阅读说明：本技术 维生素d3对绵羊的免疫力和生殖功能的影响实验方法 (Experimental method for influence of vitamin D3 on immunity and reproductive function of sheep ) 是由 庞全海 李振 王天元 陈昱筱 王帅 秦红 张娟 梁睿 李颖平 赵礼军 谢莹 赵 于 2019-12-07 设计创作，主要内容包括：本发明公开了维生素D3对绵羊的免疫力和生殖功能的影响实验方法,包括以下步骤：步骤一,试验动物分组与饲养；步骤二,绵羊组织取样；步骤三,绵羊组织总RNA提取；步骤四,cDNA合成；步骤五,细胞凋亡相关基因及VDR的引物设计；步骤六,荧光定量PCR；步骤七,数据统计与分析；对照组维生素D3的添加量为0IU/kg,试验组维生素D3的添加量分别为300IU/kg、600IU/kg、900IU/kg和1200IU/kg；该维生素D3对绵羊的免疫力和生殖功能的影响实验方法,通过微贮豆秸中添加不同浓度维生素D3饲喂绵羊探索研究维生素D3对绵羊细胞凋亡的作用机制,最后得出维生素D3添加水平在300IU/kg时有利于提高绵羊免疫力及降低组织细胞凋亡,实验过程严谨,结果可靠。(The invention discloses an experimental method for influence of vitamin D3 on immunity and reproductive function of sheep, which comprises the following steps: grouping and feeding test animals; sampling sheep tissues; step three, extracting total RNA of the sheep tissue; step four, cDNA synthesis; designing primers of apoptosis related genes and VDRs; step six, performing fluorescence quantitative PCR; seventhly, data statistics and analysis are carried out; the addition amount of the control group vitamin D3 is 0IU/kg, and the addition amounts of the test group vitamin D3 are 300IU/kg, 600IU/kg, 900IU/kg and 1200IU/kg respectively; according to the experimental method for the influence of the vitamin D3 on the immunity and reproductive function of sheep, the vitamin D3 with different concentrations is added into the micro-storage beanstalk to feed the sheep to explore and research the action mechanism of the vitamin D3 on sheep apoptosis, and finally, the vitamin D3 is obtained, when the addition level is 300IU/kg, the sheep immunity is favorably improved, the tissue apoptosis is favorably reduced, the experimental process is rigorous, and the result is reliable.)

1. Experimental method for influence of vitamin D3 on immunity and reproductive function of sheep comprises the following steps: grouping and feeding test animals; sampling sheep tissues; step three, extracting total RNA of the sheep tissue; step four, cDNA synthesis; designing primers of apoptosis related genes and VDRs; step six, performing fluorescence quantitative PCR; seventhly, data statistics and analysis are carried out; the method is characterized in that:

in the first step, the grouping and feeding of the test animals comprises the following steps:

1) selecting 25 male sheep of 2 months old, weighing 20-25kg, randomly dividing into 5 groups, each group having 5 repetitions, and each repetition having 1 sheep;

2) the pre-feeding period is 10 days, the testing period is 70 days, all groups are fed with basic daily ration and the micro-storage beanstalk, the addition amount of the vitamin D3 of the control group is 0IU/kg, and the addition amounts of the vitamin D3 of the test group are 300IU/kg, 600IU/kg, 900IU/kg and 1200IU/kg respectively;

3) kneading beanstalk into 1-3cm, uniformly mixing microbial agent, brown sugar and water, uniformly spreading on the straw, compacting, sealing and packaging by using a packaging machine during sealing, and fermenting for one month to start microbial fermentation beanstalk;

4) the daily food intake of each sheep is adjusted at any time according to the daily gain of the sheep, the ratio of semen to crude is 1: 2 (based on dry matter), the feeding is carried out for 2 times in the test period, the feeding is carried out in the order of crude first and fine first, and the water is freely drunk;

in the second step, 4 sheep are randomly selected from each group for slaughter sampling on the last day of the test period, the liver, the kidney, the spleen, the thymus and the mesenteric lymph are respectively collected and placed in a freezing storage tube, and liquid nitrogen is put for standby;

in the third step, the extraction of the total RNA of the sheep tissue comprises the following steps:

1) sterilizing a mortar at high pressure, then carrying out cooling treatment on the mortar by using liquid nitrogen, then quickly taking out sheep tissues from the liquid nitrogen, grinding the sheep tissues in the mortar, putting the ground tissues in an EP (ethylene propylene glycol) tube, adding a proper amount of Trizol, marking, vibrating and uniformly mixing with force, and standing on ice;

2) placing the mixed solution in a centrifuge at 4 deg.C, and centrifuging for 10 min; the supernatant was slowly aspirated, transferred to another EP tube and incubated at room temperature for 5 min; adding 200 μ L chloroform, shaking, and centrifuging at 4 deg.C for 15 min; slowly sucking the supernatant, transferring into another EP tube, adding equal volume of isopropanol, standing at room temperature for 10min, centrifuging at 4 deg.C for 10min, and allowing white precipitate to appear;

3) discarding isopropanol in an EP tube, adding a proper amount of 1mL of glacial ethanol, and centrifuging for 15min at 4 ℃; discarding the ethanol, drying at room temperature for 10min, adding appropriate amount of 20 μ L sterile RNase H₂Dissolving white precipitate by O; taking a proper amount of 1 mu L of extracted total RNA, determining the concentration of the total RNA by using a nucleic acid determinator, and storing the residual total RNA sample in a refrigerator at the temperature of-80 ℃ for later use;

wherein in the fourth step, the cDNA synthesis comprises the following steps:

1) the RNA sample was prepared in 4. mu.L, 2. mu.L of AccuRT Reaction Mix (4X), and 2. mu.L of nucleic-free H₂O, 2. mu.L of AccuRT Reaction Stopper, 4. mu.L of 5 × All-In-One RT MasterMix and 6. mu.L of nucleic-free H₂Preparing a cDNA reaction system by using O;

2) placing the prepared reagent into a preheated PCR instrument, and reacting at 25 ℃ for 10min, 42 ℃ for 50min and 85 ℃ for 5min respectively;

in the fifth step, NCBI searches gene sequences of sheep VDR, Bcl2, BAX, Caspase9, Caspase3 and 18s, designs primers by using Priem 5.0 software according to conserved regions (CDs) of the genes, and entrusts other bioengineering companies to synthesize the primer sequences;

in the sixth step, the fluorescent quantitative PCR comprises the following steps:

1) 10 μ L of EvaGreen 2 xqPCR MasterMix, 0.6 μ L of Forward Primer, 0.6 μ L of Reverseprimer, 2 μ L of cDNA and 6.8 μ L of RNase Free ddH₂Preparing a 20 mu L Real-time PCR reaction system according to the proportion of O;

2) diluting a cDNA sample synthesized by reverse transcription, adding the diluted cDNA sample into a reaction system, centrifuging, fully and uniformly mixing, and placing the mixture into a preheated qRT-PCR instrument, wherein the reaction steps are as follows:

① activating enzyme, at 95 deg.C for 10min, and circulating number is 1;

② modified, 95 ℃, 15secs, cycle number 40;

③ annealing and expanding, 60 ℃, 60secs, cycle number is 40;

④ the curve is melted, 55 ℃/95 ℃, 30secs/60secs, the cycle number is 81;

wherein in the seventh step, the CT value of the fluorescence quantification is 2^-ΔΔCTThe algorithm calculates the relative expression and analyzes the data using One-way analysis of variance (ANOVA) in Graphpadprism5 software.

2. Experimental method of the effect of vitamin D3 on sheep's immunity and reproductive function, according to claim 1, characterized in that: in the step one 3), the proportion of the microbial agent, the brown sugar and the water is 1: 20: 80, and the total number of probiotics of the microbial inoculum is more than or equal to 5 multiplied by 10⁷Per gram.

3. Experimental method of the effect of vitamin D3 on sheep's immunity and reproductive function, according to claim 1, characterized in that: in the step one 4), the feeding management of all the test sheep conforms to the ethical welfare protection regulations of animals.

