CN113647358A - Construction method of chicken immune stress model, chicken immune stress model and application thereof - Google Patents
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- A—HUMAN NECESSITIES
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- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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Abstract
The invention provides a method for constructing a chicken immune stress model, the chicken immune stress model and application thereof, and relates to the technical field of biology. According to the construction method of the chicken immune stress model, the chick is continuously injected by LPS to construct the chicken immune stress model, wherein the dose of each injection is 0.4-0.6 mg/kg BW independently, the continuous injection frequency is 8-12, and the continuous injection time interval is 0.5-2 days. The invention adopts a mode of continuously injecting LPS to induce and construct the chicken immune stress model for the first time, optimizes the injection dosage, the injection times and the injection time interval of the LPS and determines the optimal condition for constructing the chicken immune stress model. The constructed chicken immune stress model is high in practicability and accuracy, and theoretical guidance is provided for relieving stress in actual production and reducing the influence of LPS on the growth performance of the broiler chicken.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chicken immune stress model, a chicken immune stress model and application thereof.
Background
With the rapid development of high-density and intensive breeding mode of domestic poultry, the times of vaccine immunity and the infection rate of pathogenic microorganisms are increased, and the poultry is in an immune stress state for a long time. Particularly, due to the characteristics of high growth speed and poor adaptability of the broilers, and the adoption of a large-scale high-density feeding mode, the broilers are particularly easy to generate stress, the industry is prevented from achieving an ideal growth effect and an optimal feed utilization rate, and great potential harm is caused to the breeding industry.
The successful establishment of an immune stress model is a precondition and a basis for researching anti-immune stress response, and the establishment of a suitable animal stress model is the problem which needs to be solved firstly in the current research of an immune stress mechanism and the research and development of anti-immune stress drugs. Lipopolysaccharide (LPS) is a main component of gram-negative bacillus cell wall, has no toxic effect, can release endogenous active factors, can induce livestock and poultry to generate immune stress in a short time as a nonspecific immunogen, and is one of the most common and classical stimulation medicaments used in establishing animal immune stress models at present. The research shows that LPS is injected into livestock and poultry organisms, the content of inflammatory cytokines of the broiler chicken is increased by the stimulation of the LPS, and the poultry can be heated and anorexia can be generated due to the increase of the inflammatory factors, so that the feed intake is reduced. These inflammatory cytokines stimulate corticosterone secretion from the adrenal gland via the hypothalamic-pituitary-adrenal cortical axis, which ultimately leads to elevated corticosterone levels, increased oxidative muscle breakdown and decreased synthesis rates.
At present, the common mode of injecting LPS into broilers is intraperitoneal injection, and the method is convenient to operate and high in absorption capacity, and does not need to consider cardiac overload. However, in the experimental operation process, due to the lack of reference standards, the LPS injection dosage is different in size, the injection time interval is different in length, and the effect of the immune stress model is difficult to measure. Therefore, the invention optimizes and screens the injection dosage, injection interval and injection frequency of the LPS and the injection age of the broiler chicken, constructs an obtained broiler chicken immune stress model accurately and stably, avoids the death rate caused by insufficient LPS efficacy or excessive dosage and multiple injections, provides a new way for preclinical screening of anti-stress drugs, and lays a foundation for the use of the broiler chicken in the process of exploring an immune stress mechanism.
Disclosure of Invention
The invention aims to provide a method for constructing a chicken immune stress model, which is simple and practical, and the constructed chicken immune stress model has strong accuracy and good practicability.
The second purpose of the invention is to provide a chicken immune stress model.
The third purpose of the invention is to provide the application of the chicken immune stress model in chicken feeding.
In a first aspect, the invention provides a method for constructing a chicken immune stress model, which comprises the following steps: continuously injecting the chicken by LPS to construct a chicken immune stress model;
wherein the dose of each injection is 0.4-0.6 mg/kg BW independently;
the number of continuous injections is 8-12;
the time interval of the continuous injection is 0.5-2 days.
As a further technical solution, the dose per injection is 0.5mg/kg BW.
