CONCENTRATION OF CONNECTIVE TISSUE BIOPOLYMERS OF CERVICAL MUCUS OF COWS AT DIFFERENT STAGES OF THE ESTRUS CYCLE AND ANAPHRODISIA

Abstract

Timely detection of estrus in cows is the key to successful artificial insemination in Ukrainian farms. Until now, various methods of detecting the immobility reflex are used, including visual control of the condition of the external genitalia against the background of changes in behavior, contact with a test bull, quantitative and qualitative changes in cervical mucus. The condition of the cervical mucus is the most informative, since its physical and chemical changes do not go unnoticed when using any methods of diagnosing estrus. Despite this fact, until now many characteristics of cervical secretion remain unexplored, especially biochemical and physicochemical indicators at different stages and states of reproductive function. In order to study the regenerative and restorative processes and to diagnose infertility, the dynamics of the concentration of hexoses conjugated with protein, glycosaminoglycans and glycoproteins of the cervical mucus of the uterine herd of cows of experimental farms during different stages of the estrous cycle and during anaphrodisiacs were studied. The research was conducted on cows of the black-spotted and Swedish breeds, as well as on cows of the Simmental and brown dairy breeds. The material for research was cervical mucus, collected from the cervical canal of the uterus of cows aged 3-10 years, during estrus, the flowering of the corpus luteum, expected estrus, and from clinically healthy cows that did not show sexual cyclicity after contracting endometritis and delaying litter for more than 30 days To collect cervical mucus, a sanitary toilet of the external genitalia of cows was performed. A sterile vaginal speculum was inserted into the vagina, after which, using an additional light source, mucus was collected from the caudal part of the cervical canal using a sterile syringe and a catheter attached to it. After collection, the mucus was frozen for storage at a temperature of -20°C. Before the research, mucus samples were thawed at room temperature, homogenized with 0.5 N NaOH solution and left at +4◦С for 2 hours. Further, the homogenate was centrifuged at 3000 rpm. within 15 minutes The actual content of protein-carbohydrate compounds in samples of mucus homogenates was calculated taking into account the ratio (volume of mucus - 0.5 N NaOH solution). In the supernatant, the content of hexoses combined with proteins, glycosaminoglycans and glycoproteins was determined by the fractional method in the orcinol reaction with fractionation with ethanol and cetylpyridine chloride. Photometry of the samples was carried out at 540 nm with an optical path length of 10 mm. The actual content of the test compounds is expressed in g/l or mg/l according to the calibration graph that shows the dependence: optical density is the concentration of hexoses. Principle of the method: proteins due to hexoses are precipitated in the presence of 96% ethanol. The hexoses released as a result of hydrolysis at 80°C interact with the orcin reagent (1.6% solution of orcin and concentrated sulfuric acid), coloring the reactogenic mixture in an orange color, the saturation of which is proportional to the concentration of hexoses in the sample. Hexoses of glycosaminoglycans were determined in the same way, but were precipitated with a 1% solution of cetylpyridinium chloride. The role of protein-bound hexoses, glycosaminoglycans, and glycoproteins in the process of transformation of genital organs during their preparation for insemination and fertilization has been clarified. As a result of the research, a significant difference in the content of protein-bound hexoses, glycosaminoglycans, and glycoproteins during various stages of the estrous cycle and during anaphrodisiacs was revealed. It was found that the number of glycosaminoglycans and glycoproteins significantly increases in the cervical secretion of cows during the manifestation of the immobility reflex, due to changes in biophysical properties and an increase in the number of gonadal hormones and enzymes. Estrus is characterized by a significant increase in glycosaminoglycans and glycoproteins in the cervical secretion of cows, which is caused by a change in the biophysical properties of the cervical mucus due to the increase in enzymes and gonadal hormones. In the post-morbid period, namely after endometritis and retention of litter, the content of glycosaminoglycans and glycoproteins in the cervical secretion decreases significantly, compared to the indicator during estrus.