Effects of probiotic formulations on GIT morphological changes and gut microbiota in Hu lambs

Effects of probiotic formulations on GIT morphological changes and gut microbiota in Hu lambs | 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 Effects of probiotic formulations on GIT morphological changes and gut microbiota in Hu lambs Ting Xu, Siling Ding, Ye Xu, Siyu Yang, Yian Yao, Pengcheng Wan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7333756/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 Gut microbiota plays an important role in the digestive system of ruminants. It affects the health status of Hu sheep and their development and production rates. However, its composition may be influenced by several factors such as gender, age, and diet. In the animal husbandry industry, probiotic formulations have been widely used as alternatives to antibiotics, offering advantages such as non-toxicity, non-residue, and non-pathogenicity. This study aimed to investigate the morphological changes in the gastrointestinal tract and dynamics of gut microbiota during postnatal development of Hu lambs, and evaluated the effects of dietary supplementation with probiotic formulations on gut microbiota. Fifteen male Hu lambs were randomly divided into five groups, with three lambs per group. One blank control group did not receive food postnatally, while two control groups were fed according to standard farm practices with milk and a basic diet. Two experimental groups were administered 10 mL of probiotic formulations within 2 hours after birth, followed by the same feeding regimen as the controls. During the 60-day experiment, samples were collected from the rumen, duodenum, jejunum, and ileum at 2 hours, 20 days, and 55 days after birth. The collected samples included digestive tract tissues and their contents. High-throughput 16S rRNA gene sequencing was employed to identify and analyze microbial diversity, and morphological analysis was conducted to compare differences in the surface tissues of the digestive tract. The results indicated that, with increasing age, the length of the rumen papillae and the height of the villi in the jejunum and ileum of both control and experimental groups continued to increase. Additionally, the overall diversity of the gut microbiota exhibited a gradual upward trend. The microbial diversity indices in the rumen, duodenum, jejunum, and ileum of the experimental group were higher than those of the control group at corresponding time points, although the differences were not statistically significant ( p > 0.05). The number of beneficial bacteria, such as Ruminococci , in the gastrointestinal tract of Hu lambs in the experimental group increased, while the number of potentially pathogenic bacteria, such as Escherichia spp ., relatively decreased. The results suggest that the supplementation of probiotic formulations promotes early gastrointestinal tract development, enhances the colonization of beneficial bacteria, and improves microbial diversity in Hu lambs. This result provides valuable insights into the gastrointestinal development and microbial dynamics of Hu lambs, as well as the production of probiotic formulations for sheep. Health sciences/Gastroenterology Biological sciences/Microbiology Biological sciences/Zoology Hu lambs probiotic formulations 16S rRNA gene sequencing gut microbiota GIT morphology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Sheep are a pivotal species in global animal husbandry, being distributed worldwide and serving as a significant economic resource for herders. With the evolution and scaling of the livestock industry, sheep farming has become a cornerstone of this sector. Prolificacy is a critical determinant in the development of meat sheep farming [ 1 ] . Hu sheep are renowned for their high fertility, early sexual maturity, continuous estrous cycles, adaptability to hot and humid climates, and suitability for intensive housing systems [ 2 ] . This breed possesses one of the highest fertility rates globally, with lambing rates exceeding 280% [ 3 , 4 ] , making it a primary choice for meat sheep farming and crossbreeding programs. However, Hu sheep are characterized by a low carcass yield and inferior meat quality [ 5 ] . In sheep breeding, feed expenses can constitute up to 70% of total costs [ 6 ] , while diseases such as colds, pneumonia, and diarrhea in lambs, along with milk fever and mastitis in ewes, are prevalent [ 7 ] . Enhancing feed conversion efficiency and immune competence is therefore crucial for economic gains in sheep breeding. These factors are influenced by both genetic predisposition and gut microbiota. Nutrient absorption, energy metabolism, and immune regulation in animals are intricately linked to their gut microbiota [ 8 – 10 ] . The gut microbiota has been confirmed as a vital component in ruminant growth and health. The interaction between the gut microbiota and the host intestinal environment plays a key role in digestion, metabolism, immunity, and disease prevention [ 11 – 13 ] . Given the substantial intake of crude fiber by ruminants, the breakdown of cellulose depends on cellulolytic bacteria within the gut microbiota [ 14 ] . The gut microbiota, as a key site for digestion and absorption, exerts a substantial influence on the health and performance of the host [ 15 ] . In the animal husbandry industry, probiotic formulations have been widely employed as alternatives to antibiotics, offering advantages such as non-toxicity, non-residue, and non-pathogenicity [ 16 ] . Previous studies have demonstrated that feeding probiotic formulations enhances immune parameters and growth performance in Hu sheep pre- and post-weaning (60 days of age) [ 17 ] The addition of probiotic formulations to the diet of Hu sheep significantly increases the carcass yield and feed conversion ratio (FCR), enhances immunity and antioxidant capacity, and regulates the gut microbial homeostasis [ 18 ] . Moreover, feeding probiotics to ruminants has been shown to improve nutrient intake and digestibility, milk production, immunity, reproduction, and feed conversion efficiency [ 19 ] . Furthermore, feeding probiotic formulations significantly increases total blood protein, globulin, and weight gain in lambs [ 20 ] , as well as enhances daily weight gain, feed digestibility, and body immunity in weaned goats [ 21 ] . The effects of probiotic formulations on ruminants are primarily achieved by improving the structure and function of microbial communities and influencing animal behaviors such as feed intake [ 22 ] . Probiotic formulations adhere to the inner wall of the digestive tract, forming a colonization resistance in the intestine. Over time, a stable microbial community is established, regulating the structure and quantity of intestinal microbiota, thereby influencing the immune system and growth performance of the host [ 23 ] . Currently, commercial probiotic formulations primarily include probiotics, prebiotics, and synbiotics, which are widely used in sheep farming and offer various beneficial functions [ 17 , 24 ] , thereby improving the efficiency of sheep breeding. Commercially available probiotic formulations utilizing slow-release encapsulated multiple probiotics can enhance immunity and promote rapid growth during the short-term growth phase in Hu lambs [ 17 ] . However, the effects of such probiotic formulations on the digestive system microorganisms of lambs, as well as the alteration of gut microbiota composition, remain unclear. Therefore, this experiment provides a theoretical basis for elucidating the developmental processes of gastrointestinal tract (GIT), the basic characteristics of the gut microbiota, and the effects of probiotic formulations in the feeding of Hu lambs, thus offering a reference for the production and development of probiotic formulations for sheep. Materials and methods Experimental material The probiotic formulation used in this study was purchased from Qingdao Zhengtai United Nutritional Technology Co., Ltd. (product name “Duyikang”), provided in a cream formulation with a volume of 60 mL per tube. The labeled ingredients included Bacillus licheniformis , Bacillus subtilis , Enterococcus faecalis , and Lactobacillus rhamnosus as the main probiotics, along with prebiotics such as colostrum extract, medium-chain triglycerides, trace elements, and vitamins. Laboratory animals and sample collection This experiment was conducted from October to December in 2022 at Hangzhou Caiyang Herding Co., Ltd. (Zhejiang, China), over a 60-day period. Fifteen lambs with similar birth times and weights were selected from 56 newly born male Hu lambs that were twin-born and randomly assigned to five groups (n = 3). The groups were as follows: one blank control group (BG, Group I), two control groups (CG, Groups II and III), and two experimental groups (TG, Groups IV and V). The GIT contents of lambs in BG were sampled within 2 hours after birth. Lambs in TG IV and V were orally administered 10 mL of the probiotic formulations within 2 hours after birth, while lambs in CG II and III did not receive the probiotic formulations. All lambs in TG and CG groups were suckled naturally under identical conditions, with supplemental lamb milk substitute provided from approximately 30 days of age. Free feeding was allowed, and all experimental lambs were tagged using ear tags with wool staining for identification. All experimental procedures involving Hu sheep were conducted in accordance with the guidelines of the China Council on Animal Care. At the corresponding time of experimental designed, namely 0 day, 20 days, and 55 days after the birth of the lamb, no food was fed on the day of the sample collection. All animals from each group were euthanized by captive bolt stunning. Physical disruption methods are often followed by exsanguination as the adjunctive method to ensure humane euthanasia. The slaughter procedures were performed by trained personnel in accordance with standard animal welfare protocols. Immediately after slaughter and dissection, the organs to be sampled were rinsed clean with ice cold sterile PBS. Intestinal segments were defined as follows: the proximal third (duodenum), mid third (jejunum), and distal third (ileum) of the small intestine. For each target segment (duodenum, jejunum, ileum), a section approximately 3 cm in length was ligated proximally and distally using sterile suture material. The isolated segment was then carefully excised using sterile surgical scissors. The luminal contents within this isolated segment were gently expressed into sterile collection tubes. One section of each sample was snap-frozen in liquid nitrogen and then stored at -80°C until the time of analysis. The remaining portion was fixed in neutral buffered 10% formalin for histological analysis. Samples from the rumen, duodenum, jejunum, and ileum contents were collected from lambs in CG II and TG IV as well as those in CG III and TG V at 20 and 55 days of age, respectively, based on the rate and timing of weight gain influenced by feeding the probiotic formulations [ 17 ] . For BG I, GIT contents were similarly collected from the same anatomical sites within 2 hours after birth, snap-frozen in liquid nitrogen, and stored at − 80°C. Morphological Observations Tissue samples from the rumen, duodenum, jejunum, and ileum were collected in accordance with the GIT content sampling strategy. Morphological analysis was performed using hematoxylin-eosin (HE) staining and light microscopy. The methodology followed Wang et al. [ 25 ] , with the procedure detailed as follows: tissue samples were fixed in paraffin, and the embedded paraffin blocks were sectioned into 4 µm-thick cross-sections. The sections were then stained with HE and observed under a light microscope (Eclipse Ci, Nikon, Melville, NY). Photographs of the tissues were captured using an imaging system (Digital Sight DS-Fi2, Nikon, Melville, NY). Measurements of rumen papillae length and width, muscularis propria depth, villus height (VH), crypt depth (CD), and muscularis propria thickness were performed in the duodenum, jejunum, and ileum. The villus height to crypt depth (V/C) ratio was subsequently calculated. DNA extraction and PCR amplification of gut microbiota Total genomic DNA was extracted from samples collected from different organs using Bacterial Genome Extraction Kit (Tiangen Biotech Co.,Ltd., Beijing, China), following the manufacturer’s instructions. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, United States), and DNA integrity was assessed via 2% agarose gel electrophoresis. PCR amplification targeting the hypervariable V3-V4 region of the 16S rRNA gene was performed using the primers 515F (5’-GTGYCAGCMGCCGCGGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) [ 26 ] . Positive PCR products were purified using the GeneJET® Gel Extraction Kit (Thermo Scientific), and sequencing libraries were constructed with the Ion Plus Fragment Library Kit (Thermo Scientific). Library quality was evaluated using a Qubit 2.0 fluorometer (Thermo Scientific). 