Broiler compound feed additive and application thereof

Abstract

The application provides a broiler compound feed additive and application thereof, and belongs to the technical field of animal nutrition and feed additive. The broiler compound feed additive is prepared by mixing active ingredients, namely perilla flavonoids, L-glutamine and coated sodium butyrate, as a compound feed additive. By exerting the synergistic effects of the active ingredients, such as antioxidation, anti-inflammation, rapid repair of intestinal epithelium and tight connection, the broiler jejunum villus height is significantly improved, the crypt depth is reduced, the villus-crypt ratio is improved, the total antioxidant capacity and glutathione peroxidase activity of the jejunum are enhanced, the content of malondialdehyde is reduced, the activation of NF-kappa B, IL-1beta and TNF-alpha pro-inflammatory signals is inhibited, and the expression of the tight connection related genes ZO-2, Claudin-1 and Occludin of the jejunum is significantly up-regulated. The compound feed additive is safe and residue-free, can be applied to the preparation of feed, premix or medicine for enhancing the jejunum mucosal barrier function of broilers, and is suitable for the healthy breeding of broilers without or with reduced antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and feed additive technology, specifically relating to a compound feed additive for broilers that is particularly effective in enhancing the jejunal mucosal barrier. Background Technology

[0002] In intensive broiler production, the brooding and rearing stages are susceptible to multiple stressors, including high stocking density, temperature and humidity fluctuations, feed changes, and pathogenic microorganisms. This can lead to intestinal oxidative stress and excessive inflammation, resulting in damage to jejunal epithelial cells, loosening of tight junctions, decreased mucosal barrier function, or increased intestinal permeability. Ultimately, this can cause diarrhea, reduced feed conversion ratio, secondary infections, and decreased growth performance, severely hindering the profitability of broiler farming. Current technologies typically employ antibiotics or other drug treatments to address the problems caused by jejunal mucosal barrier damage, aiming to strengthen or restore the barrier. However, the extensive use of antibiotics and other intestinal repair drugs not only increases treatment costs but also leads to antibiotic or other drug residues in the broiler's body, significantly impacting broiler quality.

[0003] To address the aforementioned issues, existing technologies have opted to develop safer feed additives to enhance the jejunal mucosal barrier in broilers. For example, Chinese invention patent CN120514060A discloses a feed additive based on a ternary combination of olive leaf extract (oleuropein, luteolin, and crataegolic acid) that synergistically enhances broiler antioxidant stress resistance and its application. This additive utilizes specific active substances extracted from olive leaves to increase daily feed intake, reduce feed conversion ratio, improve broiler immunity, and reduce intestinal infections such as diarrhea. Therefore, developing safe, efficient, and residue-free antibiotic alternatives has become an urgent industry need. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned technical problems and provide a compound feed additive that is a safe, efficient, and residue-free antibiotic alternative. Another purpose of this invention is to provide the compound feed additive for use in broiler compound feed that enhances the jejunal mucosal barrier of broilers.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A compound feed additive whose active ingredients include perilla flavonoids, L-glutamine and coated sodium butyrate.

[0007] Furthermore, the mass ratio of the perilla flavonoids, L-glutamine and coated sodium butyrate is (1~5):(10~30):(10~30).

[0008] Furthermore, the mass ratio of the perilla flavonoids, L-glutamine, and coated sodium butyrate is 1:10:10.

[0009] Furthermore, the above-mentioned compound feed additive, based on a complete diet, has the following active ingredient concentrations: perilla flavonoids 50-200 mg / kg, L-glutamine 500-1500 mg / kg, and coated sodium butyrate 500-1500 mg / kg; preferably, perilla flavonoids 80-150 mg / kg, L-glutamine 800-1200 mg / kg, and coated sodium butyrate 800-1200 mg / kg.

[0010] Furthermore, the active ingredient concentrations are 100 mg / kg for perilla flavonoids, 1000 mg / kg for L-glutamine, and 1000 mg / kg for coated sodium butyrate.

[0011] Furthermore, the compound feed additive also includes a feed-grade acceptable carrier; the carrier is selected from one or more of silica, zeolite powder, bentonite, maltodextrin, corn starch, and dextrin.

