| The high temperature and humidity environment in summer in southern China has a serious impact on the physiological status of most dairy cows,which makes them in a long-term stress state in summer and results in the decline of antioxidant function,milk yield and quality,and the decline of reproductive performance of dairy cows,broughts serious economic losses to the dairy industry.It has caused serious losses to the dairy industry.Astragalus belongs to leguminous herbs.A large number of studies have shown that astragalus polysaccharides(APS)are the main beneficial ingredient in the traditional Chinese medicine Astragalus,which has nutritional and medicinal value and is widely praised by the aquaculture industry.However,there are few reports on the application of APS in dairy cows under stress and the mechanism of its influence.In view of this,the stress model of dairy cows was established by in vivo and in vitro experiments,and the effects of APS on production performance,antioxidant index,serum metabolites and mammary epithelial cells of dairy cows were explored in order to provide reference for the rational application of APS in dairy cows.This test is divided into three aspects:Experiment 1 Effect of astragalus polysaccharides on performance,serum metabolism index and antioxidant index of dairy cows under stressThirty healthy dairy cows were randomly divided into three groups(10 cows in each group)according to parity(1.95±0.95)、lactation days(220.34 ±42.73 d)and milk yield(34.45±4.89 kg/d).In the experimental group,30 mL/d(Treatment Ⅰ)and 50 mL/d(Treatment Ⅱ)of APS injection were injected into the neck muscle respectively.Each stage was injected with APS for 4 days(8:00 a.m.every day)and stopped for 3 days.The experiment lasted for 21 days.The rectal temperature,respiratory rate,milk yield and milk composition were measured on day 0,4,7,11,14,18 and 21 of the trial period.Blood samples were collected from tail vein to determine serum biochemical indexes,serum hormones,HSP70 and antioxidant indexes.The results showed that on the 21st day,the Treatment Ⅰ and the Treatment Ⅱ significantly reduced the rectal temperature of the cows compared with the control group(P<0.05).And the respiratory rate of dairy cows in Treatment Ⅱ decreased significantly on the 14th day.In addition,the milk yield of the three groups decreased significantly before and after the experiment(P<0.05).On the 21st day,compared with the control group,the number of somatic cells in the Treatment Ⅱ decreased significantly(P<0.05).In addition,on the 21st day,the serum globulin(GLB)concentration of dairy cows was significantly decreased in Treatment Ⅰ and the Treatment Ⅱ(P<0.05),and on the 4th day,the level of beta-hydroxybutyric acid(P-HBA)in TreatmentⅠ and the Treatment Ⅱ was significantly decreased compared with the control group(P<0.05).On the 4th,11th,18th and 21st day,the cortisol(COR)of group Ⅱ was significantly higher than that of control group and Treatment Ⅰ(P<0.05).Over time,T3 in Treatment Ⅰand Treatment Ⅱ increased significantly(P<0.05)on the 21st day compared with that before the experiment,and T3 in Treatment Ⅰ and Treatment Ⅱ increased significantly(P<0.05)on the 14th day compared with the control group(P<0.05).Thyroxine(T4)content in Treatment Ⅱ was significantly higher than that in control group(P<0.05).However,APS had no significant effect on other serum biochemical indices,INS,GC,HSP70 and antioxidant indices of dairy cows under oxidative stress(P>0.05).Therefore,APS can reduce rectal temperature,respiratory rate,number of breast somatic cells and serum globulin in cows under oxidative stress,increase serum COR,T3 and T4 levels,which had certain effects on physiological status and serum biochemical indicators of cows under oxidative stress.APS has the potential to alleviate stress in cows.Experiment 2 Effect of GS-MS non-targeted astragalus polysaccharides on serum metabolome of dairy cows under stressOn the basis of experiment 1,the cows of Treatment Ⅰ were selected as the research objects.Blood samples were collected from tail vein before(day 0),during(day 4)and after(day 21)to separate serum.Gas chromatography-mass spectrometry(GC-MS)was used to detect the changes of serum metabolites in dairy cows exposed to stress by non-targeted metabolomics.First,unsupervised principal component analysis(PCA)was used to observe the overall distribution of samples and the stability of the whole analysis process.Then,supervised(orthogonal)partial least squares(OPLS-DA)was used to distinguish the overall differences of metabolic profiles among groups and find the different metabolites among groups.Through searching and screening of Fiehn database,20 biomarkers of differential metabolites were identified.Among them,glucose-1-phosphate,glutamine,glycerol-1-phosphate,glycine,lysine,pyrophosphate,putrescine,tryptophan and tyrosine were up-regulated before and after the experiment.2-pyridinecarboxylic acid,3-aminoisobutyric acid,alanine,gamma-aminobutyric acid,glucose,glycol,nicotinamide,valine and phenylacetic acid were down-regulated before and after the experiment,catechol and methylamine were up-regulated first and then down-regulated after the injection of APS.The results showed that APS changed the serum metabolites of dairy cows under stress and regulated the energy metabolism of dairy cows under stress through glucose metabolism and amino acid metabolism.Experiment 3 Effects of astragalus polysaccharides on H2O2 induced oxidative damage and apoptosis in bovine mammary epithelial cellsThis experiment was conducted to study the effects of astragalus polysaccharides(APS)on H2O2-induced apoptosis,oxidative damage and expression of related genes and proteins in the bovine mammary epithelial cells.Bovine mammary epithelial cell line was cultured in vitro and induced by H2O2(600 M,2 h)to establish an oxidative stress model.The experiment was divided into three groups:blank control group,H2O2 group and H2O2+APS group(APS 8mg/mL).In this experiment,poptosis was detected by flow cytometry and CCK-8,the changes of ROS content in cells were detected by immunofluorescence,the activities of antioxidant enzymes were also determined,RT-qPCR was used to analyze the apoptotic gene expression of Bax,Bcl-2 and Caspase-3 and Western blot was used to detect the expression of apoptosis-related proteins.The results show that,compared with the blank control group,the cell viability decreased significantly in H2O2 group(P<0.05),the apoptosis rate and ROS content both decreased significantly(P<0.05),while the activities of SOD and GSH-Px increased significantly(P<0.05).In addition,compared with H2O2 group,the ratio of Bax/Bcl-2 mRNA and expression of Caspase-3 mRNA decreased extremely significantly,meantime,pro-apoptotic protein Bax in H2O2+APS group were significantly lower than that in H2O2 group(P<0.05).APS could increase the activity of SOD and GSH-Px in bovine mammary epithelial cells induced by H2O2,and decrease the production of MDA.In addition,APS can reduce the production of ROS in mammary epithelial cells induced by H2O2,decrease the ratio of Bax to Bcl-2,and inhibit the activity of apoptotic factor Caspase-3,thus slowing down the apoptosis of mammary epithelial cells induced by oxidative damage. |
