Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on growth performance, immunity, fecal fermentation parameters and microbiota structure of suckling foals

Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on growth performance, immunity, fecal fermentation parameters and microbiota structure of suckling foals | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on growth performance, immunity, fecal fermentation parameters and microbiota structure of suckling foals Jia-Hao WANG, Wen-Jie ZHANG, Hao LU, Hai-Feng DENG, Hai LI, Zhen LIU, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4380361/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract (1) In this study, we investigated the effects of dietary supplementation with Lactobacillus rhamnosus and compound probiotics on the growth performance and microbiota structure of suckling Ili horse foals (aged 1 month; average weight 69.83 ± 13.76 kg). (2) At d 0, d 30, d 60 and d 90 of the regular feeding period, the weights of the foals were recorded, blood samples were obtained from the jugular vein and feces were collected by rectal extraction for the determination of microbial diversity and fermentation parameters. (3) The results showed that dietary supplementation with Lactobacillus rhamnosus and compound probiotics in 1-month-old suckling foals affected the production of volatile fatty acids in the intestine by changing the composition and abundance of intestinal microorganisms, promoting the digestion and absorption of nutrients, enhancing the level of nutrient metabolism and immunity to promote growth performance. (4) In conclusion, dietary supplementation with Lactobacillus rhamnosus and compound probiotics has a time-dependent positive effect on the growth performance of suckling foals. Biological sciences/Microbiology/Applied microbiology Biological sciences/Microbiology/Bacteria Biological sciences/Zoology/Animal physiology suckling foals Lactobacillus rhamnosus Compound probiotics Growth performance Immune index Fecal fermentation parameters Structure microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The growth and development of animals in the early stages of life at have a great influence on their later productive performance. Consequently, the conditions that provide a solid foundation for the healthy growth of young animals has become a focus of research worldwide. Nutrients are digested and absorbed in the gastrointestinal tract, which is also the largest immune organ in the body. Through the interaction between the intestinal mucosa and the external environment, healthy and stable intestinal microorganisms can inhibit colonization by pathogenic bacteria, improve the absorption of nutrients in the intestine and maintain the balance of the intestinal microecology [ 1 – 2 ]. In addition, the fermentation products of intestinal microorganisms provide nutrients to the organism or interfere with its health [ 3 ]. Therefore, the establishment of a healthy and stable intestinal environment in young mammals has a critical influence on intestinal fermentation capacity. The intestinal flora then promotes the level of nutrient metabolism in the organism, thereby improving growth performance and immunity. As an active microbial feed additive often used in livestock production and breeding programs, probiotics can eliminate harmful pathogens via several molecular mechanisms and modulate the immune response of the host to promote health. Blaise et al. [ 4 ] found that the activation of probiotics promotes the differentiation of B lymphocytes to plasma cells and increased production of secretory immunoglobulin A, which aids clearance of exogenous pathogenic bacteria and toxic and harmful molecules from the intestine and provides non-specific immune protection. A study by Yin et al. [ 5 ] indicated that dietary supplementation with Lactobacillus plantarum significantly increased the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a mouse model of acute enteritis, mitigating the damaging effects of the disease on the intestine and maintaining intestinal health. Thus, evidence suggests that probiotics enhance the growth performance of young animals by improving the absorption of nutrients and enhancing feed utilization, while also playing a key dominant role in the immune capacity. During birth, mammals are exposed to the external environment. The first intake of colostrum begins the colonization of the gastrointestinal tract with microorganisms, leading ultimately to the development of the intestinal flora. In foals, the physiological functions of the intestinal tract improve during the period from 0 to 6 months of age, although the balance of the intestinal microecology remains to be fully established [ 23 ]. At this stage, foals face significant stresses such as changes in diet structure from breast milk to plant-based feed. The stability of intestinal microorganisms is easily disturbed by the external environment, and the intestinal microecology is threatened. In this study, we investigated the effects of dietary supplementation with Lactobacillus rhamnosus and compound probiotics ( Lactobacillus , Bacillus licheniformis , Saccharomyces cerevisiae and Aspergillus ) on the growth performance and microbiota structure of suckling foals aged 1–4 months to provide a reference for promoting healthy growth. 2. Materials and methods 2.1 Experimental animals and materials Eighteen healthy suckling Ili horse foals (aged 1 month) from Zhaosu horse farm in Zhaosu County, Xinjiang, China with a similar birth date (± 7 d) and a mean weight of (69.83 ± 13.76) kg were selected for this study. Lactobacillus rhamnosus (live bacteria 1×10 10 CFU/g) were purchased from Xi'an Wanfang Biotechnology Co. Compound probiotics ( Lactobacillus , Bacillus licheniformis , Saccharomyces cerevisiae and Aspergillus ; live bacteria 2×10 9 CFU/g) were purchased from Inner Mongolia Heimei Kesheng Biotechnology Co. 2.2 Experimental design Eighteen foals were randomly divided into three groups (n = 6/group). Each day, the CK group received 0.65 kg/100 kg body weight supplement concentrate, the LGG group received Lactobacillus rhamnosus 1.00 g (live bacteria 1×10 10 CFU/g) on the basis of 0.65 kg/100 kg body weight supplement concentrate per day, and the CBM group received 5.00 g compound probiotics ( Lactobacillus , Bacillus licheniformis , Saccharomyces cerevisiae , and Aspergillus ; live bacteria 2×10 9 CFU/g) on the basis of 0.65 kg/100 kg body weight supplement concentrate per day. The trial period was 100 d, with a pre-feeding period of 10 d and a regular feeding period of 90 d. The foals and their dams were all from the same grazing pasture, and all foals were fed equal amounts of concentrate supplements per horse per day, after which the supplements were adjusted every 30 d ( Table 1 ). During the regular feeding period, samples were collected on d 0, d 30, d 60 and d 90. Table 1 Feeding amount of the concentrate supplement Items Average weight (kg) Feeding amount (kg/d/horse) 0–30 d 69.83 ± 13.76 0.46 30–60 d 87.64 ± 14.42 0.58 60–90 d 103.83 ± 14.90 0.68 2.3 Feeding management During the trial period, all foals were stabled in the outdoor barn from 8:00 to 19:00. The daily supplement concentrate was divided into three equal portions and foals were fed at 9:00, 14:00 and 18:00 with an appropriately sized feed pocket to ensure normal breathing and prevent spillage. At 9:00 every day, the foal was fed with probiotics in a capsule mixed with the concentrate to ensure that the capsule is consumed by the foal. The mare was milked during this period. Foals and mares were mixed from 19:00 to 8:00 the next day, during which time foals were free to take milk and water. The composition and nutritional level of the foal concentrate supplement are shown in Table 2 . Table 2 Composition and nutrient levels of the concentrates (DM basis) Ingredients Content Nutrient levels 2 Content Corn, % 52.00 OM, % 92.80 Wheat bran, % 12.00 CP, % 16.87 Wheat middings, % 10.00 NDF, % 20.81 Soybean meal, % 20.00 ADF, % 4.62 CaHPO 4 , % 2.00 Ca, % 0.72 NaCl, % 2.00 P, % 0.31 Limestone, % 1.00 GE, MJ/kg 17.79 Premix 1 , % 1.00 Total, % 100 1 The premix provided the following per kg of concentrate supplement: FeSO 4 ·7H 2 O 135.0 mg, KI 1.5 mg, MnSO 4 60.0 mg, ZnSO 4 60.0 mg, CuSO 4 15.0 mg, vitamin A 3600 IU, vitamin D 396 IU, vitamin E 1200 IU, vitamin B 1 4.5 mg, vitamin B 2 3.0 mg. 2 Nutrient levels are measured values. 2 OM: organic matter, CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, GE: gross energy 2.4 Sample collection and processing 2.4.1 Collection of growth performance data During the sampling period (d 0, d 30, d 60 and d 90 of the regular feeding period), the foals were driven to the center of the weighbridge (ID3000 Bluetooth® from TRU-TEST) before the morning feed. Weights were recorded when the foals were standing still and the weighbridge display was stable. 2.4.2 Collection and processing of plasma samples During the sampling period (d 0, d 30, d 60 and d 90 of the regular feeding period), blood samples were collected from the jugular vein (on an empty stomach before the morning feed) into sodium heparin anticoagulation tubes, centrifuged at 3500 r/min for 15 min, and the supernatant was stored frozen at -20℃. 2.4.3 Collection and processing of fecal samples During the sampling period (d 0, d 30, d 60 and d 90 of the regular feeding period), feces were collected by rectal extraction at 19:00. A portion of the sample was packed in sterile lyophilized tubes and snap frozen in liquid nitrogen for the determination of fecal microbial diversity; the remaining samples were packed in lyophilized tubes and frozen at -20 ℃ for the determination of fermentation parameters. 2.5 Measurement indexes and methods 2.5.1 Measurement of plasma indicators The following metabolic indicators were measured in foal plasma: urea (UREA), determined by urease assay; glucose (GLU), determined by glucose oxidase assay; growth hormone (GH) and insulin-like growth factor-1 (IGF-1), determined by enzyme-linked immunosorbent assay (ELISA). The following immunological indicators were measured in foal plasma: immunoglobulin A (IgA) and immunoglobulin G (IgG), determined by immunoturbidimetry; interleukin-2 (IL-2), IL-6 and interleukin-8 (IL-8), determined by ELISA. All measurements were performed using kits from Beijing Huaying Biotechnology Co., according to the manufacturer’s instructions. 2.5.2 Measurement of manure fermentation parameters The pH of feces was determined using a FiveEasy model benchtop instrument from METTLER TOLEDO. The concentration of ammoniacal nitrogen in feces was determined with an alkaline sodium hypochlorite-phenol colorimetric method using an Infinite M200 enzyme standard [ 6 ]. The concentration of volatile fatty acids, including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and total volatile fatty acids in feces was determined with a GC-2010 gas chromatograph (Shimadzu, Japan) using a crotonic acid internal standard method [ 7 ]. 2.5.3 High-throughput sequencing analysis of fecal microbial bacteria Genomic DNA from fecal samples was extracted using QIAamp DNA stool Mini Kit (Qiagen, Hilden, Germany). After extraction, the purity and concentration of the DNA was then checked by 0.8% agarose gel electrophoresis and NanoDrop 2000(Thermo Scientific, USA). Good quality extracted genomic DNA was used as a template for PCR amplification with the 16S rDNA V4 region specific primers 515F: 5'-GTTTCGGTGCCAGCMGCCGCGGTAA-3' and 806R: 5'- GCCAATGGACTACHVGGGTWTCTAAT-3'. All PCRs were carried out in a total reaction volume of 30 µL containing 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 µM forward and reverse primers, and approximately 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98℃ for 1 min, followed by 30 cycles of denaturation at 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation at 72℃ for 30 s, with a final incubation at 72℃ for 5 min. PCR mixtures were added to an equal volume of 1× loading buffer (containing SYBR green) and separated by 2% agarose gel for detection electrophoresis. Samples with a bright band of 400–450 bp were chosen for further experiments. PCR products was mixed in equal ratios and then purified with the GeneJET Gel Extraction Kit(Thermo Scientific). The Agilent 5400 fragment analyzer was used to confirm that the integrity, concentration and the total amount of the amplification production were met the requirements for library construction. Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced using the Illumina HiSeq platform and 250 bp paired-end reads were generated. Sequencing data were analyzed by QIIME2. We then prepared the downloaded sequencing data and barcodes, installed the working configuration environment and created the directory. After standardization of the original format, data were imported. The samples were then split by barcode, and the basic data information, including minimum reads, maximum reads, reads contained in each sample and quality information were obtained. Quality control of the data and removal of chimeras were performed based on the basic data information, and the final amplicon sequence variants (ASVs)were obtained by DADA2. The data for each sample were homogenized according to the least amount of data in the sample, and the phylogenetic tree was constructed and analyzed using sequences for calculating alpha and beta diversity, while the naïve Bayes classifier was used for sequence species annotation. 2.6 Statistical analysis The raw test data were initially organized in Excel, and the data were processed using one-way ANOVA and two-way ANOVA with SPSS 25.0, with multiple comparisons and analysis of variance between groups performed by Duncan’s test. Data were expressed as mean ± standard deviation. P < 0.05 indicated significant differences, and P < 0.01 indicated highly significant differences. 3. Results 3.1 Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on the growth performance of suckling foals As shown in Table 3 , the body weight of foals increased with age and was significantly higher ( P < 0.05) in the LGG and CBM groups than that in the CK group on d 90 of the trial (18.61% and 18.05% vs. CK group, respectively). During the whole trial period, the total weight gain and mean daily weight gain of foals in both the LGG and CBM groups were significantly ( P < 0.01) higher than those in the CK group. As shown in Fig. 1 , the foals gained weight faster in the prelactation period (d 0–30 the trial period) than in the middle and late lactation periods (d 30–90 of the trial period), and the average weight gain of foals in both the LGG and CBM groups was significantly higher than that of the CK group during all trial phases ( P < 0.01). Table 3 Effect of supplementary feeding of Lactobacillus rhamnosus and compound probiotics on body weight of suckling foals Items CK Group LGG Group CBM Group d 0 70.33 ± 15.72 69.92 ± 18.05 69.25 ± 8.40 d 30 84.00 ± 15.25 89.83 ± 19.76 89.08 ± 7.70 d 60 95.58 ± 15.09 108.50 ± 18.69 107.42 ± 7.37 d 90 105.67 ± 15.35 b 125.33 ± 19.20 a 124.75 ± 7.07 a Total weight gain 35.33 ± 2.04 Bb 55.42 ± 1.93 Aa 55.50 ± 2.43 Aa Average daily gain 0.40 ± 0.03 Bb 0.62 ± 0.02 Aa 0.62 ± 0.03 Aa Note: In the same row, values with different small letter superscripts indicate a significant difference ( P <0.05); values with different capital letter superscripts indicate an extremely significant difference ( P 0.05). 