16S rRNA sequencing and analysis Sequencing was performed on the Ion S5™ XL platform, generating single-end reads of 407 to 412 base pairs. Sequencing data were processed by splitting sequences based on barcode information, followed by removal of barcode and primer sequences using the software cutadapt (v1.9). Overlapping region sequences were merged with the software FLASH (v1.2.8). Sequencing reads were subjected to quality control using a window scanning method implemented in fgtrim, with sequences shorter than 100 bp after truncation discarded. Chimeric sequences were identified and removed using Vsearch (v2.3.4). DADA2 was employed via QIIME2 for length filtering and denoising to obtain Amplicon Sequence Variant (ASV) feature sequences and abundance tables. Based on the ASV feature sequences and abundance data, α-diversity and β-diversity analyses were performed. Comparative analyses of species abundance between control and experimental groups were conducted, with a significance threshold of p 3) enabled the identification of important bacteria between groups. Statistical analysis Experimental data were organized using Excel 2021, and statistical analyses were conducted using SPSS 23.0. A one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. The Least Significant Difference (LSD) method was employed to determine statistical significance, with p < 0.05 considered indicative of a significant difference. Results are expressed as mean ± standard deviation. Results Changes in the morphological structure of GIT and effects of probiotic formulations GIT is the primary organ responsible for nutrient absorption in animals, and its developmental status directly impacts nutrient absorption efficiency. Histological and morphological analyses revealed that the length and width of the rumen papillae, as well as the thickness of the rumen musculature, were significantly greater at 55 days of age compared to 20 days ( p < 0.05), consistent with the lambs' growth. In the duodenum, the height of the villi was higher at birth, gradually decreased, and subsequently increased with age. Similarly, the depth of the crypts increased, the V/C ratio decreased, and the muscularis propria thickness increased over time. In the control groups, the villus height in the duodenum at both 20 and 55 days of age was significantly lower than at birth. The crypt depth and muscularis propria thickness were significantly greater at 55 days compared to 20 days and at birth. The V/C ratio in control lambs at both 20 and 55 days was significantly lower than at birth. In the jejunum, villus height at 55 days was significantly higher than at 20 days and at birth. The crypt depth and muscularis propria thickness at 55 days were also significantly greater than at 20 days and birth. The V/C ratio at birth and 55 days was significantly higher than at 20 days. In the ileum, villus height and crypt depth at 55 days were significantly greater than at 20 days and birth, while the muscularis propria thickness was also significantly higher at 55 days compared to earlier stages. However, no significant differences in V/C were observed among the three control groups. Comparative analysis between the experimental and control groups indicated that no significant difference ( p > 0.05) in the length of the rumen papillae at 20 days. However, at 55 days, the length of the rumen papillae in the experimental group increased significantly ( p 0.05) in the width of the rumen papillae or the thickness of the muscularis layer between the experimental and control groups at the same age. In the duodenum, the villus height showed no significant difference ( p > 0.05) between the experimental and control groups at 20 days. However, at 55 days, the villus height in the experimental group was significantly greater ( p 0.05) in crypt depth, V/C, or muscularis layer thickness in the duodenum between the experimental and control groups at any age. Similarly, no significant differences ( p > 0.05) were found in villus height, crypt depth, V/C, or muscularis layer thickness in the histomorphology of the jejunum and ileum. Table 1 Effect of probiotic formulations on rumen tissue morphology in Hu sheep. Items Groups CG20 TG20 CG55 TG55 Length of rumen papilla/µm 471.74 ± 85.33 c 484.72 ± 126.66 c 896.07 ± 194.03 b 1040.57 ± 107.72 a Width of rumen papilla/µm 176.95 ± 41.15 b 167.05 ± 31.80 b 247.28 ± 32.04 a 274.58 ± 17.62 a Muscle thickness/µm 713.73 ± 38.99 b 753.14 ± 79.16 b 940.24 ± 45.98 a 942.51 ± 44.93 a a,b,c Within a row for each item, different superscripts indicate significant differences ( p < 0.05). Table 2 Effect of probiotic formulations on the morphology of duodenum in Hu sheep. Items Groups BG00 CG20 TG20 CG55 TG55 Villus height /µm 457.57 ± 59.94 a 336.07 ± 39.64 bc 312.34 ± 39.6 c 373.74 ± 35.27 b 424.72 ± 52.33 a Crypt depth /µm 100.90 ± 15.59 d 139.07 ± 18.29 c 133.85 ± 17.22 c 175.59 ± 26.22 b 182.43 ± 22.68 b V/C 4.68 ± 0.68 a 2.30 ± 0.30 b 2.30 ± 0.24 b 2.11 ± 0.10 b 2.17 ± 0.41 b Muscle thickness /µm 78.83 ± 13.43 c 175.08 ± 56.46 b 175.53 ± 16.35 b 231.67 ± 26.73 a 240.91 ± 9.54 a a,b,c Within a row for each item, different superscripts indicate significant differences ( p < 0.05). Changes in the diversity of gut microbiota in control lambs High-throughput sequencing of the microbiota from the rumen, duodenum, jejunum, and ileum contents of the BG00, CG20, and CG55 groups revealed that Shannon curves plateaued at sequencing depths exceeding 5000 reads (Fig. 3 ). This indicated that the sequencing depth and data volume of the 16S rRNA V3-V4 gene region were sufficient for diversity analysis. A Venn diagram was used to visualize the specific and shared ASVs among the three groups. The ASV results are shown in Fig. 4 . Across all four sites, the BG00 group exhibited the lowest number of ASVs, while the CG55 group showed the highest number, demonstrating a gradual increase in ASVs with lamb growth. This finding indicates that the diversity and abundance of gut microbiota increase as lambs mature. Comparison of α diversity in different gastrointestinal regions at various growth stages (Table 3 ) showed that in the rumen, the Chao1 and Shannon indices displayed an upward trend with age. The Chao1 index increased significantly at 20 days compared to birth ( p < 0.05) and both Chao1 and Shannon indices were significantly higher at 55 days than at birth ( p < 0.05; Fig. 5 ). In the duodenum, the Chao1 index increased with age, but the change was not significant. In the jejunum, the α diversity indices followed a clear ‘V’ pattern, being higher at birth and 55 days and lowest at 20 days, though the differences were not significant. In the ileum, the Chao1 index showed a continuous increase, with significant differences at 20 and 55 days compared to birth ( p < 0.05). However, the Simpson and Shannon indices exhibited a "V-shaped" pattern, with significantly higher values at birth and 55 days than at 20 days. These findings indicate an overall increasing trend in gut microbiota diversity and abundance as lambs grow. The results of β diversity analysis using Principal Coordinates Analysis (PCoA) (Fig. 6 ) demonstrated distinct separations among the three control groups at birth, 20 days, and 55 days. Within each group, samples were aggregated, suggesting significant differences in gut microbiota composition among the three stages. This indicates that the structure of the gut microbiota undergoes substantial changes, with increased diversity during lamb growth. Table 3 Changes in α-diversity of gut microbiota of Hu lambs at 0, 20 and 55 days of age. Item Groups Observed_otus Shannon Simpson Chao1 Goods_coverage Pielou_e BG00 148 ± 82.49 b 4.37 ± 0.58 b 0.85 ± 0.11 155.56 ± 82.22 b 1 ± 0 0.62 ± 0.1 Rumen CG20 916.67 ± 418.82 ab 6.33 ± 2.37 ab 0.85 ± 0.21 1090.11 ± 491.15 a 0.98 ± 0.01 0.64 ± 0.2 CG55 1367 ± 376.76 a 9.25 ± 0.24 a 1 ± 0 1567.27 ± 506.85 a 0.98 ± 0.01 0.89 ± 0.02 BG00 275 ± 172.2 5.21 ± 1.52 0.94 ± 0.06 228.17 ± 207.76 1 ± 0 0.71 ± 0.1 Duodenum CG20 534.33 ± 427.4 5.12 ± 2.67 0.87 ± 0.11 635.33 ± 470.64 0.99 ± 0.01 0.57 ± 0.22 CG55 692 ± 362.26 6.12 ± 2.18 0.91 ± 0.09 792.69 ± 362.53 0.99 ± 0 0.65 ± 0.18 BG00 339 ± 459.67 5.85 ± 2.54 0.94 ± 0.05 356.13 ± 487.16 1 ± 0 0.78 ± 0.11 Jejunum CG20 153.67 ± 53.72 2.75 ± 1.89 0.59 ± 0.37 181.91 ± 75.25 1 ± 0 0.39 ± 0.29 CG55 370.33 ± 93.63 4.17 ± 0.91 0.77 ± 0.19 418.62 ± 113.54 0.99 ± 0 0.49 ± 0.11 BG00 116 ± 75.94 b 4.61 ± 0.77 a 0.92 ± 0.03 a 132.66 ± 95.55 b 1 ± 0 0.69 ± 0.03 Ileum CG20 219 ± 29.46 b 3.2 ± 0.64 b 0.75 ± 0.09 b 265.92 ± 27.15 b 1 ± 0 0.41 ± 0.08 CG55 618 ± 127.7 a 5.51 ± 0.61 a 0.9 ± 0.06 a 717.37 ± 133.32 a 0.99 ± 0 0.6 ± 0.08 Note: a,b Within a column for each item, different superscripts indicate significant differences ( p < 0.05). Biomarkers of gut microbiota in control lambs To analyze changes in bacterial microbiota during lamb growth at both the phylum and genus levels, LEfSe was utilized to identify significantly different bacterial taxa (LDA scores > 3) across control groups. The findings revealed (Fig. 7 ) that, in the rumen, the BG00 group was enriched with 20 bacterial taxa, including 1 phylum and 5 genera. The CG20 group exhibited enrichment in 28 bacterial taxa, comprising 2 phyla and 12 genera, while the CG55 group displayed enrichment in 40 bacterial taxa, including 1 phylum and 16 genera. In the duodenum, the BG00 group was enriched in 26 bacterial taxa, encompassing 2 phyla and 5 genera. The CG20 group was enriched in 8 bacterial taxa, including 4 genera, and the CG55 group demonstrated enrichment in 14 bacterial taxa, involving 6 genera. In the ileum, the BG00 group was enriched in 18 bacterial taxa, including 5 genera. The CG20 group exhibited enrichment in 6 bacterial taxa, comprising 4 genera, whereas the CG55 group showed enrichment in 25 bacterial taxa, encompassing 2 phyla and 7 genera. A comparative analysis of lamb growth from birth to 55 days of age identified 33 genera with significant differences in the rumen, 16 in the duodenum, 11 in the jejunum, and 16 in the ileum. The BG00 group exhibited the lowest number of genera in the rumen, suggesting that Hu lambs possess a relatively established but limited bacterial microbiota early in life. As lambs matured, the relative abundance of Ruminococcus , Prevotellaceae , Megasphaera , Fibrobacterota , Richenellaceae _ RC9_gut_group , Lachnospiraceae , Christensenellaceae_R-7_group , and Ligilactobacillus increased, while the relative abundance of Caulobacter and Citrobacter decreased. In the duodenum, the relative abundance of Firmicutes, Bacteroidetes, Prevotella , and Christensenellaceae_R-7_group increased. In the jejunum, an increase in Firmicutes and Akkermansia was observed, with a concomitant decrease in Proteobacteria. In the ileum, Proteobacteria and Actinobacteria decreased, while Firmicutes, Prevotella , and Christensenellaceae_R-7_group increased. Effect of probiotic formulations on gut microbiota diversity in Hu lambs Following administration of probiotic formulations, the results of ASV visualization analysis of 16S rRNA sequencing Venn diagrams for gastrointestinal contents in the TG20 and TG55 groups indicated that the number of ASVs in the rumen, duodenum, jejunum, and ileum increased with the growth of Hu lambs (Fig. 8 ). The diversity indices in the experimental groups were higher than those in the control groups at corresponding time points, though the differences were not statistically significant ( p > 0.05). PCoA based on Bray-Curtis distance demonstrated less pronounced separation of gut microbiota in the TG groups compared to the CG groups over the same period, with lower levels of within-group aggregation. Effects of probiotic formulations on gut microbiota structure and composition of Hu lambs A comparative analysis of microbial communities in the rumen, duodenum, jejunum, and ileum of experimental and control groups at 20 and 55 days revealed increased microbial diversity indices and relative abundances at both the phylum and genus levels (Fig. 9 , 10 ). However, these increases were not statistically significant. Firmicutes were identified as the dominant phyla across all gastrointestinal sites in both experimental and control groups at 20 and 55 days of age. Variations in microbial communities were evident among specific gastrointestinal sites. In the rumen, Firmicutes and Bacteroidetes were predominant in all groups. In the duodenum and jejunum, Firmicutes remained dominant, whereas in the ileum, Firmicutes and Proteobacteria were major phyla at 20 days, with Proteobacteria declining by 55 days as Firmicutes became predominant. At the genus level, microbial species varied across organs and groups, with no significant differences observed (Fig. 11 , 12 ). In the rumen, the relative abundance of Rikenellaceae _ RC9_gut_group , Succiniclasticum , and Prevotella increased in the experimental group, while that of Muribaculaceae _ unclassified decreased. In the duodenum, the experimental group exhibited increased relative abundance of Lactobacillus , Christensenellaceae_R-7_group , Prevotella , and Rikenellaceae_RC9_gut_group , while Clostridium _ sensu_stricto_1 decreased. In the jejunum, the experimental group demonstrated increases in Lactobacillus and Ruminococcus , with declines in Romboutsia , Clostridium_sensu_stricto_1 , Sarcina , and Candidatus _ Arthromitus . In the ileum, Lactobacillus , Candidatus _ Saccharimonas , and Lachnospiraceae _ UCG_002 showed increased relative abundance in the experimental group (Fig. 13 , 14 ). Discussion Hu sheep, a renowned prolific indigenous breed in China, provides valuable insights into ovine growth, development, and physiological processes. The prolificacy of this breed not only enhances the productivity of sheep farming but also necessitates additional investments in the rearing of multiple lambs. These investments include artificial feeding, fostering, and the development and application of various milk replacer materials. Additionally, probiotic formulations and other management strategies are employed to increase the survival and growth rates of lambs from multiple births. Commonly used probiotics in animal husbandry, such as Lactobacillus , Enterococcus , Bacillus , and Bifidobacterium , have been shown to exert beneficial effects on animal growth [ 27 ] . Combined probiotics are generally more effective