Cellulosolytic Activity of Gastrointestinal Microflora and Energy Metabolism in Yakutian Horses During Winter

The article presents a study to improve the technology for winter feeding of horses of the Yakut breed. Feeding technology affects digestion and assimilation of the carbohydrate complex of plant feed since the supply of horses with energy at negative temperatures, the physical and technical properties of wool, and the quality of meat products depend on the absorption of nutrients by the body. The article presents a comparative study of the cellulolytic activity of the microflora of the gastrointestinal tract and energy metabolism of adult horses and foals at the age of 9 months and the efficiency of including the probiotic "Sakhabactisubtil" into the compound starter feed for foals under one year of age. As a result of the research, it has been found that during the winter, when consuming the hay and oat diet, foals at the age of 9 months consume considerably more metabolic energy per kilogram of metabolic weight than mares. A mare used 0.99 MJ of energy per kilogram of metabolic weight (LW+0.75), while foals used 1.39 MJ, which was 40% more. In the experiment to establish the efficiency of using compound starter feed with the probiotic "Sakhabactisubtil", young horses of the experimental groups used the nutrients of the feed better.


INTRODUCTION
Yakutian horses are pastured all year round, consuming pasture fodder, with only 10-15% of their annual ration being hay with a small amount of oats. however, for 7 months of the year, horses consume the haylage of winter pasture which represents grassland litter or aftergrass. The chemical composition of the aftergrass is dominated by crude fibre (35%) and nitrogen-free extractive substances (44.7%). Crude fat is 4.2%, crude ash is 7.8%, and only crude protein is 8.8%. With ageing, lignin accumulates in plants, which creates a solid carbohydrate-lignin complex, 1309 which reduces the digestibility of fibre and non-natural extractive substances in farm animals. The accumulation of knowledge on the digestibility and assimilation of carbohydrate complex of plant fodder is important to improve the technology of winter feeding of Yakutian horses because it affects the energy supply of horses at subzero temperatures. a horse consumes fodder firstly using a maximum of easily accessible nutrients in the stomach and intestine: soluble proteins, starches, sugars, minerals, vitamins, and in the large intestine, with the help of residual enzymes of the small intestine and microorganisms, utilizes hardly accessible fibre, residues of protein, carbohydrates, and minerals (novakovskaya, 2015;khompodoeva et al., 2020;Ivanov et al., 2020).

Physiological Communication
Article Information:*Corresponding Author: klg_14@mail.ru received 15/07/2021 accepted after revision 28/09/2021 bIoScIence bIotechnoloGy reSearch communIcatIonS the horse's small intestine digests 60 to 70% protein, 65 to 75% soluble carbohydrates, and only 15 to 25% fibre. a large part of the diet, 75-85% fibre, 30-40% protein, and 25-35% soluble carbohydrates, is digested in the large intestine. Fibre digestion is carried out by cellulosolytic bacteria. These processes are as active as rumen digestion in corresponding cattle. According to the currently adopted technology, foals are weaned for sale for meat and for repair at the age of 6-7 months in november at a lower air temperature of 15-20°C, when the carcasses of slaughtered foals are rapidly cooled. In this case, the fatness of foals left for repair is reduced in the first days and the animals are poorly nourished due to the stress of weaning and the unfamiliar hay and oat ration. A slowdown in the rate of growth and development of foals occurs during the whole winter period with an almost complete halt in mid-winter. Meanwhile, it is known in the Yakutian horse breeding practice that foals left on a suckling with mothers without weaning are also growing well in winter. At the same time, foals' growth and development depend on the mother's milk yield (Gabyshev, 2002;Vinokurov, 2012;Sharaskina, 2019).