3.2 Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics plasma metabolic levels in suckling foals As shown in Table 4 , the plasma GLU levels decreased with increasing age in all groups of suckling foals. Plasma levels of GH and IGF-1 were highest at d 0 and lowest at d 30, and increased with increasing age thereafter. The plasma IGF-1 levels were significantly higher in the LGG group than those in the CK and CBM groups at d 0 ( P < 0.05). At d 30, the plasma UREA levels in the CBM group were significantly higher than those in the LGG group ( P < 0.05), while the plasma GH levels in the CK group were significantly higher than those in the LGG group ( P < 0.05). At d 60, the plasma levels of GLU in the LGG group were significantly higher than those in the CK group ( P < 0.05). The plasma levels of IGF-1 in the LGG group were also significantly higher than those in the CBM group ( P < 0.05). At d 90, the plasma GH levels in the LGG group were significantly higher than those in the CBM group ( P < 0.01). Treatment also had a significant effect on plasma UREA levels in suckling foals ( P < 0.05). In addition, age had a highly significant effect on plasma levels of GH, IGF-1 and GLU ( P < 0.01). Overall, we observed a significant interaction of treatment and age on plasma levels of GH and IGF-1 in suckling foals ( P < 0.05). Table 4 Effects of supplementary feeding with Lactobacillus rhamnosus and compound probiotics on plasma levels of metabolites in suckling foals Date Groups UREA, mmol/L GLU, mmol/L GH, ng/mL IGF-1, ng/mL d 0 CK 5.88 ± 1.88 6.04 ± 0.66 12.92 ± 2.21 306.36 ± 13.61 b LGG 4.73 ± 2.16 6.10 ± 0.54 15.86 ± 3.87 331.32 ± 22.08 a CBM 6.99 ± 1.30 5.96 ± 0.46 16.12 ± 2.63 307.47 ± 11.59 b d 30 CK 5.50 ± 0.54 ab 5.40 ± 0.90 4.86 ± 0.40 a 190.50 ± 14.00 LGG 4.97 ± 1.18 b 5.41 ± 1.00 4.29 ± 0.52 b 180.16 ± 18.86 CBM 6.57 ± 1.15 a 5.50 ± 0.97 4.49 ± 0.27 ab 180.65 ± 25.90 d 60 CK 4.63 ± 0.64 4.28 ± 0.53 b 5.55 ± 0.33 222.24 ± 14.44 ab LGG 4.66 ± 0.48 5.32 ± 0.63 a 5.38 ± 0.59 232.11 ± 18.90 a CBM 5.20 ± 0.71 4.83 ± 0.63 ab 5.24 ± 0.68 207.97 ± 14.95 b d 90 CK 5.82 ± 1.75 4.20 ± 0.93 6.55 ± 0.49 ABab 252.96 ± 20.02 LGG 5.57 ± 1.33 3.87 ± 0.74 7.45 ± 1.72 Aa 238.19 ± 8.34 CBM 5.76 ± 0.67 3.75 ± 0.76 5.56 ± 0.33 Bb 248.96 ± 13.17 P-value trt 0.011 0.624 0.261 0.161 day 0.072 0.001 0.001 0.001 trt*day 0.493 0.416 0.022 0.037 Note: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference ( P < 0.05); values with different capital letter superscripts indicate an extremely significant difference ( P 0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age. 3.3 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on plasma immune indexes in suckling foals As shown in Table 5 , the plasma levels of IL-2, IL-6 and IL-8 in all groups of suckling foals increased with age. There were no significant differences ( P > 0.05) in the immune indexes in the plasma of suckling foals in all groups at d 0 of the experiment. At d 30, the plasma levels of IgA and IgG were significantly ( P < 0.05) and highly significantly ( P < 0.01) increased in the LGG and CBM groups compared to those in the CK group. The plasma IL-2 levels were also significantly ( P < 0.05) higher in the LGG group than those in the CBM group and the plasma IL-6 levels were significantly ( P < 0.05) higher in the CK and CBM groups than those in the LGG group. At d 60, the plasma IL-2 levels of the LGG and CBM groups were significantly higher than those in the CK group ( P < 0.05). The plasma IL-6 levels in the CBM group were also significantly higher than those in the CK group ( P < 0.01), and the plasma IL-8 levels in the LGG group were significantly higher than those in the CK group ( P < 0.05). At d 90, plasma IL-2 levels in the CK and CBM groups were significantly higher than those in the LGG group ( P 0.05). However, there was a highly significant effect of age on all immune indexes in the plasma ( P < 0.01). Overall, there was a highly significant interaction between treatment and age on plasma levels of IL-2 ( P < 0.01) and IL-6 ( P < 0.05) in suckling foals. Table 5 Effects of supplementary feeding with Lactobacillus rhamnosus and compound probiotics on plasma immune indicators in suckling foals Date Groups IgA, g/L IgG, g/L IL-2, pg/mL IL-6, pg/mL IL-8, pg/mL d 0 CK 2.52 ± 0.86 18.23 ± 5.29 215.69 ± 20.64 116.74 ± 15.18 32.97 ± 5.17 LGG 2.66 ± 0.97 18.71 ± 5.83 229.36 ± 30.94 109.41 ± 7.36 33.09 ± 3.43 CBM 2.46 ± 0.55 19.16 ± 2.37 198.05 ± 39.24 105.58 ± 11.31 37.48 ± 2.35 d 30 CK 2.56 ± 0.71 b 17.59 ± 3.30 Bb 242.85 ± 15.75 ab 139.22 ± 5.32 a 39.85 ± 2.82 LGG 3.28 ± 0.44 a 23.40 ± 1.37 Aa 260.07 ± 16.58 a 132.31 ± 4.46 b 37.69 ± 6.40 CBM 3.47 ± 0.44 a 23.46 ± 1.30 Aa 237.41 ± 12.14 b 140.46 ± 4.79 a 38.06 ± 3.16 d 60 CK 3.34 ± 0.49 22.80 ± 1.45 252.04 ± 21.42 b 157.22 ± 10.08 Bb 39.44 ± 3.45 b LGG 3.53 ± 0.31 23.20 ± 1.39 272.27 ± 10.18 a 167.53 ± 17.37 ABab 44.60 ± 3.26 a CBM 3.30 ± 0.41 21.83 ± 1.41 277.00 ± 9.15 a 184.73 ± 14.74 Aa 42.57 ± 3.78 ab d 90 CK 3.11 ± 0.51 20.16 ± 2.83 289.44 ± 7.95 a 188.51 ± 3.89 46.60 ± 2.63 LGG 2.56 ± 0.79 19.87 ± 2.73 265.46 ± 18.44 b 190.65 ± 12.45 45.64 ± 2.25 CBM 2.89 ± 0.64 20.39 ± 1.85 284.49 ± 15.69 a 199.18 ± 19.23 45.17 ± 3.87 P-value trt 0.677 0.122 0.367 0.052 0.593 day 0.001 0.002 0.001 0.001 0.001 trt*day 0.172 0.080 0.008 0.012 0.078 Note: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference ( P < 0.05); values with different capital letter superscripts indicate an extremely significant difference ( P 0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age. 3.4 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on fermentation parameters in the feces of suckling foals 3.4.1 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on fecal pH in suckling foals As shown in Fig. 2 , the fecal pH in suckling foals ranged from 5.49 to 7.12 between 1 and 4 months of age, and increased and then decreased with age, reaching the highest value at d 30. At d 60, the fecal pH in the LGG group was significantly lower than that in the CK group ( P < 0.05). At d 90, the fecal pH in the LGG group was significantly lower than that in the CK group ( P < 0.05) and very significantly lower than that in the CBM group ( P < 0.01). 3.4.2 Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on NH 3 -N concentration in the feces of suckling foals As shown in Fig. 3 , the NH 3 -N concentration in the feces of suckling foals fluctuated with age. At d 30, the highest fecal NH 3 -N concentration was detected in the CK group, reaching the highest value at 30 d, while the lowest value was detected in the LGG and CBM groups. At d 30, the fecal NH 3 -N concentration in the LGG group was significantly lower than that in the CK group ( P < 0.01). The fecal NH 3 -N concentration in the CBM group was also significantly lower than that in the CK group ( P 0.05). 3.4.3 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on volatile fatty acids in the feces of suckling foals As shown in Table 6 , the volatile fatty acid concentrations in the feces of suckling foals fluctuated with age. It is noteworthy that the total volatile fatty acid concentrations in the LGG and CBM groups decreased by 20.61% and 11.17% at d 30, while the CK group showed an increasing trend. At d 0, the fecal concentrations of valeric acid, isobutyric acid and isovaleric acid in the CBM group were significantly higher than those in the CK group ( P 0.05). At d 30, the fecal concentrations of isobutyric acid and isovaleric acid in the CBM group were significantly higher than those in the LGG group ( P 0.05). At d 90, the fecal concentrations of acetic acid, propionic acid, valeric acid and total volatile fatty acids in the CBM group were significantly higher than those in the CK group ( P 0.05). Treatment had a highly significant effect ( P < 0.01) on isobutyric acid and isovaleric acid concentrations in the feces of suckling foals and a significant effect ( P < 0.05) on fecal concentrations of propionic acid, butyric acid, valeric acid and total volatile fatty acids. Age had a highly significant effect ( P < 0.01) on fecal butyric acid concentration and a significant effect ( P 0.05). Table 6 Effects of supplementary feeding with Lactobacillus rhamnosus and compound probiotics on fecal volatile fatty acids of suckling foals, µmol/g Date Groups Total VFA Acetate Propionate Butyrate Valerate Isobutyrate Isovalerate d 0 CK 44.26 ± 6.27 26.80 ± 4.81 9.35 ± 1.30 5.78 ± 0.71 0.35 ± 0.03 b 0.83 ± 0.08 b 1.16 ± 0.15 b LGG 54.58 ± 9.42 33.46 ± 7.25 10.90 ± 1.91 7.18 ± 1.69 0.54 ± 0.11 ab 1.02 ± 0.15 ab 1.47 ± 0.28 ab CBM 57.63 ± 18.03 34.40 ± 11.53 12.08 ± 3.28 7.67 ± 2.31 0.61 ± 0.30 a 1.16 ± 0.29 a 1.71 ± 0.59 a d 30 CK 45.05 ± 9.05 27.98 ± 6.94 9.10 ± 1.16 5.47 ± 0.70 0.44 ± 0.19 0.91 ± 0.25 ab 1.15 ± 0.37 ab LGG 43.33 ± 7.42 26.39 ± 5.39 9.21 ± 1.18 5.67 ± 0.95 0.33 ± 0.11 0.76 ± 0.07 b 0.97 ± 0.09 b CBM 51.19 ± 12.07 32.67 ± 9.65 9.47 ± 1.31 5.94 ± 0.46 0.49 ± 0.15 1.11 ± 0.24 a 1.51 ± 0.39 a d 60 CK 49.33 ± 9.57 31.97 ± 7.42 9.23 ± 0.92 5.55 ± 0.72 0.49 ± 0.16 0.89 ± 0.22 1.20 ± 0.40 LGG 52.19 ± 9.44 33.31 ± 6.68 9.79 ± 1.39 6.44 ± 1.23 0.45 ± 0.13 0.95 ± 0.16 1.24 ± 0.23 CBM 49.35 ± 5.52 31.55 ± 4.35 9.47 ± 0.73 5.70 ± 0.38 0.45 ± 0.08 0.94 ± 0.12 1.24 ± 0.24 d 90 CK 41.75 ± 5.63 b 25.21 ± 4.28 b 8.99 ± 0.65 b 5.37 ± 0.48 0.32 ± 0.08 b 0.78 ± 0.09 1.07 ± 0.20 LGG 47.95 ± 6.70 ab 29.94 ± 4.93 ab 9.66 ± 0.54 ab 5.90 ± 0.79 0.43 ± 0.13 ab 0.83 ± 0.21 1.19 ± 0.34 CBM 51.54 ± 6.81 a 32.24 ± 4.76 a 10.31 ± 1.50 a 6.17 ± 0.77 0.51 ± 0.12 a 0.93 ± 0.15 1.37 ± 0.35 P-value trt 0.037 0.064 0.030 0.017 0.024 0.002 0.004 day 0.269 0.369 0.017 0.006 0.321 0.090 0.097 trt*day 0.186 0.567 0.478 0.441 0.086 0.124 0.295 Note: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference ( P < 0.05); values with different capital letter superscripts indicate an extremely significant difference ( P 0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age. 3.5 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on fecal flora of suckling foals 3.5.1 Experimental design and sequencing depth validation Species accumulation curves are used to measure and predict the magnitude of species richness in a community as the sample size increases, and are widely used in biodiversity and community surveys to judge the adequacy of sample size and estimate community richness. As can be seen in Fig. 4 , the curve tended to be flat, confirming that the 72 samples investigated in this study are sufficient to reflect to the species composition of the community and validating our experimental design. A dilution curve is constructed by randomly selecting a certain number of individuals from the sample, counting the number of species represented by these individuals, and plotting the number of individuals versus the number of species. This method can be used to evaluate whether the sequencing volume is sufficient to cover all taxa and indirectly reflect the richness of species in the sample. As shown in Fig. 5 , the curve leveled off when the sequencing depth reached 40,000, confirming that the sequencing depth in this experiment basically covered all species in the sample and no more new species could be found by increasing the amount of sequencing data. 3.5.2 Venn diagram After obtaining the ASVs by noise reduction and according to the research requirements, the common and unique ASVs among the different groups were analyzed. The number of members in each set (i.e., the number of ASVs unique to each subgroup and common among groups) was counted according to their presence or absence among groups and the corresponding Venn diagram was constructed. As shown in Fig. 6 , at d 0 of the trial, 2,067 ASVs were specific to the CK group, 2,301 were specific to the LGG group, 2,187 were specific to the CBM group, and 1,586 were common to all three groups. At d 30, 1, 273ASVs were specific to the CK group, 2,494, were specific to the LGG group, and 1.923 were common to all three groups. At d 60, 954 ASVs were specific to the CK group, and 1,629 were specific to the LGG group. At d 30, there were 1,273 ASVs in the CK group, 2,494 in the LGG group, 1,629 in the CBM group, and 1,923 in the three groups. At d 60, there were 954 ASVs in the CK group, 2,251 in the LGG group, 1,572 in the CBM group, and 2,089 in the three groups. At d 90, there were 1,515 ASVs in the CK group, 1,515 in the LGG group, and 1,586 in the CBM group. At d 90, 1,515 ASVs were specific to the CK group, 2,117 were specific to the LGG group, 1,892 were specific to the CBM group, and 2,496 were common to all three groups. As shown in Fig. 7 , there were 2,019, 1,458, 977 and 1,785 ASVs in the CK group at d 0, d 30, d 60 and d 90, respectively, with a total of 1,212 ASVs for the whole trial period. In the LGG group, there were 2,089, 1,666, 1,998 and 1,911 ASVs at d 0, d 30, d 60 and d 90, respectively, with a total of 1,456 ASVs for the whole trial period. In the CBM group, there were 2,027, 1,324, 1,619 and 1,857 ASVs at d 0, 30, 60 and 90, respectively, with a total of 1,526 ASVs for the whole trial period. 3.5.3 Microbiota diversity analysis The alpha diversity index represents the number of species in a local homogeneous habitat as a comprehensive reflection of the richness and homogeneity. The Chao1 index is used to estimate the total number of species contained in the community sample; the Chao1 index increases with the number of species present in the community at low abundance. The Shannon index represents the total number and proportion of taxa in the sample; the Shannon index increases with the diversity of the community and the uniformity of the species distribution. The Simpson index characterized the diversity and uniformity of species distribution in the community; the Simpson index increases with the uniformity of the species distribution. As shown in Table 7 , the alpha diversity index increased with age in all groups of suckling foals. At d 0, the observed_ASV and Chao1 indexes were significantly higher in the CBM group than those in the CK group ( P < 0.05). At d 30, the observed_ASVs and Chao1 indexes of the LGG group were significantly higher than those of the CK group ( P 0.05), while the alpha diversity indexes in both the LGG and CBM groups tended to increase compared to those in the CK group. At d 90, the observed_ASVs and Chao1 indexes of the LGG group were significantly higher than those of the CK group ( P 0.05), while the indexes of both the LGG and CBM groups showed an increasing trend compared to those of the CK group. There was a highly significant effect of treatment on the observed_ASVs and Chao1 indexes in suckling foals ( P < 0.01). Although there was a highly significant effect of age on the alpha diversity index in suckling foals ( P 0.05). Table 7 Alpha diversity in sucking foals. Date Group observed_ASVs Chao1 index Shannon index Simpson index d 0 CK 1219.67 ± 317.73 b 1270.61 ± 336.66 b 7.09 ± 0.99 0.95 ± 0.04 LGG 1384.67 ± 238.67 ab 1430.95 ± 241.46 ab 7.64 ± 0.72 0.97 ± 0.01 CBM 1590.00 ± 273.32 a 1670.50 ± 298.46 a 7.88 ± 0.63 0.98 ± 0.01 d 30 CK 1479.00 ± 275.70 b 1528.86 ± 274.84 b 7.77 ± 0.81 0.97 ± 0.02 LGG 1908.50 ± 310.64 a 1988.63 ± 339.88 a 8.69 ± 0.42 0.99 ± 0.01 CBM 1588.50 ± 341.92 ab 1632.72 ± 349.09 ab 8.22 ± 1.11 0.97 ± 0.03 d 60 CK 1580.17 ± 332.92 1618.83 ± 356.23 8.79 ± 0.64 0.99 ± 0.01 LGG 1898.00 ± 608.74 1973.40 ± 662.85 8.74 ± 1.07 0.99 ± 0.02 CBM 1798.17 ± 148.40 1855.08 ± 143.77 9.04 ± 0.27 0.99 ± 0.01 d 90 CK 1816.83 ± 162.63 b 1857.82 ± 170.06 b 9.09 ± 0.20 0.99 ± 0.01 LGG 2183.33 ± 257.21 a 2277.01 ± 288.97 a 9.21 ± 0.36 0.99 ± 0.01 CBM 2030.67 ± 308.39 ab 2095.35 ± 325.13 ab 9.19 ± 0.38 0.99 ± 0.01 P-value trt 0.003 0.002 0.091 0.304 day 0.001 0.001 0.001 0.001 trt*day 0.639 0.555 0.554 0.363 Note: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference ( P < 0.05); values with different capital letter superscripts indicate an extremely significant difference ( P 0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age. Beta diversity represents the dissimilarity of species composition between communities of different habitats along an environmental gradient or the rate of species turnover along an environmental gradient, and is therefore also known as interhabitat diversity; this index is therefore used to study the relationship between communities in terms of species multiplicity. The samples were clustered based on a weighted unifrac distance matrix using unweighted pair group averaging method (UPGMA, unweighted pair group method with arithmetic mean). For each sample to be displayed, the clustering results were integrated with the relative abundance of species at the gate level. As shown in Fig. 8 , the LGG and CBM groups had shorter branch lengths than the CK group at d 0, indicating that the LGG and CBM groups had similar bacterial communities. The LGG and CBM groups clustered together first, and then clustered with the CK group as one group. At d 30, the LGG and CBM groups clustered together, while the CK and LGG groups at d 60 clustered together with the CK group at d 30 indicating that the bacterial composition of the pairs of groups was similar at both stages. At d 90, the CK and LGG groups clustered together, while the bacterial communities of the CBM groups at d 60 and d 90 were similar and clustered together. Finally, the CK, LGG and CBM groups at d 90 clustered together with the CBM group at d 60. 3.5.4 Microbiota composition analysis As shown in Table 8 , the dominant phyla of the fecal bacteria in suckling foals comprised Firmicutes , Verrucomicrobia , Bacteroidetes , Proteobacteria , Euryarchaeota , Actinobacteria and Spirochetes . In each group, the relative abundance of Bacteroidetes in the fecal bacteria of suckling foals increased with age. At d 0, the relative abundance of Spirochetes was significantly higher in the CBM group than in those in the CK and LGG groups ( P 0.05). At d 30, the relative abundance of Firmicutes in the LGG group was significantly higher than that in the CK group ( P < 0.01), and the relative abundance of Firmicutes in the CBM group was significantly higher than that in the CK group ( P < 0.05). At d 60, the relative abundance of Bacteroidetes in the CBM group was significantly higher than that in the CK group ( P < 0.05) and the relative abundance of Proteobacteria in the LGG group was significantly higher than that in the CK group ( P 0.05). There was no significant effect of treatment on the dominant phylum in the fecal bacteria of suckling foals ( P > 0.05). However, there was a highly significant effect of age on the relative abundance of the Firmicutes , Bacteroidetes , Euryarchaeota , Actinobacteria and Spirochetes in the fecal bacteria of suckling foals ( P 0.05). Table 8 The relative abundance (%) of fecal flora at the phylum level in suckling foals Date Group Firmicutes Verrucomicrobiota Bacteroidota Proteobacteria Euryarchaeota Actinobacteriota Spirochaetota D 0 CK 53.15 ± 11.60 19.04 ± 14.48 6.80 ± 5.45 4.06 ± 3.56 10.70 ± 8.81 3.69 ± 4.34 0.41 ± 0.35 b LGG 58.77 ± 14.10 10.86 ± 10.18 9.71 ± 3.67 5.13 ± 6.08 9.00 ± 7.77 3.29 ± 1.38 0.44 ± 0.30 b CBM 58.85 ± 6.92 11.58 ± 6.51 8.93 ± 3.73 2.81 ± 2.39 12.41 ± 2.11 2.70 ± 1.05 0.97 ± 0.59 a D 30 CK 47.38 ± 12.98 Bb 17.70 ± 13.80 16.67 ± 10.76 3.97 ± 5.87 7.68 ± 6.07 3.70 ± 2.90 1.35 ± 1.67 LGG 63.48 ± 3.59 Aa 9.69 ± 4.78 12.17 ± 3.73 3.17 ± 1.80 3.42 ± 2.08 4.66 ± 4.69 1.86 ± 1.29 CBM 61.63 ± 8.10 AaB 14.40 ± 7.82 14.72 ± 5.42 1.05 ± 0.79 4.35 ± 3.83 1.34 ± 0.78 1.34 ± 0.66 D 60 CK 60.62 ± 12.47 14.09 ± 6.27 15.00 ± 5.82 b 0.47 ± 0.42 b 5.87 ± 8.02 0.80 ± 0.55 2.09 ± 1.01 LGG 56.11 ± 7.18 17.44 ± 6.89 16.18 ± 8.46 ab 2.53 ± 2.15 a 2.97 ± 1.97 1.21 ± 1.02 1.33 ± 0.93 CBM 52.03 ± 6.91 17.29 ± 5.36 24.18 ± 5.55 a 1.06 ± 0.68 ab 1.94 ± 1.38 0.66 ± 0.44 1.56 ± 1.07 D 90 CK 43.44 ± 6.92 18.05 ± 8.96 26.57 ± 11.41 4.95 ± 7.27 1.05 ± 0.63 1.15 ± 0.54 2.65 ± 1.37 LGG 44.62 ± 6.85 11.52 ± 5.59 31.49 ± 7.47 3.98 ± 2.77 2.05 ± 2.46 1.11 ± 0.67 2.86 ± 1.36 CBM 48.62 ± 7.13 14.26 ± 4.43 23.36 ± 6.57 3.46 ± 2.78 4.24 ± 4.23 2.01 ± 1.72 1.88 ± 0.89 P-value trt 0.173 0.153 0.738 0.296 0.375 0.363 0.788 day 0.001 0.816 0.001 0.100 0.001 0.002 0.001 trt*day 0.071 0.612 0.092 0.904 0.519 0.378 0.407 Note: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference ( P < 0.05); values with different capital letter superscripts indicate an extremely significant difference ( P 0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age. 3.6 Correlation analysis of fecal flora and volatile fatty acids in suckling foals Spearman’s rank correlation analysis revealed that the relative abundance of the top 10 ranked bacterial genera in the feces of suckling foals in each group was correlated with the concentration of volatile fatty acids in the feces. As shown in Fig. 9 (a) , there was no significant correlation ( P > 0.05) between the concentration of volatile fatty acids and the relative abundance of the top 10 genera of bacteria ranked in the feces of foals in the CK group. As shown in Fig. 9 (b) , the relative abundance of the Christensenellaceae_R-7_group in the fecal microflora of the LGG group was highly significantly and positively correlated with the concentrations of propionic acid and butyric acid ( P < 0.01). The relative abundances of WCHB1-41 and the Rikenellaceae_RC9_gut_group were highly significantly and negatively correlated with the concentrations of propionic acid and butyric acid ( P < 0.01). The relative abundance of Methanobrevibacter was highly significantly and negatively correlated with the concentrations of valeric acid ( P < 0.01). As shown in Fig. 9 (c) , the relative abundances of Methanobrevibacter , Chlamydia and Saccharofermentans in the fecal microflora of foals in the CBM group were highly significantly and positively correlated with the concentrations of acetic acid and isovaleric acid ( P < 0.01). The relative abundances of WCHB1-41 , Rikenellaceae_RC9_gut_group and F082 were highly significantly and negatively correlated with the concentrations of acetic acid and isovaleric acid ( P < 0.01). 