延胡索酸二钠对山羊瘤胃甲烷生成的调控研究及相关瘤胃微生物菌群分析
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摘要
反刍动物瘤胃甲烷的排放,既意味着可被动物消化能量的损失,又导致了大气中温室气体的增加,因此减缓瘤胃甲烷生成已经越来越引起人们的重视。延胡索酸是瘤胃中的重要中间代谢产物,可作为电子受体用于抑制甲烷生成。本论文系统研究了延胡索酸二钠调控瘤胃甲烷生成的微生物学机理及延胡索酸还原菌的多样性。首先建立了检测瘤胃微生物代谢产物中的有机酸含量的高效液相色谱分析方法,为分析瘤胃微生物的代谢状况提供了可靠的分析手段；其次研究了在不同精粗比条件下添加延胡索酸二钠对山羊血清生化指标、甲烷生成、瘤胃发酵、瘤胃微生物区系组成及重要微生物数量的影响,从多个角度揭示了延胡索酸二钠调控瘤胃甲烷生成的机理：再就是利用编码延胡索酸还原酶的功能基因frdA基因研究了瘤胃延胡索酸还原菌的多样性；利用体外继代培养结合16S rRNA克隆库的方式研究了可培养延胡索酸利用菌在体外继代培养过程中的变化规律；最后通过厌氧滚管技术分离到两株延胡索酸还原菌并对其进行了形态学、生理生化特性以及16S rRNA序列鉴定。本研究试验共分为五个部分：
1、建立高效液相色谱法检测瘤胃微生物代谢产物中的有机酸
建立了一种同时快速分析瘤胃微生物发酵液中7种有机酸的高效液相色谱法。该方法采用色谱级甲醇和20mM磷酸氢二钠缓冲液(用磷酸调pH至2.7,v:v=1：99)作为流动相,流速采用二元泵梯度程序洗脱方法控制,紫外检测波长为215nm,柱温为30℃,能够快速、准确地分离和测定瘤胃微生物发酵液中的甲酸、乙酸、丙酸、延胡索酸、琥珀酸、苹果酸、乳酸。方法的回收率为96.40%～99.60%,相对标准偏差为1.50%～4.40%,各种有机酸的线性相关系数均大于0.9993,具有较高的精密度和准确度,可以用于瘤胃微生物发酵液中的有机酸分析。
2、不同精粗比条件下添加延胡索酸二钠对山羊血清生化指标、甲烷生成、瘤胃发酵、微生物区系组成及重要微生物数量的影响
选用4只体重约为24±1.43kg、装有永久瘘管的本地杂交阉公羊,采用2×2两因子析因设计,按一个4×4拉丁方排序,以日粮精粗比(粗料：精料分别为4]：59或57.6：42.4)和延胡索酸二钠添加量(0g/天或10g/天)作为主要影响因子,研究不同精粗比条件下添加延胡索酸二钠对山羊血清生化指标、瘤胃发酵、甲烷生成、微生物区系组成及重要菌群数量变化的影响。结果显示,日粮精粗比和延胡索酸二钠对血糖、甘油三酯、胆固醇、尿素氮、乳酸的浓度以及谷草转氨酶、谷丙转氨酶、乳酸脱氢酶的酶活无显著影响,说明添加延胡索酸对山羊健康没有明显影响。与未添加延胡索酸二钠的对照组相比,添加延胡索酸二钠显著降低了甲烷产量(P<0.05),饲喂高精料和高粗料日粮的山羊甲烷产量分别降低了8.10%、15.76%。与未添加延胡索酸二钠的对照组相比,饲喂延胡索酸二钠组的山羊瘤胃内总挥发性脂肪酸、乙酸、丙酸浓度显著增加(P<0.05)。DGGE分析结果表明,不同日粮精粗比条件下添加延胡索酸二钠对瘤胃细菌及甲烷菌区系组成没有显著影响；饲喂延胡索酸二钠的山羊瘤胃内的产甲烷菌数量要低于未添加延胡索酸二钠组(P<0.05)。添加延胡索酸二钠组中的反刍兽新月形单胞菌(一种延胡索酸还原菌)的数量要显著高于未添加延胡索酸二钠的对照组,但仅限于高粗料条件下(P<0.05)。日粮精粗比与延胡索酸二钠之间对于反刍兽新月形单胞菌的影响存在显著交互作用(P<0.05)。真菌、原虫、黄色瘤胃球菌和产琥珀酸丝状杆菌的数量不受延胡索酸二钠添加剂量的影响。日粮的精粗比的变化对于产甲烷菌、真菌和黄化瘤胃球菌影响显著(P<0.05),但是对甲烷产量(g/天)无显著影响。这结果提示延胡索酸二钠对山羊瘤胃发酵具有有益作用,结果也进一步提示延胡索酸二钠的效果不是直接作用于真菌、原虫和主要的纤维降解菌,而是由于它促进了延胡索酸还原菌的生长,抑制了产甲烷菌的生长。其中一些指标的变化,如反刍兽新月型单胞菌的数量等,与日粮精粗比的变化相关,这说明了延胡索酸二钠对甲烷的抑制效果可能与日粮的精粗比相关。
3、利用frdA基因研究延胡索酸还原菌的多样性
