【作者】 冯志华；

【导师】 李建国；

【作者基本信息】 河北农业大学 ， 饲料作物生产与利用， 2013， 博士

【摘要】 瘤胃甲烷的产生不仅导致饲料能量的损失，而且还会促使全球气候变暖。随着全球对温室气体排放的重视，有关减缓反刍动物甲烷排放的瘤胃调控技术方面的研究受到越来越多的关注。本课题选用从蒺藜科植物蒺藜中提取的蒺藜皂苷（GSTT）作为试验材料，通过瘤胃体外发酵产气试验、双外流连续培养试验和奶牛饲养试验，系统研究GSTT对奶牛瘤胃发酵、甲烷产量、营养物质消化代谢及其抗氧化能力和免疫功能的影响，并利用荧光定量PCR技术分析检测瘤胃微生态主要菌群的变化，对GSTT减少甲烷产生的机理进行探讨。试验一本试验采用Mneke的Syrnige系统研究GSTT对体外发酵特性和甲烷产量的影响。试验设五个处理水平，GSTT添加量分别为0，0.15，0.30，0.60和0.90g/L，每个处理设四个重复，同时设一个空白对照组。在此培养系统中，瘤胃培养液用量为30mL，培养底物为200mg玉米粉和羊草粉等量混合物。结果表明，体外培养24h后，除0.15g/L GSTT处理组外，其余处理组体外甲烷浓度和产量较对照组均显著降低（P<0.05），0.30，0.60和0.90g/L GSTT处理组甲烷产量分别降低26.67%，28.89%，31.11%（P<0.05）。添加GSTT对pH和挥发性脂肪酸（VFA）浓度无显著影响（P>0.05），但随着GSTT添加水平的增加，氨态氮（NH3-N）的浓度降低（P<0.05），丙酸摩尔百分比升高（P<0.05），乙酸、丁酸摩尔百分比和乙酸/丙酸（A/P）值降低（P<0.05）。体外发酵24h后，GSTT降低了瘤胃体外培养液中原虫的数量（P<0.05），且随着GSTT添加水平的增加呈线性降低（P<0.01）。0.15，0.30，0.60和0.90g/L GSTT处理组原虫数量较对照组分别降低27.03%，37.84%，60.36%和72.07%。与对照组相比，较低剂量GSTT处理组体外干物质消失率（IVDDM）无显著变化（P>0.05），但高剂量GSTT处理组（0.60和0.90g/L）显著降低（P<0.05）。试验二本试验旨在探讨利用ANKOM RFS气体测量系统研究瘤胃体外发酵的可行性，并进一步研究GSTT对体外瘤胃发酵、甲烷产量和微生物区系的影响。试验设五个处理水平，GSTT添加量分别为0，0.15，0.30，0.60和0.90g/L，每个处理设四个重复，同时设一个空白对照组。在此培养系统中，瘤胃培养液用量为100mL，培养底物由340g/kg玉米、160g/kg豆粕和500g/kg羊草草粉组成。结果表明，在任一培养时间点，随着GSTT添加水平的升高，体外甲烷排放量呈线性降低（P<0.01）。体外培养24h时，添加0.15，0.30，0.60和0.90g/L GSTT处理组甲烷产量较对照组分别降低8.72%（P>0.05），15.44%（P<0.05），21.48%（P<0.05）和24.16%（P<0.05）。添加GSTT对pH和VFA的浓度没有显著影响（P>0.05）。发酵模式发生改变，随着GSTT添加水平的增加，丙酸摩尔百分比升高，乙酸摩尔百分比和A/P值降低，NH3-N浓度下降。GSTT降低了瘤胃体外培养液中原虫相对于总菌（16S rDNA）的数量（P<0.05），且随着GSTT添加水平的提高呈线性和二次曲线规律降低（P<0.01）。甲烷菌和真菌相对于总菌（16S rDNA）的数量随着GSTT添加水平的提高呈线性降低（P<0.01）。GSTT对产琥珀酸丝状杆菌和黄色瘤胃球菌相对于总菌（16S rDNA）的数量无显著影响（P>0.05）。总体而言，GSTT对体外产气量，甲烷产量以及发酵特性的影响在Mneke的Syrnige系统和ANKOM RFS气体测量系统中的趋势基本一致，说明ANKOM RFS系统可以模拟瘤胃用以研究体外发酵。试验三本试验旨在利用DFCOS－Ⅱ型双外流连续培养系统，进一步研究日粮中添加不同浓度的GSTT对奶牛瘤胃发酵特性的影响，进而筛选适宜剂量或范围，为后续体内试验研究提供参考依据。试验设四个处理水平，GSTT添加量分别为0，0.15，0.30和0.60g/L，每个处理设三个重复，试验周期为7d，前5d为适应稳定期，最后2d为采样期。结果显示，日粮添加不同水平GSTT对瘤胃pH、总VFA浓度无显著影响（P>0.05）；随着GSTT添加水平的提高，人工瘤胃液NH3-N浓度、乙酸摩尔百分比和A/P值降低（P<0.05），丙酸摩尔百分比升高（P<0.05）。提示，GSTT可望作为一种反刍动物瘤胃发酵调控剂来改变瘤胃发酵模式，改善对饲料能量和蛋白质的利用效率。试验四本试验以健康瘘管奶牛为试验动物，进行4×4拉丁方试验，共4期，每期28d。研究GSTT对奶牛瘤胃微生物发酵、甲烷产量、营养物质消化代谢及其抗氧化能力和免疫功能的影响，以验证其实际应用效果。根据体外试验结果，确定4个处理组中GSTT的添加水平分别为每天0g（对照组），5g，10g，15g。研究结果表明：（1）日粮中添加不同水平的GSTT对奶牛DMI、瘤胃液pH和总VFA的浓度均无显著影响（P>0.05）。添加10g/d和15g/d GSTT处理组奶牛甲烷排放量较对照组显著降低（P<0.05），但两处理组之间差异不显著（P>0.05）。若以奶牛每千克干物质采食量所排出的甲烷产量来计，添加10g/d GSTT处理组最低（14.94g/kg DMI）。与对照组相比，10g/d和15g/d GSTT处理组奶牛瘤胃液NH3-N浓度降低（P<0.05），丙酸摩尔百分比升高（P<0.05），A/P值降低（P<0.05）。GSTT对奶牛瘤胃内原虫数量有显著影响，10g/d和15g/d GSTT处理组低于对照组（P<0.05）。奶牛瘤胃液中真菌和甲烷菌的数量随GSTT添加量的增加均呈线性降低趋势，但各处理组之间差异均不显著（P>0.05）。日粮中添加GSTT对奶牛瘤胃液中产琥珀酸丝状杆菌和黄色瘤胃球菌的数量没有显著影响（P>0.05）。（2）日粮中添加不同水平的GSTT对DM在奶牛瘤胃内的降解率及其有效降解率均无显著影响（P>0.05），但其有效降解率有降低趋势。GSTT对36h、48h时CP的瘤胃降解率及其有效降解率均有不同程度影响，以15g/d GSTT处理组最低，显著低于对照组（P<0.05）。GSTT对奶牛NDF和ADF瘤胃有效降解率均无显著影响（P>0.05）。