【作者】 林波；

【导师】 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 研究和开发瘤胃发酵调控剂,抑制反刍动物瘤胃甲烷的产生是近年来的研究热点,挥发油和富马酸盐是目前研究报道较多的两种新型瘤胃发酵调控剂。本试验分别采用体外和体内试验研究了挥发油与活性成分不同组合对瘤胃发酵的作用差异,及其与富马酸钠共同添加对湖羊瘤胃甲烷产生、微生物区系和营养物质消化的影响；同时结合分子生物学方法,进一步阐明了挥发油对瘤胃微生物的影响及其作用机制。为将二者的组合作为瘤胃发酵调控剂的提供了理论依据。试验一：为研究挥发油与其活性成分对瘤胃发酵的影响差异,本试验采用压力传感器式体外产气系统,分别将牛至油、肉桂油及其对应的活性成分香芹酚和肉桂醛以0、50、200和500mg/L的水平添加到体外发酵液中,研究它们对瘤胃发酵和甲烷产生的影响。结果表明,产气量、挥发性脂肪酸(VFA)和甲烷产量均随挥发油添加浓度的增加而显著降低(P<0.05),200mg/L的添加浓度下,香芹酚和肉桂醛对VFA的降低幅度均大于30%,而500mg/L的油和活性成分均降低了VFA达50%以上,表明瘤胃发酵显著抑制。同等浓度下,活性成分对瘤胃发酵的抑制幅度大于其对应的油,且甲烷抑制潜势也低于对应的油；高浓度下,香芹酚和牛至油对发酵抑制幅度大于肉桂醛和肉桂油。可见,不同挥发油对瘤胃具有不同的作用效果,而单种活性成分对甲烷抑制的效果弱于对应的天然挥发油。试验二：研究不同挥发油组合和活性成分组合组合对瘤胃发酵的影响,并筛选可降低瘤胃甲烷的优化组合。本试验将丁香油、肉桂油、牛至油和柠檬油按照1：2：3：4,2：1：4：3,3：4：1：2,4：3：2：1和1：1：1：1的比例组成5种不同的挥发油组合(EOC1, EOC2, EOC3, EOC4, EOC5),且将丁香酚、肉桂醛、香芹酚和柠檬醛按照相同的比例组成5种不同的挥发油活性成分组合(EOAC1,EOAC2,EOAC3, EOAC4, EOAC5),采用压力传感器式体外培养系统研究了这些组合对体外甲烷产量和瘤胃发酵指标的影响,各种组合的添加浓度为0、50、200和500mg/L。研究结果表明,添加EOC和EOAC对产气量、甲烷产量和VFA的产量的影响均呈一种浓度依赖趋势。在同等挥发油添加浓度下,甲烷的降低幅度的趋向明显高于VFA和产气量的降低幅度。EOC和EOAC降低了瘤胃氨氮和A/P的比值,但对pH值则无显著影响。通过氢平衡分析发现,500 mg/L的EOC5中氢被用于生成甲烷与挥发性脂肪酸的比值为各挥发油组最低,同时降低了62.3%的甲烷和17.9%的TVFA,是具有最高甲烷抑制潜势的挥发油组合；而200 mg/L的EOAC5中氢被用于生成甲烷与挥发性脂肪酸的比值为各活性成分组最低,降低了31.3%的甲烷,好12.9%的VFA,是具有最高甲烷抑制潜势的活性成分组合。总体而言,挥发油组合对甲烷的抑制潜力大于活性成分组合。试验三：为研究挥发油及其活性成分和富马酸之间的协同作用,本试验采用压力传感器式体外系统,以试验一中筛选的最优挥发油组合5(EOC5)及活性成分组合5(EOCC5)为研究对象,将500mg/L的EOC5和200mg/L的EOCC5分别将0,5,10,15 mmol/L的富马酸一钠组合添加,研究二者对甲烷产生及瘤胃发酵指标和瘤胃微生物数量的影响。结果表明,5和10mmol/L的富马酸钠可进一步降低瘤胃甲烷排放,同时增加VFA的产量,EOC5和EOAC5两组均是在添加10 mmol/L的富马酸一钠时具有最大的甲烷抑制潜势。采用RT-PCR法对该最优组合的甲烷菌、真菌、原虫和两种重要的纤维降解菌F. succinogenes与R. flavefaciens进行定量分析表明,瘤胃原虫被显著抑制,真菌和两种纤维降解菌的数量也有较大程度的降低,但甲烷菌种群的数量降低幅度则明显的低于其他微生物。这些结果表明,添加富马酸有利于挥发油对瘤胃甲烷的抑制,但挥发油添加抑制瘤胃微生物可能会降低纤维消化。试验四旨在开发以微孔淀粉吸附挥发油后,用海藻酸钠为壁材包埋挥发油的工艺,同时研究此工艺对挥发油包埋效果的影响。试验采用五种挥发油成分(丁香酚、香芹酚、肉桂醛、柠檬醛、β—蒎烯)23.75：23.75：23.75：23.75：5的比例配成混合物,以微孔淀粉吸附后作为芯材,以海藻酸钠为壁材进行包埋试验,采用气喷雾法对挥发油活性成分和微孔淀粉的复合物进行包被试验。结果表明,微孔淀粉吸附组的包封率为82.6%,挥发油含量为12.8%,利用微孔淀粉吸附植物挥发油后,再用海藻酸钠包被,与直接包被相比,可提高海藻酸钠对结构和性质不同的挥发油单体的包被完全性,同时提高包被产物的稳定性,因此微孔淀粉吸附挥发油后再进行包被是一项提高挥发油包被效率的技术。试验五本试验采用装有瘤胃、十二指肠和回肠三通瘘管的4头湖羊,以4×4拉丁方设计,研究了植物挥发油及其活性成分和富马酸钠添加对湖羊生化及抗氧化指标、瘤胃发酵指标和营养物质消化率的影响。试验设四个处理,处理—饲喂基础日粮(头日400g精料+500g粗料,C),处理二添加1g混合挥发油(牛至油、肉桂油、丁香油和柠檬油按等比例组成)与25 g富马酸—钠(EOCF),处理三添加0.5 g混合挥发油活性成分(香芹酚、肉桂醛、丁香酚和柠檬醛等比例混合)与25 g富马酸一钠(LEACF),处理四添加1g混合活性成分与25 g富马酸一钠(HEACF)。试验分4期进行,每期包括14d预试期和7d正试期。结果表明,在同时添加25g/天富马酸钠的情况下,挥发油及其活性成分的添加对湖羊精料的采食量无显著影响,1g/天的天然挥发油组合还有提高粗料采食量的趋势。添加挥发油或其活性成分并不影响湖羊机体抗氧化性力,0.5g/天挥发油活性成分添加并未影响湖羊机体健康,但湖羊可能对1g/天活性成分产生应激。瘤胃发酵方面,挥发油和其活性成分添加三组均不影响瘤胃pH值,混合挥发油组(EOCF)和高量活性成分组(HEACF)显著降低了VFA,三组均显著降低了乙酸丙酸比例、氨氮浓度、甲烷预测产量、原虫和甲烷菌数量,但活性成分两组降低纤维消化菌数量,可能不利于纤维的消化。挥发油和活性成分组合的添加不影响干物质和有机物消化道各段的有机物消化率；有利于抑制前胃蛋白的降解,降低饲料蛋白的瘤胃消化率。此外,挥发油活性成分组合不影响饲料纤维的前胃消化率,但有提高肠道消化率的趋势,总体而言对饲料湖羊饲料纤维的消化无不利作用。综上所述,将不同作用机制的挥发油或其活性成分组合是一种有效的瘤胃甲烷抑制手段,但油的效果优于活性成分。挥发油降低甲烷和氨氮的机制在于抑制瘤胃原虫和甲烷菌,但对瘤胃纤维降解可能有抑制作用。富马酸钠添加可促进挥发油对甲烷的抑制作用,动物试验表明同时添加挥发油和富马酸不影响干物质、有机物、NDF和ADF的前胃消化率,但可降低饲料蛋白的前胃消化率,总体上对动物整体营养物质消化率无显著影响。更多还原

【Abstract】 Development of some substances to manipulate ruminal fermentation and methane production is a research focus during recent years. Up-to-date, plant essential oils (EO) and fumarate are most studied substance. In this study, in vitro and in vivo trials were conducted to study the effects of different ratios of EO or their active component in combination with fumarate on rumen fermentation, methane production, and nutrients digestibility in Hu sheep. Molecular biological methods were also used to investigate the effects of EO and fumararte on ruminal microbes, aiming at elucidating their action mechanism.In Expt.1, Reading