虽然瘤胃延胡索酸还原菌对于降低甲烷生成可能具有重要作用,但是目前关于在山羊瘤胃内的延胡索酸还原菌的多样性的研究却仍不清楚。本研究利用构建延胡索酸还原酶frdA基因克隆库的方式来分析多种日粮条件下(高精料或高粗料,精粗比分别为41：59或57.6：42.4,两种日粮均添加0或10g/天延胡索酸二钠)山羊瘤胃内Proteobacteria门延胡索酸还原菌的多样性。RFLP分析结果表明206个阳性克隆可分为24个分类操作单元(OTU),表明延胡索酸还原菌的多样性非常高；frdA扩增序列所推导的氨基酸与已知氨基酸序列的相似性范围为75%-93%,这意味着瘤胃内存在许多未知的延胡索酸还原菌。它们均属于Proteobacteria;系统进化树分析表明,这些序列在遗传距离上相距较远。
4、体外继代培养研究山羊瘤胃延胡索酸利用菌的变化规律
采用PCR-DGGE结合16S rRNA克隆库技术,研究了山羊瘤胃内容物中延胡索酸利用菌菌群在体外传代过程中的变化规律。PCR-DGGE的分析结果表明,随继代培养次数的增加,细菌DGGE图谱上的条带数逐渐变少,一些条带(A、B、C、E、F、G.H)消失,一部分一直存在(D、O、P、Q、R、S),而部分条带明显得到富集(I、J、K、L、M、N)。继代培养5次后细菌条带数趋于稳定。16Sr RNA基因测序比对结果表明,所获得的88个克隆中,6个克隆属于Fusobacterium varium(相似性>97%),占克隆的6.82%；42个克隆属于Wolinella succinogenes(相似性>97%),占克隆的47.73%；4个克隆属于Clostridium cochlearium(相似性>97%),占克隆的4.55%;10个克隆属于Bacteroides pyogenes(相似性>97%),占克隆的11.36%；8个克隆属于Agrobacterium tumefaciens(相似性>97%),占克隆的9.09%；2个克隆属于Oribacterium sinus(相似性<97%),占克隆的2.27%；3个克隆属于Clostridium subterminale(相似性>97%),占克隆的3.41%；3个克隆属于Fusobacterium naviforme(相似性<97%),占克隆的3.41%；10个克隆属于Clostridium hastiforme(相似性>97%),占克隆的11.36%。系统进化树分析表明,它们分别属于Clostridiales、 Fusobacteriales、Rhizobiales、Campylobacterales、Bacteroidales。以上结果说明,混合瘤胃内容物中存在大量的延胡索酸利用菌,它们可能会参与瘤胃延胡索酸还原过程,添加延胡索酸可起到体外富集延胡索酸还原菌的作用。
5、山羊瘤胃延胡索酸利用菌的分离、鉴定
通过厌氧滚管法从健康山羊瘤胃内容物中分离得到了两株瘤胃厌氧细菌,分别命名为Y1和Y10。经HPLC法检测它们对延胡索酸二钠的代谢情况可知,它们都能够利用延胡索酸生成琥珀酸并进一步形成丙酸。经形态学和生理生化特性分析,初步鉴定Y1属于埃希氏菌属,Y10属于变形杆菌属。将两株菌的16S rRNA序列与GenBank中的已知序列相比较,其与Escherichia coli、Proteus mirabilis的相似性高达99%,进一步确定所分离到的菌株Y1为埃希氏菌属弗氏埃希氏菌(Escherichia,ergusonii),Y10为奇异变形杆菌(Proteus mirabilis).经RDP数据库分类比较,它们均属于变形杆菌(Proteobacteria)。
Methane eructated from ruminants represents an energy loss of ingested by the animal and contributes to global warming. Mitigation of rumen methane emission has received increasing attention. Fumarate is a key intermediate metabolite in the rumen and an alternative electron acceptor to methanogenesis.The microbial mechanism of ruminal methane reduction by disodium fumarate and the diversity of fumarate reducing bacteria were