Total mixed ration containing fermented cotton straw and application thereof

Abstract

The invention belongs to the technical field of livestock feed, and particularly discloses a total mixed ration containing fermented cotton straw and application thereof. The total mixed ration comprises the following raw materials on the basis of dry matter: 5-15% of fermented cotton straw, 10-20% of corn straw, 5-10% of alfalfa, 25% of corn, 9% of soybean meal, 7.5% of dry corn vinasse, 10% of corn germ meal, 15% of soybean hull, 0.9% of calcium hydrophosphate, 0.4% of ammonium chloride, 0.2% of choline chloride, 0.5% of sodium chloride and 0.5% of sodium bicarbonate. The total mixed ration can significantly improve the growth performance and feed conversion efficiency of the Hu sheep, improve the digestion and utilization rate of nutrient substances, effectively improve the health and anti-stress level of animal bodies, improve the diversity and structure of rumen microflora, promote the growth of beneficial microorganisms, and establish a more stable and efficient rumen fermentation system.

Description

Technical Field

[0001] This invention belongs to the field of poultry and livestock feed technology, specifically relating to a total mixed ration containing fermented cotton straw and its application. Background Technology

[0002] As an important meat sheep breed in my country, the profitability of Huzhou sheep farming is closely related to feed costs. Traditional total mixed rations (TMR) for Huzhou sheep primarily use conventional roughage sources such as corn stalks, wheat straw, alfalfa hay, and peanut vines. While these meet the basic fiber requirements of ruminants, large-scale farming faces challenges such as intensified competition for feed resources, unstable supply of high-quality roughage, and high costs. Therefore, developing unconventional roughage resources that are widely available, inexpensive, and have good nutritional value has become an urgent need for the sustainable development of the livestock industry.

[0003] Cotton stalks, a byproduct of the cotton industry, are produced in huge quantities annually. However, due to their low crude protein content, high lignin content, coarse texture, and poor palatability, direct feeding with cotton stalks can lead to low dry matter digestibility, reduced feed intake, and may affect rumen health and animal growth performance. Currently, the main methods for utilizing cotton stalks as feed are as follows: First, direct or simple physical processing followed by feeding, but this has limited improvement in nutritional value and palatability; second, chemical treatment (such as ammoniation), which can improve nitrogen content and fiber digestibility to some extent, but poses safety risks such as ammonia residue and the generation of harmful substances, and does not substantially improve protein nutrition; third, fermentation with a single strain followed by low-proportion addition, which often results in insufficient fermentation depth, unstable product quality, and an inability to achieve a high proportion of replacement for traditional roughage, thus the cost reduction effect is not significant, and there is a lack of systematic animal growth trials and verification of health effects. Existing technologies generally fail to simultaneously achieve the physical loosening of cotton stalks, the enrichment of nutrients, and the elimination of anti-nutritional factors through biotransformation. They also fail to conduct scientific compatibility and gradient verification in total mixed ration systems, resulting in low substitution rates and unclear overall benefits in practical applications. Summary of the Invention

[0004] The present invention aims to provide a total mixed diet containing fermented cotton straw and its application. This total mixed diet can significantly improve the growth performance and feed conversion efficiency of Hu sheep, improve the digestibility and utilization of nutrients, effectively improve animal health and stress resistance, improve the diversity and structure of rumen microbial community, promote the growth of beneficial microorganisms, and establish a more stable and efficient rumen fermentation system.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A total mixed ration containing fermented cotton straw, comprising the following ingredients on a dry matter basis: 5%-15% fermented cotton stalks, 10%-20% corn stalks, 5%-10% alfalfa, 25% corn, 9% soybean meal, 7.5% dried corn distillers grains, 10% corn germ meal, 15% soybean hulls, 0.9% dicalcium phosphate, 0.4% ammonium chloride, 0.2% choline chloride, 0.5% sodium chloride, 0.5% sodium bicarbonate.

