Feed additive for improving growth and bone health of broiler chickens in chronic stress state and application of feed additive

Abstract

The invention belongs to the technical field of poultry feed additives, provides a feed additive for improving growth and skeleton health of broiler chickens in a chronic stress state and application of the feed additive, and particularly relates to new application of the feed additive in broiler chicken feed additives with high-density stress and heat stress. And the used lipoic acid and chromium methionine are safe, free of drug resistance, free of adverse reaction and low in price. The feed additive comprises lipoic acid and chromium methionine, and the addition amount of the lipoic acid is 300mg / kg; and chromium methionine is 400 [mu] g / kg. The additive can influence the bone quality and improve the bone breaking strength, the bone-body ratio and the weight of the heat stress broiler chicken. The additive can improve the immune organ index of the broiler chicken and improve the relative weight of spleen, thymus and abdominal fat. The feed additive can improve the oxidation resistance and immune performance of the broiler chickens under high-density stress and heat stress, relieve bone injury, and possibly protect bone health of the broiler chickens by removing free radicals in the body and maintaining calcium and phosphorus metabolic balance in the body.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry feed additives, and particularly relates to a feed additive for improving the growth and bone health of broilers in a chronic stress state and an application thereof. Background Art

[0002] In the breeding and production of broilers, environmental factors have always been the focus of attention in actual production. Among the many environmental factors, stocking density and temperature are crucial. With the rapid development of my country's poultry farming industry, the stocking density of broiler farming has continued to increase. Although this breeding model can increase broiler production to a certain extent, it ignores the health of individual broilers. Such a breeding model is very likely to cause high-density stress in broilers. Secondly, poultry itself lacks sweat glands and is extremely sensitive to temperature changes. Especially in high temperatures in summer, broilers are very prone to heat stress, which leads to imbalance in the homeostasis of broiler bodies. In severe cases, it can also lead to the death of poultry, causing huge economic losses to farmers. Both density stress and heat stress are chronic stress, which will lead to a decrease in broiler growth performance, immunity and antioxidant activity, leading to oxidative stress in the body, reducing bone strength, and causing bone metabolism disorders.

[0003] The stocking density of broiler chickens can be defined based on the number of chicks per chicken per square meter. In actual production, the normal stocking density range of broiler chickens is 30-35 kg / m 2 Or 14-16 / m 2 The general high stocking density is 39kg / m 2 Or 18 / m 2 Density stress can reduce the feed intake and body weight of broilers, increase feed consumption to weight gain ratio, reduce production performance, reduce antioxidant capacity, and affect bone quality.

[0004] High-density rearing affects the bone quality of broilers, such as increased tibial curvature and reduced tibial fracture strength due to insufficient rearing space, which may lead to a series of animal welfare issues such as lameness. Stress in broilers activates the hypothalamus-pituitary-adrenal (HPA) axis, thereby increasing the synthesis of adrenal corticosterone (CORT). Excessive corticosteroids affect bone quality by promoting osteoclast proliferation, inhibiting osteoblast osteogenesis, and reducing bone density.

[0005] Lipoic acid (LA) is known as the "universal antioxidant" and is the most effective natural antioxidant. LA is a dithiol compound produced by mitochondria and widely found in animals and plants. It has multiple biological functions such as scavenging free radicals, resisting oxidative stress, and regulating immune response. LA can mediate the secretion of thyroid hormones and calcitonin in broilers, regulate calcium metabolism in the body, and thus affect bone quality.

[0006] Chromium has strong chemical stability and strong antioxidant activity, and is considered the preferred mineral in poultry diets. Chromium methionine has the advantages of low toxicity and high absorption rate, which can alleviate the decline in production performance, immune function and bone quality of animals under stress. Summary of the invention

[0007] The purpose of the present invention is to provide a feed additive for improving the growth and bone health of broilers in a chronic stress state and an application thereof, especially a new application in a feed additive for broilers under high-density stress and heat stress. The lipoic acid and chromium methionine used are safe, have no drug resistance, have no adverse reactions, and are low in price.

[0008] The present invention is realized by the following technical scheme: a feed additive for improving the growth and bone health of broilers in a chronic stress state, wherein the feed additive comprises lipoic acid and chromium methionine, and the added amount is 300 mg / kg of lipoic acid and 400 ug / kg of chromium methionine.