4. Discussion From 1 to 4 months of age is a critical stage in foal growth. At this stage, the foal grows fast, leading to high nutrient requirements. During weaning, the amount of fiber feed in the diet is gradually increased, and the intake of large amounts of fiber affects the gastrointestinal tract structure and function, leading to microbial bacteria instability, increased susceptibility to pathogenic bacteria as well as inhibition of normal bacterial colonization and proliferation, thus affecting the foal's health, growth and development. Supplemental feeding is the main approach to supporting the foal to cope with the stresses associated with these changes in diet and the feeding environment that are experienced during this stage. Probiotics can improve the activity of digestive enzymes in the gastrointestinal tract of animals, improve the absorption efficiency of nutrients, increase the conversion rate of feed, regulate the balance between the beneficial and harmful bacterial groups in the gastrointestinal tract of animals, and promote the healthy growth of animals. 4.1 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on the growth performance of suckling foals The lactation period is a critical for the growth and development of foals, with body weight representing a visual indicator of foal growth [ 8 ]. The gastrointestinal tract of foals at this stage is still developing and has a weak ability to digest and absorb nutrients from forage, and relying only on nutrients from breast milk cannot fully meet their growth needs [ 9 ]. Probiotic supplementation effectively increased the digestibility of nutrients in Holstein calves, thus improving their health and growth performance [ 10 ]. A study by Bocourt et al. [ 11 ] confirmed that supplemental feeding with Lactobacillus rhamnosus improved the average daily weight gain and meat-feed ratio of early weaned piglets. In the present study, long-term supplementation with Lactobacillus rhamnosus and compound probiotics in foals aged 1–4 months significantly increased the body weight of foals at d 90 of the trial, and significantly increased the total weight gain and average daily weight gain of foals during the whole trial period. These findings confirmed that probiotics had a positive effect on the body weight of suckling foals, and this effect increased with the supplementation time. The weight gain rate of foals aged 1–4 months was more rapid in the prelactation period than in the late lactation period, indicating that the weight gain rate slowed down with age, which is consistent with the findings of Juliand et al. [ 12 ]. 4.2 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on plasma metabolic levels in suckling foals Plasma UREA, GLU, GH and IGF-1 are not only important indicators of nutrient absorption and metabolism, but also key indicators for monitoring growth and development. UREA in plasma is a product of protein catabolism and can reflect the balance of nitrogen metabolism and amino acids [ 13 ], while GLU levels reflect the dynamic balance of sugar absorption, transport and metabolism of the organism [ 14 ] and GH and IGF-1 levels reflect the ability to promote growth [ 15 ]. In the present study, supplemental feeding with Lactobacillus rhamnosus significantly increased the plasma levels of GLU and IGF-1 in foals at d 60 and GH at d 90. Supplemental feeding with compound probiotics significantly increased the plasma levels of UREA in foals at d 30. This indicates that Lactobacillus rhamnosus plays a prominent role in improving blood glucose metabolism and regulating the levels of related growth hormones in suckling foals. Supplementation with compound probiotics enhanced protein metabolism in suckling foals. The plasma GLU levels in suckling foals decreased with age, indicating that the hindgut fermentation capacity of foals gradually increased with age and changes in feeding habits. Plasma levels of GH and IGF-1 in suckling foals decreased and then increased with age, indicating that the hormone levels in newborn foals were high and gradually decreased with age to fluctuate within the normal range, which is consistent with the results of other mammalian studies [ 16 ]. 4.3 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on plasma immune indexes in suckling foals IgA is the main component of the mucosal defense system and is the first line of defense against pathogenic invasion. Among the plasma immunoglobulins, IgG occurs at the highest concentrations, and is an important component in the resistance to infection [ 17 ]. IL-2 is an indicator of anti-tumor immune function and not only stimulates the growth, proliferation and differentiation of T cells, but also functions as an important immune enhancer [ 18 ]. IL-6 is an important cytokine expressed in the initial response of the innate immune system to injury and infection [ 19 ]. IL-8 acts as a chemoattractant and activates neutrophils, and promotes the lysosomal enzyme activity of neutrophils and phagocytosis [ 20 ]. In the present study, supplementation with Lactobacillus rhamnosus and compound probiotics significantly increased the plasma levels of IgA and IgG in foals at d 30. The plasma levels of IL-2 and IL-8 were also significantly increased in foals at d 30 and d 60. Supplementation with compound probiotics significantly increased the plasma levels of IL-2 and IL-6 in foals at d 30, d 60 and d 90. Probiotic supplementation also significantly increased the levels of IL-2 and IL-6 in the plasma of foals at 30, 60 and 90 days of the trial, indicating that prolonged supplementation with probiotics could improve the immunity of suckling foals, thus protecting them from external environmental stresses and promoting healthy growth. Furthermore, the plasma levels of IL-2, IL-6 and IL-8 in suckling foals increased with age, indicating that the foals were exposed to more antigens during their growth and development, and the antibody levels increased, thus improving their immunity. 4.4 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on fermentation parameters of manure in suckling foals The fermentation products of the intestinal microflora of suckling foals provide nutrients and metabolites, such as volatile fatty acids, which have various biological functions such as providing energy for foals, maintaining intestinal integrity, controlling the intestinal flora and regulating immune function. NH 3 -N, which is the main source of nitrogen for intestinal microflora fermentation, is a key indicator of microbial activity. NH 3 -N is also one of the metabolites of the nitrogen cycle. A stable pH is required to provide a favorable environment for intestinal microorganisms [ 21 ]. In the present study, we found that the pH of feces of suckling foals tended to increase and then decrease with time, reflecting the fluctuations in the concentrations of NH 3 -N and volatile fatty acids. The concentration of NH 3 -N and total volatile fatty acids in the feces of foals supplemented with Lactobacillus rhamnosus and compound probiotics decreased at d 30 compared with the control group, indicating that the probiotics accelerated the nitrogen cycle in the suckling foals and enhanced nitrogen utilization. Supplementation with Lactobacillus rhamnosus significantly decreased the pH of feces at d 60 and 90, while supplementation with Lactobacillus rhamnosus and compound probiotics significantly decreased the pH of feces at d 30. In the feces of foals supplemented with Lactobacillus rhamnosus , the concentrations of isobutyric acid and isovaleric acid at d 30 and the concentrations of total volatile fatty acid, acetic acid, propionic acid and valeric acid at d 90 were significantly increased, indicating that supplementation with probiotics effectively improved the intestinal fermentation capacity of suckling foals and lays the foundation for the stable development of intestinal flora. 4.5 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on the structure of the fecal flora of suckling foals ASVs are de-duplicated sequences generated by the DADA2 method after noise reduction, and replace OTUs to improve the accuracy, comprehensiveness and reproducibility of marker gene data analysis. In addition, ASVs can reflect the abundance of species or numbers of microflora in sequenced samples. Proudman et al. [ 22 ] detected OTUs in the range 1,200–3,000 in the feces of thoroughbred racehorses. In this study, the number of ASVs in the feces of foals supplemented with Lactobacillus rhamnosus and compound probiotics was found to be significantly higher than that in the control group, indicating that supplementation with probiotics increases the species and number of fecal bacteria in foals. In accordance with the findings reported by Costa et al. [ 23 ], we found that the changes in the number of ASVs in the feces of suckling foals indicated that foals are infected with bacteria after birth and, immediately after natural mechanisms are activated to remove toxic and harmful bacteria, the foals then acquired more bacteria through exposure to the external environment and forage feeding until their flora is similar to adult horses. During this process, suckling foals are affected by various external factors that may disrupt the intestinal flora, and supplemental feeding of probiotics can effectively and steadily increase the types and amounts of bacteria in the intestinal tract of suckling foals. The alpha diversity index is a comprehensive index reflecting the abundance and homogeneity of sequenced samples. In this study, supplementation with Lactobacillus rhamnosus significantly increased the observed_ASVs and Chao1 indexes of foal feces microorganisms at d 30 and d 90. The alpha diversity index of the feces of foals supplemented with compound probiotics tended to increase between d 30 and 60, indicating that dietary supplementation with probiotics can increase the diversity and richness of the intestinal microflora in suckling foals. These findings are consistent with those reported by Vladimir et al. [ 24 ] following oral administration with Lactobacillus rhamnosus and Enterococcus faecalis to foals. Costa et al. [ 24 ] showed that the intestinal microflora of foals changes over time. Similarly, we found that the diversity and richness of the intestinal microflora of suckling foals increased with age. These results are consistent with the increase in the diversity and richness of the intestinal microflora with age, indicating that the diversity and homogeneity of the intestinal flora can be optimized by long-term supplementation with probiotics. The intestinal microflora of suckling foals gradually increases in complexity, diversity and stability with age and changes in feeding habits and structure of the diet [ 25 ]. The intestinal microflora plays an important role in various physiological functions, such as promoting nutrient absorption, regulating substance metabolism, and regulating immunity and antioxidant capacity. Disturbance of the intestinal flora can lead to diarrhea and enteritis. In this study, the dominant phyla of the fecal bacteria in suckling foals were Firmicutes , Verrucomicrobia , Bacteroidetes , Proteobacteria , Euryarchaeota , Actinobacteria and Spirochetes . The relative abundances of Firmicutes and Proteobacteria in the feces of suckling foals was significantly increased at d 30 and d 60 by long-term supplementation with Lactobacillus rhamnosus . The relative abundances of the Firmicutes and Bacteroidetes in the feces of suckling foals was significantly increased at d 30 and d 60 by long-term supplementation with compound probiotics. Firmicutes and Bacteroidetes are important components of the foal gut microflora and are involved in key metabolic activities in vivo. In forage, Firmicutes acts on fibrous substances [ 26 ] and Bacteroidetes acts on starchy substances [ 27 ]. Although numerous studies have confirmed that the abundance of Proteobacteria is closely associated with microecological dysregulation in the host gut [ 28 ], Proteobacteria is also an important phylum that explains the significant functional variation in the gut microbiome [ 29 ]. The relative abundance of many relevant microbial bacteria, such as the Bacteroidetes , in foals will gradually increase with the intake of roughage and changes in the structure of the diet. At the same time, the composition and abundance of fecal microbial bacteria in suckling foals may change other external environmental influences as increasing age. Supplemental feeding with probiotics will have a significant effect on the composition and relative abundance of intestinal microbial bacteria in suckling foals, improving the digestion and absorption of nutrients, promoting healthy intestinal development and improving intestinal physiological functions. 4.6 Correlation analysis of fecal flora and volatile fatty acids in suckling foals The intestinal flora of suckling foals plays an important role in maintaining intestinal health by regulating the intestinal environment and functions of the organism through the production of volatile fatty acids, such as acetic acid, propionic acid and butyric acid, through degradation of cellulose and hemicellulose, thereby regulating intestinal pH, reducing the colonization of pathogenic bacteria and alleviating intestinal infections and inflammation caused by pathogenic bacteria [ 4 ]. At the same time, volatile fatty acids in the foal’s intestine regulate the structure and abundance of the microbial community and thus maintain the flora in benign dynamic balance. In the present study, there was no significant correlation between the relative abundance of fecal microbial bacteria and the content of volatile fatty acids in control foals. However, in the foals supplemented with Lactobacillus rhamnosus , there was a significant positive correlation between the relative abundances of the Christensenellaceae_R-7_group and the concentrations of propionic acid and butyric acid in the feces. The relative abundances of WCHB1-41 , the Rikenellaceae_RC9_gut_group and Methanobrevibacter were significantly and negatively correlated with fecal concentrations of propionic acid, butyric acid and valeric acid. The relative abundances of Methanobrevibacter , Chlamydia and Saccharofermentans in the fecal microflora of foals supplemented with compound probiotics were highly significantly and positively correlated with the concentrations of acetic acid and isovaleric acid in feces. The relative abundances of WCHB1-41 , the Rikenellaceae_RC9_gut_group and F082 were highly and negatively correlated with the concentrations of acetic acid and isovaleric acid. These findings indicate that dietary supplementation with probiotics affects the physiological function of the intestinal tract and homeostasis of the internal environmental of suckling foals by altering the composition of microbial bacterial communities in the intestine and by promoting or inhibiting the ability of the foals to produce volatile fatty acids by intestinal fermentation. Despite the significant conclusions of this study, there are still some shortcomings. First, we did not quantitatively analyze beneficial bacteria such as Bifidobacterium and harmful bacteria such as Salmonella ; second, due to the species used in our experimental animals, it was difficult to collect the contents of the various intestinal segments of the foals. In further studies, we plan to investigate the that the addition of beneficial bacteria immediately after birth may be more effective for early microbial colonization, and quantitative analysis of bacteria by collecting the intestinal contents of foals may also be more meaningful. 5. Conclusions In this study, we showed that dietary supplementation with Lactobacillus rhamnosus and compound probiotics had a positive and effect on the growth performance of suckling foals aged suckling foals aged 1–4 months, and that this effect was enhanced with time. We concluded that this effect was mediated by the production of volatile fatty acids in the intestine induced by changes in the composition and abundance of intestinal microorganisms, thereby promoting the digestion and absorption of nutrients, and enhancing nutrient metabolism and host immunity, to promote healthy growth. Declarations Acknowledgments: We would like to thank the staff at our laboratory for their ongoing assistance. Author Contributions: Conceptualization, Jiahao WANG, Wenjie ZHANG, Kailun YANG and Fengming LI; Data curation, Jiahao WANG, Wenjie ZHANG and Fengming LI; Formal analysis, Jiahao WANG, Wenjie ZHANG, Zhen LIU, Kailun YANG and Fengming LI; Funding acquisition, Kailun YANG; Investigation, Jiahao WANG, Wenjie ZHANG and Hao LU; Methodology, Jiahao WANG, Wenjie ZHANG, Haifeng DENG, Hai LI, Zhen LIU, Kailun YANG and Fengming LI; Project administration, Kailun YANG and Fengming LI; Resources, Wenjie ZHANG, Hao LU, Haifeng DENG, Hai LI, Kailun YANG and Fengming LI; Supervision, Kailun YANG and Fengming LI; Validation, Jiahao WANG, Wenjie ZHANG, Hao LU and Fengming LI; Visualization, Jiahao WANG and Fengming LI; Writing – original draft, Jiahao WANG; Writing – review & editing, Kailun YANG and Fengming LI. All authors will be informed about each step of manuscript processing including submission, revision, revision reminder, etc. via emails from our system or assigned Assistant Editor. Data Availability: The datasets analyzed in the current study are available from the corresponding author on reasonable request. Ethics statement: The authors declare that this article is reported in accordance with the ARRIVE guidelines 2.0. All procedures in this study were approved by the Animal Experiment Ethics Committee of Xinjiang Agricultural University (permit number:2018012). Conflicts of Interest: The authors declare that there are no competing interests. Funding: This study was supported by the Major Science and Technology Special Project of Xinjiang Uygur Autonomous Region ( 2022A02013-2). References Chung, H., Pamp, S. J., Hill, J. A., Surana, N. K., Edelman, S. M., Troy, E. B., Reading, N. C., Villablanca, E. J., Wang, S., Mora, J. R., Umesaki, Y., Mathis, D., Benoist, C., Relman, D. A., & Kasper, D. L. Gut immune maturation depends on colonization with a host-specific Microbiota. Cell 2012, 149(7), 1578–1593. Mao, S., Zhang, M., Liu, J., & Zhu, W. 