[0006] Preferably, the fermented cotton stalks are prepared by the following steps: S1. Mix apple pomace, corn husks and crushed cotton stalks evenly to obtain a cotton stalk mixture; S2. Spray the compound microbial agent onto the cotton straw mixture obtained in S1, adjust the moisture content, place it in a fermentation tank, seal it, and carry out solid-state aerobic fermentation. Dry the fermentation product at low temperature to obtain fermented cotton straw.

[0007] Preferably, the dry matter ratio of apple pomace, corn husks and crushed cotton stalks in S1 is 5-10:5-10:80-90.

[0008] Preferably, in S1, the length of the crushed cotton stalks is 1-3 cm.

[0009] Preferably, in S2, the compound microbial agent includes one or more of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis, and the volume ratio of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis in the compound microbial agent is 1:1:1.

[0010] Preferably, the effective viable count of the *Lactobacillus plantarum* is ≥1×10⁻⁶. 9 CFU / mL, the effective viable count of the brewer's yeast is ≥1×10⁻⁶. 9 CFU / mL, the effective viable count of the Bacillus subtilis is ≥1×10⁻⁶. 9 CFU / mL.

[0011] Preferably, in step S2, the moisture content is adjusted to 50%-60%, the fermentation temperature is 25-35℃, and the fermentation time is 7-15 days.

[0012] Preferably, in S2, the specific process parameters for low-temperature drying of the fermentation product are: drying at a temperature of 55-65℃ until the moisture content is ≤13%.

[0013] The present invention also provides the application of the total mixed ration containing fermented cotton straw in the fattening of Hu sheep.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a total mixed ration (TMR) containing fermented cotton straw and its application. This TMR significantly improves the growth performance and feed conversion efficiency of Hu sheep, while comprehensively improving the digestibility and utilization of nutrients. After feeding the feed of this invention, the apparent digestibility of nutrients in the diet by Hu sheep was significantly improved. This indicates that the TMR is more easily digested and absorbed by animals, improves the rumen environment and microbial community, promotes the growth of beneficial microorganisms, inhibits harmful bacteria, and thus promotes the synergistic digestion and utilization of other nutrients in the entire diet.

[0015] This total mixed diet can improve the serum biochemical indicators and health status of Hu sheep, help enhance their antioxidant defense capabilities, alleviate physiological stress, and thus maintain better health, providing a physiological basis for efficient growth.

[0016] This invention transforms cotton stalks, which are typically low in feed value and largely wasteful, into high-quality functional roughage rich in true protein and organic acids with a porous structure through a compound microbial fermentation process. This not only provides a practical solution for reducing feed costs in sheep farming but also opens up an effective pathway for the resource-based and high-value utilization of agricultural byproducts such as cotton stalks, yielding significant social and ecological benefits.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 The graph shows the results of rumen microbial α-diversity among Examples 3-5 and Comparative Example 1. Figure 2 This refers to the rumen microbial β-diversity among Examples 3-5 and Comparative Example 1. Figure 3 The diagram shows the phylum of rumen microbiota differences among Examples 3-5 and Comparative Example 1. Figure 3 In this context, A represents the phylum Actinobacteria. Figure 3 The B in the text represents the Bacillus phylum. Figure 3 The C in the text stands for Bacteroidetes. Figure 3 In this context, D stands for Chlorophytum. Figure 3 In this context, E stands for Cyanobacteria. Figure 3 In this context, F stands for Fibrobacteria. Figure 3 In this context, G stands for Patellar Bacteria. Figure 3 The H in the text represents the phylum Pseudomonas. Figure 3 In this context, I stands for the spirochete phylum. Figure 3 J in the text stands for Thermostobacteria. Figure 4 The diagram shows the rumen microbial differences among Examples 3-5 and Comparative Example 1. Figure 4 In this context, A represents Bifidobacteria. Figure 4The B in the text represents the phylum Saccharomyces. Figure 4 C in the text refers to the R-7 group of the Crestiaceae family. Figure 4 The D in the text represents Enterococcus spp. Figure 4 E in the text refers to the NC2004 group of the family Trichophyceae. Figure 4 F in the text refers to group NK3A20 of the family Trichophyceae. Figure 4 In this context, G represents the genus *Eurotium*. Figure 4 H in the text represents the RC9 intestinal group of the Riken Bacteriaceae family. Figure 4 The "I" in the name stands for Ruminococcus. Figure 4 J in the text stands for Xylanobacterium. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Source of experimental materials: Lactobacillus plantarum was identified as Lactobacillus plantarum CGMCC21766; Saccharomyces cerevisiae was identified as Saccharomyces cerevisiae CCTCCM2022124; and Bacillus subtilis was identified as Bacillus subtilis CICC10071.

