Application of compound probiotics to improvement of intestinal digestion ability of Debao dwarf foals and preparation of compound probiotics in improvement of intestinal digestion ability of Debao dwarf foals

Abstract

The invention discloses application of compound probiotics to improvement of intestinal digestive ability of Debao pony and a preparation. The compound probiotics are composed of bacillus, saccharomycetes and lactic acid bacteria according to the ratio of 8: 7: 0.5, and 50-70 mL / piece of compound probiotics is added into drinking water every day. By adjusting the intestinal flora structure, the relative abundance of ackermania and wart micromycota is remarkably reduced, the proliferation of Alamiaceae RC9, Heliraceae XPB1014 / AC2044, anaerobic vibrio and glycozymophyte is promoted, and the digestive ability of the foals to crude fibers and non-fibrous carbohydrates is improved. By means of the application, the apparent digestibility of CP, the apparent digestibility of NDF and the apparent digestibility of ADF are improved by 7.57%, 10.66% and 10.82% respectively, the average daily gain is improved by 8.08%, and the feed-to-weight ratio is reduced by 4.59%. The invention provides a high-efficiency and low-cost solution for large-scale healthy breeding of Debao pony.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of a composite probiotic in improving the intestinal digestion ability of a Debao pony foal and a preparation thereof. Background Art

[0002] Debao pony is one of the only two remaining dwarf pony bloodlines in the world. As a rare dwarf pony breed unique to China, it has extremely high genetic resource protection value and ecological and cultural significance. Its foals often face growth retardation and digestive disorders caused by post-weaning stress, which seriously affects the health and economic benefits of the population.

[0003] Probiotics, through their beneficial metabolites, not only increase the proportion of beneficial bacteria and improve the composition of intestinal flora but also promote digestion and absorption of feed nutrients by livestock and poultry. With the recent comprehensive ban on antibiotic use in the livestock industry, the application of probiotics as an alternative is expected to expand. Bacillus spores promote the breakdown and absorption of nutrients in feed, thereby enhancing feed utilization efficiency in livestock and poultry. Yeast can be used to produce single-cell protein feeds. Lactic acid bacteria, resident in the animal intestine, promote protein breakdown by proteases, accelerating nutrient digestion and absorption. Adding a combination of probiotics, including Lactobacillus and Lactobacillus plantarum, to the drinking water of Lingnan yellow-feathered broilers significantly increased average daily gain (ADG) and reduced feed-to-gain ratio (F / G) between 22 and 42 days of age. Li Zheng's research found that calves fed a combination of probiotics showed increased bacterial richness and diversity in their feces, altering the structure of their intestinal flora. The group supplemented with 6g of the probiotic had the lowest diarrhea rate, effectively inhibiting the proliferation of pathogens. Yuan Lihong found that adding probiotics to weaned piglet diets not only reduced feed-to-weight ratios and diarrhea rates, but also reduced the number of harmful Escherichia coli bacteria in the piglet's intestines, improving their growth performance. Adding Candida utilis and Bacillus subtilis to Hu sheep's complete mixed diets increased their average daily weight gain by 5.3% and 10.5%, respectively, compared to the control group, and increased crude protein (CP) digestibility by 9.1% and 3.7%, respectively, significantly improving feed conversion efficiency. Yang Bowen's research found that adding 150 and 200 mg / kg of a composite probiotic preparation improved intestinal absorption of protein and calcium in laying hens, increasing dietary nutrient utilization.

[0004] Probiotics are effective in commercial animals such as pigs, chickens, cattle, and sheep, but research on equines is extremely scarce. Furthermore, existing studies have mostly used feed addition methods. Feeding probiotic liquid directly to animals not only avoids the drying process and reduces costs, but also offers operational advantages in large-scale horse farming by feeding it in drinking water. The effects of different combinations also vary, and the combined addition of multiple probiotics is more effective than feeding a single strain of probiotics. The synergistic effects of compound probiotics on Debao pony foals remain unclear. Summary of the Invention

[0005] The purpose of the present invention is to provide an application and preparation of a compound probiotic in improving the intestinal digestion ability of Debao pony foals. The present invention takes Debao pony foals as the subjects and explores the effects of adding compound probiotics to drinking water on their growth performance, apparent digestibility of nutrients and intestinal flora. The present invention aims to provide a theoretical basis for the scientific breeding of rare horse breeds, and at the same time provide a reference for expanding the application of probiotics in nutritional regulation of equine animals. It is of great significance to regulate the intestinal health of foals through microorganisms and alleviate post-weaning stress.