investigated in this study. Firstly, a garadient elution high performance liquid chromatography method was established for the determination of organic acid in ruminal fluid; Secondly, the effect of disodium fumarate on blood parameters, methane emission, ruminal fermentation, microbiota composition and key microbial population abundance from goats under different forage:concentrate ratios were assessed;Then, the diversity of fumarate reducing bacteria in the rumen fluid from goats under different diet conditions was assessed; The variation of fumarate utilizing bacteria in rumen of goats after being cultured seven times in vitro was assessed. Lastly, two single fumarate reducing bacteria from rumen contents of goats were isolated and identified. The main results are shown as follows:
1. Development of gradient elution high performance liquid chromatography for detection of organic acid in ruminal contents
A high performance liquid chromatography method was developed for simultaneous determining formate, malate, lactate. acetate, succinate, propionate and fumarate in ruminal contents. ZORBAX SB-Aq column (4.6×150mm×5um) was used at30℃.Mobile phase was methanol and20mM Na2HPO4(pH2.7, v:v=1:99) with variable flow rate which controlled by program.Detection UV wavelength was at215nm.The method could fast and effectively separate and detect the organic acids in ruminal fluid. The recoveries were96.40%～99.60%and the relative standard deviations were1.50%～4.40%.All the correlation coefficients were no less than0.9993.The results indicated that the method was precise and accurate which could be applied to the determination of organic acids in ruminal fluids.
2. Effect of disodium fumarate on blood parameters, methane emission, ruminal fermentation, microbiota composition and key microbial population abundance of goats under different forage:concentrate ratios.