[0022] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0023] Lactobacillus plantarum slant culture medium: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, anhydrous sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium citrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, Tween-80 1 mL / L, agar 20 g / L, pH 6.2~6.5, sterilized at 115℃ for 20 min.

[0024] Saccharomyces cerevisiae slant culture medium: 10 g / L malt extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar, pH 6.0, sterilized at 115℃ for 20 min.

[0025] Bacillus subtilis slant culture medium: beef extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, agar 20 g / L, pH 7.0, sterilized at 121℃ for 20 min.

[0026] Liquid seed culture medium for Lactobacillus plantarum: Same as Lactobacillus plantarum slant culture medium (with agar removed), with the addition of 0.5 g / L cysteine ​​salt (anaerobic agent).

[0027] Saccharomyces cerevisiae liquid seed culture medium: same as Saccharomyces cerevisiae slant culture medium (agar removed).

[0028] Bacillus subtilis liquid seed culture medium: same as Bacillus subtilis slant culture medium (agar removed).

[0029] Secondary expansion medium for Lactobacillus plantarum: 15 g / L peptone, 8 g / L yeast extract, 20 g / L glucose, 5 g / L anhydrous sodium acetate, 2 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 1 mL / L Tween-80, 0.8 g / L L-cysteine ​​hydrochloride, pH 6.2~6.5, sterilized at 115℃ for 20 min.

[0030] Secondary culture medium for Saccharomyces cerevisiae: 15 g / L malt extract, 25 g / L peptone, 30 g / L glucose, 1 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, pH 5.8, sterilized at 115℃ for 20 min.

[0031] Secondary expansion culture medium for Bacillus subtilis: 8 g / L beef extract, 15 g / L peptone, 4 g / L glucose, 5 g / L sodium chloride, 1.5 g / L disodium hydrogen phosphate, 2 g / L calcium carbonate, pH 7.2, sterilized at 121℃ for 20 min.

[0032] Example 1 Preparation and activation of microbial agents: (1) Preparation of strains: Commercial freeze-dried Lactobacillus plantarum strain, Saccharomyces cerevisiae strain, and Bacillus subtilis strain were inoculated onto their respective slant agar plates for activation. The activation conditions were as follows: Lactobacillus plantarum was cultured anaerobically at 30℃ for 24 h, Saccharomyces cerevisiae was cultured aerobically at 28℃ for 48 h, and Bacillus subtilis was cultured aerobically at 37℃ for 24 h. After two generations of continuous activation, stable activated strains were obtained.

[0033] (2) Preparation of primary seed culture: Single colonies activated as described above were picked and inoculated into the corresponding liquid seed culture media for cultivation. The cultivation conditions were as follows: *Lactobacillus plantarum* anaerobic shaking culture at 30℃ and 150 r / min for 20 h; *Saccharomyces cerevisiae* aerobic shaking culture at 28℃ and 200 r / min for 24 h; *Bacillus subtilis* aerobic shaking culture at 37℃ and 220 r / min for 18 h; until the OD of the bacterial culture was reached... 600 When the value reaches 1.0, it is considered to be a first-grade seed solution.

