The research was conducted from December 2020 to June 2021 on a farm belonging to the joint-stock company “Plemkhoz Naro-Osanovsky” of the Odintsovsky district of the Moscow region, Russia. The experiment was conducted according to methods approved by the scientific council of the Institute of Zoology and Biology of the RSAU – MTAA (Protocol №. 198 from 12.10.2020). The animal study was reviewed and approved by the Bioethics Commission of the Institute of Animal Science and Biology of the Russian State Agrarian University — Moscow Timiryazev Agricultural Academy (Protocol № 2021-4 from 12.10.2021).

Forty highly productive Holstein dairy cows during the dry period (3 weeks before calving), giving into account their origin, age, live weight, physiological condition, and milk production. The animals were clinically healthy and kept under the same conditions throughout the experiment (3 weeks before calving until the end of the stage of lactation (120 DIM)). Cows were milked three times/day (3×).

All animals under the study consumed the same total mixed ration (TMR), which was nutritionally balanced and corresponded to the feeding rate for cows during the milking period with a milk yield of 36 kg of milk per day. The first group of animals served as control and received only the basic (TMR) consistent with the normal standards of feeding highly productive cows (Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst (19) (Tables 1, 2). while the 2nd, 3rd, and the 4th group fed the balanced (TMR) supplemented with the multicomponent synbiotic “Kormomix® Rumin” in amounts (25, 50, and 75 g/head/day) respectively until the end of the 1st phase of lactation. Administration of multicomponent synbiotic “Kormomix® Rumin” was done manually and individually to each animal after the morning feeding and mixed thoroughly and carefully together with the concentrate once/daily.

Multicomponent synbiotic “Kormomix® Rumin” consisted of an enzyme mixture that includes amylase, cellulase, xylanase, β-gluconate, protease, phytase, live probiotic cultures (B. subtilis and B. licheniformis), prebiotic culture based on mannanoligosaccharides, and mineral filler (Figure 1) (silicon dioxide). The nutritional value of “Kormomix® Rumin” up to 100 g, is very low and will not significantly affect the composition of the diet. However, it contains many micro and macro elements. Because the action of this additive is aimed to manipulate the rumen fermentation to increase the efficiency of feed conversion and animal productivity. The Multicomponent synbiotic “Kormomix® Rumin” as a product was manufactured by LLC PO “Sibbiofarm” company (Berdsk, Russia).

Animal rations were balanced using the Feed Optima program (v. 2020.8.17251) to satisfy cows' needs (energy, protein, lipids, carbohydrates, vitamins, and minerals) during the early lactation period (120 DIM). Feed samples were sent to the Cherkizovo Research and Testing Center (Yakovlevskoe village, Troitskiy Autonomous District, Moscow) for measuring the chemical composition of the (TMR) fed to the experimental animals.

Milk samples were collected (from all dairy cows under the study) in a personal container and stored in a refrigerator at 4°C until sent to the laboratory. Fat and protein content in milk was determined in the laboratory of selective milk quality control of the regional information and selective breeding center of JSC “Moskovskoe” for breeding work (Noginsk, Moscow region) on a Combi Foss FT+ device. For the objectivity of measurements, we used reference milk samples intended for metrological control (calibration), made according to international standards: the mass fraction of fat according to ISO 1211-2012. 2446-2009; mass fraction of protein according to ISO 8968-1-2008. Control milking and milk tests were measured every 10 days. The gross yield of protein and fat in the milk of cows was calculated. Daily and gross milk yields of natural and 4% FCM were estimated based on control milkings. The gross milk yield of 4% FCM was calculated according to the formula proposed by (20) based on the gross daily milk yield of natural fat and the chemical composition of milk.

At the age of 3 months of lactation (90 DIM), blood samples were collected 3 h after the morning feeding on the subcostal vein (three cows from each group) into vacuum tubes with a coagulant coagulation activator (for obtaining blood serum for estimation of some biochemical parameters by the campaign of Zhejiang Gongdong Medical Technology Co., Ltd., China) and tubes with K3 EDTA (whole blood) for obtaining whole blood in order to estimate of hematological parameters on an ABC VET analyzer (Horiba ABZ. France) using “UniGem” reagent kits (Reamed, Russia). Biochemical blood parameters were determined at a certified independent veterinary laboratory (Moscow) on a Beckman Coulter AU 480 device (Beckman Coulter. Inc., USA).

