| 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.
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