| 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 contentsA 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 dietsAlthough 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 vitroFumarate 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 goatsTwo 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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