【作者】 翁秀秀；

【导师】 李发弟； 王加启；

【作者基本信息】 甘肃农业大学 ， 动物营养与饲料科学， 2013， 博士

【摘要】 本次试验结合我国奶牛实际养殖现状，比较研究我国规模化奶牛场和散养户两种典型日粮组成对奶牛瘤胃发酵和VFA吸收影响的差异，以及从组织形态学和分子生物学角度探讨两种类型日粮对奶牛瘤胃壁VFA吸收的影响机理。共分为四个试验。试验一：旨在研究饲喂不同日粮对泌乳奶牛瘤胃发酵参数的影响。选用10头体重（BW）为542±22kg，泌乳日龄（DIM）为127±13d的头胎、健康且装有永久性瘤胃瘘管的荷斯坦奶牛，采用完全随机设计，按照体重、泌乳日龄和产奶量随机分为两组，每组5头瘘管牛，分别饲喂以玉米秸秆为唯一粗饲料来源的日粮（CS）和以苜蓿、羊草和玉米青贮为混合粗饲料来源的日粮（MF）。试验共进行9w，其中预饲期2w，正饲期7w。试验第37d开始，每隔6h通过瘤胃瘘管采集瘤胃液，共采集3d。测定各采样时间点的瘤胃液pH、氨态氮和VFA的浓度。试验结果表明，瘤胃液pH与总VFA含量在处理间差异不显著（P>0.05）。MF组奶牛瘤胃液乙酸摩尔比和乙酸/丙酸均极显著高于CS组（P<0.01），氨态氮浓度、丁酸、异丁酸、戊酸和异戊酸摩尔比均极显著低于CS组（P<0.01），丙酸浓度显著低于CS组（P<0.05）。CS组奶牛瘤胃夜氨态氮浓度极显著高于MF组，说明CS日粮组存在氮源的浪费，相比之下MF日粮组能有效的利用氮源。试验二：旨在研究饲喂不同日粮对奶牛瘤胃壁VFA吸收速率的影响。选用10头带有永久瘤胃瘘管的泌乳后期（DIM=262±13d）荷斯坦奶牛，按体重、泌乳天数和平均日产奶量随机分成两组，每组5头瘘管奶牛，分别饲喂CS和MF日粮，日粮的设计及配制同试验一。整个试验维持30d，预试期14d。试验第28d将戊酸-CoEDTA指示剂溶液与瘤胃食糜充分混匀，分别采集混匀后0、0.5、1、2、3、6、12、18和24h的瘤胃液，测定各采样时间点的瘤胃pH、VFA浓度、渗透压和Co含量，根据戊酸和Co-EDTA随时间的指数衰减率来估测瘤胃VFA的吸收速率和流通速率。试验结果表明，总VFA的吸收速率和流通速率在处理间均差异不显著（P>0.05），但MF组显著提高了乙酸清除率（mol/h，P=0.05）和VFA流通速率（mol/h，P=0.045）的估测值。试验三：旨在研究饲喂不同日粮对奶牛瘤胃壁乳头组织形态学的影响。试验动物、试验设计、饲养管理及试验日粮同试验一。试验结束经瘤胃瘘管活体采集瘤胃乳头，制备切片，应用光学显微镜和透射电镜对瘤胃乳头的组织结构及超微结构进行观察。结果表明：MF日粮组极显著降低了瘤胃乳头的宽度（P<0.01）；MF组奶牛的瘤胃乳头表面上皮的颗粒层厚度有高于CS组的趋势（P=0.17），而角质层厚度有低于CS组的趋势（P=0.18），棘层和基底层的厚度在组间差异均不显著（P>0.05）。透射电镜结果表明，CS日粮组奶牛瘤胃乳头颗粒层的细胞连接退化，细胞之间存在较大的空隙；角质层有少量的蜕皮现象，角质层下的细胞层细胞呈现加速的迁移状态。由此可见，给泌乳奶牛饲喂CS日粮，影响了瘤胃乳头的形态学结构。试验四：旨在研究饲喂不同日粮对奶牛瘤胃乳头中18个与VFA转运和代谢相关基因表达的影响。试验动物、试验设计、饲养管理及试验日粮同试验一。试验结束经瘤胃瘘管活体采集瘤胃乳头，PBS冲洗后液氮冷冻保存。提取瘤胃乳头的总RNA，利用GeneBank数据库中牛的mRNA序列设计出目的基因的引物序列，通过荧光定量PCR方法检测瘤胃乳头中18个与细胞生长、VFA转运和代谢相关基因的表达。试验结果表明，CS组显著上调了NHE-1、NHE-3、NHE-4和GPR-43基因的表达（P<0.10）；MCT-1和IGFBP-5基因的表达在MF组显著上调（P<0.10）；除ASCC-1被MF组显著下调外，日粮处理对VFA代谢相关基因的表达没有显著影响（P>0.10）。更多还原

