Compound diet for broilers in late fattening period and application thereof

Abstract

This invention relates to the field of feed technology and provides a compound diet for broilers in the late fattening stage and its application. The compound diet for broilers in the late fattening stage comprises compound additives and a basal diet. The compound additives include *Trifolium repens* leaf powder and ε-polylysine hydrochloride; *Trifolium repens* leaf powder is obtained by crushing the above-ground vine and leaves of the *Trifolium repens* plant (Vitaceae family). The content of *Trifolium repens* leaf powder is 0.3-1.2% by weight of the total basal diet, and the content of ε-polylysine hydrochloride is 0.03-0.13%. This invention combines the nutritional needs of broilers in the late fattening stage with the synergistic compounding of the natural antibacterial plant *Trifolium repens* vine and ε-polylysine hydrochloride to replace traditional antibiotic growth promoters; the synergistic effect of the two can improve the growth performance and immune function of broilers, while also improving meat flavor and intestinal health, resulting in a highly efficient, antibiotic-free, and improved quality compound diet for broilers in the late fattening stage.

Description

Technical Field

[0001] This invention belongs to the field of feed technology, and in particular relates to a compound diet for broiler chickens in the later stage of fattening and its application. Background Technology

[0002] The late fattening stage of broiler chickens (usually 2-3 weeks before slaughter) is a crucial period for weight gain, improved meat flavor, and enhanced immune health. However, existing late fattening diets for broilers still have many technical shortcomings:

[0003] (1) The ratio of dietary energy to protein is unreasonable;

[0004] (2) Poor intestinal health, resulting in a high mortality rate in aquaculture;

[0005] (3) It is difficult to take into account core quality indicators such as meat flavor and drip loss;

[0006] (4) They rely heavily on antibiotic growth promoters, which pose drug residues and food safety risks.

[0007] Therefore, there is an urgent need in poultry farming to develop a high-efficiency, low-consumption, antibiotic-free, and quality-improving compound diet for broilers in the later stages of fattening. To this end, this invention proposes a compound diet for broilers in the later stages of fattening and its application. Summary of the Invention

[0008] The purpose of this invention is to provide a compound diet for broilers in the later stages of fattening and its application, aiming to solve the problems of imbalanced energy-egg ratio, high feed conversion ratio, poor intestinal health, difficulty in achieving meat quality and flavor indicators, and reliance on antibiotics, which pose drug residues and food safety risks in existing broiler diets in the later stages of fattening.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A compound additive comprising Trifoliate orange leaf powder and ε-polylysine hydrochloride; the Trifoliate orange leaf powder is obtained by crushing the above-ground vine and leaf parts of Trifoliate orange, a plant of the Vitaceae family.

[0011] A compound diet for broiler chickens in the later stages of fattening includes the aforementioned compound additives and a basal diet.

[0012] Furthermore, based on the percentage of total mass of the basal diet, the compound additive contains 0.3-1.2% of *Ipomoea aquatica* leaf powder and 0.03-0.13% of ε-polylysine hydrochloride.

[0013] Furthermore, the basal diet comprises the following components by weight percentage: corn 65-75%, soybean meal 10-30%, wheat bran 2-10%, limestone powder 2-5%, soybean oil 2-5%, vitamin-mineral premix 1%, dicalcium phosphate 0.5-1.5%, choline chloride 0.2-1.0%, sodium chloride 0.1-0.5%, and DL-methionine 0.1-0.2%.

[0014] Furthermore, the crude protein content in the compound diet for broilers in the later stages of fattening is 14-20%, and the metabolizable energy is 10-15%.

[0015] The application of the above-mentioned compound additive or broiler compound diet in the preparation of feed that improves broiler growth performance, immune function, meat flavor and intestinal health.

[0016] Further improvements in growth performance include: increasing average daily weight gain; and increasing average daily feed intake.

[0017] Further improvements in immune function include: increasing spleen index; increasing bursal index; increasing serum TNF and IL-6 levels; and decreasing serum IL-10 and IgG levels.

