CN111772053A - Method for improving chicken immunity by adding glutamine to rapeseed meal as chicken feed protein source - Google Patents

Abstract

A method for improving chicken immunity by adding glutamine into rapeseed meal as a chicken feed protein source comprises adding 5-25% of rapeseed meal into a daily ration of broiler, adding 0.5-2% of glutamine based on the daily ration of rapeseed meal, and adding 1.0% of Gln into the daily ration to improve the immunity of the broiler and improve the intestinal health to a certain extent. Provides basis for scientific preparation of daily ration for the broiler, reasonable utilization of rapeseed dregs and safe upper limit of addition. Rapeseed meal is used as a vegetable protein source to the maximum extent in the daily feed of the broiler chicken instead of soybean meal, and Gln is added into the daily feed to reduce the toxic and side effects of the rapeseed meal to the maximum extent, so that the normal growth of the broiler chicken is not influenced, the feed cost is reduced, and the economic benefit is improved.

Description

Method for improving chicken immunity by adding glutamine to rapeseed meal as chicken feed protein source

technical field

The invention relates to the effect of adding glutamine to rapeseed meal diets on broilers, in particular to a method for adding glutamine to rapeseed meal as a feed protein source for broilers to protect the intestines of broilers and improve the immune performance of broilers.

Background technique

Rapeseed meal is a by-product of rapeseed oil extraction. Its crude protein content is 38%-40%. Its amino acid composition is relatively reasonable and its methionine content is high. It is a good source of vegetable protein. Rapeseed meal can be used more effectively. It is an effective way to solve the current shortage of feed protein in my country. However, with the increase in the amount of rapeseed meal added, the growth performance, intestinal morphology, feed conversion rate and protein utilization efficiency of animals all declined, so the application of rapeseed meal in broiler diets was limited to a certain extent. As the amount of rapeseed meal added to the diet increases, the intestinal morphology of broilers will be destroyed, thereby affecting the utilization efficiency of nutrients. Therefore, it is inevitable to use reasonable nutritional regulation methods to improve the utilization efficiency of rapeseed meal in broiler diets. Glutamine (Gln) is the most abundant amino acid in the blood, accounting for 30%-35% of the amino acid nitrogen source of the whole body, and it is an important carrier of nitrogen transport in the body. Gln is an important energy source for intestinal epithelial cells, an essential precursor for the synthesis of purines and pyrimidines, and plays an important role in maintaining intestinal epithelial integrity and nutrient absorption. The gut is the main organ for nutrient metabolism, digestion and absorption, and the largest immune organ in the body. Promoting the proliferation of small intestinal epithelial cells is of great significance for promoting intestinal development, maintaining poultry health and improving nutrient digestion and utilization.

SUMMARY OF THE INVENTION

The purpose of the present invention is to add glutamine to rapeseed meal as a method for improving chicken immunity as a chicken feed protein source, adding 5%-25% rapeseed meal to the broiler ration, and adding 5%-25% rapeseed meal to the broiler diet. Adding 0.5%-2% glutamine, the growth performance (average daily gain, average daily feed intake and feed-to-weight ratio), immune performance (including immune organ index, intestinal secretion type sIgA and Intestinal mucin 1 and 2), intestinal morphology (villus height, crypt depth and ratio of villus height to crypt depth), intestinal tight junction proteins (Occudin-1, ZO-1 and Claudin) mRNA expression, all play a role in to a better effect. The experimental results showed that dietary supplementation of 1.0% Gln could improve the immune performance of broilers and improve intestinal health to a certain extent.

Rapeseed meal is a by-product of rapeseed pressing or leaching process to produce edible oil. The crude protein content is 34.8%~40%, rich in methionine, calcium, phosphorus and B vitamins in minerals. It is an alternative to soybean meal. The preferred plant-based protein raw material. However, rapeseed meal contains anti-nutritional factors such as glucosinolate, sinapine, tannin, etc., which will have certain toxic and anti-nutritional effects on animals. Studies have shown that the addition of about 10% rapeseed meal in the early broiler diet does not affect the growth performance of broilers. However, with the increase of rapeseed meal addition, the growth performance and organ development of broilers were inhibited. Studies have shown that when the content of rapeseed meal in the rapeseed meal diet reaches or exceeds 20%, the intestinal morphology of broilers is damaged. Therefore, in order to improve the utilization of rapeseed meal, the present invention aims to select a certain exogenous nutrient regulator to overcome the above defects, which is an effective method. The exogenous nutrition regulator is glutamine (Gln).