[0006] To achieve the above objectives, the present invention provides an application of a composite probiotic in improving the intestinal digestive ability of Debao pony foals, wherein the intestinal digestive ability includes reducing the relative abundance of Akkermansia and Verrucomicrobia in the foal's feces, and increasing the relative abundance of the RC9 intestinal group of the Lachnospiraceae family, XPB1014, AC2044, Anaerobic Vibrio, and Saccharofermentative Bacteria.

[0007] It is further explained that the addition amount of the compound probiotics is 50-70 mL / pig in drinking water.

[0008] It is further stated that the Debao pony foal is a weaned foal of 8-9 months old and weighs 75±2kg.

[0009] The present invention also provides a composite probiotic preparation for improving the intestinal digestion ability of Debao pony foals, comprising bacillus, yeast and lactic acid bacteria; the total viable bacteria ratio is: bacillus: yeast: lactic acid bacteria = 8:7:0.5.

[0010] Further, the total number of viable bacteria of the Bacillus, yeast and lactic acid bacteria are: Bacillus ≥ 8×10 8 CFU / g, yeast ≥7×10 8 CFU / g, lactic acid bacteria ≥5×10 7 CFU / g.

[0011] It is further specified that the Bacillus is Bacillus subtilis, the yeast is Candida utilis, and the lactic acid bacteria is Lactobacillus plantarum.

[0012] The present invention also provides a method for healthy breeding of Debao pony foals, comprising the following steps:

[0013] (1) Add the composite probiotic preparation of claim 4 to the drinking water in a drinking bucket at a dose of 50-70 mL / pickle;

[0014] (2) Fill the drinking bucket with 1.5-2.5 kg of water per horse to ensure that the foals drink the bacteria-containing water in the morning;

[0015] (3) Feed continuously for 60 days, during which time the foals' growth performance and fecal flora changes are monitored.

[0016] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0017] (1) The present invention has obtained through experimental research that: through the synergistic effect of composite probiotics, the relative abundance of Akkermansia and Verrucomicrobiota is significantly reduced, and the proliferation of potential pathogenic bacteria is inhibited; at the same time, the reproduction of beneficial bacteria such as Rikenella RC9, Lachnospiraceae XPB1014 / AC2044, Anaerovorax and Saccharofermentans is promoted, thereby optimizing the intestinal microecological balance. The above-mentioned changes in the microbial community are significantly positively correlated with the crude fiber degradation ability and the digestion efficiency of non-fibrous carbohydrates (P<0.05). The experimental data support the direct correlation between microbial community regulation and digestibility improvement. The composite probiotics promote the decomposition of structural carbohydrates in feed by secreting active substances such as cellulase, xylanase and protease. The apparent digestibility of crude protein (CP) in the experimental group increased by 7.57%, and the apparent digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) increased by 10.66% and 10.82%, respectively, effectively improving feed utilization. The average daily gain (ADG) of the foals increased by 8.08%, and the feed-to-gain ratio (F / G) decreased by 4.59%, significantly outperforming conventional farming methods. By enhancing nutrient absorption efficiency, the fattening cycle was shortened and farming costs were reduced.

[0018] (2) The compound probiotics inhibited the overgrowth of Akkermansia, reduced abnormal mucin degradation, maintained the integrity of the intestinal mucosal barrier, and reduced the risk of pathogen invasion. The Shannon index and Chao1 index of the fecal flora of the experimental group foals were higher than those of the control group, indicating that the diversity and stability of the flora were significantly enhanced, which helped to alleviate the digestive disorders caused by post-weaning stress.

[0019] (3) By improving feed digestibility, reducing the undegraded crude fiber content in feces, and reducing breeding environmental pollution, it meets the needs of green animal husbandry development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a photo of the feeding site of Debao pony foals with added composite probiotics according to Example 2 of the present invention.

[0021] Figure 2 This is a diagram of the collection of feces samples of Debao pony foals in Example 2 of the present invention.

[0022] Figure 3 This is a diagram showing the effect of adding composite probiotics on the community composition at the classification level of microbial phylum in the feces of Debao pony foals in Example 2 of the present invention.

[0023] Figure 4 This is a diagram showing the effect of adding composite probiotics on the community composition of microbial genus classification level in the feces of Debao pony foals in Example 2 of the present invention.

[0024] Figure 5 This is a cluster heat map analysis of the correlation between bacteria at the genus level in fecal flora and the apparent digestibility of nutrients in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0026] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely one example of a series of equivalent or similar features.