[0018] Further improvements to meat flavor include: increasing the redness and yellowness values ​​of the breast muscle; increasing the shear force of the leg muscle; and reducing the shear force of the breast muscle.

[0019] Furthermore, improving gut health includes: regulating the structure of the cecal gut microbiota; and increasing the content of acetic acid, propionic acid, and isovaleric acid in the cecal contents.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. This invention can effectively increase broiler weight, daily feed intake, daily weight gain and optimize feed conversion ratio, thereby enhancing the growth performance of broilers.

[0022] 2. This invention can effectively increase the bursa of Fabricius index and spleen index of broilers; regulate serum biochemical indicators, reduce serum IgG and IL-10 levels, increase TNF and IL-6 levels, and improve the immune function of broilers.

[0023] 3. This invention can effectively improve the meat color index of broilers, reduce the shear force of the breast muscle, increase the shear force of the leg muscle, and improve the meat flavor.

[0024] 4. This invention can effectively improve the intestinal microbial community structure of the cecum of farmed broilers: at the phylum level, the relative abundance of Bacteroidota and Firmicutes is slightly increased; at the genus level, the abundance of Bacteroides and some genera related to intestinal metabolism is increased; at the same time, the content of volatile fatty acids in the intestine is increased, thus improving the intestinal metabolic environment. Attached Figure Description

[0025] Figure 1 This is a Venn diagram.

[0026] Figure 2 This is a PCoA diagram.

[0027] Figure 3 This refers to the gut microbiota structure at the phylum level.

[0028] Figure 4 This represents the gut microbiota community structure at the genus level.

[0029] Figure 5 This is a diagram showing the species differences within the genus *Sellimonas*.

[0030] Figure 6 This is a diagram showing the species differences of the GCA-900066575 genus.

[0031] Figure 7 This is a diagram showing the species differences within the genus *Campyiobacter*.

[0032] Figure 8 This is an evolutionary branch diagram of LDA Effect Size (LeFSe).

[0033] Figure 9 A bar chart showing the distribution of LDA values ​​(LDA value > 3). Detailed Implementation

[0034] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0035] This invention provides a compound diet for broiler chickens in the late fattening stage, comprising the following components by weight percentage: 65-75% corn, 10-30% soybean meal, 2-10% wheat bran, 2-5% limestone powder, 2-5% soybean oil, 1% vitamin-mineral premix, 0.5-1.5% dicalcium phosphate, 0.2-1.0% choline chloride, 0.1-0.5% sodium chloride, 0.1-0.2% DL-methionine, 0.3-1.2% trifoliate orange leaf powder, and 0.03-0.13% ε-polylysine hydrochloride.

[0036] Among them: the leaf powder of Tetrastigma hemsleyanum Diels et Gilg was obtained by crushing the above-ground vine and leaf parts of the Vitaceae plant Tetrastigma hemsleyanum Diels et Gilg; ε-polylysine hydrochloride was purchased from Zhejiang Xinyinxiang Bioengineering Co., Ltd.

[0037] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: Experimental Design;

[0039] This study was conducted at the Quzhou Longyou Ma Chicken Breeding Farm in Quzhou City, Zhejiang Province. 288 healthy broiler chickens of similar weight were selected and randomly divided into four groups (control group, *Trifolium repens* group, ε-polylysine hydrochloride group, and compound group), with six replicates per group and twelve chickens per replicate. The basal diet was formulated according to the NY / T 3645-2020 standard, and the specific formula is shown in Table 1. The control group was fed the basal diet (without additional supplementation); the *Trifolium repens* group was supplemented with 1.0% *Trifolium repens* leaf powder; the ε-polylysine hydrochloride group was supplemented with 0.05% ε-polylysine hydrochloride; and the compound group was supplemented with a compound additive, including 1.0% *Trifolium repens* leaf powder and 0.05% ε-polylysine hydrochloride. The additives and amounts for each group are shown in Table 2. All experimental feeds were mixed into the basal diet. Broilers were raised in experimental chicken houses with free access to feed and water, 24-hour lighting, regular cleaning and disinfection, and immunization and feeding management in accordance with the regulations of the experimental chicken farm.