[0009] The present invention also provides the use of the feed additive for improving the growth and bone health of broilers in a chronic stress state in feed or medicine for improving the antioxidant capacity and immunity of broilers in a heat stress and / or high density stress state.

[0010] The present invention also provides the use of the feed additive for improving the growth and bone health of broilers in a chronic stress state in feed or medicine for improving the bone health of broilers in a heat stress and / or high-density stress state.

[0011] Furthermore, the feed additive is used in feed or medicine for removing free radicals in the body, maintaining calcium-phosphorus metabolic balance in the body, and improving the bone health of broilers.

[0012] Furthermore, the feed additive is used in feed or medicine to improve the immune organ index and bone quality of broilers, improve the transport capacity of small intestine for calcium and phosphorus ions, and alleviate bone damage.

[0013] The additive of the present invention can affect the bone quality, improve the bone breaking strength, bone-to-body ratio and weight of heat-stressed broilers. The additive of the present invention can improve the immune organ index of broilers and increase the relative weight of spleen, thymus and abdominal fat. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The effect of high-density stress on the key genes for small intestinal calcium absorption in broiler chickens; in the figure: A is the relative expression of CaBP-D28k mRNA, B is the relative expression of PMCA1b mRNA, C is the relative expression of NCX1 mRNA, and D is the relative expression of NaPi-Ⅱb mRNA; Figure 2The effect of high-density heat stress on key genes for small intestinal calcium absorption in broiler chickens; in the figure: A is the relative mRNA expression level of PMCA1b, B is the relative mRNA expression level of NaPi-Ⅱb, C is the relative mRNA expression level of NCX1, and D is the relative mRNA expression level of CaBP-D28k. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0017] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0018] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0019] 1. Effects of lipoic acid and chromium methionine supplementation in feed on growth and bone health of broilers under high density 1. Experimental animals and experimental design: 444 AA + Broilers were fed uniformly from 0 to 21 days of age. At 21 days of age, chickens of similar body weight were randomly divided into 4 groups and evenly distributed to 4 feeding houses. Six replicates were raised in each house. The relative humidity of each treatment group was 50%-60%. The experimental period was 28 to 42 days of age. The 4 groups were normal density group (LSD), high density group (HSD), high density + lipoic acid group (HSD+LA) and high density + lipoic acid + chromium methionine group (HSD+LA+Cr-Met), with 6 replicates in each group; each replicate of the normal density group had 14 chickens fed with a basal diet; each replicate of the high density group had 20 chickens fed with a basal diet; the HSD+LA group and the HSD+LA+Cr-Met group were fed with 300 mg / kg lipoic acid and 300 mg / kg lipoic acid + 400 μg Cr / kg chromium methionine, respectively, added to the diet based on high-density feeding.

[0020] The experimental period was from 28 to 42 days of age. The experimental animals were caged and had free access to food and water. The experimental broilers were weighed after fasting for 8 hours at 28 and 42 days of age, and the feed consumption was counted. The total feed intake, average daily weight gain and feed consumption-to-weight ratio from 22 to 48 days of age were calculated. At 42 days of age, venous blood was collected, and serum was separated by centrifugation at 3000r / min and stored at -20℃ for the detection of serum calcium, phosphorus, alkaline phosphatase (ALP), parathyroid hormone, calcitonin and other indicators. The chickens after venous blood collection at 42 days of age were slaughtered and the left tibia was taken to measure the mass, length, diameter, strength, and calculate the bone-to-body ratio. The length and diameter were measured with a vernier caliper; the three-point bending test was used to detect bone strength.

[0021] The composition and nutritional level of the basal diet for broiler chickens are shown in Table 1.

[0022] Table 1: Composition and nutritional level of basal diet for broiler chickens (air-dry basis, %) 1: Trace mineral premix provided per kilogram of diet: copper 16 mg; zinc, 110 mg; iron, 80 mg; manganese, 120 mg; selenium, 0.3 mg; iodine, 1.5 mg; cobalt, 0.5 mg.

[0023] 2: Vitamin premix provided per kilogram of daily diet: Vitamin A, 10000IU; Vitamin D3, 2400IU; Vitamin E, 20mg; Vitamin K3, 2mg; Vitamin B1, 2mg; Vitamin B2, 6.4mg; Vitamin B6, 3mg; Vitamin B12, 0.02mg; Biotin, 0.1mg; Folic acid, 1mg; Pantothenic acid, 10mg; Niacinamide, 30mg.

