CN109601477A - A method of based on vitamin D receptor blood biochemical markers breeding Wuding Chicken - Google Patents
A method of based on vitamin D receptor blood biochemical markers breeding Wuding Chicken Download PDFInfo
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- 108050000156 vitamin D receptors Proteins 0.000 title claims abstract description 100
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- 210000004369 blood Anatomy 0.000 title claims abstract description 43
- 239000008280 blood Substances 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000009395 breeding Methods 0.000 title claims abstract description 20
- 230000001488 breeding effect Effects 0.000 title claims abstract description 20
- 238000011161 development Methods 0.000 claims abstract description 80
- 241000287828 Gallus gallus Species 0.000 claims abstract description 49
- 210000002966 serum Anatomy 0.000 claims abstract description 42
- 230000037182 bone density Effects 0.000 claims abstract description 35
- 230000037118 bone strength Effects 0.000 claims abstract description 22
- 238000005259 measurement Methods 0.000 claims abstract description 10
- 210000000988 bone and bone Anatomy 0.000 claims description 17
- 238000010219 correlation analysis Methods 0.000 claims description 8
- 210000001694 thigh bone Anatomy 0.000 claims description 8
- 238000010241 blood sampling Methods 0.000 claims description 7
- 102000005962 receptors Human genes 0.000 claims description 7
- 108020003175 receptors Proteins 0.000 claims description 7
- 230000002596 correlated effect Effects 0.000 claims description 5
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- 108090000790 Enzymes Proteins 0.000 claims description 4
- 229930003316 Vitamin D Natural products 0.000 claims description 4
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 4
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- 235000019166 vitamin D Nutrition 0.000 claims description 4
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- 150000003710 vitamin D derivatives Chemical class 0.000 claims description 4
- 229940046008 vitamin d Drugs 0.000 claims description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
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- 238000004458 analytical method Methods 0.000 claims description 2
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- 244000144977 poultry Species 0.000 abstract description 7
- 238000013459 approach Methods 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 4
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- FGRBYDKOBBBPOI-UHFFFAOYSA-N 10,10-dioxo-2-[4-(N-phenylanilino)phenyl]thioxanthen-9-one Chemical compound O=C1c2ccccc2S(=O)(=O)c2ccc(cc12)-c1ccc(cc1)N(c1ccccc1)c1ccccc1 FGRBYDKOBBBPOI-UHFFFAOYSA-N 0.000 description 1
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- 102000004067 Osteocalcin Human genes 0.000 description 1
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- 239000003446 ligand Substances 0.000 description 1
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Classifications
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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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of methods based on vitamin D receptor blood biochemical markers breeding Wuding Chicken, belong to poultry blood biochemical markers technical field, the present invention prepares serum, detects the concentration value of its vitamin D receptor (VDR) specifically includes the following steps: 1) acquire the blood sample of Wuding Chicken to be determined；2) concentration value that step 1) measures is compared with Wuding Chicken blood biochemical markers, Wuding Chicken to be measured is distinguished as skeleton development normal type Wuding Chicken and skeleton development ectype Wuding Chicken.Concentration variation of the present invention by measurement vitamin D receptor in skeleton development normal type and skeleton development ectype Wuding Chicken, bone density, bone strength in conjunction with the Wuding Chicken of detection, give skeleton development normal type and skeleton development ectype Wuding Chicken blood biochemical markers, screening high-quality chicken, it provides fundamental basis for the early stage breeding of Wuding Chicken, also it is the selection for probing into Wuding Chicken, provides new approaches in breeding work for Yunnan local chicken breeds.
Description
Technical field
The invention belongs to poultry blood biochemical markers technical fields, specifically, are related to a kind of based on vitamin D receptor blood
The method of liquid biochemical marker breeding Wuding Chicken.
Background technique
For bone as organ important in poultry body, function is to play support, protection and movement to the body of poultry
Effect.And a nearly century comes, the breeding of poultry high intensity production performance has a negative impact to poultry skeleton development, causes to increase
The disease incidence for adding the osteopathy such as skeleton development defect, osteoporosis, osteomalacia affects the sound development of domestic fowl farming.Family chicken bone is close
Degree and intensity are the important economic characters of family chicken, every year because the osteopathy such as skeleton development defect, osteoporosis, osteomalacia give family chicken industry
Cause huge economic loss.American Studies show that the death rate caused by skeleton deformity accounts for about 1.5%-3.2%, show bone
Deformity brings hundred million dollars of about 15-20 of loss (Jendral et al., 2008) to aviculture every year.China's broiler chicken is averaged osteopathy
Disease incidence is 3%-16%, and direct economic loss is about 50-80 hundred million.Wherein, since heredity and trophic factor cause skeleton development
Defect and cause the osteoporosis, osteomalacia, thigh bone of poultry the generations of osteopathy such as to become fragile, cause the huge economic damage of domestic fowl farming
It loses.