than single strains [ 28 , 29 ] , as they exhibit synergistic or cooperative effects [ 30 , 31 ] . Specifically, Bacillus licheniformis , included in probiotic supplements, has been demonstrated to promote growth, enhance antioxidant and immune functions, and increase the population of beneficial intestinal bacteria in sheep [ 32 ] . Commercial probiotic mixtures containing Bacillus licheniformis have been shown to improve growth rates, bolster immune function, and enhance the diversity of intestinal microbiota, thereby supporting metabolic homeostasis [ 20 ] . The interaction between gut microbiota and the host is essential for the development of the host's gastrointestinal, immune, metabolic, and neurological systems [ 33 ] . Numerous studies have demonstrated that early microbial colonization significantly influences long-term health across an organism's life cycle [ 34 – 36 ] . The larval stage is critical for the establishment and maturation of microbiota, with the composition of gastrointestinal microbiota playing a crucial role in growth and health [ 37 ] . The present study analyzed the intestinal microbiota of Hu lambs from birth to weaning and identified significant increases in beneficial bacterial populations alongside growth and diversification. Furthermore, the administration of probiotic formulations significantly enhanced the probiotic microbiota in the rumen, duodenum, jejunum, and ileum. These findings align with observed improvements in immunity and growth rates during the same period [ 17 ] . Microbiota analysis revealed that Firmicutes predominated in the rumen, duodenum, jejunum, and ileum throughout the growth period, alongside Bacteroidetes and Proteobacteria. The diversity of microbiota increased with the growth of the lambs, consistent with the main probiotic components of probiotic formulations used in this experiment. The formulation included strains such as Bacillus licheniformis , Bacillus subtilis , Enterococcus faecalis , and Lactobacillus rhamnosus , all of which belong to the Firmicutes phylum. In the control group, microbial diversity exhibited a "V-shaped" pattern from birth to 55 days of age, with a decline at 20 days followed by an increase. This pattern is hypothesized to be associated with the initial acquisition of microbiota exclusively from the dam. A reduction in microbial diversity occurs at 20 days as the lambs have not yet established their distinct microbiota. The original microbial populations decline, and by 55 days of age, lambs gradually develop their own microbial communities through environmental exposure and maternal milk intake, resulting in significant differences across the three periods. Following the administration of probiotic formulations, the introduction of sustained-release strains resulted in a convergence of microbial community types across different organs and between ages of 20 and 55 days. This reduced inter-group differences and led to lower intra-group clustering, suggesting consistent effects of the additive. Consequently, the study confirms that feeding probiotic formulations can enhance the population of dominant microbial species in GIT of Hu lambs, ensuring the colonization of beneficial microbes and thereby improving immune function and growth rates. Morphological analysis revealed significant differences in gastrointestinal tissue structure across different stages in the control group of Hu lambs from birth to 55 days of age. Notably, at 55 days, the rumen papillae's length and width, as well as the rumen muscle layer, were significantly greater than those observed at 20 days of age. The increase in rumen papillae dimensions is believed to enhance the absorption efficiency of volatile fatty acids (VFAs), thereby improving feed conversion rates and promoting growth and development [ 38 ] . These findings suggest significant improvements in rumen fermentation performance as Hu lambs mature. In the duodenum, V/C ratio significantly decreased from birth to 20 and 55 days of age. This reduction is likely associated with the initial necessity for maternal milk absorption in newborn lambs. The absorptive surface area of the intestine is determined by villus height, with taller villi correlating with greater absorptive capacity. Villus height, crypt depth, and the V/C ratio reflect the small intestine's capacity for nutrient digestion and absorption, while intestinal muscularis thickness indicates rhythmic contractile capability [ 39 ] . The administration of probiotic formulations significantly increased the length of rumen papillae in 55-day-old lambs ( p < 0.05), with a concurrent increase in width, although early changes were less pronounced. This may be related to the dietary transition from a milk-based diet to concentrate feeds such as cornmeal and subsequently to pelleted feed. Probiotics ingested and colonized gradually exerted their effects through probiotic formulations by breaking down complex compounds like fiber, leading to the production of VFAs that promote the growth of rumen papillae [ 38 ] . Consequently, this enhanced the growth performance of lambs, consistent with findings that lambs fed probiotic formulations exhibited higher relative growth rates after 30 days of age. Probiotic formulations also significantly increased duodenal villus height in 55-day-old lambs ( p < 0.05), potentially enhancing nutrient absorption and contributing to the observed growth improvements. The formulation had minimal impact on intestinal tissue changes in 20-day-old lambs, possibly due to the colonization and effects of probiotics being influenced by postnatal dietary structure and digestive tract development. In evaluating microbial diversity, α diversity represents the diversity within a specific environment or ecosystem, encompassing species richness and evenness. In contrast, β diversity reflects the similarity or dissimilarity between individuals or groups, where greater distances between samples indicate more pronounced differences in their microbial community structures. Together, α and β diversities define the overall diversity or biological heterogeneity of a given environmental community. In this study, analyses of α and β diversities revealed that microbial diversity in both the rumen and duodenum was lowest at birth, intermediate at 20 days of age, and highest at 55 days of age in Hu lambs. However, in the jejunum and ileum, α diversity indices at 20 days of age were lower than those observed at birth. This may be attributable to the transitional nature of microbial maturation postpartum, the singularity of a milk-based diet, and the decline of initial microbial communities as the intestinal environment develops [ 40 , 41 ] . By 55 days of age, microbial diversity had surpassed birth levels, indicating that gastrointestinal microbial diversity increases dynamically, rather than linearly, with age. Significant changes in the gastrointestinal microbial community structure were observed, suggesting that enhanced microbial diversity is correlated with healthy growth in Hu lambs [ 17 ] . Furthermore, the microbial community structures in the rumen, duodenum, jejunum, and ileum of lambs showed significant differences at 20 days of age compared to both birth and 55 days of age. These findings indicate that substantial changes in gut microbiota occur from birth to weaning, after which community structure gradually stabilizes. Supplementation with probiotic formulations was found to increase both α and β diversities in the gut microbiota of Hu lambs, although the effect was not pronounced, consistent with the observations by Wang et al [ 42 ] . This suggests that probiotic supplementation may serve as a beneficial addition to the gut microbiota of lambs or may require a longer period (beyond 30 days) for effective colonization. There is evidence that certain additives can alter the community structure and diversity of rumen microorganisms [ 43 – 45 ] , likely influenced by the composition and dosage of probiotic formulations. It is inferred that different components of probiotic formulations have varying effects on gut microbiota, with high doses of probiotics may induce rapid structural changes. However, the slow-release composite probiotic formulations used in this study was designed primarily to enhance the immunity of weak lambs and improve the colonization of beneficial gut bacteria, aligning largely with the expected outcomes. As lambs mature, notable changes occur in both microbial composition and relative abundance, particularly in the proliferation of beneficial bacterial species within different regions of GIT. Ruminococcus and Lachnospiraceae are fibrolytic bacteria capable of degrading cellulosic polysaccharides, producing beneficial metabolic byproducts, and playing critical roles in feed digestion and absorption [ 46 ] . Succiniclasticum can degrade crystalline cellulose, which other rumen bacteria cannot process, through its cellulolytic and enzyme-binding regions, thereby enhancing feed digestion and absorption [ 47 , 48 ] . The Christensenellaceae_R-7_group has been shown to improve growth performance, enhance intestinal metabolism, and increase the efficiency of protein and fiber breakdown in animals [ 49 , 50 ] . Prevotella species are associated with the degradation and utilization of starch, proteins, and polysaccharides in the rumen [ 51 ] . They play crucial roles in maintaining digestive tract balance, reducing systemic inflammatory responses, and improving digestive capabilities. Megasphaera elsdenii is notable for its ability to utilize accumulated lactic acid, thereby preventing acidosis—a significant concern in ruminant nutrition. Akkermansia contributes to gastrointestinal homeostasis and metabolic balance [ 52 ] . Rikenellaceae , which is commonly found in mammalian GIT, plays a significant role in the digestion of coarse fiber, with its abundance closely linked to the type of feed consumed [ 53 ] . This study demonstrated that as Hu lambs grow, the composition and abundance of beneficial bacterial genera increase. Moreover, the genera enriched in the treatment group supplemented with probiotic formulations overlapped with those that increased over time in the control group. These included Rikenellaceae RC9_gut_group , Prevotella , and Fibrobacter in the rumen; Christensenellaceae_R-7_group and Prevotella in the duodenum; Lactobacillus and Ruminococcus in the jejunum; and Lactobacillus and Lachnospiraceae _ UCG_002 in the ileum. These findings suggested that probiotic formulations positively influenced the formation and stabilization of gut microbiota in lambs. Extensive research has indicated that Firmicutes and Bacteroidetes are the predominant microbial phyla in the digestive tracts of ruminants, contributing to host metabolism, lipid regulation, and enhanced energy conversion efficiency [ 42 , 54 – 57 ] . Firmicutes, primarily involved in cellulose breakdown, are essential for protein and carbohydrate digestion [ 58 – 60 ] . These Gram-positive bacteria play significant roles in maintaining gut microbiota balance and boosting immunity [ 61 – 63 ] . For instance, species of Clostridiales are involved in nutrient metabolism and absorption, providing energy to intestinal epithelial cells [ 64 ] . The inclusion of Lactobacillus in feed promotes the production of adhesins that colonize the intestinal mucosa, helping maintain microbiota balance and supporting weight gain in ruminants [ 65 ] . The findings of this study further substantiate the role of Firmicutes in enhancing the metabolic efficiency and overall health of ruminants. Firmicutes are the most abundant phylum in GIT of Hu lambs, accounting for 40–90% of the relative abundance [ 66 , 67 ] . In this study, the relative abundance of Firmicutes in the jejunum of 55-day-old control group was found to be 80.35%, consistent with findings by Wang et al. [ 68 ] on the bacterial microbiota characteristics in different intestinal segments of Qinghai semi-fine wool sheep. These results suggest that the primary microbial structure of the lamb GIT forms rapidly after birth and gradually stabilizes. The supplementation of probiotic formulations increased the relative abundance of both Firmicutes and Bacteroidetes, albeit without statistical significance. This finding indicates that the preparation may enhance microbial populations without disrupting the existing balance or those such effects might require a longer duration or higher dosage to become apparent [ 69 ] . The gut microbiota of ruminants plays a crucial role in digestion, metabolism, nutritional compensation, and growth, with higher microbial diversity and richness being positively correlated with improved animal health [ 70 ] . Characterization of the Hu sheep rumen microbiota reveals a highly complex and diverse community, with Bacteroidetes and Firmicutes identified as the dominant and core phyla [ 71 ] . In the ileum, an increase in dominant phyla has been associated with reduced diarrhea in animals [ 72 ] , suggesting that probiotic formulations may be particularly effective in lambs exhibiting diarrheal symptoms [ 73 ] . In summary, significant changes in the gut microbiota of Hu lambs were observed between birth and 20 and 55 days of age, indicating an increase in microbial diversity and stability as the lambs matured. The rumen and intestines harbored distinct microbial communities due to their unique physiological functions; the rumen was dominated by fiber-degrading bacteria, while the small intestine primarily hosted short-chain fatty acid (SCFA)-producers bacteria. Supplementation with probiotic formulations after birth was shown to enhance the abundance of gut microbiota, potentially offering probiotic benefits. Conclusion Probiotic formulations represent an effective alternative to antibiotics in animal husbandry, playing a vital role in promoting animal health and productivity while exerting beneficial effects on growth. This study demonstrated that supplementation with probiotic formulations sustainably enhanced the diversity of gut microbiota, increased the abundance of beneficial bacteria, improved digestive function, and thereby elevated the health status and growth rate of Hu lambs. Declarations Acknowledgements Not applicable. Authors’ Contributions T.X. and G.S. conceived and designed the study, performed the microbiological analyses, and wrote and revised the manuscript. Y.Y. and S.Y. performed data analysis and data visualization. S.D. and Y.X. realized the animal experiments. Y.W., P.W. and J.J. supervised the project and funding acquisition and participated in the experiment. F.G. provided direction and supervision for the study, and reviewed and edited the final manuscript. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by grants from the National Natural Science Foundation of China (31672394), Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding （2021C02068-6）, Zhejiang Province Agricultural Major Technology Collaborative Promotion Plan（2023ZDXT16-3）, the Project for Zhejiang Province Basic Public Welfare Research Program (LTGN24C050001), China Agriculture Research System (CARS-39-07), Young Science and Technology Top Talent Program of Tianshan Talent Training Program in Xinjiang Province (2022TSYCCX0124), Xinjiang Agriculture Research System (XJARS-09-26), as well as Project of Corps Science and Technology in Key Areas ( 2024AB017). Data Availability The sequence datasets generated during the current study are available in the GSA repository, (https://ngdc.cncb.ac.cn/gsa) as CRA023269. All the other data are included in this published article. Ethics Approval and Consent to Participate All experimental procedures involving Hu sheep were conducted in accordance with the guidelines of the China Council on Animal Care. The protocols were approved by the Experimental Animal Management Committee of China Jiliang University (approval number: 2022-014). Consent for Publication Not applicable. Competing Interests We have no competing interests. References Fu, X. et al. Transcriptomic Study of Spermatogenesis in the Testis of Hu Sheep and Tibetan Sheep . Genes (Basel) . 13(12), 2212. https://doi.org/10.3390/genes13122212 (2022) Guo, R. et al. Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR . Genes (Basel) . 14(6), 1216. https://doi.org/10.3390/genes14061216 (2023) Li, Y. et al. Runs of Homozygosity Revealed Reproductive Traits of Hu Sheep . Genes (Basel) . 13(10), 1848. https://doi.org/10.3390/genes13101848 (2022) Guan, F., Liu, S.R., Shi, G.Q. & Yang, L.G. 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Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yimin","middleName":"","lastName":"Wang","suffix":""},{"id":512747629,"identity":"3133910d-0e9a-4990-aa5b-27408aef4d36","order_by":8,"name":"Feng Guan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYLCCCgiVeOCDgY0dcVrOQKiEgzMK0pJJ0sJwmOfDIcYGQqrlI5KfPThQccdufv+CB4dtDA4wM7AfProBnxbDG2nmBgfOPEtunPEg4XCOwR0+Bp60tBt4tcxIMJP+2HY4mVniAEjLM2YGCR4zAlrSv0kc/Hc4mQ2kxcLgMGMDIS3yEjlmEgcbDtvx8DckHGYgRosBz5syiQPHDidISAADuccgLZmNkF/k29O3SRyoOWwv338m8cGPPzZ2/OyHj+G35QCETmyQyEkAs9jwKQfb0gCh7Rn4jx8gpHgUjIJRMApGKAAAppVVxnjj1m8AAAAASUVORK5CYII=","orcid":"","institution":"China Jiliang University","correspondingAuthor":true,"prefix":"","firstName":"Feng","middleName":"","lastName":"Guan","suffix":""},{"id":512747630,"identity":"5ef4b3b6-5533-4070-af90-abb1ba4902ee","order_by":9,"name":"Junfang Jiang","email":"","orcid":"","institution":"Academy of Zhejiang Agriculture Science","correspondingAuthor":false,"prefix":"","firstName":"Junfang","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2025-08-09 12:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7333756/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7333756/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91519805,"identity":"fb6a82a8-af89-4483-a2b7-d2a73ff0f210","added_by":"auto","created_at":"2025-09-17 09:59:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84966,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of probiotic formulations on rumen morphology (10×).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/25f499b1bf56e4d97abd0321.jpg"},{"id":91519808,"identity":"b722970e-cfa9-4fb9-b59e-2080ea1dc92d","added_by":"auto","created_at":"2025-09-17 09:59:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128894,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of probiotic formulations on duodenal morphology (10×).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/08e4970d26cde060411e86ce.jpg"},{"id":91518809,"identity":"88e57ded-5975-4c68-a67a-571b17084d3b","added_by":"auto","created_at":"2025-09-17 09:51:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103736,"visible":true,"origin":"","legend":"\u003cp\u003eRarefaction curves of rumen, duodenum, jejunum and ileum in Hu lambs at 0, 20 and 55 days of age.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/73d2344f06df88786546ad31.jpg"},{"id":91520081,"identity":"f3c5a152-3fcf-42cc-8050-3d5926132278","added_by":"auto","created_at":"2025-09-17 10:07:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65794,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagrams of the composition of ASVs in gut microbiota of Hu lambs at 0, 20 and 55 days of age. (\u003cstrong\u003eA\u003c/strong\u003e) Rumen. (\u003cstrong\u003eB\u003c/strong\u003e) Duodenum. (\u003cstrong\u003eC\u003c/strong\u003e) Jejunum. (\u003cstrong\u003eD\u003c/strong\u003e) Ileum.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/43194be1d39c22c5c39c8d33.jpg"},{"id":91518812,"identity":"5635609b-34b0-4275-b512-c46d8a0ce790","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107323,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic changes in α diversity metrics of the gut microbiota of Hu lambs at 0, 20 and 55 days of age. (\u003cstrong\u003eA\u003c/strong\u003e) Chao1 index; (\u003cstrong\u003eB\u003c/strong\u003e) Shannon index; (\u003cstrong\u003eC\u003c/strong\u003e) Simpson index; (\u003cstrong\u003eD\u003c/strong\u003e) Observed_otus. All values are reported as mean ± SD, in which * indicates statistical significance (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/126f8a501dae0be72300bb7a.jpg"},{"id":91518815,"identity":"08c227f6-cb71-4fd1-ba41-201179d06dfc","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74837,"visible":true,"origin":"","legend":"\u003cp\u003ePCoA of the gut microbiota of Hu lambs at 0, 20 and 55 days of age. (\u003cstrong\u003eA\u003c/strong\u003e) Rumen. (\u003cstrong\u003eB\u003c/strong\u003e) Duodenum. (\u003cstrong\u003eC\u003c/strong\u003e) Jejunum. (\u003cstrong\u003eD\u003c/strong\u003e) Ileum.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/bef51888da3a498f58df797e.jpg"},{"id":91518821,"identity":"90c4aa78-94c9-4fb6-88f8-e0b6855e0d7e","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":146299,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of linear discriminant analysis (LDA) scores for Hu sheep at different age. The ordinate indicates the taxon with significant differences between groups; the abscissa corresponds to the logarithmic LDA scores based on taxonomic analysis. Differential microbial species in the GIT microbiota of Hu sheep between three groups. (\u003cstrong\u003eA\u003c/strong\u003e) Rumen. (\u003cstrong\u003eB\u003c/strong\u003e) Duodenum. (\u003cstrong\u003eC\u003c/strong\u003e) Jejunum. (\u003cstrong\u003eD\u003c/strong\u003e) Ileum.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/7340a54c88b749c3d596f36b.jpg"},{"id":91518823,"identity":"ecf92ac0-fe8b-489f-8254-b94de8a34f45","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":59363,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagrams of the composition of ASVs in gut microbiota of Hu lambs in the 20-day-old treatment and control groups. (\u003cstrong\u003eA\u003c/strong\u003e) Rumen. (\u003cstrong\u003eB\u003c/strong\u003e) Duodenum. (\u003cstrong\u003eC\u003c/strong\u003e) Jejunum. (\u003cstrong\u003eD\u003c/strong\u003e) Ileum.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/c45301596a2fe51d40c13782.jpg"},{"id":91518819,"identity":"1d9366de-f4fb-4d16-ad7b-4bf519608caf","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":95365,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in relative abundances of 20-day-old microbiota in treatment and control groups at the phylum level.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/9cad37b5c574f4e62be49816.jpg"},{"id":91519817,"identity":"d7bfa175-30dd-4d6f-9a07-96847fab3fe7","added_by":"auto","created_at":"2025-09-17 09:59:35","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":121123,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in relative abundances of 55-day-old microbiota in treatment and control groups at the phylum level.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/e52cbf3f3e2d161fb4fe6af6.jpg"},{"id":91518834,"identity":"7b69aa22-d73a-4a6a-b67f-16a604213e64","added_by":"auto","created_at":"2025-09-17 09:51:35","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":162040,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in relative abundances of 20-day-old microbiota in treatment and control groups at the genus level.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/b625f4019c97be10368056be.jpg"},{"id":91519812,"identity":"dd68cc4d-07e5-4550-89c5-0b29abddc1c7","added_by":"auto","created_at":"2025-09-17 09:59:34","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":164781,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in relative abundances of 55-day-old microbiota in treatment and control groups at the genus level.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/5acf82cd2fc5b2af238451dd.jpg"},{"id":91518824,"identity":"278399e8-a63f-49a8-aee6-ac257cd00df2","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":155005,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of linear discriminant analysis (LDA) scores for Hu sheep at 20 days of age. The ordinate indicates the taxon with significant differences between groups; the abscissa corresponds to the logarithmic LDA scores based on taxonomic analysis. Differential microbial species in the GIT microbiota of Hu sheep between two groups. (\u003cstrong\u003eA\u003c/strong\u003e) Rumen. (\u003cstrong\u003eB\u003c/strong\u003e) Duodenum. (\u003cstrong\u003eC\u003c/strong\u003e) Jejunum. (\u003cstrong\u003eD\u003c/strong\u003e) Ileum.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/4ab973a781f4d9ba33288cff.jpg"},{"id":91518826,"identity":"add16e11-1a42-43f6-b930-f6ccec30b7e4","added_by":"auto","created_at":"2025-09-17 09:51:34","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":189885,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of linear discriminant analysis (LDA) scores for Hu sheep at 55 days of age. The ordinate indicates the taxon with significant differences between groups; the abscissa corresponds to the logarithmic LDA scores based on taxonomic analysis. Differential microbial species in the GIT microbiota of Hu sheep between two groups. (\u003cstrong\u003eA\u003c/strong\u003e) Rumen. (\u003cstrong\u003eB\u003c/strong\u003e) Duodenum. (\u003cstrong\u003eC\u003c/strong\u003e) Jejunum. (\u003cstrong\u003eD\u003c/strong\u003e) Ileum.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/6483b9b147ba53a85e704578.jpg"},{"id":92460479,"identity":"acaa332a-461b-453d-b6a9-68c3f53015c9","added_by":"auto","created_at":"2025-09-30 03:32:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2822472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7333756/v1/a9c242b8-4a10-431b-a4fc-d1ad2b4d3cf2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of probiotic formulations on GIT morphological changes and gut microbiota in Hu lambs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSheep are a pivotal species in global animal husbandry, being distributed worldwide and serving as a significant economic resource for herders. With the evolution and scaling of the livestock industry, sheep farming has become a cornerstone of this sector. Prolificacy is a critical determinant in the development of meat sheep farming \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Hu sheep are renowned for their high fertility, early sexual maturity, continuous estrous cycles, adaptability to hot and humid climates, and suitability for intensive housing systems \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. This breed possesses one of the highest fertility rates globally, with lambing rates exceeding 280% \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, making it a primary choice for meat sheep farming and crossbreeding programs. However, Hu sheep are characterized by a low carcass yield and inferior meat quality \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn sheep breeding, feed expenses can constitute up to 70% of total costs \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, while diseases such as colds, pneumonia, and diarrhea in lambs, along with milk fever and mastitis in ewes, are prevalent \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Enhancing feed conversion efficiency and immune competence is therefore crucial for economic gains in sheep breeding. These factors are influenced by both genetic predisposition and gut microbiota. Nutrient absorption, energy metabolism, and immune regulation in animals are intricately linked to their gut microbiota \u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The gut microbiota has been confirmed