阅读说明：本技术 一种提高羊生产性能和瘤胃健康的复合微生态制剂及其制备方法和应用 (Composite microecological preparation for improving sheep production performance and rumen health and preparation method and application thereof ) 是由 左瑞雨 常娟 尹清强 韩露 王平 吕战旗 毕梦凡 王利军 李茂龙 刘超齐 党晓伟 于 2021-08-24 设计创作，主要内容包括：本发明公开了一种提高羊生产性能和瘤胃健康的复合微生态制剂及其制备方法和应用,具体涉及微生物制剂领域。所述制剂包括米曲霉1、米曲霉2和产朊假丝酵母菌。所述制剂的制备方法包括米曲霉的培养、产朊假丝酵母菌得培养和混合。所述制剂可用于提高羊的生产性能、羊的瘤胃健康和对纤维的消化。本发明制备的复合微生态制剂可以显著的提高羊的日增重,降低料肉比,提高羊的生产性能；本发明制备的复合微生态制剂可以改善羊的消化功能,提高羊对纤维成分的消化率；羊的基础饲粮中加入本发明制备的复合微生态制剂能够提高羊的血清免疫水平,提高羊的免疫力；本发明制备的复合微生态制剂可以促进羊胃肠道组织结构的完整,降低羊的消化道炎性因子表达,显著提高羊的瘤胃健康。(The invention discloses a composite microecological preparation for improving sheep production performance and rumen health, and a preparation method and application thereof, and particularly relates to the field of microbial preparations. The preparation comprises Aspergillus oryzae 1, Aspergillus oryzae 2 and Candida utilis. The preparation method comprises culturing Aspergillus oryzae, and culturing and mixing Candida utilis. The preparation can be used for improving sheep productivity, sheep rumen health, and fiber digestion. The composite microecological preparation prepared by the invention can obviously improve the daily gain of sheep, reduce the feed conversion ratio and improve the production performance of sheep; the composite microecological preparation prepared by the invention can improve the digestion function of sheep and improve the digestion rate of sheep on fiber components; the composite microecological preparation prepared by the method is added into the basic feed of the sheep, so that the serum immunity level of the sheep can be improved, and the immunity of the sheep can be improved; the composite microecological preparation prepared by the invention can promote the integrity of the tissue structure of the sheep gastrointestinal tract, reduce the expression of inflammatory factors of the sheep digestive tract and obviously improve the rumen health of the sheep.)

1. A composite microecological preparation for improving sheep productivity and rumen health is characterized in that the preparation method comprises the following steps:

step one, Aspergillus oryzae culture

Respectively culturing Aspergillus oryzae 1 and Aspergillus oryzae 2 in PDA culture medium constant temperature incubator, washing cultured spore with normal saline to obtain spore suspension, inoculating the spore suspension into Aspergillus oryzae solid fermentation culture medium, standing for culture in constant temperature incubator, taking out solid culture, and naturally air drying at below 60 deg.C; obtaining an aspergillus oryzae 1 solid culture and an aspergillus oryzae 2 solid culture;

step two, culturing candida utilis

Culturing Candida utilis in YPD culture medium liquid; centrifuging the cultured bacteria liquid, taking out the precipitated bacteria, adding a cryoprotectant, and freeze-drying in a freeze dryer to obtain candida utilis freeze-dried bacteria powder;

step three, mixing

And (3) fully and uniformly mixing the Aspergillus oryzae 1 solid culture, the Aspergillus oryzae 2 solid culture and the Candida utilis freeze-dried powder to obtain the compound microecological preparation.

2. The method for preparing a complex microecological preparation for enhancing sheep productivity and rumen health according to claim 1, wherein Aspergillus oryzae (Aspergillus oryzae)1 has a collection number of 5817; aspergillus oryzae (Aspergillus oryzae)2 has accession number 65904.

3. The method for preparing a composite microecological preparation for enhancing sheep productivity and rumen health as claimed in claim 1, wherein in the first step, the PDA culture medium comprises glucose 20.0g, soluble starch 6.0g, MgSO₄·7H₂O 0.3g，KH₂PO₄1.0g, 2g yeast, 5g soybean peptone and 15g agar powder, dissolving with distilled water, diluting to constant volume of 1L, and dissolving at 121 deg.C and 1.034 × 10⁵Autoclaving under Pa, and pouring out the solid plate.