4. Experimental method of the effect of vitamin D3 on sheep's immunity and reproductive function, according to claim 1, characterized in that: in the step one 4), the basic daily ration consists of 42% of corn, 30% of wheat bran, 24% of soybean meal and 4% of premix.

5. Experimental method of the effect of vitamin D3 on sheep's immunity and reproductive function, according to claim 1, characterized in that: in the step one 4), the feeding time is 8: 00 and 18: 00.

6. experimental method of the effect of vitamin D3 on sheep's immunity and reproductive function, according to claim 1, characterized in that: in the third step 2), the centrifugal speed is 12000 r/min.

7. Experimental method of the effect of vitamin D3 on sheep's immunity and reproductive function, according to claim 1, characterized in that: in the seventh step, the difference is significant when P is less than 0.05, and the difference is very significant when P is less than 0.01.

Technical Field

The invention relates to the technical field of vitamin D3 receptors, in particular to an experimental method for influence of vitamin D3 on sheep immunity and reproductive function.

Background

As a big agricultural country, China has abundant crop straws, and the recycling of the straws is always a social concern. The straw burning is the most economic, fastest and effective straw treatment method. However, the straw burning will cause incomplete combustion, and release a large amount of toxic air pollutants, such as volatile organic compounds, carbon monoxide, fine/inhalable particles, carcinogenic polycyclic aromatic hydrocarbons, etc., and in japan, the straw burning is found to be related to asthma in children. Straw incineration also causes environmental problems and loss of straw nutrients. Therefore, the problem of recycling straw resources is urgent. In recent years, Vitamin D3 Receptor (VDR) has been of increasing interest, and its physiological functions have been discovered in turn. The earliest reports of VDR were that it plays an important role in maintaining bone health and calcium homeostasis. Furthermore, VDR plays an important role in inflammation, diabetes, hypertension, atherosclerosis, cell proliferation and differentiation, tumor tissue, and the like. Vitamin D3 can improve the expression ability of VDR, and the level of VDR can regulate vitamin D3 after transcription and translation. It follows that the physiopathological role of vitamin D3 can be explored by studying the role of VDR in tissues. Therefore, it is necessary to design an experimental method for the influence of vitamin D3 on the immunity and reproductive function of sheep.

Disclosure of Invention

The invention aims to provide an experimental method for the influence of vitamin D3 on the immunity and reproductive function of sheep, so as to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme: experimental method for influence of vitamin D3 on immunity and reproductive function of sheep comprises the following steps: grouping and feeding test animals; sampling sheep tissues; step three, extracting total RNA of the sheep tissue; step four, cDNA synthesis; designing primers of apoptosis related genes and VDRs; step six, performing fluorescence quantitative PCR; seventhly, data statistics and analysis are carried out;

in the first step, the grouping and feeding of the test animals comprises the following steps:

1) selecting 25 male sheep of 2 months old, weighing 20-25kg, randomly dividing into 5 groups, each group having 5 repetitions, and each repetition having 1 sheep;

2) the pre-feeding period is 10 days, the testing period is 70 days, all groups are fed with basic ration and the micro-storage beanstalk, the addition amount of the vitamin D3 of the control group is OIU/kg, and the addition amounts of the vitamin D3 of the test group are 300IU/kg, 600IU/kg, 900IU/kg and 1200IU/kg respectively;

3) kneading beanstalk into 1-3cm, uniformly mixing microbial agent, brown sugar and water, uniformly spreading on the straw, compacting, sealing and packaging by using a packaging machine during sealing, and fermenting for one month to start microbial fermentation beanstalk;

4) the daily food intake of each sheep is adjusted at any time according to the daily gain of the sheep, the ratio of semen to crude is 1: 2 (based on dry matter), the feeding is carried out for 2 times in the test period, the feeding is carried out in the order of crude first and fine first, and the water is freely drunk;

in the second step, 4 sheep are randomly selected from each group for slaughter sampling on the last day of the test period, the liver, the kidney, the spleen, the thymus and the mesenteric lymph are respectively collected and placed in a freezing storage tube, and liquid nitrogen is put for standby;

in the third step, the extraction of the total RNA of the sheep tissue comprises the following steps:

1) sterilizing a mortar at high pressure, then carrying out cooling treatment on the mortar by using liquid nitrogen, then quickly taking out sheep tissues from the liquid nitrogen, grinding the sheep tissues in the mortar, putting the ground tissues in an EP (ethylene propylene glycol) tube, adding a proper amount of Trizol, marking, vibrating and uniformly mixing with force, and standing on ice;

2) placing the mixed solution in a centrifuge at 4 deg.C, and centrifuging for 10 min; the supernatant was slowly aspirated, transferred to another EP tube and incubated at room temperature for 5 min; adding 200 μ L chloroform, shaking, and centrifuging at 4 deg.C for 15 min; slowly sucking the supernatant, transferring into another EP tube, adding equal volume of isopropanol, standing at room temperature for 10min, centrifuging at 4 deg.C for 10min, and allowing white precipitate to appear;

3) discarding isopropanol in an EP tube, adding a proper amount of 1mL of glacial ethanol, and centrifuging for 15min at 4 ℃; discarding the ethanol, drying at room temperature for 10min, adding appropriate amount of 20 μ L sterile RNase H₂Dissolving white precipitate by O; taking a proper amount of 1 mu L of extracted total RNA, determining the concentration of the total RNA by using a nucleic acid determinator, and storing the residual total RNA sample in a refrigerator at the temperature of-80 ℃ for later use; wherein in the fourth step, the cDNA synthesis comprises the following steps:

1) the RNA sample was prepared in 4. mu.L, 2. mu.L of AccuRT Reaction Mix (4X), and 2. mu.L of nucleic-fresh H₂O, 2. mu.L of AccuRT Reaction Stopper, 4. mu.L of 5 × All-In-One RT MasterMix and 6. mu.L of nucleic-free H₂Preparing a cDNA reaction system by using O;

2) placing the prepared reagent into a preheated PCR instrument, and reacting at 25 ℃ for 10min, 42 ℃ for 50min and 85 ℃ for 5min respectively;

in the fifth step, NCBI searches gene sequences of sheep VDR, Bcl2, BAX, Caspase9, Caspase3 and 18s, designs primers by using Priem 5.0 software according to conserved regions (CDs) of the genes, and entrusts other bioengineering companies to synthesize the primer sequences;

in the sixth step, the fluorescent quantitative PCR comprises the following steps:

1) 10 μ L of EvaGreen 2 xqPCR MasterMix, 0.6 μ L of Forward Primer, 0.6 μ L of Reverse Primer, 2 μ L of cDNA and 6.8 μ L of RNase FreedH₂Preparing a 20 mu L Real-timePCR reaction system according to the proportion of O;

2) diluting a cDNA sample synthesized by reverse transcription, adding the diluted cDNA sample into a reaction system, centrifuging, fully and uniformly mixing, and placing the mixture into a preheated qRT-PCR instrument, wherein the reaction steps are as follows:

① activating enzyme, at 95 deg.C for 10min, and circulating number is 1;

② modified, 95 ℃, 15secs, cycle number 40;

③ annealing and expanding, 60 ℃, 60secs, cycle number is 40;

④ the curve is melted, 55 ℃/95 ℃, 30secs/60secs, the cycle number is 81;

wherein in the seventh step, the CT value of the fluorescence quantification is 2^-ΔΔCTThe algorithm calculates the relative expression and analyzes the experimental data using One-way analysis of variance (ANOVA) in Graphpad Prism5 software.

According to the technical scheme, in the step one 3), the ratio of the microbial agent to the brown sugar to the water is 1: 20: 80, and the total number of probiotics of the microbial inoculum is more than or equal to 5 multiplied by 10⁷Per gram.

According to the technical scheme, in the step one 4), the feeding management of all the test sheep conforms to the ethical welfare protection regulations of animals.

According to the technical scheme, in the step one 4), the basic daily ration consists of 42% of corn, 30% of wheat bran, 24% of soybean meal and 4% of premix.

According to the technical scheme, in the step one 4), the feeding time is 8: 00 and 18: 00.

according to the technical scheme, in the step three 2), the centrifugal speed is 12000 r/min.