as a vital component in ruminant growth and health. The interaction between the gut microbiota and the host intestinal environment plays a key role in digestion, metabolism, immunity, and disease prevention \u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Given the substantial intake of crude fiber by ruminants, the breakdown of cellulose depends on cellulolytic bacteria within the gut microbiota \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The gut microbiota, as a key site for digestion and absorption, exerts a substantial influence on the health and performance of the host \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn the animal husbandry industry, probiotic formulations have been widely employed as alternatives to antibiotics, offering advantages such as non-toxicity, non-residue, and non-pathogenicity \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Previous studies have demonstrated that feeding probiotic formulations enhances immune parameters and growth performance in Hu sheep pre- and post-weaning (60 days of age) \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e The addition of probiotic formulations to the diet of Hu sheep significantly increases the carcass yield and feed conversion ratio (FCR), enhances immunity and antioxidant capacity, and regulates the gut microbial homeostasis \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Moreover, feeding probiotics to ruminants has been shown to improve nutrient intake and digestibility, milk production, immunity, reproduction, and feed conversion efficiency \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Furthermore, feeding probiotic formulations significantly increases total blood protein, globulin, and weight gain in lambs \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, as well as enhances daily weight gain, feed digestibility, and body immunity in weaned goats \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The effects of probiotic formulations on ruminants are primarily achieved by improving the structure and function of microbial communities and influencing animal behaviors such as feed intake \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Probiotic formulations adhere to the inner wall of the digestive tract, forming a colonization resistance in the intestine. Over time, a stable microbial community is established, regulating the structure and quantity of intestinal microbiota, thereby influencing the immune system and growth performance of the host \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCurrently, commercial probiotic formulations primarily include probiotics, prebiotics, and synbiotics, which are widely used in sheep farming and offer various beneficial functions \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, thereby improving the efficiency of sheep breeding. Commercially available probiotic formulations utilizing slow-release encapsulated multiple probiotics can enhance immunity and promote rapid growth during the short-term growth phase in Hu lambs \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. However, the effects of such probiotic formulations on the digestive system microorganisms of lambs, as well as the alteration of gut microbiota composition, remain unclear. Therefore, this experiment provides a theoretical basis for elucidating the developmental processes of gastrointestinal tract (GIT), the basic characteristics of the gut microbiota, and the effects of probiotic formulations in the feeding of Hu lambs, thus offering a reference for the production and development of probiotic formulations for sheep.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental material\u003c/h2\u003e\u003cp\u003eThe probiotic formulation used in this study was purchased from Qingdao Zhengtai United Nutritional Technology Co., Ltd. (product name \u0026ldquo;Duyikang\u0026rdquo;), provided in a cream formulation with a volume of 60 mL per tube. The labeled ingredients included \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e as the main probiotics, along with prebiotics such as colostrum extract, medium-chain triglycerides, trace elements, and vitamins.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLaboratory animals and sample collection\u003c/h3\u003e\n\u003cp\u003eThis experiment was conducted from October to December in 2022 at Hangzhou Caiyang Herding Co., Ltd. (Zhejiang, China), over a 60-day period. Fifteen lambs with similar birth times and weights were selected from 56 newly born male Hu lambs that were twin-born and randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;3). The groups were as follows: one blank control group (BG, Group I), two control groups (CG, Groups II and III), and two experimental groups (TG, Groups IV and V).\u003c/p\u003e\u003cp\u003eThe GIT contents of lambs in BG were sampled within 2 hours after birth. Lambs in TG IV and V were orally administered 10 mL of the probiotic formulations within 2 hours after birth, while lambs in CG II and III did not receive the probiotic formulations. All lambs in TG and CG groups were suckled naturally under identical conditions, with supplemental lamb milk substitute provided from approximately 30 days of age. Free feeding was allowed, and all experimental lambs were tagged using ear tags with wool staining for identification.\u003c/p\u003e\u003cp\u003e All experimental procedures involving Hu sheep were conducted in accordance with the guidelines of the China Council on Animal Care. At the corresponding time of experimental designed, namely 0 day, 20 days, and 55 days after the birth of the lamb, no food was fed on the day of the sample collection. All animals from each group were euthanized by captive bolt stunning. Physical disruption methods are often followed by exsanguination as the adjunctive method to ensure humane euthanasia. The slaughter procedures were performed by trained personnel in accordance with standard animal welfare protocols. Immediately after slaughter and dissection, the organs to be sampled were rinsed clean with ice cold sterile PBS. Intestinal segments were defined as follows: the proximal third (duodenum), mid third (jejunum), and distal third (ileum) of the small intestine. For each target segment (duodenum, jejunum, ileum), a section approximately 3 cm in length was ligated proximally and distally using sterile suture material. The isolated segment was then carefully excised using sterile surgical scissors. The luminal contents within this isolated segment were gently expressed into sterile collection tubes. One section of each sample was snap-frozen in liquid nitrogen and then stored at -80\u0026deg;C until the time of analysis. The remaining portion was fixed in neutral buffered 10% formalin for histological analysis. Samples from the rumen, duodenum, jejunum, and ileum contents were collected from lambs in CG II and TG IV as well as those in CG III and TG V at 20 and 55 days of age, respectively, based on the rate and timing of weight gain influenced by feeding the probiotic formulations \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. For BG I, GIT contents were similarly collected from the same anatomical sites within 2 hours after birth, snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eMorphological Observations\u003c/h3\u003e\n\u003cp\u003eTissue samples from the rumen, duodenum, jejunum, and ileum were collected in accordance with the GIT content sampling strategy. Morphological analysis was performed using hematoxylin-eosin (HE) staining and light microscopy. The methodology followed Wang et al. \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, with the procedure detailed as follows: tissue samples were fixed in paraffin, and the embedded paraffin blocks were sectioned into 4 \u0026micro;m-thick cross-sections. The sections were then stained with HE and observed under a light microscope (Eclipse Ci, Nikon, Melville, NY). Photographs of the tissues were captured using an imaging system (Digital Sight DS-Fi2, Nikon, Melville, NY). Measurements of rumen papillae length and width, muscularis propria depth, villus height (VH), crypt depth (CD), and muscularis propria thickness were performed in the duodenum, jejunum, and ileum. The villus height to crypt depth (V/C) ratio was subsequently calculated.\u003c/p\u003e\n\u003ch3\u003eDNA extraction and PCR amplification of gut microbiota\u003c/h3\u003e\n\u003cp\u003eTotal genomic DNA was extracted from samples collected from different organs using Bacterial Genome Extraction Kit (Tiangen Biotech Co.,Ltd., Beijing, China), following the manufacturer\u0026rsquo;s instructions. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, United States), and DNA integrity was assessed via 2% agarose gel electrophoresis.\u003c/p\u003e\u003cp\u003ePCR amplification targeting the hypervariable V3-V4 region of the 16S rRNA gene was performed using the primers 515F (5\u0026rsquo;-GTGYCAGCMGCCGCGGGTAA-3\u0026rsquo;) and 806R (5\u0026rsquo;-GGACTACHVGGGTWTCTAAT-3\u0026rsquo;) \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Positive PCR products were purified using the GeneJET\u0026reg; Gel Extraction Kit (Thermo Scientific), and sequencing libraries were constructed with the Ion Plus Fragment Library Kit (Thermo Scientific). Library quality was evaluated using a Qubit 2.0 fluorometer (Thermo Scientific).\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S rRNA sequencing and analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSequencing was performed on the Ion S5\u0026trade; XL platform, generating single-end reads of 407 to 412 base pairs. Sequencing data were processed by splitting sequences based on barcode information, followed by removal of barcode and primer sequences using the software cutadapt (v1.9). Overlapping region sequences were merged with the software FLASH (v1.2.8). Sequencing reads were subjected to quality control using a window scanning method implemented in fgtrim, with sequences shorter than 100 bp after truncation discarded. Chimeric sequences were identified and removed using Vsearch (v2.3.4). DADA2 was employed via QIIME2 for length filtering and denoising to obtain Amplicon Sequence Variant (ASV) feature sequences and abundance tables.\u003c/p\u003e\u003cp\u003eBased on the ASV feature sequences and abundance data, α-diversity and β-diversity analyses were performed. Comparative analyses of species abundance between control and experimental groups were conducted, with a significance threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Additionally, the linear discriminant analysis effect size (LEfSe, LDA scores\u0026thinsp;\u0026gt;\u0026thinsp;3) enabled the identification of important bacteria between groups.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eExperimental data were organized using Excel 2021, and statistical analyses were conducted using SPSS 23.0. A one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. The Least Significant Difference (LSD) method was employed to determine statistical significance, with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered indicative of a significant difference. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eChanges in the morphological structure of GIT and effects of probiotic formulations\u003c/h2\u003e\u003cp\u003eGIT is the primary organ responsible for nutrient absorption in animals, and its developmental status directly impacts nutrient absorption efficiency. Histological and morphological analyses revealed that the length and width of the rumen papillae, as well as the thickness of the rumen musculature, were significantly greater at 55 days of age compared to 20 days (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), consistent with the lambs' growth. In the duodenum, the height of the villi was higher at birth, gradually decreased, and subsequently increased with age. Similarly, the depth of the crypts increased, the V/C ratio decreased, and the muscularis propria thickness increased over time.\u003c/p\u003e\u003cp\u003eIn the control groups, the villus height in the duodenum at both 20 and 55 days of age was significantly lower than at birth. The crypt depth and muscularis propria thickness were significantly greater at 55 days compared to 20 days and at birth. The V/C ratio in control lambs at both 20 and 55 days was significantly lower than at birth. In the jejunum, villus height at 55 days was significantly higher than at 20 days and at birth. The crypt depth and muscularis propria thickness at 55 days were also significantly greater than at 20 days and birth. The V/C ratio at birth and 55 days was significantly higher than at 20 days. In the ileum, villus height and crypt depth at 55 days were significantly greater than at 20 days and birth, while the muscularis propria thickness was also significantly higher at 55 days compared to earlier stages. However, no significant differences in V/C were observed among the three control groups.\u003c/p\u003e\u003cp\u003eComparative analysis between the experimental and control groups indicated that no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the length of the rumen papillae at 20 days. However, at 55 days, the length of the rumen papillae in the experimental group increased significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Rumen morphology data are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, with representative images shown in Fig.\u0026nbsp;1. There was no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the width of the rumen papillae or the thickness of the muscularis layer between the experimental and control groups at the same age. In the duodenum, the villus height showed no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the experimental and control groups at 20 days. However, at 55 days, the villus height in the experimental group was significantly greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The morphology of the duodenum is depicted in Fig.