4. The method for preparing a composite microecological preparation for enhancing sheep productivity and rumen health according to claim 1, wherein in the first step, the PDA culture medium is cultured in a constant temperature incubator at 30 ℃ for 3 days; the Aspergillus oryzae solid fermentation culture medium is bran: bean pulp: adding 40% distilled water into corn at ratio of 7:2:1, stirring, sterilizing with high pressure steam at 121 deg.C under 1.034 × 105Pa for 20min, and cooling.

5. The method for preparing a composite microecological preparation for enhancing production performance and rumen health of sheep according to claim 1, wherein in the first step, the normal saline is sterilized normal saline containing 0.05% Tween 80 by volume.

6. The method for preparing a composite microecological preparation for enhancing sheep productivity and rumen health as claimed in claim 1, wherein in the first step, the spore suspension is inoculated by inoculating Aspergillus oryzae solid fermentation medium with 1% of spore suspension; standing and culturing in a constant temperature incubator in an Aspergillus oryzae solid fermentation culture medium for 5d under the condition of 30 ℃.

7. The method according to claim 1, wherein in the second step, the YPD medium comprises peptone 20g, yeast extract powder 10g and glucose 20g, and distilled water is added to the YPD medium, and the volume is adjusted to 1L after the YPD medium is fully stirred and dissolved; the cryoprotectant is 10% of skimmed milk, the skimmed milk is 10% of skimmed milk powder, 10% of sucrose, 1% of sodium glutamate and 1% of trehalose are dissolved in 78% of distilled water, and the cryoprotectant is obtained by uniformly mixing.

8. The method for preparing a composite microecological preparation for enhancing sheep productivity and rumen health according to claim 1, wherein in the second step, the Candida utilis is cultured in YPD medium for 3 days; the temperature of the freeze-drying was-68 ℃.

9. A complex probiotic prepared according to the process of any of claims 1 to 8, characterised in that it comprises Aspergillus oryzae 1, Aspergillus oryzae 2 and Candida utilis.

10. The complex microecological formulation prepared by the method of any one of claims 1 to 8, which is useful for improving the productivity and rumen health of sheep.

Technical Field

The invention relates to the field of microbial preparations, in particular to a composite microbial ecological preparation for improving sheep production performance and rumen health, and a preparation method and application thereof.

Background

In recent years, the sheep raising industry in China is rapidly developed, but the problems of unbalanced proportion of fine and coarse fodder, low utilization efficiency of green and coarse fodder, low economic benefit and the like exist in production. The rumen is a main place for digesting the green and coarse fodder of the sheep, the feeding proportion of the green and coarse fodder is improper, and intensive feeding conditions are adopted, so that various problems are easy to occur in the digestion and health of the rumen of the sheep, and the key technology for feeding the sheep in a large scale and standardized way is how to effectively improve the health condition of the rumen of the sheep, improve the utilization rate of the green and coarse fodder and promote the healthy growth of animals.

The preparation method has the advantages that the preparation method is used for developing and developing the composite microecological preparation which is specially used for sheep and has the functions of improving the rumen health and the production performance, has important significance for the health and the sustainable development of the sheep industry, and has fewer effective products aiming at improving the rumen health, fiber digestion and animal production of sheep at home and abroad at present. The invention uses two kinds of aspergillus oryzae and yeast which can produce a plurality of cellulase, protease and amylase to prepare the composite microecological preparation, thereby effectively regulating the health of sheep rumen, promoting the utilization of roughage and improving the production performance and the immunity level of sheep.

Disclosure of Invention

Therefore, the invention provides a composite microecological preparation for improving sheep production performance and rumen health, and a preparation method and application thereof, so as to improve sheep rumen health and production performance.

