【作者】 毛胜勇；

【导师】 朱伟云；

【作者基本信息】 南京农业大学 ， 预防兽医， 2006， 博士

【摘要】 本课题以延胡索酸及其钠盐为试验材料，研究了其对瘤胃体外发酵和甲烷产量以及瘤胃细菌区系变化的影响。通过比较延胡索酸及其钠盐对瘤胃发酵参数的影响的差异，初步确定了延胡索酸二钠改善瘤胃发酵的适宜添加水平及其效果；而后以延胡索酸二钠为手段，系统的探讨了不同日粮条件下（粗饲料、全精料和不同精粗比饲料等）添加延胡索酸二钠对瘤胃体外发酵和甲烷产量的作用；在此基础上，以南京本土山羊为试验动物，探讨了延胡索酸二钠对山羊瘤胃代谢、血液生化指标和瘤胃细菌区系结构的影响。试验共分7部分进行：1延胡索酸对瘤胃微生物体外发酵影响的研究分别以0.6g玉米粉+0.4g稻草片段（2mm）为底物，收集来自于三头装有永久性瘤胃瘘管本地山羊的瘤胃液，在体外厌氧条件下，研究了不同浓度延胡索酸(0、4、8、12mmol·L^-1)对瘤胃微生物发酵活力的影响。结果表明，与对照组相比，延胡索酸处理显著提高了体外培养体系中总产气量、总挥发性脂肪酸（TVFA）和丙酸产量（P＜0.01），降低了pH值和乙丙比值（P＜0.01），且对各指标的影响呈线性效应。结果显示，延胡索酸可促进瘤胃微生物发酵，但极显著地降低了发酵体系中的pH值（P＜0.01）。2延胡索酸及其钠盐对瘤胃微生物体外发酵影响的比较研究以玉米、豆粕和黑麦草粉的混合物为底物，体外发酵法比较了延胡索酸及其钠盐(0、4、7和10mmol·L^-1)对瘤胃微生物体外发酵的影响。结果显示，延胡索酸及其钠盐均可显著提高体外发酵体系中的总产气量、TVFA及丙酸产量（P＜0.05），降低了乙丙比（P＜0.05）；但延胡索酸及其钠盐在对累计产气量、pH值和丁酸浓度的影响程度方面存在差异（P＜0.05）。在培养初始及培养结束时，同浓度条件下，各延胡索酸及其钠盐处理间，延胡索酸二钠组的pH值最高，延胡索酸一钠次之，延胡索酸最低，三者之间差异显著（P＜0.05）。试验结果表明，延胡索酸及其钠盐皆可促进瘤胃发酵活力，但三者在对一些瘤胃发酵参数的影响方面存在差异，比较而言，延胡索酸二钠作为瘤胃调控剂的作用效果可能最佳。3高精料条件下添加延胡索酸二钠对瘤胃发酵的影响以混合精料（0.7g玉米粉，0.3g豆粕粉和0.43g高羊茅草粉）、玉米或小麦粉（1g）为底物，研究了延胡索酸二钠(4、7和10mmo·l·L^-1)对瘤胃微生物体外发酵及动态过程的影响，同时探讨了应用延胡索酸二钠预防急性酸中毒的可行性。混合精料发酵试验结果表明，与对照组相比，延胡索酸二钠处理组的pH值、总产气量、总挥发性脂肪酸浓度（TVFA）、丙酸比例和表观干物质消失率皆显著升高（P＜0.05），而乙／丙比显著降低（P＜0.05），添加延胡索酸二钠有使乳酸浓度降低的趋势（P＜0.10）。动态研究表明，与对照相比，添加延胡索酸二钠显著提高了累计产气量、pH值、TVFA产量、丙酸浓度及CMcase酶活（P＜0.05），而乙丙比皆显著降低（P＜0.05），但对NH₃-N和乙酸浓度及淀粉酶酶活无显著影响（P＞0.05）。除TVFA外，对其他发酵参数，延胡索酸二钠处理与发酵时间之间存在显著的互作效应（P＜0.05）。以玉米及小麦为底物时结果表明，延胡索酸二钠显著提高了累计产气量（P＜0.01）和pH值（P＜0.05）；但对其他指标的影响不一致。乳酸大量累积条件下添加延胡索酸二钠对发酵参数的影响的结果表明，延胡索酸二钠处理组的总产气量、pH值、TVFA浓度和丙酸浓度皆显著增加（P＜0.05），而乳酸浓度和乙丙比则极显著地降低（P＜0.01）。结果显示，在底物为高精料日粮条件下，添加延胡索酸二钠可提高瘤胃微生物的发酵活力，降低发酵液中的乳酸浓度，但对一些发酵参数如挥发性脂肪酸组成及乳酸浓度的影响程度与日粮的天然属性有关。4延胡索酸二钠对混合瘤胃微生物、瘤胃纤维降解细菌及瘤胃真菌发酵粗饲料活力的影响采用批次培养技术，探讨了延胡索酸二钠对瘤胃微生物发酵黑麦草粉、高羊茅草粉和稻草粉的影响，同时研究了延胡索酸二钠对黄化瘤胃球菌和瘤胃真菌发酵粗饲料活力的影响。结果表明，与对照组相比，延胡索酸二钠处理组的累计产气量显著增加（P＜0.01），黑麦草、高羊茅和稻草的底物干物质消失率分别较对照组提高了26.22％（P＜0.05）、14.16％（P＜0.05）和28.54％（P＜0.01）；延胡索酸二钠对黄化瘤胃球菌发酵活力的影响的试验结果表明，添加延胡索酸二钠显著提高了该纤维降解菌对黑麦草的降解率（P＜0.05），同时发酵液中纤维降解菌的数量也显著增多（P＜0.05）；延胡索酸二钠对厌氧真菌发酵活力影响的试验结果表明，延胡索酸二钠处理组的总产气量与发酵底物的干物质消失率显著低于对照组（P＜0.05）。结果说明，延胡索酸二钠对可提高瘤胃细菌与纤维降解菌发酵底物的能力，但对瘤胃真菌的发酵活力具有抑制效应。5日粮精料水平逐渐提高条件下添加延胡索酸二钠对瘤胃微生物生长、甲烷产量及发酵的影响采用体外批次培养，以三种日粮（低精料日粮、中等水平精料的日粮和高精料日粮）为底物，比较了不同日粮条件下添加延胡索酸二钠(0、4和7mmol·L^-1)对山羊瘤胃微生物发酵及甲烷产量的影响。结果表明，较对照相比，添加延胡索酸二钠显著提高了累计产气量、pH值、TVFA产量和微生物蛋白量（P＜0.05），降低了甲烷产量（P＜0.05），其中低精料日粮组下降幅度最大。结果表明，延胡索酸二钠在降低甲烷产量方面与发酵底物的天然特性有关，其中对低精料日粮的作用效应最为显著。6不同类型日粮条件下添加延胡索酸二钠对瘤胃微生物体外发酵活力及瘤胃细菌区系结构的影响采用体外批次培养和PCR／DGGE技术，并结合16S rDNA序列分析方法，比较了不同日粮条件下（黑麦草、混合日粮和精料）添加延胡索酸二钠对瘤胃微生物体外发酵活力及瘤胃细菌区系结构的影响。结果表明，对上述三种底物，添加延胡索酸二钠显著提高了累计产气量、pH值和TVFA产量（P＜0.05），同时乳酸浓度显著降低（P＜0.05）。发酵动力学试验结果显示，添加延胡索酸二钠显著提高了最大产气速率及渐近产气量（P＜0.05）。DGGE图谱及相似性分析结果表明，在上述三种底物条件下，添加延胡索酸二钠后瘤胃细菌区系结构发生了显著改变。结果显示，延胡索酸二钠可促进瘤胃微生物发酵，并显著地影响了瘤胃细菌区系结构。7高精料日粮添加延胡索酸二钠对山羊瘤胃代谢、血液生化指标及瘤胃细菌区系的影响采用4×4拉丁方设计，利用常规分析法及PCR／DGGE技术，研究了延胡索酸二钠(0g·d^-1、5g·d^-1、10g·d^-1、15g·d^-1)对山羊瘤胃代谢、血液生化指标和瘤胃细菌区系变化的影响。结果表明，添加延胡索酸二钠显著提高了瘤胃液中的pH值（P＜0.05），降低了瘤胃中的乳酸浓度（P＜0.01），但对乙酸、丙酸、丁酸、TVFA和NH₃-N浓度未产生显著影响（P＞0.05）。对血液指标测定结果表明，添加延胡索酸二钠显著降低了血液中的乳酸浓度（P＜0.05），但对乳酸脱氢酶（LDH）、血糖、胆固醇、尿素氮及甘油三酯浓度无显著影响（P＞0.05）。DGGE图谱分析表明，添加延胡索酸二钠后，DGGE图谱上瘤胃细菌的优势条带数较对照显著增多（P＜0.05），同时瘤胃细菌的多样性也显著增加（P＜0.05）。基因序列分析表明，DGGE图谱中优势条带的16srDNA基因序列中有4个基因序列与基因数据库登录的相关序列的相似性大于97％，8个基因序列的相似性在88～96％。相似性大于97％的4个克隆中，2个被鉴定为牛链球菌及溶糊精琥珀酸弧菌，另外两个属于未鉴定菌；添加延胡索酸二钠后，溶糊精琥珀酸弧菌、产丁酸菌相似菌、未培养瘤胃细菌、真杆菌相似菌、唾液普雷沃菌相似菌成为优势菌。结果说明，添加延胡索酸二钠可提高瘤胃pH值，降低瘤胃及血液中的乳酸浓度，促进一些瘤胃细菌的增殖，但对动物机体氮代谢无明显影响。更多还原