Vitamin D receptor (VDR) belongs to nuclear receptor superfamily, inherently a kind of nuclear factor for relying on ligand,
As long as research finds that karyocyte can express VDR.VDR is present in osteoblast and osteoclast.Therefore, VDR is to body
Interior calcium and phosphate radical, bone metabolism, mineral homeostasis and various histiocytic growths, differentiation and immunological regulation etc. have weight
The effect wanted.
Wuding Chicken mainly originates in the Wuding county of Yunnan Province Chuxiong autonomous prefecture, and figure is larger, and delicious meat is strong and brisk in taste, standby
Liked by people, becomes exclusive one " business card " in Wuding county or even Yunnan Province.Not yet in relation to vitamin D receptor to Wuding Chicken bone
The research of bone development impact, to meet the needs of people are to Local chicken breeds, it is necessary to propose that a kind of energy breeding skeleton development is good
The new method of Wuding, Yunnan chicken one blood biochemical markers is established by the research to vitamin D receptor and skeleton development,
It lays the groundwork for the early stage breeding of Wuding, Yunnan chicken.
Summary of the invention
In order to solve the problems, such as background technique, the present invention provides one kind to be based on vitamin D receptor blood biochemical
The method for marking breeding Wuding Chicken, by measurement vitamin D receptor in skeleton development normal type and skeleton development ectype Wuding County
Concentration variation in chicken, bone density, bone strength in conjunction with the Wuding Chicken of detection give skeleton development normal type and skeleton development
Ectype Wuding Chicken blood biochemical markers, screening high-quality chicken are provided fundamental basis for the early stage breeding of Wuding Chicken, also to probe into
The selection of Wuding Chicken provides new approaches in working in the rain for Yunnan local chicken breeds.
To achieve the above object, the present invention is achieved through the following technical solutions:
The method based on vitamin D receptor blood biochemical markers breeding Wuding Chicken, specifically includes the following steps:
1) blood sample for acquiring Wuding Chicken to be determined, prepares serum, detects the concentration value of its vitamin D receptor (VDR)；
2) concentration value that step 1) measures is compared with Wuding Chicken blood biochemical markers, Wuding Chicken to be measured is distinguished as
Skeleton development normal type Wuding Chicken and skeleton development ectype Wuding Chicken.
Further, Wuding Chicken blood biochemical markers establishment specifically includes the following steps:
A. skeleton development normal type Wuding Chicken family and skeleton development ectype are randomly choosed from Wuding Chicken sources group
Wuding Chicken family detects its thigh bone bone density, bone strength respectively, acquires blood sample, prepares serum；
B. Serum Vitamin D receptor (VDR) content assaying method: vitamin D receptor (VDR) ELISA reagent is selected
Box, using the level of chicken vitamin D receptor (VDR) in double antibody sandwich method measurement sample.With the chicken vitamin D receptor of purifying
(VDR) antibody is coated with microwell plate, and solid phase antibody is made, and sequentially adds vitamin D receptor into the microwell plate of coating monoclonal antibody
(VDR), then in conjunction with vitamin D receptor (VDR) antibody of HRP label, antibody-antigene-hrp-antibody complex is formed, is passed through
Thoroughly add substrate TMB colour developing after washing.TMB converts au bleu under the catalysis of HRP enzyme, and is converted under the action of an acid final
Yellow.Vitamin D receptor (VDR) is positively correlated in the depth and sample of color.Suction is measured under 450nm wavelength with microplate reader
Luminosity (OD value) calculates vitamin D receptor (VDR) concentration in sample by standard curve；
C. according to step b the data obtained, skeleton development normal type Wuding Chicken and skeleton development ectype Wuding County chicken blood are obtained
The concentration value of clear vitamin D receptor carries out Serum Vitamin D further according to the thigh bone bone density of selected Wuding Chicken, bone strength
The correlation analysis of receptor and bone density, bone strength；
D. according to the obtained analysis of step c as a result, determining the blood biochemical markers of Wuding Chicken.