[0024] 3: Metabolizable energy is a calculated value, others are analyzed values.

[0025] 2. Measurement indicators and methods A. Growth performance: The experimental broilers were weighed after fasting for 8 hours at 28 and 42 days of age, and the feeding amount and residual feed weight were counted. The total feed intake, average daily weight gain and feed consumption-weight gain ratio during the period of 22-48 days of age were calculated.

[0026] B. Serum indexes: At 42 days of age, 10 mL of blood was collected from the wing vein of a broiler chicken with a body weight close to the average from each replicate, centrifuged at 3000 r / min for 10 min, and the upper serum was collected and stored at -20°C. The following indexes were tested: total antioxidant capacity (T-AOC), corticosterone, serum calcium, phosphorus, alkaline phosphatase (ALP), and parathyroid hormone. The above kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and all the above determination steps were strictly carried out according to the instructions. Serum insulin and calcitonin were measured by Shanghai Enzyme-linked Immunoassay (Shanghai, China) (ELISA test).

[0027] C. Determination of bone indexes: After collecting venous blood at 42 days of age, the left tibia was slaughtered and the mass, length, diameter and strength were measured. The cartilage, fat, muscle tissue and other attachments on the bone were carefully removed, the fresh weight of the tibia was measured, and the bone weight rate (BWR) was calculated. The length and diameter were measured with a vernier caliper. The three-point bending test was used to test the bone strength.

[0028] D. Key genes for small intestinal calcium absorption: Primer sequences are shown in Table 2.

[0029] Table 2: Primer sequences and parameters E. Data statistics and result analysis: The experimental data were statistically analyzed using SPSS26.0 software, and the normal density group and high density group were analyzed by independent sample t test. One-way analysis of variance (ANOVA) was performed for high density, high density plus lipoic acid, and lipoic acid + chromium methionine. Tukey multiple comparisons were used to analyze the differences between the groups, and P was considered significant.

[0030] 3. Results and analysis A. Effect of adding lipoic acid and chromium methionine to feed on the production performance of high-density broilers: The results are shown in Table 3. The results show that compared with normal density, high density significantly reduced the body weight at 42 days of age, body weight gain at 28-42 days of age and feed intake of broilers (P<0.05); and significantly reduced the feed consumption to weight gain ratio (P<0.05). Adding lipoic acid and chromium methionine had no significant effect on the average body weight at 42 days of age, total weight gain at 28-42 days of age, and total feed intake at 28-42 days of age (P>0.05).

[0031] Table 3: Effects of lipoic acid and chromium methionine on the production performance of 42-day-old broilers under high density * indicates the independent sample t-test results between the LSD group and the HSD group (*P<0.05).

[0032] B. Effects of adding lipoic acid and chromium methionine to feed on organ indexes of high-density broilers: The results are shown in Table 4. The results show that compared with normal density, high density significantly increased the bursa index (P<0.05); significantly reduced the abdominal fat index (P<0.05); adding lipoic acid to feed significantly increased the relative weight of spleen, thymus and abdominal fat of high-density broilers (P<0.05); compared with high-density controls, the mixed addition of lipoic acid and chromium methionine significantly increased the relative weight of bursa, abdominal fat and thymus (P<0.05).

[0033] Table 4: Effects of lipoic acid and chromium methionine supplementation on organ indices of 42-day-old broilers raised at high density * indicates the independent sample t-test results of the LSD group and the HSD group (*P<0.05). ac indicates the ANOVA analysis results of the HSD group, the HSD+LA group and the HSD+LA+Cr-Met group. Different lowercase letters in the same row indicate significant differences (P<0.05).

[0034] C. Effects of adding lipoic acid and chromium methionine to feed on serum glucose and lipid metabolism indices of high-density broilers: The results are shown in Table 5. It can be seen from Table 5 that compared with normal density, high density significantly increased serum triglyceride content (P<0.05); serum insulin in the high-density + lipoic acid + chromium methionine group was significantly higher than that in the high-density group and high-density + lipoic acid group; the other indicators were not significant (P<0.05).

