RU2402205C1 - Method for improvement of animal stress resistance - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to livestock farming. Method includes parenteral administration of lithium salt gamma-aminobutyric acid in amount from 15 to 30 mg per kg of body weight.

EFFECT: method application prevents development of stress reactions, softens and eliminates their consequences.

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Description

The invention relates to agriculture, in particular to veterinary medicine, and can be used in livestock for parenteral administration and in the diets of animals and birds in order to prevent the harmful effects of stress factors of any etiology on the body.

The introduction of intensive industrial technologies for the production of livestock products is often associated with the emergence of contradictions between the biological and technological aspects of the same problem. Facilitating the working conditions, a person often makes the living conditions of animals less comfortable. There are a number of new stressors called technological. An absolute consequence of the stresses of any etiology is the excessive formation of free radicals, which adversely affects the metabolism, and consequently, the health of animals, their productivity, quality of products and the cost of its production.

The prior art knows a large number of all kinds of neurotropic substances for both humans and animals. These biologically active substances are intended to prevent, and if this was not possible in a timely manner, then to mitigate and eliminate the effects of stressful effects on the body. They are designed to help the animal normalize responses to inappropriate stimuli that initiate metabolic abnormalities that go beyond the fluctuations of the natural biological parameters of dynamic homeostasis. All these substances of various nature are mainly products of chemical synthesis. All of them are more or less alien to a living organism, all of them are far from indifferent to animal health, almost all of them cannot be used in the production of environmentally friendly food for humans (Fomichev Yu.P. Beef biotechnology. M .: Rosselkhozizdat, 1984. - 238 pp .; Baidevlyatov AB, Nikolaenko VP Prevention of stress of movement and veterinary treatments of poultry. Scientific and technical bulletin. Kharkov. (BUT Ukraine. Research Institute of Poultry). - 1983. - No. 15. - P.37-39. Afanasyeva A.I., Oguy V.G., Myakushk about N.V., Taranenko V.N. Technological methods for adaptive methods of calf rearing (Barnaul, AGAU, 2006, 319 pp., prototype).

The present invention is aimed at solving the technical problem of developing a new, more physiological and more effective way to deal with any form of stress by preventing the development of stress reactions or to mitigate and eliminate the consequences, if the reaction to the stress factor could not be prevented, in farm animals and poultry different species and different age and gender groups. The need to develop new, more advanced ways to increase stress resistance of agricultural workers animals and poultry is due to closely related to this criterion problems of increasing animal productivity, improving the quality of products, reducing the cost of feed, labor and financial resources for its production.

The essence of the author’s working hypothesis is as follows: gamma-aminobutyric acid (GABA) is one of the main natural mediators of inhibition in the central nervous system of all higher animals. Excitability, nervousness, sensitivity of animals and humans to stress are primarily associated with this acid. Difficulties arise due to the fact that the GABA molecule is a typical zwitterion, and due to its pronounced polarity and hydrophilicity, under normal conditions, it practically does not penetrate the blood-brain barrier and acts mainly either peripherally or directly in the synthesis sites (Sytinsky I.A. Gamma-aminobutyric acid - a mediator of inhibition. L .: Nauka, 1977 .-- 139 p.).

In search of GABA analogues that can penetrate the blood-brain barrier and have a predominantly central effect, medicine in the late sixties of the last century focused on gamma-hydroxybutyric acid, which in the experiment and clinic showed a clear hypnotic, tranquilizing and antihypoxic effect. In the form of sodium salt, the drug, called sodium oxybutyrate, has found wide application in surgery, anesthesiology, ophthalmology, neurology and psychiatry (Zakusov V.V., ed. Sodium oxybutyrate. Neuropharmacological and clinical research. M: Medicine, 1968, p. 134). A valuable property of sodium oxybutyrate was its ability to enhance the effect of narcotic and analgesic substances, to exert antihypoxic and antitoxic effects. In psychiatric practice, the tranquilizing and hypnotic effects of the drug are used.

A few years later, lithium salt of gamma-hydroxybutyric acid was clinically used as a psychotropic drug (Lyubimov B.I., Tolmacheva N.S., Ostrovskaya R.U., Mitrofanov BC An experimental study of the neurotropic activity of lithium oxybutyrate. Pharmacol. And toxicol., 1980, 43, 395-401, prototype). Pharmacological and clinical studies of the drug showed that its effect is not a simple sum of the effects of lithium cation and anion oxybutyrate, but is characterized by its own spectrum combining the antimaniacal effect of lithium with the tranquilizing effect of gamma-hydroxybutyric acid. The effect of lithium in this case is enhanced due to greater accumulation in the brain tissue, which allows it to be used in smaller doses. In the clinic, the drug exhibits a distinct prophylactic effect in depressive states, while having its own sedative effect (Raevsky KS, Georgiev VP Mediator amino acids. M: Medicine, 1986, p.239). Since the body of ruminant farm animals constantly produces a huge amount of short-chain fatty acids, including butyric with its many interconversions, including hydroxybutyrate, it does not make sense to test the lithium salt with hydroxybutyric acid existing in medicine on ruminants.