[0034] (3) Secondary expansion culture: The primary seed culture was inoculated into the corresponding secondary expansion culture medium at an inoculation rate of 5% (v / v) for high-density fermentation culture. The culture conditions were as follows: Lactobacillus plantarum was cultured anaerobically at 30℃ and 180 r / min for 24 h; Saccharomyces cerevisiae was cultured aerobically at 28℃ and 220 r / min for 36 h, with dissolved oxygen ≥20% throughout the process; and Bacillus subtilis was cultured aerobically at 37℃ and 250 r / min for 20 h to obtain the secondary expansion culture.

[0035] (4) Microbial agent detection: The viable cell count of the above-mentioned secondary culture solution was performed by plate dilution and plating method. The viable cell counts of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis were all ≥1×10⁻⁶. 9 A high concentration of liquid bacterial agent can be obtained by using CFU / mL.

[0036] (5) Mix the high-concentration liquid bacterial agents of Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis prepared above at a volume ratio of 1:1:1 to obtain a compound bacterial agent.

[0037] Example 2 A method for preparing fermented cotton stalks includes the following steps: S1. Mix 5% apple pomace (crushed and dried), 5% corn husk (crushed and dried) and 90% cotton stalks crushed to 1-3 cm evenly to obtain a cotton stalk mixture; S2. The compound microbial agent prepared in Example 1 (based on the dry weight of the cotton straw mixture, the spraying amount of the compound microbial agent is 0.10%) is sprayed onto the cotton straw mixture obtained in S1. The moisture content is adjusted to 50%-60%, placed in a fermentation tank, compacted, sealed with 8 layers of sterile gauze, and solid-state fermented at 25-35℃ for 10 days. The fermentation product is dried at 60℃ until the moisture content is 13% to obtain fermented cotton straw.

[0038] Total mixed rations (TMRs) for Hu sheep were prepared. In Example 3, 5% of the fermented cotton straw prepared in Example 2 was added; in Example 4, 10% of the fermented cotton straw prepared in Example 2 was added; and in Example 5, 15% of the fermented cotton straw prepared in Example 2 was added. Comparative Example 1 did not include the fermented cotton straw prepared in Example 2. The groupings and ration formulations for each group are shown in Table 1.

[0039] Table 1 Dietary Formulas

[0040] Each kilogram of premix contains the following ingredients: 2.5 grams of iron; 3.5 grams of copper; 2.5 grams of zinc; 1.5 grams of manganese; 0.15 grams of iodine; 0.03 grams of selenium; 0.03 grams of cobalt; 7000 IU of vitamin A; 700 IU of vitamin D; and 60 IU of vitamin E.

[0041] Preparation method: Crush the corn stalks and alfalfa (2-4 cm in length); crush all concentrated feed ingredients (corn, soybean meal, etc.) and additives and mix them evenly; mix the treated corn stalks and alfalfa, fermented cotton stalks (no need for additional crushing) with the mixed concentrated feed in a TMR mixer, and add water to adjust the overall moisture content to about 45-50%, thus obtaining the total mixed diet of Examples 3-5 and Comparative Example 1.

[0042] The effects of Examples 3-5 and Comparative Example 1 were verified.

[0043] Animal husbandry and management: Animal selection and grouping: One hundred Hu sheep rams of similar weight (21.63 kg ± 3.55 kg), good health, and consistent age were selected as experimental animals. A completely randomized block design was used, and the sheep were randomly divided into four groups of 25 animals each: the control group (CON), and the experimental groups FCS-5, FCS-10, and FCS-15. The control group was fed the diet of Comparative Example 1, while the FCS groups were fed the diets provided in Examples 3, 4, and 5, respectively.

[0044] Experimental Period and Feeding Management: The experimental period lasted 95 days, including a 10-day pre-feeding period and an 85-day main trial period. On the first day of the pre-feeding period, sheep were fed the corresponding group's rations to allow them to adapt to the ration formula and feeding environment. During the experiment, all sheep were housed in the same standardized sheepfold, fed individually, with free access to water. Rations were given daily at morning feeding (08:00) and evening feeding (18:00), and the daily feed intake of each sheep was recorded. The feeding amount was adjusted based on the sheep's feed intake.