[0027] Example 1:

[0028] The combined effects of different composite microbial agents also vary. The applicant conducted the following feeding experiments to study the effects of a combined microbial agent of Bacillus, yeast, and lactic acid bacteria on the apparent digestibility of nutrients in Debao ponies:

[0029] 1.1 Experimental design and diet composition

[0030] Eighty-four healthy 8- to 9-month-old Debao pony foals of similar weight (76 ± 2.00 catties) were randomly divided into 28 groups: one blank control group and three experimental groups (groups 1-27). Each treatment group had three replicates. The control group had free access to water, while experimental groups 1-27 received 50 mL / foal of the probiotics listed in Table 2.

[0031] Among them, Bacillus subtilis, Bacillus licheniformis, Saccharomyces cerevisiae, Candida utilis, Lactobacillus casei, and Lactobacillus plantarum were purchased from the market. The total number of viable Bacillus subtilis cells was ≥8×10 8 CFU / g, total number of viable Bacillus licheniformis ≥8×10 8 CFU / g, total number of viable Saccharomyces cerevisiae ≥7×10 8 CFU / g, total number of viable Candida utilis ≥7×10 8 CFU / g, total number of viable Lactobacillus casei ≥5×10 7 CFU / g, total number of viable Lactobacillus plantarum ≥5×10 7 CFU / g.

[0032] The experimental group used drinking buckets filled with 1.5-2.5 kg of water per horse to ensure that the foals could drink it all in the morning and drink water freely in the afternoon. The three groups were fed a complete mixed basal diet with a concentrate to roughage ratio of 4:6 (see Table 1). The concentrate supplement was made into pellets. The actual amount of concentrate supplement was calculated according to the formula of the concentrate supplement and the weight of the foals. The horses were raised in a fenced house with a pre-trial period of 7 days and a main trial period of 60 days. The complete mixed diet was fed once at 09:00 and 16:00 every day, and the feed amount was adjusted based on the principle that the leftover feed did not exceed 5% of the feed amount. The feed amount and leftover feed amount were recorded daily in the pen. Clean and sufficient drinking water was provided to the horses throughout the experiment. Daily feeding management, immunization procedures and disease treatment were carried out according to the regulations of the farm.

[0033] Table 1 Composition and nutritional level of experimental diets (dry matter basis)

[0034]

[0035] Note: Nutritional levels are measured values.

[0036] Table 2 Effects of the combination of compound microbial agents on the level of orthogonal experimental factors

[0037]

[0038]

[0039] 1.2 Sample collection and processing

[0040] After morning feeding in the last week of the feeding period, the total mixed ration (TMR) of each experimental horse was collected for 3 consecutive days. Feces were collected rectally once while wearing disposable long-arm gloves. After mixing the fecal samples collected for 3 days for each horse, 10 g was taken and placed in a 50 mL sterile centrifuge tube, numbered, and quickly placed in a liquid nitrogen tank for storage before testing the fecal microbial flora. The remaining fecal samples were dried at 65°C for sample preparation.

[0041] 1.3 Nutrient Apparent Digestibility Index

[0042] The apparent digestibility of nutrients (crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), etc.) in the experimental diet and fecal samples of Debao pony foals was determined using the acid-insoluble ash method (2 mol / L hydrochloric acid insoluble ash). The specific determination method is based on Zhang Liying's "Feed Analysis and Feed Quality Testing Technology (4th Edition)." The formula for calculating the apparent digestibility of each nutrient is as follows:

[0043] Nutrient apparent digestibility = 100% - 100% × [(A1 / A2) × (F2 / F1)]

[0044] Where: A1 is the AIA (%) content of ash insoluble in 2 mol / L hydrochloric acid in feed; A2 is the AIA content in feces; F1 is the nutrient content in feed; F2 is the nutrient content in feces.

[0045] Table 3 Effect of adding compound probiotics on the apparent digestibility of nutrients in Debao pony foals (%)

[0046]

[0047]

[0048] It can be seen from the above table that the combinations of the six probiotics studied have different effects on the apparent digestibility of CP, ADF and NDF of Debao pony foals. According to the results of this experiment, the combination of Bacillus + yeast + lactic acid bacteria has a certain improvement, but not all probiotic species can improve the apparent digestibility of CP, ADF and NDF of Debao pony foals. From the experimental data, the combination of experimental group 23 is the best option, that is, the combination of Bacillus subtilis + Candida utilis + Lactobacillus plantarum can effectively improve the apparent digestibility of CP, ADF and NDF of Debao pony foals.