【Abstract】 The present research was conducted to investigate the influence of fumarate and itssodium on rumen fermentation and ruminal bacterial communities of goat. By comparingthe effects of fumarate and its sodium （fumarate,sodium fumarate and disodium fumarate）on rumen fermentation, the proper adding levels of fumarate in vitro was established. Byusing different incubation condition （including concentrate, forages and differentconcentrate: forage ratio diets）, we evaluated the effects of disodium fumarate on rumenfermentation and methane production in vitro. Furthermore, the effect of disodium fumarateaddition on rumen fermentation, plasma metabolites and ruminal bacterial community invivo was investigated. This dissertation was described in the following seven sections.1 Effect of fumarate addition on rumen fermentation in vitroRumen contents from three local male goats with permanent rumen fistulae wereincubated in anaerobic media with substrate consisting of rice straw powder （0.4 g） andcorn （0.6g）. The effects of fumarate with final concentrations at 4,8 and 12 mmol·L^-1 onthe ruminal fermentation were investigated. Results showed that, as compared with thecontrol, total volatile fatty acids（TVFA）, propionate production（P%） and total gasproduction for the group treated with fumarate increased （P＜0.05）, while the ration ofacetate to propionate and pH decreased （P＜0.05）. The effect of fumarate was moresignificant as its concentration increased, suggesting its dose dependent effect. The resultsindicated that fumarate addition stimulated the fermentation activity of rumen microbesfrom goat, while lowered the pH of the medium （P＜0.01）.2 Comparisons of effects resulting from fumarate and its saltsaddition on fermentation by rumen micro-organisms in vitroRumen contents from three local male goats with permanent rumen fistulae wereincubated in anaerobic media with substrate consisting of cracked corn, soybean meal andryegrass particle. The effects of fumarate, sodium fumarate or disodium fumarate with final concentrations at 4, 7 and 10 mmol·L^-1 on the in vitro fermentation were compared. Resultsshowed that, as compared with the control, total volatile fatty acids （TVFA）, propionateproduction （P%） and total gas production for all treatments increased significantly, while theratio of acetate to propionate and lactic acid production decreased significantly（P＜0.05）. At0 h or 24 h of incubation, the pH value in the group treated with disodium