\u0026nbsp;2 and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Additionally, there were no significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in crypt depth, V/C, or muscularis layer thickness in the duodenum between the experimental and control groups at any age. Similarly, no significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were found in villus height, crypt depth, V/C, or muscularis layer thickness in the histomorphology of the jejunum and ileum.\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\u003eEffect of probiotic formulations on rumen tissue morphology in Hu sheep.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG20\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTG20\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCG55\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTG55\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLength of rumen papilla/\u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e471.74\u0026thinsp;\u0026plusmn;\u0026thinsp;85.33\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e484.72\u0026thinsp;\u0026plusmn;\u0026thinsp;126.66\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e896.07\u0026thinsp;\u0026plusmn;\u0026thinsp;194.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1040.57\u0026thinsp;\u0026plusmn;\u0026thinsp;107.72\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWidth of rumen papilla/\u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e176.95\u0026thinsp;\u0026plusmn;\u0026thinsp;41.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e167.05\u0026thinsp;\u0026plusmn;\u0026thinsp;31.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e247.28\u0026thinsp;\u0026plusmn;\u0026thinsp;32.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e274.58\u0026thinsp;\u0026plusmn;\u0026thinsp;17.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMuscle thickness/\u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e713.73\u0026thinsp;\u0026plusmn;\u0026thinsp;38.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e753.14\u0026thinsp;\u0026plusmn;\u0026thinsp;79.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e940.24\u0026thinsp;\u0026plusmn;\u0026thinsp;45.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e942.51\u0026thinsp;\u0026plusmn;\u0026thinsp;44.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea,b,c\u003c/sup\u003e Within a row for each item, different superscripts indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\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\u003eEffect of probiotic formulations on the morphology of duodenum in Hu sheep.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBG00\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCG20\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTG20\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCG55\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTG55\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVillus height /\u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e457.57\u0026thinsp;\u0026plusmn;\u0026thinsp;59.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e336.07\u0026thinsp;\u0026plusmn;\u0026thinsp;39.64\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e312.34\u0026thinsp;\u0026plusmn;\u0026thinsp;39.6\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e373.74\u0026thinsp;\u0026plusmn;\u0026thinsp;35.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e424.72\u0026thinsp;\u0026plusmn;\u0026thinsp;52.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrypt depth /\u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100.90\u0026thinsp;\u0026plusmn;\u0026thinsp;15.59\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e139.07\u0026thinsp;\u0026plusmn;\u0026thinsp;18.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e133.85\u0026thinsp;\u0026plusmn;\u0026thinsp;17.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e175.59\u0026thinsp;\u0026plusmn;\u0026thinsp;26.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e182.43\u0026thinsp;\u0026plusmn;\u0026thinsp;22.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV/C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMuscle thickness /\u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e78.83\u0026thinsp;\u0026plusmn;\u0026thinsp;13.43\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e175.08\u0026thinsp;\u0026plusmn;\u0026thinsp;56.46\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e175.53\u0026thinsp;\u0026plusmn;\u0026thinsp;16.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e231.67\u0026thinsp;\u0026plusmn;\u0026thinsp;26.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e240.91\u0026thinsp;\u0026plusmn;\u0026thinsp;9.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea,b,c\u003c/sup\u003e Within a row for each item, different superscripts indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChanges in the diversity of gut microbiota in control lambs\u003c/h3\u003e\n\u003cp\u003eHigh-throughput sequencing of the microbiota from the rumen, duodenum, jejunum, and ileum contents of the BG00, CG20, and CG55 groups revealed that Shannon curves plateaued at sequencing depths exceeding 5000 reads (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This indicated that the sequencing depth and data volume of the 16S rRNA V3-V4 gene region were sufficient for diversity analysis. A Venn diagram was used to visualize the specific and shared ASVs among the three groups. The ASV results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Across all four sites, the BG00 group exhibited the lowest number of ASVs, while the CG55 group showed the highest number, demonstrating a gradual increase in ASVs with lamb growth. This finding indicates that the diversity and abundance of gut microbiota increase as lambs mature.\u003c/p\u003e\u003cp\u003eComparison of α diversity in different gastrointestinal regions at various growth stages (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed that in the rumen, the Chao1 and Shannon indices displayed an upward trend with age. The Chao1 index increased significantly at 20 days compared to birth (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and both Chao1 and Shannon indices were significantly higher at 55 days than at birth (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the duodenum, the Chao1 index increased with age, but the change was not significant. In the jejunum, the α diversity indices followed a clear \u0026lsquo;V\u0026rsquo; pattern, being higher at birth and 55 days and lowest at 20 days, though the differences were not significant. In the ileum, the Chao1 index showed a continuous increase, with significant differences at 20 and 55 days compared to birth (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the Simpson and Shannon indices exhibited a \"V-shaped\" pattern, with significantly higher values at birth and 55 days than at 20 days. These findings indicate an overall increasing trend in gut microbiota diversity and abundance as lambs grow.\u003c/p\u003e\u003cp\u003eThe results of β diversity analysis using Principal Coordinates Analysis (PCoA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e) demonstrated distinct separations among the three control groups at birth, 20 days, and 55 days. Within each group, samples were aggregated, suggesting significant differences in gut microbiota composition among the three stages. This indicates that the structure of the gut microbiota undergoes substantial changes, with increased diversity during lamb growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChanges in α-diversity of gut microbiota of Hu lambs at 0, 20 and 55 days of age.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItem\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eObserved_otus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eShannon\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSimpson\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChao1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGoods_coverage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePielou_e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBG00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e148\u0026thinsp;\u0026plusmn;\u0026thinsp;82.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e155.56\u0026thinsp;\u0026plusmn;\u0026thinsp;82.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRumen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e916.67\u0026thinsp;\u0026plusmn;\u0026thinsp;418.82\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1090.11\u0026thinsp;\u0026plusmn;\u0026thinsp;491.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1367\u0026thinsp;\u0026plusmn;\u0026thinsp;376.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1567.27\u0026thinsp;\u0026plusmn;\u0026thinsp;506.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBG00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e275\u0026thinsp;\u0026plusmn;\u0026thinsp;172.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e228.17\u0026thinsp;\u0026plusmn;\u0026thinsp;207.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDuodenum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e534.33\u0026thinsp;\u0026plusmn;\u0026thinsp;427.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e635.33\u0026thinsp;\u0026plusmn;\u0026thinsp;470.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e692\u0026thinsp;\u0026plusmn;\u0026thinsp;362.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e792.69\u0026thinsp;\u0026plusmn;\u0026thinsp;362.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBG00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e339\u0026thinsp;\u0026plusmn;\u0026thinsp;459.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e356.13\u0026thinsp;\u0026plusmn;\u0026thinsp;487.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJejunum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e153.67\u0026thinsp;\u0026plusmn;\u0026thinsp;53.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e181.91\u0026thinsp;\u0026plusmn;\u0026thinsp;75.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e370.33\u0026thinsp;\u0026plusmn;\u0026thinsp;93.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e418.62\u0026thinsp;\u0026plusmn;\u0026thinsp;113.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBG00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e116\u0026thinsp;\u0026plusmn;\u0026thinsp;75.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e132.66\u0026thinsp;\u0026plusmn;\u0026thinsp;95.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIleum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e219\u0026thinsp;\u0026plusmn;\u0026thinsp;29.46\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e265.92\u0026thinsp;\u0026plusmn;\u0026thinsp;27.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCG55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e618\u0026thinsp;\u0026plusmn;\u0026thinsp;127.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e717.37\u0026thinsp;\u0026plusmn;\u0026thinsp;133.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: \u003csup\u003ea,b\u003c/sup\u003e Within a column for each item, different superscripts indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBiomarkers of gut microbiota in control lambs\u003c/h2\u003e\u003cp\u003eTo analyze changes in bacterial microbiota during lamb growth at both the phylum and genus levels, LEfSe was utilized to identify significantly different bacterial taxa (LDA scores\u0026thinsp;\u0026gt;\u0026thinsp;3) across control groups. The findings revealed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e) that, in the rumen, the BG00 group was enriched with 20 bacterial taxa, including 1 phylum and 5 genera. The CG20 group exhibited enrichment in 28 bacterial taxa, comprising 2 phyla and 12 genera, while the CG55 group displayed enrichment in 40 bacterial taxa, including 1 phylum and 16 genera. In the duodenum, the BG00 group was enriched in 26 bacterial taxa, encompassing 2 phyla and 5 genera. The CG20 group was enriched in 8 bacterial taxa, including 4 genera, and the CG55 group demonstrated enrichment in 14 bacterial taxa, involving 6 genera. In the ileum, the BG00 group was enriched in 18 bacterial taxa, including 5 genera. The CG20 group exhibited enrichment in 6 bacterial taxa, comprising 4 genera, whereas the CG55 group showed enrichment in 25 bacterial taxa, encompassing 2 phyla and 7 genera.\u003c/p\u003e\u003cp\u003eA comparative analysis of lamb growth from birth to 55 days of age identified 33 genera with significant differences in the rumen, 16 in the duodenum, 11 in the jejunum, and 16 in the ileum. The BG00 group exhibited the lowest number of genera in the rumen, suggesting that Hu lambs possess a relatively established but limited bacterial microbiota early in life. As lambs matured, the relative abundance of \u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003ePrevotellaceae\u003c/em\u003e, \u003cem\u003eMegasphaera\u003c/em\u003e, \u003cem\u003eFibrobacterota\u003c/em\u003e, \u003cem\u003eRichenellaceae\u003c/em\u003e_\u003cem\u003eRC9_gut_group\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e, \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e, and \u003cem\u003eLigilactobacillus\u003c/em\u003e increased, while the relative abundance of \u003cem\u003eCaulobacter\u003c/em\u003e and \u003cem\u003eCitrobacter\u003c/em\u003e decreased. In the duodenum, the relative abundance of Firmicutes, Bacteroidetes, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e increased. In the jejunum, an increase in Firmicutes and \u003cem\u003eAkkermansia\u003c/em\u003e was observed, with a concomitant decrease in Proteobacteria. In the ileum, Proteobacteria and Actinobacteria decreased, while Firmicutes, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e increased.