Effect Of Alfalfa Flavonoids On Production Performance,Ruminal Metabolism And Immunity

Alfalfa(Medicago sativa L.),as a cultivated forage grass with merits of good quality,high production and well-adapted characteristic,is called "a king of pastures"which planted widely in northwest and north China.The flavonoids are a kind ofbioactive components in alfalfa.Many researches on flavonoids had been reported,but the studies on alfalfa flavonoids applied in animals were still less.The mechanisms of action of alfalfa flavonoids were still unclear in many aspects.Atpresent,few studies had been reported on the effect of alfalfa flavonoids on production performance and its mechanisms study.Therefore,the experiment was aim to study the effect of alfalfa flavonoids on production performance,ruminal metabolism and immunity of dairy cows and its mechanisms.It provided the theoretical basis to development and utilization of alfalfa flavonoids.Experiment one:Effects of alfalfa flavonoids on production performance,and blood biochemical indexes.The aim of this study was to investigate the effects of alfalfa flavonoids(AF)on production performance,and blood biochemical indexes.Four primiparous lactating cows(average weight:500±25 kg;lactation day:79±6 d)with a permanent fistula installed in the rumen were used in a 4x4 Latin square design experiment.Dietary treatment groups comprised a control group receiving the total mixed ration(TMR)feed without alfalfa flavonoids(A)and groups receiving supplementation with 20 g(B),60 g(C)and 100 g(D)AF,respectively.The experimental period was four stages and each stage was 24 d contained 10 d of preliminary feed.The results were as follow:1)Compared with the A treatment group,the DM intake and rate of lactose in C treatment group were significantly increased(P<0.05),whereas the rate of milk fat was significantly decreased(P<0.05).The rate of milk protein and total milk solids,the somatic cell counts had a tendency to decrease with increasing the level of AF supplementation(0.05<P ? 0.10).The milk yield,4%fat corrected milk and the ratio of milk to feed were enhanced by AF,but it was no significant difference in each group.2)The digestibility of crude protein and neutral detergent fiber showed a tendency of linear increase with increasing the dosage of AF supplementation(0.05<P ? 0.10).3)The content of TP and ALP in serum showed a tendency to decrease with elevating the level of AF supplementation(0.05<P ? 0.10).The content of TC and HDL in C treatment group was the highest.4)The concentration of prolactin(PRL)in serum was increased with increasing the level of AF supplementation.Compared with the A treatment group,the concentration of T3 and T4 in D treatment group was significantly increased(P<0.05).The results showed that AF could increase feed intake and the digestibility of nutritive material,regulate the hormone secretion and the synthesis of milk fat,milk protein and lactose.Experiment two:Effects of alfalfa flavonoids on fermentation and the structure and composition rumen bacterial community of dairy cows.The aim of the experiment was to study the effect of alfalfa flavonoids on fermentation and the structure and composition rumen bacterial community of dairy cows.The design of this experiment was the same with the experiment one.The results showed:1)the concentration of acetic acid,propionic acid,butyric acid and lactic acid was reduced by AF,but each treatment group showed no significant difference.2)the sum of phylum and genus and indexes of ACE,Chao 1 and Shannon in C treatment group were significantly higher than that of A and D treatment groups(P<0.05).3)At the phylum and class level,the abundance of Mollicutes,Tenericutes,Euryarchaeota and Methanomicrobia had a tendency to increase with increasing the level of AF supplementation(0.05<P ? 0.10),whereas the abundance of Fusobacteria and 4C0d-2 had a contrary result.The abundance of Armatimonadetes in C treatment group was significantly higher than that of A and B treatment groups(P<0.05).4)At the genus level,compared with C treatment group,the abundance of Mogibacterium and Pyramidobacter in A treatment group was increased(P<0.05),whereas the abundance of Pseudomonas in B treatment group and the abundance of Roseburia in D treatment group were decreased(P<0.05).The abundance of Sutterella in D treatment group was significantly lower than that of B treatment group(P<0.05).The abundance of Asteroleplasma,Succinivibrio,Suttonella and Spirochaeta showed a tendency to linear decrease with increasing the level of AF supplementation(0.05<P<0.10).In conclusion,AF could improve the abundance and diversity of ruminal bacteria,whereas it has no significant effect on the rumenfermentation.Experiment three:Effects of alfalfa flavonoids on oxidation resistance and immune functions of dairy cows.The aim of this study was to investigate the effects of alfalfa flavonoids onimmune functions.The design of this experiment was the same with the experiment one.The results showed:1)the neutrophile granulocyte counts,the percentage of neutrophile granulocyte,the platelet counts and thrombocytocrit was increased,whereas the lymphocyte counts were reduced with increasing the level of AF supplementation.2)The Ig G content,proportion of CD4+ T lymphocyte,ratio of CD4+to CD8+ and the relative expression of GM-CSF,IL-4 and IFN-? in B treatment group was significantly higher than that of other groups(P<0.05),whereas the proportion of CD8+ T lymphocyte showed the opposite result.3)Compared