Pressure Transfer (RPT) system was adopted to study effects of two essential oil (cinnamon oil and oregano oil) and their major component (cinnamaldehyde and carvacrol) on rumen fermentation and methanogensis in vitro. The EO addition levels were 0,50,200 or 500 mg/L, respectively. Gas production, volatile fatty acid (VFA) and methane production were decreased with the increasing level of EO addition (P<0.05). At level of 200 mg/L, carvacrol and cinnamaldehyde decreased VFA by more than 30%, while 500mg/L level of oil or active components decreased VFA more than 50%, indicating that ruminal fermentation was substantially inhibited. When used at the same addition level, fermentation-inhibiting effects of cinnamaldehyde and carvacrol were larger than their correspondent oils. At the high level, carvacrol and oregano oil had higher fermentation inhibition effects than cinnamaldehyde and cinnamon oil. Results in this study indicated that effect of EO on rumen fermentation varied with their type, while methane inhibition effect of a single EO active component was weaker than their correspondent natural EO.The objectives of Expt.2 were to study the effects of different ratios of EO or EO active components on in vitro rumen fermentation, and to screen the optimal combinations to decreased methane production. Five combinations were formed by mixing oil from clove, origano, cinnamon,and lemon were mixed at ratios of 1:2:3:4, 2:1:4:3,3:4:1:2,4:3:2:1,1:1:1:1, respectively, to make up five combinations (EOC1, EOC2, EOC3, EOC4 and EOC5). Similarly, eugenol, carvacrol, citral and cinnamaldehyde were mixed to make another five combinations (EOAC1, EOAC 2, EOAC3, EOAC4, EOAC5). The mixtures were supplied at levels of 0,50,200 or 500 mg/L into the incubation fluid to screen the optimal combination for methane reduction. The RPT system was adopted to determine the effects of these combinations on rumen fermentation and methane production in vitro. Effect of EOC and EOAC on gas, methane and VFA production appeared in a dose-dependent manner. In the same addition level, methane decreasing extent tended to lower than the content to which total VFA and gas production decreased. Both EOC and EOAC decreased rumen ammonia and acetate to propionate (A/P) value, but had no influence on pH value. Hydrogen balance analysis showed that the ratio of hydrogen consumed via methane to hydrogen via VFA was the lowest at 500 mg/L EOC5 among EOC groups. At this group methane and total VFA were decreased by 62.2 and 17.9%, respectively. This level was, therefore, screened as optimal EO combination to decease methane. In 200 mg/L of EOAC5, the ratio of hydrogen consumed via methane to hydrogen via VFA was the lowest among EOAC groups, with methane and total VFA reduced by 31.3 and 12.9%, respectively. This level was screened as optimal EO combination to decease methane.In Expt.3, the 500mg/L EOC5 and 200 mg/L of EOAC5, which were screened from Expt.2, was added with 0,5,10 and 15 mmol/L monosodium fumarate, respectively, to see if fumarate had further effect on rumen fermentationt. Addition of fumarate further decreased methane production, but increased VFA production compare with EOC or EOAC added solely. The highest relative methane reduction potential (ratio of methane production reduction relative to reduced total VFA production due to the added inhibitory compounds) was at 10 mmol/L of fumarate in both EOAC and EOC groups. Quantification of microbial populations of rumen fluids by RT-PCR method showed that methanogen, protozoa, fungi, Fibrobacter