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEffect of probiotic formulations on gut microbiota diversity in Hu lambs\u003c/h2\u003e\u003cp\u003eFollowing administration of probiotic formulations, the results of ASV visualization analysis of 16S rRNA sequencing Venn diagrams for gastrointestinal contents in the TG20 and TG55 groups indicated that the number of ASVs in the rumen, duodenum, jejunum, and ileum increased with the growth of Hu lambs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The diversity indices in the experimental groups were higher than those in the control groups at corresponding time points, though the differences were not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). PCoA based on Bray-Curtis distance demonstrated less pronounced separation of gut microbiota in the TG groups compared to the CG groups over the same period, with lower levels of within-group aggregation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEffects of probiotic formulations on gut microbiota structure and composition of Hu lambs\u003c/h2\u003e\u003cp\u003eA comparative analysis of microbial communities in the rumen, duodenum, jejunum, and ileum of experimental and control groups at 20 and 55 days revealed increased microbial diversity indices and relative abundances at both the phylum and genus levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e). However, these increases were not statistically significant. Firmicutes were identified as the dominant phyla across all gastrointestinal sites in both experimental and control groups at 20 and 55 days of age. Variations in microbial communities were evident among specific gastrointestinal sites. In the rumen, Firmicutes and Bacteroidetes were predominant in all groups. In the duodenum and jejunum, Firmicutes remained dominant, whereas in the ileum, Firmicutes and Proteobacteria were major phyla at 20 days, with Proteobacteria declining by 55 days as Firmicutes became predominant.\u003c/p\u003e\u003cp\u003eAt the genus level, microbial species varied across organs and groups, with no significant differences observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e11\u003c/span\u003e, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e12\u003c/span\u003e). In the rumen, the relative abundance of \u003cem\u003eRikenellaceae\u003c/em\u003e_\u003cem\u003eRC9_gut_group\u003c/em\u003e, \u003cem\u003eSucciniclasticum\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e increased in the experimental group, while that of \u003cem\u003eMuribaculaceae\u003c/em\u003e_\u003cem\u003eunclassified\u003c/em\u003e decreased. In the duodenum, the experimental group exhibited increased relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003eRikenellaceae_RC9_gut_group\u003c/em\u003e, while \u003cem\u003eClostridium\u003c/em\u003e_\u003cem\u003esensu_stricto_1\u003c/em\u003e decreased. In the jejunum, the experimental group demonstrated increases in \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e, with declines in \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eClostridium_sensu_stricto_1\u003c/em\u003e, \u003cem\u003eSarcina\u003c/em\u003e, and \u003cem\u003eCandidatus\u003c/em\u003e_\u003cem\u003eArthromitus\u003c/em\u003e. In the ileum, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eCandidatus\u003c/em\u003e_\u003cem\u003eSaccharimonas\u003c/em\u003e, and \u003cem\u003eLachnospiraceae\u003c/em\u003e_\u003cem\u003eUCG_002\u003c/em\u003e showed increased relative abundance in the experimental group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e13\u003c/span\u003e, \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHu sheep, a renowned prolific indigenous breed in China, provides valuable insights into ovine growth, development, and physiological processes. The prolificacy of this breed not only enhances the productivity of sheep farming but also necessitates additional investments in the rearing of multiple lambs. These investments include artificial feeding, fostering, and the development and application of various milk replacer materials. Additionally, probiotic formulations and other management strategies are employed to increase the survival and growth rates of lambs from multiple births. Commonly used probiotics in animal husbandry, such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eBifidobacterium\u003c/em\u003e, have been shown to exert beneficial effects on animal growth \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Combined probiotics are generally more effective than single strains \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, as they exhibit synergistic or cooperative effects \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Specifically, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, included in probiotic supplements, has been demonstrated to promote growth, enhance antioxidant and immune functions, and increase the population of beneficial intestinal bacteria in sheep \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Commercial probiotic mixtures containing \u003cem\u003eBacillus licheniformis\u003c/em\u003e have been shown to improve growth rates, bolster immune function, and enhance the diversity of intestinal microbiota, thereby supporting metabolic homeostasis \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe interaction between gut microbiota and the host is essential for the development of the host's gastrointestinal, immune, metabolic, and neurological systems \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have demonstrated that early microbial colonization significantly influences long-term health across an organism's life cycle \u003csup\u003e[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The larval stage is critical for the establishment and maturation of microbiota, with the composition of gastrointestinal microbiota playing a crucial role in growth and health \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. The present study analyzed the intestinal microbiota of Hu lambs from birth to weaning and identified significant increases in beneficial bacterial populations alongside growth and diversification. Furthermore, the administration of probiotic formulations significantly enhanced the probiotic microbiota in the rumen, duodenum, jejunum, and ileum. These findings align with observed improvements in immunity and growth rates during the same period \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMicrobiota analysis revealed that Firmicutes predominated in the rumen, duodenum, jejunum, and ileum throughout the growth period, alongside Bacteroidetes and Proteobacteria. The diversity of microbiota increased with the growth of the lambs, consistent with the main probiotic components of probiotic formulations used in this experiment. The formulation included strains such as \u003cem\u003eBacillus licheniformis\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, all of which belong to the Firmicutes phylum. In the control group, microbial diversity exhibited a \"V-shaped\" pattern from birth to 55 days of age, with a decline at 20 days followed by an increase. This pattern is hypothesized to be associated with the initial acquisition of microbiota exclusively from the dam. A reduction in microbial diversity occurs at 20 days as the lambs have not yet established their distinct microbiota. The original microbial populations decline, and by 55 days of age, lambs gradually develop their own microbial communities through environmental exposure and maternal milk intake, resulting in significant differences across the three periods.\u003c/p\u003e\u003cp\u003eFollowing the administration of probiotic formulations, the introduction of sustained-release strains resulted in a convergence of microbial community types across different organs and between ages of 20 and 55 days. This reduced inter-group differences and led to lower intra-group clustering, suggesting consistent effects of the additive. Consequently, the study confirms that feeding probiotic formulations can enhance the population of dominant microbial species in GIT of Hu lambs, ensuring the colonization of beneficial microbes and thereby improving immune function and growth rates.\u003c/p\u003e\u003cp\u003eMorphological analysis revealed significant differences in gastrointestinal tissue structure across different stages in the control group of Hu lambs from birth to 55 days of age. Notably, at 55 days, the rumen papillae's length and width, as well as the rumen muscle layer, were significantly greater than those observed at 20 days of age. The increase in rumen papillae dimensions is believed to enhance the absorption efficiency of volatile fatty acids (VFAs), thereby improving feed conversion rates and promoting growth and development \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. These findings suggest significant improvements in rumen fermentation performance as Hu lambs mature.\u003c/p\u003e\u003cp\u003eIn the duodenum, V/C ratio significantly decreased from birth to 20 and 55 days of age. This reduction is likely associated with the initial necessity for maternal milk absorption in newborn lambs. The absorptive surface area of the intestine is determined by villus height, with taller villi correlating with greater absorptive capacity. Villus height, crypt depth, and the V/C ratio reflect the small intestine's capacity for nutrient digestion and absorption, while intestinal muscularis thickness indicates rhythmic contractile capability \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe administration of probiotic formulations significantly increased the length of rumen papillae in 55-day-old lambs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a concurrent increase in width, although early changes were less pronounced. This may be related to the dietary transition from a milk-based diet to concentrate feeds such as cornmeal and subsequently to pelleted feed. Probiotics ingested and colonized gradually exerted their effects through probiotic formulations by breaking down complex compounds like fiber, leading to the production of VFAs that promote the growth of rumen papillae \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Consequently, this enhanced the growth performance of lambs, consistent with findings that lambs fed probiotic formulations exhibited higher relative growth rates after 30 days of age.\u003c/p\u003e\u003cp\u003eProbiotic formulations also significantly increased duodenal villus height in 55-day-old lambs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), potentially enhancing nutrient absorption and contributing to the observed growth improvements. The formulation had minimal impact on intestinal tissue changes in 20-day-old lambs, possibly due to the colonization and effects of probiotics being influenced by postnatal dietary structure and digestive tract development.\u003c/p\u003e\u003cp\u003eIn evaluating microbial diversity, α diversity represents the diversity within a specific environment or ecosystem, encompassing species richness and evenness. In contrast, β diversity reflects the similarity or dissimilarity between individuals or groups, where greater distances between samples indicate more pronounced differences in their microbial community structures. Together, α and β diversities define the overall diversity or biological heterogeneity of a given environmental community.\u003c/p\u003e\u003cp\u003eIn this study, analyses of α and β diversities revealed that microbial diversity in both the rumen and duodenum was lowest at birth, intermediate at 20 days of age, and highest at 55 days of age in Hu lambs. However, in the jejunum and ileum, α diversity indices at 20 days of age were lower than those observed at birth. This may be attributable to the transitional nature of microbial maturation postpartum, the singularity of a milk-based diet, and the decline of initial microbial communities as the intestinal environment develops \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. By 55 days of age, microbial diversity had surpassed birth levels, indicating that gastrointestinal microbial diversity increases dynamically, rather than linearly, with age. Significant changes in the gastrointestinal microbial community structure were observed, suggesting that enhanced microbial diversity is correlated with healthy growth in Hu lambs \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFurthermore, the microbial community structures in the rumen, duodenum, jejunum, and ileum of lambs showed significant differences at 20 days of age compared to both birth and 55 days of age. These findings indicate that substantial changes in gut microbiota occur from birth to weaning, after which community structure gradually stabilizes. Supplementation with probiotic formulations was found to increase both α and β diversities in the gut microbiota of Hu lambs, although the effect was not pronounced, consistent with the observations by Wang et al \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. This suggests that probiotic supplementation may serve as a beneficial addition to the gut microbiota of lambs or may require a longer period (beyond 30 days) for effective colonization.