GSTT对奶牛DM、OM、GE、CP、NDF和ADF的全消化道表观消化率亦无显著影响（P>0.05）。各处理组之间奶牛瘤胃滤纸纤维素酶、羧甲基纤维素酶和水杨苷酶的活性均无显著差异（P>0.05），但15g/d GSTT处理组奶牛瘤胃木聚糖酶活性较对照组显著降低（P<0.05）。（3）日粮中添加GSTT对奶牛血清中总蛋白（TP）、白蛋白（ALB）、球蛋白（GLB）的浓度和白球比（A/G）均无显著影响（P>0.05），但血清TP浓度随GSTT添加水平的增加有升高的趋势（P=0.09）。日粮中添加GSTT可显著降低奶牛血清尿素氮（SUN）（P<0.05）、甘油三酯（TG）（P<0.01）和总胆固醇（TC）（P<0.05）的浓度。奶牛血清中高密度胆固醇（HDL-C）的浓度随GSTT添加量的增加呈线性提高（P=0.03），但各处理组之间差异未达显著水平（P>0.05）。添加GSTT对奶牛血清中谷草转氨酶（GOT）、谷丙转氨酶（GPT）活性和碱性磷酸酶（ALP）的活性及其肌酐、葡萄糖、钙和磷的浓度均无显著影响（P>0.05）。（4）与对照组相比，10g/d和15g/d GSTT处理组奶牛血清总抗氧化能力（T-AOC）、谷胱甘肽过氧化物酶（GSH-Px）活性显著升高（P<0.05），血清丙二醛（MDA）水平显著降低（P<0.05），但两处理组之间差异不显著（P>0.05）。奶牛血清超氧化物歧化酶（SOD）活性随着GSTT添加剂量的提高有升高趋势（P=0.06）。由此可见，日粮中添加GSTT对奶牛的抗氧化能力有一定的改善作用。（5）日粮中添加GSTT对奶牛血清IgA、白介素-2（IL-2）和肿瘤坏死因子-（TNF-a）含量均无显著影响（P>0.05），但10g/d和15g/d GSTT处理组奶牛血清IgG、IgM的含量较对照组显著提高（P<0.05）。随着GSTT添加剂量的增加，奶牛血清中IgA含量和IL-2的水平亦呈线性升高趋势（P=0.05）。提示GSTT对奶牛的免疫功能有一定的改善作用。综上所述，添加适量GSTT对奶牛瘤胃发酵具有调控作用，可去除部分瘤胃原虫，适度降低瘤胃液NH3-N的浓度，抑制奶牛甲烷的生成，并对奶牛的抗氧化能力和免疫功能具有一定的改善作用。综合考虑，在本试验条件下，日粮中GSTT适宜添加量为10g/d。更多还原

【Abstract】 Methane production in the rumen represents as a loss of energy for the host animal,and, in addition, methane emission by ruminants may contribute to a greenhouse effect orglobal warming. Scientists have been recently paying much attention to studymethnogen-inhibitors with the global concerning on greenhouse gas emission. The grosssaponin of Tribulus terrestris（GSTT）were selected to systematically study its effects onrumen fermentation, methane production, digestion and utilization of nutriment,antioxidant capacity and immune function of dairy cows by using artificial gas productiontrial, dual outflow continuous culture trial and feeding ruminally cannulated trial.Real-Time PCR technique to monitor ruminal microbial population to discuss themicrobiology mechanism for decreased methane production by GSTT.Exp.1The objective of this study was to determine preliminarily the effect of GSTTon ruminal fermentation and methane production in in vitro gas production test by usingMenke’s syringe system.The GSTT were added at levels of0,0.15,0.30,0.60and0.90g/Lin30mL culture medium. Substrate was200mg mixture of corn grain and Chinese wildryeon an equal mass basis.Each treatment （every dose of GSTT） was incubated inquadruplicate, and fermentations were repeated on two separate days. The set free of GSTTbut containing of the substrate was used as control. After24h of incubation, methaneproduction were reduced with increasing levels of GSTT, however, it was similar to that ofthe control treatment when addited with the lowest level（0.15g/L）.Compared to control,methane production were decreased by26.67%,28.89%,31.11%, addited with0.15,0.30,0.60and0.90g/L of GSTT, respectively. GSTT had little effect on pH and theconcentration of volatile fatty