According to the technical scheme, in the seventh step, the difference is significant when P is less than 0.05, and the difference is very significant when P is less than 0.01.

Compared with the prior art, the invention has the following beneficial effects: according to the experimental method for the influence of the vitamin D3 on the immunity and reproductive function of sheep, the vitamin D3 with different concentrations is added into the micro-storage beanstalk to feed the sheep to explore and research the action mechanism of the vitamin D3 on sheep apoptosis, and finally, the vitamin D3 is obtained, when the addition level is 300IU/kg, the sheep immunity is favorably improved, the tissue apoptosis is favorably reduced, the experimental process is rigorous, and the result is reliable.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIGS. 1, 2, 3, 4 and 5 are schematic diagrams showing the effect of vitamin D3 on the expression of apoptotic genes and VDRs in liver in the experiment of the present invention;

FIGS. 6, 7, 8, 9 and 10 are graphs showing the effect of vitamin D3 on the expression of apoptotic genes and VDRs in the kidney in experiments of the invention;

FIGS. 11, 12, 13, 14 and 15 are graphs showing the effect of vitamin D3 on the expression of apoptotic genes and VDRs in the spleen in experiments of the invention;

FIGS. 16, 17, 18, 19 and 20 are graphs showing the effect of vitamin D3 on the expression of the apoptotic gene and VDR in the thymus in the experiments of the present invention;

FIGS. 21, 22, 23, 24 and 25 are graphs showing the effect of vitamin D3 on the expression of the apoptotic gene and VDR in mesenteric lymph in experiments of the invention;

FIG. 26 is an overall flow chart of the present invention;

in the figure: p < 0.05, P < 0.01, P < 0.001.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

Referring to fig. 1-26, the present invention provides a technical solution: experimental method for influence of vitamin D3 on immunity and reproductive function of sheep comprises the following steps: grouping and feeding test animals; sampling sheep tissues; step three, extracting total RNA of the sheep tissue; step four, cDNA synthesis; designing primers of apoptosis related genes and VDRs; step six, performing fluorescence quantitative PCR; seventhly, data statistics and analysis are carried out;

in the first step, the grouping and feeding of the test animals comprises the following steps:

1) selecting 25 male sheep of 2 months old, weighing 20-25kg, randomly dividing into 5 groups, each group having 5 repetitions, and each repetition having 1 sheep;

2) the pre-feeding period is 10 days, the testing period is 70 days, all groups are fed with basic ration and the micro-storage beanstalk, the addition amount of the vitamin D3 of the control group is OIU/kg, and the addition amounts of the vitamin D3 of the test group are 300IU/kg, 600IU/kg, 900IU/kg and 1200IU/kg respectively;

3) kneading beanstalk into 1-3cm length, uniformly mixing microbial agent, brown sugar and water according to the ratio of 1: 20: 80, uniformly spreading on the straw, pressing, sealing and packaging by using a packaging machine during sealing, and fermenting for one month to start and use the microbial fermentation beanstalk;

4) the daily feed intake of each sheep was adjusted at any time according to the daily gain of the sheep, the ratio of sperm to gross (dry matter basis) was 1: 2, the test period was 2 times, the feeding time was 8: 00 and 18: 00, feeding the test sheep in a coarse-then-fine sequence, freely drinking water, enabling the feeding management of all the test sheep to meet the ethical welfare protection regulations of animals, enabling the composition and the nutritional level of basic ration to be shown in a table 1, and enabling the nutritional ingredients of microbial fermentation beanstalk to be shown in a table 2;

TABLE 1 basal diet composition and Nutrition level (Dry matter basis)

Raw materials	Content (wt.)	Nutritional levels	Content (wt.)
				Corn (corn)	42.00	Dry matter DM	89.79
Wheat bran	30.00	Crude protein CP	18.14
				Bean pulp	24.00	Crude fat EE	2.29
Premix compound	4.00	Neutral detergent fiber NDF	13.36
				Total up to	100.00	Acid detergent fiber ADF	5.66
		Calcium Ca	0.86
						Total phosphorus	0.68

Note: crude protein, crude fat, neutral detergent fiber, acid detergent fiber, calcium, total phosphorus were measured values, and dry matter was calculated value. The following table is the same.

