【作者】 丁耿芝；

【导师】 孟庆翔； 周振明；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 本论文目的是研究活性酵母菌添加对饲喂不同精粗比饲粮的肉牛瘤胃发酵和养分降解的影响,并从瘤胃微生物区系和纤维素酶活两个角度探索酵母菌影响养分降解的机理；同时本试验还对酵母添加对饲喂不同精粗比饲粮肉牛血浆代谢组的影响进行了探索。试验选择10头装有永久性瘤胃瘘管的西门塔尔×本地牛阉牛(450±50kg BW),随机分为对照组和酵母添加组,两组饲喂相同的基础饲粮,酵母添加组每天晨饲前通过瘘管投喂活性酵母菌,投喂量为0.8g/d。采用了交叉试验设计,共分为两期试验,每期包含4个阶段,从第1阶段到第4阶段,饲粮精粗比逐渐提高,分别为30：70、50：50、70：30和90：10,每个阶段持续17天,即10天预饲期,6天尼龙袋试验期,最后1天晨饲前采集颈静脉血液,晨饲后3h采集瘤胃内容物。试验采用了MIXED模型对试验结果进行了统计分析,并对饲粮精粗比的线性效应和二次曲线效应采用了正交多项式对比分析(orthogonal polynomial contrast)。具体的试验结果如下：试验一：本试验目的是研究活性酵母菌添加对饲喂不同精粗比饲粮肉牛瘤胃发酵参数的影响。结果表明,精粗比和酵母菌添加没有互作。随饲粮精粗比的提高,各瘤胃发酵参数分别呈线性或者二次曲线趋势变化,其中瘤胃pH、乙酸摩尔比例、异丁酸摩尔比例和乙丙比(A：P)呈线性趋势降低(L;P<0.01)；TVFA浓度、丙酸摩尔比例、丁酸摩尔比例、戊酸摩尔比例和异戊酸摩尔比例呈线性趋势升高(L；P<0.01)；氨态氮和乳酸浓度呈二次曲线趋势变化(Q；P<0.01)。不考虑饲粮精粗比的影响,酵母菌添加组瘤胃液pH显著高于对照组(P<0.01),酵母添加组TVFA浓度、氨态氮浓度和乳酸浓度与对照组之间差异不显著(P>0.05),各挥发酸的摩尔比例两组之间也没有显著差异(P>0.05)。试验结果表明：饲粮精粗比显著影响瘤胃发酵参数；活性酵母菌具稳定瘤胃pH的作用；活性酵母菌对瘤胃发酵模式无显著影响。试验二：本试验目的是研究活性酵母菌添加对饲喂不同精粗比饲粮肉牛瘤胃养分降解的影响。试验采用尼龙袋法测定了蒸汽压片玉米干物质(DM)的消失率与羊草、苜蓿颗粒和青贮玉米秸DM和NDF降解参数。统计结果结果表明：(1)随饲粮精粗比的提高,蒸汽压片玉米干物质各时间点的消失率均呈现二次曲线趋势变化(Q；P<0.01),三种纤维饲料DM和NDF快速降解成分(aDM,aNDF)呈二次曲线趋势降低(Q；P<0.01),降解速率(cDM,cNDF)和有效降解率(EDDM,EDNDF)呈线性趋势降低(Linear, P<0.01);(2)不考虑饲粮精粗比的影响,酵母菌添加组蒸汽压片玉米3h,6h,12h的DM消失率显著低于对照组(P<0.05)；24h,48h的消失率与对照组无显著差异(P>0.05)；酵母菌添加组羊草aDM,bDM,aNDF与对照组之间无显著差异(P>0.05),bNDF显著低于对照组(P<0.05), cDM,EDDM,ENDF和EDNDF显著高于对照组(P<0.05)；酵母添加组苜蓿颗粒aDM,bDM和EDDM与对照组无显著差异(P>0.05), CDM极显著高于对照组(P<0.01), aNDF,bNDF与对照组无显著差异(P>0.05), cNDF显著高于对照组(P<0.05), EDNDF极显著高于对照组(P<0.01)；酵母添加组青贮玉米秸的各个降解参数与对照组之间无显著差异(P>0.05)。试验结果表明：饲粮精粗比显著影响养分瘤胃降解；酵母菌添加降低了蒸汽压片玉米干物质消失速率,提高了纤维饲料养分的降解速率和有效降解率；酵母菌添加对纤维饲料降解的影响与纤维饲料本身的性质有关。试验三：本试验目的是通过瘤胃微生物区系和酶活两类指标来研究活性酵母菌添加影响饲喂不同精粗比饲粮肉牛瘤胃养分降解的机理。试验采用了Real-time PCR测定了瘤胃total bacteria、rumen fungi和protozoa的拷贝数及纤维降解菌、淀粉降解菌、乳酸利用菌和乳酸产生菌的百分比；同时采用了DNS比色法测定了测定了木聚糖酶活、羧甲基纤维素酶活、微晶纤维素酶活和β-葡萄糖苷酶活。统计结果表明,(1)随饲粮精粗比的提高,Total bacteria, Rumen fungi, Protozoa的拷贝数和StrBov的百分比呈二次曲线趋势变化(Q；P<0.01),4种纤维降解菌(R. flavefaciens, R.ablus, FibSuc, ButFib)的百分比、RumAmy和SelRum的百分比呈线性趋势降低(L;P<0.01),而Lactobacillus的百分比呈线性趋势增加(L；P<0.01)；4种纤维素酶均呈线性趋势增加(L；P<0.01)。(2)不考虑饲粮精粗比的影响,酵母添加组Total bacteria的拷贝数显著高于对照组(P<0.05), Rumen fungi和Protozoa的拷贝数有高于对照组的趋势(P<0.1), R. flavefaciens, R.ablus, FibSuc, ButFib, StrBov, Lactobacillus的百分比与对照组无显著差异(P>0.05), RumAmy的百分比显著低于对照组(P<0.05),SelRum的百分比显著高于对照组(P<0.05)；(3)不考虑饲粮精粗比的影响,酵母添加组木聚糖酶活、微晶纤维素酶活和β-葡萄糖苷酶活菌显著高于对照组(P<0.05),羧甲基纤维素酶活与对照组无显著差异(P>0.05)。试验结果表明,饲粮精粗比显著影响瘤胃微生物区系；酵母菌添加降低蒸汽压片玉米瘤胃消失速率的机理与其增加原虫数量、降低淀粉降解菌百分比有关；酵母菌促进纤维饲料降解的机理与其增加瘤胃真菌数量和纤维素酶活有关。试验四：本试验目的是研究酵母菌添加对饲喂不同精粗比饲粮肉牛血浆代谢组的影响。试验采用超高效液相色谱四级杆飞行时间质谱联用仪(UPLC-QTOFMS)对采集的血浆样品代谢组分进行了分析,应用PCA、PLS-DA和OPLS-DA等模型对不同精粗比饲粮的生物标志物和酵母添加组的生物标志物进行了筛选。试验结果为不同饲粮精粗比组的生物标志物有胞嘧啶、二氢胸腺嘧啶、软脂酰胺、硬脂酰胺、油脂酰胺和磷酸泛酸；精粗比为30：70时,酵母添加组的生物标志物有鞘氨醇、植物鞘氨醇和卵磷脂(18：0),饲粮精粗比为50：50时,酵母添加组的生物标志物有芥酰胺、鞘氨醇和卵磷脂(16：0),饲粮精粗比为70：30时,酵母添加组的生物标志物有甘油一脂、甘油二脂、甘油三脂、卵磷脂(17：0)和卵磷脂(18：0),饲粮精粗比为90：10时,酵母添加组的生物标志物有芥酰胺、胆酸、卵磷脂(18：2)和卵磷脂(20：2)。试验结果表明：随饲粮精粗比提高,机体的脂肪代谢途径得到改善,有利于提高动物的生产性能；但是动物瘤胃壁和肠道内壁的完整性受损可能加重；酵母添加改善了动物脂肪和磷脂代谢,有利于提高动物的生产性能并有利于维护消化道内壁细胞的完整性。更多还原