As a further technical scheme, the number of the continuous injections is 10.
As a further technical scheme, the time interval of the continuous injection is 1 day.
As a further technical scheme, the injection method comprises intraperitoneal injection.
As a further technical scheme, the broiler is a broiler.
According to a further technical scheme, the day age of the chicks is 12-16 days.
As a further technical scheme, the day age of the chicks is 14 days.
In a second aspect, the invention provides a chicken immune stress model.
In a third aspect, the invention provides an application of a chicken immune stress model in chicken feeding.
Compared with the prior art, the invention has the following beneficial effects:
according to the construction method of the chicken immune stress model, the chick is continuously injected by LPS to construct the chicken immune stress model, wherein the dose of each injection is 0.4-0.6 mg/kg BW independently, the continuous injection frequency is 8-12, and the continuous injection time interval is 0.5-2 days. The invention firstly adopts a mode of continuously injecting LPS to induce and construct a chicken immune stress model, optimizes the injection dosage, the injection times, the injection time interval and the experimental broiler age in days of the LPS and determines the optimal conditions for constructing the chicken immune stress model. The constructed chicken immune stress model has the characteristics of high practicability and high accuracy, and provides theoretical guidance for slowing down stress in actual production and reducing the influence of LPS on the growth performance of the broiler chickens.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of the effect of LPS treatment on broiler body weight gain;
FIG. 2 is the effect of LPS treatment on average broiler feed intake;
FIG. 3 is the effect of LPS treatment on feed conversion ratio of broiler chickens;
FIG. 4 is a graph of the effect of LPS treatment on broiler serum corticosterone;
FIG. 5 is the effect of LPS treatment on thymus index of broiler chickens;
FIG. 6 is the effect of LPS treatment on broiler spleen index;
FIG. 7 is a graph of the effect of LPS treatment on the bursal index of broiler chickens;
FIG. 8 is a graph of the effect of LPS treatment on TNF- α in broiler serum;
FIG. 9 is a graph showing the effect of LPS treatment on IL-1. beta. in broiler serum.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The term "LPS" refers to Lipopolysaccharide (Lipopolysaccharide), which is a constituent of the outer wall of the cell wall of gram-negative bacteria, a substance composed of lipids and polysaccharides (glycolipids). Lipopolysaccharide is an Endotoxin (Endotoxin) which exhibits various biological activities when it acts on other biological cells such as human or animals.
In a first aspect, the invention provides a method for constructing a chicken immune stress model, which comprises the following steps: continuously injecting the chicken by LPS to construct a chicken immune stress model;
wherein the dose for each injection is 0.4-0.6 mg/kg BW, such as but not limited to 0.4mg/kg BW, 0.42mg/kg BW, 0.44mg/kg BW, 0.46mg/kg BW, 0.48mg/kg BW, 0.5mg/kg BW, 0.52mg/kg BW, 0.54mg/kg BW, 0.56mg/kg BW, 0.58mg/kg BW or 0.6mg/kg BW.
The number of consecutive injections is 8 to 12, and for example, but not limited to, 8, 9, 10, 11 or 12.
The time interval of the continuous injection is 0.5-2 days, such as but not limited to 0.5 day, 1 day, 1.5 days or 2 days.
The invention adopts a mode of continuously injecting LPS to induce and construct the chicken immune stress model for the first time, the construction method is simple and effective, the injection dosage, the injection times and the injection time interval of the LPS are optimized, and the optimal condition for constructing the chicken immune stress model is determined. The constructed chicken immune stress model has the characteristics of high practicability and high accuracy, and provides theoretical guidance for slowing down stress in actual production and reducing the influence of LPS on the growth performance of the broiler chickens.
In some preferred embodiments, the dose per injection is 0.5mg/kg BW.
In some preferred embodiments, the number of consecutive injections is 10.
In some preferred embodiments, the time interval between consecutive injections is 1 day.