[0049] Example 2:

[0050] In this example, based on the study of Example 1, it was found that the probiotic combination of Bacillus subtilis + Candida utilis + Lactobacillus plantarum had a good effect on the apparent digestibility of CP, ADF, and NDF in Debao pony foals. The applicant further conducted feeding trials on Debao pony foals with this group of probiotics and added testing studies on other projects. The specific experiments are as follows:

[0051] 1. Materials

[0052] 1.1 Experimental animals, time and location

[0053] Eighteen healthy Debao pony foals aged 8-9 months and weighing (75.01±1.39) were selected and provided by the Debao Pony Breeding Farm of the Special Animal Research Laboratory, Animal Husbandry Research Institute, Guangxi Agricultural Vocational and Technical University. The experiment was conducted at the experimental base from September to November 2024.

[0054] 1.2 Compound probiotics

[0055] The selected drinking water-specific composite probiotics consist of Bacillus subtilis with a total number of viable bacteria ≥ 8×10 8 CFU / g, total number of viable Candida utilis ≥7×10 8 CFU / g, total number of viable Lactobacillus plantarum ≥5×10 7 The above-mentioned bacterial agent is composed of CFU / g core bacteria. The compound probiotics for drinking water are provided by Guangxi Lvye Biotechnology Co., Ltd.

[0056] 2 Methods

[0057] 2.1 Experimental design and diet composition

[0058] Eighteen Debao pony foals of similar weight were randomly divided into control group, experimental group 1 and experimental group 2 (see Figure 1 ). Each treatment group had 6 replicates, with 1 horse in each replicate. The control group had free access to water, while the experimental group 1 had 50 mL / horse of compound probiotics added to its drinking water, and the experimental group 2 had 60 mL / horse of compound probiotics added to its drinking water. To ensure the feeding effect, the experimental group used 1.5-2.5 kg / horse of water to ensure that the foals could finish drinking it in the morning and then had free access to water in the afternoon. The three groups were fed a complete mixed basal diet with a concentrate to roughage ratio of 4:6 (see Table 1). The concentrate supplement was made into pellets. The actual amount of concentrate supplement was calculated according to the formula of the concentrate supplement and the weight of the foal. The foals were raised in a pen, with a pre-test period of 7 days and a main test period of 60 days. The complete mixed diet was fed once at 09:00 and 16:00 every day, and the amount of feed was adjusted based on the principle that the amount of leftover feed did not exceed 5% of the amount of feed. The amount of feed and the amount of leftover feed were recorded daily in the pen. The horses were provided with clean and sufficient drinking water throughout the experiment, and daily feeding management, immunization procedures and disease treatment were carried out in accordance with the regulations of the farm.

[0059] 2.2 Sample collection and processing

[0060] After morning feeding in the last week of the feeding period, the total mixed ration (TMR) of each group of experimental horses was collected for 3 consecutive days. Feces were collected rectally once wearing disposable long-arm gloves. After mixing the fecal samples collected for 3 days for each horse, 10 g was taken and placed in a 50 mL sterile centrifuge tube, numbered, and quickly placed in a liquid nitrogen tank for storage for fecal microbial flora testing (see Figure 2 ), and the remaining fecal samples were dried at 65℃ for sample preparation.

[0061] 2.3 Detection indicators

[0062] 2.3.1 Growth performance indicators

[0063] On days 0 and 60 of the experiment, the horses were weighed on an empty stomach, recorded as initial and final weights, and average daily gain (ADG) was calculated. Before each morning feeding, the feed trough was cleaned and weighed to record any remaining feed. Average daily dry matter intake (ADDI) was calculated. Based on ADDI and ADG, the feed-to-gain ratio (F / G) was calculated. The calculation formulas were: ADDI = total feed intake / number of experimental days; ADG = (final weight - initial weight) / number of experimental days; and F / G ratio = average daily dry matter intake / average daily gain.

[0064] 2.3.2 Nutrient Apparent Digestibility Index

[0065] The calculation formula for the apparent digestibility of each nutrient is the same as that in Example 1.

[0066] 2.3.3 Fecal microbial flora indicators

[0067] Sequencing and bioinformatics analysis were commissioned to Hangzhou Lianchuan Biotechnology Co., Ltd. Based on species annotation results, the alpha diversity index of OTUs was calculated. Species with the highest relative abundance at the phylum and genus levels in each sample group were selected for comparative analysis. The annotation results were presented as stacked bar charts. The Lianchuan Biotechnology Cloud Platform was used for image processing and correlation heatmap creation. Differences between samples were compared and statistically tested. The Kruskal-Wallis rank sum test was used to analyze differences between multiple groups, and the Wilcoxon sum test was used to analyze differences between two groups.