Glutamine (Gln) is an important nutrient in the body, which is abundant in muscle and blood, and is an important functional amino acid. Gln is an important precursor for the synthesis of proteins, amino acids, purines and other biological macromolecules, and it is also the main energy substance for the rapid division and proliferation of intestinal mucosal epithelial cells. It plays an important role in improving the function of the small intestinal mucosa and maintaining the health of the intestinal mucosa. A large number of studies have shown that dietary supplementation of 1% Gln significantly increases the villus height of the duodenum and jejunum of broilers, and increases the ratio of intestinal villus height to crypt depth, which is conducive to maintaining the integrity of the intestinal morphological structure and improving the intestinal mucosa. immune level. Adding 0%, 0.5%, 1% and 1.5% Gln to the broiler diets, it was found that the addition of Gln in the diet significantly increased the thymus index and spleen index of broilers, and the addition of 1% Gln had the best effect. But to the best of our knowledge, there is no report on the effect of Gln on the immune performance and intestinal immune function of rapeseed meal-fed broiler chickens. Therefore, this method uses chickens as objects, and by adding 20% rapeseed meal and different proportions of Gln to their diets, the effects on the immune performance and intestinal immune function of broilers are expected to provide scientific formulation of broiler diets and rapeseed seeds. The rational use of meal and the addition of safety upper limit provide the basis.

The beneficial effects of the method are as follows: in the broiler chicken feed diet, rapeseed meal is used to replace soybean meal to the greatest extent as a vegetable protein source, and Gln is added to the diet to minimize the toxic and side effects of rapeseed meal, without affecting the normal growth of broilers, reducing Feed cost and improve economic efficiency is one of the effective methods.

Description of drawings

Figure 1 shows the effect of 20% rapeseed meal and Gln supplementation on the content of sIgA in chicken jejunum mucosa.

Figure 2 shows the effect of 20% rapeseed meal supplementation on the expression of MUC1 mRNA in jejunal mucosa.

Figure 3 is a graph showing the effect of 20% Gln addition on MUC2 mRNA expression in jejunal mucosa.

Detailed ways

In the method for improving chicken immunity by adding glutamine to rapeseed meal as a chicken feed protein source of the present invention, 5%-25% rapeseed meal is added to the broiler diet, and 0.5% of rapeseed meal is added on the basis of the rapeseed meal. %-2% glutamine, on 1-42-day-old broiler growth performance (average daily gain, average daily feed intake and feed-to-weight ratio), immune performance (including immune organ index, intestinal secretory sIgA and intestinal Mucin 1 and 2), gut morphology (villus height, crypt depth and ratio of villus height to crypt depth), and mRNA expression of gut tight junction proteins (Occudin-1, ZO-1, and Claudin) all played a role in the comparison. Good results. The experimental results showed that dietary supplementation of 1.0% Gln could improve the immune performance of broilers and improve intestinal health to a certain extent. The specific materials and methods are detailed as follows:

1.1 Test materials and sources

The rapeseed meal used in the experiment was purchased from Zunyi Jinding Agricultural Technology Co., Ltd. (crude protein content was 35.0%, glucosinolate content was 34.5 μmol/g); glutamine (Gln) was purchased from Shanghai Feiya Technology Co., Ltd. 99.10%.

1.2 Experimental design and feeding management

A total of 120 healthy Qiandongnan Xiaoxiang chickens with similar body weight on 1 day were selected and randomly divided into 4 treatments (A, B, C and D), with 5 replicates for each treatment and 6 chickens for each replicate. Among them, group A was fed the basal diet (control group, without rapeseed meal), group B was fed the diet supplemented with 20% rapeseed meal, and groups C and D were fed with an additional 0.5% of the diet of group B, respectively. and 1% Gln, and the experimental period was 42 d. The specific dietary formula and nutritional level are shown in Table 1.

