RU2689709C1 - Method of poultry feeding, which promotes increase of beta-defensins expression - Google Patents

Abstract

FIELD: biochemistry; agriculture.

SUBSTANCE: invention relates to agriculture and biochemistry, and also can be used in poultry keeping during poultry feeding. Method of poultry feeding consists in the fact that one introduces into the fodder a mixture of essential oils of eucalyptus, garlic and lemon, taken at ratio of 1:3:1, applied on wheat bran and dried to produce a dry concentrate in powder form. Powder is added to the fodder in amount of 20 g/ton of fodder once a day during the entire poultry breeding period.

EFFECT: invention provides enhanced immune response of organism with simultaneous increase of zootechnical indices of poultry.

1 cl, 4 tbl

Description

The invention relates to agricultural biotechnology, in particular to a method of feeding poultry, contributing to the enhancement of the immune response of the body expressed in increasing expression of beta-defensin genes.

Defensins are cationic peptides of the immune system that are active against bacteria, fungi, and many envelope and non-enveloped viruses. Immune cells use defensins to kill bacteria absorbed in phagocytosis. Vertebrate defensins are divided into alpha and beta defensins by the difference in the pattern of disulfide bonds. Birds contain beta defensins (gallinacins), which play a vital role in the innate immune response to bacterial infections. The expression of β-defensins is induced by the action of pro-inflammatory cytokines and various microorganisms. β-defensin is an integral part of the innate immune system and due to its antibacterial action contributes to the barrier function of intestinal epithelial cells.

The invention relates to agriculture and biochemistry, and can also be used in poultry farming for feeding birds.

Purpose of the invention

The purpose of the invention is the development of a highly efficient method of feeding birds, which allows simultaneously with an increase in zootechnical indicators, to evaluate the immune response of the body by increasing the expression of beta-defensin genes.

An original method of feeding poultry has been developed, which includes components from a mixture of vegetable oils with high phytobiotic activity. A mixture of oils is given with food once a day for the entire period of poultry rearing. The mixture of essential oils consists of eucalyptus, garlic and lemon at a ratio of 1: 3: 1, applied to wheat bran and dried to obtain a dry concentrate in powder form. All components are taken in certain quantities and introduced into the feed in the amount of 20 g / ton.

The method of feeding poultry with the introduction of a mixture of oils into the ration provides an increase in bird weight, and also has an immunomodulatory and antimicrobial effect, causes an immune response of the body, expressed in enhancing the expression of beta-defensins, detected using real-time PCR.

The basis of the present invention is to solve the problem of enhancing the immune response of the body while improving the zootechnical indicators of poultry. The immune response of the body is detected by molecular genetic methods - an increase in the expression of cytokines and beta-defensin genes. The initiation of the claimed molecular genetic and biochemical processes occurs due to the effect on the macroorganism by introducing the developed feed mixture into food.

Identifying the effect on increasing the expression of beta-defensin genes in poultry when feeding a mixture of eucalyptus, garlic and lemon essential oils, which also have immunomodulatory effects, allows us to obtain a new way to stimulate the immune response through the correction of poultry diets, expressed in biochemical processes associated with immunity.

There are other feeding methods and methods for increasing the expression of defensins in the body of animals and humans, but the applicant is not aware of any publications or patents that would contain information about the simultaneous effect of a mixture of vegetable oils, both on increasing the zootechnical indicators of agricultural poultry and on clearly pronounced The body’s immune response is an increase in beta-defensin gene expression.

Known feed additive for poultry, containing biologically active substances, which are used as extracts from plants: ginseng, nettle and other similar plants that help restore the reproductive function of the body of agricultural birds, RU No. 2202223 C2, А23К 1/16, А23К 1 / 175, April 20, 2003.

Known feed additive containing a biologically active substance - extracts of coniferous greenery, RU №2304397 C1, А23К 1/00, А23К 1/12, А23К 1/14, А23К 1/16, 20.08.2007.

Known means for the prevention of colibacillosis of birds, containing a biologically active substance, including plant extracts from the leaves of sea buckthorn leaves (Hippophae rhamnoides L.), and / or extract of the herb Macleia heart-shaped (Macleaya cordata Will L.) or small-fruited (M. microcarpas), and / or milk thistle extract (Silybum marianum (L.) Gaertn), and / or echinacea purpurea root and herb extract (Echinacea purpurea L.), RU No. 2355410 C2, A61K 36/00, 20.05.2009.

