Emission from ruminants has become one of the largest sources of anthropogenic methane emission in China. The structure of the rumen flora has a significant effect on methane production. To establish a more accurate prediction model for methane production, the rumen flora should be one of the most important parameters. The objective of the present study was to investigate the relationship among changes in rumen flora, nutrient levels, and methane production in sheep fed with the diets of different forage-to-concentration ratios, as well as to screen for significantly different dominant genera. Nine rumen-cannulated hybrid sheep were separated into three groups and fed three diets with forage-to-concentration ratios of 50:50, 70:30, and 90:10. Three proportions of the diets were fed according to a 3 × 3 incomplete Latin square, design during three periods of 15d each. The ruminal fluid was collected for real-time polymerase chain reaction (real-time PCR), high-throughput sequencing and in vitro rumen fermentation in a new real-time fermentation system wit. Twenty-two genera were screened, the abundance of which varied linearly with forage-to-concentration ratios and methane production. In addition, during the 12-hour in vitro fermentation, the appearance of peak concentration was delayed by 26-27min with the different structure of rumen bacteria. The fiber-degrading bacteria were positively correlated with this phenomenon, but starch-degrading and protein-degrading bacteria were negative correlated. These results would facilitate macro-control of rumen microorganisms and better management of diets for improved nutrition in ruminants. In addition, our findings would help in screening bacterial genera that are highly correlated with methane production.

18Emission from ruminants has become the largest source of anthropogenic emission of 19 methane in China. The structure of the rumen flora has a significant effect on methane 20 production. To establish a more accurate prediction model for methane production, the rumen 21 flora should be one of the most important parameters. The objective of the present study was 22 to investigate the relationship among changes in rumen flora, nutrient levels, and methane 23 production in sheep fed with the diets of different forage-to-concentration ratios, as well as to 24 screen for significantly different dominant genera. Nine rumen-cannulated hybrid sheep were 25 separated into three groups and fed three diets with forage-to-concentration ratios of 50:50, 26 70:30, and 90:10. Three proportions of the diets were fed according to a 3 × 3 incomplete 27 Latin square, design during three periods of 15 d each. The ruminal fluid was collected for 28 real-time qPCR, high-throughput sequencing and in vitro rumen fermentation in a new 29 real-time fermentation system wit. Twenty-two genera were screened, the abundance of 30 which varied linearly with forage-to-concentration ratios and methane production. In 31 addition, during the 12-hour in vitro fermentation, the appearance of peak concentration was 32 delayed by 26-27 min with the different structure of rumen bacteria. The fiber-degrading 33 bacteria were positively correlated with this phenomenon, but starch-degrading and 34 protein-degrading bacteria were negative correlated. These results would facilitate 35 macro-control of rumen microorganisms and better management of diets for improved 36 nutrition in ruminants. In addition, our findings would help in screening bacterial genera that 37 are highly correlated with methane production. 38 Keywords: greenhouse gas, high-throughput sequencing, ruminant, rumen bacteria, rumen 3 39 fermentation 40 Introduction 41 Of the total methane emission in China, the emission from ruminants was estimated to 42 be approximately 17%, turning them into the largest anthropogenic source of methane 43 emission [1]. The emission of methane associated with agriculture is expected to see a 44 significant increase. Therefore, new strategies were needed for reducing the emission and 45 improving livestock productivity, which had been extensively studied and reviewed [2]. 46 Rumen is the main site of methane production [3], which provided a habitat for a 47 variety of microbes, including numerous species of bacteria, archaea, viruses, protozoa and 48 fungi [4]. In the anaerobic environment of the rumen, several organic compounds present 49 could eventually be decomposed into methane by a number of microorganisms [5]. The 50 composition of ruminal microbiome was affected by different factors, such as age, breed, 51 general well-being of the animal, its location as well as administration of feed and antibiotics 52 [6-8]. Furthermore, feedstuffs were the main factors regulating the composition and 53 functional patterns of ruminal microbiome [9-11]. Among t...