The feeding and management are as follows: the temperature in the chicken house is 32-34°C on the 1st to 3rd day of the experiment, and then decreases by 2-3°C every week, so that the final room temperature is maintained at about 26°C. The humidity in the chicken house was maintained at about 70% for 1~3 days of the experiment, and maintained at about 60%~65% in the future. Feeding was fed 3 times a day (1 time in the morning, 1 time at noon, and 1 time in the evening), and the chickens ate and drank freely throughout the experimental period. Immunization was carried out according to the routine chicken immunization procedure, and the experimental site was Guizhou Rongjiang Shannong Development Co., Ltd.

1.3 The main instrument of the test

VELP ZX3 Vortex Mixer (Southeast Key Instrument); UV-5200 UV-Vis Spectrophotometer (Shanghai Yuanyan Instrument Co., Ltd.); HL-7K Palm Centrifuge (Shanghai Huxi Industrial Co., Ltd.); R 134a-Cryogenic High-speed centrifuge (Effendorf, USA); CB 1600-H ultra-clean workbench (Su Jie Medical Instruments (Suzhou) Co., Ltd.); ND2000 ultra-trace nucleic acid and protein analyzer (Thermo, Germany); CFX Connect-three-channel gradient fluorescence Quantitative PCR machine (Bio-rad, USA).

1.4 Collection of test samples

At the end of the experiment, 10 chickens from each treatment were selected for fasting weighing (close to the average body weight of each treatment group) and slaughtered, and the jugular vein blood was quickly collected in a 10 mL centrifuge tube, which was placed obliquely for the collection of serum. After blood collection was completed, the abdominal cavity was opened immediately, and the spleen, thymus and bursa of Fabricius were extracted and weighed and recorded. At the same time, about 5 cm of jejunum was collected, longitudinally dissected, rinsed with pre-cooled normal saline, and the intestinal mucosa was gently scraped with a glass slide and placed in a cryopreservation tube, and stored in liquid nitrogen for testing.

1.5 Measurement indicators and methods

1.5.1 Gut index

After all the chickens were weighed on an empty stomach, 10 broilers in each treatment group were selected to be weighed and slaughtered. The abdominal cavity was opened immediately after slaughter, and the duodenum, jejunum and ileum were collected for the determination of intestinal index.

Relative length of intestine = length of each intestinal segment (cm)/live weight before slaughter (kg).

Relative intestinal weight = weight of each intestinal segment (g)/live weight before slaughter (kg).

1.5.2 Immune Organ Index

The spleen, thymus and French sac were collected, and their fresh weight was quickly weighed with a precision balance, and the immune organ index was calculated. The calculation formula is as follows:

Immune organ index (g/kg) = immune organ weight (g) / pre-slaughter weight (kg).

1.5.3 Serum immune protein assay

Serum IgA, IgG and IgM contents were measured according to the kit's operating instructions, and the required kits were purchased from Nanjing Jiancheng Bioengineering Institute.

1.5.4 Determination of secretory immunoglobulin A (sIgA) in jejunum mucosa

The sIgA content of jejunum mucosa was determined by a kit, and the specific operation was carried out according to the instructions of the kit. The kit was purchased from Nanjing Jiancheng Bioengineering Institute.

1.5.5 MUC1 and MUC2 mRNA expression in jejunal mucosa

The expression of MUC1 and MUC2 mRNA was measured by RT-PCR. The main steps are as follows: Weigh a certain quality of jejunal mucosa sample, put it into a centrifuge tube pre-treated with 5 mL DEPC of pre-cooled RNAiso Plus (Product No.: D9108A, TaKaRa), homogenize it in an ice bath until the liquid is clarified, and then divide it into a centrifuge tube. Phase, RNA precipitation, RNA washing and RNA resolubilization, and the OD260/280 value of the extracted RNA was determined by microspectrophotometer. RNA was transcribed using PrimeScript™ RT master Mix inversion kit, and the transcribed cDNA was stored at -20°C. Then use TB Green Premix Ex Taq Kit (No.RR420A) to carry out RT-PCR test. The PCR reaction program is as follows: 95°C for 30 s (1 cycle), 95°C for 5 s and 60°C for 34 s (40 cycles) cycle). The calculation of relative quantification of gene expression was performed according to the method of Livak and Schmittgen [18].