[0035] Table 5: Effects of lipoic acid and chromium methionine on serum biochemical parameters of 42-day-old broilers under high density * indicates the independent sample t-test results of the LSD group and the HSD group (*P<0.05). ab indicates the ANOVA analysis results of the HSD group, the HSD+LA group, and the HSD+LA+Cr-Met group. Data in the same column with different lowercase letters indicate significant differences (P<0.05).

[0036] D. Effect of adding lipoic acid and chromium methionine to feed on serum antioxidant indices of high-density broilers: The results are shown in Table 6. The results showed that compared with normal density, high density significantly increased the serum corticosterone content (P<0.05); the total serum antioxidant capacity of the lipoic acid and lipoic acid + chromium methionine groups was significantly higher than that of the normal temperature high-density group (P<0.05), and the lipoic acid group and the lipoic acid + chromium methionine group could significantly reduce the serum corticosterone content (P<0.05).

[0037] Table 6: Effects of lipoic acid and chromium methionine on serum antioxidant indexes of 42-day-old broilers under high density E. Effects of adding lipoic acid and methionine chromium to feed on bone indexes of high-density broilers: The results are shown in Tables 7 and 8. It can be seen from Tables 7 and 8 that compared with normal density, high density significantly reduced the weight, fracture strength, bone density and serum calcium content of tibia (P<0.05), and significantly increased serum parathyroid hormone and calcitonin concentrations (P<0.05); adding lipoic acid or lipoic acid + methionine chromium under high density had no significant effect on serum phosphorus (P>0.05); the group with lipoic acid alone significantly increased serum calcium content compared with the other two groups (P<0.05). Compared with the high-density group, the weight and bone-to-body ratio of the high-density + lipoic acid and high-density + lipoic acid + methionine chromium groups showed an upward trend (P = 0.087), but the changes were not significant (P>0.05). Both the high density + lipoic acid and high density + lipoic acid + chromium methionine groups significantly increased the level of serum alkaline phosphatase and decreased serum parathyroid hormone (P<0.05), while the other indicators showed no significant changes (P>0.05).

[0038] Table 7: Effects of lipoic acid and chromium methionine on bone parameters of 42-day-old broilers under high density Table 8: Effects of lipoic acid and chromium methionine on bone metabolism indices of 42-day-old broilers under high density * indicates the results of independent sample t-test between LSD group and HSD group (*P<0.05). ac indicates the results of ANOVA analysis between HSD group, HSD+LA group and HSD+LA+Cr-Met group. Different lowercase letters in the same row indicate significant differences (P<0.05).

[0039] F. Effects of dietary lipoic acid and chromium methionine on key genes for small intestinal calcium absorption in broilers under high density conditions: Figure 1 As shown in the data, compared with the control group, high density significantly reduced the mRNA expression of NCX1 and NaPi-Ⅱb in the small intestine of broiler chickens (P<0.05); the addition of lipoic acid or a mixture of lipoic acid + methionine chromium could significantly increase the mRNA expression of PMCA1b (P<0.05), but had no significant effect on the expression of NCX1 (P>0.05); although the addition of lipoic acid or lipoic acid + methionine chromium could increase the mRNA expression of CaBP-D28k and PMCA1b in the small intestine, the difference was not significant (P>0.05).

[0040] The results showed that the addition of lipoic acid and chromium methionine in the feed had no positive effect on the growth performance of broilers under high-density stress. The immune organ index can be used to evaluate the immune function of broilers. The greater the absolute weight and relative weight, the stronger the cellular and humoral immune functions of the body. The high-density addition of lipoic acid in the present invention significantly increased the relative weight of the spleen and thymus. Under stress, the body can produce a large amount of glucocorticoids to activate glucocorticoid receptors and exert anti-inflammatory functions, thereby exerting the body's internal environment to resist the external stimulus environment; however, when the stress time is too long or the stress is excessive, the function of glucocorticoid receptors decreases, glucocorticoid resistance, and the body's inflammatory response and immune organ dysfunction. Therefore, the positive effects of lipoic acid and chromium (III) on the immune organ index may be due to the anti-inflammatory and antioxidant functions of the two; the addition of lipoic acid and a mixture of lipoic acid + chromium methionine can significantly increase the content of serum insulin, but has no positive effect on the skeletal muscle of broilers under oxidative stress.