[0012] The mass ratio of the carrier perilla flavonoids, L-glutamine, coated sodium butyrate and the carrier is (1~5):(10~30):(10~30):(40~70).

[0013] Furthermore, the compound feed additive is formulated as a powder, granule, capsule, microencapsulated form, or liquid; the powder is an adsorbent, coated, or microencapsulated powder.

[0014] Furthermore, the preparation method of the powder is as follows: weigh the perilla flavonoids, L-glutamine, coated sodium butyrate and carrier according to the formula and put them into a V-type mixer or a horizontal ribbon mixer, mix for 10~20 minutes until uniformly made into powder, and the compound jejunal mucosal barrier enhancing feed additive for broilers is obtained.

[0015] This invention provides the application of the above-mentioned compound feed additive in the preparation of feed, premix or medicine that enhances the jejunal mucosal barrier function of broilers.

[0016] This invention provides a complete feed, premix, or drug for broilers, comprising the above-mentioned compound feed additive for broilers.

[0017] This invention provides a complete broiler feed comprising a basal diet and the aforementioned compound broiler feed additive; the basal diet comprises the following components in parts by weight: corn 59.00~62.80; wheat bran 2.50~8.00; soybean meal 22.80~32.50; soybean oil 0.70~1.00; limestone powder 0.00~0.70; calcium bicarbonate 0.90~1.10; premix 3.00~5.00; the premix provides per kilogram VA 8000~12000 IU, VD3 2000~4000 IU, VE 20~40 mg, VK3 1~2 mg, VB1 1.0~3.0 mg, VB2 6.0~8.0 mg, niacin 20~40 mg, pantothenic acid 5~20 mg, VB6 3~4 mg, and biotin 0.1~0.4 mg. mg, folic acid 0.5~2mg, VB 12 0.01~0.03 mg, choline 400~600 mg, Fe 70~90 mg, Cu 7~9 mg, Zn 70~90 mg, Mn 70~90 mg, I 0.30~0.40 mg and Se 0.20~0.40 mg.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) Through research, this invention found that mixing the active ingredients perilla flavonoids, L-glutamine and sodium butyrate as a compound feed additive can exert the synergistic effect of each active ingredient, increase the antioxidant, anti-inflammatory, rapid intestinal epithelial repair and tight junction synergistic effects of single or two components, significantly increase the height of jejunal villi, reduce crypt depth, increase villi-crypt ratio, enhance the total antioxidant capacity and glutathione peroxidase activity of the jejunum, reduce malondialdehyde (MDA) content, inhibit the activation of NF-κB, IL-1β and TNF-α pro-inflammatory signals, and significantly upregulate the expression of jejunal tight junction related genes ZO-2, Claudin-1 and Occludin.

[0020] (2) This compound feed additive is safe and residue-free. It can be used to prepare feed, premix or drugs that enhance the jejunal mucosal barrier function of broilers. It is suitable for antibiotic-free and antibiotic-reduced healthy breeding of broilers. Attached Figure Description

[0021] Figure 1 shows the effect of the composite additive of the present invention on the morphology of broiler small intestine tissue.

[0022] Figure 2 shows the effects of the compound additive of the present invention on the antioxidant capacity, immunoglobulins and immune factors of broiler jejunum.

[0023] Figure 3 shows the effect of the compound additive of the present invention on the relative expression levels of genes related to tight junctions in the jejunum of broilers. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] Example 1: Broiler Compound Feed Additive and its Preparation

[0026] This embodiment provides a compound feed additive whose active ingredients include perilla flavonoids, L-glutamine, and coated sodium butyrate. The specific preparation method is as follows: Weigh out 2 parts of perilla flavonoids (purity ≥95%), 25 parts of L-glutamine, 25 parts of coated sodium butyrate, 25 parts of maltodextrin, and 23 parts of silica. Mix them in a V-type mixer for 15 minutes to make a uniform powder, which is the compound jejunal mucosal barrier enhancing feed additive for broilers.