[0068] 2.4 Statistical analysis of data

[0069] The experimental data were sorted using Microsoft Excel 2021 software, and the One-Way ANOVA method in SPSS17.0 software was used for significance analysis. Duncan's method was used for multiple comparisons. The results were expressed as "mean ± standard error", and P < 0.05 indicated a significant difference.

[0070] 3 Results and Analysis

[0071] 3.1 Effects of adding compound probiotics on the growth performance of Debao pony foals

[0072] Table 4 shows that compared with the control group, the average daily dry matter intake and average daily weight gain of the foals in the experimental groups increased. The average daily dry matter intake and average daily weight gain of the foals in Experimental Group 1 increased by 2.21% and 3.84%, respectively, compared with the control group; the average daily dry matter intake and average daily weight gain of the foals in Experimental Group 2 increased by 3.15% and 8.08%, respectively, compared with the control group. Compared with the control group, the feed-to-gain ratio of Experimental Groups 1 and 2 decreased by 1.63% and 4.59%, respectively, with the feed-to-gain ratio in Experimental Group 2 being significantly lower (P < 0.05).

[0073] Table 4 Effects of adding compound probiotics on the growth performance of Debao pony foals

[0074]

[0075] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05), while data with the same lowercase letters or no letters indicate no significant differences (P>0.05). The same applies to the following tables.

[0076] 3.2 Effect of adding compound probiotics on the apparent digestibility of nutrients in Debao pony foals

[0077] As shown in Table 5, compared with the control group, the apparent digestibility of CP, ADF and NDF of the foals in the experimental group 2 was significantly improved (P < 0.05), and the apparent digestibility of CP, ADF and NDF in the experimental group 1 showed an increasing trend, but the difference was not significant (P > 0.05). The apparent digestibility of CP in the experimental groups 1 and 2 was 1.0% and 7.57% higher than that in the control group; the apparent digestibility of ADF and NDF in the experimental groups 1 and 2 was 6.02% and 3.55% higher than that in the control group, and 10.82% and 10.66% higher than that in the control group, respectively.

[0078] Table 5 Effect of adding compound probiotics on the apparent digestibility of nutrients in Debao pony foals (%)

[0079]

[0080] 3.3 Effects of adding compound probiotics on fecal microflora of Debao pony foals

[0081] 3.3.1 Analysis of α-diversity of fecal microbial flora

[0082] As shown in Table 6, compared with the control group, the observed_species, Shannon, and chao1 indices of the fecal microbial flora of the experimental groups 1 and 2 were increased, but the differences were not significant (P>0.05). The ranking was: experimental 2>experiment 1>control group. The coverage of the three groups was 0.99, indicating that the data of each group can accurately reflect the composition of the foal fecal microbial flora, and the authenticity of the sequencing samples is high.

[0083] Table 6 Effects of adding compound probiotics on the α diversity of fecal microbial flora in Debao pony foals

[0084]

[0085] 3.3.2. Door horizontal structure analysis

[0086] The bacterial composition and relative abundance of foal fecal bacteria at the phylum level are shown in Tables 7 and Figure 3A total of 30 bacterial phyla were annotated, of which six had mean relative abundances greater than 1%. Firmicutes, Verrucomicrobiota, and Bacteroidetes were the top three phyla, with a combined relative abundance exceeding 90%. Other dominant phyla in the foal fecal microbiome included Actinobacteriota, Proteobacteria, and Spirochaetes. Compared with the control group, the relative abundances of Firmicutes and Actinobacteria increased in the experimental groups (with the highest relative abundances in Experiment 2), while the relative abundances of Verrucomicrobiota and Bacteroidetes decreased, but the differences were not significant (P>0.05). The F / B ratios of Firmicutes to Bacteroidetes were 4.05, 4.88, and 6.35 in the control group, Experiment 1, and Experiment 2, respectively.

[0087] Table 7 Bacterial phyla with relative abundance > 1.00% in feces of Debao pony foals

[0088]

[0089] 3.3.3 Genus-level structural analysis

[0090] The bacterial colony composition and relative abundance of foal fecal bacteria at the genus level are shown in Tables 8 and Figure 4 A total of 633 bacterial genera were annotated in this experiment, of which 22 had a relative abundance greater than 1%. Akkermansia, WCHB1-41_unclassified, and Christensenellaceae_R-7 were the top three genera, with a combined relative abundance exceeding 28%. Compared with the control group, the relative abundance of Akkermansia was significantly decreased in Experimental Groups 1 and 2 (P < 0.05), while the difference between Experimental Groups 1 and 2 was not significant. Compared with the control group and Experimental Group 1, the relative abundance of Rikenellaceae RC9, Lachnospiraceae XPB1014 and AC2044, Anaerovorax, and Saccharofermentans was significantly increased in Experimental Group 2 (P < 0.05). The relative abundance of Experimental Group 1 was higher than that of the control group, but the difference was not significant.