[0040] Table 1. Basic Diet Formula and Nutrient Content

[0041] Raw material composition, % (by weight) corn 67.50 Soybean meal (crude protein 44.2%) 20.95 soybean oil 4.00 wheat bran 2.55 stone powder 2.07 Calcium hydrogen phosphate 1.00 choline chloride 0.50 Sodium chloride 0.30 DL-methionine 0.13 <![CDATA[Vitamin - Mineral Premix ① > 1.00 total 100.00 <![CDATA[Nutritional components ② > Metabolizable energy, MJ / kg 12.77 Crude fat, % 7.5 Crude protein, % 15.2 calcium,% 0.994 Total phosphorus, % 0.53 Non-phytic acid phosphorus, % 0.19 Lysine, % 0.77 Methionine, % 0.34

[0042] Note: ① Vitamin-mineral premix can provide the following per kilogram of complete feed: Vitamin A 9750 IU, Vitamin B1 2.6 mg, Vitamin B2 6.25 mg, Vitamin B6 4 mg, Vitamin B1 4 mg, Vitamin B2 ... 120.025 mg, Vitamin D3 2200 IU, Vitamin E 25 IU, Vitamin K3 3 mg, D-Biotin 0.3 mg, D-Pantothenic Acid 8.75 mg, Nicotinamide 37.5 mg, Copper 11 mg, Iron 64 mg, Manganese 96 mg, Zinc 80 mg. ② Metabolizable energy and nonphytate phosphorus are calculated values, the rest are measured values.

[0043] Table 2. Feed additives and dosages for each experimental group

[0044] control group none 0% Three-leaf clover group Three-leaf ivy leaf powder 1.0% ε-polylysine hydrochloride group ε-polylysine hydrochloride 0.05% Composite group Trifolium leaf powder + ε-polylysine hydrochloride <![CDATA[1.05% ① ]]>

[0045] Note: ① The compound group consists of 1.0% *Trifolium repens* leaf powder and 0.05% ε-polylysine hydrochloride, totaling 1.05%.

[0046] Example 2: Effects of compound additives on the growth performance of broilers in the later stages of fattening;

[0047] Broiler growth performance was evaluated by measuring indicators such as daily weight gain, daily feed intake, and feed conversion ratio (FCR). The experimental period lasted 44 days, with a pre-feeding period of 90-100 days of age (feeding only the basal diet) and a formal trial period of 101-133 days of age (feeding experimental feeds containing different additives). Measurements of all indicators began at day 101. On days 101 and 133 of the experiment, broilers were weighed on an empty stomach in replicates and the weight was recorded. The amount of feed given and scattered throughout the rearing period was also accurately recorded. Broiler production performance was determined according to the People's Republic of China Agricultural Industry Standard (NY / T 823-2020 - Terminology and Measurement Methods for Poultry Production Performance). The calculation formulas were: Daily feed intake (g / d / bird) = Total feed consumption / Number of days reared; Daily weight gain (g / d / bird) = Total weight gain / Number of days reared; FCR = Total feed consumption / Total weight gain. The effects of each experimental diet on the growth performance of Longyou Ma chickens are shown in Table 3.

[0048] Table 3. Effects of compound additives on the growth performance of Longyou Ma chickens

[0049]

[0050] Note: Different lowercase letters in the superscript of data in the same row in the table indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0051] As shown in Table 3, during the period of 101-133 days of age, compared with the ε-polylysine hydrochloride group (ε-PLH 0.05%), the compound group (THL 1.0% + ε-PLH 0.05%) of Longyou Ma chickens showed significantly higher body weight, average daily feed intake, and average daily weight gain at 133 days of age (P < 0.05), while the feed conversion ratio showed no significant difference. Compared with the *Trifolium repens* group (THL 1.0%), the average daily feed intake of the compound group was also significantly higher (P < 0.05). These results indicate that adding compound additives to the diets of broilers in the later stages of fattening can significantly improve broiler body weight, daily feed intake, and daily weight gain, while maintaining a good feed conversion ratio.