[0027] Example 2: Broiler Compound Feed Additive and its Preparation

[0028] This embodiment provides a compound feed additive whose active ingredients include perilla flavonoids, L-glutamine, and coated sodium butyrate. The specific preparation method is as follows: Weigh out 3 parts of perilla flavonoids (purity ≥95%), 25 parts of L-glutamine, 25 parts of coated sodium butyrate, 25 parts of maltodextrin, and 23 parts of silica. Mix them in a V-type mixer for 15 minutes to make a uniform powder, which is the compound jejunal mucosal barrier enhancing feed additive for broilers.

[0029] Example 3: Broiler Compound Feed Additive and its Preparation

[0030] This embodiment provides a compound feed additive whose active ingredients include perilla flavonoids, L-glutamine, and coated sodium butyrate. The specific preparation method is as follows: Weigh out 5 parts of perilla flavonoids (purity ≥95%), 30 parts of L-glutamine, 30 parts of coated sodium butyrate, and 70 parts of maltodextrin. Mix them in a V-type mixer for 15 minutes to make a uniform powder, which is the compound jejunal mucosal barrier enhancing feed additive for broilers.

[0031] Example 4

[0032] 1. Experimental Methods

[0033] (1) Experimental design

[0034] Nine hundred one-day-old Arbor Acqua (AA) roosters were randomly divided into 15 groups, with 6 replicates per group and 10 roosters per replicate: 1) CON group: basal diet; 2) PF group: basal diet + 100 mg / kg perilla flavonoids; 3) Gln group: basal diet + 1000 mg / kg L-glutamine; 4) SB group: basal diet + 1000 mg / kg coated sodium butyrate; 5) Two-group PF + Gln group: basal diet + 100 mg / kg perilla flavonoids + 1000 mg / kg L-glutamine; 6) Two-group PF + SB group: basal diet + 100 mg / kg perilla flavonoids + 1000 mg / kg coated sodium butyrate; 7) Ratio A group (1:1:1): basal diet + 100 mg / kg perilla flavonoids + 100 mg / kg L-glutamine + 100 mg / kg perilla flavonoids + 1000 mg / kg coated sodium butyrate; 8) Group B (1:5:5): Basal diet + 100 mg / kg perilla flavonoids + 500 mg / kg L-glutamine + 500 mg / kg coated sodium butyrate; 9) Optimal ratio group (1:10:10): Basal diet + 100 mg / kg perilla flavonoids + 1000 mg / kg L-glutamine + 1000 mg / kg coated sodium butyrate; 10) High ratio group (1:20:20): Basal diet + 100 mg / kg perilla flavonoids + 2000 mg / kg L-glutamine + 2000 mg / kg coated sodium butyrate; 11) Perilla flavonoid replacement group (PF replacement group): Basal diet + 100 mg / kg quercetin + 1000 mg / kg perilla flavonoids ... 12) Glutamine replacement group (Gln replacement group): basal diet + 100 mg / kg perilla flavonoids + 1000 mg / kg L-alanine + 1000 mg / kg coated sodium butyrate; 13) Coated sodium butyrate replacement group (SB replacement group): basal diet + 100 mg / kg perilla flavonoids + 1000 mg / kg L-glutamine + 1000 mg / kg ordinary sodium butyrate; 14) Three-component same-type replacement group (three-component replacement group): basal diet + 100 mg / kg quercetin (replacing perilla flavonoids) + 1000 mg / kg L-alanine (replacing L-glutamine) + 1000 mg / kg ordinary sodium butyrate (replacing coated sodium butyrate); 15) Same-type replacement group (basal diet + 100 mg / kg quercetin + 1000 mg / kg L-alanine + 1000 mg / kg coated sodium butyrate); mg / kg coated sodium butyrate (simultaneously replacing perilla flavonoids and L-glutamine, while retaining coated sodium butyrate)). The experiment lasted 120 days, with free access to feed and water, and routine immunization and feeding management. The basal diet ratio is shown in Table 1.