The sequence of the target gene was designed according to the gene sequence published by NCBI, and β-actin was used as an internal reference, which was synthesized by Shenggong Bio. The internal reference and target gene sequence are shown in Table 2.

1.6 Data Statistics and Analysis

One-way variance procedure in SPSS 20.0 statistical software was used for data processing. If the difference was significant, Duncan's was used for multiple comparison. The results were expressed as mean ± standard deviation, and P<0.05 indicated a significant difference.

Table 1 Basic diets and nutritional levels

Note: Each kilogram of diet provides: Vitamin A 6 000 IU, Vitamin B1 3.0 mg, Vitamin B2 5 mg, Vitamin B5 42 mg, Vitamin B6 4 mg, Vitamin B12 0.01 mg, Vitamin D3 1 000 IU, Vitamin E 30 mg, Vitamin B K3 2.8 mg, calcium pantothenate 10 mg, biotin 0.15 mg, folic acid 0.85 mg, iron (as ferrous sulfate) 80 mg, copper (as copper sulfate) 8 mg, manganese (as manganese sulfate) 80 mg, zinc (calculated as zinc sulfate) 65 mg, iodine (calculated as sodium iodide) 0.50 mg, selenium Se (calculated as sodium selenate) 0.25 mg. Among them, crude protein is the measured value, and the rest are calculated values.

Note: Provided by diets per kilogram: VA 6 000 IU, VB1 3.0 mg, VB2 5 mg, VB5 42 mg, VB6 4 mg, VB12 0.01 mg, VD3 1 000 IU, VE 30 mg, VK3 2.8 mg, calciumpantothenate 10 mg, biotin 0.15 mg, folic acid 0.85 mg, Fe (as ferrous sulfate) 80 mg, Cu (as copper sulfate) 8 mg, Mn (as manganese sulfate) 80 mg, Zn (as zincsulfate) 65 mg, I (as potassium iodide) 0.50 mg, Se (as sodium selenite) 0.25 mg. CP was a measured value, while the others were calculated values.

Table 2 RT-PCR primer sequences

2 Results and Analysis

2.1 The effect of 20% rapeseed meal and Gln supplementation on intestinal index

As shown in Table 3, compared with the control group, dietary supplementation of 20% rapeseed meal significantly reduced the relative length of duodenum, relative weight of jejunum, and relative length and weight of ileum in Qiandongnan Xiaoxiang chicken (P<0.01 ), significantly decreased the relative weight of the duodenum and the relative length of the jejunum (P<0.05). Compared with 20% rapeseed meal, dietary supplementation of 0.5% Gln had no significant effect on the relative length and relative weight of duodenum, jejunum and ileum (P>0.05). In addition, the relative length of duodenum, relative weight of jejunum and relative weight of ileum in the diet supplemented with 0.5% Gln were significantly lower than those in the control group (P<0.05), but the relative weight of duodenum, relative length of jejunum and relative weight of ileum were significantly lower than those in the control group (P<0.05). The length was not significantly different from the control group (P>0.05). Compared with the 20% rapeseed meal supplemented group, dietary supplementation of 1% Gln significantly increased the relative length and weight of duodenum, jejunum, and ileum (P<0.01), and significantly increased the relative length and weight of duodenum. The relative length of jejunum (P<0.05) was not significantly different from the control group (P>0.05). Compared with 0.5% Gln supplementation, dietary supplementation of 1% Gln significantly increased the relative length and weight of the duodenum (P<0.01), but had no effect on the relative length and weight of the jejunum and ileum (P>0.05). .

