【作者】 彭华；

【导师】 王加启；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2011， 博士

【摘要】 本研究通过四个试验研究纳豆枯草芽孢杆菌在奶牛胃肠道存活效率,流通规律及其对泌乳初期奶牛生产性能,瘤胃发酵、血液代谢产物和血液蛋白组的影响。试验一:本试验旨在通过静态发酵试验研究纳豆枯草芽孢杆菌在奶牛瘤胃和十二指肠中的存活率,确定其能否耐受奶牛胃肠道环境。采集1头健康瘘管牛瘤胃液和十二指肠液过滤后,加入105/mL纳豆枯草芽孢杆菌芽孢,对照组不添加,于39℃厌氧培养,瘤胃液发酵0、6、12、24、48、72 h后检测VFA和芽孢数。十二指肠液发酵0,1,2,3,4和6 h后检测芽孢数。瘤胃发酵结果表明,纳豆枯草芽孢杆菌在瘤胃液发酵72 h过程中,芽孢数呈先增高后降低的趋势,发酵24 h和72 h的芽孢数增加了将近2倍,促进了瘤胃液丙酸和丁酸的产生(P < 0.05),减少乙酸、异丁酸和异戊酸的产生(P < 0.01)。十二指肠液发酵结果表明,纳豆枯草芽孢杆菌芽孢数在十二指肠液中发酵3 h内芽孢数几乎无变化,发酵3 h后急剧降低(P < 0.001),发酵4 h后几乎不再变化,对照组始终无法检测出芽孢。试验二:本试验旨在通过研究纳豆枯草芽孢杆菌投喂奶牛后在其胃肠道的停留时间,确定其能否在奶牛胃肠道定植。试验选取7头瘘管牛随机分成两组:处理组(4头)通过瘤胃瘘管投放1010纳豆枯草芽孢杆菌芽孢,对照组(3头)不投放。投放后6,12,24,48和72 h采集瘤胃液、十二指肠液和粪样,测定芽孢数。结果表明瘤胃液、十二指肠液和粪便中的芽孢数均随投喂时间的延长而逐渐降低,投喂48 h后三个位点很难检出芽孢。试验三:本试验旨在探讨日粮中添加纳豆枯草芽孢杆菌发酵物对泌乳初期荷斯坦奶牛产奶量、乳成分、血液代谢产物和瘤胃发酵的影响。以胎次和泌乳日龄为区组,将36头奶牛(DIM = 29±6 d,胎次= 2.8±1.1)随机分为三组(对照,DFM1,DFM2)进行为期9周的饲养试验。对照,DFM1和DFM2三个处理组添加纳豆枯草芽孢杆菌发酵物的量分别为0,6和12 g/d。结果表明:DFM1和DFM2处理组血浆NEFA浓度低于对照组(P = 0.03)(685 vs. 639和633μmol/L)。随着纳豆枯草芽孢杆菌发酵物添加浓度的增加,瘤胃丙酸浓度升高(P = 0.02) (23.9 vs. 26.3和26.9 mol/100 mol,对照vs. DFM1和DFM2),乙酸浓度降低(P < 0.05()64.2 vs. 62.7和62.1 mol/100 mol,对照vs. DFM1和DFM2)。发酵物的添加对DMI无影响,但DFM1和DFM2处理组奶牛产奶量分别比对照组提高3.2和3.1 kg/d(P < 0.06),显著差异出现在试验第5～9周,最终导致DFM1和DFM2处理组的饲料转化率分别比对照组高9.5%和11.7%(P < 0.06)。发酵物的添加对乳脂率和乳蛋白产量无影响,但增加了乳脂产量(P < 0.05)和乳糖含量(P < 0.01),并有降低乳蛋白率的趋势(P = 0.06)。试验四:本试验旨在研究纳豆枯草芽孢杆菌发酵物对奶牛血液蛋白数量和结构的影响。以胎次和泌乳天数为区组将24头奶牛随机分成两组,对照组和DFM2组分别饲喂纳豆枯草芽孢杆菌发酵物0,12 g。采用二维凝胶电泳技术分离了饲喂发酵物8周奶牛的血浆蛋白,经考马斯亮蓝染色后PDQuest 7.4软件匹配、检测凝胶中差异表达的蛋白点,对差异蛋白点采用MALDI-TOF-TOF质谱分析,SEQUEST软件搜索NCBInr数据库,结果表明,DFM2组奶牛血浆蛋白有3个蛋白点表达量升高,但未得到有效鉴定,1个蛋白点表达量降低,经鉴定为转甲状腺素蛋白。纳豆枯草芽孢杆菌能长时间耐受奶牛瘤胃环境,发酵24 h,芽孢数约增加了1倍,能促进丙酸的发酵,能在十二指肠液中耐受3 h,但不能在奶牛胃肠道定植,投喂48 h后,瘤胃、十二指肠和粪便中几乎检测不到芽孢。为了在胃肠道发挥代谢活性,应在日粮中持续添加。在对泌乳初期奶牛血液代谢产物不产生负面影响的情况下,该菌的固体发酵物可提高奶牛产奶量3 kg/d,饲料利用率约10%,其机制可能是改变了瘤胃发酵模式,并与血液中转甲状腺素蛋白等蛋白的表达有关。更多还原