[0052] Example 3: Effects of compound additives on the immune function of broilers in the later stages of fattening;

[0053] (1) Immune function is an important indicator for evaluating the health level of broilers. Enhancing the immunity of broilers can effectively resist the invasion of pathogenic microorganisms such as bacteria and viruses from the outside world. In this example, broiler breeding experiments were conducted according to Example 2. On the 133rd day of age, 6 chickens were randomly selected from each group and weighed. The heart, liver, spleen, lungs, kidneys, and bursa of Fabricius were weighed and the immune organ index was calculated. The formula for calculating the immune organ index is: Immune organ index = organ weight / carcass weight * 100%. The results of the effect of each experimental feed on the immune organ index of Longyou Ma chickens are shown in Table 4.

[0054] Table 4. Effects of compound additives on immune organ indices of Longyou Ma chickens

[0055] Note: Different lowercase letters in the superscript of data in the same row in the table indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0056] The spleen and bursa of Fabricius are important immune organs in poultry. Immune organ indices can be used to preliminarily measure the body's immune function. As shown in Table 4, compared with the control group (CON), the spleen and bursa of Fabricius indices of the compound additive group were significantly increased. These results indicate that the compound additive can stimulate the development of immune organs and improve the body's immune function.

[0057] (2) Serum immunoglobulin G (IgG), interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor (TNF), and interferon-γ levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit. All kits were purchased from Nanjing Jiancheng Biotechnology Research Institute. The effects of each experimental diet on the immune indicators of Longyou Ma chickens are shown in Table 5.

[0058] Table 5. Effects of compound additives on immune indicators of Longyou Ma chickens

[0059]

[0060] Note: Different lowercase letters in the superscript of data in the same row in the table indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0061] As shown in Table 5, compared with the control group, the serum TNF and IL-6 levels in the compound group were significantly increased (P < 0.05). Compared with the *Trifolium repens* group, the IL-10 level in the compound group was significantly decreased (P < 0.05). Compared with the ε-polylysine hydrochloride group, the IgG level in the compound group was significantly decreased (P < 0.05). These results indicate that the compound additive can improve the immune function of broilers.

[0062] Example 4: Effects of compound additives on the meat quality and flavor of broilers in the later stages of fattening;

[0063] Forty-five minutes post-slaughter, three points were selected on the right pectoral muscle, biceps femoris muscle, and gastrocnemius muscle of broilers. Using a meat color analyzer (TS7036, Guangdong Sanenshi Technology Co., Ltd.) and a pH meter (PH818M, Dongguan Wanchuang Electronic Products Co., Ltd.), the muscle brightness (L*), redness (a*), yellowness (b*), and pH value were measured three times, and the average value was taken as the final result. The measurement sites were the pectoral and leg muscles, and the measurement time was 24 hours post-slaughter. After the cooking loss was measured, the meat samples were cut into 1.0cm × 1.0cm × 3.0cm rectangular strips. A muscle tenderness analyzer was used for measurement, with the cutting direction perpendicular to the muscle fiber direction. The instrument blade cut the sample at a constant speed, and the maximum shear force required was recorded. Each sample was measured three times, and the average value was calculated as the final result. Twenty-four hours post-slaughter, 1.0cm × 3.0cm × 1.0cm pieces of the right pectoral and biceps femoris muscles were cut and trimmed to remove external fat and connective tissue. After weighing, the samples were suspended in 550 mL sealed, dry plastic bottles and placed in a 4 ℃ refrigerator. They were weighed again after 24 h and 48 h, and the drip loss rate at 24 h and 48 h was calculated. The percentage of drip loss was calculated using the formula: Drip loss (%) = (Before hanging the meat sample - After hanging the meat sample) / Before hanging the meat sample × 100. The effects of each experimental diet on the flavor of Longyou Ma chicken are shown in Table 6.

