Application of a polypeptide in the preparation of a feed additive for reducing poultry expulsion stress response

Abstract

The present invention provides an application of a polypeptide in the preparation of a feed additive for reducing the expulsion stress response of poultry, and belongs to the field of feed technology. The present invention finds through in vitro tests that duck heat shock regulatory factor 3 polypeptide can significantly reduce the heat stress response of duck small intestinal epithelial cells, and improve the ability of duck small intestinal epithelial cells to cope with cell apoptosis caused by heat stress; and can improve the oxidative stress of duck small intestinal epithelial cells caused by ultraviolet light irradiation, reduce the cell MDA level, and increase the cell SOD level. The present invention finds through clinical trials that duck heat shock regulatory factor 3 polypeptide as a feed additive can effectively reduce the impact of expulsion stress on the egg laying rate of chickens, ducks, and geese, significantly improve the egg laying rate of laying poultry under expulsion stress environment, and increase the benefits of laying poultry breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of feed, and in particular relates to application of a polypeptide in preparing a feed additive for reducing driving stress response of poultry. Background Art

[0002] Egg products are the main food for people's daily consumption. At present, caged laying hens have a dominant position in the fresh egg market due to their advantages such as high stocking density, high egg production rate and low egg price. However, with the improvement of people's living standards, free-range eggs, duck eggs and goose eggs are favored by high-end consumers because they are rich in higher nutrients and have a better taste. The market demand for free-range eggs, duck eggs and goose eggs is increasing day by day, with high unit prices, high profits and good breeding benefits. In the breeding and management of free-range chickens, ducks and geese, it is inevitable to drive away laying poultry in the links of feeding, watering, collecting eggs and cleaning feces. In particular, ducks and geese are timid by nature, and driving stress has a great impact on their growth and production. The decrease in egg production rate caused by driving stress during the peak egg-laying period will seriously affect the egg production rate of free-range chickens, ducks and geese, and reduce breeding efficiency. In addition to minimizing driving stress, it is necessary to develop effective drugs, feed additives and other products to reduce the decrease in egg production rate caused by driving stress of laying poultry.

[0003] Bioactive peptides are small molecule active peptides that are directly formed by organisms or produced by the degradation of protein molecules. Bioactive peptides are usually closely related to nutrition, hormones, immunity, antibacterial, antiviral, and antioxidant processes. Heat shock proteins are involved in the normal physiological processes of cells, and are also involved in the response or defense response of cells to changes in the external environment or external stimuli. Heat shock regulatory factors produced by cells when they are subjected to various stresses, such as high fever and hypoxia, can enhance the tolerance of cells to the next injury, maintain normal metabolism of cells, and improve survival rate. Therefore, exploring the functional peptides present in heat shock regulatory factors to reduce the expulsion stress response of free-range laying poultry has a good market prospect. Summary of the invention

[0004] The invention aims to provide an application of a polypeptide in the preparation of a feed additive for reducing the expulsion stress response of poultry, belonging to the technical field of feed.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Firstly, the present invention provides an application of a polypeptide in the preparation of a feed additive for reducing the expulsion stress response of poultry, wherein the polypeptide is duck heat shock regulatory factor 3 polypeptide BFI72978.1.

[0007] Furthermore, the amino acid sequence of the duck heat shock regulatory factor 3 polypeptide BFI72978.1 is LFPLLLLSPVANFI.

[0008] Furthermore, the feed additive can be used to reduce the impact of driving stress on the egg production rate of chickens, ducks and geese, and to increase the egg production rate of laying fowl under driving stress environment.

[0009] Secondly, the present invention provides a polypeptide for reducing the heat stress response of duck small intestinal epithelial cells, wherein the polypeptide is duck heat shock regulatory factor 3 polypeptide BFI72978.1, and the amino acid sequence of the duck heat shock regulatory factor 3 polypeptide BFI72978.1 is LFPLLLLSPVANFI.

