【作者】 王新峰；

【导师】 朱伟云；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 瘤胃生成的CH4是饲料能量的一种损失,同时也造成大气环境的污染。因此,调控瘤胃发酵,减少CH4释放,提高动物生产性能越来越受到重视。天然植物提取物皂甙具有抑制瘤胃原虫生长,减少CH4产生的作用。开发植物皂甙调节瘤胃微生物发酵减少CH4产生及其对调控的机理的研究成为热点。本试验选用具有达玛烷结构三萜类植物提取物-绞股蓝皂甙,分析其对瘤胃微生物体内和体外CH4产量及对瘤胃微生物区系构成和数量的影响,从微生物区系和数量的变化来阐述绞股蓝皂甙减少CH4产生的机理。并从耐受性试验中,分析绞股蓝皂甙减少CH4生成具有时效性的可能原因。为绞股蓝皂甙在调控反刍动物瘤胃发酵,减少CH4生成中的应用提供理论依据。1绞股蓝皂甙对瘤胃微生物体外甲烷产量及发酵特性的影响利用体外产气量法研究绞股蓝皂甙对瘤胃微生物CH4产量的影响。试验包括两个部分,试验一研究了绞股蓝皂甙对瘤胃微生物发酵的影响,试验二分析绞股蓝皂甙对瘤胃微生物发酵动力学参数的影响。试验以0.42g羊草+0.126g玉米+0.054g豆粕为发酵底物,绞股蓝皂甙添加量分别为0(对照)、5、10、20和40mg/60mL,发酵24h。结果显示,与对照组比较,各处理组CH4摩尔产量显著下降(P<0.05),CH4产量与皂甙之间有显著的线性效应。对氢的利用率仅有10mg组显著低于对照组,其它试验组无明显变化。10mg绞股蓝皂甙水平与对照组比较,显著提高了TVFA产量及乙酸、丙酸和丁酸的比例(P<0.05),乙丙比无显著变化,乙酸,丁酸,异丁酸,戊酸,异戊酸,支链脂肪酸百分比及乙丙比和TVFA浓度随着皂甙剂量增加呈现显著的二次方效应,丁酸同时具有显著的线性效应。处理组原虫数量显著下降(P<0.05),有着显著的线性和二次方效应(P<0.05),而微生物蛋白含量没有显著变化,但呈现上升趋势。高剂量皂甙组的氨态氮浓度显著高于对照组(P<0.05),与绞股蓝皂甙之间有显著的线性效应。高剂量绞股蓝皂甙降低了微生物发酵的理论产气量与实际产气量,并呈现显著的线性和二次方效应,产气速率与皂甙之间有着显著的线性效应。以上结果表明,绞股蓝皂甙能改变瘤胃微生物发酵模式,提高VFA的生成,抑杀瘤胃原虫,降低CH4产量,提高饲料能量利用效率,减少CH4释放。2绞股蓝皂甙对山羊瘤胃微生物发酵及血液生化指标的影响选用4只体重约为30±3kg的波尔山羊与本地山羊杂交的一岁阉公羊,采用4×4拉丁方试验设计,分成4组,研究绞股蓝皂甙对瘤胃发酵特性和微生物区系的影响。每天08：00和17：00进行饲喂,饲料为70%羊草和30%精料(玉米：豆粕=7：3)。每天早晚分两次经瘤胃瘘管共向瘤胃中灌注0g(对照),2g,4g和8g/d.h-1绞股蓝皂甙。采食前Oh和采食后2,4和8小时通过瘤胃瘘管负压采集瘤胃内容物。结果显示,饲喂高剂量的皂甙后,瘤胃氨态氮浓度趋于显著下降(P=0.057),但对微生物蛋白、CH4产量及氢的利用无显著影响(P>0.05),但CH4产量有下降的趋势,氢的利用率提高。乙酸、支链脂肪酸浓度及乙丙比略微下降,而采食量和丙酸浓度有所提高(P>0.05)。血清中的血糖、甘油三酯、总胆固醇、血液尿素氮和总蛋白浓度无显著变化(P>0.05)。瘤胃原虫总数及Entodinium spp和Diplodinium spp数量显著线性下降。从瘤胃内容物中提取基因组DNA,对微生物数量进行定量分析。产甲烷菌、真菌、黄化瘤胃球菌和产琥珀酸丝状杆菌数量以占总菌16S rDNA的比例来表示。产甲烷菌数量急剧下降,而真菌、纤维降解菌在低剂量2g/d.h-1条件下,与对照组比较比例显著提高。在高剂量皂甙的添加量下,几种微生物的数量显著下降。使用PCR/DGGE技术追踪了瘤胃原虫、细菌和产甲烷菌区系的变化,每只羊处理前后瘤胃原虫区系的相似性在48-75%之间变化,产甲烷菌的相似性在66～87%之间。对7种原虫上共生产甲烷菌的区系分析发现,不同原虫上产甲烷菌区系结构存在差异,且同种原虫在绞股蓝皂甙处理后,其共生产甲烷菌区系结构发生改变。切胶克隆测序发现原虫上共同存在的优势产甲烷菌为产甲烷短杆菌(Methanobrevibacter sp.)。以上结果表明,添加绞股蓝皂甙影响瘤胃发酵,减少产甲烷菌、原虫总数及单个种原虫数量,促进两种纤维降解菌及真菌的生长,改变了瘤胃微生物区系及与原虫共生产甲烷菌的区系。3瘤胃微生物的绞股蓝皂甙耐受性研究本试验通过体外批次培养,分析多次传代后,绞股蓝皂甙对微生物发酵特性及细菌和产甲烷菌区系的影响。结果显示,绞股蓝皂甙提高了发酵体系的pH值,在对产气量的影响上,除第1代5mg和10mg组产气量高于对照组外,其它试验组的产气量与对照组比较都有所下降,其中有些组的产气量显著或极显著下降,并与绞股蓝皂甙剂量之间存在显著或极显著的线性或二次方效应。CH4产量下降,与皂甙剂量之间有显著和极显著的线性和二次方效应(P<0.05或P<0.01)。氨态氮浓度均有所提高,MCP在第1代提高外,其它试验组都与对照组相比有所下降,有些剂量和不同代次达到显著或极显著效应。原虫数量在第1和2代均显著下降,与绞股蓝皂甙添加量之间有显著的线性效应,到第3代原虫数量很少,因此未做计数。绞股蓝皂甙明显降低了VFA的浓度,乙酸,丙酸和丁酸浓度下降。添加皂甙后,细菌和产甲烷菌区系发生变化,优势条带如溶纤维丁酸弧菌和产琥珀酸丝状杆菌在皂甙处理后消失,同时一些新的优势条带如欧文氏菌和未知菌成为优势菌。这些变化表明,绞股蓝皂甙能够抑制一些微生物生长,同时,一些能够适应或降解绞股蓝皂甙的微生物得以成为发酵体系中的优势菌群。以上结果表明,在多次传代中,绞股蓝皂甙改变了瘤胃微生物的发酵,微生物区系逐渐发生变化,以适应有皂甙存在的生长环境。4绞股蓝皂甙对真菌与产甲烷菌共培养甲烷的产量及发酵特性的影响以青链霉素和氯霉素为抑制剂分别制备真菌与产甲烷菌共培养(体系Ⅰ)及以真菌为主(体系Ⅱ)的两种体外培养体系,研究添加不同剂量绞股蓝皂甙对两种培养体系CH4产量及体外发酵特性的影响。试验结果表明,(1)在体系Ⅰ中添加绞股蓝皂甙,CH4产量和产甲烷菌数量极显著下降,与绞股蓝皂甙剂量之间有极显著的线性效应(P<0.01)；产气量和真菌数量极显著下降,与绞股蓝皂甙剂量之间有极显著的线性和二次方效应(P<0.01)；TVFA和乙酸浓度显著下降,并与绞股蓝皂甙剂量之间存在极显著的二次方效应(P<0.01)；pH值显著上升,并与绞股蓝皂甙剂量之间呈极显著的二次方效应(P<0.01)。(2)在体系Ⅱ中添加绞股蓝皂甙,产气量呈极显的线性和二次方下降(P<0.01)；真菌和产甲烷菌数量呈极显著的线性下降(P<0.01)；TVFA显著下降(P<0.05),乙酸极显著下降,并与绞股蓝皂甙剂量有显著的线性和二次方效应；pH值显著上升,并与剂量之间有极显著的线性和二次方效应(P<0.01)。以上结果显示,绞股蓝皂甙导致pH上升,产气量和CH4产量的下降主要是由于绞股蓝皂甙抑制了厌氧真菌数量或活性及产甲烷菌数量所致,表明绞股蓝皂甙对共培养真菌和产甲烷菌产生抑制作用。更多还原