[0064] Table 6. Effects of compound additives on the flavor of Longyou Ma chicken.

[0065]

[0066] Note: Different lowercase letters in the superscript of data in the same row in the table indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0067] As shown in Table 6, compared with the control group, the compound group showed significantly improved a and b values ​​of breast muscle color at 45 min (P < 0.05). Compared with the Trifoliate Orange group, the compound group showed a significant increase in leg muscle shear force at 45 min (improving flavor, P < 0.05). Compared with the ε-polylysine hydrochloride group, the breast muscle shear force was significantly reduced (improving meat quality, P < 0.05). These results indicate that the compound additive can effectively improve the meat quality and flavor of broiler chickens.

[0068] Example 5: The effect of compound additives on the intestinal health of broilers in the later stages of fattening;

[0069] (1) Experimental design and detection methods;

[0070] As the main site of digestion and absorption and an important immune organ, the intestine is responsible for the body's normal nutrient digestion and absorption, immune regulation, and decomposition of toxic and harmful substances. Therefore, intestinal health is closely related to growth performance and immune function, hence this embodiment was designed.

[0071] At day 133, cecal contents samples were randomly selected from nine chickens in the composite group and sent to Shanghai MajorBio Biotechnology Co., Ltd. for 16S rRNA sequencing of gut microbiota. The sequencing region was V3-V4 (338F / 806R), the sequencing platform was Nextseq2000, and the species annotation database was silva138 / 16s_bacteria. ASV analysis, β-diversity analysis, community composition analysis, and species difference analysis of the gut microbiota from the chicken cecal contents were performed on the MajorBio Biotechnology website (https: / / www.majorbio.com). Functional prediction was performed using PICRUSt2 based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Welch's t-test was used to perform Welch's t-test on the predicted KEGG modules between different groups, and the results were plotted.

[0072] (2) Analysis of ASV and β diversity in gut microbiota;

[0073] Figure 1 The study showed a significant proportion of shared ASVs between the two groups, while each group also possessed a certain number of unique bacteria. Therefore, it is evident that the addition of the compound additive had some impact on the bacterial community composition but did not alter its core bacterial structure.

[0074] Figure 2Further analysis using PCoA based on the Bray-Curtis algorithm revealed no significant differences in gut microbial composition between the two groups. This indicates that the addition of the compound additive did not alter the β-diversity of the broiler gut microbiota.

[0075] (3) phylum-level gut microbiota community composition;

[0076] Figure 3 At the phylum level, both the CG group (control group) and the EG group (combined group) showed that Firmicutes and Bacteroidetes were the dominant phyla in their gut microbiota, accounting for the vast majority of relative abundance. Compared with the CG group, the relative abundance of Firmicutes was slightly decreased in the EG group, while the relative abundance of Bacteroidetes was slightly increased, resulting in a lower Firmicutes / Bacteroidetes (F / B) ratio in the EG group. Furthermore, Actinobacteriota, Desulfobacterota, Verrucomicrobiota, Synergistota, WPS-2, Proteobacteria, and Campilobacterota were present at low abundance in both groups, with no significant differences between the groups. This indicates that the gut microbiota structure at the phylum level is quite similar between the two groups, with only slight changes in the proportion of dominant phyla.