This peculiarity of foals' growth and development during the first winter shows how important the quality of foals' nutrition is. It seems to us that the cellulosolytic activity of the microflora of the gastrointestinal tract and the energy metabolism of the foals are important as well. At this time, the foals are transitioning from a diet of mother's milk and green pasture grass to a hay and oat diet, i.e. a transition from an easily digestible carbohydrate diet to the digestion of feed with hard-to-digest fibre, with a high content of almost indigestible lignin. We also suggest that in young horses aged 7-11 months, the cellulosolytic activity of the gastrointestinal tract is less active than in adult animals. The important thing in the nutrition of young foals under 1 year of age is energy supply. both the integument and, most importantly, the subcutaneous and intra-abdominal fat insulate the body less from the cold than in adult animals because the coat in foals is shorter and fat deposits are thinner than in adult animals. equally important is the duration of feed intake, as it generates heat, defined as a specific dynamic action of food or incremental heat production (Novakovskaya, 2015;Ivanov, 2018;Ivanov et al., 2021).
This heat under conditions of stress from freezing helps to maintain the body temperature of the animal. based on the results of many years of research, it has been established that bacteria of the genus bacillus dominate in the microbiocenosis of frozen soils in Yakutia. The bacterial strains b. subtilis tnp-3 and b. subtilis tnp-5 isolated from perennially frozen soils possess a wide range of unique biological properties: pronounced antagonistic action against context-dependent pathogenic and pathogenic microorganisms (bacteria, fungi, and viruses), interferoninducing activity, immunostimulating effect, antibiotic resistance, ability to stimulate growth and development of beneficial intestinal microflora, in particular, bacteria of lactobacillus and bifidobacterium. the unique biologically active properties of strains b. subtilis tnp-3 and b. subtilis TNP-5 have led to the development of the biopreparation "Sakhabactisubtil". the preparation is widely used in northern animal breeding: in respiratory diseases, digestion, reproduction, dysbacteriosis, and mycotoxicosis, also as a part of hygienic and polishing agents, is effective for silage and haying of forages, for rehabilitation of oil-polluted soils, besides, it is a part of inactivated vaccines against horse infectious diseases (Vladimirov et al., 2020;Neustroev et al., 2020b, Neustroev andTarabukina, 2021).
the studied strains of b. subtilis tnp-3, tnp-5, and their combination were found to possess cellulase activity. according to russian and foreign scientists, bacteria of the genus bacillus are characterized by polyenzymatic properties. bacillus cells include a set of enzymes of different classes, which provides them the ability to exist in different substrates (laktionov, 2012;Skryabina et al., 2020;Abdelfattah et al., 2015;Murad and Azzaz, 2010). these enzymes can significantly affect feed digestibility and nutrient uptake, improve chemical composition, physical and technical properties of wool, and enhance the quality of meat products. In this respect, our task was to compare the cellulosolytic activity of the microflora of the gastrointestinal tract and energy metabolism of adult horses and young animals at the age of 9 months and to determine the efficiency of including the probiotic "Sakhabactisubtil" into the compound starter feed for young animals up to one year of age.

