Influence of probiotic preparations on metabolic processes, enterobiasis and the level of endogenous intoxication of animals

. The article presents the results of studies of the probiotic preparations influence, including bacteria of various strains, on the microflora, biochemical blood homeostasis, the level of endogenous intoxication of young pigs, as well as on their productivity and safety. It was determined that feeding a polyassociative probiotic preparation containing a consortium of strains of lactic acid and propionic acid bacteria (Biovet-2), as well as the microbial mass of living bacteria B. subtilis and B. licheniformis (Subtilis), had a positive effect on the formation of microbiocenosis in the intestines of piglets, contributing to a stable clinical result, characterized by an increase in the content of lactobacilli in the intestinal chyme. Their introduction into feed increased the body weight gain of animals by 20.9% and 19.2% with 100% safety of animals. The use of probiotics contributed to the intensification of metabolic processes in the body of young pigs, in particular, an increase in serum of total protein by 7.1% and 10.6%, an increase of glucose by 12.8% and 10.9%, as well as a decrease in the level of endogenous intoxication. Thus, the inclusion of probiotic preparations in the technology of young pigs rearing can become an effective way to prevent gastrointestinal diseases based on environmentally friendly mechanisms for maintaining a high level of intestinal colonization resistance.


Introduction
One of the factors that determine the productivity of pigs is the completeness of their feeding, which is achieved not only by a set of feed products, but also by the inclusion of biologically active substances such as vitamins and minerals in the diets.Their deficiency in rations leads to a decrease in the growth rate of young animals, an increase in feed payment, a deterioration in the reproductive qualities of the main herd, and therefore, a decrease in the results of fattening and the quality of the produced pork.
The new updated data on the needs of animals for nutrients and biologically active substances, especially protein, essential amino acids and minerals, are intended to increase the usefulness of the fed rations, which is the main method of increasing the growth rate of young animals and the economical consumption of feed resources during rearing [1][2][3].
However, the high productivity of pigs is accompanied by an increased intensity of metabolic processes and intense functional activity of all organs and systems.In highly productive animals, more and more undesirable qualities began to appear, such as effeminacy, increased stress sensitivity, pathological response even to slightly changing conditions and adverse effects of the external environment [4], which leads to a decrease in productivity and product quality, the emergence of an immunodeficiency and autoimmune state in animals, the development of infectious, parasitic and non-infectious diseases on this background, as well as a high death rate of young animals.
In sow litters, from 16 to 45% of piglets are born physiologically immature.Newborn piglets have relatively well developed intestines, but an underdeveloped stomach.Suckers have little pepsin in the gastric juice and there is not enough free hydrochloric acid until the age of three weeks.For this reason, the body cannot counteract the pathogenic microflora, since non-acidic gastric juice is devoid of bactericidal properties.Physiological immaturity of the stomach at this time is compensated by the increased activity of the pancreas (trypsin).The absence of hydrochloric acid deprives the gastric juice of its protective properties, which is why errors in feeding and keeping of suckers often lead to disorders, protracted diseases of the gastrointestinal tract, sometimes with a subsequent increased lethal outcome [5,6].Under these conditions only with the help of pharmacological prophylaxis and often pharmacological therapy it is possible to stop the development of pathological processes in the intestines of piglets, restoring the physiological state of the animal organism.
Since 2004, feed antibiotics have been completely prohibited in the EU countries.Therefore, in recent years, the interest of scientists and practitioners in the use of microorganisms in agricultural production has significantly increased.
In the natural conditions the determination of a microbial population in the digestive tract of all warm-blooded animals soon after birth is inevitable.The microbial species that are first established are in most cases the precursors of the final microorganisms that will colonize the digestive tract during the life of the animal [7][8][9].
There are usually two different types of bacterial populations that can be established in the digestive tract.The first exists in close connection with the intestinal epithelium, and the second is found in the intestinal contents.Populations that "spontaneously" establish themselves in the digestive tract can be beneficial or harmful to the host.If beneficial bacteria are established, they have a beneficial effect on the host animal.The ideal situation for an animal to live is to maintain specific amounts of beneficial bacteria in the digestive tract.
Based on this, it becomes obvious that veterinary specialists have an important goal -to eliminate the deficiency of intestinal microflora and maintain its protective effect at an optimally high level [10][11][12].This is possible with the help of probiotics which are the microorganisms used for therapeutic purposes to renew and maintain the intestinal microbial balance in the body.
If microbial feed additives have been used for a long time, then living microorganisms and probiotics in particular as regulators of metabolic functions, began to be used relatively recently.Nevertheless, it is already clear that they can become an alternative to antibiotics in the prevention and treatment of gastrointestinal diseases of an infectious nature in young farm animals and poultry, as well as they can stimulate nonspecific immunity, preventing and treating the disorders of the digestive tract of alimentary etiology (diarrhea, dysbacteriosis, acute lactic acidosis, depression), improving the processes of food-rhenium, accelerating the adaptation of animals to high-energy diets, increasing the efficiency of feed use and animal productivity [13][14][15][16][17].
In this connection, the aim of the research was to assess the effect of probiotic preparations, including bacteria of various strains, on microflora, biochemical blood homeostasis, the level of endogenous intoxication of young pigs, as well as on their productivity and safety.