[0077] (4) It belongs to the horizontal gut microbiota community composition;

[0078] Figure 4At the genus level, both the CG and EG groups exhibited high complexity in their gut microbiota composition, consisting of multiple genera. Among these, *Bacteroides*, *Rikenellaceae_RC9_gut_group*, *Ruminococcus_torques_group*, *unclassified_f_Lachnospiraceae*, *Phascolarctobacterium*, and *Oscillospira* were the dominant genera. Compared to the CG group, the relative abundance of *Bacteroides* was slightly increased in the EG group, while some genera related to gut metabolism (such as *Rikenellaceae_RC9_gut_group* and *Phascolarctobacterium*) also showed an upward trend. Conversely, the relative abundance of some genera, such as *Ruminococcus_torques_group*, showed a slight decrease. Furthermore, the remaining low-abundance genera (such as Alistipes, Blautia, Faecalibacterium, Lactobacillus, Prevotellaceae_UCG-001, etc.) showed similar overall distributions between the two groups, with no significant structural differences observed. This indicates that the EG group exerted a certain degree of regulatory effect on the gut microbiota structure at the genus level, mainly manifested as slight changes in the proportion of dominant genera, while the overall community structure remained relatively stable.

[0079] (5) Species differentiation analysis at the genus level;

[0080] Figure 5 At the genus level, Sellimonas showed a significant difference between the CG and EG groups (Wilcoxon rank-sum test, P = 0.04203). Specifically, the relative abundance of Sellimonas in the CG group was significantly higher than that in the EG group, with its median and overall abundance also being higher. In contrast, the abundance of this genus in the EG group was generally lower, and the data distribution was more concentrated in the low-value range. Furthermore, the CG group showed greater inter-individual variability and contained outliers with higher values, while the EG group exhibited a relatively smaller range of variation. Therefore, the compound additive significantly reduced the relative abundance of Sellimonas in the gut microbiota.

[0081] Figure 6At the genus level, GCA-900066575 showed a significant difference between the CG and EG groups (Wilcoxon rank-sum test, P = 0.04203). Specifically, the relative abundance of GCA-900066575 in the CG group was significantly lower than that in the EG group, and its median and overall levels were also lower. In contrast, the abundance of this genus was generally higher in the EG group, and the data distribution was more concentrated in the high-value range. Furthermore, the EG group showed greater inter-individual variability and contained high-value outliers, while the CG group exhibited a relatively smaller range of variation. Therefore, the compound additive significantly increased the relative abundance of GCA-900066575 in the gut microbiota.

[0082] Figure 7 At the genus level, there was a significant difference in *Campylobacter* between the CG and EG groups (Wilcoxon rank-sum test, P = 0.04483). Specifically, the relative abundance of *Campylobacter* was slightly higher in the CG group than in the EG group, and some samples with high abundance values ​​(outliers) were observed in the CG group, while the abundance of this genus in the EG group was almost zero, and the overall distribution was more concentrated. Although the overall abundance of this genus was low in both groups, statistical analysis showed that the compound additive had a significant impact on the relative abundance of *Campylobacter*, significantly reducing it in the EG group. Therefore, the compound additive significantly reduced the relative abundance of *Campylobacter* in the gut microbiota.

[0083] (6) LEfSe multilevel species differential enrichment analysis;

[0084] The specific gut microbiota of the composite group and the control group were further analyzed using LeFSe, and the results are as follows: Figure 8 and Figure 9 As shown, the two groups exhibited significant taxonomic enrichment in terms of bacterial community differences. Based on the LDA score (LDA score > 3), the CG group showed significant enrichment in multiple differentially expressed bacterial communities, while the EG group showed enrichment in only a few. In the CG group, the significantly enriched bacterial communities mainly included:

[0085] Genus level: g__Sellimonas, g__Campylobacter;

[0086] Level: c__Campylobacteria;

[0087] Science level: f__Campylobacteraceae;

[0088] Target level: o__Campylobacterales;

[0089] Gate level: p__Campilobacterota;

[0090] The above results indicate that Campylobacter-related taxa were significantly enriched at multiple phylogenetic levels in the CG group, suggesting that this bacterial community is highly representative of the control group. In contrast, the EG group only showed significant enrichment of g__GCA-900066575 at the genus level. Therefore, compared to the CG group, the EG group intervention reduced the enrichment of Campylobacter-related bacterial communities and led to specific changes in the community structure.