[0010] Thirdly, the present invention provides a polypeptide for reducing the oxidative stress response of duck small intestinal epithelial cells, wherein the polypeptide is duck heat shock regulatory factor 3 polypeptide, and the amino acid sequence of the duck heat shock regulatory factor 3 polypeptide is LFPLLLLSPVANFI.

[0011] Beneficial effects: The present invention found through in vitro experiments that duck heat shock regulatory factor 3 polypeptide can significantly reduce the heat stress response of duck small intestinal epithelial cells, improve the ability of duck small intestinal epithelial cells to cope with cell apoptosis caused by heat stress; and can improve the oxidative stress of duck small intestinal epithelial cells caused by ultraviolet light irradiation, reduce the cell MDA level, and increase the cell SOD level. Therefore, it can be used to prepare feed additives that reduce the expulsion stress response of chickens, ducks, and geese.

[0012] Secondly, through clinical trials, the present invention found that duck heat shock regulatory factor 3 polypeptide as a feed additive effectively reduced the impact of driving stress on the egg production rate of chickens, ducks and geese, significantly improved the egg production rate of chickens, ducks and geese under driving stress environment, and increased the efficiency of egg-laying poultry farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Results of mass spectrometry detection of duck heat shock regulatory factor 3 polypeptide;

[0014] Figure 2 Effects of peptides on cell viability of duck intestinal epithelial cells under heat stress;

[0015] Figure 3 Effects of peptides on MDA levels in duck intestinal epithelial cells under oxidative stress;

[0016] Figure 4 Effects of peptides on SOD levels in duck intestinal epithelial cells under oxidative stress. DETAILED DESCRIPTION

[0017] Example 1: Mining of active polypeptide sequences of duck heat shock regulatory factor 3BFI72978.1

[0018] The amino acid sequence of duck heat shock regulatory factor 3BFI72978.1 was analyzed. Its carboxyl end was truncated from the α-helix region. The aliphatic amino acid leucine-rich region was selected. The 14 amino acid peptide at positions 528 to 541 had the amino acid sequence: LFPLLLLSPVANFI. It was commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. Figure 1 The results of mass spectrometry detection of synthetic peptides.

[0019] Example 2: Detection of the effect of polypeptides on the heat stress response of duck small intestinal epithelial cells

[0020] (1) Isolation and culture of duck small intestinal epithelial cells: Take three duck embryos at 25 embryonic days, remove the small intestinal tissue of the duck embryos by aseptic operation in a clean bench, put them in PBS solution, wash the small intestinal tissue with PBS solution, remove the pancreas and mesentery, and then cut the small intestine open. Wash the inner wall of the small intestine with PBS solution until the upper liquid is clear. Cut the cleaned small intestine into 1-2 mm tissue blocks and put them in PBS solution. Use 20 mL of 1% type I collagenase digestion solution to digest the small intestinal tissue blocks, shake and digest at 37°C and 80 r / min for 60 min, and then wash them with PBS three times. Use PBS solution to gently blow the small intestinal tissue blocks, collect the upper cell suspension, and keep the tissue blocks and continue to add PBS to blow, repeat the above steps 5 times until the upper liquid is clear. Centrifuge the obtained cell suspension at 1000 r / min for 10 min, discard the supernatant, and resuspend the obtained cell clusters with complete culture medium and filter them with a 100 μm cell sieve. The complete culture medium for duck intestinal epithelial cells is: DMEM medium with 10% fetal bovine serum added. The sieved cells are suspended in the complete culture medium and blown evenly, and then inoculated into a cell culture bottle and cultured in a 37°C, 5% carbon dioxide incubator.

[0021] (2) According to 1×10 per hole 5 Duck intestinal epithelial cells in logarithmic growth phase were inoculated into 96-well plates and cultured in a 37°C, 5% carbon dioxide incubator.

[0022] (3) When the cells were cultured in a cell culture incubator until the cell density was close to 80%, DMEM complete medium containing the polypeptide at final concentrations of 0 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, 160 μg / ml, 320 μg / ml and 640 μg / ml was added. Each group had 4 parallels and a blank control group was also set up.