The study was carried out to investigate the effect of disodium fumarate on blood parameters, ruminal fermentation, methane emission, microbiota composition and key microbial population abundance of goats under different forage:concentrate ratios. Four ruminally fistulated, castrated male goats were used in a4×4Latin square design with a2x2factorial arrangement of treatments, and the main factors being the forage:concentrate ratios (forage:concentrate=41:59or57.6:42.4) and disodium fumarate supplementation (0g/day or10g/day). Results showed that there were no significant effect of disodium fumarate on blood parameters, such as concentration of blood glucose, triglyceride, cholesterol, urea nitrogen, lactate and activities of glutamic pyruvic transaminase, glutamate oxaloacetate transaminase, lactate dehydrogenase. Disodium fumarate significantly reduced methane production (P<0.05), with the methane production in high-concentrate diet and high-forage diet reduced by8.10%and15.76%, respectively, as compared with controls. The concentrations of total VFA, acetate and propionate were greater in the rumen of goats fed with disodium fumarate as compared with controls (P<0.05).DGGE profiles analysis showed that there were no special bands appeared for the composition of total bacteria and methanogens after supplementation of disodium fumarate under different forage:concentrate ratios.The population abundance of methanogens was lower in the rumen of goats fed with disodium fumarate as compared with those without disodium fumarate (P<0.05). The population abundance of Selenomonas ruminantium, a fumarate reducing bacteria, was greater in the rumen of goats fed with disodium fumarate as compared with control, but this response was present only within the high-forage diet (P <0.05). The population abundance of fungi, protozoa, Ruminococus flavefaciens and Fibrobacter succinogenes were not affected by the addition of disodium fumarate. Variable forage:concentrate ratios affected the population abundance of methanogens, fungi and R.flavefaciens (P<0.05), but did not significantly affect methane emission (g/day). The result implied that disodium fumarate had a beneficial effect on the in vivo rumen fermentation of the goats fed diets with different forage:concentrate ratios and that this effect was not a direct action on anaerobic fungi, protozoa and fibrolytic bacteria, the generally recognized fibre-degrading and hydrogen-producing microorganisms, but due to the stimulation of fumarate-reducing bacteria and the depression of methanogens. The reduction of population abundance of methanogens and the increase of population abundance of S. ruminantium was related to the forage:concentrate ratios, suggesting that disodium fumarate reduction in vivo could depend on the forage:concentrate ratios of diet.
3. Diversity analysis of fumarate reducing bacteria in the rumen contents of goats fed with different diets
Although fumarate-reducing bacteria may play an important role for methane reduction, the diversity of fumarate-reducing bacteria in the rumen of goats is still unclear. In this study, the diversity of fumarate-reducing bacteria in the mixed rumen contents of four goats fed with different diets (high-concentrate diet or high-forage diet, both diets either were supplemented with disodium fumarate at10g/day or received no supplement) was analyzed using frdA gene clone library analysis. The results indicated that the fumarate reducing bacteria diversity in the rumen contents was very high, where206positive clones belong to24different OTU by RFLP analysis. Identities of deduced amino acid sequences of cloned frdA amplicons against known sequences ranged from75-93%suggesting the presence of unknown fumarate reducing bacteria in rumen of goats. All of them were belonged to Proteobacteria phylum and the phylogenetic analysis results also demonstrated high bacteria diversity in the rumen contents of goats with distant phylogenetic position.
4. The variation of fumarate utilizing bacteria in rumen of goats after being cultured seven times in vitro
Fumarate utilizing bacteria community changes in the ruminal contents during subcultures was investigated by PCR-DGGE and16S rRNA clone library analysis. PCR-DGGE analysis showed that the number of DGGE bands decreased with the culture times increasing. Some DGGE bands in the culture disappeared (labelled as A、B、C、E、 F、G、H),and other bands (labeled as I、J、K、L、M、N) enriched after7subcultures.After five subcultures,the bands tend to keep stable. The results of16S rRNA clone library analysis showed that the dominant bands remained in the culture after7subculturing had their16S rRNA sequences most close to species of Fusobacterium varium (6of88clones, similarity>97%),Wolinella succinogenes (42of88clones,similarity>97%),Clostridium cochlearium (4of88clones, similarity>97%), Bacteroides pyogenes (10of88clones, similarity>97%), Agrobacterium tumefaciens (8of88clones, similarity>97%), Oribacterium sinus (2of88clones, similarity<97%), Clostridium subterminale (3of88clones, similarity>97%), Fusobacterium naviforme (3of88clones, similarity<97%), respectively. These clones belonged to Clostridiales, Fusobacteriales, Rhizobiales, Campy lobacterales, Bacteroidales five orders, respectively. The results indicated that there plenty of fumarate utilizing bacteria survivie in the subcultures and they may be involed to fumarate reduction and they could be enriched in vitro by fumarate supplementation.