Further, the blood biochemical markers of the Wuding Chicken are the concentration value of vitamin D receptor, when a monthly age, work as dimension
The concentration value of raw element D receptor is determined as skeleton development normal type Wuding Chicken in 10.36ng/ml or more, otherwise different for skeleton development
Normal type Wuding Chicken；When two monthly ages, when the concentration value of blood calcium is in 10.25ng/ml or more, it is determined as skeleton development normal type Wuding County
Chicken, on the contrary it is skeleton development ectype Wuding Chicken.
Further, it cuts off the water supply before blood sampling, is 12 hours jejunitas, carrying out wing venous blood sampling the next morning；It is collected with centrifuge tube
Blood sample, standing 30min, 3000rpm is centrifuged 20min at room temperature, takes supernatant, obtains serum.
Beneficial effects of the present invention:
The present invention is dense in skeleton development normal type and skeleton development ectype Wuding Chicken by measurement vitamin D receptor
Degree variation, bone density, bone strength in conjunction with the Wuding Chicken of detection, give skeleton development normal type and skeleton development ectype is military
Determine chicken blood biochemical marker, screening high-quality chicken is provided fundamental basis for the early stage breeding of Wuding Chicken, also to probe into Wuding Chicken
Selection provides new approaches in working in the rain for Yunnan local chicken breeds.
Specific embodiment
It, below will be to preferred reality of the invention in order to keep the purpose of the present invention, technical scheme and beneficial effects clearer
It applies example to be described in detail, to facilitate the technical staff to understand.
Embodiment 1
1. the acquisition and preservation of laboratory sample
(1) preparation of serum
A monthly age is randomly selected in skeleton development normal type Wuding Chicken family and skeleton development ectype Wuding Chicken family
With two monthly age skeleton development normal types and skeleton development ectype Wuding Chicken each 20, its blood measuring bone metabolism phase is acquired respectively
Close biochemical indicator.It cuts off the water supply before blood sampling, is 12 hours jejunitas, carrying out wing venous blood sampling the next morning.Blood is collected with 10ml centrifuge tube
Sample 3-4ml, standing 30min, 3000rpm is centrifuged 20min at room temperature, takes supernatant (serum) in the centrifuge tube of 1.5ml, puts
Enter and is saved backup in -20 DEG C of refrigerators.
(2) acquisition of thigh bone sample
20 are respectively butchered respectively at 1,2 monthly age skeleton development normal types and skeleton development ectype Wuding Chicken, acquisition test
The femur and shin bone of the left and right leg of chicken.The soft tissue and connective tissue for removing bone surface, it is dry with 0.75% normal saline flushing
Only, and with the gauze for being moistened with physiological saline wrap up, be tamping with valve bag, -20 DEG C save backup in measurement bone density and bone strength
Index.
2. thigh bone Bone mineral density
After test chicken left femur and left tibia thaw, using QDR-4500 type Dual-energy X-rays absorptionmetry (Hologic company,
The U.S.) measurement bone density.
3. thigh bone bone strength detects
After test chicken left femur and left tibia thaw, by bone samples be placed in electronic universal tester (AG-IS, day the island proper
Saliva company) under carry out three point bending test, to detect bone strength index: elasticity modulus, yield stress, bursting stress, fracture are answered
Power.Span calculation formula used by this test are as follows: L=(a+3b) ± 0.5b, wherein a is the diameter of supporting rail, and b is bone
Diameter.Loading velocity is 2mm/min.Detection unit is Kunming University of Science and Technology's Materials Academy experimental center.
4. blood parameters detect
The measuring method of vitamin D receptor (VDR) is according to (Nanjing Bo Pai Bioisystech Co., Ltd, kit production firm
(U.S. R&D dispense kit)) operating method that provides of operation instructions carries out:
Serum Vitamin D receptor (VDR) content assaying method: using chicken vitamin D in double antibody sandwich method measurement sample
The level of receptor (VDR).It is coated with microwell plate with chicken vitamin D receptor (VDR) antibody of purifying, solid phase antibody is made, toward coating
It is sequentially added in the microwell plate of monoclonal antibody vitamin D receptor (VDR), then in conjunction with vitamin D receptor (VDR) antibody of HRP label,
Antibody-antigene-hrp-antibody complex is formed, after thoroughly washing plus substrate TMB develops the color.TMB turns under the catalysis of HRP enzyme
Chemical conversion blue, and it is converted to final yellow under the action of an acid.Vitamin D receptor (VDR) is in the depth and sample of color
It is positively correlated.Measured under 450nm wavelength with microplate reader absorbance (OD value), by standard curve calculate sample in vitamin D by
Body (VDR) concentration.