In order to achieve the above purpose, the invention provides the following technical scheme:

according to a first aspect of the present invention, there is provided a method for preparing a complex microecological formulation for improving sheep productivity and rumen health, the method comprising the steps of:

step one, Aspergillus oryzae culture

Respectively culturing Aspergillus oryzae 1 and Aspergillus oryzae 2 in PDA culture medium constant temperature incubator, washing cultured spore with normal saline to obtain spore suspension, inoculating the spore suspension into Aspergillus oryzae solid fermentation culture medium, standing for culture in constant temperature incubator, taking out solid culture, and naturally air drying at below 60 deg.C; obtaining an aspergillus oryzae 1 solid culture and an aspergillus oryzae 2 solid culture;

step two, culturing candida utilis

Culturing Candida utilis in YPD culture medium liquid; centrifuging the cultured bacteria liquid, taking out the precipitated bacteria, adding a cryoprotectant, and freeze-drying in a freeze dryer to obtain candida utilis freeze-dried bacteria powder;

step three, mixing

And (3) fully and uniformly mixing the Aspergillus oryzae 1 solid culture, the Aspergillus oryzae 2 solid culture and the Candida utilis freeze-dried powder to obtain the compound microecological preparation.

Further, Aspergillus oryzae (Aspergillus oryzae)1 has accession number 5817; aspergillus oryzae (Aspergillus oryzae)2 has accession number 65904.

Further, in the first step, the PDA culture medium is 20.0g of glucose, 6.0g of soluble starch, and MgSO4 & 7H₂O 0.3g，KH₂PO41.0g, yeast 2g, soybean peptone 5g, agar powder 15g, dissolving with distilled water, diluting to constant volume of 1L, and dissolving at 121 deg.C and 1.034 × 10⁵Autoclaving under Pa, and pouring out the solid plate.

Further, in the first step, the PDA culture medium is cultured in a constant temperature incubator for 3d under the condition of a constant temperature incubator at 30 ℃; the Aspergillus oryzae solid fermentation culture medium is bran: bean pulp: adding 40% distilled water to corn at 7:2:1, stirring, and heating at 121 deg.C and 1.034 × 10⁵Sterilizing with high pressure steam under Pa for 20min, and cooling.

Further, in the first step, the normal saline is sterilized normal saline containing 0.05% of Tween 80 by volume fraction.

Further, in the first step, the Aspergillus oryzae is statically cultured in a constant temperature incubator in a solid fermentation medium for 5 days under the condition of 30 ℃.

In the second step, 20g of peptone, 10g of yeast extract and 20g of glucose are added into the YPD medium, and the volume is determined to be 1L after the YPD medium is fully stirred and dissolved; the cryoprotectant is 10% of skimmed milk, the skimmed milk is 10% of skimmed milk powder, 10% of sucrose, 1% of sodium glutamate and 1% of trehalose are dissolved in 78% of distilled water, and the cryoprotectant is obtained by uniformly mixing.

Further, in the second step, the liquid culture time of the YPD culture medium for the candida utilis is 3 d; the temperature of the freeze-drying was-68 ℃.

Preferably, the number of the spores of the preparation aspergillus oryzae 1 is 1.0 multiplied by 10^4.45CFU/g, Aspergillus oryzae 2 spore number of 1.0 × 10⁵CFU/g, viable count of Candida utilis of 1.0 × 10⁵CFU/g。

According to the second aspect of the invention, the composite microecological preparation prepared by the method comprises aspergillus oryzae 1, aspergillus oryzae 2 and candida utilis.

The composite microecological preparation prepared by the method provided by the third aspect of the invention can be used for improving the production performance and the rumen health of sheep.

The invention has the following advantages:

the composite microecological preparation prepared by the invention can obviously improve the daily gain of sheep, reduce the feed conversion ratio and improve the production performance of sheep; the composite microecological preparation prepared by the method can improve the digestibility of fiber components such as neutral detergent fiber and acidic detergent fiber in the sheep feed, and improve the utilization rate of the sheep on coarse feed; the composite microecological preparation prepared by the invention can improve the activity of sheep rumen cellulase and improve the digestion function of sheep rumen; the composite microecological preparation prepared by the method is added into the basic feed of the sheep, so that the serum immunity level of the sheep can be improved, and the immunity of the sheep can be improved; the composite microecological preparation prepared by the invention is added into the basic feed of sheep, so that the completeness and health of the tissue morphology of jejunum and rumen of sheep can be improved; the composite microecological preparation prepared by the invention can relieve the expression of inflammatory factors Caspase-3 and NF-kappa B, TNF-alpha in liver, jejunum and rumen, and promote the health of liver, jejunum and rumen tissues.

