日粮精粗比对舍饲育肥牦牛瘤胃菌群结构、挥发性脂肪酸及其转运载体表达量的影响

Effects of dietary concentrate to roughage ratio on the structure of rumen bacterial flora,volatile fatty acids and the expression of their transport vectors in fattened yaks

[目的]本试验以舍饲育肥牦牛为研究对象,探究日粮精粗比对舍饲育肥牦牛瘤胃代谢的影响,期望为青海牦牛规模化、产业化和现代化养殖过程中日粮精粗比调控提供参考。[方法]对15头试验牛群进行分组,分别饲喂精粗(质量)比为3∶7(A组)、5∶5(B组)、7∶3(C组)的全混合日粮(total mixed ration,TMR)。饲喂90 d后于凌晨空腹屠宰,采集瘤胃液和瘤胃上皮组织,通过16S核糖体RNA(16S ribosomal RNA,16S rRNA)高通量测序、气相色谱、RT-qPCR以及Western blot试验方法,探究不同精粗比日粮饲喂下,舍饲育肥牦牛瘤胃细菌菌群结构、瘤胃发酵参数以及瘤胃上皮中挥发性脂肪酸(VFA)转运载体表达量的变化。[结果]日粮精粗比显著影响瘤胃细菌菌群结构,随着精料占比升高,在门水平上,拟杆菌门(Bacteroidetes)、螺旋体门(Spirochaetes)和黏胶球形菌门(Lentisphaerae)相对丰度显著上升(P<0.05),厚壁菌门(Firmicutes)和软壁菌门(Tenericutes)相对丰度显著下降(P<0.05);属水平上,随着精料比例提高,理研菌科RC9肠道群(Rikenellaceae RC9 gut group)、克里斯腾森菌科R7群(Christensenellaceae R7 group)、Saccharofermentans、产琥珀酸菌属(Succiniclasticum)和Lachnoclostridium相对丰度显著升高(P<0.05),C组牦牛瘤胃球菌属(Ruminococcus)和拟杆菌属(Bacteroides)相对丰度显著低于A组(P<0.05),B组和C组牦牛普氏菌属(Prevotella)、假丁酸弧菌(Pseudobutyrivibrio)和密螺旋体属(Treponema)相对丰度显著高于A组(P<0.05)。日粮精粗比对瘤胃pH值及VFA产量影响显著,精粗比上升显著下调瘤胃内容物pH值(P<0.05),乙酸浓度、乙酸浓度/丙酸浓度显著降低(P<0.05),丙酸、戊酸和总VFA浓度显著升高(P<0.05),与A、B组相比,C组丁酸和异丁酸浓度显著升高(P<0.05)。日粮精粗比对瘤胃上皮VFA转运载体表达量影响显著。随着日粮精料比例升高,腺瘤下调蛋白基因(DRA)、假定阴离子转运载体1基因(PAT1)、单羧酸转运载体1基因(MCT1)和单羧酸转运载体4基因(MCT4)在mRNA水平的相对表达量显著升高(P<0.05),而阴离子交换蛋白2基因(AE2)在mRNA水平和蛋白水平的相对表达量则显著下降(P<0.05),上述基因的蛋白表达趋势与mRNA的一致。随着日粮精粗比例升高,牦牛的生产性能,即平均日增重、饲料转化率、屠宰率也随之提高,且具有显著差异(P<0.05)。[结论]饲喂高精料日粮可提升牦牛瘤胃中非纤维素降解菌的相对丰度,使大量能量饲料和蛋白饲料快速转化为VFA,为机体供能。激活瘤胃上皮细胞中VFA转运载体,提高其表达量,加快VFA转运入血的速度,以提高脂肪代谢底物,最终达到快速育肥的效果。

[Objectives]This experiment was based on the fertilizer yak as the research object,to explore different dietary concentrate to roughage ratio effect on the body metabolism of fattening yaks.It was expected to provide reference for the regulation and control of diet in Qinghai yaks in the process of large-scale,industrial and modern breeding.[Methods]15 yaks had been randomly divided into 3 groups.The yaks had been fed with different concentrates to roughage ratios(group A:concentrate∶roughage=3∶7,group B:concentrate∶roughage=5∶5,group C:concentrate∶roughage=7∶3).After regular feeding 90 days,these fasting yaks had been slaughtered during early morning.Then we collected the rumen fluid and the rumen epithelial tissue through 16S rRNA high-throughput sequencing,gas chromatography,RT-qPCR,and Western blot test methods to explore the changes of the structure of rumen bacterial flora,rumen fermentation parameters and the expression of volatile fatty acid(VFA)transport vector in rumen epithelium of house fed fattening yak in diets with different concentrate crude ratio.[Results]The ratio of concentrate to roughage significantly affected the structure of rumen bacterial flora.The results showed that the ratio of concentrate to roughage had significant effects on Firmicutes,Bacteroidetes,Spirochaetes,Lentisphaerae and Tenericutes in rumen bacteria of yaks.The relative abundances of Bacteroidetes,Spirochaetes and Lentisphaerae significantly increased with the increase of the proportion of concentrate(P<0.05).The relative abundances of Firmicutes and Tenericutes significantly decreased(P<0.05).On the level of the genus,with the increase of concentrate proportion,the relative abundance of Rikenellaceae RC9 gut group,Christensenellaceae R7 group,Saccharofermentans,Succiniclasticum and Lachnoclostridium significantly increased(P<0.05).Ruminococcus and Bacteroides in group C had a relative abundance significantly lower than group A(P<0.05).Prevotella,Pseudobutyrivibrio and Treponema in group B and group C had a relative abundance significantly higher than group A(P<0.05).The rumen pH and VFA were significantly affected by different concentrate-to-forage ratio diets.With the increase of concentrate-to-forage ratio,the rumen pH value of yak decreased significantly(P<0.05).The concentration of propionic acid,valeric acid and total VFA significantly increased(P<0.05),while the concentration of acetic acid and acetic acid/propionic acid significantly decreased(P<0.05).The concentration of butyric acid and isobutyric acid in group C were significantly higher than those in group A and group B(P<0.05).The results showed that downregulated in adenoma gene(DRA),putative anion transporter 1 gene(PAT1),monocarboxylate transporter 1 gene(MCT1)and monocarboxylate transporter 4 gene(MCT4),which were involved in the absorption and transport of VFA in the rumen epithelium increased significantly in mRNA level with the increase of the proportion of dietary concentrate(P<0.05).The anion exchanger 2 gene(AE2)significantly decreased(P<0.05).And the protein levels were consistent with mRNA.With the increase of the proportion of concentrate and roughage in the diet,the production performance of yaks,including the average daily gain,feed conversion ration and slaughter percentage also increased.And there was a significant difference among them(P<0.05).[Conclusions]High-concentrate diet could increase the relative abundance of non-cellulolytic bacteria in rumen of yak,and rapidly convert a large amount of energy feed and protein feed into VFA,which could provide energy for the body.VFA transporters in rumen epithelial cells are activated to increase their expression,and VFA transporters into the blood to enhance the lipid metabolism substrate,achieving the effect of rapid fattening ultimately.