[0091] Table 8 Bacterial genera with relative abundance > 1.00% in feces of Debao pony foals

[0092]

[0093]

[0094] 3.3.4 Correlation analysis between bacterial genus levels in fecal flora and apparent digestibility of nutrients

[0095] like Figure 5 As shown in the data, anaerobic Vibrio, sugar-fermenting bacteria, NK4A214_group, Lachnospiraceae (AC2044) and (XPB1014) were positively correlated with the apparent digestibility of CP, NDF and ADF nutrients; unclassified coproposterol-producing Eubacterium group, unclassified Ruminococcaceae, unclassified F082 and unclassified LD1-PB3 were negatively correlated with the apparent digestibility of NDF and ADF nutrients.

[0096] 4. Discussion

[0097] 4.1 Effects of adding compound probiotics on growth performance and apparent digestibility of Debao pony foals

[0098] Stunted growth in foals can affect the production performance, sales price, and ornamental value of adult horses. Average daily gain (ADG) is an important indicator of animal growth performance. This study found that the average daily feed intake and DG of foals in Experimental Group 1 increased by 2.21% and 3.84%, respectively, compared with the control group; while the average daily feed intake and DG of foals in Experimental Group 2 increased by 3.15% and 8.08%, respectively, compared with the control group. Compared with the control group, the feed-to-gain ratio in Experimental Groups 1 and 2 decreased by 1.63% and 4.59%, respectively, with a significant decrease in the feed-to-gain ratio in Experimental Group 2 (P < 0.05). Similar to Yan Xiaogang's research showing that daily addition of probiotics to a pelleted concentrate diet can increase calves' average daily feed intake and average daily weight gain, Li Zheng's research also showed that feeding calves a combination of probiotics significantly increased their average daily weight gain (ADG). The ADG of the three experimental groups increased by 18.42%, 18.42%, and 21.05%, respectively, compared to the control group. Pellet intake in the experimental groups was unaffected by the diet. It is speculated that the differences in feed intake may be related to the growth stage of the animals, in addition to the amount of inoculant added and the type of experimental animal. Dietary digestibility is a key indicator of animal growth performance, reflecting the conversion rate of nutrients in the feed within the animal's body. The digestibility of ADF and NDF reflects the animal's ability to digest and utilize cellulose feeds. The cecum and colon of equines function similarly to the rumen of ruminants such as cattle and sheep, enabling efficient digestion of roughage through complex microbial fermentation processes. In this experiment, the apparent digestibility of CP in groups 1 and 2 increased by 1.0% and 7.57% compared with the control group. The apparent digestibility of ADF and NDF in groups 1 and 2 increased by 6.02% and 3.55% and 10.82% and 10.66% respectively compared with the control group. This indicates that the addition of compound probiotics to drinking water can improve the digestibility of CP, ADF, and NDF in foals' feed, and the effect of using compound probiotics in group 2 is better than that of group 1, improving the utilization of feed nutrients by foals. This is consistent with the results of a study on beef cattle by Peng Zhongli et al., which showed that the addition of compound microorganisms such as Bacillus, yeast, and lactic acid bacteria to concentrate can increase the digestibility of crude protein and significantly improve the digestibility of crude fiber. It is speculated that Bacillus and yeast enter the foal's body and metabolize to produce substances such as amylase, cellulase and protease, which promote the animal's digestion and metabolism of feed nutrients. Lactic acid bacteria can produce bacteriocins and lactic acid, inhibit the growth of harmful bacteria, and have a positive effect on maintaining the microecological balance of the foal's digestive tract, promoting the improvement of digestive function, and improving the foal's digestion and absorption of feed.