5. Isolation and identification of fumarate reducing bacteria from rumen contents of goats
Two single bacterial strains (named Y1and Y10, respectively) were isolated by Hungate method from rumen contents of goats. Both of them could transform disodium fumarate into succinic acid and further transform to propionic acid which have validated by HPLC methods. Based on morphological, physiological characteristics identification and16S rRNA gene sequence analysis, Y1was identified as Escherichia fergusonii, while Y10was Proteus mirabilis. RDP database classifier analysis showed that both of them were belonged to Proteobacteria phylum.
1、建立高效液相色谱法检测瘤胃微生物代谢产物中的有机酸
建立了一种同时快速分析瘤胃微生物发酵液中7种有机酸的高效液相色谱法。该方法采用色谱级甲醇和20mM磷酸氢二钠缓冲液(用磷酸调pH至2.7,v:v=1：99)作为流动相,流速采用二元泵梯度程序洗脱方法控制,紫外检测波长为215nm,柱温为30℃,能够快速、准确地分离和测定瘤胃微生物发酵液中的甲酸、乙酸、丙酸、延胡索酸、琥珀酸、苹果酸、乳酸。方法的回收率为96.40%～99.60%,相对标准偏差为1.50%～4.40%,各种有机酸的线性相关系数均大于0.9993,具有较高的精密度和准确度,可以用于瘤胃微生物发酵液中的有机酸分析。
2、不同精粗比条件下添加延胡索酸二钠对山羊血清生化指标、甲烷生成、瘤胃发酵、微生物区系组成及重要微生物数量的影响
选用4只体重约为24±1.43kg、装有永久瘘管的本地杂交阉公羊,采用2×2两因子析因设计,按一个4×4拉丁方排序,以日粮精粗比(粗料：精料分别为4]：59或57.6：42.4)和延胡索酸二钠添加量(0g/天或10g/天)作为主要影响因子,研究不同精粗比条件下添加延胡索酸二钠对山羊血清生化指标、瘤胃发酵、甲烷生成、微生物区系组成及重要菌群数量变化的影响。结果显示,日粮精粗比和延胡索酸二钠对血糖、甘油三酯、胆固醇、尿素氮、乳酸的浓度以及谷草转氨酶、谷丙转氨酶、乳酸脱氢酶的酶活无显著影响,说明添加延胡索酸对山羊健康没有明显影响。与未添加延胡索酸二钠的对照组相比,添加延胡索酸二钠显著降低了甲烷产量(P<0.05),饲喂高精料和高粗料日粮的山羊甲烷产量分别降低了8.10%、15.76%。与未添加延胡索酸二钠的对照组相比,饲喂延胡索酸二钠组的山羊瘤胃内总挥发性脂肪酸、乙酸、丙酸浓度显著增加(P<0.05)。DGGE分析结果表明,不同日粮精粗比条件下添加延胡索酸二钠对瘤胃细菌及甲烷菌区系组成没有显著影响；饲喂延胡索酸二钠的山羊瘤胃内的产甲烷菌数量要低于未添加延胡索酸二钠组(P<0.05)。添加延胡索酸二钠组中的反刍兽新月形单胞菌(一种延胡索酸还原菌)的数量要显著高于未添加延胡索酸二钠的对照组,但仅限于高粗料条件下(P<0.05)。日粮精粗比与延胡索酸二钠之间对于反刍兽新月形单胞菌的影响存在显著交互作用(P<0.05)。真菌、原虫、黄色瘤胃球菌和产琥珀酸丝状杆菌的数量不受延胡索酸二钠添加剂量的影响。日粮的精粗比的变化对于产甲烷菌、真菌和黄化瘤胃球菌影响显著(P<0.05),但是对甲烷产量(g/天)无显著影响。这结果提示延胡索酸二钠对山羊瘤胃发酵具有有益作用,结果也进一步提示延胡索酸二钠的效果不是直接作用于真菌、原虫和主要的纤维降解菌,而是由于它促进了延胡索酸还原菌的生长,抑制了产甲烷菌的生长。其中一些指标的变化,如反刍兽新月型单胞菌的数量等,与日粮精粗比的变化相关,这说明了延胡索酸二钠对甲烷的抑制效果可能与日粮的精粗比相关。
3、利用frdA基因研究延胡索酸还原菌的多样性
虽然瘤胃延胡索酸还原菌对于降低甲烷生成可能具有重要作用,但是目前关于在山羊瘤胃内的延胡索酸还原菌的多样性的研究却仍不清楚。本研究利用构建延胡索酸还原酶frdA基因克隆库的方式来分析多种日粮条件下(高精料或高粗料,精粗比分别为41：59或57.6：42.4,两种日粮均添加0或10g/天延胡索酸二钠)山羊瘤胃内Proteobacteria门延胡索酸还原菌的多样性。RFLP分析结果表明206个阳性克隆可分为24个分类操作单元(OTU),表明延胡索酸还原菌的多样性非常高；frdA扩增序列所推导的氨基酸与已知氨基酸序列的相似性范围为75%-93%,这意味着瘤胃内存在许多未知的延胡索酸还原菌。它们均属于Proteobacteria;系统进化树分析表明,这些序列在遗传距离上相距较远。
4、体外继代培养研究山羊瘤胃延胡索酸利用菌的变化规律
采用PCR-DGGE结合16S rRNA克隆库技术,研究了山羊瘤胃内容物中延胡索酸利用菌菌群在体外传代过程中的变化规律。PCR-DGGE的分析结果表明,随继代培养次数的增加,细菌DGGE图谱上的条带数逐渐变少,一些条带(A、B、C、E、F、G.H)消失,一部分一直存在(D、O、P、Q、R、S),而部分条带明显得到富集(I、J、K、L、M、N)。继代培养5次后细菌条带数趋于稳定。16Sr RNA基因测序比对结果表明,所获得的88个克隆中,6个克隆属于Fusobacterium varium(相似性>97%),占克隆的6.82%；42个克隆属于Wolinella succinogenes(相似性>97%),占克隆的47.73%；4个克隆属于Clostridium cochlearium(相似性>97%),占克隆的4.55%;10个克隆属于Bacteroides pyogenes(相似性>97%),占克隆的11.36%；8个克隆属于Agrobacterium tumefaciens(相似性>97%),占克隆的9.09%；2个克隆属于Oribacterium sinus(相似性<97%),占克隆的2.27%；3个克隆属于Clostridium subterminale(相似性>97%),占克隆的3.41%；3个克隆属于Fusobacterium naviforme(相似性<97%),占克隆的3.41%；10个克隆属于Clostridium hastiforme(相似性>97%),占克隆的11.36%。系统进化树分析表明,它们分别属于Clostridiales、 Fusobacteriales、Rhizobiales、Campylobacterales、Bacteroidales。以上结果说明,混合瘤胃内容物中存在大量的延胡索酸利用菌,它们可能会参与瘤胃延胡索酸还原过程,添加延胡索酸可起到体外富集延胡索酸还原菌的作用。