[0035] Table 1. Composition and nutrient levels of the basal diet (%)

[0036] Table 1, A, indicates that the premix provides the following per kilogram of diet in the early stage: Vitamin A 10000 IU, Vitamin D3 3000 IU, Vitamin E 30 mg, Vitamin K3 1.5 mg, Vitamin B1 2.0 mg, Vitamin B2 7.0 mg, Niacin 30 mg, Pantothenic Acid 10 mg, Vitamin B6 3.5 mg, Biotin 0.25 mg, Folic Acid 1.0 mg, and Vitamin B1... 12 0.02 mg, choline 500 mg, Fe 80 mg, Cu 8 mg, Zn 80 mg, Mn 80 mg, I 0.35 mg, and Se 0.30 mg. B indicates that the premix provides per kilogram of diet in the later stages: VA 10000 IU, VD3 3000 IU, VE 25 mg, VK3 1.5 mg, VB1 2.0 mg, VB2 7.0 mg, niacin 30 mg, pantothenic acid 10 mg, VB6 3.0 mg, biotin 0.24 mg, folic acid 1.0 mg, and VB... 12 0.02 mg, choline 450 mg, Fe 80 mg, Cu 8 mg, Zn 80 mg, Mn 80 mg, I 0.40 mg, and Se 0.30 mg. ME and AP were calculated with reference to the "Chinese Feed Composition and Nutrition Value Table" (31st edition, 2020), while CP, Ca, and TP were measured values.

[0037] (2) Measurement indicators

[0038] 1) Growth performance: Average daily feed intake (ADFI), average daily weight gain (ADG), and feed conversion ratio (F / G) were measured. On days 1, 42, and 120 of the experiment, the weight of broilers in each replicate was measured on an empty stomach in the morning. The amount of feed fed and the amount of feed remaining in each replicate were recorded at the same time each day. If mortality occurred, the weight of the deceased chickens was recorded, and feed intake and weight gain were adjusted accordingly. The calculation formula is as follows: Replicas are used as the statistical unit.

[0039] ADFI = Feed consumption during the trial period / (Number of surviving chickens × Number of trial days);

[0040] ADG = (Ending body weight - Beginning body weight) / (Number of surviving chickens × Number of days in the experiment).

[0041] F / G = Material consumption during the test period / Weight gain during the test period.

[0042] 2) Intestinal morphology: Jejunal villus height, crypt depth, and villus-to-crypt ratio were measured. On day 120 of the experiment, one broiler chicken close to the average weight of that replicate was selected from each replicate. After fasting for 12 hours, the chicken was sacrificed, and a 2 cm segment of the mid-jejunum was harvested. The contents were gently rinsed with pre-cooled physiological saline and fixed in 4% paraformaldehyde for at least 24 hours. After dehydration with graded ethanol, clearing with xylene, paraffin embedding, sectioning (approximately 5 μm thick), and HE staining, images were acquired under an optical microscope. For each sample, 10 intact villi and 10 intact crypts were selected. The distance from the villus tip to the crypt opening was measured using image analysis software as villus height, and the distance from the crypt opening to the crypt bottom was measured as crypt depth. The villus-to-crypt ratio was calculated using the following formula:

[0043] The ratio of the number of fibers to the number of crypts is calculated as: fiber height / crypt depth.

[0044] 3) Antioxidant indicators: Total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content of jejunal mucosa were measured. The mid-segment of the jejunum was harvested and the mucosa scraped, flash-frozen in liquid nitrogen, and stored at -80 °C. Before testing, a homogenate was prepared by mixing tissue mass with pre-cooled physiological saline at a ratio of 1:9 (g / mL), centrifuged at 4 °C and 3500 r / min for 10 min, and the supernatant was collected. Colorimetric detection was performed using commercially available kits according to the manufacturer's instructions. Protein concentration was determined using the BCA method. T-AOC and GSH-Px results are expressed as U / mg protein, and MDA results are expressed as nmol / mg protein.

[0045] 4) Inflammatory factors: The levels of NF-κB, IL-1β, and TNF-α in jejunal mucosa were measured. The supernatant of the homogenate from the above jejunal mucosa was used to detect the levels of NF-κB, IL-1β, and TNF-α using a chicken-derived ELISA kit. All samples and standards were tested in parallel wells. A standard curve was plotted based on the absorbance at 450 nm using an ELISA reader, and the sample concentration was calculated. The results were corrected for protein content and expressed as pg / mg prot.