【Abstract】 Methane is a loss of feed energy for ruminants, and a pollution resource for atmosphere environment. So, the more attention were highlighted for modulating ruminal fermentation, reducing ruminal methane emission and prompting the ruminants performance. Saponin, as one of plant extracts, can inhibit the growth of protozoa and reduce the methane emission. The common interesting were attracted for exploitation of new natural plant saponin for reducing methane emission and mechanisms of reducing methane were investigated. The effects of gypenosides with dammarane structural triterpenes saponin were selected to investigate the effects on in vitro and in vivo methane production of goat rumen. Effects of gypenosides on community of rumen microbe and mechanisms of decreased methane were analysed by the shifts of community of microbes when saponin was usded to reduce methane. The reasons of reducing methane with short time effect were investigated by the experiment of microbial resistance to gypenosides. The theoretical evidences were explained for regulating ruminal fermentation and reducing methane emission.1 Effects of gypenosides on in vitro fermentation and fermentation kinetic parameters by mixed ruminal microorganismsEffects of Gypenosides on rumen methane prodcution were investigated by in vitro gas technique using mixed rumen microorganisms from goats as inoculum. The study includes two experiments. Experiment 1 was investigated to estimate its effects on fermentation characteristics of rumen microorganisms. Experiment 2 was conducted to assay its effects on fermentation kinetic parameters. The diet consisted of Leymus chinensis (70%), ground corn grain (21%) and soybean meal (9%)(1 mm screen). Five different gypenosides doses were used for each compound:0,5,10,20 and 40 mg/60 ml of the total culture medium. The results showed that as compared with the control, methane production and concentration were reduced by gypenosides addition (p<0.05) and there was a linear effects on methane production between control and treatments. The hydrogen utilization was lower than control at 10mg level. The TVFA concentration, acetate, propionate and butyrate acids proportion of TVFA were significantly increased (P<0.05) at 10mg level, and there was a significant quadratic effects on acetate, butyrate, isobutyrate, valerate, isovalerate, BCP proportion, acetate to propionate ratio and TVFA concentration with the gypenosides doses. There was a linear effect on butyrate with gypenosides addition. The protozoa counts for treatment groups decreased with a significantly linear and quadratic effects (P<0.05). Microbial protein was not changed abruptly, but with a increasing trend. Ammonia nitrogen concentration was significantly higher than control at highest saponin level and there was a linear effect on ammonia concentration with gypenosides addition. The theoretic and observed gas production decreased at high saponin level with a significant linear and quadratic effects. There was a significant linear effect on rate of gas production with saponin doses increasing. Above results indicated that gypenosides addition could modify the microorganisms fermentation pattern, reduce protozoa numbers and methane emission, abate pollution of methane derived from ruminants to environment. The VFA production and feed conversion efficiency were promoted.2 Effects of gypenosides on rumen fermentation, microorganisms population and blood parameters in goatsFour Boer crossbred with local goat wether male goats (30±2.3 kg) were