5、山羊瘤胃延胡索酸利用菌的分离、鉴定
通过厌氧滚管法从健康山羊瘤胃内容物中分离得到了两株瘤胃厌氧细菌,分别命名为Y1和Y10。经HPLC法检测它们对延胡索酸二钠的代谢情况可知,它们都能够利用延胡索酸生成琥珀酸并进一步形成丙酸。经形态学和生理生化特性分析,初步鉴定Y1属于埃希氏菌属,Y10属于变形杆菌属。将两株菌的16S rRNA序列与GenBank中的已知序列相比较,其与Escherichia coli、Proteus mirabilis的相似性高达99%,进一步确定所分离到的菌株Y1为埃希氏菌属弗氏埃希氏菌(Escherichia,ergusonii),Y10为奇异变形杆菌(Proteus mirabilis).经RDP数据库分类比较,它们均属于变形杆菌(Proteobacteria)。
Methane eructated from ruminants represents an energy loss of ingested by the animal and contributes to global warming. Mitigation of rumen methane emission has received increasing attention. Fumarate is a key intermediate metabolite in the rumen and an alternative electron acceptor to methanogenesis.The microbial mechanism of ruminal methane reduction by disodium fumarate and the diversity of fumarate reducing bacteria were investigated in this study. Firstly, a garadient elution high performance liquid chromatography method was established for the determination of organic acid in ruminal fluid; Secondly, the effect of disodium fumarate on blood parameters, methane emission, ruminal fermentation, microbiota composition and key microbial population abundance from goats under different forage:concentrate ratios were assessed;Then, the diversity of fumarate reducing bacteria in the rumen fluid from goats under different diet conditions was assessed; The variation of fumarate utilizing bacteria in rumen of goats after being cultured seven times in vitro was assessed. Lastly, two single fumarate reducing bacteria from rumen contents of goats were isolated and identified. The main results are shown as follows:
1. Development of gradient elution high performance liquid chromatography for detection of organic acid in ruminal contents
A high performance liquid chromatography method was developed for simultaneous determining formate, malate, lactate. acetate, succinate, propionate and fumarate in ruminal contents. ZORBAX SB-Aq column (4.6×150mm×5um) was used at30℃.Mobile phase was methanol and20mM Na2HPO4(pH2.7, v:v=1:99) with variable flow rate which controlled by program.Detection UV wavelength was at215nm.The method could fast and effectively separate and detect the organic acids in ruminal fluid. The recoveries were96.40%～99.60%and the relative standard deviations were1.50%～4.40%.All the correlation coefficients were no less than0.9993.The results indicated that the method was precise and accurate which could be applied to the determination of organic acids in ruminal fluids.
2. Effect of disodium fumarate on blood parameters, methane emission, ruminal fermentation, microbiota composition and key microbial population abundance of goats under different forage:concentrate ratios.