Materials and methods
An experiment was carried out in the conditions of a pig complex on weaned pigs to study the effectiveness of using the probiotic preparation Subtilis and the associated probiotic Biovet-2 (based on strains of several types of microorganisms) in the feeding of young pigs lagging behind in growth and development.
The probiotic preparation Subtilis is a microbial mass of live bacteria B. subtilis and B. licheniformis in the form of spores with a content of at least 5x109 colony-forming units (CFU) per 1 cm3 with a natural active base.The effect of the preparation is based on the antagonistic influence on a wide range of pathogenic and opportunistic microorganisms, causative agents of the gastrointestinal tract diseases, lung infections, improving the microflora in the growing area, as well as the growth-stimulating effect on the body through the production of digestive enzymes, which makes it possible to shorten the feeding period, reduce feed costs, etc.
The probiotic Biovet-2 includes strains of lactic acid and propionic acid bacteria, consisting of three complexes: the first complex is an association of strains of lactic acid bacteria Streptococcus salivarius-LT1, Streptococcus thermophilus-LT9, LT10, LT11 and the propionic acid bacteria in the ratio of 5:1, which is mixed with cereals or legumes in a ratio of 1:3.The second complex is an association of strains of lactic acid bacteria Lactobacillus plantarum-LT7, Lactobacillus acidophilus-LT12 and a strain of propionic acid bacteria Propionibacterium freidenreichii-LT8 in the ratio of 2:1.The third complex is an association of a strain of propionic acid bacteria Propionibacterium freidenreichii-LT8 and the strains of lactic acid bacteria Streptococcus salivarius-LT1, Lactobacillus plantarum LT7, Streptococcus thermophilus-LT9, LT10, LT1, Lactobacillus acidophilus-ЛТ12 in the ratio of 7:2, prepared in the same way as the first complex.The resulting complex fermented pasteurized milk with a fat content of 1.5-2.5%, in a ratio of 1:30.This ensures the production of a probiotic starter culture enriched with vitamin B12 with the high acid-forming, enzymatic and antibiotic activity, capable of suppressing opportunistic microflora in the gastrointestinal tract of animals, and significantly reducing the mortality of young animals from infectious diseases.
The study of the influence of probiotic preparations of various composition and form on the growth, development and biological status of animals was carried out on young pigs of the large white breed, lagging behind their analogues in growth and development by 30-40 %.
For the experiment, three groups of animals were formed: piglets of the first group were the control group receiving the basic diet without probiotic supplements.During the entire experimental period the probiotic Subtilis (in the form of a powder) was introduced into the diet of the second group at a dose of 0.4 g per 1 kg of feed.In the third group in the first 10 days of the experimental period, piglets were fed with the liquid probiotic preparation Biovet-2 with the compound feed in a dose of 20 ml per head and in the subsequent period -at a dose of 10 ml per head for five days with 5-day interval (Table 1).
The duration of the experiment was 60 days.The conditions of keeping and feeding the experimental animals corresponded to the recommended standards.At the same time, regardless of the research scheme, all veterinary preventive measures in the farm were carried out.
The influence of probiotic supplements on the metabolic processes of animals was assessed by the change in the biochemical parameters of blood, which was taken at the end of the experiment from five pigs from each group.Biochemical studies were carried out on an automated Vitalab Selectra Junior analyzer with software version 1.0.(an open system for photometric tests, manufactured by Vital Scientific N. V. Netherlands) using reagents from ELITech Clinical Systems (France) and Analyticon biotechnologies AG (Germany).It is known that molecules of medium mass (MMM) are universal markers of endotoxicosis, and their measurement in various biological fluids has entered the practice of doctors in many specialties.The weakening of the detoxifying functions of the body with various metabolic disorders and pathological processes leads to an increase in the concentration of MMM in the blood plasma (serum).In this research, the concentration of MMM in the blood serum of piglets was determined using the screening method of N.I.Gabrielyan and V.I.Lipatova at two wavelengths λ = 254 nm (MMM 254) and λ = 280 nm (MMM 280).To register the optical density in the ultraviolet region of the spectrum, an Ecoview UV-1100 spectrophotometer was used.
Statistical processing of the obtained results was carried out using the Statistica 10 software.Quantitative characteristics were assessed by comparing the mean values of sample populations with the determination of the Student's t -test, the difference in the arithmetic mean was recognized as statistically significant at p≤0.05.