[0045] Sample collection and processing: (1) Weight measurement: All experimental sheep were weighed on an empty stomach before morning feeding on the first day of the pre-feeding period and before morning feeding on the first day after the end of the trial period, respectively, to calculate growth performance indicators such as average daily weight gain (ADG).

[0046] (2) Rumen fluid collection: On the first morning feeding after the end of the trial period, rumen fluid of each experimental sheep was collected by oral rumen catheterization. Before collection, the catheter was disinfected, slowly inserted into the rumen, and about 50 mL of rumen fluid was extracted. After filtration through 4 layers of sterile gauze, a portion was immediately used to determine the pH value; the other portion was aliquoted into sterile centrifuge tubes, quickly frozen in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80℃ for storage. This was used for subsequent determination of rumen fermentation parameters, rumen metagenomic analysis, rumen microbial diversity (16S+ITS) analysis, and rumen metabolomics analysis.

[0047] (3) Blood sample collection: On the first day after the end of the trial period, before morning feeding, blood was collected from the jugular vein of the experimental sheep. 10 mL of blood was collected using vacuum blood collection tubes (with and without anticoagulant). The blood sample with anticoagulant was gently inverted and mixed, and used for subsequent determination of immune indicators; the blood sample without anticoagulant was allowed to stand at room temperature for 30 min, then centrifuged at 3000 r / min for 10 min to separate the serum. The serum was aliquoted into sterile centrifuge tubes and stored at -20℃ for determination of serum biochemical indicators, antioxidant indicators, and stress indicators.

[0048] (4) Fecal sample collection: On the first morning before feeding after the end of the trial period, approximately 200g of fresh fecal samples were collected from each group of experimental sheep using the rectal fecal collection method. The samples were placed in sterile sealed bags, and a portion was immediately used for the determination of apparent digestibility-related indicators; the other portion was frozen and stored in a -20℃ freezer for subsequent analysis of related indicators. Fecal contamination was avoided during the collection process.

[0049] Measurement indicators and methods: (1) Growth performance determination: Based on the body weight data measured during the pre-feeding period and the trial period, the average daily weight gain (ADG) of each group of experimental sheep was calculated as (weight at the end of the trial period - initial weight during the pre-feeding period) / number of days in the trial period; the daily feed intake and remaining amount of each group of experimental sheep were recorded, the dry matter intake (DMI) was calculated, and the feed conversion efficiency (FCE) was calculated based on ADG and DMI as ADG / DMI.

[0050] (2) Apparent digestibility determination: The apparent digestibility of nutrients was determined using the acid-insoluble ash method. The collected feed and fecal samples were dried to constant weight at 65℃, pulverized, and passed through a 40-mesh sieve. The contents of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude fat (EE), and crude ash (Ash) in the feed and feces were determined, as well as the acid-insoluble ash (AIA) content. The apparent digestibility of each nutrient was calculated using the following formula: %; In the formula: = Content of a certain nutrient in feces; C = Content of AIA in feed; D = Content of a certain nutrient in feed; E = Content of AIA in feces.

[0051] The determination methods for dry matter, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber are based on national standards GB / T 6435-2014, GB / T 40747-2021, GB / T 6432-2018, GB / T 20806-2022, and NY / T1459-2022, respectively.

[0052] Serum biochemical index determination: Using a fully automated biochemical analyzer, the following biochemical indexes in serum were measured in accordance with the instructions of the corresponding reagent kits (Nanjing Jiancheng Bioengineering Institute): total protein (TP), albumin (ALB), globulin (GLB), blood urea nitrogen (UREA), glucose (GLU), total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

[0053] Serum antioxidant index determination: Serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, and catalase (CAT) activity were determined by colorimetric method. Malondialdehyde (MDA) content was determined by thiobarbituric acid method. All operations were strictly performed in accordance with the instructions of the kit (Nanjing Jiancheng Bioengineering Institute).