MATERIAL AND METHODS
The work was carried out at the research and production farm of the horse Selection and breeding laboratory "olbuordaakh", amga ulus, of the m.G. Safronov yakut Scientific research Institute of agriculture during the winter of 2019 and 2020. The studies were carried out based on a permit to conduct research work issued by a commission consisting of the head of the nasleg I.V. Semenov and the commission members p.p. arsentieva and I.V. kuzmina no. 1-002 dated 16.02.2019. during the experiments, humane research methods were used in relation to animals. Selection and chemical analysis of winter pasture fodder and hay and oat rations were carried out according to the methods of "modern biological and biochemical research methods in Zootechnics" (burtseva and rudishin, 2014). laboratory research was carried out on an infrared analyser nIr Scanner model 4250 in the laboratory of agricultural product processing and biochemical analyses of the Yakut Scientific research Institute of agriculture. the formulation of compound starter feed for foals was developed based on establishing energy, nutrient, and mineral deficiencies and following the foals' needs for basic nutrients (Draganov et al., 2010). The experiment on the digestibility of nutrients in the hay and oat ration was conducted on 4 mares of Yakutian breed aged 10 years with approximately the same live weight. The horses consumed 14 kg of hay and 2 kg of oats per day per head. The duration of the preparatory period was 4 days, the accounting period 6 days. the experiment on the determination of the efficiency of mixed fodders was carried out on 45 foals at the age of 9 months during stationary winter feeding. Three groups of animals were formed, selected on the principle of analogue pairs by live weight, age, and body type. Differences between the pairs in body weight and age did not exceed 10%. The control group received a diet consisting of 5 kg of hay and 2 kg of oats. In the first experimental group, the oats were replaced by compound starter feed. The second experimental group received compound starter feed with the inclusion of the probiotic "Sakhabactisubtil". the experimental and control groups of animals were kept in separate barns and the duration of the experiment was 60 days. At the beginning and end of the experiments, animals were weighed and blood samples were taken for analysis. At the end of the feeding experiments, tests were carried out on the digestibility of feed nutrients. To conduct digestibility experiments, 6 horses from each group were placed in special barns to collect faeces and record the feed fed. The duration of the preliminary period was 3 days and the accounting period was 6 days. fodder and faeces sampling and storage were conducted according to the methods of "modern biological and biochemical research methods in Zootechnics" (burtseva and rudishin, 2014). energy value of fodder -by the regression equation of the Allrussian research Institute of horse breeding (kosharov et al., 1983). me = 19.46dp+35.43df+15.95dfb+15.95dnfeS, where me -metabolic energy of fodder (mJ) dP -digestible protein, g dF -digestible fat, g dfb -digestible fibre, g dnfeS -digestible nitrogen-free extractive substances, g The content of total energy in diets was calculated according to the equations developed by l.k. ernst federal research center for animal husbandry and Institute of animal physiology, biochemistry and nutrition: te = 24.24cp+38.87cf+18.39cfb+17.14cnfeS; de = 23.93dp+32.66df+18.5dfb+17dnfeS; where cp, cf, cfb, cnfeS are "crude" nutrients, g; dp, df, dfb, dnfeS are digestible nutrients, g (nadalyak et al., 1986;Shcheglov, 1991). biochemical analyses of fodder, faeces, blood were carried out in the biochemistry laboratory of the m.G. Safronov yakut Scientific research Institute of agriculture. the preparation Sakhabactisubtil was developed from an equal combination of strains of Bacillus subtilis TNP-3 and Bacillus subtilis tnp-5 with b. subtilis content of 5x109 CFU in 1 ml. The strains of Bacillus subtilis TNP-3 and Bacillus subtilis TNP-5 were extracted from frozen soils of yakutia and deposited in the all-russian collection of microorganisms used in animal husbandry and veterinary medicine (the russian State center for animal feed and drug Standardization and Quality, moscow) (Vladimirov et al., 2020). the drug was given at a dose of 50x109 cfu/ ml at the rate of 10 ml per 1 head daily in combination with hay and mixed fodder for 10 days. The preparation Sakhabactisubtil was manufactured by the Scientific and production center hotu-bact according to tu 9384-003-00670203-06 and was used according to the instructions approved by the rosselkhoznadzor on 06.07.2012. Samples of faeces from foals for microbiological studies were taken from three groups -before the experiment and after the experiment. Microbiological studies were carried out in the laboratory for the development of microbial preparations at m.G. Safronov yakut Scientific research Institute of agriculture -division of federal research centre "the yakut Scientific centre of the Siberian branch of the russian academy of Sciences. the following media were used to study the intestinal microbiota of young horses and for the quantitative accounting of isolation and quantification of bacteria: lactobacagar -for lactic acid microorganisms, bifidum medium -for bifidobacteria, azide medium -for enterococci, MAP (after heating to 85ºC for 15 minutes) -for spore-forming aerobic bacteria, endo -for escherichia, baird-parker -for staphylococci, medium with bromothymol blue -for yersinia, Chapek -for microscopic fungi. Microbial growth was recorded after 18, 24, and 48 hours for bacteria and 5 days for fungi. The number of microorganisms was determined in colony-forming units (cfu) per 1 g. Genus and species identification of microorganisms was carried out according to the "handbook on microbiological and virological research methods" (1982), "Identification of zoopathogenic microorganisms" (1995), and "bergi's bacteria identifier" (1997). The main numerical data obtained in the studies were processed by a biometric method using the Microsoft excel computer program. The table shows that digestibility ratios for all components were higher in foals, except crude fibre and nitrogen-free extractable substances. Comparatively high ratios of crude protein and fat in foals, probably, were connected with the fact that their diet contained relatively more oats than the diet of mares (foals had 2 kg of oats in 5 kg of hay, mares had 2 kg of oats in 14 kg of hay). The chemical composition of oats in both experiments was practically the same. Еру сomposition of hay in the experiments is given in Table 2. Table 2 shows that the hay consumed by the foals had more crude protein, fibre, and nitrogen-free extractive substances. This is why the digestibility of protein was higher, while fibre and nfeS were lower in the foals.