[0041] The addition of lipoic acid and the lipoic acid + methionine chromium mixed group can improve the antioxidant capacity of serum, reduce serum corticosterone content and relieve stress. As a strong oxidant, lipoic acid can inhibit the production of free radicals, remove free radicals, reduce oxidative stress, and reduce oxidative damage. Lipoic acid and methionine chromium have strong antioxidant activity and can improve the antioxidant performance of stressed broilers.

[0042] In this experiment, the addition of lipoic acid and the mixture of lipoic acid and chromium methionine had no significant effect on serum phosphorus content. The reason should be that most of the phosphorus in serum exists in the form of inorganic salts and is not greatly affected by changes in blood pH. The significant increase in ALP in the lipoic acid group and the mixed group indicates that both lipoic acid and chromium methionine can promote bone calcification and formation. The secretion of PTH is determined by the calcium content in the blood. In this experiment, the high-density addition of lipoic acid significantly increased the serum calcium content, corresponding to the significant decrease in PTH content. In summary, the addition of lipoic acid can alleviate the effects of high-density stress on broiler bones.

[0043] In this study, high density significantly reduced the expression level of intestinal NaPi-IIb protein, and the fracture strength of high-density bones was significantly reduced, indicating that density stress affects the bone mineralization of broilers and affects bone quality. The addition of lipoic acid and chromium methionine significantly increased the expression level of NaPi-IIb protein, indicating that these two additives can significantly alleviate the effects of density stress on calcium and phosphorus absorption in broilers, maintain the body's normal bone metabolism, and improve the bone quality of broilers.

[0044] The present invention shows that adding lipoic acid and a mixture of lipoic acid + methionine chromium can improve the antioxidant capacity and immunity of high-density stress; high density significantly reduces the ability of the NCX1 gene of the small intestinal basement membrane of broilers to transport calcium ions and NaPi-Ⅱb to transport phosphorus ions, reduces the intestinal absorption of calcium and phosphorus transport, reduces blood phosphorus and thus affects tibia calcium and phosphorus deposition; adding lipoic acid or a mixture of lipoic acid + methionine chromium can significantly improve the ability of basement membrane PMCA1b to transport calcium ions; alleviate the impact of density stress on broiler calcium and phosphorus absorption, maintain the normal bone metabolism process of the body, and improve the bone quality of broilers. In summary, in actual production, the damage to bones caused by high-density stress in broilers can be alleviated by improving antioxidant properties.

[0045] Effects of dietary lipoic acid and chromium methionine supplementation on growth and bone health of broilers under high density and heat stress conditions 1. Experimental animals and experimental design: Based on the previous experiments, this experiment increased the ambient temperature to simulate the temperature in the broiler house in summer and cause heat stress to broilers. 480 AA+ broilers were selected and fed uniformly from 0 to 21 days of age. Chickens of similar weight were selected at 21 days of age and randomly divided into 4 groups. They were evenly distributed to 4 environmental control cabins, with 6 replicate groups in each cabin for feeding. The relative humidity of each treatment group was 50%-60%. The experimental period was 28 to 42 days of age. The temperature of the normal temperature cabin was maintained at 24 ± 1℃ every day, and the high temperature cabin was set at 32 ± 1℃ from 9:00 to 17:00 every day, and the rest of the time was restored to normal temperature of 24±1℃. The stocking density was 20 / m 2 The broilers in the treatment groups were fed a corn-soybean meal based diet supplemented with 300 mg / kg lipoic acid and 400 μg Cr / kg methionine chromium + 300 mg / kg lipoic acid, respectively.

[0046] The composition and nutritional content of the diet were the same as those described in the previous experiment.

[0047] 2. Measurement indicators and methods: A. Growth performance: The test broilers were weighed at 28 and 42 days of age after fasting for 8 hours, and the feeding amount and residual feed weight were counted. The total feed intake, average daily weight gain and feed consumption-weight gain ratio during the period of 22-48 days of age were calculated.

[0048] B. Serum indexes: At 42 days of age, 10 mL of blood was collected from the wing vein of a broiler chicken with a body weight close to the average from each replicate, centrifuged at 3000 r / min for 10 min, the upper serum was removed, and stored at -20°C for the following indexes: total antioxidant capacity (T-AOC), corticosterone, serum calcium, phosphorus, alkaline phosphatase (ALP), parathyroid hormone. The above kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and all the above determination steps were strictly carried out according to the instructions. Serum insulin and calcitonin were measured by Shanghai ELISA (Shanghai, China).