[0054] Serum stress markers were measured using enzyme-linked immunosorbent assay (ELISA) to determine the levels of stress markers such as cortisol (COR), epinephrine (EPI), and norepinephrine (NE) in serum. All kits were purchased from a reputable manufacturer (Nanjing Jiancheng Bioengineering Institute), and the procedures were performed according to the kit instructions.

[0055] Serum immune markers were measured using ELISA to determine the levels of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), as well as the levels of cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), strictly following the operating procedures of the kit (Nanjing Jiancheng Bioengineering Institute).

[0056] Rumen fermentation parameters were determined as follows: the pH value of rumen fluid was measured on-site using a portable pH meter; the content of ammonia nitrogen (NH3-N) in rumen fluid was determined using the phenol-sodium hypochlorite colorimetric method; and the content and proportion of volatile fatty acids (VFAs) such as acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and valeric acid in rumen fluid were determined using gas chromatography.

[0057] Rumen metagenomic analysis: Total DNA from rumen microorganisms was extracted, and DNA purity and integrity were detected by agarose gel electrophoresis. Library construction was then performed after the DNA passed the tests. High-throughput sequencing was conducted using the Illumina sequencing platform. The sequencing data underwent quality control, assembly, gene prediction, and functional annotation to explore the gene functions and metabolic pathways of rumen microorganisms.

[0058] Rumen microbial diversity (16S+ITS) analysis: Specific primers were designed targeting bacterial 16S rRNA genes and fungal ITS sequences for PCR amplification. The amplified products were purified, quantified, and used to construct sequencing libraries for high-throughput sequencing using the Illumina MiSeq platform. After optimizing the sequencing data, operational taxonomic unit (OTU) clustering analysis, species annotation, and α- and β-diversity analyses were performed to reveal the differences in rumen bacterial and fungal community structure and diversity.

[0059] Data Statistics and Analysis: Experimental data were organized using Excel 2019 and statistically analyzed using SPSS 26.0 software. All data are expressed as mean ± standard error (Mean ± SE). One-way ANOVA was used to test the significance of differences between groups. P < 0.05 was considered significant, and P < 0.01 was considered highly significant. The results are shown in Table 2 below: Table 2 Effects of different groups on growth performance of meat sheep

[0060] As shown in Table 2, the final body weight, average daily gain (ADG), and dry matter intake (DMI) of the sheep in Examples 4 and 5 were significantly higher than those in Control Group 1. P <0.05). This result indicates that adding 10% to 15% fermented cotton straw to the diet can significantly improve the palatability of meat sheep, promote feed intake, and ultimately achieve a significant improvement in growth performance.

[0061] Table 3. Effects of different groups on the apparent digestibility of nutrients in meat sheep

[0062] As shown in Table 3, the apparent digestibility of dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) in the sheep of each of the example groups (Examples 3, 4, and 5) was significantly higher than that of control group 1. P <0.05). This proves that the present invention, through the fermentation process of cotton stalks, effectively degrades anti-nutritional factors such as lignin, and significantly improves the digestion and absorption efficiency of various nutrients, especially fibrous substances, in meat sheep.

[0063] Table 4. Effects of different groups on rumen fermentation parameters in meat sheep

[0064] As shown in Table 4, the concentrations of butyric acid and isovaleric acid in the rumen fluid of sheep in Examples 4 and 5 were significantly higher than those in Comparative Example 1 (P<0.05). The increase in volatile fatty acid concentration indicates that fermentation of cotton straw significantly improved the fermentation environment of the sheep rumen, enhanced the metabolic activity of rumen microorganisms, and provided the body with a richer energy source.