Samples of ruminal fluid were drawn after feeding using a transesophageal tube (rumen probe) according to the method of (21) approximately 3 h after feeding, considering that the maximum amount of saliva enters the ruminal fluid sample at the beginning of probing (Figure 2). The first portion (200 ml) was not used for analysis to determine volatile fatty acids (VFA) concentration, ammonia, and pH. Physio-chemical parameters of rumen fluid were determined in the laboratory of chemical and analytical studies of the department of physiology and biochemistry of farm animals of the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst (Podolsk, Moscow region). In the rumen content, pH was determined directly after collection on an Aquilon 420 pH meter (Aquilon, Russia). During samples transportation from the farm to the lab, the samples were placed in a bag with refrigerating elements. The level of volatile fatty acids and ammonia was determined 2 h after sample collection. The rumen contents were then filtered through four layers of gauze and in the liquid part, the total amount of volatile fatty acids was determined by steam distillation in a Markham distillation unit apparatus and the concentration of ammonia nitrogen by the micro-diffusion method according to (22).

Data were statistically analyzed using the SPSS program (23), according to the following statistical model:

Yij = μ + Gi + Eij

Yij, is an observed value of the dependent variable; μ, is a constant common to all observations; Gi, is an effect due to ith treatment (1^st = 0 g Kormomix® Rumin), (2^nd = 25 g Kormomix® Rumin), (3^rd = 50 g Kormomix® Rumin), (4^th = 75 g Kormomix® Rumin); Eij, A random deviation due to unexplained sources of variation.

Percentage data were subjected to the arcsine value. Duncan's multiple range tests were used for multiple comparisons among means at (P < 0.05) (24). Kolmogorov–Smirnov's test was used to test the data's normal distribution.

Results of milk productivity indicators in the early lactation period when supplementing multicomponent synbiotic “Kormomix® Rumin” are presented in Table 3. The average daily milk yield and gross milk yield of natural fat were not significantly different (P > 0.05) among treatments, with the highest values at (33.87 & 4,064.80 kg) in the 2nd experimental group while the lowest values at (31.62 & 3,794.60 kg) in the control group Table 3. The same pattern was recorded in the case of the gross milk fat yield and milk protein yield.

The highest value for the average daily milk yield and gross milk yield of 4 FCM% was recorded in the 2nd experimental group and represented (34.12 & 4,094.22 kg) while the lowest values were recorded in the control group and represented (31.75 & 3,810.22 kg), respectively. Similarly, in the case of mass fraction of milk fat and protein % also, the highest values were recorded by the cows of the 2nd experimental group and represented (4.05 & 3.29 %). While the lowest were recorded by the control group and represented (4.02 & 3.23%), respectively, and the differences were not significantly differed (P > 0.05).

Results that summarize the effect of multicomponent synbiotic “Kormomix® Rumin” inclusion on rumen fermentation indicators are presented in Table 4.

The rumen fluid pH of all cows was within 6.87–7.10, which is located within the physiological norm. The total VFA content in the rumen of supplemented cows with multicomponent synbiotic “Kormomix® Rumin” exceeded the control group by 2.34, 1.36, and 1.82 mmol/100 ml, respectively. In the 2nd group, the cows were fed (25 g/head/day) multicomponent synbiotic “Kormomix® Rumin” in their diet had the highest VFA concentration, which represented (9.38 mmol/100 ml) and reliable difference (P < 0.05) compared to the control group (7.04 mmol/100 ml). Moreover, the level of ammonia in the ruminal content of supplemented groups was not significantly different among treatments, with the highest value at (10.01 mg/dl) in the 4th experimental group while the lowest at (7.65 mg/dl) in the control group.

The inclusion of multicomponent synbiotic “Kormomix® Rumin” in the diets of Russian Holstein cows under the study affected non significantly the fat and protein content in milk. Our results disagree with those obtained by (25, 26); they recorded an increase in the percentage and yield of milk protein in early lactating cows fed supplemental B. subtilis. Feeding the multicomponent synbiotic “Kormomix® Rumin” produced an additional 4% FCM in supplemented groups of Holstein dairy cows when compared with the control. Still, the difference among treatments was not significantly different. Consequently, we can assume that the inclusion of multicomponent synbiotic “Kormomix® Rumin” had no impact on milk productivity parameters compared with the control group. Our results come in the opposite direction from those obtained by (27), who reported that milk yield increased in response to supplementation of B. subtilis. They mentioned that cows fed 2 × 10¹¹ CFU of B. subtilis daily produced 1.7 kg more milk than the control group. Our results also did not match those obtained by (25, 26) they reported that the milk yield increased by (3.3 and 3.4 kg/day), respectively, compared to the control group when early lactating cows' diets.