[0049] C. Sample collection and storage: Serum samples were collected and stored at -80°C for analysis of serum biochemical indicators. After blood sampling, broilers were bled to death through the carotid artery, slaughtered, and then dissected. The spleen, thymus, bursa of Fabricius, pectoral muscle, and abdominal fat were weighed to calculate organ indexes. Intestinal mucosal samples were collected and stored at -80°C for the determination of intestinal calcium absorption gene-related indicators; the left tibia was taken, the muscles and fascia attached to the tibia were removed, and the tibia was placed in a plastic bag and stored at -20°C for subsequent measurement of indicators such as breaking strength.

[0050] D. Determination of bone indexes: After collecting venous blood at 42 days of age, the left tibia was slaughtered and the mass, length, diameter and breaking strength were measured. The cartilage, fat, muscle tissue and other attachments on the bone were carefully removed, the fresh weight of the tibia was measured, and the bone weight rate (BWR) was calculated; the length and diameter were measured 3 times with a vernier caliper, and the average was taken as the final result; the three-point bending test was used to detect bone strength.

[0051] E. Key genes for small intestinal calcium absorption are shown in Table 2.

[0052] F. Data statistics and analysis methods: The experimental data were analyzed using SPSS 26.0 software. Independent t-tests were performed on high density and heat stress high density. One-way analysis of variance (ANOVA) was performed on heat stress high density, heat stress high density plus lipoic acid and methionine chromium. Tukey's multiple comparisons were used to analyze the differences between the groups. P < 0.05 was considered significant.

[0053] 3. Results and Discussion A. Effect of adding lipoic acid and chromium methionine to the diet under high-density conditions on the production performance of heat-stressed broilers: The results are shown in Table 9. It can be seen from Table 9 that the total feed intake of the heat-stressed high-density group during the 28-42 days of age was significantly lower than that of the normal temperature high-density group (P<0.05); there was no significant effect on the average body weight at 42 days of age, the total weight gain at 28-42 days of age, and the feed consumption-to-weight ratio (P>0.05); the addition of lipoic acid and chromium methionine had no significant improvement effect on the total feed intake at 28-42 days of age, the average body weight at 42 days of age, the total weight gain at 28-42 days of age, and the feed consumption-to-weight ratio (P>0.05).

[0054] Table 9: Effects of dietary lipoic acid and chromium methionine supplementation on the performance of heat-stressed broilers fed high density * indicates the results of independent sample t-test between HSD group and HSD+HS group (*P<0.05). ab indicates the results of ANOVA analysis between HS+HSD group, HS+HSD+LA group and HS+HSD+LA+Met-Cr group. Data in the same column with different lowercase letters indicate significant differences (P<0.05). Same below.

[0055] B. Effects of adding lipoic acid and chromium methionine to the diet under high-density conditions on the organ indexes of heat-stressed broilers: The results are shown in Table 10. It can be seen from Table 10 that the thymus index at normal temperature high density is significantly higher than that at heat stress high density (P<0.05); heat stress high density + lipoic acid and heat stress high density + lipoic acid and chromium methionine have significant improvements on the spleen, pectoral muscle, thymus and abdominal fat (P<0.05).

[0056] Table 10: Effects of dietary lipoic acid and chromium methionine supplementation on visceral indices of heat-stressed broilers under high density conditions Same as above.

[0057] C. Effect of adding lipoic acid and chromium methionine to the diet on the antioxidant capacity of heat stressed broilers under high-density conditions: The results are shown in Table 11. It can be seen that the serum corticosterone content in the normal temperature high-density group was significantly lower than that in the heat stress high-density group (P<0.05). The serum corticosterone content in the lipoic acid-added group and the lipoic acid + chromium methionine-added group both significantly decreased (P<0.05).

[0058] Table 11: Effects of dietary lipoic acid and chromium methionine on antioxidant parameters of heat-stressed broilers under high density conditions * indicates the results of independent sample t-test between LSD group and HSD group (*P<0.05). ab indicates the results of ANOVA analysis of HSD+HS group, HSD+HS+LA group and HSD+HS+LA+Cr-Met group. Different lowercase letters in the same row indicate significant differences (P<0.05). Same below.