RESULTS AND DISCUSSION
The carbohydrate-lignin complex of foals' hay was less accessible to the cellulosolytic bacteria in the colostrum of foals compared to mares. The relatively low digestibility of the carbohydrate part of the diet can also be explained by the insufficient establishment of cellulosolytic activity of the gastrointestinal tract microflora of foals at 9 months of age, or simply by the insufficient number of cellulosolytic bacteria in the large intestine of foals compared to the intestines of adult horses. In the conditions of the Yakut winter at minus 45-55 degrees C, the knowledge of the amount of metabolic energy for life sustenance and supersupport energy, not only theoretical but also practical, is of great importance.
mares was on average 425kg, the foals were 208kg, i.e. the mares weighed more than twice as much as the foals. however, metabolic energy intake was only 33% higher. This can probably be explained by the fact that the foals also had a comparatively higher energy expenditure to sustain life. According to our calculations, the difference in metabolic energy expenditure for life sustenance in mares and foals was only 20%. This can be explained by the fact that foals expend more sustainable energy in winter than adult animals. This is due to the fact that they seem to have worse thermal insulation than adult animals. Foals have shorter coats, and their skin and subcutaneous fat are thinner than in mares. They also have much less body fat and internal fat in the abdomen. Therefore, energy losses in foals are higher than in mares, and there is more expenditure on heat production as well. It should be noted that the expenditure of super-support energy already differed by 50%, which is probably due to the fact that at this time

Table 3. Energy consumption and utilisation by mares and foals, MJ
The quantitative characteristic of energy of urine and methane was determined according to the reference book "farm animals. physiological and biochemical parameters of the organism" (reshetov, 2002). the live weight of the It seems to us that one of the reasons why the growth and development of foals after weaning at the age of 6-7 months is reduced is that at this time, the diet is changing from a milk ration combined with green grass to hay and oats. The microbiota of the gastrointestinal tract must be adapted to digest fodder with high fibre content. the gastrointestinal tract of foals, including the large intestine where fibre digestion by cellulosolytic bacteria occurs, is small compared to that of adult horses. Apparently, for this reason, the fibre digestion of the hay and oat ration is not high enough, resulting in a low energy intake, which leads to a delay in the growth and development of the foals in the  first wintering period. therefore, we set ourselves the task of developing a formula for compound starter feed to increase the growth and development of foals up to a year old in winter stationary feeding. besides, considering probiotic, cellulosolytic activity, the preparation "Sakhabactisubtil" was included in the recipe of the second experimental group of foals. To compose the formula for compound starter feed, the deficit in energy, protein, fibre, minerals, and vitamins in young horses under 1 year of age was calculated (Table 4).