The study was carried out to investigate the effect of disodium fumarate on blood parameters, ruminal fermentation, methane emission, microbiota composition and key microbial population abundance of goats under different forage:concentrate ratios. Four ruminally fistulated, castrated male goats were used in a4×4Latin square design with a2x2factorial arrangement of treatments, and the main factors being the forage:concentrate ratios (forage:concentrate=41:59or57.6:42.4) and disodium fumarate supplementation (0g/day or10g/day). Results showed that there were no significant effect of disodium fumarate on blood parameters, such as concentration of blood glucose, triglyceride, cholesterol, urea nitrogen, lactate and activities of glutamic pyruvic transaminase, glutamate oxaloacetate transaminase, lactate dehydrogenase. Disodium fumarate significantly reduced methane production (P<0.05), with the methane production in high-concentrate diet and high-forage diet reduced by8.10%and15.76%, respectively, as compared with controls. The concentrations of total VFA, acetate and propionate were greater in the rumen of goats fed with disodium fumarate as compared with controls (P<0.05).DGGE profiles analysis showed that there were no special bands appeared for the composition of total bacteria and methanogens after supplementation of disodium fumarate under different forage:concentrate ratios.The population abundance of methanogens was lower in the rumen of goats fed with disodium fumarate as compared with those without disodium fumarate (P<0.05). The population abundance of Selenomonas ruminantium, a fumarate reducing bacteria, was greater in the rumen of goats fed with disodium fumarate as compared with control, but this response was present only within the high-forage diet (P <0.05). The population abundance of fungi, protozoa, Ruminococus flavefaciens and Fibrobacter succinogenes were not affected by the addition of disodium fumarate. Variable forage:concentrate ratios affected the population abundance of methanogens, fungi and R.flavefaciens (P<0.05), but did not significantly affect methane emission (g/day). The result implied that disodium fumarate had a beneficial effect on the in vivo rumen fermentation of the goats fed diets with different forage:concentrate ratios and that this effect was not a direct action on anaerobic fungi, protozoa and fibrolytic bacteria, the generally recognized fibre-degrading and hydrogen-producing microorganisms, but due to the stimulation of fumarate-reducing bacteria and the depression of methanogens. The reduction of population abundance of methanogens and the increase of population abundance of S. ruminantium was related to the forage:concentrate ratios, suggesting that disodium fumarate reduction in vivo could depend on the forage:concentrate ratios of diet.