Table 3 Effects of 20% rapeseed meal and Gln supplementation on intestinal index of Qiandongnan Xiaoxiang chicken

Note: Different capital letters in the corners of abc data indicate extremely significant differences, and different lowercase letters indicate significant differences (P<0.05). Group A is the basal diet, group B is the 20% rapeseed meal supplemented group, and group C is B. 0.5% Gln was added on the basis of group D, and 1% Gln was added on the basis of group B. The table below is the same.

Note: abcValues with different capital letters in the same line means extremely significant difference (P<0.01) and values with small lettersuperscripts in the same line means significantly different (P<0.05). Group Awas fed with the basal diet; Group B was supplemented with 20% rapeseed meal, Group C and Group D were separately supplemented with 0.5% or 1% Gln. Thesame as below.

2.2 The effect of 20% rapeseed meal and Gln supplementation on immune organ index

As shown in Table 4, compared with the control group, dietary supplementation of 20% rapeseed meal significantly reduced the thymus index (P<0.01), but had no significant effect on the spleen index and bursa index (P>0.05). Compared with 20% rapeseed meal, dietary supplementation of 0.5% Gln had no significant effect on spleen index, thymus index and bursa index (P>0.05). However, 1% Gln addition significantly increased the thymus index (P<0.01), but had no significant effect on the spleen index and bursa index (P>0.05), and was not significantly different from the control group (P>0.05). Compared with the 0.5% Gln supplemented group, dietary supplementation of 1% Gln significantly increased the thymus index (P<0.01), but had no effect on the spleen index and bursa index (P>0.05).

Table 4 Effects of 20% rapeseed meal and Gln supplementation on immune organ index of Qiandongnan Xiaoxiang chicken

2.3 The effect of 20% rapeseed meal and Gln supplementation on serum immunoglobulin content

As shown in Table 5, compared with the control group, the addition of 20% rapeseed meal significantly reduced the serum immune proteins IgA, IgG and IgM (P<0.01). Compared with the addition of 20% rapeseed meal, the addition of 0.5% Gln significantly increased the immune proteins IgA, IgG and IgM (P<0.05), but the IgG and IgM in the 0.5% Gln addition group were significantly lower than those in the control group (P<0.01). ). However, compared with the 20% rapeseed meal group, 1% Gln supplementation significantly increased the levels of serum immune proteins IgA, IgG and IgM (P<0.01), and the serum IgM in the 1% Gln supplemented group was significantly lower than that in the control group (P<0.01). <0.05), but the serum IgA and IgG levels were not significantly different from the control group (P>0.05). Compared with the 0.5% Gln supplemented group, dietary supplementation of 1% Gln significantly increased the serum immune protein IgG and IgM content (P<0.01), but had no effect on the IgA content (P>0.05).

Table 5 Effects of 20% rapeseed meal and Gln supplementation on serum immunoglobulins in Qiandongnan Xiaoxiang chicken

2.4 The effect of 20% rapeseed meal and Gln supplementation on sIgA content in jejunal mucosa

As shown in Figure 1, compared with the control group, the addition of 20% rapeseed meal significantly reduced the content of sIgA in the jejunum mucosa of broilers (P<0.01). Compared with the addition of 20% rapeseed meal, the addition of 0.5% Gln to the diet had no significant effect on the sIgA content of the jejunum mucosa (P>0.05), but was significantly lower than the control group (P<0.01). However, adding 1% Gln significantly increased the content of sIgA in the jejunal mucosa (P<0.01), and the difference was not significant compared with the control group (P>0.05). Compared with 0.5% Gln supplementation, dietary supplementation of 1% Gln significantly increased the content of sIgA in the jejunal mucosa (P>0.01).

Fig.1 Effects of 20% rapeseed meal and Gln supplementation on sIgA content in jejunum mucosa of Qiandongnan Xiaoxiang chicken

Fig.1 Effect of 20% rapeseed meal and glutamine on the content of sIgA injejunum mucosa of Qiandongnan Xiaoxiang chicken

Note: Different capital letters on the abc histogram indicate extremely significant differences (P<0.01), and lowercase letters indicate significant differences (P<0.05). Among them, A was the basal diet (control group); group B was supplemented with 20% rapeseed meal; group C was supplemented with 0.5% Gln on the basis of group B; group D was supplemented with 1% Gln on the basis of group B.