table 4 shows that when consuming 7.58-7.64 kg of dry substance of the hay and oat ration, young horses up to one year of age have a deficit of almost all basic nutrients, macro-and microelements, and vitamins, except for crude fibre, magnesium, and manganese. to compensate for the deficiency of the main nutrients, macro-, and microelements for young horses up to one year old, we worked out a formula of compound starter feed ( Table 5). Weighing of foals at the beginning and the end of the experiment showed that during the period of the experiment, the live weight increased from 201.44 kg to 217.0 kg (+15.56 kg) in animals of the first experimental group, the increase was from 200.5 kg to 220.3 kg (+19.8 kg) in animals of the second experimental group, and from 202.53 kg to 208.46 kg (+5.93 kg) in the control group. The difference in live weight between the animals of the control and experimental groups at the time of setting of the experiment was minimal and amounted to 1090 and 2030 grams, but at the end of the experiment, it was 8.54 and 11.8 kg or an absolute gain in the animals of experimental groups at the end of the experiment was 2.6 and 3.3 times more than the control group (table 6). * without a probiotic culture ** with a probiotic culture The average daily gain of the young Yakutian horses during the period of the experiment, in animals of experimental groups was 280 and 360 g, in the control group -108 g. Thus, a reliable increase in live weight was observed in the animals of both experimental groups 7,17 and 8,99 % (P ≥ 0,999) respectively, the control group animals showed a non-significant difference in growth (2,84 %).  Table 9. Digestibility coefficients of the main nutrients in the diet of young animals during the experiment period, % It was found that the height at withers in young animals of the first experimental group was increased by 3.3 % and was 126.1±2.43 cm (p ≥ 0.999), chest circumference was increased by 2.4 % and was 143.9±4.55 cm (p ≥ 0.95), in young animals of the second experimental group the height at withers increased by 2.6% to 128.7±2.82 cm (p ≥ 0.999), chest circumference increased by 4.2% to 145.1±3.50 cm (p ≥ 0.95), in the control group -123.7±2.45 cm and 143.3±1.58 cm respectively. When studying the effect of the feed factor on the animal's organism, the assessment and analysis of the indices of digestibility and use of nutrients in the feed is of great importance. We calculated the amount of nutrients and energy ingested by the experimental animals during the day based on the chemical composition and the amount of feed that was eaten. Table 8 shows that the amount of dry substance, organic substance, and crude protein consumed in all groups was approximately equal. The daily amount of nutrients ingested with the diet is not fully absorbed by the animals, and a certain part of them is excreted with the faeces. The difference between the amount of nutrients entering the gastrointestinal tract of the animals and the amount excreted in the faeces characterizes the amount of digested nutrients.
the data we obtained during the scientific and economic experiment show that the greatest amount of nutrients in the diets was digested by the animals fed with compound feed with the probiotic culture. At the same time, young animals of the first experimental group surpassed their control group counterparts by 0.6% in the digestion of dry substance, organic substance also by 0.6%, protein by 1.1%, fat by 1.55%, and fibre by 0.8%. the analyzed data show superiority in digestibility of young animals of experimental groups over their counterparts from the control group.
As for the youngsters of the second experimental group that consumed compound feed with the probiotic culture, they surpassed their counterparts of the control and first experimental group on all indicators, namely, by 2.8% on dry matter of the first experimental group and by 3.4% of the control, organic matter by 1.3% of the first experimental group and by 1.9% of the control, protein by 0.7% and 1.8% respectively, fat by 2.08% and by 3.63%, and fibre by 2.36% and 3.16% -the control group.
A relatively high intake of nutrients throughout the experiment provided the young horses of the experimental groups with higher coefficients of digestibility of the main nutrients. remarkably, young horses of the control group were inferior to their counterparts of experimental group I in terms of nfeS digestibility coefficient by 1.96% (p ≥ 0.95), and those of experimental group II by 3.34%. Inclusion of compound feed into the winter ration at stationary feeding of foals had a positive effect on feed intake, digestibility and assimilability of nutrients in the ration, while compound feed with the addition of the probiotic "Sakhabactisubtil" promotes even more successful assimilation of fodder in the ration of horses.