\u003c/p\u003e\u003cp\u003eThere is evidence that certain additives can alter the community structure and diversity of rumen microorganisms \u003csup\u003e[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, likely influenced by the composition and dosage of probiotic formulations. It is inferred that different components of probiotic formulations have varying effects on gut microbiota, with high doses of probiotics may induce rapid structural changes. However, the slow-release composite probiotic formulations used in this study was designed primarily to enhance the immunity of weak lambs and improve the colonization of beneficial gut bacteria, aligning largely with the expected outcomes.\u003c/p\u003e\u003cp\u003eAs lambs mature, notable changes occur in both microbial composition and relative abundance, particularly in the proliferation of beneficial bacterial species within different regions of GIT. \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003eLachnospiraceae\u003c/em\u003e are fibrolytic bacteria capable of degrading cellulosic polysaccharides, producing beneficial metabolic byproducts, and playing critical roles in feed digestion and absorption \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eSucciniclasticum\u003c/em\u003e can degrade crystalline cellulose, which other rumen bacteria cannot process, through its cellulolytic and enzyme-binding regions, thereby enhancing feed digestion and absorption \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e has been shown to improve growth performance, enhance intestinal metabolism, and increase the efficiency of protein and fiber breakdown in animals \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003ePrevotella\u003c/em\u003e species are associated with the degradation and utilization of starch, proteins, and polysaccharides in the rumen \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. They play crucial roles in maintaining digestive tract balance, reducing systemic inflammatory responses, and improving digestive capabilities. \u003cem\u003eMegasphaera elsdenii\u003c/em\u003e is notable for its ability to utilize accumulated lactic acid, thereby preventing acidosis\u0026mdash;a significant concern in ruminant nutrition. \u003cem\u003eAkkermansia\u003c/em\u003e contributes to gastrointestinal homeostasis and metabolic balance \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eRikenellaceae\u003c/em\u003e, which is commonly found in mammalian GIT, plays a significant role in the digestion of coarse fiber, with its abundance closely linked to the type of feed consumed \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study demonstrated that as Hu lambs grow, the composition and abundance of beneficial bacterial genera increase. Moreover, the genera enriched in the treatment group supplemented with probiotic formulations overlapped with those that increased over time in the control group. These included \u003cem\u003eRikenellaceae RC9_gut_group\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003eFibrobacter\u003c/em\u003e in the rumen; \u003cem\u003eChristensenellaceae_R-7_group\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e in the duodenum; \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e in the jejunum; and \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eLachnospiraceae\u003c/em\u003e_\u003cem\u003eUCG_002\u003c/em\u003e in the ileum. These findings suggested that probiotic formulations positively influenced the formation and stabilization of gut microbiota in lambs.\u003c/p\u003e\u003cp\u003eExtensive research has indicated that Firmicutes and Bacteroidetes are the predominant microbial phyla in the digestive tracts of ruminants, contributing to host metabolism, lipid regulation, and enhanced energy conversion efficiency \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. Firmicutes, primarily involved in cellulose breakdown, are essential for protein and carbohydrate digestion \u003csup\u003e[\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. These Gram-positive bacteria play significant roles in maintaining gut microbiota balance and boosting immunity \u003csup\u003e[\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. For instance, species of \u003cem\u003eClostridiales\u003c/em\u003e are involved in nutrient metabolism and absorption, providing energy to intestinal epithelial cells \u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe inclusion of \u003cem\u003eLactobacillus\u003c/em\u003e in feed promotes the production of adhesins that colonize the intestinal mucosa, helping maintain microbiota balance and supporting weight gain in ruminants \u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. The findings of this study further substantiate the role of Firmicutes in enhancing the metabolic efficiency and overall health of ruminants.\u003c/p\u003e\u003cp\u003eFirmicutes are the most abundant phylum in GIT of Hu lambs, accounting for 40\u0026ndash;90% of the relative abundance \u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e. In this study, the relative abundance of Firmicutes in the jejunum of 55-day-old control group was found to be 80.35%, consistent with findings by Wang et al. \u003csup\u003e[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e on the bacterial microbiota characteristics in different intestinal segments of Qinghai semi-fine wool sheep. These results suggest that the primary microbial structure of the lamb GIT forms rapidly after birth and gradually stabilizes. The supplementation of probiotic formulations increased the relative abundance of both Firmicutes and Bacteroidetes, albeit without statistical significance. This finding indicates that the preparation may enhance microbial populations without disrupting the existing balance or those such effects might require a longer duration or higher dosage to become apparent \u003csup\u003e[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe gut microbiota of ruminants plays a crucial role in digestion, metabolism, nutritional compensation, and growth, with higher microbial diversity and richness being positively correlated with improved animal health \u003csup\u003e[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e. Characterization of the Hu sheep rumen microbiota reveals a highly complex and diverse community, with Bacteroidetes and Firmicutes identified as the dominant and core phyla \u003csup\u003e[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]\u003c/sup\u003e. In the ileum, an increase in dominant phyla has been associated with reduced diarrhea in animals \u003csup\u003e[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]\u003c/sup\u003e, suggesting that probiotic formulations may be particularly effective in lambs exhibiting diarrheal symptoms \u003csup\u003e[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn summary, significant changes in the gut microbiota of Hu lambs were observed between birth and 20 and 55 days of age, indicating an increase in microbial diversity and stability as the lambs matured. The rumen and intestines harbored distinct microbial communities due to their unique physiological functions; the rumen was dominated by fiber-degrading bacteria, while the small intestine primarily hosted short-chain fatty acid (SCFA)-producers bacteria. Supplementation with probiotic formulations after birth was shown to enhance the abundance of gut microbiota, potentially offering probiotic benefits.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eProbiotic formulations represent an effective alternative to antibiotics in animal husbandry, playing a vital role in promoting animal health and productivity while exerting beneficial effects on growth. This study demonstrated that supplementation with probiotic formulations sustainably enhanced the diversity of gut microbiota, increased the abundance of beneficial bacteria, improved digestive function, and thereby elevated the health status and growth rate of Hu lambs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.X. and G.S. conceived and designed the study, performed the microbiological analyses, and wrote and revised the manuscript. Y.Y. and S.Y. performed data analysis and data visualization. S.D. and Y.X. realized the animal experiments. Y.W., P.W. and J.J. supervised the project and funding acquisition and participated in the experiment. F.G. provided direction and supervision for the study, and reviewed and edited the final manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (31672394), Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding\u0026nbsp;（2021C02068-6）, Zhejiang Province Agricultural Major Technology Collaborative Promotion Plan（2023ZDXT16-3）, the Project for Zhejiang Province Basic Public Welfare Research Program (LTGN24C050001), China Agriculture Research System (CARS-39-07), Young Science and Technology Top Talent Program of Tianshan Talent Training Program in Xinjiang Province (2022TSYCCX0124), Xinjiang Agriculture Research System (XJARS-09-26), as well as Project of Corps Science and Technology in Key Areas ( 2024AB017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence datasets generated during the current study are available in the GSA repository, (https://ngdc.cncb.ac.cn/gsa) as CRA023269. All the other data are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving Hu sheep were conducted in accordance with the guidelines of the China Council on Animal Care. The protocols were approved by the Experimental Animal Management Committee of China Jiliang University (approval number: 2022-014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFu, X. et al. 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A multi-omic assessment of the mechanisms of intestinal microbes used to treat diarrhea in early-weaned lambs\u003cem\u003e.\u003c/em\u003e \u003cem\u003emSystems\u003c/em\u003e. 9(2), e0095323. https://doi.org/10.1128/msystems.00953-23 (2024)\u003cstrong\u003e\u003c/strong\u003e\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":"Hu lambs, probiotic formulations, 16S rRNA gene sequencing, gut microbiota, GIT morphology","lastPublishedDoi":"10.21203/rs.3.rs-7333756/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7333756/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGut microbiota plays an important role in the digestive system of ruminants. It affects the health status of Hu sheep and their development and production rates. However, its composition may be influenced by several factors such as gender, age, and diet. In the animal husbandry industry, probiotic formulations have been widely used as alternatives to antibiotics, offering advantages such as non-toxicity, non-residue, and non-pathogenicity. This study aimed to investigate the morphological changes in the gastrointestinal tract and dynamics of gut microbiota during postnatal development of Hu lambs, and evaluated the effects of dietary supplementation with probiotic formulations on gut microbiota. Fifteen male Hu lambs were randomly divided into five groups, with three lambs per group. One blank control group did not receive food postnatally, while two control groups were fed according to standard farm practices with milk and a basic diet. Two experimental groups were administered 10 mL of probiotic formulations within 2 hours after birth, followed by the same feeding regimen as the controls. During the 60-day experiment, samples were collected from the rumen, duodenum, jejunum, and ileum at 2 hours, 20 days, and 55 days after birth. The collected samples included digestive tract tissues and their contents. High-throughput 16S rRNA gene sequencing was employed to identify and analyze microbial diversity, and morphological analysis was conducted to compare differences in the surface tissues of the digestive tract. The results indicated that, with increasing age, the length of the rumen papillae and the height of the villi in the jejunum and ileum of both control and experimental groups continued to increase. Additionally, the overall diversity of the gut microbiota exhibited a gradual upward trend. The microbial diversity indices in the rumen, duodenum, jejunum, and ileum of the experimental group were higher than those of the control group at corresponding time points, although the differences were not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The number of beneficial bacteria, such as \u003cem\u003eRuminococci\u003c/em\u003e, in the gastrointestinal tract of Hu lambs in the experimental group increased, while the number of potentially pathogenic bacteria, such as \u003cem\u003eEscherichia spp\u003c/em\u003e., relatively decreased. The results suggest that the supplementation of probiotic formulations promotes early gastrointestinal tract development, enhances the colonization of beneficial bacteria, and improves microbial diversity in Hu lambs. This result provides valuable insights into the gastrointestinal development and microbial dynamics of Hu lambs, as well as the production of probiotic formulations for sheep.\u003c/p\u003e","manuscriptTitle":"Effects of probiotic formulations on GIT morphological changes and gut microbiota in Hu lambs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 09:51:29","doi":"10.21203/rs.3.rs-7333756/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":"218ab817-bbb7-437b-ad1c-edc71d5cecea","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54467018,"name":"Health sciences/Gastroenterology"},{"id":54467019,"name":"Biological sciences/Microbiology"},{"id":54467020,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2025-09-30T03:23:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 09:51:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7333756","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7333756","identity":"rs-7333756","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}