Note: abc Values above column with different capital letters meansignificant difference (P<0.01) and values with different letters meansignificant difference (P<0.05 ). Group A was fed with the basal diet; GroupB was supplemented with 20% rapeseed meal, Group C and Group D were separately supplemented with 0.5% or 1% Gln. The same as below.

2.5 The effect of 20% rapeseed meal and Gln supplementation on the expression of MUC1 and MUC2 mRNA in jejunal mucosa

As shown in Figure 2 and Figure 3, compared with the control group, the addition of 20% rapeseed meal significantly reduced the mRNA expression levels of MUC1 (Figure 2) and MUC2 (Figure 3) in the jejunal mucosa (P<0.01). Compared with adding 20% rapeseed meal, adding 0.5% Gln had no significant effect on the expression levels of MUC1 and MUC2 mRNA in the jejunal mucosa of broilers (P>0.05), but both were significantly lower than those in the control group (P<0.01). However, compared with the 20% rapeseed meal supplementation group, 1% Gln supplementation significantly increased the expression levels of MUC1 and MUC2 mRNA in the jejunal mucosa (P<0.01), and was not significantly different from the control group (P>0.05). Compared with the 0.5% Gln supplemented group, dietary supplementation of 1% Gln had no significant effect on the expression of MUC1 mRNA in the jejunal mucosa (P>0.05), but significantly increased the expression of MUC2 mRNA in the jejunal mucosa (P<0.05).

Figure 2, Figure 3 Effects of 20% rapeseed meal and Gln supplementation on the expression of MUC1 and MUC2 mRNA in jejunal mucosa

Fig.2 Effect of 20% rapeseed meal and glutamine supplementation on mRNAexpression of MUC1 and MUC2 in jejunum mucosa of Qiandongnan Xiaoxiangchicken

3 Effects on chicken growth and development

3.1 The effect of 20% rapeseed meal and Gln supplementation on intestinal index

The growth, development and health of the intestinal tract can reflect the overall growth performance and physiological status of the animal to a certain extent. The results of this study showed that dietary supplementation of 20% rapeseed meal significantly reduced the relative length and weight of the duodenum, jejunum and ileum in Qiandongnan Xiaoxiang chickens. So far, there has been no report on the effect of rapeseed meal on the development of intestinal index, but a large number of studies have shown that with the increase of the dosage of rapeseed meal, the intestinal morphology of broiler chickens is damaged, which is manifested in the height of intestinal villi. Decreased and increased crypt depth. This means that dietary supplementation of high-dose rapeseed meal inhibited the growth and development of the intestine to a certain extent. Furthermore, our previous study found that dietary supplementation with 20% rapeseed meal significantly reduced the relative length and weight of duodenum, jejunum and ileum in 21-day-old Qiandongnan Xiaoxiang chickens (unpublished data). However, dietary supplementation of 1% Gln significantly increased the relative length and relative weight of the duodenum, jejunum, and ileum. Similarly, Huang Xiaoliang et al. [20] also showed that dietary supplementation of Gln significantly increased the weight and length of the duodenum, jejunum and ileum in broilers. This may be due to the fact that Gln provides energy for intestinal epithelial cells and promotes the promotion of cell proliferation and differentiation, thereby promoting intestinal morphology and mucosal development [21]. This indicated that Gln had a certain ameliorating effect on the inhibition of intestinal development caused by the addition of rapeseed meal.