acids（VFA）in culture fluid（P>0.05）, while NH3-Nconcentrations were decreased（P<0.05）, by addition of GSTT. Molar proportion of acetateand butyrate was reduced and propionate was increased with a corresponding reduction inA/P ratio（P<0.05）. Total protozoal counts showed a linear decrease（P<0.01）withincreasing levels of GSTT. Compared to control, total protozoal counts were reduced by27.03%,37.84%,60.36%and72.07%for0.15,0.30,0.60and0.90g/L of GSTT（P<0.05）,respectively. Excluding the lower levels（0.15and0.30g/L）of GSTT, IVDDM wasdecreased（P<0.05）at higher levels（0.60and0.90g/L）.Exp.2The objective of this study was to further research on ruminal fermentation, methane production and microbial community in in vitro gas production test by usingANKOM RFS gas measurement system. The GSTT were added at levels of0,0.15,0.30,0.60and0.90g/L in100mL culture medium. The substrate was composed on a dry matter（DM）basis of340g/kg corn grain,160g/kg soybean meal, and500g/kg Chinese wildryegrass hay.Each treatment（every dose of GSTT）was incubated in quadruplicate, andfermentations were repeated on two separate days. The set free of GSTT but containing ofthe substrate was used as control. Results showed that methane emission decreased（P<0.01）in a line manner at all incubation times.Addition with0.15,0.30,0.60and0.90g/L of GSTT decreased methane production by8.72%（P>0.05）,15.44%（P<0.05）,21.48%（P<0.05）and24.16%（P<0.05）relative to controls at24h, respectively. GSTT hadlittle effect on pH and the concentration of total VFA in culture fluid（P>0.05）. However,the fermentation patterns were modified, with higher propionate molar proportion, andlower acetate molar proportion, A/P ratio as well as NH3-N concentration. In addition,GSTT also altered the population of rumen microbes relative to total bacterial16SrDNA.The abundance of rumen protozoa were significantly decreased by addition ofGSTT,in general, linear and quadratic（P<0.01）. The abundance of methanogens and fungidecreased by GSTT addition with linear dose–response effects.However, Fibrobactersuccinogenes and Ruminococcus falvefaciens were not affected. In summary, there were nosignificant difference in in vitro rumen fermentation, gas production or methane productionbetween Menke’s syringe system and ANKOM RFS gas measurement system,indicatedthat both systems could be used to simulate the rumen fermentation.Exp.3The effects of different levels of GSTT on rumen fermentation characteristicwere examined in DFCOS－Ⅱdual-flow continuous culture system. Furethermore, thepoper adding levels of GSTT in vitro was estabilished to provide reference basis forsubsequent experiment in vivo study. The GSTT were added at levels of0,0.15,0.30and0.60g/L in culture medium. Each