Known drug for improving the viability of chickens, which is a water-ammonia extract from a mixture of plant components, containing birch buds, immortelle flowers, calendula petals, herb, mildew, nettle, St. John's wort, hollow-leaf flowers, mother-and-frost leaves, mother-and-frost leaves, mother-and-frost leaves, mother-to-bee, small-leaved leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-frost flowers, small-leaved leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-frost leaves, mother-to-bee flowers, small linden leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-jungle, small-leaved leaves, mother-and-frost leaves, mother-and-frost leaves, mother-and-toe mache. medicinal, large plantain, heartwort, pine buds, fennel fruits, common thyme, tripartite sequence, dried forest and kelp, prickly Eleutherococcus root, Ropsha, tansy flowers, corn stigmas and coriander fruits, RU # 2428998 C1, А61К 36/00, 20.09.2011.

All presented feeding methods and feed additives for poultry contribute to an increase in zootechnical indicators and immunity, however, in this invention for the first time a new property appears, namely, an increase in the expression of beta-defensins genes when feeding this mixture of vegetable oils in a fixed ratio.

The applicant has not identified sources that contain information about solutions that are identical to the present invention, which allows to make a conclusion about its compliance with the criterion "novelty".

The feeding method can be useful both in agricultural production of poultry to increase important zootechnical indicators, such as productivity and safety of livestock.

The feeding method can be useful for molecular genetic studies in assessing the immune response associated with an increase in the expression of beta-defensin genes: Gal-9, Gal-10, Gal-11 in poultry.

A mixture of essential oils used in the composition of feed for farm animals and poultry.

Studies on the use of a mixture of essential oils in the proposed ratios are presented in the example.

The experimental part of the study was carried out in vivarium VNIVIP, where experience was given to raising chickens using a mixture of essential oils. An analysis was also made of the effect of a dry concentrate of a mixture of essential oils when a bird is infected with the epizootic strain Salmonella enteritidis, since according to the Rosselkhoznadzor, salmonellosis is one of the most frequent infectious diseases in the poultry industry (Petrova, 2011).

For the study, conditions were created for growing, vaccination and feeding of birds, as close as possible to production. The investigated bird - Ross-308 cross-country chickens in the amount of 180 heads were kept from the 1st day to the 42-day-old age, divided into groups of analogs:

1. control;

2. dry concentrate blend of essential oils.

Later, after infecting a part of the livestock, the bird was further divided into the following groups:

1. control;

2. control + infection with S. enteritidis;

3. dry concentrate mixture of essential oils;

4. dry concentrate of a mixture of essential oils + S. enteritidis infection.

All birds at the age of one day were vaccinated subcutaneously in the upper third with the vaccine against Gumboro disease. Giving of a dry concentrate of a mixture of essential oils of essential oils began to be carried out from the first day to 20 g / ton of feed.

At the age of 19 days, part of the livestock was infected with an epizootic strain of Salmonella enteritidis in the amount of 500 million CFU intramuscularly into the pectoral muscle, after which further infection was made into groups according to infection.

Once a week, polls were weighed and sampled for further analysis of gene expression, beta-defensins.

Sampling was carried out subject to strict adherence to sterility.

Sample preparation was carried out in accordance with the requirements of the “Instructions for the Sanitary-Microbiological Control of Carcasses, Poultry Meat, Poultry Products, Eggs and Egg Products at Poultry and Processing Enterprises”.

Molecular genetic studies were carried out in accordance with the protocols developed during the 1 year of the project under grant number 14. W03.31.0013 dated February 20, 2017

Laboratory testing of samples was carried out in the company BIOTROF +, where a laboratory of molecular genetic research was created, including the use of a set of techniques based on molecular genetic approaches, which allow the composition of the intestinal microflora of farm animals to be as completely and accurately as possible.

The analysis of the expression of the genes responsible for the immune response - beta-defensins (Table 1) was carried out. The study was performed on gene expression in an experiment conducted on chickens infected with Salmonella ssp., And receiving, in addition to the basic ration, a dry concentrate of a mixture of essential oils one day after infection (19-day-old chickens). RNA was isolated from the walls of the blind processes of chickens, a reverse transcription reaction was performed, and cDNA was obtained. Then, gene expression analysis was performed using real-time PCR. The beta-actin gene was selected as the reference gene. Analysis of gene expression allows you to detect which genes are activated in response to a particular effect on the macroorganism. Analysis of gene expression was performed using real-time PCR. The relative expression was calculated using the 2 -ΔΔCt method [delta delta Ct] (Livak, Schmittgen, 2001).