[0046] 5) Tight junction genes: The relative expression levels of ZO-1, ZO-2, Claudin-1, and Occludin in jejunal mucosa were determined. Jejunal mucosa samples were collected, and total RNA was extracted using the TRIzol method. After determining the RNA concentration and purity, it was reverse transcribed into cDNA. Using β-actin as an internal control, the expression of the target genes was detected by SYBR Green real-time quantitative PCR. The reaction system was 20 μL, and the program was 95℃ pre-denaturation for 30 s, followed by 95℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles. Melting curve analysis was performed to confirm amplification specificity. Each sample was tested in triplicate, and the relative expression levels were calculated using the 2^-ΔΔCt method.

[0047] 6) Statistical analysis: Data were organized with repetitions as the statistical unit. One-way ANOVA was used to compare the differences between the treatment groups, and Duncan's method was used for multiple comparisons. The results are expressed as mean values. P<0.05 was considered statistically significant, and P<0.01 was considered extremely statistically significant.

[0048] 2. Experimental Results

[0049] (1) Effect of composite additives on growth performance

[0050] As shown in Table 2, the results of the effect of the compound additive on growth performance showed that the optimal ratio group (1:10:10) had significantly higher body weight at 120 days, ADG at 43-120 days, and ADG at 1-120 days than other treatment groups, and a significantly lower F / G ratio at 43-120 days (P<0.01). The single-component group showed improvement compared to the CON group, and the two-component compound group was generally better than the single-component group. Among the three-component groups, the ratios A (1:1:1) and B (1:5:5) were lower than the optimal ratio group. Although the high ratio group (1:20:20) maintained a certain improvement effect, it did not further improve to the optimal ratio level, indicating that there is a dosage window for the three components. When perilla flavonoids, L-glutamine, or coated sodium butyrate were replaced individually by similar components, the growth performance was lower than that of the optimal ratio group. The effect further decreased after all three components were replaced simultaneously, indicating that the combination of the core components of this invention has a synergistic effect that cannot be easily replaced.

[0051] (2) Effects of compound additives on intestinal morphology

[0052] The effects of compound additives on intestinal morphology are as follows: Figure 1 As shown, the optimal formulation group had significantly higher jejunal villus height and villus-to-crypt ratio than other groups, while the crypt depth was relatively lower (P<0.05). The two-component combination group had higher values ​​than the single-component group, while the villus-to-crypt ratios of formulations A, B, and the same substitution group were all lower than the optimal formulation group. These results indicate that perilla flavonoids, L-glutamine, and sodium butyrate can synergistically promote the restoration of jejunal mucosal structural integrity at a specific mass ratio, and deviating from the optimal ratio or replacing the core component weakens this effect.

[0053] (3) Compound additives have antioxidant and immune indicators

[0054] The effects of compound additives on antioxidant and immune indicators are as follows: Figure 2As shown, the optimal ratio group showed significantly increased jejunal T-AOC and GSH-Px activities and significantly decreased MDA content (P<0.05), with better antioxidant improvement than the single-component group, the two-component group, other ratio groups, and the replacement group. No significant differences were found among the groups in immunoglobulins or immune factors such as IgA, IgG, IgM, and IL-2, suggesting that this compound additive mainly enhances the jejunal mucosal barrier by improving local antioxidant capacity and reducing oxidative damage, rather than significantly altering the basic immunoglobulin levels.

[0055] (4) Compound additives on anti-inflammatory markers and tight junction gene expression

[0056] The effects of compound additives on anti-inflammatory markers and tight junction gene expression are as follows: Figure 2 and Figure 3 ,like Figure 2 As shown, the optimal formulation group exhibited significantly reduced levels of NF-κB, IL-1β, and TNF-α (P<0.05), indicating its ability to inhibit jejunal pro-inflammatory signaling activation; Figure 3 As shown, the relative expression levels of ZO-2, Claudin-1, and Occludin were significantly upregulated in the optimal ratio group (P<0.01), with ZO-1 also showing an upregulation trend, while Claudin-2 showed no significant change. The upregulation levels in the two-component combination group, other ratio groups, and replacement group were all lower than those in the optimal ratio group, further demonstrating that the three core active ingredients have a synergistic advantage in repairing jejunal tight junctions at a ratio of 1:10:10.