[0099] 4.2 Effects of adding compound probiotics on fecal microflora of Debao pony foals

[0100] In this experiment, under consistent feeding and dietary management conditions, the Shannon, observed_species, and chao1 indices increased in both groups 1 and 2, with the addition of compound probiotics to the drinking water. The ranking was: group 2 > group 1 > control group. This suggests that the addition of compound probiotics to the drinking water increased the richness and diversity of the foals' fecal microbiota, altered the structure of their cecal microbiota, and played a positive role in maintaining the dynamic balance of the intestinal microbiota. The species richness and diversity of the intestinal microorganisms in the control group showed a downward trend, suggesting that the microbial homeostasis may have become imbalanced, which may have affected the control group's digestion and absorption of nutrients and lower production performance. The use of probiotic preparations during the feeding of the foals in this experiment artificially increased the number of beneficial bacteria in the intestine, competitively inhibiting the number of some pathogenic bacteria, increasing the abundance of beneficial bacteria, and promoting the animals to achieve a new microbial balance, improving intestinal health, and reducing the occurrence of intestinal diseases (diarrhea or constipation).

[0101] This study found that the dominant bacterial phyla in the foal's hindgut were Firmicutes, Verrucomicrobia, and Bacteroidetes, as well as Actinobacteria, Proteobacteria, and Spirochaetes. This suggests that the six bacterial phyla identified in this study are important components of the equine gut microbiome, consistent with the typical characteristic of a fiber-degrading microbiome. This suggests that adding a compound probiotic to drinking water does not alter the dominant bacterial phyla in foal feces, but does alter their abundance. In this study, compared with the control group, the relative abundance of Verrucomicrobia and Bacteroidetes decreased in the experimental group, while the relative abundance of Firmicutes and Actinobacteria increased (with the highest relative abundance in experimental group 2). Firmicutes and Bacteroidetes play a dominant role in the intestinal immune response and the conversion of dietary fiber to short-chain fatty acids. A balanced F / B ratio between Firmicutes and Bacteroidetes is crucial for maintaining intestinal homeostasis and intestinal health. A higher ratio facilitates energy storage and nutrient absorption, while a lower ratio is generally associated with inflammatory bowel disease. In this experiment, the F / B ratios of the control group, test group 1, and test group 2 were 4.05, 4.88, and 6.35, respectively. The control group was lower than test groups 1 and 2, which means that the intestinal environment of the control group was unbalanced, which may have caused the imbalance of the intestinal environment of the control group, thereby affecting the body's digestion and absorption of feed nutrients. The abundance of Firmicutes in test group 2 was the highest, while the abundance of Bacteroidetes was the lowest, indicating that the addition of a certain compound bacterial agent to test group 2 can promote the utilization of carbohydrates and the degradation of crude fiber by the intestinal flora of foals. Actinobacteria synthesize organic acids (such as propionic acid and succinic acid) and various functional enzymes such as glycoside hydrolases and proteases in the intestine. Their metabolic activities are of great significance for the regulation of intestinal acid-base balance and the inhibition of pathogenic microorganisms. In this experiment, the addition of the compound probiotic preparation caused the relative abundance of intestinal Actinobacteria to show an upward trend, indicating that the preparation may have a positive regulatory effect on the stability of the intestinal environment and the health of the body by promoting the proliferation of Actinobacteria. It can be seen that adding a combined bacterial agent of Bacillus + yeast + lactic acid bacteria to drinking water can effectively improve the ability of the fecal bacterial community of Debao pony foals to degrade crude fiber and the body's ability to digest carbohydrates, and has a positive effect on maintaining the balance of the intestinal flora structure and the balance of environmental homeostasis after weaning foals, improving intestinal health, and allowing foals to obtain more nutrition from food, which can further promote fat deposition and thereby improve the growth performance of foals.

[0102] This study found that at the genus level, Akkermansia was the dominant genus in the fecal microorganisms of foals. Compared with the control group, the relative abundance of Akkermansia in groups 1 and 2 was significantly reduced, and the difference between the two groups was not significant. At the same time, the relative abundance of Verrucomicrobia in the control group showed an increasing trend compared with the experimental group, indicating that the addition of compound probiotics to drinking water has a positive effect on maintaining the balance of the intestinal environment of foals and ensuring the normal physiological function of the intestine.

[0103] This experiment also found that the relative abundance of the Rikenella RC9 intestinal group in the feces of the foals in the experimental group 2 was significantly higher than that in the experimental group 1 and the control group, which indicated that the fiber digestibility and protein utilization rate of the experimental group 2 were higher than those in the experimental group 1 and the control group. The significant increase in its relative abundance increased the apparent digestibility of CP, ADF and NDF, promoted the digestion and absorption of nutrients, and the gastrointestinal health of the foals in the experimental group 2 was higher than that in the control group and the experimental group 1.