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. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 shows the jejunum tissue structure of Hu sheep (50X) according to the present invention; wherein A is a control group and B is a test group;

FIG. 2 shows the rumen tissue structure of Hu sheep (50X); wherein A is a control group and B is a test group;

FIG. 3 shows the expression effect (50 ×) of Caspase-3 in Hu sheep liver tissue; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 4 shows the expression effect (50X) of inflammatory factor NF- κ B in Hu sheep liver tissue according to the present invention; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 5 shows the expression effect (50X) of inflammatory factor TNF- α in Hu sheep liver tissue according to the present invention; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 6 shows the expression effect (50X) of Caspase-3 in the jejunal tissue of Hu sheep; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 7 shows the expression effect (50X) of inflammatory factor NF- κ B in the jejunal tissue of Hu sheep; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 8 shows the expression effect (50X) of inflammatory factor TNF- α in the jejunal tissue of Hu sheep; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 9 shows the expression effect (50 x) of Caspase-3, a rumen inflammatory factor of Hu sheep; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 10 shows the expression effect (50X) of the rumen inflammatory factor NF- κ B of Hu sheep; wherein A is a control group and B is a test group; arrows point to sites of inflammatory factor expression;

FIG. 11 shows the expression effect (50X) of the ruminal inflammatory factor TNF-alpha of Hu sheep; wherein A is a control group and B is a test group; the arrow points to where the inflammatory factor is expressed.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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.

Example a composite micro-ecological preparation for improving sheep productivity and rumen health and a preparation method thereof

PDA solid medium: glucose 20.0g, soluble starch 6.0g, MgSO₄·7H₂O 0.3g，KH₂PO₄1.0g, 2g of yeast, 5g of soybean peptone and 15g of agar powder, dissolving the mixture with distilled water, fixing the volume to 1L, sterilizing the mixture under high pressure at the temperature of 121 ℃ and the pressure of 1.034 multiplied by 105Pa, and pouring the solid plate to obtain the agar gel.

YPD medium: 20g of peptone, 10g of yeast extract powder and 20g of glucose, fully stirring and dissolving, and fixing the volume to 1L.

Preparation of skim milk: 10% of skimmed milk powder, 10% of cane sugar, 1% of sodium glutamate, 1% of trehalose and distilled water as a solvent; dissolving the skim milk powder, sucrose, sodium glutamate and trehalose with distilled water to obtain skim milk solution.

Two kinds of aspergillus oryzae: aspergillus oryzae 1(Aspergillus oryzae CGMCC5817) and Aspergillus oryzae 2(Aspergillus oryzae CGMCC 65904) were cultured in a PDA solid medium at 30 deg.C for 3 days. An appropriate amount of sterile saline with a volume fraction of 0.05% Tween 80 was added to the plate, and the spores were scraped off with a spreading bar to prepare a spore suspension.

The Aspergillus oryzae solid fermentation culture medium is bran: bean pulp: adding 40% distilled water to corn at 7:2:1, stirring, and heating at 121 deg.C and 1.034 × 10⁵Sterilizing with high pressure steam under Pa for 20min, cooling, adding spore suspension at an inoculum size of 1%, standing in 30 deg.C constant temperature incubator for 5d, taking out solid culture, and naturally air drying at 60 deg.C or below for storage.

The candida utilis is cultured in YPD medium liquid for 3 d. Centrifuging the cultured bacteria liquid, taking out the precipitated bacteria, adding 10% skimmed milk as a freezing protective agent, and placing in a freeze dryer at-68 deg.C to obtain lyophilized bacteria powder.