TABLE 2 nutrient components of microbial fermentation beanstalk (Dry matter basis)

In the second step, 4 sheep are randomly selected from each group for slaughter sampling on the last day of the test period, the liver, the kidney, the spleen, the thymus and the mesenteric lymph are respectively collected and placed in a freezing storage tube, and liquid nitrogen is put for standby;

in the third step, the extraction of the total RNA of the sheep tissue comprises the following steps:

1) sterilizing a mortar at high pressure, then carrying out cooling treatment on the mortar by using liquid nitrogen, then quickly taking out sheep tissues from the liquid nitrogen, grinding the sheep tissues in the mortar, putting the ground tissues in an EP (ethylene propylene glycol) tube, adding a proper amount of Trizol, marking, vibrating and uniformly mixing with force, and standing on ice;

2) placing the mixed solution in a centrifuge at 4 ℃, and centrifuging for 10min at the speed of 12000 r/min; the supernatant was slowly aspirated, transferred to another EP tube and incubated at room temperature for 5 min; adding 200 μ L chloroform, shaking, and centrifuging at 4 deg.C for 15min at 12000 r/min; slowly sucking the supernatant, transferring into another EP tube, adding equal volume of isopropanol, standing at room temperature for 10min, centrifuging at 4 deg.C for 10min at 12000r/min to obtain white precipitate;

3) discarding isopropanol in an EP tube, adding a proper amount of 1mL of glacial ethanol, and centrifuging at 4 ℃ for 15min at a centrifugation speed of 12000 r/min; discarding the ethanol, drying at room temperature for 10min, adding appropriate amount of 20 μ L sterile RNase H₂Dissolving white precipitate by O; taking a proper amount of 1 mu L of extracted total RNA, determining the concentration of the total RNA by using a nucleic acid determinator, and storing the residual total RNA sample in a refrigerator at the temperature of-80 ℃ for later use;

in the fourth step, the reaction system is prepared according to the table 3 and placed in a preheated PCR instrument, and the reaction conditions are shown in table 4:

TABLE 3 cDNA reaction System

TABLE 4 PCR reaction conditions

Temperature (. degree.C.)	Time (min)
		25	10
42	50
		85	5

In the fifth step, NCBI searches gene sequences of sheep VDR, Bcl2, BAX, Caspase9, Caspase3 and 18s, then utilizes Priem 5.0 software to design primers according to the conserved regions (CDs) of each gene, and entrusts other bioengineering companies to synthesize primer sequences, as shown in Table 5:

TABLE 5 information on the genes of interest and reference genes

In the sixth step, the fluorescent quantitative PCR comprises the following steps:

1) the cDNA sample synthesized by reverse transcription was diluted and qRT-PCR reaction was carried out using a fluorescent quantitation kit, and the reaction system is shown in Table 6.

TABLE 6 Real-time PCR reaction System

Reagent	Volume (μ L)
		EvaGreen 2×qPCR MasterMix	10
Forward Primer	0.6
		Reverse Primer	0.6
cDNA	2
		RNase Free ddH2O	6.8

2) Preparing a 20 mu L reaction system according to the proportion, centrifuging, fully mixing uniformly, placing in a preheated qRT-PCR instrument, wherein the reaction conditions are shown in Table 7:

TABLE 7 Real-time PCR reaction conditions

Wherein in the seventh step, the CT value of the fluorescence quantification is 2^-ΔΔCTThe algorithm calculates the relative expression amount, and the One-way analysis of variance (ANOVA) in Graphpad Prism5 software is adopted to analyze the test data, wherein the difference is obvious when P < 0.05 and extremely obvious when P < 0.01.

Wherein the total probiotic count of the micro-storage microbial inoculum is more than or equal to 5 multiplied by 10⁷Per gram.