succinogenes and Ruminococcus flavefaciens were significantly decreased in EOAC5-added groups. In summary, addition of fumarate has beneficial effect on decreasing methane production by EOC and EOAC, while the EOC and EOAC have inhibitory effects on rumen microbes.Expt.4 was conducted to explore the techniques for use of sodium alginate to encapsulate the microporous starch (MPS) in which essential oil is absorbed. Eugenol, carvacrol, cinnamaldehyde, citral and (3-pinene were mixed at a proportion of 23.75, 23.75,23.75,23.75 and 5%, respectively, then MPS was adopted to pre-absorb the complex of five pure EO components. The absorption complex was encapsulated by sodium alginate using high gas pressure spray method and formed into calcium alginate products. Adsorption rate of MPS with essential oil absorbed was 82.6%, and the EO content was 12.8%. Compare with direct encapsulate of EO, the EO components absorbed with MPS had an increased inclusion completeness of EO components, and improved stability of EO component. Therefore, absorption of EO with MPS before encapsulation is an effective technique to improve inclusion rate and increase encapsulation completeness of EO components.In Expt 5, four rumen fistulated Hu sheep were used to investigate effect of mixture of EOs or their active components along with fumarate on anti-oxidative status, blood biochemical parameters, rumen fermentation and nutrient digestibility. A 4×4 Latin Square design involved four treatments:basal diet added with 25 g/day monosodium fumarate (Control, C); C plus 1 g/d of EO combinations (mixture of equal proportion of oil from clove, oregano, cinnamon and lemon) (EOCF); C plus 1.0 (HEACF) or 0.5 g/day EO active component combinations (mixture of equal portion of eugenol, carvacrol, citral and cinnamaldehyde) (LEACF), respectively. Each period lasted for 21 days including 14 days for adaptation. With 25g/day monofumarate added, addition of EO and their active components did not have influence on concentrate intake of Hu sheep, but addition of EOCF tended to increase forage intake. The EO had a limited effect on anti-oxidant ability. Low dose (0.5g/day) of EO does not exert an influence on sheep health, while 1 g/d of EOAC would result in some stress to sheep. As for the rumen fermentation, pH value of incubation fluid was not influenced. The A/P ratio, ammonia-N, protozoa, methanogen were significantly decreased, indicating that methane would be decreased. However, fibrolytic bacteria were also decreased, suggesting that fiber digestion would be influenced by such addition. Addition of EO and their components did not have influence on digestibility of dry matter and organic matter in every segment of digestive tracts, but was beneficial for inhibition of forestomach protein degradation. Besides, EO and their components combination did not have effect on forestomach digestibility of feed fiber, but tended to increase fiber digestibility in the duodenum. Generally, addition of EO and their components had no harmful effects on fiber digestion of Hu sheep.In summary, Combination of different EO or EO components would be a promising measure to inhibit methane production, while the effect of EO was better than components. Addition of EO would inhibi protozoa and methanogen, but would also have a negative effect on fiber digestion. Addition of fumarate with EO and their components is beneficial to decrease methane production, and did not have farmful influence on DM, OM, NDF and ADF digestibility in Hu sheep.更多还原