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Equine Veterinary Journal 2015 , 47(5), 580–586. Costa, M. C., Stämpfli, H. R., Allen-Vercoe, E., & Weese, J. S. Development of the faecal microbiota in foals. Equine Veterinary Journal 2016 , 48(6), 681–688. Urubschurov, V., Stroebel, C., Günther, E., Romanowski, K., Büsing, K., & Zeyner, A. Effect of oral supplementation of probiotic strains of Lactobacillus rhamnosus and Enterococcus faecium on the composition of the faecal microbiota of foals. Journal of Animal Physiology and Animal Nutrition 2019 , 103(3), 915–924. Dougal, K., de la Fuente, G., Harris, P. A., Girdwood, S. E., Pinloche, E., & Newbold, C. J. Identification of a core bacterial community within the large intestine of the horse. PloS One 2013 , 8(10), e77660. Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R., & Gordon, J. I. Worlds within worlds: evolution of the vertebrate gut microbiota. Nature Reviews. Microbiology 2008 , 6(10), 776–788. Qin, J., MetaHIT Consortium, Li, R., Raes, J., Arumugam, M., Burgdorf, K. S., Manichanh, C., Nielsen, T., Pons, N., Levenez, F., Yamada, T., Mende, D. R., Li, J., Xu, J., Li, S., Li, D., Cao, J., Wang, B., Liang, H., … Wang, J. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 2010 , 464(7285), 59–65. Shin, N.-R., Whon, T. W., & Bae, J.-W. Proteobacteria: microbial signature of dysbiosis in gut microbiota. Trends in Biotechnology 2015 , 33(9), 496–503. Mukhopadhya, I., Hansen, R., El-Omar, E. M., & Hold, G. L. IBD-what role do Proteobacteria play? Nature Reviews. Gastroenterology & Hepatology 2012 , 9(4), 219–230. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4380361","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":309835418,"identity":"cc446c48-ad57-4966-9ff3-3ca954b14dd3","order_by":0,"name":"Jia-Hao WANG","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Hao","middleName":"","lastName":"WANG","suffix":""},{"id":309835419,"identity":"0da38f12-b5fd-408f-920d-78425c11d839","order_by":1,"name":"Wen-Jie ZHANG","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Jie","middleName":"","lastName":"ZHANG","suffix":""},{"id":309835422,"identity":"3030d9e5-e634-4dcb-9b60-a775f1e94c23","order_by":2,"name":"Hao LU","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"LU","suffix":""},{"id":309835423,"identity":"d0b25450-c1ec-4a93-ae7e-ec5732e3b326","order_by":3,"name":"Hai-Feng DENG","email":"","orcid":"","institution":"Zhaosu Racecourse of Yili Kazak Autonomous Prefecture","correspondingAuthor":false,"prefix":"","firstName":"Hai-Feng","middleName":"","lastName":"DENG","suffix":""},{"id":309835424,"identity":"4c5c2701-1f60-42b3-b48a-be74e564bc02","order_by":4,"name":"Hai LI","email":"","orcid":"","institution":"Animal Husbandry and Veterinary Bureau of Xinjiang Zhaosu County","correspondingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"LI","suffix":""},{"id":309835425,"identity":"511890f8-74db-44a0-ad8f-63d4e2c62e76","order_by":5,"name":"Zhen LIU","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"LIU","suffix":""},{"id":309835426,"identity":"50b88b98-cf5e-435a-b204-695be4ba1ecf","order_by":6,"name":"Kai-Lun YANG","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kai-Lun","middleName":"","lastName":"YANG","suffix":""},{"id":309835427,"identity":"39ede797-f7ca-4bb1-aade-116762ea5d77","order_by":7,"name":"Feng-Ming LI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie3RIQvCQBTA8TcOtzJcvaJ+hclgCPsyu7TiLBbDHJeuLu9bzGY8GdzK2WfTYjYJFvGG5u1sgvdrD+7PO3gAhvGDxsg6QwwwBeDdjIYTGyG/S4IvElCJQuh71kkcNKou+21SHeorhk1EqHPkQx+zWyKbtKQixCATQt1VPJB4oiVMpAXwEFusJhS7vsYWJhIbmju2nvpJFnsg1Raqn/B5SeV6EYskYO6yP/G82j49WD7z22bX3rJoUjiyP/moATCH7qbqUnpytY9qvjUMw/g7L4HsQshiEATfAAAAAElFTkSuQmCC","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Feng-Ming","middleName":"","lastName":"LI","suffix":""}],"badges":[],"createdAt":"2024-05-07 05:27:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4380361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4380361/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57716224,"identity":"5cb50220-2a59-4be0-9352-321923594d86","added_by":"auto","created_at":"2024-06-04 17:12:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55133,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of supplementary feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on average weight gain of suckling foals at various stages. The CK group received 0.65 kg/100 kg body weight supplement concentrate per day, the LGG group received \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e 1.00 g (live bacteria 1×10\u003csup\u003e10\u003c/sup\u003e CFU/g) on the basis of 0.65 kg/100 kg body weight supplement concentrate per day and the CBM group received 5.00 g compound probiotics (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eSaccharomyces\u003c/em\u003e \u003cem\u003ecerevisiae\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e; live bacteria 2×10\u003csup\u003e9\u003c/sup\u003e CFU/g) on the basis of 0.65 kg/100 kg body weight supplement concentrate per day. The abscissa represents foals at different age stages, and the ordinate represents the average weight gain of foals within each stage.Data represent the mean ± standard deviation. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, * *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0l\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/6c861e61bb5139aa2a479235.jpg"},{"id":57716552,"identity":"dc26da8d-3903-47b0-acc6-4dccdf391d80","added_by":"auto","created_at":"2024-06-04 17:20:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61136,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of supplementary feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on fecal pH in suckling foals\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/319ae95b933ec63ae0c0ba0b.jpg"},{"id":57716553,"identity":"6ea87a4e-f8a0-4550-b9fd-1a8e314a587b","added_by":"auto","created_at":"2024-06-04 17:20:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45873,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of supplementary feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on fecal NH\u003csub\u003e3\u003c/sub\u003e-N in suckling foals.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/138d05e0bc34dbbb93348b71.jpg"},{"id":57716225,"identity":"04542c06-25cd-427b-b805-8f539b674112","added_by":"auto","created_at":"2024-06-04 17:12:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37205,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies accumulation curves.The horizontal axis denotes the sample size, the vertical axis denotes the observed species ASVs at this sample size when sampled 100 times are, the black dashed line is the median line, and the boxplot reflects the numerical distribution of the number of species observed.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/47abde51f5bb301c2494836c.jpg"},{"id":57716230,"identity":"c9e52800-2dfc-4dbe-b7f0-c31088b1d765","added_by":"auto","created_at":"2024-06-04 17:12:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100154,"visible":true,"origin":"","legend":"\u003cp\u003eRarefaction curve.CK.0.1 and CK.0.2 represent the fecal samples collected from foal numbers 1 and 2 at d 0 etc. The horizontal axis denoted the amount of sequencing data, and the vertical axis denotes the corresponding alpha diversity index.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/f11da0d78d08c727e96b605c.jpg"},{"id":57716227,"identity":"21b03f9e-5cc9-48ce-8d1f-d095f7311f53","added_by":"auto","created_at":"2024-06-04 17:12:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41284,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of the ASVs in the groups of suckling foals during the trial period. (a): ASVs at day 0; (b):ASVs at day 30; (c):ASVs at day 60; (d): ASVs at day 90.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/3b45bddd7bd2646430ea1b2f.jpg"},{"id":57716229,"identity":"4c10676c-dc21-4e92-93d7-d4cc156e6f8d","added_by":"auto","created_at":"2024-06-04 17:12:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":44397,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of the ASVs in the groups of suckling foals during the trial period. (a) ASVs in the CK group. (b) ASVs in the LGG group; (c) ASVs in the CBM group.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/c780577490605cad662f46fa.jpg"},{"id":57716232,"identity":"d956be61-7c9b-4baf-8cc0-aca48d189cdd","added_by":"auto","created_at":"2024-06-04 17:12:52","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":68215,"visible":true,"origin":"","legend":"\u003cp\u003eWeighted pair group method with arithmetic mean. The UPGMA clustering tree structure is shown on the left and the relative abundance distribution of species at the phylum level is plotted for each sample on the right. The branch lengths in the clustering tree structure on the left represent similarity. Branch length is inversely proportional to the degree of similarity; different colors in the relative abundance on the right represent the different bacteria at the phylum level.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/8ae48f00802ef72091aca3d6.jpg"},{"id":57716554,"identity":"eb6ada2f-1c73-45b5-9ff9-d3b2445b47ca","added_by":"auto","created_at":"2024-06-04 17:20:52","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":63954,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of the microbial community and VFA concentrations in the feces of suckling foals. (a) Correlation analysis in the CK group. (b) Correlation analysis in the LGG group. (c) Correlation analysis in the CBM group.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/e9ed6878dde527ae83a1cbf9.jpg"},{"id":78701075,"identity":"5a821eab-54ec-45b3-bd52-752a8dcf1d5e","added_by":"auto","created_at":"2025-03-17 19:08:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2921326,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4380361/v1/585facfd-5834-4918-ae7e-1e3f7424e542.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on growth performance, immunity, fecal fermentation parameters and microbiota structure of suckling foals","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe growth and development of animals in the early stages of life at have a great influence on their later productive performance. Consequently, the conditions that provide a solid foundation for the healthy growth of young animals has become a focus\u003c/p\u003e \u003cp\u003eof research worldwide.\u003c/p\u003e \u003cp\u003eNutrients are digested and absorbed in the gastrointestinal tract, which is also the largest immune organ in the body. Through the interaction between the intestinal mucosa and the external environment, healthy and stable intestinal microorganisms can inhibit colonization by pathogenic bacteria, improve the absorption of nutrients in the intestine and maintain the balance of the intestinal microecology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition, the fermentation products of intestinal microorganisms provide nutrients to the organism or interfere with its health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, the establishment of a healthy and stable intestinal environment in young mammals has a critical influence on intestinal fermentation capacity. The intestinal flora then promotes the level of nutrient metabolism in the organism, thereby improving growth performance and immunity. As an active microbial feed additive often used in livestock production and breeding programs, probiotics can eliminate harmful pathogens via several molecular mechanisms and modulate the immune response of the host to promote health. Blaise et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] found that the activation of probiotics promotes the differentiation of B lymphocytes to plasma cells and increased production of secretory immunoglobulin A, which aids clearance of exogenous pathogenic bacteria and toxic and harmful molecules from the intestine and provides non-specific immune protection. A study by Yin et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] indicated that dietary supplementation with \u003cem\u003eLactobacillus plantarum\u003c/em\u003e significantly increased the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in a mouse model of acute enteritis, mitigating the damaging effects of the disease on the intestine and maintaining intestinal health. Thus, evidence suggests that probiotics enhance the growth performance of young animals by improving the absorption of nutrients and enhancing feed utilization, while also playing a key dominant role in the immune capacity.\u003c/p\u003e \u003cp\u003eDuring birth, mammals are exposed to the external environment. The first intake of colostrum begins the colonization of the gastrointestinal tract with microorganisms, leading ultimately to the development of the intestinal flora. In foals, the physiological functions of the intestinal tract improve during the period from 0 to 6 months of age, although the balance of the intestinal microecology remains to be fully established [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. At this stage, foals face significant stresses such as changes in diet structure from breast milk to plant-based feed. The stability of intestinal microorganisms is easily disturbed by the external environment, and the intestinal microecology is threatened.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effects of dietary supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e) on the growth performance and microbiota structure of suckling foals aged 1\u0026ndash;4 months to provide a reference for promoting healthy growth.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental animals and materials\u003c/h2\u003e \u003cp\u003eEighteen healthy suckling Ili horse foals (aged 1 month) from Zhaosu horse farm in Zhaosu County, Xinjiang, China with a similar birth date (\u0026plusmn;\u0026thinsp;7 d) and a mean weight of (69.83\u0026thinsp;\u0026plusmn;\u0026thinsp;13.76) kg were selected for this study.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e (live bacteria 1\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/g) were purchased from Xi'an Wanfang Biotechnology Co.\u003c/p\u003e \u003cp\u003eCompound probiotics (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e; live bacteria 2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g) were purchased from Inner Mongolia Heimei Kesheng Biotechnology Co.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design\u003c/h2\u003e \u003cp\u003eEighteen foals were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;6/group). Each day, the CK group received 0.65 kg/100 kg body weight supplement concentrate, the LGG group received \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e 1.00 g (live bacteria 1\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU/g) on the basis of 0.65 kg/100 kg body weight supplement concentrate per day, and the CBM group received 5.00 g compound probiotics (\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e; live bacteria 2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/g) on the basis of 0.65 kg/100 kg body weight supplement concentrate per day. The trial period was 100 d, with a pre-feeding period of 10 d and a regular feeding period of 90 d.\u003c/p\u003e \u003cp\u003eThe foals and their dams were all from the same grazing pasture, and all foals were fed equal amounts of concentrate supplements per horse per day, after which the supplements were adjusted every 30 d ( Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). During the regular feeding period, samples were collected on d 0, d 30, d 60 and d 90.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFeeding amount of the concentrate supplement\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage weight (kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFeeding amount (kg/d/horse)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026ndash;30 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e69.83\u0026thinsp;\u0026plusmn;\u0026thinsp;13.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u0026ndash;60 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e87.64\u0026thinsp;\u0026plusmn;\u0026thinsp;14.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u0026ndash;90 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e103.83\u0026thinsp;\u0026plusmn;\u0026thinsp;14.