Microbial Underpinning Of The Differential Methane Emission Between Hu Sheep And New Zealand White Rabbits

Mitigating methane emission from ruminants benefits for both environment and livestock production.Many strategies have been reported to mitigate ruminal methane emission recently,however,most of them have side-effects on animal health or feed digestibility.Although both ruminants and non-ruminant herbivores produce methane during fermentation,the former produces much more than the later.The distinct microbiota in their respective digestive organs may be the main reason for the differential methane emission.In the present study,we tested this hypothesis using 5 Hu sheep as ruminants and 15 New Zealand White rabbits as non-ruminant herbivores,with alfalfa hay as the only diet.Greenhouse gases emission(methane and carbon dioxide),fermentation characteristics,microbial communities(based on both DNA and RNA levels),gene expression of Carbohydrate-Active Enzymes,and related microbiota in the sheep rumen and the rabbit cecum were comparatively analyzed.The differences determined in the above measurements will help understand the physiological and microbial underpinnings of methane emission by ruminants and non-ruminant herbivores,and the knowledge on correlations between microbiota and methane emission may be useful for targeted modification of rumen microbiota to mitigate methane emission from ruminants.1 Physiological underpinning of the differential methane emissionEach of the sheep emitted substantially more methane than each rabbit per day per unit of metabolic body weight(P<0.01),dry matter intake(P<0.01),digestible neutral detergent fiber(P<0.01),and digestible acid detergent fiber(P<0.01).However,the sheep emitted less CO2 per unit of metabolic body weight than each rabbit.The pH in the rabbit cecum(pH 5.8)was 1.3 unit lower than that in the sheep rumen(pH 7.1).No significant difference in total volatile fatty acid concentration was observed between the two digestive organs.However,the acetate to propionate ratio in the rabbit cecum was more than threefold greater than that in the sheep rumen.A higher activity of carboxymethyl cellulase(P<0.01),microcrystalline cellulase(P<0.01),and pectinase(P<0.01)was observed in the rabbit cecum than in the sheep rumen either per g freeze-dried sample or mg their microbial crude protein.The lower pH,a higher redox potential,and a greater digesta passage rate in the rabbit cecum may be major chemical factors attributable to the lower methane emission from the rabbits.The significantly greater activities of carboxymethyl cellulase,microcrystalline cellulase,and pectinase in the rabbit cecum than in the sheep rumen,suggesting an enrichment of fibrolytic and pectinolytic microbes in the rabbit cecum.2 Archaeal underpinning of the differential methane emissionIn the present study,we quantified the abundance of methanogens,analyzed the diversity,structure,and gene expression of the archaeal microbiota,as well as the differential expressed genes involved in methanogenesis pathways to understand the archaeal underpinning of the differential methane emission between the sheep and the rabbits.The sheep rumen had a greater abundance of RCC methanogens(P<0.01),non-RCC methanogens(P<0.01),and total methanogens(as quantified as mcr A gene copies/g freeze-dried sample,P<0.01)than the rabbit cecum.The two digestive organs each harbored a distinct archaeal microbiota,with Methanobrevibracter woesei being the dominant species in the rabbit cecum,whereas the sheep rumen containing Methanobrevibracter thaueri as the most predominant known species followed by Methanobrevibracter millerae and Methanobrevibracter woesei.Based on transcriptomic sequencing,the unigenes assigned to archaea in the sheep rumen accounted for 1.14%of the total sequencing reads,with hydrogenotrophic Methanobrevibacter and methylotrophic unclassified Thermoplasmatales being dominant.While the unigenes assigned to archaea in the rabbit cecum only accounted for 0.02%of the total sequencing reads which was great lower than that in the sheep rumen.In addition,the numbers of the differential expressed genes involved in H2/CO2 methanogenesis pathway was larger in the sheep rumen than in the rabbit cecum,especially genes encoding mcr.The differential abundance,predominance,and gene expression of methanogens in the sheep rumen and the rabbit cecum should be one explanation of the differential methane emission seen between the sheep and the rabbits.3 Hydrogen metabolic microbial underpinning of the differential methane emissionIn the present study,we quantified the abundance of hydrogen-producing and hydrogen-utilizing microbes,analyzed the diversity,structure,and gene expression of the bacterial microbiota to understand the hydrogen metabolic microbial underpinning of the differential methane emission between the sheep and the rabbits.As determined by qPCR,the sheep rumen had a greater abundance of fungi(P<0.01),protozoa(P<0.01),Ruminococcus ahlus(P<0.01),Ruminococcus flavefaciens(P<0.01),Butyrivibrio fibrisolvens(P<0.01),all of which can produce hydrogen during feed fermentation,than the rabbit cecum.While the rabbit cecum had a greater abundance of fhs(P<0.01)involved in homoacetogenesis pathway than the sheep rumen.Combined analysis of the bacterial microbiota revealed Ruminococcus,Prevotella,Butyrivibrio,Succiniclasticum were unique and/or predominant hydrogen-producing bacteria in the sheep rumen,whose relative abundance was strongly and positively correlated with methane emission.Among them,gene expression of Ruminococcus and Prevotella were most abundant.While Blautia(contain acetogens),Bacteroides(contain succinate producers),and Oscillospira(contain butyrate producers)were predominant in the rabbit cecum,whose relative abundance was strongly and negatively correlated with methane emission.Among them,gene expression of Bacteroides was most abundant.The differential abundance,predominance,and gene expression of hydrogen-producing and hydrogen-utilizing microbiota residing in the sheep rumen and the rabbit cecum can explain the differential methane emission between the sheep and the rabbits.Blautia,Bacteroides,and Oscillospira are potential targeting microbiota to mitigate methane emission from ruminants.4 The diversity of Carbohydrate-Active Enzymes(CAZymes)related microbiota underpinning of the differential methane emissionAs determined by transcriptomic sequencing,the diversity of Glycoside Hydrolase,Carbohydrate Esterase,and Polysaccharide Lyase was higher in the rabbit cecum than in the sheep rumen,among them,the relative abundance of unigenes assigned to Glycoside Hydrolase was significantly higher in the rabbit cecum than in the sheep rumen(P<0.01).Clostridiales,Bacteroidales,Prevotella,Ruminococcus,Bacteroides,Fibrobacter succinogenes,and Ruminococcus flavefaciens play an important role in polysaccharide degradation in the digestive organs.The abundance and predominance of the CAZymes related microbiota were different between the sheep rumen and the rabbit cecum,whereas the sheep rumen contained more hydrogen-producing CAZymes related microbiota,resulting in more methane emission from the sheep.In addition,CAZymes related Bacteroides may be an ideal potential targeting microbiota to mitigate methane emission from ruminants without side-effects on animal health or feed digestibility.The present study demonstrates that different methane emission between the sheep and the rabbits can be explained by the different physiological environments of their respective digestive organs and the microbiota residing therein.The lower pH in the rabbit cecum is probably a major chemical factor attributable to the low methane emission from the rabbits.Lower abundance,predominance,and gene expression of CAZymes related hydrogen-producing microbiota and methanogens,and increased homoacetogenesis as an alternative hydrogen-utilizing pathway in the rabbit cecum might result in lower methane emission from the rabbits.The rabbit cecum is potentially a rich resource to fibrolytic bacteria and cellulolytic enzymes,especially Glycoside Hydrolase.Bacteroides may be an ideal potential targeting microbiota to mitigate methane emission from ruminants without side-effects on animal health or feed digestibility.