fumarate washigher than that of fumarate or disodium fumarate treatment. No significant difference wasobserved in other ruminal characteristics among the three treatments. The results showedthat fumarate and its salts addition could stimulate the rumen fermentation, while thedisodium fumarate had a better beneficial effect on the in vitro fermentation by rumenmicroorganisms.3 Influence of disodium fumarate addition on the fermentation ofconcentrates by rumen microorganisms in vitroFour experiments were conducted to investigate the effects of disodium fumarate onthe in vitro fermentation profiles of concentrate feed. In experiment 1, mixed concentratediets （0.7 g cracked corn, 0.3 g soybean meal and 0.43 g festuca elata, DM basis） werefermented in vitro by rumen microorganisms from local goats. Results showed that ascompared with the control, total volatile fatty acids（TVFA）, the percentage of propionate（P%） , total gas production and the dry matter loss for the disodium fumarate treatmentincreased （P＜0.05）,while the ratio of acetate to propionate and lactate production decreasedsignificantly. In experiment 2, a time course study was conducted to investigate the effect ofdisodium fumarate at 7 mmol·L^-1 on rumen fermentation in vitro. The results showed thatdisodium fumarate addition increased the cumulative gas production, pH, propionate andTVFA production, and reduced the ratio of acetate to propionate, whereas no treatmenteffect was observed for the NH₃-N concentration and the amylase activity. Disodiumfumarate addition tended to lower the lactate production（P＜0.05）.With the exception ofTVFA production, there was significant interr-reaction effect between the disodiumfumarate treatment and fermentation time（P＜0.05）. In experiment 3, influences of disodiumfumaratc addition on fermentation of cracked corn and cracked wheat by rumenmicroorganisms in vitro were investigated. The results showed that during 24 h ofincubations, disodium fumarate addition increased （P＜0.05） the gas production and the pHfor the two substrates, but this effect was not observed for other fermentation characteristics.In experiment 4, the effect of disodium fumarate addition on rumen lactic acidosis in vitro was investigated. Lactic acidosis was induced by in vitro fermentation at 39℃for 24 hwith a 25 g·L^-1 test diet. The result showed disodium fumarate addition decreased lactateconcentration （P＜0.01）. These results suggest that the addition of disodium fumarate toruminant feed could stimulate the in vitro fermentation of rumen microbes from goat,whereas this effect on changes of ruminal character tics may largely depend on the generalnature of the substrates.4 Influence of disodium fumarate addition on the fermentation offorages by mixed rumen microorganism, Ruminococcus flavefaciensand rumen fungusThree experiments were conducted to investigate the effect of disodium fumarate