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Feeding management\u003c/h2\u003e \u003cp\u003eDuring the trial period, all foals were stabled in the outdoor barn from 8:00 to 19:00. The daily supplement concentrate was divided into three equal portions and foals were fed at 9:00, 14:00 and 18:00 with an appropriately sized feed pocket to ensure normal breathing and prevent spillage. At 9:00 every day, the foal was fed with probiotics in a capsule mixed with the concentrate to ensure that the capsule is consumed by the foal. The mare was milked during this period. Foals and mares were mixed from 19:00 to 8:00 the next day, during which time foals were free to take milk and water. The composition and nutritional level of the foal concentrate supplement are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition and nutrient levels of the concentrates (DM basis)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNutrient levels \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eContent\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOM, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat bran, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat middings, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNDF, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eADF, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaHPO\u003csub\u003e4\u003c/sub\u003e, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCa, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimestone, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGE, MJ/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremix \u003csup\u003e1\u003c/sup\u003e, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003e The premix provided the following per kg of concentrate supplement: FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 135.0 mg, KI 1.5 mg, MnSO\u003csub\u003e4\u003c/sub\u003e 60.0 mg, ZnSO\u003csub\u003e4\u003c/sub\u003e 60.0 mg, CuSO\u003csub\u003e4\u003c/sub\u003e 15.0 mg, vitamin A 3600 IU, vitamin D 396 IU, vitamin E 1200 IU, vitamin B\u003csub\u003e1\u003c/sub\u003e 4.5 mg, vitamin B\u003csub\u003e2\u003c/sub\u003e 3.0 mg. \u003csup\u003e2\u003c/sup\u003e Nutrient levels are measured values.\u003c/p\u003e \u003cp\u003e \u003csup\u003e2\u003c/sup\u003e OM: organic matter, CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, GE: gross energy\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Sample collection and processing\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Collection of growth performance data\u003c/h2\u003e \u003cp\u003eDuring the sampling period (d 0, d 30, d 60 and d 90 of the regular feeding period), the foals were driven to the center of the weighbridge (ID3000 Bluetooth\u0026reg; from TRU-TEST) before the morning feed. Weights were recorded when the foals were standing still and the weighbridge display was stable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Collection and processing of plasma samples\u003c/h2\u003e \u003cp\u003eDuring the sampling period (d 0, d 30, d 60 and d 90 of the regular feeding period), blood samples were collected from the jugular vein (on an empty stomach before the morning feed) into sodium heparin anticoagulation tubes, centrifuged at 3500 r/min for 15 min, and the supernatant was stored frozen at -20℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Collection and processing of fecal samples\u003c/h2\u003e \u003cp\u003eDuring the sampling period (d 0, d 30, d 60 and d 90 of the regular feeding period), feces were collected by rectal extraction at 19:00. A portion of the sample was packed in sterile lyophilized tubes and snap frozen in liquid nitrogen for the determination of fecal microbial diversity; the remaining samples were packed in lyophilized tubes and frozen at -20 ℃ for the determination of fermentation parameters.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Measurement indexes and methods\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Measurement of plasma indicators\u003c/h2\u003e \u003cp\u003eThe following metabolic indicators were measured in foal plasma: urea (UREA), determined by urease assay; glucose (GLU), determined by glucose oxidase assay; growth hormone (GH) and insulin-like growth factor-1 (IGF-1), determined by enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e \u003cp\u003eThe following immunological indicators were measured in foal plasma: immunoglobulin A (IgA) and immunoglobulin G (IgG), determined by immunoturbidimetry; interleukin-2 (IL-2), IL-6 and interleukin-8 (IL-8), determined by ELISA.\u003c/p\u003e \u003cp\u003eAll measurements were performed using kits from Beijing Huaying Biotechnology Co., according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Measurement of manure fermentation parameters\u003c/h2\u003e \u003cp\u003eThe pH of feces was determined using a FiveEasy model benchtop instrument from METTLER TOLEDO. The concentration of ammoniacal nitrogen in feces was determined with an alkaline sodium hypochlorite-phenol colorimetric method using an Infinite M200 enzyme standard [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The concentration of volatile fatty acids, including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and total volatile fatty acids in feces was determined with a GC-2010 gas chromatograph (Shimadzu, Japan) using a crotonic acid internal standard method [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 High-throughput sequencing analysis of fecal microbial bacteria\u003c/h2\u003e \u003cp\u003eGenomic DNA from fecal samples was extracted using QIAamp DNA stool Mini Kit (Qiagen, Hilden, Germany). After extraction, the purity and concentration of the DNA was then checked by 0.8% agarose gel electrophoresis and NanoDrop 2000(Thermo Scientific, USA). Good quality extracted genomic DNA was used as a template for PCR amplification with the 16S rDNA V4 region specific primers 515F: 5'-GTTTCGGTGCCAGCMGCCGCGGTAA-3' and 806R: 5'- GCCAATGGACTACHVGGGTWTCTAAT-3'. All PCRs were carried out in a total reaction volume of 30 \u0026micro;L containing 15 \u0026micro;L of Phusion\u0026reg; High-Fidelity PCR Master Mix (New England Biolabs), 0.2 \u0026micro;M forward and reverse primers, and approximately 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98℃ for 1 min, followed by 30 cycles of denaturation at 98℃ for 10 s, annealing at 50℃ for 30 s, and elongation at 72℃ for 30 s, with a final incubation at 72℃ for 5 min. PCR mixtures were added to an equal volume of 1\u0026times; loading buffer (containing SYBR green) and separated by 2% agarose gel for detection electrophoresis. Samples with a bright band of 400\u0026ndash;450 bp were chosen for further experiments. PCR products was mixed in equal ratios and then purified with the GeneJET Gel Extraction Kit(Thermo Scientific). The Agilent 5400 fragment analyzer was used to confirm that the integrity, concentration and the total amount of the amplification production were met the requirements for library construction.\u003c/p\u003e \u003cp\u003e Sequencing libraries were generated using NEB Next\u0026reg; Ultra\u0026trade; DNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer\u0026rsquo;s recommendations and index codes were added.\u003c/p\u003e \u003cp\u003eThe library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced using the Illumina HiSeq platform and 250 bp paired-end reads were generated.\u003c/p\u003e \u003cp\u003eSequencing data were analyzed by QIIME2. We then prepared the downloaded sequencing data and barcodes, installed the working configuration environment and created the directory. After standardization of the original format, data were imported. The samples were then split by barcode, and the basic data information, including minimum reads, maximum reads, reads contained in each sample and quality information were obtained. Quality control of the data and removal of chimeras were performed based on the basic data information, and the final amplicon sequence variants (ASVs)were obtained by DADA2. The data for each sample were homogenized according to the least amount of data in the sample, and the phylogenetic tree was constructed and analyzed using sequences for calculating alpha and beta diversity, while the na\u0026iuml;ve Bayes classifier was used for sequence species annotation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe raw test data were initially organized in Excel, and the data were processed using one-way ANOVA and two-way ANOVA with SPSS 25.0, with multiple comparisons and analysis of variance between groups performed by Duncan\u0026rsquo;s test. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated significant differences, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 indicated highly significant differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Effect of supplemental feeding of\u003c/strong\u003e \u003cstrong\u003eLactobacillus rhamnosus\u003c/strong\u003e \u003cstrong\u003eand compound probiotics on the growth performance of suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the body weight of foals increased with age and was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the LGG and CBM groups than that in the CK group on d 90 of the trial (18.61% and 18.05% vs. CK group, respectively). During the whole trial period, the total weight gain and mean daily weight gain of foals in both the LGG and CBM groups were significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) higher than those in the CK group. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the foals gained weight faster in the prelactation period (d 0\u0026ndash;30 the trial period) than in the middle and late lactation periods (d 30\u0026ndash;90 of the trial period), and the average weight gain of foals in both the LGG and CBM groups was significantly higher than that of the CK group during all trial phases (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of supplementary feeding of \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on body weight of suckling foals\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItems\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCK Group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLGG Group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCBM Group\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70.33\u0026thinsp;\u0026plusmn;\u0026thinsp;15.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.92\u0026thinsp;\u0026plusmn;\u0026thinsp;18.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.25\u0026thinsp;\u0026plusmn;\u0026thinsp;8.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.00\u0026thinsp;\u0026plusmn;\u0026thinsp;15.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.83\u0026thinsp;\u0026plusmn;\u0026thinsp;19.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.08\u0026thinsp;\u0026plusmn;\u0026thinsp;7.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.58\u0026thinsp;\u0026plusmn;\u0026thinsp;15.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e108.50\u0026thinsp;\u0026plusmn;\u0026thinsp;18.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e107.42\u0026thinsp;\u0026plusmn;\u0026thinsp;7.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ed 90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.67\u0026thinsp;\u0026plusmn;\u0026thinsp;15.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.33\u0026thinsp;\u0026plusmn;\u0026thinsp;19.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124.75\u0026thinsp;\u0026plusmn;\u0026thinsp;7.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal weight gain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage daily gain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eNote: In the same row, values with different small letter superscripts indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05); values with different capital letter superscripts indicate an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01); values with the same or no letter superscripts indicate no significant difference (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Effect of supplemental feeding of\u003c/strong\u003e \u003cstrong\u003eLactobacillus rhamnosus\u003c/strong\u003e \u003cstrong\u003eand compound probiotics plasma metabolic levels in suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the plasma GLU levels decreased with increasing age in all groups of suckling foals. Plasma levels of GH and IGF-1 were highest at d 0 and lowest at d 30, and increased with increasing age thereafter. The plasma IGF-1 levels were significantly higher in the LGG group than those in the CK and CBM groups at d 0 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 30, the plasma UREA levels in the CBM group were significantly higher than those in the LGG group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the plasma GH levels in the CK group were significantly higher than those in the LGG group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 60, the plasma levels of GLU in the LGG group were significantly higher than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The plasma levels of IGF-1 in the LGG group were also significantly higher than those in the CBM group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 90, the plasma GH levels in the LGG group were significantly higher than those in the CBM group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003eTreatment also had a significant effect on plasma UREA levels in suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, age had a highly significant effect on plasma levels of GH, IGF-1 and GLU (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Overall, we observed a significant interaction of treatment and age on plasma levels of GH and IGF-1 in suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of supplementary feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on plasma levels of metabolites in suckling foals\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUREA, mmol/L\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGLU, mmol/L\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGH, ng/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIGF-1, ng/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e306.36\u0026thinsp;\u0026plusmn;\u0026thinsp;13.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e331.32\u0026thinsp;\u0026plusmn;\u0026thinsp;22.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e307.47\u0026thinsp;\u0026plusmn;\u0026thinsp;11.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e190.50\u0026thinsp;\u0026plusmn;\u0026thinsp;14.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e180.16\u0026thinsp;\u0026plusmn;\u0026thinsp;18.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e180.65\u0026thinsp;\u0026plusmn;\u0026thinsp;25.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e222.24\u0026thinsp;\u0026plusmn;\u0026thinsp;14.44\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.11\u0026thinsp;\u0026plusmn;\u0026thinsp;18.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e207.97\u0026thinsp;\u0026plusmn;\u0026thinsp;14.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eABab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e252.96\u0026thinsp;\u0026plusmn;\u0026thinsp;20.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e238.19\u0026thinsp;\u0026plusmn;\u0026thinsp;8.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e248.96\u0026thinsp;\u0026plusmn;\u0026thinsp;13.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.161\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt*day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eNote: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); values with different capital letter superscripts indicate an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); values with the same or no letter superscripts indicate no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Effect of supplemental feeding with\u003c/strong\u003e \u003cstrong\u003eLactobacillus rhamnosus\u003c/strong\u003e \u003cstrong\u003eand compound probiotics on plasma immune indexes in suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the plasma levels of IL-2, IL-6 and IL-8 in all groups of suckling foals increased with age. There were no significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the immune indexes in the plasma of suckling foals in all groups at d 0 of the experiment. At d 30, the plasma levels of IgA and IgG were significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and highly significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) increased in the LGG and CBM groups compared to those in the CK group. The plasma IL-2 levels were also significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in the LGG group than those in the CBM group and the plasma IL-6 levels were significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in the CK and CBM groups than those in the LGG group. At d 60, the plasma IL-2 levels of the LGG and CBM groups were significantly higher than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The plasma IL-6 levels in the CBM group were also significantly higher than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the plasma IL-8 levels in the LGG group were significantly higher than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 90, plasma IL-2 levels in the CK and CBM groups were significantly higher than those in the LGG group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eThere was no significant effect of treatment on immune indexes in the plasma of suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, there was a highly significant effect of age on all immune indexes in the plasma (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Overall, there was a highly significant interaction between treatment and age on plasma levels of IL-2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and IL-6 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in suckling foals.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of supplementary feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on plasma immune indicators in suckling foals\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgA, g/L\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgG, g/L\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-2, pg/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-6, pg/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-8, pg/mL\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.23\u0026thinsp;\u0026plusmn;\u0026thinsp;5.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e215.69\u0026thinsp;\u0026plusmn;\u0026thinsp;20.