3.2 Effects of 20% rapeseed meal and Gln supplementation on immune organ development

The Immune Organ Index is a generally accepted indicator for evaluating the immune status of livestock and poultry. The development of immune organs can affect the body's immunity and the ability to resist bacterial infection. Spleen, thymus and bursa are the main immune organs of poultry. Among them, the spleen is involved in humoral immunity and cellular immunity, and is the largest peripheral organ of birds. Bursa of Fabricius is a unique central lymphoid organ in birds, and it is the main place for the development and maturation of B lymphocytes. Among them, the bursa is mainly involved in humoral immunity, while the thymus is mainly involved in cellular immunity. The results of this experiment showed that the addition of 20% rapeseed meal significantly reduced the thymus index and bursa index, but had no significant effect on the spleen index. However, the results of the study showed that dietary supplementation of 12% rapeseed meal had no significant effect on the thymus index, bursa index and spleen index of broilers. Studies have shown that dietary supplementation of 15% rapeseed meal significantly reduces broiler thymus index, but has no effect on spleen index and bursa index. This is not exactly the same as the results of this experiment, and the reason for the difference may be caused by the difference in the amount of rapeseed meal added. This also suggests that high-dose rapeseed meal supplementation may reduce the development of immune organs in the body, thereby affecting immune performance. Previous studies have shown that dietary supplementation of Gln provides abundant purine, pyrimidine and other active precursors for the development of immune organs, which is beneficial to promote the growth and development of immune organs. Moreover, Gln supplementation can stimulate the proliferation of peripheral T lymphocytes in broilers, thereby increasing the weight of immune organs. In addition, the development of immune organs can reflect the state of the body's immune function to a certain extent. This study found that dietary supplementation of 1% Gln increased the thymus index and bursa index of Qiandongnan Xiaoxiang chickens. This is similar to the findings of Xiao Shuangshuang [32], who showed that adding Gln could significantly increase the bursa index of broilers. This may be because Gln stimulates the proliferation of lymphocytes and promotes the development and maturation of immune organs, thereby improving the immune function of the body. The above results suggest that dietary supplementation of Gln can promote the development of immune organs to a certain extent, which is beneficial to improve the immune performance of the body.

3.3 The effect of 20% rapeseed meal and Gln supplementation on serum immunoglobulin content

The immunoglobulins in broiler serum include IgA, IgG and IgM, which are important immune molecules in humoral immunity. Among them, IgA directly enters the blood circulation after being produced by the bone marrow, IgG is secreted by the spleen and synthesized by plasma cells in the lymph nodes, and IgM is the main immunoglobulin expressed on the surface of chicken B lymphocytes. The level of the body's immunoglobulin content reflects the state of the body's immune function to a certain extent []. The results of this study showed that the addition of 20% rapeseed meal significantly reduced the serum IgA, IgG and IgM content of broilers. However, the study by Drazbo et al. [37] showed that the addition of 15% rapeseed meal to the diet significantly reduced the IgM content of broilers, but had no effect on the IgA content. The reason for the different results may be due to the different amount of rapeseed meal added. However, the addition of 0.5% Gln and 1% Gln both increased serum IgA, IgG and IgM levels. Similarly, Bartell and Batal [38] showed that dietary supplementation of 1% Gln significantly increased serum IgA and IgG levels in broilers. Previous studies reported that IgG and IgM are T helper cell-dependent antibodies and indicators of T helper cell responses. However, Gln can promote the proliferation of CD4+ (a kind of T-helper cells), which suggests that Gln may increase the content of serum IgG or IgM by increasing the proliferation of T-helper cells. Therefore, dietary supplementation of Gln significantly increased the serum immunoglobulin content and the humoral immunity level of the body.