[0059] D. Effect of adding lipoic acid and chromium methionine to the diet under high-density conditions on the glucose and lipid metabolism of heat-stressed broilers: The results are shown in Table 12. The serum triglyceride content in the heat-stress high-density group was significantly higher than that in the normal temperature high-density group (P<0.05), and the addition of lipoic acid and chromium methionine significantly reduced the triglyceride content in the group (P<0.05).

[0060] Table 12: Effects of dietary lipoic acid and chromium methionine on serum parameters of heat-stressed broilers under high density conditions Same as above.

[0061] E. Effects of adding lipoic acid and methionine chromium to the diet on bone indexes of heat-stressed broilers under high density conditions: The results are shown in Tables 13 and 14. The serum phosphorus content of normal temperature high density was higher than that of heat stress high density, but the difference was not significant (P>0.05). The serum phosphorus content of the groups with lipoic acid and lipoic acid and methionine chromium was significantly increased (P<0.05). Compared with the HD group, the bone mineral content, serum alkaline phosphatase and parathyroid hormone content of the HS+HSD group were significantly decreased (P<0.05). Although the weight, diameter, length and breaking strength of the HS+HD group showed a downward trend, there was no significant change (P>0.05). The univariate analysis of the three groups of HS+HSD, HS+HSD+lipoic acid, and HS+HSD+lipoic acid+methionine chromium showed that the tibia weight, breaking strength, bone-to-body ratio, bone mineral content, serum alkaline phosphatase and parathyroid hormone content were significantly increased (P<0.05); the bone length of the group with lipoic acid was significantly reduced (P<0.05).

[0062] Table 13: Effects of dietary lipoic acid and chromium methionine supplementation on bone appearance in heat-stressed broilers under high density conditions Same as above.

[0063] Table 14: Effects of dietary lipoic acid and chromium methionine on bone metabolic parameters in heat-stressed broilers fed high density * indicates the independent sample t-test results of the HSD group and the HSD+HS group (*P<0.05). ac indicates the ANOVA analysis results of the HS+HSD group, the HS+HSD+LA group, and the HS+HSD+LA+Met-Cr group. The data with different lowercase letters in the same row indicate significant differences (P<0.05).

[0064] F. Effects of dietary lipoic acid and chromium methionine on key genes for small intestinal calcium absorption in heat-stressed broilers under high-density conditions: Figure 2The results showed that compared with the high-density control group, heat stress significantly reduced the mRNA expression of PMCA1b, NCX1, and NaPi-Ⅱb in the small intestine of broiler chickens (P<0.05); while the addition of lipoic acid or a mixture of lipoic acid and methionine chromium could significantly increase the mRNA expression of CaBP-D28k, PMCA1b, and NaPi-Ⅱb (P<0.05). For the mRNA expression of NCX1, the addition of lipoic acid and methionine chromium could significantly increase its mRNA expression (P<0.05). Although the addition of lipoic acid alone showed an upward trend, the difference was not significant (P>0.05).

[0065] The results of the effects of adding lipoic acid and methionine chromium to the diet on the production performance and organ indexes of heat-stressed broilers under high-density conditions showed that compared with the high-density group, the heat-stressed high-density group had a further negative impact on the production performance of broilers; the addition of lipoic acid and the combination of lipoic acid + methionine chromium also had no significant effect on growth performance. The addition of lipoic acid and the co-addition of lipoic acid and methionine chromium significantly increased the spleen and thymus indexes, indicating that even under dual stress, lipoic acid and methionine chromium can still have a positive effect on the immune organs of broilers.

[0066] The effects of adding lipoic acid and chromium methionine to the diet under high-density conditions on the glucose and lipid metabolism of heat-stressed broilers indicate that oxidative stress can cause insulin resistance and lead to abnormal insulin secretion in the body. Double stress conditions will further affect the glucose metabolism in broilers.

[0067] In the present invention, heat stress has a tendency to increase blood sugar, but the difference is not significant, and the content of serum insulin is significantly increased; after adding lipoic acid, the glucose and insulin contents in the serum of broilers have no significant changes. It shows that under the conditions of this experiment, chronic heat stress may lead to an increase in serum glucose, but an increase in insulin content reduces the content of serum glucose, and an excessively high insulin content may also cause insulin resistance, leading to abnormal insulin secretion in the body and causing glucose metabolism disorders.