[0057] Table 2. Effects of dietary compound additives on broiler growth performance

[0058] Note: Different superscript letters in the same data indicate significant differences (P<0.05); SEM represents the standard error.

[0059] Conclusion: The active substances of this invention exhibit significant synergistic effects, with the optimal three-component ratio showing the best effect > two-component combination > single component. The ratio significantly affects the effect, with 100+1000+1000 mg / kg being the optimal ratio. Deviating from this ratio at low and high doses reduces the effect, but it is still better than the effect without this component. The core component is irreplaceable; replacing perilla flavonoids, L-glutamine, or coated sodium butyrate individually with similar components weakens the effect, and replacing all three components simultaneously further deteriorates the effect. In summary, this compound additive can comprehensively enhance the jejunal mucosal barrier function of broilers by improving intestinal morphology, enhancing antioxidant capacity, inhibiting inflammatory factors, and upregulating the expression of tight junction-related genes.

[0060] 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 the solutions. 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 be made to the technical solutions of the present invention based on the understanding of the solutions, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the protection scope of the present invention.

Claims

1. A compound feed additive, characterized in that, The active ingredients include perilla flavonoids, L-glutamine, and coated sodium butyrate.

2. The compound feed additive according to claim 1, characterized in that, The mass ratio of perilla flavonoids, L-glutamine and coated sodium butyrate is (1~5):(10~30):(10~30).

3. The compound feed additive according to claim 2, characterized in that, The mass ratio of perilla flavonoids, L-glutamine, and coated sodium butyrate is 1:10:

10.

4. The compound feed additive according to claim 1, characterized in that, Based on the complete feed, the mass concentration of the active ingredients is 50-200 mg / kg of perilla flavonoids, 500-1500 mg / kg of L-glutamine, and 500-1500 mg / kg of coated sodium butyrate; preferably, the mass concentration of the active ingredients is 80-150 mg / kg of perilla flavonoids, 800-1200 mg / kg of L-glutamine, and 800-1200 mg / kg of coated sodium butyrate.

5. The compound feed additive according to claim 4, characterized in that, Based on the complete feed, the active ingredient concentrations are 100 mg / kg for perilla flavonoids, 1000 mg / kg for L-glutamine, and 1000 mg / kg for coated sodium butyrate.

6. The compound feed additive according to claim 1, characterized in that, The compound feed additive also includes a feed-grade acceptable carrier; the carrier is selected from one or more of silica, zeolite powder, bentonite, maltodextrin, corn starch, and dextrin.

7. The compound feed additive according to claim 1, characterized in that, The compound feed additive is formulated as a powder, granule, capsule, microencapsulated form, or liquid; the powder is an adsorbent, coated, or microencapsulated powder.

8. The use of the compound feed additive according to any one of claims 1 to 7 in the preparation of feed, premix or medicine that enhances the jejunal mucosal barrier function of broilers.

9. A complete feed, premix, or medicine for broilers, characterized in that, It includes the broiler compound feed additive as described in any one of claims 1 to 7.

10. A complete feed for broilers, characterized in that, The feed includes a basal diet and the broiler compound feed additive as described in any one of claims 1 to 7; the basal diet comprises the following components in parts by weight: corn 59.00-62.80; wheat bran 2.50-8.00; soybean meal 22.80-32.50; soybean oil 0.70-1.00; limestone 0.00-0.70; calcium bicarbonate 0.90-1.10; and premix 3.00-5.00; the premix provides per kilogram of VA 8000-12000 IU, VD3 2000-4000 IU, VE 20-40 mg, VK3 1-2 mg, VB1 1.0-3.0 mg, VB2 6.0-8.0 mg, niacin 20-40 mg, pantothenic acid 5-20 mg, VB6 3-4 mg, biotin 0.1-0.4 mg, folic acid 0.5-2 mg, and VB... 12 0.01~0.03 mg, choline 400~600 mg, Fe 70~90 mg, Cu 7~9 mg, Zn 70~90 mg, Mn 70~90 mg, I 0.30~0.40 mg and Se 0.20~0.40 mg.

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