Mixing the obtained solid culture and lyophilized powder completely to obtain composite microecological preparation containing Aspergillus oryzae 1 spore 1 of 1.0 × 10 per gram^4.45CFU/g, Aspergillus oryzae 2 spore number of 1.0 × 10⁵CFU/g, viable count of Candida utilis of 1.0 × 10⁵CFU/g。

Experimental example 1 Effect of Compound Microecological preparation on sheep Productivity

1. Animal test design and grouping

60 Hu sheep with the weight of about 20kg and good health state are selected in the test and divided into 2 groups, each group has 5 repetitions, and each repetition has 6 sheep. The grouping is as follows: control group (group a): basal diet fed, test group (group B): adding compound microecological preparation into basic diet at a ratio of 400g/t (Aspergillus oryzae 1 spore number of 4.0 × 10 per kg of diet)^4.45CFU/kg, Aspergillus oryzae 2 spore number of 4.0 × 10⁴CFU/kg, Candida utilisThe number of viable mother bacteria is 4.0 × 10⁴CFU/kg). The test period was 60 days. The experimental diet composition and nutritional ingredients are shown in table 1.

Table 1 diet composition and nutritional levels (%, dry matter basis)

Note: 1) the premix provides 16.0mg of Cu, 60.0mg of Fe, 59.0mg of Mn, 120.0mg of Zn, 1.4mg of I, 0.10mg of Se, 0.03mg of Co, 14400IU of VA, 4400IU of VD and 30mg of VE for each kilogram of diet. 2) The nutrient level is the measured value.

2. Feeding management

Test Hu sheep were housed in the same sheep pen and each replicate sheep was housed in a pen. The sheep pen is kept clean and disinfected regularly in the test period. Test Hu sheep were fed twice a day at 07:00 am and 16:00 pm, the daily feed rate was recorded, and the trough residue was cleaned and weighed at 15:00 am and 06:00 am on the following day.

3. Method and apparatus for measuring index

Measurement of growth Performance: each sheep was weighed on an empty stomach on day 1, day 30 and morning on day 60 at the beginning of the positive session, the body weight was recorded, and the daily gain, daily feed intake, feed conversion ratio were calculated.

Daily gain is total gain/days of feed.

Daily feed intake-the amount of feed fed per day-the amount of feed remaining per day.

The feed-meat ratio is the total amount of the feed intake/the total weight gain.

4. Statistical analysis

The test data are preliminarily arranged by Excel 2010, then are analyzed by SPSS 22.0 software, and are subjected to multiple comparison by a Duncan method, wherein the difference is obvious when P <0.05, and the result is expressed by 'mean value plus or minus standard deviation'.

5. Analysis and discussion of results

As can be seen from Table 2, there was no significant difference in the average daily food intake of the Hu sheep groups over the test period (P > 0.05).

In the early stage of the test (1-30 d), the average daily gain and weight of each group have no significant difference (P is more than 0.05). In the later period of the test (31-60 d), the average daily gain of the group B is obviously higher than that of the group A (P < 0.05). The average daily gain of the group B was significantly higher than that of the group A (P <0.05) throughout the test period (1-60 d).

In the later test period (31-60 d) and the whole test period (1-60 d), the feed-meat ratio of the Hu sheep is remarkably reduced by the group B added with the composite microecologics (P is less than 0.05).

TABLE 2 influence of the Complex Microecological preparation on the Hu sheep Productivity

The test result shows that: the average daily gain can be improved and the feed conversion ratio can be reduced by adding the composite microecological preparation into the Hu sheep feed. This may be related to the beneficial effects of aspergillus oryzae in the composite microecological preparation with various enzyme activities and yeasts, and is consistent with the result of adding composite probiotics to improve the digestibility of feed nutrients.

Experimental example 2 Effect of Compound Microecological preparation on nutrient digestibility and rumen health of sheep

1. Collection of samples

Collecting a feces sample: and (3) selecting one Hu sheep with equal weight from each repetition of each group to carry out single-circle feeding by adopting a total feces collection method at 26-28d and 57-58d of animal experiments, accurately recording the feed intake every day, completely collecting feces every day, and weighing. After weighing, about 150g of feces is taken out from each repetition of each group, and the feces is added with 10 percent sulfuric acid for nitrogen fixation treatment and then stored at the temperature of minus 20 ℃. And (3) after the test is finished, uniformly mixing the excrement collected by repeating 3d in each group, drying at 65 ℃, recording the weight after 24h of moisture regain in a natural environment, and crushing for later use.

