm resulted in a decrease in the morphofunctional parameters of frozen/thawed spermatozoa.

Low-temperature semen storage technologies are already being used in poultry conservation programs, but the quality of reproductive material stored in cryobanks varies greatly and cannot always be successfully used for practical purposes. Therefore, it is necessary to improve the compositions of cryoprotective media to improve their quality. This study aimed to investigate the composition of membrane lipids and carbohydrates in the cytosol of rooster spermatozoa, to explain the dose-dependent effect of a combination of trehalose and fructose in cryoprotective media on the preservation of their morphological and kinetic parameters during freezing/thawing, and to determine the most effective diluent composition. Ejaculates were collected from Rhode Island Red roosters (n = 10). The effectiveness of three diluents containing trehalose was evaluated: LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). Chromatographic analysis of membrane lipids, carbohydrates, and polyols of the spermatozoa cytosol was performed. A decrease in the content of glycolipids in the plasma membranes of spermatozoa from 2.0% in native spermatozoa to 1.1–1.4% (frozen/thawed) and phospholipids from 71.2% (native) to 70.5% (frozen/thawed) reduced the progressive sperm motility from 65.7% in native spermatozoa to 12.6–27.6% (frozen/thawed). The same dynamics were observed for the viability parameter of 90.4% (native) and 27.0–41.2% (frozen/thawed). The Treh20 diluent, using a combination of fructose (36 mM) and trehalose (9.5 mM) saccharides, maximally preserved the lipid profile of plasma membranes and the composition of the cytosol of frozen/thawed rooster spermatozoa, which positively affected the indicators of general and progressive mobility and viability.

Цель: изучение влияния уровня активных форм кислорода в нативных сперматозоидах петухов на качественные показатели свежеразбавленных и деконсервированных индивидуальных эякулятов (подвижность, повреждение клеточных мембран спермиев) и определение допустимого уровня генерации АФК для совершенствования протокола криоконсервации. Материалы и методы. Объектом исследования являлись петухи породы род-айланд красный (n= 20) в возрасте 44 недели жизни. Все самцы содержались в индивидуальных клетках при принятой в БРК «Генетическая коллекция редких и исчезающих пород кур» ВНИИГРЖ системе кормления, поения и светового режима. Сперму получали в пенициллиновые флаконы объемом 10 мл при помощи метода абдоминального массажа. Измеряли объем каждого индивидуального эякулята, проводили оценку подвижности сперматозоидов, концентрацию. Криоконсервация производилась в гранулах. Оттаивание гранул проводилось при t 60°С в щелевом оттаивателе. Поврежденность плазматических мембран сперматозоидов в нативном и деконсервированном семени оценивали при помощи метода суправитальной окраски по Блюму. Сперматозоиды с поврежденными мембранами окрашивались в красный цвет, интактные клетки оставались белыми (бесцветными). В каждом препарате оценивалось не менее 200 клеток. Для определения уровней генерации АФК в сперматозоидах петухов использовали метод, основанный на люминол-пероксидазной хемилюминесценции, которую измеряли на хемилюминометре Lum-1200. Время проведения каждого измерения составляло 3 часа, основываясь на кривой хемилюминисценции активных форм кислорода (учитывая рост показателя, пик и спад). Концентрация клеток (7х106 кл./мл) для проведения измерений подбиралась экспериментальным путем по результатам серии предварительных экспериментов. Результаты. В результате исследования впервые получены данные, основанные на методе люминолпероксидазной хемилюминесценции по допустимому уровню АФК в нативной сперме петухов. Установлен диапазон активных форм кислорода (от 75 до 249 Вольт*сек), при котором клетки не получают значительных повреждений мембранных структур при криоконсервации. При превышении порога равного 250 Вольт*сек количество клеток с поврежденными мембранами резко увеличивается с 17,19% до 62,87%. Полученные данные позволяют проводить оценку и отбор петухов по качеству их спермы для целей криоконсервации и формирования криобанков репродуктивных клеток.

Result of the work, lipid profile (triglycerides (TG), low-density and low-density lipoproteins (LDL, HDL), cholesterol), total protein, macroelements (calcium, phosphorus) in follicular fluid, dynamics of growth and development ovarian follicles different diameters (5 mm) of pigs are determined. A violation of TG health was revealed with a decrease in follicles with a diameter of 3‒5 mm (at 0.044 mmol/L; P<0.05). Also, the increase in HDL was noted, least according to follicle growth: from <3 mm to 3‒5 mm ‒ by 0.145 mmol/L (P5 mm ‒ by 0,236 mmol/L (P<0.001), while the concentration of LDL was determined by the completion follicle growth (from 5 mm) by 0.066 mmol/L (P<0.05). Concentration total protein, glucose and gradually increased according to minimum increase in follicles from <3 to 3-5 mm by 2.181 g/L (P<0.05), 1.313 mmol/L (P<0.05) and 0.279 mmol/L (P<0.001), respectively. At the same time, phosphorus value in relation to follicles with a diameter of ˃5 mm decreased by 0.699 mmol/L (P<0.05). The results studies show a change in concentration of follicular fluid components, changing depending on stage of follicle development, which can be used in future to detect composition of nutrient medium for in vitro cell culture and duration of oocytes in pigs.