【Abstract】 The objective of the current study was to evaluate the effect of live yeast (5. cerevisiae, CNCM1-1077, Lallemand, Toulouse, France) on rumen fermentation and nutrient degradation in steers fed diets with different concentrate to forage ratios (CTFR); the mechnism of S. cerevisiae (SC) affect nutrient degradation was studied through the effect of SC on fibrolytic activities and rumen microbial populations. Effect of SC on plasma metabonomics in steers fed diets with different concentrate to forage ratios (CTFR) was studied at the same time. Ten Simental x Local breed steers (450±50kg BW) fitted with rumen fistulas (10cm diameter) were assigned to control and treatment groups. Steers in the two groups were fed the same basal diets but the treatment groups received supplementation with SC (8×109cfu/h/day through the ruminal fistula) following a2-period crossover design. Each period consisted of four phases, each of which lasted17days:16days for dietary adaptation, and1day for rumen sample collection. From the1st to the4th phase, steers were fed in a stepwise fashion with increasing CTFRs, i.e.,30:70,50:50,70:30, and90:10. MIXED procedure was used to analysis effect of dietary CTFR and SC supplementation on peremeters. Linear and quadratic responses for dietary CTFR were assessed using orthogonal polynomial contrast statements.Experiment1:The objective was to assess the effects of S. cerevisiae on rumen fermentation characteristics of steers fed diets with different CTFRs. With CTFR increasing, rumen fermentation characteristics presented quadratic variation trend. Ruminal pH, molar proportion of acetate and isobutyrate, and A:P linearly decreased (L; P<0.01); TVFA concentration, molar proportion of propionate, butyrate, valerate and isovalerate linearly increased (L; P<0.01); ammonia N and lactate concentration presented quadratic variation (Q; P<0.01). Ruminal pH in the SC groups was higher than the control (P<0.05). There were no difference in concentrations of TVFA, ammonia N and D+L-lactate, and in VFA molar proportion between SC supplementation and control groups. These results showed that dietary concentrate to forage ratios significantly affect rumen fermentation characteristics; SC supplementation increased rumen pH, but has no effect on rumen ammonia N, TVFA and lactate concentration or rumen fermentation pattern.Experiment2:The objective was to assess the effects of S. cerevisiae supplementation on nutrient degradation of steers fed diets with different CTFR. With CTFR increasing, steam-flaked corn DM disappearance rate at each time point presented quadratic variation trend (Q; P<0.01); rapidly degradable fraction (aDM,aNDF) of the three fibrous feedstuffs quadraticly decreased (Q; P<0.01); degradation rate (cDM, cNDF) and effective degradability (EDDM, EDND) linearly decreased (L; P<0.01). SC supplementation decreased3h,6h and12h corn DM disappearance rate (P<0.05), but had no effect on24h and48h disappearance rates (P>0.05). There were no difference in CRH