This circumstance has become one of the motives for creating a new drug - lithium salts with GABA.

It is known from the prior art that lithium mineral salts also have nootropic (psychotropic) properties, while they freely cross the blood-brain barrier and penetrate the brain (Schou M. Lithium studies. Acta Pharmacol. Toxicol, 1998, 45 (2): 115- 124). Therefore, it was logical to assume that when gamma-aminobutyric acid is introduced in combination with lithium, GABA will be metabolized more slowly by a specific aminotransferase (the main enzyme of GABA degradation), it is easier to overcome the blood-brain barrier and its concentration in the brain will increase. In turn, an increase in the concentration of GABA, based on knowledge of its physiological effect, will entail a simultaneous decrease in the excitability of animals with an increase in stress resistance.

Based on these data, it can be said that the specific goal of creating a complex compound of lithium with gamma-aminobutyric acid is the need to obtain such a preparation, on the basis of which it will be possible to obtain a more physiological and higher positive effect than is the case when animals separately use lithium and gamma-aminobutyric acid. We synthesized such a compound, application No. 2009116486 dated April 29, 2009. The method of synthesis of organic compounds enhancing stress resistance of animals.

Both components that make up the new organic salt (lithium mineral salt and gamma-aminobutyric acid) are legally approved for use in medical practice.

In the patent and scientific literature there is no description of a method for increasing the stress resistance of animals using the lithium salt of gamma-aminobutyric acid. This circumstance allows us to consider the claimed invention patentable as meeting the condition of patentability “inventive step” (part four of the Civil Code of the Russian Federation No. 230-FZ of 12/18/2007).

The method of synthesis of lithium salts with gamma-aminobutyric acid.

We used lithium sulfate (Li ₂ SO ₄ ), sodium hydroxide (NaOH), potassium hydroxide (KOH), gamma-aminobutyric acid, ethyl alcohol (98%). All reagents brand XC.

A weighed portion of lithium sulfate salt weighing 90 g was dissolved in 100 ml of distilled water to obtain a clear solution saturated at a temperature of 40 ° C. After dissolution, it was controlled that the pH of the solution was 7. To the resulting solution, weighed portions of sodium hydroxide weighing 50 g and potassium hydroxide weighing 50 g. As a result of the substitution reaction, a white precipitate of lithium oxide hydrate precipitates.

The precipitate formed was filtered off from the supernatant on a glass filter under vacuum. The resulting white powder of lithium hydroxide was washed with ethanol (98%) and transferred to a desiccator until completely dry. Next, a sample of dried lithium hydroxide weighing 60 g was dissolved in 250 ml of distilled water at a temperature of 25 ° C. In this case, the solution acquires a pH value of 10. Dissolved a sample of gamma-aminobutyric acid 50 g in 100 ml of distilled water. Both solutions were poured into a round-bottom flask, placed in a water bath with a temperature of 45 ° C and distillation was carried out using a rotary evaporator under vacuum (P = 97 Pa). Within 6.5 hours, a precipitate forms. The resulting pasty mass of white was transferred to a filter mass (98%) and placed in a desiccator until completely dry. Received 62.04 g of dry matter, which is the final desired product - lithium salt of GABA.

The main physicochemical parameters of GABA - Li

Nomenclature name - lithium salt of gamma-aminobutyric acid

The empirical formula is NH ₂ —CH ₂ —CH ₂ —CH ₂ —COOLi;

The gross formula is C ₄ H ₈ O ₂ NLi;

Elemental composition in percent:

C - 42.8

H - 7.1

O - 28.5

N - 12.4

Li - 6.2

Mass fraction of the main substance - 97%

Molecular Weight - 108.9

Melting point - 209 ° C

Decomposition temperature - salt melts with decomposition.

The salt is soluble in water-alcohol mixtures and water.

Harmless to animals - GABA-Li withstands the test, belongs to the third hazard class according to GOST 12.1. 007-76.

