Histological investigation of the slow freezing and melatonin effect on bos taurus embryos obtained through assisted reproductive techniques

Usage of assisted reproductive techniques (ART) in cattle livestock embryo production in vitro ensure the continuity of animal breeds with high yield capability. Many studies have been carried out by using slaughterhouse-derived oocytes from different animal species with different cryopreservation methods by using assisted reproductive techniques in vitro, so embryos can be stored for a long time and a high fertility rate is targeted. By working/experimenting with various animal species, different cryopreservation methods and applications, main goal of storing embryos for long periods with high proliferation is targeted. However, due to the exposure times to cryoprotectants and differences in their toxicity levels, embryos due to ice crystals that can form during freezing-warming, breakage in zona pellucida, damage to cell membranes and skeleton may occur in the embryo formation. The effects of melatonin after slow freezing on Bos taurus embryos obtained by ART in this study were investigated by histological methods to be able to attract the damages to small amounts. Oocytes obtained by follicle aspiration from ovaries acquired from healthy cows in Holstein (Bos taurus) genus 4-8 years old procured from slaughterhouse were taken into in vitro maturation process. Commercially purchased frozen bull sperm and IVF (In vitro fertilization) treated oocytes were stored in liquid nitrogen at -196 ° C for 1 day following melatonin supplementation and slow freezing. To examine the possible effects of cryopreservation by histological methods, the dissolved embryos after 1 day were observed under the light microscope with Azide II-Methylene Blue-Basic Fuchsin staining for lipid determination and Toluidine Blue staining for nucleus and organelle distributions. With hoescht staining for embryo viability and total cell count determinations were also examined by fluorescence microscopy. The control group embryos were found to have distinct, nearly equal size blastomeres with large subzonal areas, clear borders of surrounding zona pellucida and without any fragmentations. In slow freezing group embryos; Disorganization, fragmentation-like occurrences and/or degenerations have been observed at the blastomeric borders. In the embryos of the slow freezing group, vacuolarizations in the cytoplasm, intercellular wastes in the interblastomer and subzonal spaces were determined and scattered lipid droplets of different size and numbers were observed in the blastomeres. By comparing melatonin application and non-melatonin application groups; Embryo cleavage and blastocyst progression rates after slow freezing were investigated and numerical data were presented. Viability rate data were determined by fluorescence microscope. It has been thought that the addition of antioxidant melatonin at the appropriate dose and amount into the culture medium may be beneficial for embryo development to reduce the irreversible degradation of the membrane permeability due to the damage caused by the ROS (Reactive Oxygen Species) products occuring during IVF and slow freezing processes. Further research is needed to produce more data and achieve even more reliable results.