| In ruminants,rumen is the major organ for diet fermentation and nutrients supply due to the huge amount of microbial ecosystem.In practice,it’s common strategy to feed ruminants especially lactating dairy cow with high concentrate diet to improve the productivity.In addition,feeding high concentrate diet can cause chronic stress to ruminants,and paralleled the activation of stress axis(hypothalamus-pituitary-adrenal axis)and high level of cortisol in the circulating system.Therefore,the purpose of this study was to investigate the changes of long-term or short-term feeding high concentrate diet to lactating dairy goats on the composition of ruminal microbiota,ruminal metabolites and functional genes expression in the epithelial tissues of rumen.Moreover,the effects of stress hormone dexamethasone on rumen fermentation was also determined in vitro.1 Effect of long-term or short-term feeding high concentrate diet to lactating dairy goats on the composition of ruminal bacteria flora,ruminal metabolites and functional genes expression in ruminal epitheliumFifteen lactating goats with similar body weight and milk production were randomly divided into control group feeding low concentrate diet(concentrate:forage 35:65,LC,n ＝10),long-term feeding high concentrate diet group(concentrate:forage 65:35,HL,n = 5).After feeding 15 wks,LC group goats were randomly divided into two groups,control group goats were still fed with LC diet and short-term feeding high concentrate diet for 4 wks(concentrate:forage 65:35,HS,n = 5).HS and HL group goats were fed with high concentrate diet for 4 and 19 wks,respectively.At the end of the experiment,rumen fluid was collected for biochemical analysis,pyrosequencing,metabolites analysis and absolute quantitative PCR for detecting the number of microbiota copies.Plasma and ruminal epithelial tissues were collected for biochemical analysis and genes expression analysis.Volatile fatty acids in ruminal lumen were detected by meteorological chromatography.The level of cortisol in blood were measured by radioimmunoassay(RIA)method.Plasma concentration of histamine and ruminal concentration of lactic acid and LPS,microbial protein(MCP)and Ammonia nitrogen(NH3-N)were measured by commercial kits.The expression of genes involved in inflammation,VFA transportation and cells apoptosis in ruminal epithelium tissues were detected by real-time PCR.The RIA results showed that short-term feeding high concentrate had no effect on plasma cortisol concentration,while HL goats showed significantly lower level of cortisol than LC and HS(P<0.05).The concentration of histamine in HL plasma was significantly higher than that of LC(P<0.05),but there was no difference between LC and HS(P>0.05).The meteorological chromatography results showed that diet had no effect on the concentrations of acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and the total volatile fatty acid.However,the ratio of acetate to propionate in HL was significantly lower than LC(P<0.05).Lactic acid content in rumen fluid of HL goats was significantly higher than that of LC(P<0.05).The concentration of NH3-N,MCP and LPS in LC were significantly lower than that in HS and HL(P<0.05),while there was no difference between HS and HL(P>0.05).Metabonomics analysis of rumen fluid showed that there was significantly different between two of three groups(P<0.05).The pyrosequencing results showed that the duration of feeding high concentrate could affect the rumen bacteria flora diversity.Compared to LC,HS and HL had lower Chao value,ACE index as well as Shannon index(P<0.05).The percentage of Cyanobacteria in HS was markedly lower than LC(P<0.05),and the percentage of Verrucomicrobia in HL was significantly lower than LC(P<0.05).However,diet markedly affected bacteria composition at the genus level,but there was no difference between HS and HL(P>0.05).Real-time PCR results showed that duration of feeding high concentrate had some certain impacts on related gene expression in ruminal epithelial tissues.(1)Compared to LC,HS and HL significantly increased TLR4 mRNA expression(P<0.05)and down-regulated IL-8 mRNA expression(P<0.05).Compared to LC,MyD88 mRNA expression in HS was significantly decreased(P<0.05),while IL-10 mRNA expression in HL was increased significantly(P<0.05).IL-1β mRNA expression in HL was significantly lower than that in HS(P<0.05).Diet had no effect on TNF-α,TLR2,INF-γ or IL-1αmRNA expression(P>0.05).(2)Compared to LC,NHE2,NEH3 and Na/K ATPase mRNA expression were significantly increased in HS and HL(P<0.05),but no change was observed between HS and HL(P>0.05).There also was no effect of diet on MCT1 and MCT4 mRNA expression(P>0.05).However,high concentrate diet could markedly increase caspase-3 and caspase-8 mRNA expression(P<0.05),but Bax mRNA expression was only increased in HL goats(P<0.05).(3)Moreover,HS goats showed significantly higher level of caspase-3 activity when compared to LC group(P<0.05).These results indicate that both long-term and short-term feeding high concentrate diet to lactating goats led to some certain disorders in ruminal microbiota composition,fermentation,stress response as well as epithelial functions.2 Effect of dexamethasone on rumen fermentation in vitroRuminal fermentation was mimicked in vitro,and the effects of stress hormone dexamethasone on ruminal fermentation were evaluated using a 24 h batch culture of rumen fluid in vitro,which was based on the basic diet of forage-to-concentrate ratio of 40:60.The groups were assigned as follows:control group(CON),ethanol group(ETH,negetive control),and three doses of dexamethasone groups treated with 1(low dosage,LD),5(middle dosage,MD)and 10 ng/mL(high dosage,HD)dexamethasone,respectively.The pH value was immediately measured at 0,2,4,8,12 and 24 h after fermentation,respectively.Samples were collected and analyzed for concentrations of volatile fatty acids(VFA),lactic acid,Microbial crude protein(MCP),ammonia nitrogen(NH3-N)and DNA isolation.The results showed that dexamethasone treatments,fermentation time and their interaction significantly affected ruminal pH value,and ruminal pH was gradually decreased during fermentation(P<0.01).At 12 h pH in HD group was significantly lower than CON group(P<0.01)and lower than ETH group(P<0.05),respectively.At 24 h pH in HD group was significantly lower than CON group(P<0.05).The content of total volatile fatty acid was significantly affected by treatments and time(P<0.01).At 24 h the concentration of total volatile fatty acid in HD was higher than CON group(P<0.05).The content of acetic acid was significantly affected by treatments,time and their interaction(P<0.01).At 24 h the concentration of acetic acid in HD was higher than any other groups(P<0.05),however,the concentration of propionic acid in HD group was not changed compared to control group and the concentration of butyrate was lower than CON and ETH groups(P<0.05).Lactic acid concentration was significantly affected by treatments and time,which showed increased during 1～12 h fermentation,and then decreased during 12～24 h.The content of NH3-N was only significantly affected by fermentation time(P<0.01).The NH3-N concentration in each group was increased gradually.However,there was no significant difference between treated and control group during 12 to 24 h.Theconcentration of MCP was significantly affected by treatment,time and their interaction(P<0.01).The concentration of MCp was increased in the first stage,and then decreased.The results of absolute quantitative PCR showed that the number of total bacteria was gradually increased during the-experiment.At the same time,the number of Fibrobacter succinogenes,Ruminococcus albus,Streptococcus bovis and Prevotella genus concentration of rumen fluid was not changed by the treatment.The proportion of runinococcus flavefaciens was increased and the proportion in HD was higher than that of CON,ETH and LD at 24 h(P<0.05).These results suggested that the hormone glucocorticoids can directly promote rumen microbial fermentation and growth. |
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