Assessment of the cytological integrity and functional usefulness of thawed rooster (Gallus gallus domesticus L.) spermatozoa under the influence of antioxidant components of cryoprotective media – inositol and zinc oxide nanoparticles (ZnO NP)

Damage to spermatozoa during the freeze-thaw cycle leads to reduced functionality and a decrease in the number of offspring produced. Therefore, ensuring the preservation of cytological integrity of spermatozoa is a prerequisite for the practical use of cryopreservation in poultry reproduction. In the presented work it is shown for the first time that when ZnO nanoparticles (NP) (concentration 50 µg/100 ml) are used in the composition of cryoprotective media for freezing the semen of roosters, nanoparticles can have a contraceptive effect and reduce the functionality of thawed spermatozoa, and the advantage of using inositol in the composition of cryoprotective media is proved. The aim of this work was to evaluate the effect of antioxidants of natural origin (inositol) and/or NP ZnO in cryoprotective media on the structural and functional state of frozen-thawed rooster spermatozoa. Studies were carried out in 2023 on roosters ( Gallus gallus domesticus L.) of Russian White egg laying chicken breed (n = 23) at the age of 39-44 weeks from the bioresource collection Genetic Collection of Rare and Endangered Breeds of Chickens (RRIFAGB, St. Petersburg). Selection of roosters by seed quality was carried out in accordance with GOST 27267-2017 (M., 2017). The volume of ejaculate (ml) was measured with a graduated pipette, sperm concentration (billion/ml) was measured using a photometer (Accuread Photometer, IMV Technologies, UK), and total motility (%) was measured using a Micromed MS-12 microscope (Ningbo Sheng Heng Optics & Electronics Co Ltd., China) at a magnification of ×100. The obtained ejaculates were pooled and divided into 4 equal aliquots (according to the number of diluents), then diluted with cryoprotective media in a 1:1 ratio. The following cryoprotective media were used: LCM (control), Mal20, Mal20 + Inositol (inositol content 11.25 mmol), Mal20 + ZnO NPs (NP size 14 nm; 50 μg). Frozen semen samples were stored in Dewar vessels for at least 55 days. Total and progressive sperm motility were analysed using the CASA ArgusSoft-Poultry imaging system (ООО ArgusSoft, Russia). Viability was assessed by staining with eosin-nigrosin (EN) dye and visualised on a Motic BA410E phase-contrast microscope (Motic, China) at a magnification of ×1000. The acrosome integrity of spermatozoa from native and frozen-thawed semen was determined. V-AF488 flow cytometry kit (ООО Lumiprobe RUS, Russia) was used to assess live cells and cells in the state of apoptosis. The mitochondrial marker Mito TMRE (tetramethylrhodamine, ethyl ester; ООО Lumiprobe RUS, Russia) was used on a Cytoflex flow cytometer (Beckman Coulter, Inc., USA) to stain active mitochondria and measure the mitochondrial membrane potential in living cells. The H2DCFDA (2¢,7¢-dichlorodihydrofluorescein diacetate) flow cytometry kit (ООО Lumiprobe RUS, Russia) was used to determine the content of intracellular ROS (H2O2) in live spermatozoa. The fluorescence intensity of thawed spermatozoa was determined using the Flow Cytometry Analysis platform (https://floreada.io/). Fertility of frozen-thawed semen was evaluated by artificial insemination of hens. Three groups of hens (at least 15 hens in each group) were formed, inseminated 3 times according to the scheme: two consecutive days and after 1 day. Semen frozen in LCM (control), Mal20 + Inositol, Mal20 + NP ZnO was used for insemination. The use of Mal20 + Inositol medium increased the viability of thawed spermatozoa by ~ 4 % (p function show_eabstract() { $('#eabstract1').hide(); $('#eabstract2').show(); $('#eabstract_expand').hide(); }