Characteristics And Regulation Of Enteric Methane Emissions From Lactating Holstein Dairy Cows

Environmental problems such as global warming caused by anthropogenic greenhouse gas emissions have gradually attracted people’s attention,and China has taken measures to address them.As a greenhouse gas,methane（CH₄）has the characteristics of greater global-warming potential and shorter life,so the reduction of CH₄ emission is a fast and effective way to mitigate climate change.Ruminants are the main source of CH₄ emissions in livestock industry,and the methanogenesis will also cause the loss of dietary energy.There is a lack of systematic greenhouse gas emission data and local emission factors in livestock industry,and CH₄ emission reduction strategies are urgently needed.In this study,the Green Feed system was used to measure CH₄ emissions of a large herd of lactating Holstein dairy cows under normal feeding conditions to study the CH₄ emission characteristics of dairy cows.Based on CH₄emission characteristics,BS and MCE were selected as dietary additives to study their effects on CH₄ emission of dairy cows.Rumen metagenomics was used to reveal the CH₄ emission characteristics of dairy cows and the inhibition mechanisms of these two additives on CH₄ emission.This study includes the following four parts:1.Characteristics of greenhouse gas emissions from lactating Holstein dairy cowsGreen Feed system was used to measure the amount of CH₄ and carbon dioxide（CO₂）emitted by the lactating Holstein dairy cows through respiration and eructation.Data from a commercial cow farm were used to observe the effects of parity,body weight,milk yield,and milk component yield on CH₄ and CO₂ emissions.Mean herd responses throughout the study were as follows:111 cows completed all experimental processes,while 42 cows were rejected because they were sick or had not visited the Green Feed system 20 times.On average,days in milk of cows was 138±19.04 d,metabolic weight was 136.5±9.5 kg,parity was 2.8±1.0,dry matter intake（DMI）was23.1±2.6 kg/d,and milk yield was 38.1±6.9 kg/d.The Green Feed system revealed that CH₄ production was found to be 332 g/d,CH₄/DMI was 14.4 g/kg,CH₄/energy-corrected milk（ECM）was 9.18 g/kg,total CO₂ production（CH₄ production plus CO₂production）was 19 201 g/d,total CO₂/DMI was 831 g/kg,and total CO₂/ECM was 531g/kg.The parity and metabolic weight of cows had no significant effect on total CO₂emissions（P>0.05）.Cows with high milk yield,milk fat yield,milk protein yield,and total milk solids yield produced more total CO₂ production（P<0.05）,but their total CO₂ production per kg of ECM was low（P<0.05）.The total CO₂/ECM of the medium and high milk yield groups was 17%and 27%lower than that of the low milk yield group,respectively.The parity and body weight had no effect on total CO₂ emissions,while the total CO₂/ECM was negatively correlated with milk yield,milk fat yield,milk protein yield,and total milk solids yield in lactating Holstein dairy cows.2.Rumen metagenome reveals ruminal microbial features associated with enteric methane production of dairy cowsBased on CH₄ emissions from 111 lactating Holstein dairy cows that previously measured using the Green Feed system,we used rumen metagenomics to identify potential microbial features that underpins CH₄ production in cows.Based on the enteric CH₄ production from cows,9 cows with the highest CH₄ production and the 9cows with the lowest CH₄ production were selected to group the high-methane-production cows（HMP,CH₄=403±22.06 g/d）and the low-methane-production cows（LMP,CH₄=261±22.27 g/d）,respectively.Comparison of metagenomic data revealed that hydrogen-producing bacteria and ciliate protozoa were enriched in HMP cows while hydrogen-utilizing bacteria were enriched in LMP cows（P<0.05）.The EC:2.3.1.12（K00627）,EC:1.2.1.2（K00123）,and EC:6.2.1.1（K01895）involved in the formate metabolism and acetoclastic pathways were enriched in HMP cows（P<0.05）.Co-occurrence network analysis of the rumen microbiome revealed some negative relationships among methanogens,in which Methanobrevibacter was negatively associated with Methanothermobacter,Methanobacterium,Methanosphaera,Methanomassiliicoccus,Methanoculleus,Candidatus Methanomethylophilus,Methanomicrobium,Methanococcus,Methanocaldococcus,Methanolobus,and Methanoplanus.Random forest model analysis screened 34 genera of microorganisms（32 bacteria and 2 archaea）as biomarkers of dairy cows,and these microorganisms were significantly correlated with acetate,propionate,valerate,and CH₄ production（P<0.05）.This study enlightens the causal associations that exist for rumen microbiome composition and rumen methanogenesis leading to two significantly different enteric CH₄ productions in dairy cows,further identifying microbial biomarkers of rumen methanogenesis.3.Diets supplementation with Bacillus