[0023] (4) After culturing in a 39°C, 5% carbon dioxide incubator for 24 h, the OD450 value was determined using CCK-8 and the cell survival rate was calculated. Cell survival rate = (OD450 of the experimental group - OD450 of the blank control group) / (OD450nm of the control group - OD450 of the blank control group).

[0024] from Figure 2 It can be seen that under 39°C heat stress conditions for 24h, within the concentration range of 20μg / ml-640μg / ml, the synthetic polypeptide has a significant promoting effect on the cell viability of duck small intestinal epithelial cells. The promoting effect of 20μg / ml-320μg / ml increases with the increase of polypeptide concentration, and there is no significant enhancement at 320μg / ml-640μg / ml.

[0025] Example 3: Detection of the effect of polypeptides on oxidative stress in duck intestinal epithelial cells

[0026] (1) According to 1×10 per hole 5 Duck intestinal epithelial cells in logarithmic growth phase were seeded in 96-well plates;

[0027] (2) Cultivate in a cell culture incubator until the cell density is close to 80%, then add DMEM medium containing synthetic polypeptides at final concentrations of 0 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, 160 μg / ml, 320 μg / ml and 640 μg / ml. Each group has 4 parallels, and a blank control group is also set up;

[0028] (3) The control group was placed in a cell culture incubator and cultured for 24 h; the cells in the oxidative stress group were placed in a biosafety cabinet under ultraviolet light for 30 min and then transferred to a cell culture incubator and cultured for 24 h;

[0029] (4) The culture medium was removed, the cells were washed with PBS, and the superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected using superoxide dismutase (SOD) and malondialdehyde (MDA) kits.

[0030] from Figure 3 It can be seen that within the concentration range of 40μg / ml-640μg / ml, the synthetic peptide can significantly reduce the MDA level of duck small intestinal epithelial cells, the effect is the largest at 320μg / ml, and the effect of 640μg / ml is equivalent to that of 320μg / ml. Figure 4 It can be seen that within the concentration range of 40μg / ml-640μg / ml, the synthetic polypeptide can significantly increase the SOD level of duck intestinal epithelial cells, the effect reaches a peak at 320μg / ml, and the effect of 640μg / ml is equivalent to 320μg / ml. Ozone produced by ultraviolet light irradiation causes oxidative stress in duck intestinal epithelial cells. After adding the polypeptide to the culture medium, the MDA level is significantly reduced, the SOD level is significantly increased, and the oxidative stress reaction is improved. Therefore, using the polypeptide to treat duck intestinal epithelial cells can effectively reduce oxidative stress.

[0031] Example 4: Effect of duck heat shock regulatory factor 3 polypeptide on driving stress in laying hens

[0032] (1) Add duck myogenic regulatory factor WOB19092.1 No. 3 polypeptide to the laying hen basic feed in different proportions and mix thoroughly. Prepare three types of feeds at a ratio of 1:1000, 1:10000, and 1:100000 based on the weight of polypeptide feed additive and the weight of basic feed;

[0033] (2) 400 285-day-old high-production triple-yellow laying hens (with similar egg production rates) were randomly divided into four groups, 100 per group, and raised in a free-range field isolated by wire mesh;

[0034] (3) The chickens were fed once at 8:00 and 16:00 each day, and were allowed to eat and drink freely. The chickens were driven to run for 10 minutes before each feeding. The experimental group was fed with feed containing polypeptide feed additives, while the control group was fed with basic feed only. The experimental period was 30 days, and the egg production of each group was counted.

[0035] Table 1 Effect of different proportions of peptide feed additives on egg production rate of laying hens

[0036] Add Scale Egg production rate (%) 1:1000 84.6 1:10000 82.2 1:100000 78.7 No Addition 78.4

[0037] As can be seen from Table 1, the duck heat shock regulatory factor 3 polypeptide of the present invention is added to the basic feed at a ratio of 1:1000 and 1:10000 as a feed additive to feed laying hens, which can significantly improve the effect of driving stress on the egg production rate of laying hens, and the egg production rate is increased by 4.85% to 7.91% compared with the group without addition.

