Screening Of Chinese Herbal Medicine And Mechanisms Of Target Herb On Methane Mitigation In Rumen

Reduction of methane emission from ruminants is meaningful for the mitigation of greenhouse effect as well as the improvement of feed utilization. Chinese medicinal herbs are sources of natural substances and compounds that can potentially be used to modulate rumen microbial populations to enhance feed digestion and decrease methane emission. In this study, extracts from 100 medicinal herbs were assessed for their ability to decrease methane production by rumen microbiota in vitro. The most effective dosage form of the most promising herbs (P. frutescens seeds, PFS) was determined. The dose responses of P. frutescens seeds extract (PFSE)were further examined in vitro with respect to rumen microbiota populations and microbiota composition using real time PCR and 16S rRNA gene sequencing. Future feeding trials were conducted with Hu sheep to evaluate the effects of PFSE supplementation on ruminal digestion ability, methane production and rumen fermentation in vivo.1 Screening of Chinese herbal medicine1.1 Preliminary evaluation for promising herbal extractsPreliminary evaluation of the 100 herbal extracts was conducted using in vitro rumen fermentation cultures, with 0.1 g fermentation substrate consisting of Chinese wild rye grass and corn meal in a 70:30 ratio. For the treatment group, each herbal extract was added at a concentration of 40 mg/mL and tested in triplicate. Three replicate control cultures were included that did not receive any herbal extract but the same volume of DMSO (100 μL). After 24 h of incubation, the gas pressure in each culture tube was recorded and CH4 concentration in the headspace of each culture tube was measured. Compared to the control,31 of the herbal extracts decreased CH4 production by more than 10%. The 10 most CH4-inhibiting herbal extracts were from the pericarp of Garcinia mangostana; stem, root and twig peels of Magnolia officinalis; resin of Boswellia carterii; heartwood of Caesalpinia sappan; rhizomes of Curcuma longa; velamen of Acanthopanax gracilistylus; seeds of Cnidium monnieri; roots of Polygala tenuifolia; seeds of P. frutescens; and flower buds of Magnolia biondii.1.2 Effect of select herbal extracts on CH4 production and other fermentation characteristicsBased on the results of the preliminary evaluation experiment described above,31 herbal extracts that decreased CH4 production the most were selected for further evaluation using in vitro rumen cultures. The in vitro cultures were prepared and incubated essentially the same as described in the preliminary evaluation experiment, except for an increased culture volume (50 mL each in 120-mL serum bottles each containing 0.5 g of the same fermentation substrate). Each treatment and the control that received no herbal extract had four replicates and the evaluation experiment was repeated once. At the end of the 24 h incubation, total gas production, CH4 concentration and volatile fatty acids (VFA) concentrations were determined.Ten of these herbal extracts decreased total gas production, while another six increased total gas production. Twelve of these herbal extracts significantly decreased CH4 production, but one (i.e., the extract from Amygdalus persica) increased CH4 production. With the exception of the extract from Polygala tenuifolia, all the 10 most CH4-inhibiting herbal extracts were confirmed to suppress CH4 production significantly (P＜0.05). Two (i.e., Magnolia officinalis and Ziziphus jujube) of the 12 CH4-inhibiting herbal extracts did not decrease (P>0.05) gas production. Only one herbal extract (from Magnolia biondii) decreased (P<0.05) total VFAs production, but 9 of them decreased (P<0.05) the A:P ratio, including the seven of the 12 CH4-inhibiting extracts.The extract of PFS, exhibited the strongest inhibition to CH4 production (63.6%), were selected for further evaluation.2 Effects of P. frutescens seed extract on rumen fermentation and CH4 productionChemical compositions of three forms of PFS were determined, which was PFSE, PFS ground at 80 mesh level (PFS80) and PFS ground at 600 mesh level (PFS600). Long chain fatty acids were the major compounds of all three forms, with highest in PFSE (675 μg/mg). In vitro rumen fermentation results showed that, compared to the high dose of PFSE (2%,0.2 mg/ml, DM), PFS80 and PFS600, the low dose of PFSE (0.99%,4.96mg, DM)significantly reduced methane production(P<0.05), increased the effective gas production (P＜0.05) and with no significant effects on other