| In this study,the variation of methane production and the number and diversity of Methanogens and Protozoa in rumen of high-yield and low-yield dairy cows at different lactation stages was explored,and the mechanisms for the different methane production of high-yield and low-yield dairy cows were revealed from the perspective of the change of rumen microbial community,meanwhile the correlation between the relative abundance of Methanogens and Protozoa in rumen and methane production,milk production and milk quality was analyzed,so as to provide theoretical basis for efficient breeding of dairy cows.The experiment adopted a two-factor experimental design.Thirty-six Holstein cows with good health,similar weight,parity and feeding management conditions were divided into high-yield early lactation group and high-yield middle lactation group according to lactation performance [high yield(40.27±3.76 kg/d)and low yield(20.71±4.32 kg/d)] and lactation stage [prophase(0-100 d),metaphase(101-200 d)and anaphase(201-300 d)].All cows were fed the same total mixed ration.The prediction model of methane production was selected to calculate methane production,the number of Methanogens and Protozoa in rumen was determined by real-time fluorescence quantitative PCR,and the diversity of Methanogens and Protozoa in rumen was determined by high-throughput sequencing technique.The results showed that:(1)The methane production of high-yield dairy cows was significantly lower than that of low-yield dairy cows(P<0.05),and the methane production of dairy cows in late lactation was significantly higher than that in early and middle lactation(P<0.05).(2)The activities of carboxymethyl cellulase,β-glucosidase,xylanase and filter paper cellulase in high-yield dairy cows were significantly higher than those in low-yield dairy cows(P<0.05).The activities of carboxymethyl cellulase,xylanase and filter paper cellulase in the middle lactation period were significantly higher than those in the early and late lactation period(P<0.05),and the activities of β-glucosidase in the early lactation period were significantly higher than those in the middle and late lactation period(P<0.05).(3)The number of Methanobrevibacter and Methanosphaera in ruminants of high-yield dairy cows was significantly higher than that of low-yield dairy cows(P<0.05),while the total number of Methanogens,Methanogens from RCC and Methanogens from non-RCC and the expression of mcr A gene were significantly lower than that of low-yield dairy cows(P<0.05).The number of total Methanogens,RCC Methanogens and non-RCC Methanogens in early lactation and the expression of mcr A gene were significantly higher than those in middle and late lactation(P<0.05).The interaction between lactation performance and lactation stage had significant effects on the total Methanogens,non-RCC Methanogens,Methanogens and mcr A gene expression(P<0.05),but had no significant effects on the Methanogens of RCC Methanogens and Methanobrevibacter(P>0.05).(4)The relative abundance of Methanosphaera in rumen of high-yield dairy cows was significantly higher than that of low-yield dairy cows(P<0.05),while the relative abundance of Methanobrevibacter was significantly lower than that of low-yield dairy cows(P<0.05).The relative abundance of Methanosphaera in the middle lactation period was significantly higher than that in the early and late lactation period(P<0.05),and the relative abundance of Methanobrevibacter in the late lactation period was significantly higher than that in the early lactation period(P<0.05).The interaction between lactation performance and lactation stage significantly affected the relative abundance of Methanobrevibacter(P<0.05),but had no significant effect on the relative abundance of Methanosphaera(P>0.05).(5)The number of total Protozoa,Entodinium in the rumen of high-yield dairy cows was significantly higher than that of low-yield dairy cows(P<0.05),but there was no significant difference between the number of Dasytrichas and low-yield dairy cows(P>0.05).The number of total Protozoa,Entodinium and Dasytrichas in the rumen of dairy cows in the middle lactation period was significantly higher than that in the early and late lactation period(P<0.05),and the interaction between lactation performance and lactation stage had significant effects on the number of Dasytrichas(P<0.05),The relative abundance of Isotricha in rumen of high-yield dairy cows was significantly lower than that of low-yield dairy cows,while that of Entodinium was significantly higher than that of low-yield dairy cows(P<0.05).The relative abundance of Isotricha in the middle and late lactation period of dairy cows was significantly higher than that in the early lactation period(P<0.05),but there was no significant difference in the relative abundance of Isotricha in the three lactation stages(P>0.05).The interaction between lactation performance and lactation stage significantly affected the relative abundance of Isotricha(P<0.05),but had no significant effect on the relative abundance of Entodinium(P>0.05).In conclusion,the structure changes of Methanogens and Protozoa community in the rumen of dairy cows had significant effects on their lactation performance.The number and diversity of Methanogens and Protozoa in the rumen of high-yielding dairy cows were lower than those of low-yielding dairy cows,while the activity of cellulase was higher than that of low-yielding dairy cows,and the yield of methane was lower than that of low-yielding dairy cows,methane production is lower than that of low-yield dairy cows,suggesting that high-yielding dairy cows had a more simple and efficient structure of rumen Microbial community,which could reduce energy waste and improve the absorption and utilization efficiency of nutrients in the diet. |
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