In the invention, the injection dosage, the injection times and the injection time interval of the LPS are further optimized and adjusted by observing the production performance, the stress response index and the growth index change of the broiler chicken during the test, so that the constructed chicken immune stress model has higher accuracy.
In some preferred embodiments, the method of injection includes, but is not limited to, intraperitoneal injection.
In the invention, the mode of injecting LPS into the abdominal cavity is adopted, which is more beneficial to the construction of the chicken immune stress model.
In some preferred embodiments, the broiler chicken includes, but is not limited to, broiler chicken or egg chicken, wherein the change of broiler growth index is more obvious than that of egg chicken, so that the immune stress model is preferably constructed by taking the broiler chicken as a subject.
In some preferred embodiments, the day age of the chicks is 12-16 days old, and may be, for example, but not limited to, 12 days old, 13 days old, 14 days old, 15 days old, or 16 days old.
In some preferred embodiments, the day-old of the chicks is 14 days old.
The day age of the chicks influences the construction effect of the immune stress model, the chicks with small day age are not suitable for being injected by an injector, and the LPS has large damage to the chicks; the old chicks have strong immunity to LPS, and influence the construction of immune stress models.
In a second aspect, the invention provides a chicken immune stress model.
The chicken immune stress model is constructed by adopting a mode of continuously injecting the LPS into the chickens, has the characteristics of high practicability and high accuracy, and provides theoretical guidance for relieving stress in actual production and reducing the influence of the LPS on the growth performance of the chickens.
In a third aspect, the invention provides an application of a chicken immune stress model in chicken feeding.
The chicken immune stress model provided by the invention can be used for researching the influence of LPS on the growth performance of the broiler chicken, and a method for reducing the influence of LPS on the growth performance of the broiler chicken is searched, so that guidance is provided for feeding the chicken.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
A chicken immune stress model is constructed by the following steps:
and (3) injecting LPS into the abdominal cavity of 14-day-old chicks for 10 days continuously, wherein the dose of the LPS injected into the abdominal cavity is 0.5mg/Kg BW once a day, and constructing to obtain a chicken immune stress model.
Example 2
A chicken immune stress model is constructed by the following steps:
and (3) injecting LPS into the abdominal cavity of 14-day-old chicks for 8 days continuously, twice a day, wherein the dosage of the LPS is 0.4mg/Kg BW, and constructing to obtain a chicken immune stress model.
Example 3
A chicken immune stress model is constructed by the following steps:
and (3) injecting LPS into the abdominal cavity of 14-day-old chicks for 12 days, once every two days, and setting the dosage of the injected LPS to be 0.6mg/Kg BW to construct a chicken immune stress model.
Test example 1
1. Experimental animal and grouping design
192 AA male broilers with good health conditions and similar body weights and age of 14 days are randomly divided into 2 groups, each group is 8 in number, each group is 12 in number, and basic daily ration is fed. The LPS group was constructed according to example 1, and the saline group was treated in a manner different from example 1 in that the injected LPS was replaced with an equal amount of physiological saline. And (4) carrying out statistics on the production performance, stress, immunity indexes and the like of the broiler chickens in the experimental period.
Data are presented as mean ± SEM, single factor independent T-test analysis using the sps 13.0 software, with P <0.05 being significantly different.
2. Test daily ration
The basic ration is corn-soybean meal type ration, and the composition components and the nutritional level of the basic ration are shown in table 1.
TABLE 1 basic diet composition and nutritional level
1) The microelement premix is provided for each kilogram of compound feed: copper 8mg (CuSO)4·5H2O)；Iron 80mg (FeSO)4) (ii) a Manganese 100mg (MnSO)4·H2O); selenium 0.15mg (Na)2SeO3) (ii) a Iodine 0.35mg (KI).
2) The vitamin premix is provided for each kilogram of compound feed: VA 9500IU, VD362.5ug, VE 30IU, VK 32.65mg, VB12 mg, VB 66 mg, VB120.025mg, biotin 0.0325mg, folic acid 1.25mg, pantothenic acid 12mg, nicotinic acid 50 mg.