5. data statistics
1 one monthly age of table Wuding Chicken bone density, bone strength data
Note: colleague's data, " * " indicate significant difference (P < 0.05).
2 two monthly age of table Wuding Chicken bone density, bone strength data
Note: colleague's data, " * " indicate significant difference (P < 0.05).
The content of 3 one monthly age of table Wuding Chicken Serum Vitamin D receptor
Note: colleague's data, " * " indicate significant difference (P < 0.05).
The content of 4 two monthly age of table Wuding Chicken Serum Vitamin D receptor
Note: colleague's data, " * " indicate significant difference (P < 0.05).
5 one monthly age of table skeleton development large fowl Serum Vitamin D receptor and bone density, bone strength correlation analysis
Note: " * * " indicates that, in 0.01 horizontal significant correlation, " * " is indicated in 0.05 horizontal significant correlation.
6 one monthly age of table skeleton development ectype chicken serum vitamin D receptor and bone density, bone strength correlation analysis
Note: " * * " indicates that, in 0.01 horizontal significant correlation, " * " is indicated in 0.05 horizontal significant correlation.
7 two monthly age of table skeleton development large fowl Serum Vitamin D receptor and bone density, bone strength correlation analysis
Note: " * * " indicates that, in 0.01 horizontal significant correlation, " * " is indicated in 0.05 horizontal significant correlation.
8 two monthly age of table skeleton development ectype chicken serum vitamin D receptor and bone density, bone strength correlation analysis
Note: " * * " indicates that, in 0.01 horizontal significant correlation, " * " is indicated in 0.05 horizontal significant correlation.
6. interpretation of result
By Tables 1 and 2 it is found that bone density data when one, two monthly age: the femur bone of skeleton development normal type Wuding Chicken is close
Degree content, shin bone bone density are above skeleton development ectype Wuding Chicken, wherein two time differences at monthly age are different significant (P < 0.05)；
One, bone strength data when two monthly ages: femur backbone elasticity modulus, the tibial bone dry elasticity mould of skeleton development normal type Wuding Chicken
Amount, femur backbone yield stress, shin bone backbone yield stress, femur backbone maximum stress, shin bone backbone maximum stress, femur bone
Dry fracture strength, shin bone backbone fracture strength are all remarkably higher than skeleton development ectype Wuding Chicken (P < 0.05).
By table 3 and table 4 it is found that VDR content data in Wuding County's chicken serum when one, two monthly age: skeleton development normal type is military
Determine VDR content in chicken serum and is significantly higher than skeleton development ectype Wuding Chicken (P < 0.05).
By the correlation analysis of one, two monthly age Wuding Chicken bone densities and bone strength it is found that when one, two monthly age, bone hair
Educate the femoral bmd content of normal type Wuding Chicken and skeleton development ectype Wuding Chicken with shin bone bone density, femur bone
Dry elasticity modulus, tibial bone dry elasticity modulus, femur backbone yield stress, shin bone backbone yield stress, femur backbone maximum are answered
Power, shin bone backbone maximum stress, femur backbone fracture strength, shin bone backbone fracture strength are positively correlated and (are shown in Table 5, table 6, table
7, table 8).By the correlation analysis of VDR content in one, two monthly age Wuding Chicken serum and bone density and bone strength it is found that one, two
When the monthly age, VDR content is close with femur bone in the serum of skeleton development normal type Wuding Chicken and skeleton development ectype Wuding Chicken
Spend content, shin bone bone density, femur backbone elasticity modulus, tibial bone dry elasticity modulus, femur backbone yield stress, shin bone
Backbone yield stress, femur backbone maximum stress, shin bone backbone maximum stress, femur backbone fracture strength, the fracture of shin bone backbone
Stress is positively correlated and (is shown in Table 5, table 6, table 7, table 8), wherein VDR content and femoral bmd related coefficient exist in serum
0.475~0.663, with shin bone bone density related coefficient 0.351~0.694；It is obtained by above-mentioned correlation data result, serum
Middle VDR content has decisive role to early stage Wuding Chicken skeleton development, can be used as the excellent skeleton development Wuding Chicken of early stage breeding
Blood biochemical markers.