treatment（every dose of GSTT）was incubated intriplicate. The entire experimental period was7days, including5days for adjustment andthe last2days for sample collection. It was concluded that addition with0.15,0.30and0.60g/L of GSTT had little effect on ruminal pH and the concentration of total VFA（P>0.05）, while molar proportion of acetate, A/P ratio as well as NH3-N concentrationsignificantly decreased（P<0.05）,molar proportion of propionate significantly increased（P<0.05）. Theses results indicated that GSTT has a potential to be used as a rumenmodulator to improve the utilization efficiency of energy and protein.Exp.4The experiment was conducted to study the effects of GSTT on rumenmicrobial fermentation,methane production, digestion and utilization of nutriment,antioxidant capacity and immune function of dairy cows. Four healthy ruminally fistulateddairy cows were randomly assigned in a4×4Latin square design. The animals receivedthe same basal diets and added with the GSTT at levels of0（control）,5,10and15g d1,respectively.The experiment lasted for four periods, each feeding period lasting for28days. The results showed as follows：（1）Addition with different levels of GSTT had little effect on dry matter intakes（DMI）, ruminal pH and the concentration of total VFA（P>0.05）. Compared with thecontrol treatment,methane emission of dairy cows,added with the GSTT at levels of10and15g d1,were decreased（P<0.05）,but there were no difference between the twotreatments（P>0.05）.When expressed on the basis of DMI，methane production was foundlowest（14.94g/kg DMI）for dairy cows with GSTT supplement at the dose of10g d1.Compared with the control treatment,rumen NH3-N concentration and A/P ratio was lowerfor dairy cows with GSTT supplement at the dose of10and15g d1（P<0.05）.However,addition with GSTT significantly enhanced molar proportion of propionate（P>0.05）.Inaddition, feeding GSTT decreased significantly rumen total protozoal counts,the totalprotozoal counts for dairy cows added with the GSTT at levels of10and15g d1werelower than those of dairy cows in control treatment（P<0.05）.Moreover, The rumenmethanogens and fungi counts of dairy cows tend to decrease linearly with increasinglevels of GSTT,but there were no significant differences between different treatments（P>0.05）. The rumen Fibrobacter succinogenes and Ruminococcus falvefaciens were notaffected significantly by addition with GSTT（P>0.05）.