Results and analysis

1. Influence of different concentrations of VD3 added in micro-storage beanstalk on mRNA level of liver apoptosis gene

As can be seen from FIGS. 1, 2, 3, 4 and 5, the relative expression levels of BAX and Caspase9 in sheep livers are remarkably increased (P is less than 0.01) compared with that in a control group when the addition level of vitamin D3 is 600IU/kg, 900IU/kg and 1200 IU/kg; the relative expression level of Caspase3 in each experimental group is very different from that in the control group (P is less than 0.01); the relative expression level of VDR is significant (P < 0.05) or extremely significant (P < 0.01) when the vitamin D3 is added at 900IU/kg and 1200IU/kg, which is higher than that of the control group.

2. Influence of different concentrations of VD3 added in micro-storage beanstalk on mRNA level of apoptosis gene of kidney

As can be seen from fig. 6, 7, 8, 9 and 10, when the vitamin D3 addition level was 600, 900, 1200IU/kg, the relative expression level of sheep kidney BAX was significantly increased (P < 0.01) compared to the control group; the relative expression level of Bcl2 in each test group is reduced remarkably (P < 0.05) or extremely remarkably (P < 0.01) compared with that in a control group; when the addition level of the vitamin D3 is 600IU/kg and 900IU/kg, the relative expression quantity of Caspase9 and Caspase3 is obviously (P < 0.05) or extremely (P < 0.01) increased compared with that of a control group; when the addition level of the vitamin D3 is 600IU/kg, the relative expression level of VDR is obviously increased (P is less than 0.05) compared with that of a control group.

3. Influence of different concentrations of VD3 added in micro-storage beanstalk on spleen apoptosis gene mRNA level

As can be seen from FIGS. 11, 12, 13, 14 and 15, the relative expression levels of sheep spleen BAX, Caspase9, Caspase3 and VDR were significantly (P < 0.01) increased in the levels of 600, 900 and 1200IU/kg of vitamin D3 addition compared with the control group; when the addition level of the vitamin D3 is 1200IU/kg, the relative expression level of the Bcl2 is obviously increased (P < 0.05) compared with that of a control group.

4. Influence of different concentrations of VD3 added in micro-storage beanstalk on thymus apoptosis gene mRNA level

As can be seen from FIGS. 16, 17, 18, 19 and 20, the relative expression level of Caspase9 in sheep was significantly increased (P < 0.01) compared to the control group at vitamin D3 addition levels of 600, 900 and 1200 IU/kg; when the addition level of the vitamin D3 is 600IU/kg, the relative expression levels of BAX and Caspase3 are remarkably increased (P is less than 0.01) compared with that of a control group; when the vitamin D3 is added at the level of 600IU/kg and 900IU/kg, the relative expression level of VDR is remarkably increased (P is less than 0.01) compared with that of a control group.

5. Influence of different concentrations of VD3 added in micro-storage beanstalk on mesenteric apoptosis gene mRNA level

As can be seen from FIGS. 21, 22, 23, 24 and 25, the relative expression levels of sheep mesenteric lymph BAX, Bcl2, Caspase3, Caspase9 and VDR were significantly increased (P < 0.01) in comparison with the control group at vitamin D3 addition levels of 600, 900 and 1200 IU/kg.

In conclusion, when the addition level of the vitamin D3 is 600IU/kg, 900IU/kg and 1200IU/kg, the relative expression levels of the liver, the kidney and the spleen BAX, Caspase3 and Caspase9 of the sheep are remarkably increased compared with those of a control group; when the addition level of the vitamin D3 is 900IU/kg and 1200IU/kg, the relative expression amount of VDR is obviously higher than that of a control group; when the addition level of the vitamin D3 is 1200IU/kg, the relative expression level of the sheep spleen Bcl2 is obviously increased compared with that of a control group. When the addition level of the vitamin D3 is 600IU/kg and 900IU/kg, the relative expression level of the sheep thymus VDR is greatly increased compared with that of a control group; the relative expression levels of the sheep mesenteric lymph BAX, Bcl2, Caspase3, Caspase9 and VDR are all remarkably increased compared with the control group; the vitamin D3 is beneficial to improving sheep immunity and reducing tissue apoptosis when the addition level of the vitamin D3 is 300 IU/kg.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.