[0038] Example 5: Effect of duck heat shock regulatory factor 3 polypeptide on driving stress in laying ducks

[0039] (1) Add duck myogenic regulatory factor WOB19092.1 No. 3 polypeptide into the basic feed of laying ducks in different proportions and stir thoroughly. Prepare three types of feeds according to the weight of polypeptide feed additive: weight of basic feed, 1:1000, 1:10000, and 1:100000;

[0040] (2) 400 220-day-old high-production Mallard ducks (with similar egg production rates) were randomly divided into four groups, 100 ducks per group, and raised in a free-range enclosure separated by wire mesh;

[0041] (3) Feed the ducks once at 8:00 and 16:00 every day. Feed and drink freely. Drive the ducks to run for 10 minutes before each feeding. The experimental group was fed with feed containing polypeptide feed additives, while the control group was fed with basic feed only. The experimental period was 30 days, and the egg production of each group was counted.

[0042] Table 2 Effects of different proportions of peptide feed additives on egg production rate of ducks

[0043] Add Scale Egg production rate (%) 1:1000 85.1 1:10000 83.7 1:100000 79.7 No Addition 79.2

[0044] As can be seen from Table 2, the duck heat shock regulatory factor 3 polypeptide of the present invention is added to the basic feed at a ratio of 1:1000 and 1:10000 as a feed additive to feed laying ducks, which can significantly improve the effect of driving stress on the egg production rate of laying hens, and the egg production rate is increased by 5.68% to 7.45% compared with the group without addition.

[0045] Example 6: Effect of duck heat shock regulatory factor 3 polypeptide on driving stress in laying geese

[0046] (1) Add duck myogenic regulatory factor WOB19092.1 No. 3 polypeptide to goose basic feed in different proportions and mix thoroughly. Prepare three types of feed according to the weight of polypeptide feed additive: weight of basic feed, 1:1000, 1:10000, and 1:100000;

[0047] (2) 200 740-day-old high-production geese (with similar egg production rates) were randomly divided into 4 groups, 50 geese per group, and raised in a free-range field isolated by wire mesh;

[0048] (2) The geese were fed once at 8:00 and 16:00 each day, and were allowed to eat and drink freely. The geese were driven to run for 10 minutes before each feeding. The experimental group was fed with feed containing polypeptide feed additives, while the control group was only fed with basic feed. The experimental period was 30 days, and the egg production of each group was counted.

[0049] Table 3 Effects of different proportions of peptide feed additives on egg laying rate of Huoyan geese

[0050] Add Scale Egg production rate (%) 1:1000 50.7 1:10000 49.4 1:100000 48.1 No Addition 47.2

[0051] As can be seen from Table 3, the duck heat shock regulatory factor 3 polypeptide of the present invention is added to the basic feed at a ratio of 1:1000 and 1:10000 as a feed additive to feed laying geese, which can significantly improve the effect of driving stress on the egg laying rate of laying geese, and the egg laying rate is increased by 4.66% to 7.42% compared with the group without addition.

Claims

1. Use of a polypeptide in the preparation of a feed additive for improving the egg production rate of poultry under driving stress conditions, characterized in that: The polypeptide is duck heat shock regulatory factor 3 polypeptide BFI72978.1, and the amino acid sequence of the duck heat shock regulatory factor 3 polypeptide BFI72978.1 is LFPLLLLSPVANFI; the addition ratio of the feed additive in the poultry basic feed is not less than 1:10000 by weight.

2. The use according to claim 1, characterized in that: The feed additive can be used to reduce the influence of driving stress on the egg laying rate of chickens, ducks and geese.

3. A polypeptide for reducing heat stress and oxidative stress in duck small intestinal epithelial cells, characterized in that: The polypeptide is duck heat shock regulatory factor 3 polypeptide BFI72978.1, and the amino acid sequence of the duck heat shock regulatory factor 3 polypeptide BFI72978.1 is LFPLLLLSPVANFI.

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