fermentation parameters.The extract from PF seeds was evaluated at five doses (0,0.1,0.2,0.3, and 0.4 mg/mL culture) for its dose effects on in vitro fermentation and CH4 production. The results showed that all the measured fermentation characteristics, including gas production (both total and effective), CH4 production, total VFAs, A:P ratio, and DMD, were decreased linearly (P＜0.05) with increasing doses of the PF seed extract.As quantified by real time PCR, the PF seed extract did not affect the population of total bacteria as well as total methanogens (P>0.05) irrespective of the doses, while that of protozoa was lowered (.P<0.05). The activity of cellulase, CMCase, and xylanse was not decreased either (.P>0.05).Illumina Miseq sequencing platform were used to generate large-scale 16S rRNA gene sequencing data to investigate the ruminal microbal communities of the samples obtained in the experimental groups of control (0%,0 mg/mL), low dose (2%, 0.2mg/mL) and high dose (4%,0.4 mg/mL). (1) Effects of PFSE on bacterial communities:Chao 1 estimate indicates was not affected, whereas Shannon-Wiener diversity index was decreased (P<0.05) in a dose-dependent manner. The number of observed OTUs decreased with increasing doses of the PF seed extract. At OTU level, the PCoA based on weighted UniFrac distances also showed that the PF seed extract affected the bacterial community at 0.4 mg/mL but not at 0.2 mg/mL. (2) Effects of PFSE on archaeal communities:Shannon-Wiener diversity index decreased linearly with the increased doses of the herbal extract, but no significant difference in Chao 1 estimate was noted. The qPCR data showed that the total population of archaea was not affected by the PF seed extract. The sequencing data revealed increase in relative abundance of Methanobrevibacter and Methanosphaera, while that of unclassified Methanobacteriaceae was lowered in a dose-dependent manner. The relative abundance of Methanobrevibacter, the dominant methanogens in the rumen, was increased by the PF seed extract.Correlation was found between a number of genera/group of rumen microbes and several fermentation characteristics. Based on the correlations, five groups of microbes were found. Group I was positively (P＜0.05, r>0.607) associated with production of both CH4 and total VFA. Included in this group were Fibrobacter, Sphaerochaeta, and eight groups of unclassified bacteria and one group of unclassified Methanobacteriaceae. Containing 14 genera/groups of bacteria, including Clostridium, Pedobacter, Anaeroplasma, Paludibacter, Ruminococcus, Zymomonas, Luteimonas, and seven unclassified bacterial groups, group Ⅱ was positively correlated (P＜0.05, r>0.602) to CH4 production but not to total VFA production, either positively or negatively. In contrast to group Ⅰ, group Ⅲ was negatively (P＜0.05, r>-0.598) correlated to production of both CH4 and VFA, and this group included Roseomonas, Selenomonas, Shuttleworthia, Pseudobutyrivibrio, Anaerovibrio, Ruminobacter, Succinivibrio, Methanosphaera, and three groups of unclassified bacteria. Group IV included unclassified Sinobacteraceae and Methanobrevibacter, and it only exhibited negative (P<0.05, r>-0.650) correlation to CH4 production. Group V included two groups of unclassified bacteria, and it was not correlated to production of either CH4 or total VFA.3 Effects of P. frutescens seed extract on CH4 production in Hu sheep.Using cross-over study design,8 male sheep were divided into two dietary treatments with supplementation of 0 and 1% PFSE to a basal diet. Addition of fructus did not affect BW, DMI, digestibility of DM, crude protein, NDF, and ADF (P>0.05). Addition of fructus tended to decrease methane production per gram feed intake (DM basis, P=0.086), but not per gram digestible DM nor digestible fiber (P>0.1); Addition of PFSE had no significant effect on ruminal pH, NH3-N, acetate, propionate, butyrate, A:P ratio and total VFA (P>0.1).Medicinal herbs are potential sources that can be used to mitigate CH4 emission from ruminant animals. Extract of PFS decreased CH4 production without adversely affecting feed fermentation when included at relatively low concentration. Given the high content of protein and lipid, PFS may be directly incorporated into rations to both enhance nutrition and mitigate CH4 emission. A number of bacteria and methanogens were found to be correlated to CH4 production, a better understanding of these bacteria and methanogens may help further develop new strategies to mitigate CH4 emission without adversely affecting rumen fermentation.