[0104] This study found that compared with the control group and test group 1, the relative abundance of Lachnospiraceae XPB1014 and AC2044, Anaerobic Vibrio, and Saccharofermentation Bacteria in test group 2 was significantly increased (P < 0.05), with test group 1 showing higher abundance than the control group. This suggests that the addition of compound probiotics to drinking water promotes the growth and reproduction of beneficial bacteria such as Lachnospiraceae, Anaerobic Vibrio, and Saccharofermentation Bacteria in the intestines of foals in the experimental group, improving feed intake and the efficiency of intestinal decomposition of plant fiber and protein, enabling better nutrient absorption. Furthermore, the abundance of Lachnospiraceae XPB1014 and AC2044, Anaerobic Vibrio, and Saccharofermentation Bacteria was positively correlated with the apparent digestibility of CP, NDF, and ADF nutrients, further demonstrating that the addition of compound probiotics such as Bacillus, yeast, and lactic acid bacteria to drinking water improves the digestion of beneficial bacteria such as Lachnospiraceae XPB1014 and AC2044, Anaerobic Vibrio, and Saccharofermentation Bacteria in the intestines of foals. This study found that compared with the control group, the feed-to-weight ratios of Experiment 1 and Experiment 2 decreased by 1.63% and 4.59%, respectively, with a significant reduction in the feed-to-weight ratio in Experiment 2 (P < 0.05), demonstrating the key role of microbiota in optimizing feed utilization. It is hypothesized that intestinal commensal bacteria, such as Lachnospiraceae, Anaerobic Vibrio, and Saccharofermentobacteria, significantly enhance the foals' efficiency in degrading structural carbohydrates by secreting carbohydrate-active enzymes such as cellulase and xylanase. This enzymatic hydrolysis not only enhances the conversion of dietary fiber into short-chain fatty acids but also indirectly promotes animal growth performance by optimizing intestinal energy metabolism and nutrient absorption. Therefore, the addition of a Bacillus, Yeast, and Lactobacillus complex probiotic to drinking water effectively improves the intestinal ability of Debao pony foals to degrade crude fiber and digest non-fibrous carbohydrates, promoting food digestion and effective nutrient absorption, thereby improving growth performance and having a positive effect on the growth and health of Debao pony foals.

[0105] 5 Conclusion

[0106] Adding compound probiotics to drinking water increased the richness and diversity of bacterial flora in Debao pony foals' feces, reduced the relative abundance of Akkermansia and Verrucomicrobia, and promoted the growth of beneficial bacteria such as the RC9 enteric group of the family Cynobacteria, XPB1014 and AC2044 of the family Lachnospiraceae, Anaerobic Vibrio, and Saccharofermentobacter. This improved the foals' intestinal ability to degrade crude fiber and digest non-fiber carbohydrates, enhancing food digestion and effective nutrient absorption, and promoting healthy growth. Adding 60 mL / horse of compound probiotics to drinking water daily was most effective.

[0107] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. An application of a composite probiotic in improving the intestinal digestive ability of Debao pony foals, characterized by: The intestinal digestion capacity includes reducing the relative abundance of Akkermansia and Verrucomicrobia in foal feces, and increasing the relative abundance of the RC9 intestinal group of the Lachnospiraceae family, XPB1014, AC2044, Anaerobic Vibrio, and Saccharofermenting Bacteria.

2. The use according to claim 1, characterized in that: The addition amount of the compound probiotics is 50-70 mL / pig in drinking water.

3. The use according to claim 1, characterized in that: The Debao pony foal was an 8-9 month old weaned foal with a body weight of 75±2 kg.

4. A composite probiotic preparation for improving the intestinal digestion ability of Debao pony foals, characterized by: Including Bacillus, yeast and lactic acid bacteria; the ratio of total viable bacteria is: Bacillus: yeast: lactic acid bacteria = 8:7:0.

5.

5. The composite probiotic preparation according to claim 4, wherein: The total number of viable bacteria of the Bacillus, yeast and lactic acid bacteria are: Bacillus ≥ 8×10 8 CFU / g, yeast ≥7×10 8 CFU / g, lactic acid bacteria ≥5×10 7 CFU / g.

6. The composite probiotic preparation according to claim 4, wherein: The bacillus is Bacillus subtilis, the yeast is Candida utilis, and the lactic acid bacteria is Lactobacillus plantarum.

7. A method for healthy breeding of Debao pony foals, characterized in that: The following steps are involved: (1) Add the composite probiotic preparation of claim 4 to the drinking water in a drinking bucket at a dose of 50-70 mL / pickle; (2) Fill the drinking bucket with 1.5-2.5 kg of water per horse to ensure that the foals drink the bacteria-containing water in the morning; (3) Feeding was continued for 60 days, during which the growth performance and fecal flora changes of the foals were monitored.

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