Blood sample collection: one Hu sheep with equal body weight was selected from each replicate in each group in the morning of 60d, and blood was collected via fasting jugular vein, and the collected blood was centrifuged at 3500r/min, and the separated serum was stored at-20 ℃.

Collecting a tissue sample: after the completion of the 60d test, 3 Hu sheep with equal body weight were selected from each of the A and B groups and slaughtered. Flushing the jejunum section with normal saline, and cutting 1cm of jejunum section with a scalpel; then, the peripheral tissues of the liver and the inner wall of the rumen with the area of about 3 multiplied by 3cm are cut off. The collected tissue samples were placed in 10% formalin fixation and labeled.

2. Method and apparatus for measuring index

And (3) determination of nutrient digestibility: the content of crude protein in feed and feces is determined according to GB/T6432-.

Determination of serum biochemical indexes: the contents of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), Total Protein (TP), Albumin (ALB), Globulin (GLO), urea nitrogen (BUN), Glucose (GLU), total Cholesterol (CHO), Triglyceride (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), and Lactate Dehydrogenase (LDH) in serum were measured using a fully automatic biochemical analyzer (BK-Au 5800).

The content of Malondialdehyde (MDA), superoxide dismutase (SOD) and reduced Glutathione (GSH) in serum is measured by visible spectrophotometry. The above kits were purchased from Solebao Biotechnology Ltd.

The contents of serum immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interleukin 2(IL-2), interleukin 4(IL-4), interleukin 6(IL-6), interleukin 10(IL-10), interferon (IFN-. gamma.), CD4+ and CD8+ were determined by an enzyme-linked immunoassay. The above kits were purchased from enzyme-linked biotechnology limited.

And (3) observing a tissue morphological structure: using HE staining method. Rumen, liver and jejunum tissue are washed, dehydrated and embedded to be made into 6 μm thick slices, and then the slices are stained by hematoxylin-eosin, and then are transparent by xylene, finally the slices are sealed by neutral resin and observed by a biological optical microscope.

Immunohistochemical assay: sp method is adopted. Dewaxing the slices, washing 3 times with PBS (5 min/time); repairing citric acid antigen for 10min, and soaking in PBS for 3 times (5 min/time); 3% H₂O₂Sealing endogenous peroxidase, soaking in PBS for 3 times (5 min/time) at room temperature for 20 min; dropping goat serum (50 μ L/tablet), sealing endogenous biotin at room temperature for 20 min; removing the blocking solution, adding primary antibody (1:100, 50 μ L/tablet), and standing at 4 deg.C overnight; removing primary antibody, soaking in PBS for 3 times (5 min/time), adding secondary antibody (50 μ L/piece), soaking in PBS for 3 times (5 min/time) at 37 deg.C for 30 min; dripping horseradish enzyme labeled protease chain albumin (50 mu L/piece), soaking for 3 times (5 min/time) in PBS at 37 ℃, developing DAB, stopping after brown appears, counterstaining with hematoxylin, decolorizing with gradient alcohol, performing xylene transparent treatment, sealing with neutral resin, and observing with a biological optical microscope.

3. Statistical analysis

Analysis of immunohistochemical results: and comprehensively judging by adopting the number of positive cells and the color of the positive cells. The number of positive cells was divided into four grades: the total number of positive cells is less than 5%, and the score is 0; the total number of positive cells is 6-20%, and the score is 1; the total number of positive cells is 21% -50%, and the score is 2; the total number of positive cells was greater than 50% and scored 3 points. Positive cell color was also divided into four grades: colorless (0 min), yellowish (1 min), tan (2 min), tan (3 min). The product of the number of the positive cells and the color score of the positive cells is a total score, and when the total score is 0, the cells are not damaged; when the total score is 1-2, the cells are slightly damaged; when the total score is 3-4, the degree of cell damage is higher; when the total score is greater than 6, apoptosis and necrosis are serious. In summary, a higher total score indicates a severe degree of cell damage.