The study revealed a significant difference in the expression level of beta-defensins, as well as in the weight gain of the studied birds.

The averaged data over groups is presented in table 2.

If at the beginning of the experiment, the whole bird was about the same weight, then after two weeks there is a strong difference in weight gain. Thus, the chickens of the control group had an average weight of 322 g, while the group receiving a dry concentrate of an essential oil mixture was 360 g. By the end of the experiment, the best weight indicator among non-infected poultry was in the group receiving a dry concentrate of an essential oil mixture weighing 2403 g, against 2294 g in the control group.

Among the birds infected with the virulent strain of S. enteritidis, the weight result of the groups that received and did not receive the dry concentrate of the mixture of essential oils did not differ statistically.

Samples for the analysis of gene expression were collected from birds a day after infection (20 day experience). Samples were taken from the following groups: control, control + S. enteritidis, dry concentrate of a mixture of essential oils and dry concentrate of a mixture of essential oils + S. enteritidis.

The results of the evaluation of gene expression are presented in tables 3 and 4.

In general, Salmonella infection contributed to an increase in the expression of beta-defensins relative to the control one day after infection. At the same time, a significant increase in the expression of beta-defensins in poultry was found as a result of the use of a dry concentrate of a mixture of essential oils, both against the background of infection and without infection with salmonella.

Thus, the lowest expression of beta-defensin 10 was observed in birds of the control group. The expression of the beta-defensin 10 gene increased 1.84 times after Salmonella infection. The introduction of a dry concentrate of a mixture of essential oils in the diet of chickens led to an increase in the expression of beta-defensin 17.51 times without infection with salmonella and an increase by 2.1 times with infection with salmonella.

The expression level of beta-defensins 9 and 11 increased as a result of infection of the bird with salmonella by 1.19 and 0.72 times, respectively. As a result of the use of a dry concentrate of a mixture of essential oils, the expression of beta defensins 9 and 11 was increased by 1.57 and 1.70 times, respectively, against infection, and without infection, by 1.46 and 1.29 times, respectively.

This may indicate immunomodulating properties of the supplement. Probably, a high level of expression in the group in whose diet a dry concentrate of a mixture of essential oils (without infection) was administered indicates a higher level of activation of innate immunity.

An increase in the expression of gallinacin 3 and gallinacin 6 has been previously shown in birds when infected with a number of pathogens (Zhao et al., 2001; van Dijk et al., 2007). The increase in cytokines and immune proteins, such as IL1β, IL6, IL8, IL12, IL17, IL18, IL22, IL23, IFNγ, LITAF after infection with Salmonella chicks has been repeatedly reported in many studies (Berndt et al, 2007; Crhanova et al., 2011). The data obtained by a number of researchers largely confirm the role of hollinacins as protective molecules against intestinal pathogens (Hasenstein & Lamont, 2007; Mukhopadhyay et al., 2010). Some researchers suggest using gallinacin as a potential marker against a gram-negative infection (Mukhopadhyay et al., 2010).

Thus, against the background of the use of a dry concentrate of a mixture of essential oils, a higher activity of these genes associated with immunity was observed, which indicates a more rapid development of the immune response in response to infection with Salmonella.

Analysis of gene expression three weeks after infection (42 daily birds) showed a tendency to reduce the level of expression of genes associated with the immune system. At the same time, against the background of infection, higher gene activity was noted.

The use of a dry concentrate of a mixture of essential oils on average contributed to a lower level of gene expression compared with the control at this time with and without Salmonella infection. This may indicate an earlier attenuation of the inflammatory response and the restoration of the health of infected birds.

Note: at p≤0.05 with respect to the control group

Note: at p≤0.05 with respect to the control group

Claims (1)

A method for feeding poultry to increase the expression of beta defensins, characterized in that a mixture of essential oils of eucalyptus, garlic and lemon, taken at a ratio of 1: 3: 1, applied to wheat bran and dried to obtain a dry concentrate in powder form is introduced into the feed. in the amount of 20 g / ton, and give once a day during the entire period of breeding poultry.