Embodiment 2
In the monthly age Wuding Chicken 100 that Wuding, Yunnan and farm select uniformity of type, are in a good state of health, examine respectively
VDR concentration value in its blood is surveyed, statistical packet is carried out according to gained concentration value in a certain range, obtains following statistical form.
The content of 9 one monthly age of table Wuding Chicken VDR counts
By this 5 group of monthly age Wuding Chicken in strict accordance with immune programme, raised under identical rearing conditions to two monthly ages, difference
Its shin bone bone density (being detected using portable borne densitometers) is measured, each group statistical result is following (being shown in Table 10).
10 1 monthly age of table Wuding Chicken mean tibial bone density statistics
As seen from Table 10, the 4th group of mean tibial bone density is significantly increased compared with first three groups, and the five, the six groups successively rise
Height, reasoning out the 4th group of interval range is a significant break range, i.e. when a monthly age, VDR content range in serum
(10.33-10.41ng/ml) is break range.
Above-mentioned steps are being repeated to the chicken within the scope of the break, finally obtaining 10.36ng/ml is that VDR contains in serum
The significant break of amount.
The VDR content in Wuding County's chicken serum at above-mentioned two monthly age is detected, as a result following (being shown in Table 11)
The content of 11 2 monthly age of table Wuding Chicken VDR counts
By this 5 group of two monthly age Wuding Chicken in strict accordance with immune programme, raised under identical rearing conditions to three monthly ages, difference
Its shin bone bone density (wherein there are 2 Wuding Chickens because of leg joint injury, to be not counted in statistical result) is measured, each group statistical result is as follows
(being shown in Table 12).
The Wuding Chicken mean tibial bone density accrual accounting of 12 3 monthly age of table
As seen from Table 12, third group mean tibial bone density increment significantly increases for two groups earlier above, and the 4th group to the 6th
Group Wuding Chicken mean tibial bone density increment is in raising trend, and reasoning out 3rd interval range is a significant break range,
That is when two monthly age, VDR content (10.17-10.27ng/ml) is break range in serum.
By within the scope of two breaks Wuding Chicken and adjacent interval in VDR concentration value in 10.17-10.27ng/ml
Chicken combination, then repeats above-mentioned steps, finally obtaining 10.25ng/ml is the significant of VDR content in serum
Break.
In Yunnan, experiment, conclusion and above-mentioned experiment knot is repeated several times by the above experimental procedure in other Wuding Chicken farms
By identical, conclusion are as follows: when a monthly age, VDR content range (10.33-10.41ng/ml) is significant break range in serum, and
10.36ng/ml is the significant break of VDR content in serum；When two monthly ages, VDR content (10.17-10.27ng/ml) in serum
It is break range, and 10.25ng/ml is the significant break of VDR content in serum.
Embodiment 3
1. the preparation of experimental material
In Wuding, Yunnan county, each Wuding Chicken farm sampling one monthly age Wuding Chicken totally 120 (includes skeleton development normal type
With skeleton development ectype), two monthly age Wuding Chickens totally 120 (include skeleton development normal type and skeleton development ectype), point
Its shin bone bone density is not measured；Wing venous blood sampling is carried out, blood sample is collected with 5ml centrifuge tube, stands 30min at room temperature
3000rpm is centrifuged 20min, takes supernatant, extremely serum.
2. detecting the concentration value of VDR in serum
It carries out strictly regulating operation according to VDR ELISA (ELISA) kit, be measured using double antibody sandwich method
The level of chicken osteocalcin (VDR) in sample is measured absorbance (OD value) under 450nm wavelength with microplate reader, passes through standard curve
Calculate osteocalcin (VDR) content in sample.
3. comparative analysis
When one monthly age, shin bone bone density is in 4.4176g/cm2Below is 31, in 0.4356g/cm2It is above shared
72, i.e. in 120 Wuding Chickens, skeleton development ectype Wuding Chicken 31, skeleton development normal type Wuding Chicken 72.At 31
In skeleton development ectype Wuding Chicken, there is in 29 Wuding County's chicken serums VDR content in 10.36ng/ml hereinafter, to account for skeleton development different
The 93.55% of normal type Wuding Chicken.In 72 skeleton development normal type Wuding Chickens, there is VDR content in 67 Wuding County's chicken serums to exist
10.36ng/ml or more accounts for the 93.06% of skeleton development normal type Wuding Chicken.