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.74\u0026thinsp;\u0026plusmn;\u0026thinsp;15.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.97\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.71\u0026thinsp;\u0026plusmn;\u0026thinsp;5.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e229.36\u0026thinsp;\u0026plusmn;\u0026thinsp;30.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.41\u0026thinsp;\u0026plusmn;\u0026thinsp;7.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.09\u0026thinsp;\u0026plusmn;\u0026thinsp;3.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198.05\u0026thinsp;\u0026plusmn;\u0026thinsp;39.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.58\u0026thinsp;\u0026plusmn;\u0026thinsp;11.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.59\u0026thinsp;\u0026plusmn;\u0026thinsp;3.30\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e242.85\u0026thinsp;\u0026plusmn;\u0026thinsp;15.75\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.22\u0026thinsp;\u0026plusmn;\u0026thinsp;5.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e260.07\u0026thinsp;\u0026plusmn;\u0026thinsp;16.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e132.31\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.69\u0026thinsp;\u0026plusmn;\u0026thinsp;6.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e237.41\u0026thinsp;\u0026plusmn;\u0026thinsp;12.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e140.46\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e252.04\u0026thinsp;\u0026plusmn;\u0026thinsp;21.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e157.22\u0026thinsp;\u0026plusmn;\u0026thinsp;10.08\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e272.27\u0026thinsp;\u0026plusmn;\u0026thinsp;10.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e167.53\u0026thinsp;\u0026plusmn;\u0026thinsp;17.37\u003csup\u003eABab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e277.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e184.73\u0026thinsp;\u0026plusmn;\u0026thinsp;14.74\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e289.44\u0026thinsp;\u0026plusmn;\u0026thinsp;7.95\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e188.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e265.46\u0026thinsp;\u0026plusmn;\u0026thinsp;18.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e190.65\u0026thinsp;\u0026plusmn;\u0026thinsp;12.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e284.49\u0026thinsp;\u0026plusmn;\u0026thinsp;15.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e199.18\u0026thinsp;\u0026plusmn;\u0026thinsp;19.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.677\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.593\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt*day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); values with different capital letter superscripts indicate an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); values with the same or no letter superscripts indicate no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Effect of supplemental feeding with\u003c/strong\u003e \u003cstrong\u003eLactobacillus rhamnosus\u003c/strong\u003e \u003cstrong\u003eand compound probiotics on fermentation parameters in the feces of suckling foals\u003c/strong\u003e\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.4.1 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on fecal pH in suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003c/span\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the fecal pH in suckling foals ranged from 5.49 to 7.12 between 1 and 4 months of age, and increased and then decreased with age, reaching the highest value at d 30. At d 60, the fecal pH in the LGG group was significantly lower than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 90, the fecal pH in the LGG group was significantly lower than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and very significantly lower than that in the CBM group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on NH\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003e3\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003e-N concentration in the feces of suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the NH\u003csub\u003e3\u003c/sub\u003e-N concentration in the feces of suckling foals fluctuated with age. At d 30, the highest fecal NH\u003csub\u003e3\u003c/sub\u003e-N concentration was detected in the CK group, reaching the highest value at 30 d, while the lowest value was detected in the LGG and CBM groups. At d 30, the fecal NH\u003csub\u003e3\u003c/sub\u003e-N concentration in the LGG group was significantly lower than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The fecal NH\u003csub\u003e3\u003c/sub\u003e-N concentration in the CBM group was also significantly lower than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 0, d 60 and d 90, there were no significant differences in the fecal NH\u003csub\u003e3\u003c/sub\u003e-N concentrations of foals within each group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.3 Effect of supplemental feeding with Lactobacillus rhamnosus and compound probiotics on volatile fatty acids in the feces of suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the volatile fatty acid concentrations in the feces of suckling foals fluctuated with age. It is noteworthy that the total volatile fatty acid concentrations in the LGG and CBM groups decreased by 20.61% and 11.17% at d 30, while the CK group showed an increasing trend. At d 0, the fecal concentrations of valeric acid, isobutyric acid and isovaleric acid in the CBM group were significantly higher than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there were no significant differences in the fecal concentrations of the remaining volatile fatty acids between the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At d 30, the fecal concentrations of isobutyric acid and isovaleric acid in the CBM group were significantly higher than those in the LGG group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 60, there were no significant differences in the fecal concentrations of volatile fatty acids among the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At d 90, the fecal concentrations of acetic acid, propionic acid, valeric acid and total volatile fatty acids in the CBM group were significantly higher than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not significantly different from those in the LGG group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eTreatment had a highly significant effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) on isobutyric acid and isovaleric acid concentrations in the feces of suckling foals and a significant effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) on fecal concentrations of propionic acid, butyric acid, valeric acid and total volatile fatty acids. Age had a highly significant effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) on fecal butyric acid concentration and a significant effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) on the propionic acid concentration. There was no significant interaction between treatment and age on the concentration of volatile fatty acids contained in the feces of suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of supplementary feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on fecal volatile fatty acids of suckling foals, \u0026micro;mol/g\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal VFA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcetate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePropionate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eButyrate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eValerate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsobutyrate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsovalerate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.26\u0026thinsp;\u0026plusmn;\u0026thinsp;6.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.58\u0026thinsp;\u0026plusmn;\u0026thinsp;9.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.46\u0026thinsp;\u0026plusmn;\u0026thinsp;7.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.63\u0026thinsp;\u0026plusmn;\u0026thinsp;18.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.40\u0026thinsp;\u0026plusmn;\u0026thinsp;11.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.05\u0026thinsp;\u0026plusmn;\u0026thinsp;9.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.98\u0026thinsp;\u0026plusmn;\u0026thinsp;6.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.39\u0026thinsp;\u0026plusmn;\u0026thinsp;5.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.19\u0026thinsp;\u0026plusmn;\u0026thinsp;12.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.67\u0026thinsp;\u0026plusmn;\u0026thinsp;9.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.33\u0026thinsp;\u0026plusmn;\u0026thinsp;9.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.97\u0026thinsp;\u0026plusmn;\u0026thinsp;7.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.19\u0026thinsp;\u0026plusmn;\u0026thinsp;9.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.31\u0026thinsp;\u0026plusmn;\u0026thinsp;6.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.35\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.75\u0026thinsp;\u0026plusmn;\u0026thinsp;5.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.21\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.95\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.94\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.54\u0026thinsp;\u0026plusmn;\u0026thinsp;6.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.097\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt*day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.295\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNote: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); values with different capital letter superscripts indicate an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); values with the same or no letter superscripts indicate no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Effect of supplemental feeding with\u003c/strong\u003e \u003cstrong\u003eLactobacillus rhamnosus\u003c/strong\u003e \u003cstrong\u003eand compound probiotics on fecal flora of suckling foals\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5.1 Experimental design and sequencing depth validation\u003c/h2\u003e\n \u003cp\u003eSpecies accumulation curves are used to measure and predict the magnitude of species richness in a community as the sample size increases, and are widely used in biodiversity and community surveys to judge the adequacy of sample size and estimate community richness. As can be seen in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the curve tended to be flat, confirming that the 72 samples investigated in this study are sufficient to reflect to the species composition of the community and validating our experimental design.\u003c/p\u003e\n \u003cp\u003eA dilution curve is constructed by randomly selecting a certain number of individuals from the sample, counting the number of species represented by these individuals, and plotting the number of individuals versus the number of species. This method can be used to evaluate whether the sequencing volume is sufficient to cover all taxa and indirectly reflect the richness of species in the sample. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the curve leveled off when the sequencing depth reached 40,000, confirming that the sequencing depth in this experiment basically covered all species in the sample and no more new species could be found by increasing the amount of sequencing data.\u003c/p\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.5.2 Venn diagram\u003c/h2\u003e\n \u003cp\u003eAfter obtaining the ASVs by noise reduction and according to the research requirements, the common and unique ASVs among the different groups were analyzed. The number of members in each set (i.e., the number of ASVs unique to each subgroup and common among groups) was counted according to their presence or absence among groups and the corresponding Venn diagram was constructed.\u003c/p\u003e\n \u003cp\u003eAs shown in \u003cstrong\u003eFig.\u0026nbsp;6\u003c/strong\u003e, at d 0 of the trial, 2,067 ASVs were specific to the CK group, 2,301 were specific to the LGG group, 2,187 were specific to the CBM group, and 1,586 were common to all three groups. At d 30, 1, 273ASVs were specific to the CK group, 2,494, were specific to the LGG group, and 1.923 were common to all three groups. At d 60, 954 ASVs were specific to the CK group, and 1,629 were specific to the LGG group. At d 30, there were 1,273 ASVs in the CK group, 2,494 in the LGG group, 1,629 in the CBM group, and 1,923 in the three groups. At d 60, there were 954 ASVs in the CK group, 2,251 in the LGG group, 1,572 in the CBM group, and 2,089 in the three groups. At d 90, there were 1,515 ASVs in the CK group, 1,515 in the LGG group, and 1,586 in the CBM group. At d 90, 1,515 ASVs were specific to the CK group, 2,117 were specific to the LGG group, 1,892 were specific to the CBM group, and 2,496 were common to all three groups.\u003c/p\u003e\n \u003cp\u003eAs shown in \u003cstrong\u003eFig.\u0026nbsp;7\u003c/strong\u003e, there were 2,019, 1,458, 977 and 1,785 ASVs in the CK group at d 0, d 30, d 60 and d 90, respectively, with a total of 1,212 ASVs for the whole trial period. In the LGG group, there were 2,089, 1,666, 1,998 and 1,911 ASVs at d 0, d 30, d 60 and d 90, respectively, with a total of 1,456 ASVs for the whole trial period. In the CBM group, there were 2,027, 1,324, 1,619 and 1,857 ASVs at d 0, 30, 60 and 90, respectively, with a total of 1,526 ASVs for the whole trial period.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.5.3 Microbiota diversity analysis\u003c/h2\u003e\n \u003cp\u003eThe alpha diversity index represents the number of species in a local homogeneous habitat as a comprehensive reflection of the richness and homogeneity. The Chao1 index is used to estimate the total number of species contained in the community sample; the Chao1 index increases with the number of species present in the community at low abundance. The Shannon index represents the total number and proportion of taxa in the sample; the Shannon index increases with the diversity of the community and the uniformity of the species distribution. The Simpson index characterized the diversity and uniformity of species distribution in the community; the Simpson index increases with the uniformity of the species distribution.\u003c/p\u003e\n \u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, the alpha diversity index increased with age in all groups of suckling foals. At d 0, the observed_ASV and Chao1 indexes were significantly higher in the CBM group than those in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 30, the observed_ASVs and Chao1 indexes of the LGG group were significantly higher than those of the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 60, there were no significant differences in the alpha diversity indexes among the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the alpha diversity indexes in both the LGG and CBM groups tended to increase compared to those in the CK group. At d 90, the observed_ASVs and Chao1 indexes of the LGG group were significantly higher than those of the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). During the whole trial period, there were no significant differences in the Shannon and Simpson indexes among the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the indexes of both the LGG and CBM groups showed an increasing trend compared to those of the CK group.\u003c/p\u003e\n \u003cp\u003eThere was a highly significant effect of treatment on the observed_ASVs and Chao1 indexes in suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Although there was a highly significant effect of age on the alpha diversity index in suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), there was no significant interaction between treatment and age on the alpha diversity index (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAlpha diversity in sucking foals.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eobserved_ASVs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChao1 index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eShannon index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSimpson index\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1219.67\u0026thinsp;\u0026plusmn;\u0026thinsp;317.73\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1270.61\u0026thinsp;\u0026plusmn;\u0026thinsp;336.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1384.67\u0026thinsp;\u0026plusmn;\u0026thinsp;238.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1430.95\u0026thinsp;\u0026plusmn;\u0026thinsp;241.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1590.00\u0026thinsp;\u0026plusmn;\u0026thinsp;273.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1670.50\u0026thinsp;\u0026plusmn;\u0026thinsp;298.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1479.00\u0026thinsp;\u0026plusmn;\u0026thinsp;275.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1528.86\u0026thinsp;\u0026plusmn;\u0026thinsp;274.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1908.50\u0026thinsp;\u0026plusmn;\u0026thinsp;310.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1988.63\u0026thinsp;\u0026plusmn;\u0026thinsp;339.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1588.50\u0026thinsp;\u0026plusmn;\u0026thinsp;341.92\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1632.72\u0026thinsp;\u0026plusmn;\u0026thinsp;349.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1580.17\u0026thinsp;\u0026plusmn;\u0026thinsp;332.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1618.83\u0026thinsp;\u0026plusmn;\u0026thinsp;356.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1898.00\u0026thinsp;\u0026plusmn;\u0026thinsp;608.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1973.40\u0026thinsp;\u0026plusmn;\u0026thinsp;662.