3.4 Effect of 20% rapeseed meal and Gln supplementation on sIgA content in jejunum mucosa

The intestine is considered to be the largest immune organ in the body, and sIgA is the key to intestinal immunity. It is secreted by plasma cells. The immunoglobulin (IgA) binds to the secretory fragments produced by the epithelial cells through intestinal epithelial cells or in the intercellular space. form. sIgA has the function of clearing antigens in intestinal immunity, directly blocking the contact of antigens or pathogens with the epithelial surface and thus protecting the epithelial cell surface from damage]. This experimental study showed that the addition of 20% rapeseed meal significantly decreased the sIgA content of the jejunal mucosa. So far, there has been no report on the effect of high-dose rapeseed meal supplementation on intestinal sIgA content. Studies have shown that sIgA is the main immune effector produced by plasma cells in the lamina propria of the intestinal mucosa, and is the most abundant immunoglobulin secreted by the body. In addition, sIgA-based humoral immunity plays a major role in intestinal immunity and plays an important protective role in intestinal mucosal immunity [43]. Moreover, the reduction of intestinal sIgA content in this study is consistent with the reduction of serum immunoglobulin (IgA, IgG and IgM) content, which means that 20% rapeseed meal supplementation reduces the body's humoral immunity level. However, dietary supplementation of 1% significantly increased jejunal sIgA content. Li Hu's study also showed that dietary supplementation of Gln significantly increased the content of sIgA in the duodenum of broilers. It has been previously reported that Gln can promote the proliferation and differentiation of macrophages and lymphocytes, and increase the level of sIgA, thereby improving intestinal immune function. And Gln addition can increase the number of sIgA cells in the intestinal mucosa and promote the secretion of sIgA [16], while the content of sIgA decreases when Gln is deficient or insufficient. Therefore, dietary supplementation of Gln is beneficial to enhance the immune barrier function of intestinal mucosa.

3.5 Effects of 20% rapeseed meal and Gln supplementation on the expression of MUC1 and MUC2 in jejunal mucosa

Mucin is a kind of high molecular weight glycoprotein secreted by epithelial cells. MUC1 mainly exists in the tissues of the body. The epithelial cells in the organs are close to the lumen and have functions such as anti-adhesion and immunosuppression. MUC2 is the main component of the intestinal mucus layer of the body, which mainly forms a mucus layer through the surface of the intestinal tract, which has the functions of lubricating and antagonizing the adhesion and invasion of pathogenic bacteria to the intestinal tract. The results of this experiment showed that the addition of 20% rapeseed meal significantly decreased the expression of jejunal mucosal proteins MUC1 and MUC2, while the addition of 1% Gln significantly increased the expression of MUC1 and MUC2. So far, there is no report on the effect of Gln on MUC1 and MUC2 of broiler intestinal mucosa. However, previous studies in mice and rabbits found that Gln addition could significantly increase the expression levels of MUC1 and MUC2 in the intestinal mucosa. Moreover, the study of Xing Shen [49] in piglets showed that Gln addition increased the number of goblet cells in the small intestine and increased the expression level of MUC2 in the jejunal mucosa. The above results indicate that dietary supplementation of Gln helps to promote intestinal mucin secretion and enhance the function of intestinal mucosal immune barrier.

4 Conclusion

The addition of 20% rapeseed meal in the diet reduced the immune performance and intestinal immunity of Qiandongnan Xiaoxiang chickens, but the addition of 1% Gln in the diet had a certain improvement on the immune function of broilers fed the high-dose rapeseed meal diet.

Claims (3)

1. Rapeseed meal adds glutamine as the method for chicken feed protein source to improve chicken immunity, it is characterized in that: in broiler chicken ration, add the rapeseed meal of 5%-25%, and in the rapeseed meal ration basis Adding 0.5%-2% glutamine, the growth performance of 1-42-day-old broilers includes: average daily gain, average daily feed intake and feed-to-weight ratio, immune performance including immune organ index, intestinal secretory sIgA and Intestinal mucin 1 and 2, intestinal morphology including villus height, crypt depth and ratio of villus height to crypt depth, and the mRNA expression of intestinal tight junction proteins Occudin-1, ZO-1 and Claudin, all played a better role. The results showed that adding 1.0% Gln to the diet could improve the immune performance of broilers and improve the intestinal health to a certain extent. 2. rapeseed meal according to claim 1 adds glutamine to make the method for chicken feed protein source to improve chicken immunity, it is characterized in that: the preferred mixing ratio of the rapeseed meal and glutamine added in broiler diet It is rapeseed meal 20% glutamine 1%. 3. the method that rapeseed meal according to claim 1 adds glutamine as chicken feed protein source and improves the method for chicken immunity, it is characterized in that: in broiler chicken ration feed ingredients, rapeseed meal replaces soybean meal feed protein source maximum amount It is 25%, that is, the mixing ratio of rapeseed meal and glutamine is 25% of rapeseed meal and 1-2% of glutamine.

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