The test data are preliminarily arranged by Excel 2010, then are analyzed by SPSS 22.0 software, and are subjected to multiple comparison by a Duncan method, wherein the difference is obvious when P <0.05, and the result is expressed by 'mean value plus or minus standard deviation'.

4. Analysis and discussion of results

4.1 Effect of Compound Microecological preparation on Hu sheep nutrient digestibility

As can be seen from table 3, in the early stage (1-30 d) and the later stage (31-60 d) of the experiment, the compound microecologics added into the feed significantly improve the digestibility (P <0.05) of Neutral Detergent Fiber (NDF) and Acidic Detergent Fiber (ADF) in the feed, the fiber components are main nutritional components in the forage grass, and aspergillus oryzae 1 and aspergillus oryzae 2 both have high cellulase activity in the early stage of the laboratory research, which shows that the digestion of the feed fiber components by adding the compound microecologics plays a significant role.

TABLE 3 influence of the Complex micro-ecological preparation on Hu sheep nutrient digestibility (%)

4.2 Effect of Compound Microecological preparation on Hu sheep rumen digestive enzyme Activity

As can be seen from Table 4, the addition of the composite microecologics in the feed significantly improves the cellulase activity of the Hu sheep digestive tract, and the results further reveal the reasons and effects of the composite microecologics in improving the fiber digestibility of the Hu sheep feed.

TABLE 4 influence of the Complex Microecological preparation on the rumen digestive enzyme Activity of Hu sheep

4.3 Effect of Compound Microecological preparation on Hu sheep serum Biochemical index

As can be seen from Table 5, the addition of the composite microecological preparation has no significant influence on the conventional biochemical indexes of the serum of the Hu sheep, but the content of CD4+ and the ratio of CD4+/CD8+ are significantly improved (P < 0.05). CD4+/CD8+ is an important index for evaluating cellular immune function, and the increase of CD4+/CD8+ indicates that the cellular immune function is improved. The results show that: the addition of the composite microecological preparation into the basic feed of the Hu sheep can improve the serum immune level of the Hu sheep.

TABLE 5 influence of the Complex Microecological preparation on Hu sheep serum antioxidant and immune indices

4.4 Effect of Compound Microecological preparation on Hu sheep jejunum tissue Structure

As can be seen from FIG. 1, the jejunal villi of group A are normal in morphology, long in villi length and orderly arranged. Compared with the group A, the group B has relatively more fluff quantity and no obvious difference in length and shape. There was no significant difference in crypt depth between group a and group B. As can be seen from table 6, the ratio of villus height to crypt depth was significantly higher in group B than in group a (P < 0.05).

TABLE 6 influence of the Complex Microecological preparation on the height of Hu sheep jejunum villi and crypt depth

As can be seen from fig. 2, group a had intact rumen morphology, abundant rumen muscle layer, and more pronounced rumen papilla. Group B had a thicker ruminal layer, greater number of papillae and longer length than group a. The addition of the composite microecological preparation improves the health of the intestinal and rumen tissues of the Hu sheep.

4.5 Effect of Complex Microecological Agents on inflammatory factor expression in Hu sheep tissue

It can be seen from the combination of Table 7 and FIGS. 3, 4 and 5 that the addition of 400g/t of the complex microecologics in liver tissue, both in terms of the number of positive cells and the color of positive cells, eases the expression of the liver inflammatory factors Caspase-3 and NF-kappa B, TNF-alpha and promotes the health of the liver tissue structure; it can be seen from the comprehensive table 7 and the combination of fig. 6, 7 and 8 that the addition of 400g/t of the complex microecologics in the jejunal tissue both in terms of the number of positive cells and the color of the positive cells eases the expression of the jejunal inflammatory factors Caspase-3 and NF-kappa B, TNF-alpha and promotes the health of the tissue structure; as can be seen from table 7 taken together with fig. 9, 10, 11, in rumen tissue; the added 400g/t composite microecologics both relieve the expression of rumen inflammatory factors Caspase-3 and NF-kappa B, TNF-alpha and promote the health of tissue structure in terms of the number of positive cells and the color of the positive cells.

TABLE 7 Hu sheep tissue immunohistochemical scoring sheet

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.