【Abstract】 This study was carried out to investage the effect of two different types of dietson rumen fermentation, fractional absorption rate of volatile fatty acids, ruminalpapillae morphology and expression of genes involved in VFA transporters, cellulargrowth, and VFA metabolism in ruminal papillae of dairy cows.Experiment1: An experiment was conducted to investigate responses in rumenfermentation of dairy cows receiving a corn straw or mixed forage diet. Tenprimiparous and rumen-cannulated Holstein dairy cows averaging127±13d in milk(mean±SD) were randomly assigned to CS (37.1%corn straw as sole forage) or MFdiet (3.7%Chinese wildrye+28.4%alfalfa hay+26.5%corn silage as mixed forage).The trial lasted for63d. Samples of rumen fluid were collected via cannula every6hover a72-h duration starting on day37to analyze pH, ammonia-N and VFAconcentrations. Results showed that cows fed MF diet had higher (P<0.01) ruminalacetate concentrate and ratio of acetate to propionate, and lower concentrations ofammonia-N (P<0.01), propionate (P<0.05), butyrate (P<0.01), isobutyrate (P<0.01),valerate (P<0.01) and isovalerate (P<0.01) than cows fed CS diet. Collectively, theseresults found that CS group had higher rumen ammonia nitrogen concentration whichmay suggest that CS group can not efficiently make use of nitrogen sources from diet.Experiment2: Objective of this experiment was to investigate effects of two types offorage diets on fractional absorption rate of volatile fatty acids (VFA) through therumen wall. Ten primiparous, rumen-cannulated Holstein dairy cows in late-lactationwere randomly assigned to CS (37.1%corn straw as sole roughage) or MF diet (3.7%Chinese wildrye+28.4%alfalfa hay+26.5%corn silage as mixed forage). Cowswere fed their respective diet for28d, following which n-valeric acid and Co-EDTAwere used as markers for measuring ruminal VFA absorption and passage rates.Rumen fluid was collected from ventral sac of the rumen. Results showed thatfractional rates of total VFA absorption and passage from the rumen were not affectedby dietary treatments, averaging14.78and13.64%/h, respectively. However, cows fed MF diet had higher (P<0.05) estimated clearance rate of acetate, as well as totalVFA passage rate (mol/h).Experiment3: The objective of this study was to determine the effect of differenttypes of diets on ruminal papillae morphology in dairy cows. Ten primiparous andrumen-cannulated Holstein dairy cows averaging127±13d in milk (mean±SD)were randomly assigned to CS (37.1%corn straw as sole forage and62.9%concentrate) or MF (3.7%Chinese wildrye+28.4%alfalfa hay+26.5%corn silageas mixed forage and41.4%concentrate) diet. The experiment lasted for63days. Atthe end of experiment, rumen papillae were biopsied from the ventral sac andobserved using light and transmission electron microscope. Microscopic examinationrevealed a reduction in the width of papillae (P<0.01) and the thickness of stratumcorneum (P=0.18), and an increase in the thickness of stratum granulosum (P=0.17)when cows fed MF diet. No significant differences (P>0.05) were found for thethickness of stratum spinosum and stratum basale between dietary treatments.Biopsied papillae exhibited a decline in cellular junctions and large spaces betweencells of stratum granulosum, moderate sloughing of the stratum corneum, and anaccelerated migratory state in all strata below the stratum corneum in cows fed CSdiet. These results suggest that it affects ruminal papillae morphological structurewhen cows were fed CS diet.Experiment4: This study was conducted to determine the effect of different types ofdiets on expression of18genes involved in VFA transporters, cellular growth, andVFA metabolism in ruminal papillae of dairy cows. Ten primiparous andrumen-cannulated Holstein dairy cows averaging127±13d in milk (mean±SD)were randomly assigned to CS (37.1%corn straw as sole forage and62.9%concentrate) or MF (3.7%Chinese wildrye+28.4%alfalfa hay+26.5%corn silageas mixed forage and41.4%concentrate) diet. The experiment lasted for63days. Atthe end of experiment, rumen papillae were biopsied from the ventral sac, and genesexpression was evaluated using quantitative real-time PCR. Compared with CS group, the mRNA expression of NHE-1、NHE-3、NHE-4、GPR-43and ASCC-1in ruminalpapillae was decreased in MF group (P<0.10), and the mRNA expression of MCT-1and IGFBP-5was increased in MF group (P<0.10).更多还原