onfermentation of forage by mixed rumen microorganism, Ruminococcus flavefaciens andrumen fungus. In experiment 1, rumen contents from three local male goats with permanentrumen fistulae were incubated in anaerobic media with substrate including rice straw,festuca elara and ryegrass, and effects of disodium fumarate with final concentrations at7mmol·L^-1 on the in vitro fermentation was investigated. The results showed that disodiumfumarate addition increased the total gas production for all the substrates. As comparedwith the control, the addition of disodium fumarate increased the dry matter loss by 26.22% （P＜0.05）, 14.16% （P＜0.05） and 28.54%（P＜0.01）, receptively. In experiment 2, theeffect of disodium fumarate on the fermentation activity of Ruminococcus flavefaciens wasinvestigated. Disodium fumarate was added to the incubation bottles to achieve finalconcentrations of 0, 4, 7 and 10 mmol·L^-1. Results showed that disodium fumarate additionincreased the dry matter loss, TVFA production and the counts of Ruminococcusflavefaciens（P＜0.05）. In experiment 3, the effect of disodium fumarate on the ryegrass offermentation by the rumen fungus was investigated. The results showed that the disodiumfumarate addition (0, 4, 7 and 10mmol·L^-1) reduced the dry matter loss and the cumulativegas production （P＜0.01）.In conclusions, the diosodium fumarate addition could stimulate.the fermentation activity of the mixed rumen micro-organism and Ruminococcusflavefaciens, whereas reduced the fermentation activity of the rumen fungus.5 Effects of disodium fumarate on in vitro methane production andfermentation of diets differing in their forage:concentrate ratioEffects of disodium fumarate on CH₄ production and fermentation of three diets differing in their concentrate content by rumen micro-organisms were investigated by batchcultures. Rumen contents were collected from three local goats. Disodium fumarate wasadded to the incubation bottles to achieve final concentrations of 0, 4 and8mmol.L^-1 -fumarate.Results showed that disodium fumarate addition increased linearly（P＜0.001） the final pH, acetate and propionate production, and reduced the amount ofNH₃-N in the cultures. As compared with the control, adding disodium fumarate to batchcultures reduced CH₄ production. Disodium fumarate tended to increase （P＜0.05） theMCP（rumen microbial protein） for the high-forage diet, but no differences （P＜0.05） wereobserved for the high-concentrate diets. These results indicate that the effects of disodiumfumarate on rumen fermentation mainly depend on the nature of the incubated substrate,with the high-forage diet showing the greatest response.6 Influence of disodium fumarate addition on rumen fermentationand ruminal bacterial community in vitroTwo experiments were conducted to investigate the effect of disodium fumarateaddition on the ruminal fermentation of different kinds of diet （festuca elata, mixed diet andconcentrate diet） in vitro. In experiment 1, effects of disodium fumarate with finalconcentrations at 7mmol.L^-1, on the in vitro fermentation and the changes of rumen bacteriawas investigated. Results showed that disodium fumarate addition increased （P＜0.05） thecumulative gas production, pH and the TVFA production, and reduced the lactic acidproduction （P＜0.05）. The analysis of DGGE profiles revealed that disodium fumarateaddition affected the composition of the rumen bacteria communality. In experiment 2, gasproduction was monitored and the cumulative values were fitted to a mono-phasic curve.For all substrates, disodium fumarate treatment increased the asymptotic gas production （A）and the max rate of gas production （P＜0.05）, whereas no treated effects （P＞0.10） wereobserved for the half-time C with the exception of mixed diets （5:3:2, DM basis）.Collectively, these results suggest that disodium fumarate is effective in increasing the pHand gas production for the diets differing in forage: concentrate ratio in vitro, whereas itseffect on changes of ruminal microbial community may largely depend on the generalnature of the substrate.7 Effect of disodium fumarate on ruminal and plasm metabolites andthe ruminal bacterial communities in goats Effects of disodium fumarate on ruminal metabolism and changes of rumen microbialcommunity were investigated. Results showed that disodium fumarate treatment increasedthe pH （P＜0.05） and reduced the lactate production （P＜0.05）, whereas did not affect theproduction of acetate, propionate, butyrate and TVFA production significantly （P＜0.05）.Disodium fumarate addition did not influence the plasma of concentrations of glucose, ureaN, cholesterol and triacylglycerol, but reduced the concentration of lactate. The analysis ofDGGE profile revealed that, as compared with the control, disodium fumarate treatmentshowed a higher degree of bacterial diversity （P＜0.05）. A clone library was created fromcomplete 16S rDNA. From the library, 12 clones had their V6-V8 regions matchedpredominant bands on the DGGE gel and their 16S rDNA were then sequenced andsubjected to an online similarity search. Four of the clones showed their similarities over97% with database sequences, with two sequences similar to Succinivibrio dextrinosolvensand Streptococcus bovis, with other two were similar to those unidentified rumen bacteria,Others clones had their similarities in the range of 88%～96% with database sequences. Ascompared with the control, Succinivibrio dextrinosolvens, Butyrate-producing bacteriumSL7/1, Uncultured rumen bacteria, Clostridium hathewayi and Prevotella salivae becamethe predominant bands after disodium fumarate addition. Results indicated that disodiumfumarate addition was effective in improving the pH value and lowering the lactate in therumen, and stimulating the growth of some ruminal bacteria.更多还原