Example 1. An experiment was conducted to study the effect of lithium salt of gamma-aminobutyric acid (GABA-lithium) on the stress resistance of rats. The study was conducted on a generally accepted experimental model of rat adaptogenicity and stress tolerance, because this model is very sensitive to the action of pharmacological agents that are GABA agonists and GABA antagonists.

In the experiment, male Wistar rats of 2 months of age were used. The animals were kept under standard vivarium conditions and were on a typical commercial full-grain granular feed for linear rats. Three similar groups were formed (10 goals per group).

1. Group - control, intraperitoneal injection of placebo.

2. Group - an experimental, intraperitoneal injection of GABA-lithium at a dose of 15 mg / kg body weight.

3. A group of experimental, intraperitoneal injection of GABA-lithium at a dose of 30 mg / kg body weight.

During the experiment, the animals were subjected to various types of stress: social stress (cell neighbors changed); feed stress (animals were withdrawn for 48 hours). Animals were weighed every three days. Two weeks after the start of the experiment, the animals were euthanized with ether, euthanized, and the mass of internal organs was determined: kidneys; liver; spleen; lungs; heart; adrenal glands; thymus. No significant difference in the size of the internal organs was noted. This indicates the absence of adverse effects of this drug on the body of experimental animals. Data on live weight are presented in table 1.

Table 1 Live weight of experimental animals 2 weeks after the start of the experiment Groups The average live weight at the beginning of the experiment The average live weight at the end of the experiment % to control The control 192.1 ± 16.2 216.8 ± 14.6 one hundred% 2nd group 174.8 ± 12.1 240.4 ± 18.7 111% 3rd group 184.6 ± 10.7 236.5 ± 11.3 109%

As follows from the materials of table 1, in the experimental groups of animals relative to the control group, the live weight was 9-11% higher. From the above data it can also be established that for the lithium salt of gamma-aminobutyric acid, a dose of 15 mg / kg of live weight of the animal is more effective. Thus, a single injection of GABA lithium leads to a decrease in the effect of a stressor on the body and an improvement in the growth of experimental animals. The drug was dose dependent.

Example 2. The study of the adaptogenic properties of GABA-lithium (general physical endurance, swimming test).

The study of adaptogenic properties was carried out using male Wistar laboratory rats. For this purpose, three groups of analog rats were formed with 10 animals per group. The animals in the control group were given placebo. The rats of the experimental group received intraperitoneally one injection of GABA-lithium at a dose of 15 mg / kg body weight 30 minutes before the experiment. The third group was intact - not subjected to any treatment.

All animals were subjected to a standard test for general physical endurance by prolonged swimming in water at a temperature of 20 ° C until they were completely tired (which required an average of about 1 hour for animals). After completion of the test, all animals were euthanized with ether and decapitated. After autopsy, control animals showed 15 ± 4.1 ulcers in the stomach. In the experimental groups, at autopsy, five times less ulcers were observed (3 ± 1.0).

Example 3. General physical endurance. Suspension test.

Used the same way as in the previous experiment, 3 similar groups of rats with 10 animals each. The dose of GABA-lithium in the experimental group was also 15 mg / kg body weight and was administered intraperitoneally 30 minutes before the test. After 30 minutes, the rats were suspended by the forelimbs and remained in this position for 24 hours. After the test was completed, the animals were removed, euthanized with ether, decapitated, then the number of ulcers in the stomach was opened and counted. The number of ulcers in the control group was 19 ± 4.6, which is almost four times more than in the experimental group (5 ± 1.9). In the intact group, ulceration of the stomach was absent.

It is known from the prior art that eosinophils are a fairly informative indicator associated with a change in the concentration of corticosteroids in the blood, which, in turn, is a reliable criterion for the magnitude of the stressor exposure. An increase in blood “corticosteroids - stress hormones” causes a decrease in the concentration of eosinophils in the blood. Eosinopenia (a decrease in the concentration of eosinophils in the blood) has long been qualified as a significant criterion for the magnitude of the stress response in the body. This was confirmed in our experiment. In the native group of animals, the concentration of eosinophils was 974 ± 47/1 mm ^{3 of} blood). In the control group of animals, the concentration of these formed blood elements more than halved and became 435 ± 26/1 mm ³ , while in the experimental group the number of eosinophils decreased by less than 20% and remained within the physiological norm: 793 ± 38/1 mm ³ .

The data obtained confirm the conclusion that the tested GABA-lithium salt has pronounced stress-protective and adaptogenic properties.

Claims (1)

A method of increasing the stress resistance of animals, including the introduction of a biologically active substance, characterized in that the lithium salt of gamma-aminobutyric acid is administered parenterally in an amount of 15 to 30 mg per kilogram of body weight as a biologically active substance.