【Abstract】 Four experiments were conducted to study survival of Bacillus subtilis natto in rumen and duodenum, dispear time and its effects on the performance, rumen fermentation, blood meterbolites and blood proteiniom.In experiment 1, experiment in vitro was conducted to evaluate the survival laws of Bacillus subtilis natto (BSN) in rumen and duodenum of Holstein dairy cows. BSN spores were added at 105/mL to strained rumen fluid or duodenum fluid taken from a healthy dairy cow and incubated in vitro at 39°C. Strained rumen fluid or duodenum fluid with no BSN inoculants served as controls. Changes of BSN spore counts and volatile fatty acid in rumen fluid were monitored at 0, 6, 12, 24, 48 and 72 h. Changes of BSN spore counts in duodenum fluid were monitored at 0, 1, 2, 3, 4, 5 and 6 h. The results of rumen fermentation showed that spores increased in the first 24 h, and then decreased. The spores at 24 h were two times of 0 h. In addition, BSN increased the concentration of propionate and butyrate in rumen fluid (P < 0.05), but reduced the concentration of acetic acid (P < 0.01). The results of duodenum fermentation showed that spore counts tended to increase in the first 2 h, and then decreased gradually.In experiment 2: seven cows were randomly assigned to two groups. Four cows were infused with 1010 spores of BSN into rumen through rumen cannula and the other three cows received no infusion. Rumen fluid, duodenum fluid and feces were collected at 0, 6, 12, 24, 48, 72 h after the infusion. Spore counts in rumen, duodenum and feces continued to decrease and almost can’t be detected at 48 h after infusion in all of the location.In experiment 3: This experiment was conducted to determine the effect of B. subtillis natto fermentation product supplementation on blood metabolites, rumen fermentation, milk production and composition in early lactation dairy cows. Thirty-six multiparous Holstein cows (DIM = 29±6 d, parity = 2.8±1.1) were blocked by DIM and parity and then randomly assigned to 3 treatments (12 per treatment) in a 9-wk trial. Cows in control, DFM1 and DFM2 were fed TMR diets supplemented with 0, 6 or 12 gram of B. subtillis natto solid state fermentation product per day per cow respectively. Plasma nonesterified fatty acids were lower (P = 0.03) in DFM1 and DFM2 compared with control cows (633 and 639 vs. 685μmol/l). Ruminal propionate increased (P = 0.02) (23.9 vs. 26.3 and 26.9 mol/100 mol, control vs. DFM1 and DFM2, respectively) and acetate decreased (P < 0.05) (64.2 vs. 62.7 and 62.1 mol/100 mol, control vs. DFM1 and DFM2, respectively) with increasing B. subtillis natto fermentation product supplementation. DMI of the cows in three treatments was not affected by B. subtillis natto fermentation product supplementation, but milk yield was 3.1 kg/day and 3.2 kg/day higher for DFM1 and DFM2 than control cows on average across the 9-wk trial and significant differences were observed during wk 5 to 9 of the trial, which resulted in 9.5% and 11.7% increase in feed efficiency. B. subtillis natto fermentation product supplementation did not affect milk fat percentage and protein yield, but increased (P < 0.05) milk fat yield and lactose percentage (P < 0.01) and tended to decrease protein percentage (P = 0.06).In experiment 4: This experiment was conducted to study the effect of Bacillus subtilis natto on blood protein quantity and construction. Tweenty-four multiparous Holstein cows (DIM = 29±6 d, parity = 2.8±1.1) were blocked by DIM and parity and randomly assigned to 2 treatments (12 per treatment). Cows in control and DFM2 were fed TMR diets supplemented with 0 and 12 gram of B. subtillis natto solid state fermentation product per day per cow respectively. Changes of plasma proteins on the 8 wk after the fermentation product was fed were separated by two-dimensional electrophoresis providing a platform for parallel analysis. After proteins were stained with Coomassie Blue G-250, differentially expressed proteins were analyzed by PDQuest 7.4 software, and identified by HPLC equipped with ion trap mass spectrometer. The result showed that three proteins were up-regulated for the feeding of fermentation product; however, they were not identified. Transthyretin was down-regulated.In conclusion, B. subtillis natto spores have the ability to survive in rumen and alter rumen fermentation and survive in duodenum fluid for 3 h but can not permanently colonize in the gastrointestinal tract of Holstein dairy cows. The supplementation should be repeated in order to keep metabolic activities of B. subtillis natto additives and exert their effects on the ruminal microflora. When B. subtillis natto fermentation product was fed to dairy cows in early lactation, it was found that fermentation product was effective in increasing lactation performance of early lactation dairy cows possibly by altering the rumen fermentation pattern and down-regulat the express of transthyretin without any negative effects on blood metabolites.更多还原