3. Diversity analysis of fumarate reducing bacteria in the rumen contents of goats fed with different diets
Although fumarate-reducing bacteria may play an important role for methane reduction, the diversity of fumarate-reducing bacteria in the rumen of goats is still unclear. In this study, the diversity of fumarate-reducing bacteria in the mixed rumen contents of four goats fed with different diets (high-concentrate diet or high-forage diet, both diets either were supplemented with disodium fumarate at10g/day or received no supplement) was analyzed using frdA gene clone library analysis. The results indicated that the fumarate reducing bacteria diversity in the rumen contents was very high, where206positive clones belong to24different OTU by RFLP analysis. Identities of deduced amino acid sequences of cloned frdA amplicons against known sequences ranged from75-93%suggesting the presence of unknown fumarate reducing bacteria in rumen of goats. All of them were belonged to Proteobacteria phylum and the phylogenetic analysis results also demonstrated high bacteria diversity in the rumen contents of goats with distant phylogenetic position.
4. The variation of fumarate utilizing bacteria in rumen of goats after being cultured seven times in vitro
Fumarate utilizing bacteria community changes in the ruminal contents during subcultures was investigated by PCR-DGGE and16S rRNA clone library analysis. PCR-DGGE analysis showed that the number of DGGE bands decreased with the culture times increasing. Some DGGE bands in the culture disappeared (labelled as A、B、C、E、 F、G、H),and other bands (labeled as I、J、K、L、M、N) enriched after7subcultures.After five subcultures,the bands tend to keep stable. The results of16S rRNA clone library analysis showed that the dominant bands remained in the culture after7subculturing had their16S rRNA sequences most close to species of Fusobacterium varium (6of88clones, similarity>97%),Wolinella succinogenes (42of88clones,similarity>97%),Clostridium cochlearium (4of88clones, similarity>97%), Bacteroides pyogenes (10of88clones, similarity>97%), Agrobacterium tumefaciens (8of88clones, similarity>97%), Oribacterium sinus (2of88clones, similarity<97%), Clostridium subterminale (3of88clones, similarity>97%), Fusobacterium naviforme (3of88clones, similarity<97%), respectively. These clones belonged to Clostridiales, Fusobacteriales, Rhizobiales, Campy lobacterales, Bacteroidales five orders, respectively. The results indicated that there plenty of fumarate utilizing bacteria survivie in the subcultures and they may be involed to fumarate reduction and they could be enriched in vitro by fumarate supplementation.
5. Isolation and identification of fumarate reducing bacteria from rumen contents of goats
Two single bacterial strains (named Y1and Y10, respectively) were isolated by Hungate method from rumen contents of goats. Both of them could transform disodium fumarate into succinic acid and further transform to propionic acid which have validated by HPLC methods. Based on morphological, physiological characteristics identification and16S rRNA gene sequence analysis, Y1was identified as Escherichia fergusonii, while Y10was Proteus mirabilis. RDP database classifier analysis showed that both of them were belonged to Proteobacteria phylum.
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