（2）Feeding GSTT did not affect the apparent degradabity or the effectivedegradation（ED）of dry matte（rDM）（P>0.05）.However, the ED of DM tend to decrease.Addition with GSTT declined the apparent degradabity and ED of CP at36h and48h,andthe numerical value of dairy cows added with the GSTT at levels of15g d1was thelowest,which was lower significantly than that of the control treatment（P<0.05）.FeedingGSTT had little effect on the ED of NDF and（P>0.05）.In addition, the apparent total tractdigestibilities of DM,OM,GE,CP,NDF and ADF were unaffected by the supplementationwith GSTT（P>0.05）. There were no significant differences in rumen cellulase, CMCaseor salicinase activites of dairy cows between different groups（P>0.05）.However, thexylanase activities of dairy cows added with the GSTT at levels of15g d1were lowerthan those of the control treatment（P<0.05）.（3）The serum concentration of total proteins（TP）, albumin（ALB）, globulin（GLB）and ratio of albumin to globulin （A/G） ratio was unaffected significantly by thesupplementation with GSTT（P>0.05）,but the serum concentration of TP tend to enhancewith the increasing levels of GSTT（P=0.09）. Addition with GSTT reduced the serumconcentration of serum urea nitrogen（SUN）（P<0.05）, triacylglycerol（TG）（P<0.01）and cholesterol（TC）（P<0.05）.Moreover, The serum concentration of HDL cholesterol（HDL-C）was increased linearly with the increasing levels of GSTT（P=0.03）,however,there were no significant differences among groups（P>0.05）.There was no significantdifference in the enzyme activties of glutamic-oxalacetic transaminase （GOT）, glutamic-pyruvic transaminase（GPT）, alkaline phosphatase（ALP）and in the serumconcentration of creatinine, glueose, Ca and P among treatments（P>0.05）.（4）Compared to the control treatment, the enzyme activities of serum T-AOC andGSH-Px for dairy cows added with the GSTT at levels of10and15g d1were improved（P<0.05）, the serum concentration of MDA was declined（P<0.05）,but there were nodifferences between the two treatments（P>0.05）.The enzyme activities of serum SODtend to improve with the increasing levels of GSTT（P=0.06）.It was concluded thataddition with GSTT contribute to improve the antioxdtion capability of dairy cows.（5）The serum concentrations of IgA, IL-2or TNF-a did not differ significantlyamong the gruops（P>0.05）. However, the serum concentrations of IgG and IgM of dairycows added with the GSTT at levels of10and15g d1were higher than those of thecontrol treatment（P<0.05）.In addition,the serum concentrations of IgA and IL-2wereimproved in a linear manner with the increasing levels of GSTT（P=0.05）.It was indicatedthat the supplement of GSTT could improve immune functions of dairy cows.In summary, proper leves of GSTT may moderate rumen fermentation, includinginhibited rumen protozoa,decreased rumen NH3-N concentrations moderately and methaneemission of dairy cows. Moreover, antioxidant capacity and immune function of dairycows were enhanced. Ovell, under the study conditions, the optimum adding level ofGSTT to the diet in dairy cows was10g/d.更多还原