When two monthly ages, shin bone bone density is in 0.4066g/cm2Below is 33, in 0.5124g/cm2It is above shared
76, i.e. in 120 Wuding Chickens, skeleton development ectype Wuding Chicken 33, skeleton development normal type Wuding Chicken 76.At 33
In skeleton development ectype Wuding Chicken, there is in 28 Wuding County's chicken serums VDR content in 10.25ng/ml hereinafter, to account for skeleton development different
The 84.85% of normal type Wuding Chicken.In 76 skeleton development normal type Wuding Chickens, there is VDR content in 71 Wuding County's chicken serums to exist
10.25ng/ml or more accounts for the 93.42% of skeleton development normal type Wuding Chicken.
In conclusion the present invention is by VDR content in measurement blood in skeleton development normal type and skeleton development ectype
Concentration variation in Wuding Chicken, Wuding Chicken bone density, bone strength index in conjunction with measurement give skeleton development normal type and bone
Blood biochemical markers in bone dysplastic type Wuding Chicken, screening high-quality chicken are provided fundamental basis, together for the early stage breeding of Wuding Chicken
When be also to explore the high-quality indigenous a breed of chicken new method in plateau, new approaches are provided in breeding work for Yunnan local chicken breeds.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention rather than limits, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (4)
1. a kind of method based on vitamin D receptor blood biochemical markers breeding Wuding Chicken, it is characterised in that: specifically include following
Step:
1) blood sample for acquiring Wuding Chicken to be determined, prepares serum, detects the concentration value of its vitamin D receptor (VDR)；
2) concentration value that step 1) measures is compared with Wuding Chicken blood biochemical markers, Wuding Chicken to be measured is distinguished as bone
Develop normal type Wuding Chicken and skeleton development ectype Wuding Chicken.
2. a kind of method based on vitamin D receptor blood biochemical markers breeding Wuding Chicken according to claim 1, special
Sign is: the establishments of Wuding Chicken blood biochemical markers specifically includes the following steps:
A. skeleton development normal type Wuding Chicken family and skeleton development ectype Wuding County are randomly choosed from Wuding Chicken sources group
Chicken family detects its thigh bone bone density, bone strength respectively, acquires blood sample, prepares serum；
B. Serum Vitamin D receptor (VDR) content assaying method: vitamin D receptor (VDR) enzyme linked immunological kit is selected, is answered
With the level of chicken vitamin D receptor in double antibody sandwich method measurement sample；Micropore is coated with the chicken vitamin D receptor antibody of purifying
Solid phase antibody is made in plate, and vitamin D receptor, then the vitamin D with HRP label are sequentially added into the microwell plate of coating monoclonal antibody
Receptor antibody combines, and forms antibody-antigene-hrp-antibody complex, and after thoroughly washing plus substrate TMB develops the color；TMB exists
Au bleu is converted under the catalysis of HRP enzyme, and is converted to final yellow under the action of an acid；Life is tieed up in the depth and sample of color
Plain D receptor is positively correlated；It is measured under 450nm wavelength with microplate reader absorbance (OD value), is calculated in sample by standard curve
Vitamin D receptor concentration；
C. according to step b the data obtained, skeleton development normal type Wuding Chicken and skeleton development ectype Wuding County's chicken serum dimension are obtained
The concentration value of raw element D receptor carries out Serum Vitamin D receptor further according to the thigh bone bone density of selected Wuding Chicken, bone strength
With the correlation analysis of bone density, bone strength；
D. according to the obtained analysis of step c as a result, determining the blood biochemical markers of Wuding Chicken.
3. -2 is described in any item a kind of based on vitamin D receptor blood biochemical markers breeding Wuding Chicken according to claim 1
Method, it is characterised in that: the blood biochemical markers of the Wuding Chicken are the concentration value of vitamin D receptor, when a monthly age, when dimension is given birth to
The concentration value of plain D receptor is determined as skeleton development normal type Wuding Chicken in 10.36 ng/ml or more, otherwise different for skeleton development
Normal type Wuding Chicken；When two monthly ages, when the concentration value of blood calcium is in 10.25 ng/ml or more, it is determined as skeleton development normal type Wuding County
Chicken, on the contrary it is skeleton development ectype Wuding Chicken.
4. -2 is described in any item a kind of based on vitamin D receptor blood biochemical markers breeding Wuding Chicken according to claim 1
Method, it is characterised in that: cut off the water supply before blood sampling, is 12 hours jejunitas, carrying out wing venous blood sampling the next morning；Blood is collected with centrifuge tube
Sample, standing 30min, 3000rpm is centrifuged 20min at room temperature, takes supernatant, obtains serum.
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