subtilis and Macleaya cordata extract improve production performance and the metabolism of energy and nitrogen,while reduce enteric methane emissions in dairy cowsThe objective of this experiment was to evaluate the effectiveness of Bacillus subtilis and Macleaya cordata extract on production performance,enteric CH₄emissions,nutrient digestion and utilization of lactating Holstein dairy cows.Sixty healthy lactating Holstein dairy cows averaged（mean±SD）145±12.5 of days in milk,38.3±3.3 kg/d of milk production,and 2.5±0.9 in parity were used in the experiment.Cows were assigned to 1 of 3 treatments:（1）control diet（CON）,（2）control diet plus Bacillus subtilis（BS）dosed at 50 g/head/d,（3）control diet plus Macleaya cordata extract（MCE）dosed at 450 mg/head/d.The entire experimental was 75 d in length,using Green Feed system measured enteric CH₄ and CO₂ emissions for individual cows.Six cows from each treatment were randomly selected to measure nutrient digestion and utilization.Compared with CON,BS and MCE diets increased DMI by 14%and11%,as well as milk yield（6.9%and 4.7%）,milk fat yield,milk protein yield,and milk lactose yield（P<0.05）.Furthermore,BS and MCE diets reduced（P<0.05）CH₄ yield（g/kg·DMI）by 10%and 9%compared with CON,respectively.Based on fat-corrected milk（FCM）or energy-corrected milk（ECM）yield,CH₄ intensity was also reduced（P<0.05）by 6.8%and 5.3%（g/kg·FCM）,or 6.8%and 5.3%（g/kg·ECM）,respectively.BS diet had greater（P<0.05）neutral detergent fiber and acid detergent fiber digestibility than CON and MCE diets.Compared with CON,BS and MCE diets both increased（P<0.05）digestible energy,metabolizable energy,digestible nitrogen（N）,and retained N,whereas it reduced（P<0.05）CH₄ energy as a percent of gross energy.Cows offered the BS had greater（P<0.05）energy and N digestibility,as well as retained N and it as a percent of N intake or digestible N,compared with CON and MCE.Results suggested that implementation of these dietary interventions had potential to improve animal productivity led to a decrease in enteric CH₄ yield or intensity.4.Rumen metagenomics reveals Bacillus subtilis and Macleaya cordata extract influence enteric methane emission in dairy cowsThe objective of this study was to elucidate the potential regulatory mechanisms of methanogenesis by BS and MCE in lactating Holstein dairy cows using rumen metagenomics.On day 75 of the third experiment,12 cows were selected from each group to collect rumen content for metagenomic sequencing.Results showed that BS decreased ruminal acetate and butyrate,and increased propionate concentrations,resulting in decreased acetate:propionate ratio（P<0.05）.Metagenomics analysis revealed that MCE reduced relative abundances of nine archaeal species,including Methanosphaera sp.WGK6、Methanosphaera stadtmanae、Methanobrevibacter wolinii、Methanobrevibacter sp.Ab M4、Candidatus Methanomassiliicoccus intestinalis、Methanobrevibacter cuticularis、Methanomicrobium mobile、Methanobacterium formicicum,and Methanobacterium congolense（P<0.05）;BS reduced relative abundances of Methanosphaera sp.WGK6 and Methanosphaera stadtmanae.Co?occurrence network analysis of the bacteria and archaea in the rumen,revealing differential microbial interaction patterns among groups,with BS and MCE cows having more and stronger associations than CON cows.Random forest and heatmaps analysis revealed that the Halopenitus persicus was positively correlated with fat-and protein-corrected milk yield（P<0.05）;Clostridium sp.CAG 269,Clostridium sp.27 14,Haloarcula rubripromontorii,and Methanobrevibacter curvatus were negatively correlated with acetate,butyrate,and acetate:propionate ratio（P<0.05）,whereas Selenomonas rumiantium was positively correlated with those（P<0.05）.These findings of the present study provided a fundamental understanding of how the microbiome-dependent mechanisms contribute to methanogenesis mitigation of dairy cows feed BS and MCE.In conclusion,this study successfully measured CH₄ and CO₂ emissions of 111lactating Holstein dairy cows under normal feeding conditions using the Green Feed system,and successively analyzed the negative correlations between milk yield,milk fat yield,milk protein yield,and total milk solids yield and total CO₂/ECM emissions;while parity and metabolic weight had no effect on total CO₂ emissions.Rumen metagenomics revealed that hydrogen-producing bacteria,ciliate protozoa,formate metabolism pathway,and acetoclastic pathway played an important role in rumen methanogenesis.In addition,it was proved that BS and MCE improved the lactation performance and nutrient digestion and utilization of Holstein dairy cows,while reduced the CH₄ yield and intensity.BS decreased the acetate/propionate ratio and MCE decreased the relative abundance of 9 archaeal species,which supported the inhibition of methanogenesis by these two additives.