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1798.17\u0026thinsp;\u0026plusmn;\u0026thinsp;148.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1855.08\u0026thinsp;\u0026plusmn;\u0026thinsp;143.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ed 90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1816.83\u0026thinsp;\u0026plusmn;\u0026thinsp;162.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1857.82\u0026thinsp;\u0026plusmn;\u0026thinsp;170.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2183.33\u0026thinsp;\u0026plusmn;\u0026thinsp;257.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2277.01\u0026thinsp;\u0026plusmn;\u0026thinsp;288.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2030.67\u0026thinsp;\u0026plusmn;\u0026thinsp;308.39\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2095.35\u0026thinsp;\u0026plusmn;\u0026thinsp;325.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.304\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt*day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.363\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003eNote: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); values with different capital letter superscripts indicate an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); values with the same or no letter superscripts indicate no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eBeta diversity represents the dissimilarity of species composition between communities of different habitats along an environmental gradient or the rate of species turnover along an environmental gradient, and is therefore also known as interhabitat diversity; this index is therefore used to study the relationship between communities in terms of species multiplicity. The samples were clustered based on a weighted unifrac distance matrix using unweighted pair group averaging method (UPGMA, unweighted pair group method with arithmetic mean). For each sample to be displayed, the clustering results were integrated with the relative abundance of species at the gate level.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, the LGG and CBM groups had shorter branch lengths than the CK group at d 0, indicating that the LGG and CBM groups had similar bacterial communities. The LGG and CBM groups clustered together first, and then clustered with the CK group as one group. At d 30, the LGG and CBM groups clustered together, while the CK and LGG groups at d 60 clustered together with the CK group at d 30 indicating that the bacterial composition of the pairs of groups was similar at both stages. At d 90, the CK and LGG groups clustered together, while the bacterial communities of the CBM groups at d 60 and d 90 were similar and clustered together. Finally, the CK, LGG and CBM groups at d 90 clustered together with the CBM group at d 60.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.5.4 Microbiota composition analysis\u003c/h2\u003e\n \u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, the dominant phyla of the fecal bacteria in suckling foals comprised \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eVerrucomicrobia\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eEuryarchaeota\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eSpirochetes\u003c/em\u003e. In each group, the relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e in the fecal bacteria of suckling foals increased with age. At d 0, the relative abundance of \u003cem\u003eSpirochetes\u003c/em\u003e was significantly higher in the CBM group than in those in the CK and LGG groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there were no significant differences in the relative abundances of the remaining bacteria at the phylum level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). At d 30, the relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e in the LGG group was significantly higher than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e in the CBM group was significantly higher than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 60, the relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e in the CBM group was significantly higher than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the relative abundance of \u003cem\u003eProteobacteria\u003c/em\u003e in the LGG group was significantly higher than that in the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At d 90, none of the differences in the relative abundance of bacteria at the phylum level were significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eThere was no significant effect of treatment on the dominant phylum in the fecal bacteria of suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, there was a highly significant effect of age on the relative abundance of the \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eEuryarchaeota\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eSpirochetes\u003c/em\u003e in the fecal bacteria of suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Overall, there was no significant interaction between treatment and age on the dominant phylum in the fecal bacteria of suckling foals (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe relative abundance (%) of fecal flora at the phylum level in suckling foals\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eFirmicutes\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eVerrucomicrobiota\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBacteroidota\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eProteobacteria\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEuryarchaeota\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eActinobacteriota\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSpirochaetota\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eD 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.15\u0026thinsp;\u0026plusmn;\u0026thinsp;11.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.04\u0026thinsp;\u0026plusmn;\u0026thinsp;14.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.80\u0026thinsp;\u0026plusmn;\u0026thinsp;5.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;8.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.77\u0026thinsp;\u0026plusmn;\u0026thinsp;14.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.86\u0026thinsp;\u0026plusmn;\u0026thinsp;10.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.71\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;6.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.85\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eD 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.38\u0026thinsp;\u0026plusmn;\u0026thinsp;12.98\u003csup\u003eBb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.70\u0026thinsp;\u0026plusmn;\u0026thinsp;13.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;10.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.97\u0026thinsp;\u0026plusmn;\u0026thinsp;5.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.68\u0026thinsp;\u0026plusmn;\u0026thinsp;6.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.48\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.63\u0026thinsp;\u0026plusmn;\u0026thinsp;8.10\u003csup\u003eAaB\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.40\u0026thinsp;\u0026plusmn;\u0026thinsp;7.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.72\u0026thinsp;\u0026plusmn;\u0026thinsp;5.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eD 60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.62\u0026thinsp;\u0026plusmn;\u0026thinsp;12.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.09\u0026thinsp;\u0026plusmn;\u0026thinsp;6.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;8.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.11\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.18\u0026thinsp;\u0026plusmn;\u0026thinsp;8.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.03\u0026thinsp;\u0026plusmn;\u0026thinsp;6.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.29\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.18\u0026thinsp;\u0026plusmn;\u0026thinsp;5.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eD 90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.05\u0026thinsp;\u0026plusmn;\u0026thinsp;8.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.57\u0026thinsp;\u0026plusmn;\u0026thinsp;11.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.95\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.62\u0026thinsp;\u0026plusmn;\u0026thinsp;6.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.52\u0026thinsp;\u0026plusmn;\u0026thinsp;5.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.49\u0026thinsp;\u0026plusmn;\u0026thinsp;7.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.62\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.26\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.36\u0026thinsp;\u0026plusmn;\u0026thinsp;6.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.363\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.788\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eday\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.816\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003etrt*day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.407\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eNote: The top half of the table shows the results of one-way ANOVA of each group at each stage of the trial. In the same column, values with different small letter superscripts indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); values with different capital letter superscripts indicate an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); values with the same or no letter superscripts indicate no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The bottom half of the table shows the results of two-way ANOVA under the combined influence of treatment and age. Trt refers to different test treatments; day refers to different stages of age.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Correlation analysis of fecal flora and volatile fatty acids in suckling foals\u003c/h2\u003e\n \u003cp\u003eSpearman\u0026rsquo;s rank correlation analysis revealed that the relative abundance of the top 10 ranked bacterial genera in the feces of suckling foals in each group was correlated with the concentration of volatile fatty acids in the feces. As shown in \u003cstrong\u003eFig.\u0026nbsp;9 (a)\u003c/strong\u003e, there was no significant correlation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the concentration of volatile fatty acids and the relative abundance of the top 10 genera of bacteria ranked in the feces of foals in the CK group. As shown in \u003cstrong\u003eFig.\u0026nbsp;9 (b)\u003c/strong\u003e, the relative abundance of the \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e in the fecal microflora of the LGG group was highly significantly and positively correlated with the concentrations of propionic acid and butyric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The relative abundances of \u003cem\u003eWCHB1-41\u003c/em\u003e and the \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e were highly significantly and negatively correlated with the concentrations of propionic acid and butyric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The relative abundance of \u003cem\u003eMethanobrevibacter\u003c/em\u003e was highly significantly and negatively correlated with the concentrations of valeric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). As shown in \u003cstrong\u003eFig.\u0026nbsp;9 (c)\u003c/strong\u003e, the relative abundances of \u003cem\u003eMethanobrevibacter\u003c/em\u003e, \u003cem\u003eChlamydia\u003c/em\u003e and \u003cem\u003eSaccharofermentans\u003c/em\u003e in the fecal microflora of foals in the CBM group were highly significantly and positively correlated with the concentrations of acetic acid and isovaleric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The relative abundances of \u003cem\u003eWCHB1-41\u003c/em\u003e, \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e and \u003cem\u003eF082\u003c/em\u003e were highly significantly and negatively correlated with the concentrations of acetic acid and isovaleric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFrom 1 to 4 months of age is a critical stage in foal growth. At this stage, the foal grows fast, leading to high nutrient requirements. During weaning, the amount of fiber feed in the diet is gradually increased, and the intake of large amounts of fiber affects the gastrointestinal tract structure and function, leading to microbial bacteria instability, increased susceptibility to pathogenic bacteria as well as inhibition of normal bacterial colonization and proliferation, thus affecting the foal's health, growth and development. Supplemental feeding is the main approach to supporting the foal to cope with the stresses associated with these changes in diet and the feeding environment that are experienced during this stage. Probiotics can improve the activity of digestive enzymes in the gastrointestinal tract of animals, improve the absorption efficiency of nutrients, increase the conversion rate of feed, regulate the balance between the beneficial and harmful bacterial groups in the gastrointestinal tract of animals, and promote the healthy growth of animals.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.1 Effect of supplemental feeding with\u003c/b\u003e \u003cb\u003eLactobacillus rhamnosus\u003c/b\u003e \u003cb\u003eand compound probiotics on the growth performance of suckling foals\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe lactation period is a critical for the growth and development of foals, with body weight representing a visual indicator of foal growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The gastrointestinal tract of foals at this stage is still developing and has a weak ability to digest and absorb nutrients from forage, and relying only on nutrients from breast milk cannot fully meet their growth needs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Probiotic supplementation effectively increased the digestibility of nutrients in Holstein calves, thus improving their health and growth performance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A study by Bocourt et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] confirmed that supplemental feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e improved the average daily weight gain and meat-feed ratio of early weaned piglets. In the present study, long-term supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics in foals aged 1\u0026ndash;4 months significantly increased the body weight of foals at d 90 of the trial, and significantly increased the total weight gain and average daily weight gain of foals during the whole trial period. These findings confirmed that probiotics had a positive effect on the body weight of suckling foals, and this effect increased with the supplementation time. The weight gain rate of foals aged 1\u0026ndash;4 months was more rapid in the prelactation period than in the late lactation period, indicating that the weight gain rate slowed down with age, which is consistent with the findings of Juliand et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2 Effect of supplemental feeding with\u003c/b\u003e \u003cb\u003eLactobacillus rhamnosus\u003c/b\u003e \u003cb\u003eand compound probiotics on plasma metabolic levels in suckling foals\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePlasma UREA, GLU, GH and IGF-1 are not only important indicators of nutrient absorption and metabolism, but also key indicators for monitoring growth and development. UREA in plasma is a product of protein catabolism and can reflect the balance of nitrogen metabolism and amino acids [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], while GLU levels reflect the dynamic balance of sugar absorption, transport and metabolism of the organism [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and GH and IGF-1 levels reflect the ability to promote growth [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the present study, supplemental feeding with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e significantly increased the plasma levels of GLU and IGF-1 in foals at d 60 and GH at d 90. Supplemental feeding with compound probiotics significantly increased the plasma levels of UREA in foals at d 30. This indicates that \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e plays a prominent role in improving blood glucose metabolism and regulating the levels of related growth hormones in suckling foals. Supplementation with compound probiotics enhanced protein metabolism in suckling foals. The plasma GLU levels in suckling foals decreased with age, indicating that the hindgut fermentation capacity of foals gradually increased with age and changes in feeding habits. Plasma levels of GH and IGF-1 in suckling foals decreased and then increased with age, indicating that the hormone levels in newborn foals were high and gradually decreased with age to fluctuate within the normal range, which is consistent with the results of other mammalian studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.3 Effect of supplemental feeding with\u003c/b\u003e \u003cb\u003eLactobacillus rhamnosus\u003c/b\u003e \u003cb\u003eand compound probiotics on plasma immune indexes in suckling foals\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIgA is the main component of the mucosal defense system and is the first line of defense against pathogenic invasion. Among the plasma immunoglobulins, IgG occurs at the highest concentrations, and is an important component in the resistance to infection [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. IL-2 is an indicator of anti-tumor immune function and not only stimulates the growth, proliferation and differentiation of T cells, but also functions as an important immune enhancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. IL-6 is an important cytokine expressed in the initial response of the innate immune system to injury and infection [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. IL-8 acts as a chemoattractant and activates neutrophils, and promotes the lysosomal enzyme activity of neutrophils and phagocytosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the present study, supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics significantly increased the plasma levels of IgA and IgG in foals at d 30. The plasma levels of IL-2 and IL-8 were also significantly increased in foals at d 30 and d 60. Supplementation with compound probiotics significantly increased the plasma levels of IL-2 and IL-6 in foals at d 30, d 60 and d 90. Probiotic supplementation also significantly increased the levels of IL-2 and IL-6 in the plasma of foals at 30, 60 and 90 days of the trial, indicating that prolonged supplementation with probiotics could improve the immunity of suckling foals, thus protecting them from external environmental stresses and promoting healthy growth. Furthermore, the plasma levels of IL-2, IL-6 and IL-8 in suckling foals increased with age, indicating that the foals were exposed to more antigens during their growth and development, and the antibody levels increased, thus improving their immunity.