[0068] The effect of adding lipoic acid and chromium methionine to the diet on the bones of heat-stressed broilers under high-density conditions shows that compared with the heat-stressed high-density group, the addition of lipoic acid and the mixture of lipoic acid + chromium methionine can significantly increase the weight, length, breaking strength and bone-to-body ratio of broilers, indicating that the two additives are more effective under dual stress. The present invention shows that the addition of octanoic acid and the mixture of lipoic acid + chromium methionine can significantly increase the secretion of PTH in serum, but the serum calcium in this experiment has an increasing trend but the difference is not significant. This may be because lipoic acid and chromium methionine inhibit the osteofusion of osteoclasts, inhibit the entry of bone calcium into the blood, increase the activity of osteoblasts, strengthen the mineralization of bone matrix, and improve bone quality. Heat stress significantly reduced the activity of serum alkaline phosphatase, and the fracture strength of bones tended to decrease, indicating that low levels of alkaline phosphatase activity affected bone calcification and osteoblast maturation; adding lipoic acid or lipoic acid and chromium methionine significantly increased serum alkaline phosphatase activity and fracture strength, indicating that both additives can alleviate the effects of stress on bone calcification and osteoblasts, and play a vital role in protecting bones. In summary, even under dual stress, adding lipoic acid and a mixture of lipoic acid + chromium methionine can still significantly improve the bone quality of broilers.

[0069] The effects of adding lipoic acid and methionine chromium to the diet under high density conditions on the key genes for calcium absorption in the small intestine of heat-stressed broilers showed that: CaBP-D28k is expressed in the cytoplasm of poultry intestinal cells and is responsible for transporting Ca 2+ from the apical membrane to the basal membrane; PMCA1b and NCX1 are located in the basal membrane of the cells and are responsible for transporting Ca 2+ absorbed by CaBP-D28k into the cell basal membrane of intestinal cells to the blood. Under the dual stress of density stress and heat stress, the gene expression of CaD28K, PMCA1b, and NCX1 in the jejunum of broilers was significantly reduced; the addition of lipoic acid and methionine chromium can significantly increase the expression of these three genes, which may be because lipoic acid and methionine chromium act as antioxidants to remove free radicals produced by oxidative stress in the body.

[0070] Heat stress high density significantly reduced the expression level of intestinal NaPi-IIb protein, and heat stress high density bone fracture strength was significantly reduced, indicating that density stress and heat stress further affected the bone mineralization of broilers and affected bone quality. Adding lipoic acid and chromium methionine significantly increased the expression level of NaPi-IIb protein, indicating that these two additives can significantly alleviate the effects of heat stress and density stress on broiler calcium and phosphorus absorption, maintain the body's normal bone metabolism process, and improve the bone quality of broilers.

[0071] Increasing the feeding temperature at high density to cause heat stress can still significantly reduce the growth performance, immune organ index and bone quality of broilers, and significantly reduce the absorption of calcium ions by the small intestine. However, adding lipoic acid or a mixture of lipoic acid and chromium methionine can significantly improve the immune organ index and bone quality of broilers, which is related to the improvement of the small intestine's ability to transport calcium and phosphorus ions.

[0072] Whether under single oxidative stress or double stress, adding lipoic acid and chromium methionine can improve the antioxidant and immune functions of broilers under high-density stress and heat stress, alleviate bone damage, and protect the bone health of broilers by removing free radicals in the body and maintaining the balance of calcium and phosphorus metabolism in the body.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A feed additive for improving growth and bone health of broiler chickens in chronic stress state, characterized in that: The feed additives are lipoic acid and chromium methionine, and the added amounts are 300 mg / kg of lipoic acid and 400 ug / kg of chromium methionine.

2. Use of the feed additive for improving the growth and bone health of broilers in chronic stress state as claimed in claim 1 in feed or medicine for improving the antioxidant capacity and immunity of broilers under heat stress and / or high-density stress.

3. Use of the feed additive for improving the growth and bone health of broilers under chronic stress as claimed in claim 1 in feed or medicine for improving the bone health of broilers under heat stress and / or high-density stress.

4. The use according to claim 3, characterized in that: The feed additive is used in feed or medicine for removing free radicals in the body, maintaining calcium-phosphorus metabolic balance in the body and improving the bone health of broilers.

5. The use according to claim 3, characterized in that: The feed additive is used in feed or medicine for improving the immune organ index and bone quality of broilers, improving the transport capacity of the small intestine for calcium and phosphorus ions, and alleviating bone damage.

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