When studying the metabolism of substances and energy in the body, as well as evaluating the nutritional value of feed and rationing of animals, the following types of energy are distinguished: total, digestible, metabolic (or physiological), the energy of heat production, and energy released during production. The transformation of feed energy into animal products is significantly affected by the level of feeding, the structure of the ration, the concentration of energy per unit of dry substance, as well as the balance of the ration on mineral elements and biologically active substances (kalashnikov et al, 2011, Ivanov et al, 2020, Ivanov, et al, 2021. The experimental data we obtained in the physiological experiment testify to the positive effect of compound starter feed when including it instead of oats in the oat and hay ration during winter stationary feeding of young horses under one year of age on energy intake and utilization. The amount of total energy consumed by young animals of experimental and control groups was comparatively equal, the indicators of experimental group I and II exceeded the control group counterparts in this indicator by only 1.1 MJ (0.80%). At the same time, the young animals of experimental group I exceeded their control group counterparts in digestible energy by -5.52% and metabolizable energy by -6.81%. experimental group II outperformed the control group in digestible energy by -7.85%, metabolizable energy by -9.03%. As a result, the metabolism total energy in young animals of experimental group I that consumed compound starter feed was higher -by 6.14% than in the control group counterparts. as for the young animals of the experimental group II, which consumed compound feed with probiotic culture, they surpassed their counterparts from the control group by 8.39%. The analyzed data show that the metabolism rate of the total energy of the experimental group II, which consumed the compound feed with the probiotic culture, is the highest. Thus, the obtained data indicate that feeding young animals with compound feed with the addition of the probiotic culture "Sakhabactisubtil" in the spring-winter period contributed to a significant increase in the productive use of energy (koilybaeva et al., 2018;neustroev et al., 2020a;Vinokurova et al., 2021).
currently, spore-forming bacteria of the genus bacillus are widely used as probiotic additives, as well as feed additives for farm animals (koilybaeva et al., 2018;Vinokurova et al., 2021Vinokurova et al., , neustroev et al., 2020b. Such qualities of bacilli as high and varied biological activity, the ability to survive in the gastrointestinal tract of animals, and the thermal resistance of spores make these bacteria attractive as probiotics and appealing for further search for active strains and the creation of new highly effective drugs. As a result of the studies carried out by Neustroev, it was found that when using the drug Sakhabactisubtil (single intragastric administration to mice of the DM 1 line), mortality or any signs of intoxication in animals were absent when the drug was administered to them in the maximum allowable volumes for animals of this type. Therefore, the drug does not have a potential toxic effect (Neustroev et al., 2020a).

CONCLUSION
experimental data show that in winter when consuming a hay and oats diet, foals at 9 months of age consume significantly more metabolic energy per kilogram of metabolic weight than mares. Per kilo of metabolic weight (LW+0.75) mares have 0.99 MJ of energy consumption, while foals have 1.39 MJ, which is 40% more. In the experiment on establishing the efficiency of using the compound feed starter with the probiotic "Sakhabactisubtil", young horses of the experimental groups used the nutrients in the feed better. reliably high coefficients of digestibility were observed in the experimental group II on dry matter by 5.1% (66.5±0.08%), on organic matter by 2.7% (70.1±0.20%), on crude fibre by 6.0% (52.66±0.28%), and on beb by 4.5% (73.48±1.10%). young animals of the experimental groups I and II outperformed their counterparts in digestible energy by -5.52 and 7.85% (136.3 ± 0.17 and 136.3 ± 2.14 mJ), metabolism energy by -6.81 and 9.03% (77.8 ± 1.25 and 79.7 ± 1.19 mJ), respectively (p ≥ 0.95). the metabolism of total energy in young animals of experimental groups I and II was higher, by -6.14 and 8.39% than in the control group counterparts. The obtained data indicate that replacement of oats in the hay and oat ration during winter stationary feeding of young animals with the compound starter feed offered by us contributed to a significant increase in the growth and live weight of foals of experimental groups due to a significant increase in the productive use of energy.