aDM, bDM,aNDF between SC supplementation and control groups; CRH BNDF was lower than control groups (P<0.05); CRH cDM, EDDM,cNDF and EDNDF were higher than control groups (P<0.05). There were no differences (P>0.05) in alfalfa aDM, bDM, EDDM, aNDF and bNDF between the two groups, but cDM, cNDF and EDNDF were higher (P<0.05) than control groups.There was no differences (P>0.05) for silage maize stalk degradation characters between two groups. The results showed that dietary CTFR significantly affect nutrient degradation. SC supplementation enhanced fibrous feedstuff degradation rate and effective degradability.Experiment3:The objective was to assess the effects of S. cerevisiae supplementation on fibrolytic activities and rumen microbial populations of steers fed diets with different CTFR. With CTFR increasing, microbial popolations presented in linear or quadratic variation trend but fibrolytic activities linearly increased. Compared with the control group, there was an increasing trend of rumen fungi and protozoa in SC group (P<0.1); copies of total bacteria in SC group were significantly higher (P<0.05). Additionally, percentage of Ruminobacter amylophilus was significantly lower (P<0.05) but percentage of Selenomonas ruminantium was significantly higher (P<0.05) in the SC group. Compared with control,S. cerevisiae supplementation gave higher activities of xylanase and avicelase (P<0.05) as well as β-glucanase activity (P<0.1), but had no effect on carboxymethyl cellulose activity (P>0.05). Overall, S. cerevisiae supplementation increased rumen total bacteria, fungi, protozoa, and lactate-utilizing bacteria but reduced starch-degrading and lactate-producing bacteria; S. cerevisiae supplementation increase enzyme fibrotic activities.Experiment4:The objective was to assess the effects of S. cerevisiae supplementation on plasma metabonomics of steers fed diets with different CTFR. UPLC-QTOFMS was used to detecte plasma metabonomics. PCA, PLS-DA and OPLS-DA was used to filtrate biomarkers.The results showed that biomarkers among different dietary CTFR groups were cytosine, dihydrothymine, palmitic amide, stearamide, oleamide, D-4’-phosphopantothenate. Dietary CTFR at30:70, biomarkers between SC supplementation and control groups were Sphinganine, Phytosphingosine, PC (18:0). Dietary CTFR at50:50, biomarkers between the two groups were13E-docosenamide,1-monoacylglycerol, PC(16:0). Dietary CTFR at70:30, biomarkers between the two groups were diacylglycerol,1-monoacylglycerol, triacylglycerol, PC (18:0), PC (17:0). Dietary CTFR at90:10, biomarkers between the two groups were13E-docosenamide, glycocholic acid, PC (18:2), PC (20:2). The results show that with concentrate level increasing, fat metabolism was improved, which was benifical to improve production performance, but rumen wall damage may be aggravating; SC supplementation improved phospholipid and fat metabolism, which is beneficial to maitain animal health status and improve production performance.更多还原