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.4 Effect of supplemental feeding with\u003c/b\u003e \u003cb\u003eLactobacillus rhamnosus\u003c/b\u003e \u003cb\u003eand compound probiotics on fermentation parameters of manure in suckling foals\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe fermentation products of the intestinal microflora of suckling foals provide nutrients and metabolites, such as volatile fatty acids, which have various biological functions such as providing energy for foals, maintaining intestinal integrity, controlling the intestinal flora and regulating immune function. NH\u003csub\u003e3\u003c/sub\u003e-N, which is the main source of nitrogen for intestinal microflora fermentation, is a key indicator of microbial activity. NH\u003csub\u003e3\u003c/sub\u003e-N is also one of the metabolites of the nitrogen cycle. A stable pH is required to provide a favorable environment for intestinal microorganisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, we found that the pH of feces of suckling foals tended to increase and then decrease with time, reflecting the fluctuations in the concentrations of NH\u003csub\u003e3\u003c/sub\u003e-N and volatile fatty acids. The concentration of NH\u003csub\u003e3\u003c/sub\u003e-N and total volatile fatty acids in the feces of foals supplemented with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics decreased at d 30 compared with the control group, indicating that the probiotics accelerated the nitrogen cycle in the suckling foals and enhanced nitrogen utilization. Supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e significantly decreased the pH of feces at d 60 and 90, while supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics significantly decreased the pH of feces at d 30. In the feces of foals supplemented with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, the concentrations of isobutyric acid and isovaleric acid at d 30 and the concentrations of total volatile fatty acid, acetic acid, propionic acid and valeric acid at d 90 were significantly increased, indicating that supplementation with probiotics effectively improved the intestinal fermentation capacity of suckling foals and lays the foundation for the stable development of intestinal flora.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.5 Effect of supplemental feeding with\u003c/b\u003e \u003cb\u003eLactobacillus rhamnosus\u003c/b\u003e \u003cb\u003eand compound probiotics on the structure of the fecal flora of suckling foals\u003c/b\u003e\u003c/p\u003e \u003cp\u003eASVs are de-duplicated sequences generated by the DADA2 method after noise reduction, and replace OTUs to improve the accuracy, comprehensiveness and reproducibility of marker gene data analysis. In addition, ASVs can reflect the abundance of species or numbers of microflora in sequenced samples. Proudman et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] detected OTUs in the range 1,200\u0026ndash;3,000 in the feces of thoroughbred racehorses. In this study, the number of ASVs in the feces of foals supplemented with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics was found to be significantly higher than that in the control group, indicating that supplementation with probiotics increases the species and number of fecal bacteria in foals. In accordance with the findings reported by Costa et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], we found that the changes in the number of ASVs in the feces of suckling foals indicated that foals are infected with bacteria after birth and, immediately after natural mechanisms are activated to remove toxic and harmful bacteria, the foals then acquired more bacteria through exposure to the external environment and forage feeding until their flora is similar to adult horses. During this process, suckling foals are affected by various external factors that may disrupt the intestinal flora, and supplemental feeding of probiotics can effectively and steadily increase the types and amounts of bacteria in the intestinal tract of suckling foals.\u003c/p\u003e \u003cp\u003eThe alpha diversity index is a comprehensive index reflecting the abundance and homogeneity of sequenced samples. In this study, supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e significantly increased the observed_ASVs and Chao1 indexes of foal feces microorganisms at d 30 and d 90. The alpha diversity index of the feces of foals supplemented with compound probiotics tended to increase between d 30 and 60, indicating that dietary supplementation with probiotics can increase the diversity and richness of the intestinal microflora in suckling foals. These findings are consistent with those reported by Vladimir et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] following oral administration with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and Enterococcus faecalis to foals. Costa et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] showed that the intestinal microflora of foals changes over time. Similarly, we found that the diversity and richness of the intestinal microflora of suckling foals increased with age. These results are consistent with the increase in the diversity and richness of the intestinal microflora with age, indicating that the diversity and homogeneity of the intestinal flora can be optimized by long-term supplementation with probiotics.\u003c/p\u003e \u003cp\u003eThe intestinal microflora of suckling foals gradually increases in complexity, diversity and stability with age and changes in feeding habits and structure of the diet [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The intestinal microflora plays an important role in various physiological functions, such as promoting nutrient absorption, regulating substance metabolism, and regulating immunity and antioxidant capacity. Disturbance of the intestinal flora can lead to diarrhea and enteritis. In this study, the dominant phyla of the fecal bacteria in suckling foals were \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eVerrucomicrobia\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eEuryarchaeota\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eSpirochetes\u003c/em\u003e. The relative abundances of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e in the feces of suckling foals was significantly increased at d 30 and d 60 by long-term supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e. The relative abundances of the \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e in the feces of suckling foals was significantly increased at d 30 and d 60 by long-term supplementation with compound probiotics. \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e are important components of the foal gut microflora and are involved in key metabolic activities in vivo. In forage, \u003cem\u003eFirmicutes\u003c/em\u003e acts on fibrous substances [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and \u003cem\u003eBacteroidetes\u003c/em\u003e acts on starchy substances [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although numerous studies have confirmed that the abundance of \u003cem\u003eProteobacteria\u003c/em\u003e is closely associated with microecological dysregulation in the host gut [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eProteobacteria\u003c/em\u003e is also an important phylum that explains the significant functional variation in the gut microbiome [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The relative abundance of many relevant microbial bacteria, such as the \u003cem\u003eBacteroidetes\u003c/em\u003e, in foals will gradually increase with the intake of roughage and changes in the structure of the diet. At the same time, the composition and abundance of fecal microbial bacteria in suckling foals may change other external environmental influences as increasing age. Supplemental feeding with probiotics will have a significant effect on the composition and relative abundance of intestinal microbial bacteria in suckling foals, improving the digestion and absorption of nutrients, promoting healthy intestinal development and improving intestinal physiological functions.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Correlation analysis of fecal flora and volatile fatty acids in suckling foals\u003c/h2\u003e \u003cp\u003eThe intestinal flora of suckling foals plays an important role in maintaining intestinal health by regulating the intestinal environment and functions of the organism through the production of volatile fatty acids, such as acetic acid, propionic acid and butyric acid, through degradation of cellulose and hemicellulose, thereby regulating intestinal pH, reducing the colonization of pathogenic bacteria and alleviating intestinal infections and inflammation caused by pathogenic bacteria [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. At the same time, volatile fatty acids in the foal\u0026rsquo;s intestine regulate the structure and abundance of the microbial community and thus maintain the flora in benign dynamic balance. In the present study, there was no significant correlation between the relative abundance of fecal microbial bacteria and the content of volatile fatty acids in control foals. However, in the foals supplemented with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, there was a significant positive correlation between the relative abundances of the \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e and the concentrations of propionic acid and butyric acid in the feces. The relative abundances of \u003cem\u003eWCHB1-41\u003c/em\u003e, the \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e and \u003cem\u003eMethanobrevibacter\u003c/em\u003e were significantly and negatively correlated with fecal concentrations of propionic acid, butyric acid and valeric acid. The relative abundances of \u003cem\u003eMethanobrevibacter\u003c/em\u003e, \u003cem\u003eChlamydia\u003c/em\u003e and \u003cem\u003eSaccharofermentans\u003c/em\u003e in the fecal microflora of foals supplemented with compound probiotics were highly significantly and positively correlated with the concentrations of acetic acid and isovaleric acid in feces. The relative abundances of \u003cem\u003eWCHB1-41\u003c/em\u003e, the \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e and \u003cem\u003eF082\u003c/em\u003e were highly and negatively correlated with the concentrations of acetic acid and isovaleric acid. These findings indicate that dietary supplementation with probiotics affects the physiological function of the intestinal tract and homeostasis of the internal environmental of suckling foals by altering the composition of microbial bacterial communities in the intestine and by promoting or inhibiting the ability of the foals to produce volatile fatty acids by intestinal fermentation.\u003c/p\u003e \u003cp\u003eDespite the significant conclusions of this study, there are still some shortcomings. First, we did not quantitatively analyze beneficial bacteria such as \u003cem\u003eBifidobacterium\u003c/em\u003e and harmful bacteria such as \u003cem\u003eSalmonella\u003c/em\u003e; second, due to the species used in our experimental animals, it was difficult to collect the contents of the various intestinal segments of the foals. In further studies, we plan to investigate the that the addition of beneficial bacteria immediately after birth may be more effective for early microbial colonization, and quantitative analysis of bacteria by collecting the intestinal contents of foals may also be more meaningful.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we showed that dietary supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics had a positive and effect on the growth performance of suckling foals aged suckling foals aged 1\u0026ndash;4 months, and that this effect was enhanced with time. We concluded that this effect was mediated by the production of volatile fatty acids in the intestine induced by changes in the composition and abundance of intestinal microorganisms, thereby promoting the digestion and absorption of nutrients, and enhancing nutrient metabolism and host immunity, to promote healthy growth.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe would like to thank the staff at our laboratory for their ongoing assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Jiahao WANG, Wenjie ZHANG, Kailun YANG and Fengming LI; Data curation, Jiahao WANG, Wenjie ZHANG and Fengming LI; Formal analysis, Jiahao WANG, Wenjie ZHANG, Zhen LIU, Kailun YANG and Fengming LI; Funding acquisition, Kailun YANG; Investigation, Jiahao WANG, Wenjie ZHANG and Hao LU; Methodology, Jiahao WANG, Wenjie ZHANG, Haifeng DENG, Hai LI, Zhen LIU, Kailun YANG and Fengming LI; Project administration, Kailun YANG and Fengming LI; Resources, Wenjie ZHANG, Hao LU, Haifeng DENG, Hai LI, Kailun YANG and Fengming LI; Supervision, Kailun YANG and Fengming LI; Validation, Jiahao WANG, Wenjie ZHANG, Hao LU and Fengming LI; Visualization, Jiahao WANG and Fengming LI; Writing \u0026ndash; original draft, Jiahao WANG; Writing \u0026ndash; review \u0026amp; editing, Kailun YANG and Fengming LI.\u003c/p\u003e\n\u003cp\u003eAll authors will be informed about each step of manuscript processing including submission, revision, revision reminder, etc. via emails from our system or assigned Assistant Editor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe datasets analyzed in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement:\u0026nbsp;\u003c/strong\u003eThe authors declare that this article is reported in accordance with the ARRIVE guidelines 2.0. All procedures in this study were approved by the Animal Experiment Ethics Committee of Xinjiang Agricultural University (permit number:2018012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by the Major Science and Technology Special Project of Xinjiang Uygur Autonomous Region ( 2022A02013-2).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChung, H., Pamp, S. J., Hill, J. A., Surana, N. K., Edelman, S. M., Troy, E. B., Reading, N. C., Villablanca, E. J., Wang, S., Mora, J. R., Umesaki, Y., Mathis, D., Benoist, C., Relman, D. A., \u0026amp; Kasper, D. L. Gut immune maturation depends on colonization with a host-specific Microbiota. 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Gastroenterology \u0026amp; Hepatology\u003cstrong\u003e 2012\u003c/strong\u003e, 9(4), 219\u0026ndash;230.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"suckling foals, Lactobacillus rhamnosus, Compound probiotics, Growth performance, Immune index, Fecal fermentation parameters, Structure microbiota","lastPublishedDoi":"10.21203/rs.3.rs-4380361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4380361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e(1) In this study, we investigated the effects of dietary supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics on the growth performance and microbiota structure of suckling \u003cem\u003eIli\u003c/em\u003e horse foals (aged 1 month; average weight 69.83\u0026thinsp;\u0026plusmn;\u0026thinsp;13.76 kg). (2) At d 0, d 30, d 60 and d 90 of the regular feeding period, the weights of the foals were recorded, blood samples were obtained from the jugular vein and feces were collected by rectal extraction for the determination of microbial diversity and fermentation parameters. (3) The results showed that dietary supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics in 1-month-old suckling foals affected the production of volatile fatty acids in the intestine by changing the composition and abundance of intestinal microorganisms, promoting the digestion and absorption of nutrients, enhancing the level of nutrient metabolism and immunity to promote growth performance. (4) In conclusion, dietary supplementation with \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and compound probiotics has a time-dependent positive effect on the growth performance of suckling foals.\u003c/p\u003e","manuscriptTitle":"Effect of supplemental feeding of Lactobacillus rhamnosus and compound probiotics on growth performance, immunity, fecal fermentation parameters and microbiota structure of suckling foals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 17:12:47","doi":"10.21203/rs.3.rs-4380361/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96f944f2-baec-4f77-a093-4950b446932b","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32734378,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":32734379,"name":"Biological sciences/Microbiology/Bacteria"},{"id":32734380,"name":"Biological sciences/Zoology/Animal physiology"}],"tags":[],"updatedAt":"2025-03-17T19:08:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-04 17:12:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4380361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4380361","identity":"rs-4380361","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}