In the rumen content such indices as the concentration of hydrogen ions (pH), the total amount of VFA and ammonia from different sides reflect the efficiency of the fermentation process. There was no impact on ruminal pH in supplemented cows with multicomponent synbiotic “Kormomix® Rumin” which may be due to more intensive carbohydrate fermentation with the formation of VFA as well, the ruminal fluid was likely taken in the morning where there was an insufficient number of carbonates in the saliva. Thus, we assumed that the inclusion of multicomponent synbiotic “Kormomix® Rumin” did not affect ruminal pH. This result follows those obtained by (18) who found no effects on ruminal pH when bacterial probiotics were administered to dairy cows suffering subacute ruminal acidosis.

Russian Holstein dairy cows fed (25 g/head/day) multicomponent synbiotic “Kormomix® Rumin” in their diet had the highest VFA concentration which differed (P < 0.05) from the control group. This may be attributed to the more intense digestibility of carbohydrates in the rumen of these cows. This result comes in the same way as those obtained by (7) they recorded a significant difference in total VFA between supplemented dairy cows with the 40 g of Saccharomyces cerevisiae in addition to 40 g of Lactobacillus plantarum and the control group. Conversely, the present finding contrasts with those obtained by (28). They mentioned that supplementation of bacteria-based probiotics revealed no difference in VFA concentration among experimental groups.

With regard to the level of ammonia in the ruminal content of supplemented groups, it was not significantly different among groups when compared to the control group. This finding disagrees with those obtained by (29) which recorded that treatment with feeding Lactobacilli (LAB) probiotic of the diet revealed significantly higher concentrations of ruminal ammonia when compared with non-supplemented animals.

The quantitative characteristics of biochemical blood parameters are one of the tools for controlling the nutrient and energy requirements of the body of animals. Blood glucose concentration recorded non-significant differences between different groups. Total alpha-amylase as a criterion for energy metabolism recorded the highest value in the blood of the control group and the lowest value in the 2nd experimental group. At the same time, the differences between the two groups were significant (30) mentioned that, the normal physiological limit of alpha-amylase in the blood of dairy cows (15–107 U/L). The addition of dietary digestive enzymes may improve the digestion of intestinal nutrients and provide additional nutrients for the development of normal microbiota. Intestinal microorganisms also play a vital role in the process of nutrient digestion, and can also affect the secretion of digestive enzymes (31).

Probably, the decrease in total alpha-amylase content is due to the optimal ratio of physiologically beneficial and pathogenic bacteria, the immune system, and the intestinal epithelial barrier, which in case of feeding disorders can lead to dysbiosis (disruption of the microbiota homeostasis). Due to the violation of the integrity of the intestinal mucosa, pathogenic microflora can enter the pancreas. The pancreas does not have its own microbiota, and therefore inflammatory and tumor processes affecting the gland may be associated with intestinal dysbiosis (32–34), which in turn is the cause of increases in the content of pancreatic enzymes, including amylase, in the blood. The inclusion of an additive based on probiotic and prebiotic cultures contributes to the improvement of the physico-chemical parameters of the rumen and in further work, the microbial community of the rumen of lactating cows will be studied. There is the fact that amylase in the blood when increased, it indicates a destructive effect on the intestines or pancreas. Thus, the lower level of amylase in the blood of supplemented cows represented a positive effect of the supplemental feed additive which means that the cows' bodies received additional metabolites of rumen fermentation, strengthening the metabolic and immune status of animals (35).

The observed creatinine concentrations indicate an increase in nitrogen metabolism in general, which is due to the physiological state of animals and is consistent with data on milk productivity and the chemical composition of milk. The decrease in the urea/creatinine ratio in the blood during the period is probably due to the high consumption of urea for the synthesis of microbial protein in the rumen and the use of muscle proteins for synthetic processes in the mammary gland of highly productive cows (3). The ratio of urea/creatinine intensively affects nitrogen metabolism and reflects the state of the kidneys the higher the ratio, the higher the probability of kidney disease due to impaired excretion of creatinine and urea.

The morphological studies of blood at the early lactation period revealed the superiority of Russian Holstein dairy cows supplemented with multicomponent synbiotic “Kormomix® Rumin” in their diets compared to the control group. Thus, we conclude that the inclusion of multicomponent synbiotic “Kormomix® Rumin” in diets of Russian Holstein dairy cows, in general, did not deteriorate the blood characteristics. Our results match those obtained by (27); they mentioned that hematological and biochemical parameters did not vary between dairy cows fed different treatments of B. subtilis.