[0065] Table 5. Effects of different groups on serum biochemical indicators of meat sheep

[0066] As shown in Table 5, all example groups significantly increased the levels of total protein (TP) and albumin (ALB) in sheep serum. P <0.05); meanwhile, the total cholesterol (TC) content in the serum of Example 5 group was significantly lower than that of Comparative Example 1 group ( P <0.05). The changes in the above indicators indicate that the addition of fermented cotton straw in this invention can effectively improve the protein synthesis metabolism level of meat sheep and optimize the lipid metabolism pattern.

[0067] Table 6. Effects of different groups on serum antioxidant and stress indicators in meat sheep

[0068] As shown in Table 6, the levels of glutathione peroxidase (GSH-Px) in the serum of sheep in each of the example groups were significantly higher than those in the control group 1. P <0.05). The elevated enzyme level demonstrates that the diet provided by this invention can significantly enhance the antioxidant capacity of sheep, scavenge free radicals in the body, and thus improve the health of sheep.

[0069] Table 7. Effects of different groups on serum immune indicators in meat sheep

[0070] As shown in Table 7, the levels of D-lactic acid (D-LA) and diamine oxidase (DAO) in the serum of sheep in each of the example groups were significantly lower than those in control group 1. P <0.05). D-LA and DAO are key indicators for measuring the degree of intestinal mucosal damage. The significant decrease in their levels indicates that the addition of fermented cotton straw in this invention effectively protects the integrity of the intestinal mucosa, reduces the entry of enterogenic toxins into the blood, and significantly improves the immune level and intestinal barrier function of meat sheep.

[0071] Results of rumen microbial community in meat sheep as follows Figures 1-4 As shown.

[0072] Depend on Figure 1It can be seen that, compared with Comparative Example 1, Examples 3 and 4 maintained a high level of microbial richness, while the ACE index of Example 5 showed a certain degree of adjustment and decrease. This change indicates that the addition of a high proportion (15%) of fermented cotton straw exerts significant selective pressure on rumen microorganisms, achieving precise enrichment of specific functional degrading microorganisms by reducing the abundance of non-core bacteria. This moderate adjustment of diversity is conducive to building a more efficient and specific fiber-degrading microecological system, thereby improving feed conversion efficiency.

[0073] Depend on Figure 2 As can be seen from the PCoA analysis results, the samples in each group exhibited significant differences in cluster distribution on the coordinate axes. Specifically, Examples 4 and 5 showed significant vertical separation from Comparative Examples 1 and 3 on the PC2 axis (P = 0.001), indicating that adding 10% or more of fermented cotton straw can significantly reshape the rumen microbial community structure. This significant differentiation in β-diversity proves that the fermentation of cotton straw in this invention does not simply change the quantity of microorganisms, but fundamentally promotes the evolution of the rumen microecology towards a new, more degradative microbial community pattern, laying a solid biological foundation for improving the utilization of crude fiber resources by ruminants.

[0074] Depend on Figure 3 It can be seen that with the increase of fermented cotton straw addition, the abundance of dominant intestinal flora exhibits a regular evolution. Compared with the comparative example, each example can specifically increase the proportion of flora related to cellulose degradation while maintaining stable flora diversity. Among them, Example 5 (15% addition) showed the most significant effect, demonstrating the positive optimization effect of fermented cotton straw on the intestinal microecological environment. Experimental data show that in Example 5... Bacillota The relative abundance was significantly increased compared to the control ratio. P<0.05), because this phylum contains a large number of probiotics (such as Ruminococcus and Clostridium) that degrade complex polysaccharides and cellulose, the increase in its abundance directly proves that the fermented cotton straw of this invention can effectively improve the digestibility and utilization rate of crude fiber feed in animals. In Example 5, the proportion of Bacteroidota was significantly lower than that of the control group. This increase in the Bacillota / Bacteroidota (F / B) ratio is an important indicator of enhanced intestinal energy acquisition capacity, indicating that this invention, through the intervention of fermented cotton straw, successfully guides the intestinal flora to shift towards efficient energy metabolism, thereby achieving better growth performance or feed conversion ratio. In addition, in each example, the abundance of bacteria in phyla such as Actinomycetota and Pseudomonadota remained within a stable and reasonable range compared with the control group, and no abnormal proliferation of pathogenic bacteria (such as Proteobacteria) was observed. This further proves that the fermented cotton stalks used in this invention not only improve digestive performance but also have extremely high biosafety, do not disrupt the healthy balance of the intestinal microecology, and all indicators are superior to the comparative group without added fermented stalks.

