UA108932U - METHOD OF SUSTAINABLE SUSTAINABILITY FOR CRYCONSERVATION - Google Patents

Abstract

A method of preserving the viability of sperm by cryopreservation involves the use of choline chloride, glycerol. Lactose, egg yolk, Decomson antibiotic and 1.5 ml of 20% choline chloride are additionally introduced into the sperm thinner.

Description

A useful model belongs to the field of agriculture and biotechnology, in particular to increasing the resistance of sperm to deep freezing of sperm, namely to the balancing of dilution media and cryopreservation of bull ejaculates. A useful model can be used in the work of breeding enterprises in animal husbandry to dilute ejaculates and increase the resistance of bull sperm to the negative effects of ultra-low temperatures (-196 C) cryopreservation and increase the number of living viable cells with high fertilizing ability.

There are known methods of increasing the resistance of sperm of agricultural animals, in particular, bulls, to the action of extremely low temperatures (-196 C; during deep freezing;) |Ostashko Ff.Yi. etc.

Kharkiv technology of aseptic obtaining, dilution, cryopreservation of sperm of breeding bulls / F.I. Ostashko, M.P. Pavlenko, G.M. Kuznetsov, E.F. Isachenko. // Development - production / Ed. M.V. Zubtsia / UAAN - K: Agrarian science, 1999. - P. 282-283). There are also known methods of adding various compounds with antioxidant properties to the dilution medium of ejaculates both before and after freezing (Ostapiv D.D., Klevets /L.0O., Vashchyshina O.G.,

Kava S.Y. "Method of sperm protection during freezing of ejaculates of bulls". Stalemate. of Ukraine for invention (USA). - Me 20900 A; AbIO 7/02, Mo. ts96124651, Application. 13.12.1996, Publ. 07.10.1997;

SI Tsekhmistrenko, VA Koberska. "A way to increase sperm survival". Stalemate. Ukraine, АЭ1О 19/02 (2006.01) - Mo и201413151 Application. 08.12.2014, Publ. 10.04.2015).

The disadvantages of these methods are that their use inhibits or stimulates a certain link of metabolism in sperm and, accordingly, disrupts the physiological course of metabolic processes in sperm after thawing.

The most promising method for use is the method of increasing the effectiveness of known cryoprotectants, which would enhance their effect and not show a negative impact on metabolic processes.

The closest in essence to the claimed method is the method using 20 95 choline chloride in certain concentrations in a diluent for diluting and cryopreservation of male sperm (V.Y. Hryshchenko, Yu.S. Parashchuk, F.V. Dakhno, G. Yurchenko .H. Cryobiology and the problem of infertility. - K: Naukova dumka, 1990. - 136 p. However, the disadvantage of this method is the use of both high concentrations of glycerol (10 95) and glucose (40 95) as a diluent. the use of the specified method ensures preservation of only 20-30 95

From the cells after thawing of the sperm and cannot, due to its composition of energy substrates and concentration of glycerol, be used for cryopreservation of bull sperm.

The method proposed by us eliminates the shortcomings of the prototype and provides higher: almost twice the survival of sperm and 29.0 95 activity of the enzyme-marker of the fertilizing capacity of germ cells - succinate dehydrogenase (SDH) than in the prototype.

The basis of a useful model is the task of developing a method that would ensure higher preservation of bull gametes during deep freezing, improve the physiological properties and increase the fertilizing ability of sperm, reduce costs for the production of a diluent, and be easy to manufacture and use.

The task is solved by the fact that the method of preserving the viability of sperm during cryopreservation, which includes the use of choline chloride, glycerin, according to a useful model, lactose, chicken egg yolk, the antibiotic "Dekomsan" and 1.5 ml of 20 95 are additionally introduced into the composition of the sperm thinner choline chloride.

The technical result is achieved by adding choline chloride to the lactose-yolk-glycerol thinner, which is one of the fractions of lecithin, performs the function of a methylating agent in biological systems and enhances the cryoprotective properties of glycerol. To perform the method, prepare the initial 20 95 solution of choline chloride and add 1.5 ml of this solution to 100 ml of lactose-yolk-glycerol ejaculate thinner.

The effectiveness of the method is based on the ability of choline chloride to supply methyl groups to glycerol, which ensures an increase in the proton-accepting capacity of the hydroxyl groups of the cryoprotectant and increases, in an optimal ratio, the protective properties of sperm.

The protective properties are provided by the components of the thinner (egg yolk and glycerin, the energy substrate - lactose) and added choline chloride, which have a cryoprotective effect and protect the germ cells of bulls from extremely low temperatures (-196,275).

Additional introduction into the diluent in a dose of 1.5 ml of 20 95 choline chloride solution in 100 ml of diluent provides protection and preservation after thawing and increases the fertilizing ability of sperm. Sperm are able to incorporate chicken egg yolk lecithin into their structures, and choline chloride, which is a component of lecithin, enhances the interaction between the components of the diluent and, together with glycerol, provides an increase in the proton-accepting capacity of the hydroxyl groups of the indicated cryoprotectant. With this method of liquefaction of bull ejaculates, sperms increase their resistance to freezing, and after thawing, a larger number of cells show higher motility and survival, and the proposed thickener of bull ejaculates is a simple way to ensure sperm resistance to deep freezing, prolong survival and increase the fertilizing ability of sperm and use in the practice of artificial insemination.