Influence of LPS stimulation on broiler Productivity
At test 0 (before injection), 1, 3, 5, 7 and 9 days, broiler chickens are weighed by taking a repeat group as a unit after fasting for 8 hours, the weights of 14, 15, 17, 19, 21 and 23 days of age are recorded, and the average daily feed intake, the average daily gain and the feed weight ratio of 14-15, 15-17, 17-19, 19-21 and 21-23 days of age are calculated. The results are shown in FIGS. 1 to 3.
The results show that the LPS obviously reduces the feed intake of the broiler chickens of 15, 17 and 23 days old and the body weight gain of the broiler chickens of 15 and 17 days old, and obviously increases the feed conversion ratio of the broiler chickens of 15 days old.
Effect of LPS stimulation on broiler serum corticosterone
The content of CORT in the serum samples was determined by chicken CORT ELISA kit, and the results are shown in FIG. 4.
As can be seen from the figure, LPS significantly elevated broilers serum corticosterone 72h after the first LPS injection (17 days of age).
Effect of LPS stimulation on broiler immune function
5.1 immune organ index
One average weight chicken was selected from each replicate group on days 0 (pre-injection), 1, 3, 5, 7, 9 and 10, and the thymus, spleen and bursa of fabricius were removed after necropsy by jugular vein bleeding, and weighed after fat removal. Thymus index, spleen index and bursa of Fabricius index were calculated and expressed as the fresh weight (g) of immune organs in the weight (kg) of broiler chickens, and the results are shown in FIGS. 5 to 7.
The results show that the spleen index of 19, 21 and 24 day old broiler chickens is obviously increased by LPS. The thymus index of the broiler chicken of 23 days old is obviously reduced.
5.2 inflammatory cytokine content
The contents of TNF-alpha and IL-1 beta in the serum sample were sequentially determined by ELISA detection kits for chicken TNF-alpha and IL-1 beta, respectively, and the results are shown in FIG. 8 and FIG. 9.
The result shows that LPS can obviously increase the content of TNF-alpha and IL-1 beta in the serum of the broiler chicken 72h (17 days old) after the first injection of LPS.
Conclusion of the study
According to experimental results, stress indexes and immunity indexes of the stress model constructed by the method are obviously changed. 72h after the first injection of the LPS, the LPS enables the corticosterone of the broiler chicken to be obviously increased, and the stress model is successfully constructed; the secretion level of TNF-alpha and IL-1 beta of the stress group is also obviously increased; meanwhile, LPS in the model has adverse effect on the production performance of the broiler chicken. Therefore, LPS with the dose of 0.5mg/Kg BW is injected into the abdominal cavity of 14-day old broilers for 10 consecutive days, and the LPS is injected once a day for 10 consecutive days, which can be used as the optimal condition for successfully constructing the chicken immune stress model.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for constructing a chicken immune stress model is characterized by comprising the following steps: continuously injecting the chicken by LPS to construct a chicken immune stress model;
wherein the dose of each injection is 0.4-0.6 mg/kg BW independently;
the number of continuous injections is 8-12;
the time interval of the continuous injection is 0.5-2 days.
2. The method for constructing chicken immune stress model according to claim 1, wherein the dose per injection is 0.5mg/kg BW.
3. The method for constructing chicken immune stress model according to claim 1, wherein the number of consecutive injections is 10.
4. The method for constructing chicken immune stress model according to claim 1, wherein the time interval of the continuous injection is 1 day.
5. The method for constructing chicken immune stress model according to claim 1, wherein the injection method comprises intraperitoneal injection.
6. The method for constructing a chicken immune stress model according to claim 1, wherein the chicken is a broiler chicken.
7. The method for constructing a chicken immune stress model according to claim 1, wherein the day age of the chicks is 12-16 days old.
8. The method for constructing a chicken immune stress model according to claim 7, wherein the day-old of the chicks is 14 days-old.
9. The chicken immune stress model constructed by the method for constructing the chicken immune stress model according to any one of claims 1 to 8.
10. Use of the chicken immune stress model of claim 9 in chicken feeding.
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