[0075] Depend on Figure 4 It can be seen that in Example 4 Lachnospiraceae NK3A20 group The abundance of (NK3A20 genus of Trichophyceae family) was significantly increased compared to control example 1. P <0.05), this genus, as an important butyrate-producing bacterium, directly enhances the rumen's conversion efficiency of fiber components in fermented straw. Microecological barrier optimization: In Example 5 Enterococcus The relative abundance of Enterococcus was significantly higher than that of control group 1. P <0.05), as a key probiotic group, its significant increase helps improve the gut microenvironment and enhance the body's immunity. (Example 5) Rikenellaceae RC9 gut group The abundance was significantly lower than that of control group 1, combined with Bifidobacterium The moderate modulation of (Bifidobacterium) indicates that this invention successfully guides the rumen microbiota to shift its metabolic mode from processing simple sugars to processing complex structural carbohydrates. In summary, the differential analysis at the genus level further confirms that this invention, by adding fermented cotton straw, precisely upregulates the core genera associated with crude fiber degradation and gut health, achieving significantly better technical results than the control group without addition.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A total mixed ration containing fermented cotton stalks, characterized in that, On a dry matter basis, it includes the following raw materials: 5%-15% fermented cotton stalks, 10%-20% corn stalks, 5%-10% alfalfa, 25% corn, 9% soybean meal, 7.5% dried corn distillers grains, 10% corn germ meal, 15% soybean hulls, 0.9% dicalcium phosphate, 0.4% ammonium chloride, 0.2% choline chloride, 0.5% sodium chloride, 0.5% sodium bicarbonate.

2. The total mixed ration according to claim 1, characterized in that, The fermented cotton stalks are prepared by the following steps: S1. Mix apple pomace, corn husks and crushed cotton stalks evenly to obtain a cotton stalk mixture; S2. Spray the compound microbial agent onto the cotton straw mixture obtained in S1, adjust the moisture content, place it in a fermentation tank, seal it, and carry out solid-state aerobic fermentation. Dry the fermentation product at low temperature to obtain fermented cotton straw.

3. The total mixed ration according to claim 2, characterized in that, The dry matter ratio of apple pomace, corn husks and crushed cotton stalks mentioned in S1 is 5-10:5-10:80-90.

4. The total mixed ration according to claim 2, characterized in that, In S1, the length of the crushed cotton stalks is 1-3 cm.

5. The total mixed ration according to claim 1, characterized in that, In S2, the compound microbial agent includes one or more of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis, and the volume ratio of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis in the compound microbial agent is 1:1:

1.

6. The total mixed ration according to claim 5, characterized in that, The effective viable count of *Lactobacillus plantarum* is ≥1×10⁻⁶. 9 CFU / mL, the effective viable count of the brewer's yeast is ≥1×10⁻⁶. 9 CFU / mL, the effective viable count of the Bacillus subtilis is ≥1×10⁻⁶. 9 CFU / mL.

7. The total mixed ration according to claim 2, characterized in that, In S2, the moisture content is adjusted to 50-60%, the fermentation temperature is 25-35℃, and the fermentation time is 7-15 days.

8. The total mixed ration according to claim 2, characterized in that, In S2, the specific process parameters for low-temperature drying of the fermentation product are: drying at a temperature of 55-65℃ until the moisture content is ≤13%.

9. The application of the total mixed ration containing fermented cotton straw as described in claim 1 in the fattening of Hu sheep.

Images