When the applicant conducted a patent information search, a technical solution was found, which has a significant feature in common with the claimed one - the use of choline chloride in a male sperm thinner (V.Y. Hryshchenko, Yu.S. Paraschuk, F.V. Dakhno, G. Yurchenko. G. Cryobiology and the problem of infertility. - K: Naukova dumka, 1990. - 136 p...

However, this is not enough to obtain the technical result of the declared solution.

No technical solutions have been found that would fully match the features of the claimed method of increasing the stability and physiological characteristics of bull sperm. This allows us to draw a conclusion about the conformity of the declared technical solution with the "novelty" criterion of the invention (utility model).

In the sources of patent and scientific and technical information, no information was found about the methods of preservation and protection of sperm during cryopreservation, which would contain features that distinguish the claimed invention (utility model) from the prototype: the use of a lactose-galactose-glycerin thinner with an additional introduction to the composition 1, 5 ml of 20 95 choline chloride.

The useful model can be used in the practice of artificial insemination and reproductive biotechnology for the production of diluents for ejaculates of bulls, the preservation of sperm through cryopreservation, increasing the survival and fertilizing ability of germ cells and the fecundity of cows and therefore meets the criterion of the invention (useful model) "industrial applicability".

To implement the declared useful model, it is necessary to prepare the initial 20 95 solution of choline chloride and add 1.5 ml of this solution to 100 ml of lactose-yolk-glycerine diluent of the following composition: lactose - 11.5 g, chicken egg yolk - 20 ml, glycerin - 5 ml and antibiotic - Decomsan. Freshly obtained ejaculates of bulls, after assessment of physiological characteristics and suitability for cryopreservation, in accordance with the activity and concentration of spermatozoa, are diluted with a lactose-yolk-glycerol thinner with the addition of 1.5 ml of 20 95 choline chloride solution. According to the technological process of preparation of diluted sperm

Before freezing, it is equilibrated for 4 hours. at a temperature of 4 "C, frozen - first in pairs and then immersed in liquid nitrogen (temperature - 196 7C). If it is necessary to use frozen spermatozoa, they are thawed in a water bath at a temperature of 38-40 "C in accordance with

Instructions for artificial insemination of cows and heifers (Kyiv, 2001. - 38 p.)

In order to evaluate the effectiveness of the declared "Method of preserving the viability of sperm through cryopreservation", an experiment was conducted on the specific implementation of the solution.

At the breeding enterprise, ejaculates of bulls were selected, which were characterized by: volume - 3.25:0.28 ml, concentration - 0.95:20.10x109 cells x ml and number of live sperm - 69.88:-2.56 95. In laboratory conditions, freshly obtained ejaculates were divided into four parts: one control and three experimental. To establish the optimal content of choline chloride in the diluent, the control part was diluted with a lactose-yolk-glycerin diluent, and the experimental ones - with a similar diluent with the addition of 20 95 choline chloride solution: in the first - 1.5 ml, in the second - 3.0 ml, and in the third - 6.0 ml per 100 ml of diluent.

Sperm survival and activity of succinate dehydrogenase (SDH) enzyme, a marker of the fertilizing capacity of germ cells, were evaluated in thawed sperm after dilution with the medium according to the prototype and according to the stated method. It was established that the addition of 1.5 ml of 20 95 choline chloride solution to the diluent provides almost two times higher sperm survival in thawed sperm compared to the prototype (table). An increase in the dose of choline chloride in the diluent to 3.0 ml and 6.0 ml is accompanied by a decrease in sperm survival compared to a dose of 1.5 ml, respectively, by 8.9 and 25.5 95. However, the value of the physiological indicator is higher at 75.6 and 47.5 95 than in the prototype.

Table

Sperm quality of thawed bull semen due to the addition of choline chloride, p-30, Mat-tyaya - to the diluent of ejaculates

Similar changes were found when studying the activity of SDH - an enzyme-marker of the fertilizing ability of sperm. The highest activity of the enzyme was established when adding 20 95 choline chloride solution to the lactose-yolk-glycerol thinner in a dose of 1.5 ml (33,225.46 units) and lower values, respectively, by 13.3 and 22.9 95 when using 3, 0 ml and 6.0 ml. The lowest (23.6:25.03 units) SDH activity was found in the control. The difference between the maximum and minimum values is 29.0 95.

Therefore, when using the claimed method for cryopreservation of bull ejaculates, it is necessary to add 2095 choline chloride solution in a dose of 1.5 ml to sperm diluted with lactose-yolk-glycerin thinner, which ensures almost two times higher sperm survival and 29.0 95 SDH activity , than in the prototype.

Claims (1)

USEFUL MODEL FORMULA A method of preserving the viability of sperm during cryopreservation, which includes the use of choline chloride, glycerin, which differs in that lactose, chicken egg yolk, the antibiotic "Decomsan" and 1.5 ml of 20 95 choline chloride are additionally introduced into the composition of the sperm thinner .