used in a 4×4 Latin square experiment to determine effects of gypenosides from Gynostemma Pentaphyllum Makino on ruminal fermentation characteristics and ruminal microbial community. The goats fed a 70%Leymus chinensis:30%concentrate (corn:soybean meal=2:1) were intraruminally given with suspensioned gypenosides at 0800 and 1700 daily. Doses were 0 (control),2,4 and 8g/d.head-1. Ruminal contents were sampled at 0(immediately prior to feeding),2,4 and 8 h after feeding in the morning. The results showed that ruminal ammonium concentration in gypenosides treatments decreased (P=0.057), while there was no significant effect on microbial crude protein, methane emission and hydrogen recovery (P>0.05). But, there was a decreasing trend for methane and a increasing rate of hydrogen utilization Acetate, BCVFA concentration and ratio of acetate to propionate decreased slightly, whereas feed intake (low and high dose), propionate concentration increased than control group (P>0.05). There were no differences for blood parameters such as blood serum glucose, TG, TC, BUN and TP concentration between control and treatments (P>0.05). The numbers of total protozoa, Entodinium spp. and Diplodinium spp. in rumen with gypenosides addition were significantly lower (P<0.05) than those in control group, with a dose-dependent manner. Total genomic DNA were extracted from ruminal microbes, and populations of rumen microbes were determined by a real-time PCR. Populations of total methanogens, anaerobic fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes were expressed as a proportion of total ruminal bacterial 16S rDNA. The number of methanogens sharply reduced. While fungi, fibrolytic bacteria percentage of total bacteria in rumen markedly increased at 2 g/dh-1. The numbers of all microbial groups decreased obviously at high dose of gypenosides addition. DGGE was used to monitor the shifts of protozoal, methanogenic and bacterial population after gypenosides treatment. The similarity varied from 48%to 75%for protozoa,66 to 87%for methanogens and 40 to 82%for bacteria. The community assay of methanogens from seven protozoal species found that there were difference for the different protozoa. The shift of methanogenic population from single protozoal species was observed before and after treatment with gypenosides. The common dominant methanogen in all protozoa collected was Methanobrevibacter sp. after treat or untreat by gypenosides. These results suggest that the gypenosides supplementation could influence on the rumen fermentation, and reduce methanogens, total protozoal and single protozoal species number Gypenosides could promote the growth of two fibrolytic bacteria and fungi, change the microorganism community and the methanogens community associated with protozoa. 