with the A treatment group,the activity of GSH-Px in D treatment group and the activity of CAT in B treatment group were increased(P<0.05),whereas the MDA content in C treatment group were decreased(P<0.05).In conclusion,AF could change immunity capability by regulating blood cell counts and cytokines activity and enhancing antioxidant capacity.Experiment four:Effect of alfalfa flavonoids on cell proliferation and synthesis of milk compositionThe aim of this study was to investigate the effect of alfalfa flavonoids on proliferation of BMECs and synthesis of milk composition.The BMECs were divided into five groups with the medium contained 0(Con),25 50,75 and 100 ?g/mL of alfalfa flavonoids,respectively.The BMECs were cultured in cell incubator at the condition of 37 ?,5%CO2.The results were as follow:1)AF could not affect the proliferation of BMECs cultured for 48 h,whereas 75 ?g/mL of AF could increase significantly the proliferation of BMECs cultured for 96 h(P<0.05).2)Compared with the control group,the 50 ?g/mL treatment group showed increased significantly the activity of GSH-Px,CAT and NO concentration(P<0.01 or P<0.05),but decreased significantly the activity of LDH(P<0.01);the MDA concentration in 75?g/mL treatment group was decreased significantly(P<0.01).3)Compared with the control group,the 25 ?gg/mL treatment group showed reduced significantly the relative expression of LAT1,JAK2,S6K1,mTOR,4EBPI,eIF4E,FATP1,FATP4,?-1,4-Gal T and HK2(P<0.01),but increased significantly the relative expression of ACACA and Glut8(P<0.01 or P<0.05);the 50 ?g/mL treatment group showed increased significantly the relative expression of PPAR-?,FATP4,SCD1,FABP3 and FAS(P<0.01).The results showed that AF could improve proliferation and oxidation resistance of cells and affect milk composition by regulating the relative expression of genes of milk fat,lactoprotein and lactose synthesis.Experiment five:Effect of alfalfa flavonoids on bovine mammary epithelial cells cultured in vitro under heat shockThe aim of this study was to evaluate the effect of alfalfa flavonoids on in vitro BMECs under heat shock.BMECs were cultured on media with different concentrations of AF(0,25,50,75,and 100 ?g/mL)at 37 ? with 5%CO2 for 48 h.Then,the cultured cells were subjected to a heat-shock treatment(42? for 1 h)before being returned to 37? with 5%CO2 for 12 h.The cell activity,antioxidant index,and gene transcript levels were evaluated.The results showed that the cell activity was significantly higher in the 25 ?g/mL treatment group than in the control group and 50 ?g/mL treatment group(P<0.05),but cell activity did not differ significantly among the other groups.Compared with the control group,the 50,75,and 100 ?gg/mL treatment groups showed significantly higher GSH-Px activity(P<0.01)and significantly lower LDH activity and MDA contents(P<0.01 or P<0.05),respectively.The CAT activity did not differ significantly among the five groups.Compared with the control group,the 75 ?g/mL treatment group showed significantly reduced transcript levels of Caspase3,P53 and Socs3(P<0.01),but significantly increased transcript levels of Stat3,and Socsl(P<0.05).The transcript level of Statlwas significantly increased in 25 ?g/ml,treatment group compared to the control group(P<0.01or P<0.05).The transcript levels of Bcl-2 and Fas did not differ significantly among the groups.Together,these results show that AF can enhance antioxidant capacity of BMECs,inhibit cell apoptosis and increase cell activity.Experiment six:Effect of alfalfa flavonoids on apoptosis and synthesis of milk composition of bovine mammary epithelial cells induced by lipopolysacchrideThe aim of this experiment was to study the effect of alfalfa flavonoids on cell apoptosis and synthesis of milk composition of BMECs induced by lipopolysaccharide(LPS).The BMECs were divided into three treatments with the medium contained 0 ?g/mL LPS and AF(Con),1?g/mL LPS(L)and 1 ?g/mL LPS and 75 ?g/mL AF(L+F),respectively.The BMECs were cultured in cell incubator at the condition of 37 ?,5%CO2.The results were as follow:1)LPS reduced significantly the viability of cell cultured for 12 h and 24 h(P<0.05 or P<0.01),whereas the AF could increase significantly the viability of cell cultured for 12 h(P<0.05).2)LPS could increase significantly the ROS concentration of cells(P<0.01),whereas the AF could decrease significantly the ROS concentration(P<0.01).3)LPS increased the expression of IL-1?,IL-6,TNF-?,TLR4 and MyD88 in cells(P<0.01),whereas the AF supplementation reduced the expression of IL-1? and TLR2(P<0.01).4)The expression quantity of P53,P38 and Caspase 3 was increased significantly when the cells were stimulated by LPS(P<0.01).However,the AF could reduce the expression quantity of P53 and P38(P<0.05).5)LPS reduced significantly the expression of PPAR-?,SCD1,FABP3,?-CS,JAK2 and mTOR(P<0.01)and enhanced significantly the expression of SREBP1,CAT1 and eIF4E(P<O.Olor P<0.05),whereas the AF could increase the expression of PPAR-?,S6K1,Glut1,Glut4,Glut8(P<0.01or P<0.05)and decrease the expression of FATP1,eIF4E(P<0.05).In conclusion,LPS could promote the inflammation of cells and reduce the cell viability,whereas the AF could inhibit cell apoptosis and improve the cell viability.It showed the AF has anti-inflammatory effect.LPS could inhbit the synthesis of milk protein and fat,but the AF could promote the synthesis of milk protein and unaffect the synthesis of milk fat.The AF could promote the synthesis of lactose in cells stimulated by LPS.