Results
As a result of the research it was determined that feeding the studied probiotic supplements had a different effect on the growth rate of young pigs (Table 2).Differences are significant * р≤0.05, ** р≤0.01 in relation to the control group It was determined that with an equal initial weight of piglets (after a 12-day equalization period), the gross increase in the live weight in young animals of the second group for the first 30 days of the experiment increased by 20.2% (p≤0.05).In the third group, the use of the probiotic Biovet-2 to piglets lagging behind in growth also contributed to an increase in the intensity of their growth -the increase in the group was 18.3% (p≤0.05) in comparison with the indicators of the control group.It should be noted that in the control group six pigs were diagnosed with diarrhea, from which one piglet died, while in the experimental groups there was no deaths.
In the next rearing period (4 months), the gain in the live weight of piglets in the second group exceeded the indicators of the control analogs by 24.5% (p≤0.01), in the third group the live weight exceeded by 20.0%.During this period one piglet died in the control group and one piglet died in the first experimental group (pulmonary dystrophy).In general, over the experiment the gross live weight gain in piglets of the second group was higher by 20.9%, and in the third group it was higher by 19.2%, in comparison with the control analogs.
Evaluating the average daily weight gain of piglets over the entire period of the experiment, it should be noted that the maximum indicator was recorded in the second group -481.7 g, while in the third group the increase was 475 g, and in the control group it was 398.3 g.
In general, it can be concluded that piglets, lagging behind in growth, due to probiotic supplements, use diet nutrients more efficiently than their analogues from the control group, however, they did not manage to achieve the indicators of healthy piglets by the end of the research period, because in the same period with a similar level of feeding, the average daily gains of healthy young animals were more than 550 g.
At the age of 100 days fecal samples from three piglets from each group were taken into sterile containers to determine the amount and composition of the microflora of the lower gastrointestinal tract.The research results are presented in Table 3. Differences are significant * р≤0.05 in relation to the control group The extremely important role of lactobacilli in the vital activity of the macroorganism is determined by the variety of functions they perform.They take part in the regulation of optimal levels of metabolic processes, the creation of high colonization resistance of mucous membranes, inhibit the adhesion, penetration and reproduction of pathogenic and opportunistic microorganisms, have a wide range of antimicrobial mechanisms and produce biologically active substances of protein and non-protein nature.First of all, lactobacilli have a modulating effect on the immune functions of the macroorganism, including the intestine.In the experimental groups, there was a tendency to an increase in the concentration of lactobacilli in the chyme, especially in the third group the increase was by 21.4%, where the animals were fed the multicomponent probiotic Biovet-2.In the second group the concentration of lactobacilli increased by 15.6%.In all studied samples, there was no growth of clostridia and staphylococci, which indicates compliance with the established procedure for disinfection and veterinary preventive measures on the farm.
Enterococci are part of the normal microflora of the gastrointestinal tract of most vertebrates; they play an important role in ensuring the colonization resistance of the mucous membrane.Enterococci colonize mainly the small intestine, but also occur in a noticeable amount in the large intestine.The absence of enterococci in the inoculum of the control group indicates undesirable changes in the composition of the populations of the microbiocenosis of the intestine of pigs, especially in the presence of this group of enterobacteria, yeasts and molds in the studied feces.
In general, enrichment of the diets for young pigs with probiotic supplements had a positive effect on intestinal microbiocenosis, especially on the level of lactobacilli.
Biochemical studies (Table 4) determined that the concentration of total protein in the blood serum revealed certain changes, characterized by its increase in the experimental groups by 7.1% and 10.6%.At the same time, in pigs of these groups in the protein spectrum, the largest specific weight was occupied by the fraction of albumin, which is responsible for the active transport of most chemical compounds dissolved in the blood, which indicates more intense synthesis-forming processes in the body of piglets that received supplements of probiotic preparations, as well as better use of amino acids for protein synthesis.The differences between the control and experimental groups were in 1.59 (group 2) and in 1.47 (group 3) times.The use of probiotic preparations in the experimental groups made it possible to preserve the constancy of energy reserves in the piglets' body and to ensure a more efficient use of metabolic energy due to the utilization of exogenous and endogenous fat in the process of metabolism.And although in all groups the glucose concentration was significantly high, which may be associated with the stress in the process of taking blood, however, in the experimental piglets this indicator was closer to the upper limit of the norm, while in the control group the glucose concentration was lower than the values of the third group by 12.8 %, the second group it was lower by 10.9%.
All piglets participating in the experiment showed a high level of urea in the blood, which correlates with the indicators of total protein, since urea is the final metabolite of protein metabolism.The maximum values of the indicator were noted in the first experimental group.
Intergroup differences with the control analogs were 2.6%, with the second experimental group they were 8.3%.
The functional state of the liver of animals was assessed by the dynamics of changes in transamination enzymes such as aspartate aminotransferase and alanine aminotransferase.In all groups a high activity of AST was noted, which is associated with the growth of young animals with a priority in the experimental groups.The difference in the second group with control indicators was 20.1%, in the third it was 22.4%.It should be noted that the activity of AST in the blood of pigs is physiologically lower than the activity of ALT; therefore, the high values of this enzyme in the experimental groups up to the maximum permissible values can be explained by the intensification of metabolic processes in the process of gaining muscle mass in piglets on the background of the high value of protein nutrition.The dynamics of ALT activity was practically similar to the AST.However, according to this indicator, the highest activity was characteristic for the animals of the control group.Differences with piglets from the experimental groups were 30.3% and 41.6%.
Analyzing the dynamics of the concentration of calcium and phosphorus, it should be noted the tendency of a decrease in the content of phosphorus in the blood serum of experimental piglets, which, obviously, was due to the positive balance of phosphorus because of the larger percentage of its use in the body and the normalization of the calciumphosphorus ratio.
Evaluating the state of biochemical homeostasis of the blood of animals it can be concluded that the introduction of probiotics into the compound feed of piglets lagging behind in growth and development has a positive effect on the metabolic processes of the body, manifested by the activation of protein, carbohydrate and mineral metabolism.
When determining the level of endogenous intoxication in the body of piglets, it was found that all indicators of MMM in the blood serum of animals of the experimental groups receiving probiotic preparations were lower than the data of intact control (Table 5).Differences are significant * р≤0.05 in relation to the control group Thus, in the fraction of MMM λ-280 the difference was 6.4% in the second group and 10.9% in the third group.At the wavelength of 280 nm the maximum absorption of aromatic non-toxic amino acids, which are mainly contained in peptides, was recorded.Normally, aromatic amino acids are decarboxylated in the intestine under the action of bacterial decarboxylases to form biologically active amines, which, entering the liver through the portal vein, undergo the oxidative deamination [18].
The decrease in the blood serum pool of MMM λ 254 in animals of the experimental groups relative to the control was by 7.1% and 13.7%, respectively.The MMM λ 254 index is considered as an integral criterion for the content of UV-absorbing substances of low and medium molecular weight from 500 to 5000 D, which, with the exception of peptides, include about 200 compounds of normal and abnormal metabolism.On average, up to 80% of MMM in this group belong to products of impaired protein metabolism, which have a toxic effect, and up to 20% belong to biologically active substances and compounds of intermediate metabolism.
Consequently, the use of probiotic complexes in pigs has an optimizing effect on metabolism, helping to reduce the endotoxicosis of the body with the most pronounced effect on MMM λ 254 (toxic fraction).