3 The study of ruminal microbial resistance to gypenosidesConsecutive batch cultures, involving six serial transfer were established to investigate the effects of gypenosides on ruminal microbial fermentation properties, and the communities of bacteria and methanogens from rumen digesta. The results showed that pH value increased after gypenosides treatment. Compared with the control group, the gas production of different gypenosides groups in different transfers was low excepted for the 5mg and 10mg groups of first transfer with higher gas production. Gas production of some treatment groups decreased with significant linear and quadratic effects. There was a significant reducing with linear and quadratic effects between methane production and gypenosides doses (P<0.05 or P<0.01). Ammonia nitrogen concentration of treatment were higher than that of control. MCP concentration of most of treatment groups decreased than that of control excepted for first transfer with gypenosides increasing. There were significant decrease for some treatments and different transfers(P<0.05). Protozoal numbers reduced significantly with a linear effect for first transfer and second transfer after treatment by gypenosides. Protozoa were not detected under microscopy for third transfer. There were significantly decreasing for TVFA, acetate, propionate and butyrate concentration after gypenosides addition. The shifts of communities of bacteria and methanogen were observed after treatment. Some predominant bands such as Butyrivibrio fibrisolvens and Fibrobacter succinogenes were gradually disappeared and a few new predominant bands such as Erwinia alni and uncultured bacterium were observed after treatment by gypenosides. These data indicated that the growth of some microbes were inhibited, and others bacteria with capability of adaptation to gypenosides and degradation of gypenosides became the predominant bacteria in fermentation systems. The results suggested that gypenosides affected the ruminal microbial fermentation. In turn, the community of bacteria were gradually formed to adapt to the survival environment with gypenosides.4 Effects of gypenosides on fungi co-culture with methanogens methane production and fermentation characteristicsThis study were conducted to investigate the effects of gypenosides on methane production and fermentation property under fungi co-culture methanogens (Ⅰ) and fungi as dominant species (Ⅱ). The formation of two culture system of fungi associated with methanogens and fungi as main species were treated by penicilline & streptomycin (I) and chloramphenicol (Ⅱ), respectively. The results showed that, in system I after treatment by gypenosdies, there were significantly linearly decrease for methane production and methanogenic numbers (P<0.01). Gas production and fungi numbers were siginificantly reduced with a linear or quadratic effects (P<0.01). pH value increased with significant quadratic effect (P<0.05). TVFA and acetate concentration decreased significantly with a significant quadratic effect. In system II after treatment by gypenosides, methane production were not detected for minor production. Gas production decreased with a linear or quadratic effects (P<0.01). Fungi and methanogens numbers were significantly reduced with a linear effect. TVFA (P<0.05) and acetate concentration (p<0.01) decreased with linear or quadratic effects. pH value increased (P<0.05) with linear or quadratic effects (P<0.01). These results indicated that the pH value increasing, gas and methane production decreasing were induced by the decreased numbers of fungi or methanogens, and inhibitation of fungi and methanogenic activity. Growth of fungi and methanogens in two fermentation systems were depressed by gypenosides.更多还原