[0091] (7) Determination of volatile fatty acids in the cecum;

[0092] The concentration of volatile fatty acids (VFAs) in the cecum was determined as follows: A cecum sample (0.5 g) was diluted with 1.5 mL of distilled water and then centrifuged at 12000 r / min for 10 min. Subsequently, 1 mL of the clear supernatant was mixed with 0.2 mL of 25% metaphosphoric acid solution. The mixture was incubated at 4 °C for 30 min and then centrifuged again at 12000 r / min for 10 min. Finally, 1 mL of the clear supernatant was filtered through a 0.22 μm membrane filter and transferred to a 2 mL gas chromatography vial. The concentrations of volatile fatty acids (acetic acid, butyric acid, valeric acid, propionic acid, isovaleric acid, and isobutyric acid) were determined using a capillary gas chromatography system (GC-2010 Pro) and a column of HPINNOWax (19091 n-133) and a capillary column (30 m × 0.25 mm × 0.25 μm). The effects of the compound additives on the volatile fatty acids of Longyou Ma chickens are shown in Table 7.

[0093] Table 7. Effects of compound additives on volatile fatty acids in Longyou Ma chickens

[0094]

[0095] Note: Different lowercase letters in the superscript of data in the same row in the table indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0096] As shown in Table 7, the volatile fatty acids produced in the cecal contents of broilers were mainly acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid. Compared with the control group, the content of acetic acid, propionic acid, and isovaleric acid in the compound group of broilers was significantly increased.

[0097] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention.

Claims

1. A composite additive, characterized in that, It includes powdered leaves of *Trifolium repens* and ε-polylysine hydrochloride; the powdered leaves of *Trifolium repens* are obtained by crushing the above-ground vine and leaf parts of the Vitaceae plant *Trifolium repens*.

2. A compound diet for broiler chickens in the late fattening stage, characterized in that, It includes the compound additive and basal diet as described in claim 1.

3. The compound diet for broiler chickens in the later stage of fattening according to claim 2, characterized in that, The compound additive contains 0.3-1.2% of *Ipomoea aquatica* leaf powder and 0.03-0.13% of ε-polylysine hydrochloride, based on a percentage of the total mass of the basal diet.

4. The compound diet for broiler chickens in the later stage of fattening according to claim 2, characterized in that, The basal diet comprises the following components by weight percentage: corn 65-75%, soybean meal 10-30%, wheat bran 2-10%, limestone powder 2-5%, soybean oil 2-5%, vitamin-mineral premix 1%, dicalcium phosphate 0.5-1.5%, choline chloride 0.2-1.0%, sodium chloride 0.1-0.5%, and DL-methionine 0.1-0.2%.

5. The compound diet for broiler chickens in the later stage of fattening according to claim 2, characterized in that, The crude protein content of the compound diet for broilers in the later stage of fattening is 14-20%; the metabolizable energy is 10-15%.

6. The application of the compound additive according to claim 1 or the compound diet for broiler chickens in the late fattening stage according to any one of claims 2 to 5 in the preparation of feed for improving the growth performance, immune function, meat flavor and intestinal health of broilers.

7. The application according to claim 6, characterized in that, Improving the growth performance includes: increasing average daily weight gain; increasing average daily feed intake.

8. The application according to claim 6, characterized in that, Improving the immune function includes: increasing the spleen index; increasing the bursa of Fabricius index; increasing serum TNF and IL-6 levels; and decreasing serum IL-10 and IgG levels.

9. The application according to claim 6, characterized in that, Improving the meat flavor includes: increasing the redness and yellowness values ​​of the breast muscle; increasing the shear force of the leg muscle; and decreasing the shear force of the breast muscle.

10. The application according to claim 6, characterized in that, Improving gut health includes: regulating the cecal gut microbiota structure; and increasing the levels of acetic acid, propionic acid, and isovaleric acid in the cecal contents.

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