Conclusion
The presented data indicate that feeding young pigs, lagging behind in growth and development, with the diets balanced in accordance with detailed feeding norms and enriched with probiotic preparations, ensures that weaned piglets receive high average daily gains in the live weight.Thus, the introduction of the preparation based on the spore-forming bacteria Subtilis into the feed made it possible to ensure an increase in body weight of 481.7 g, exceeding the values of the control group by 20.9%, and the use of the associated probiotic Biovet-2 increased the growth of experimental piglets by 19.2 % with 100% safety of animals.In the second group, the safety was 92.9%, and in the group of control analogs it was 85.7%.
The polyassociative probiotic preparation, containing a consortium of strains of lactic acid and propionic acid bacteria (Biovet-2), as well as the microbial mass of live bacteria B. subtilis and B. licheniformis (Subtilis), had a positive influence on the formation of microbiocenosis in the intestines of piglets, contributing to stable clinical result, characterized by an increase in the concentration of lactobacilli in the intestinal chyme.
The use of probiotics contributed to the intensification of metabolic processes in the body of young pigs by stimulating metabolic processes, in particular protein metabolism, which is characterized by an increase in serum total protein by 7.1% and 10.6% and increase in its albumin fraction in 1.59 and 1.47 times and carbohydrate one -an increase in glucose concentration by 12.8% and 10.9%.
The level of endogenous intoxication in the piglets' organism was lower than the data of the intact control: MMM λ-280by 6.4% (second group) and 10.9% (third group); MMM λ 254by 7.1% and 13.7%, respectively.
All of the above indicates that the inclusion of probiotics in the technology of young pigs' rearing can become an effective way to prevent gastrointestinal diseases based on environmentally friendly mechanisms for maintaining a high level of intestinal colonization resistance.

Table 2 .
Dynamics of body weight and growth of piglets in the experiment.

Table 3 .
Results of the study of microbiocenosis in pigs, lg CFU/g.