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Effects of combined addition of 3-nitrooxypropanol and vitamin B12 on methane and propionate production in dairy cows by in vitro-simulated fermentation

Open AccessPublished:November 07, 2022DOI:https://doi.org/10.3168/jds.2022-22207

      ABSTRACT

      The compound 3-nitrooxypropanol (3-NOP) is a promising methane inhibitor, which performs well in inhibiting methane emission and does not affect animal feed intake and digestibility. However, it causes a significant increase in hydrogen production while suppressing methane emission, resulting in a waste of feed energy. Vitamin B12 is a key factor in the propionate production pathway and thus plays an important role in regulating the hydrogen utilization pathway. In this study, the effects of 3-NOP combined with vitamin B12 supplementation on rumen fermentation and microbial compositional structure in dairy cattle were investigated by simulating rumen fermentation in vitro. Experiments were performed using a 2 × 2-factorial design: two 3-NOP levels (0 or 2 mg/g dry matter) and 2 vitamin B12 levels (0 or 2 mg/g dry matter). Three experiments were performed, each consisting of 4 treatments, 4 replicates, and 4 blanks containing only inoculum. The combined supplementation of 3-NOP and vitamin B12 reduced methane emission by 12% without affecting dry matter digestibility. The combined addition of 3-NOP and vitamin B12 significantly increased the concentration of propionate and reduced the concentration of acetate and the acetate to propionate ratio. At the bacterial level, 3-NOP increased the relative abundances of Christensenellaceae_R-7_group and Lachnospiraceae_NK3A20_group. Vitamin B12 increased the relative abundances of unclassified_f__Prevotellaceae and Prevotellaceae_UCG-003 and decreased the relative abundance of Lachnospiraceae_NK3A20_group. At the archaeal level, the combination of 3-NOP and vitamin B12 increased the relative abundances of Methanobrevibacter_ sp._ Abm4, OTU1125, and OTU95 and decreased the relative abundances of uncultured_methanogenic_archaeon_g__Methanobrevibacter, OTU1147, OTU1056, and OTU55. The results indicated that 3-NOP combined with vitamin B12 could alleviate rumen hydrogen emission and enhance the inhibition of methane emission compared with 3-NOP alone.

      Key words

      INTRODUCTION

      The greenhouse effect is an urgent global environmental problem. Greenhouse gases including carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and ozone (O3) can absorb atmospheric infrared radiation to cause climate change and global warming (
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      The compound 3-nitrooxypropanol (3-NOP) is a CH4 inhibitor with molecular structure similar to methyl coenzyme M designed and developed by

      Duval, S., and M. Kindermann, inventors. 2012. Use of nitrooxy organic molecules in feed for reducing methane emission in ruminants, and/or to improve ruminant performance. World Intellectual Property Organization, Geneva, Switzerland Pat. No. WO 2012/084629 A1.

      . It inactivates the key enzyme methyl coenzyme M reductase required for CH4 production by oxidizing the active site Ni (I) of methyl coenzyme M, so as to inhibit CH4 production (
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      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      , 3-NOP reduced rumen CH4 emission by 30% without affecting DMI, milk yield, and fiber digestibility, and this significant effect remained throughout the 12-wk experiment. Although 3-NOP can effectively reduce CH4 emission, it also causes a significant increase in hydrogen (H2) emission (
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      • Duval S.
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      Enteric methane emission, milk production, and composition of dairy cows fed 3-nitrooxypropanol.
      ). The results of a study by
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.W.
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      • Weeks H.L.
      • Branco A.F.
      • Moate P.J.
      • Deighton M.H.
      • Williams S.R.
      • Kindermann M.
      • Duval S.
      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      showed that addition of 3-NOP at 40–80 mg/kg DM of feed decreased CH4 emission by 30% in high-yielding cows, but increased H2 emission by 64-fold, although the intensity of H2 emission declined after a period of time. Hydrogen, which exists in the rumen as a gas (H2) or in liquid ([H]) state (
      • Janssen P.H.
      Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics.
      ), is a key intermediate in rumen microbial fermentation (
      • Hungate R.E.
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      ) and an energy substrate for CH4 production by rumen archaea (
      • Ellis J.L.
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      ). In cows fed 3-NOP, the production of CH4 was inhibited, and the increased hydrogen was excreted in the form of H2 or accumulated in the rumen in the form of [H] (
      • Melgar A.
      • Harper M.T.
      • Oh J.
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      Effects of 3-nitrooxypropanol on rumen fermentation, lactational performance, and resumption of ovarian cyclicity in dairy cows.
      ). However, either form is a waste of feed energy. Furthermore, the daily increased H2 emission of dairy cows fed with 3-NOP is much smaller than that produced by [H] but not used for CH4 production, so a large amount of [H] may be accumulated in rumen (
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.W.
      • Harper M.T.
      • Weeks H.L.
      • Branco A.F.
      • Moate P.J.
      • Deighton M.H.
      • Williams S.R.
      • Kindermann M.
      • Duval S.
      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      ;
      • Melgar A.
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      • Oh J.
      • Giallongo F.
      • Young M.E.
      • Ott T.L.
      • Duval S.
      • Hristov A.N.
      Effects of 3-nitrooxypropanol on rumen fermentation, lactational performance, and resumption of ovarian cyclicity in dairy cows.
      ). Therefore, when using 3-NOP to inhibit CH4 production, how to reasonably allocate the fate of [H] in rumen is still an important scientific problem to be solved.
      Transferring [H] to metabolic processes that have nutritional value for animals is an ideal strategy to suppress the loss of digestible energy in the process of CH4 production, while avoiding fermentation inhibition (
      • Lan W.
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      Ruminal methane production: Associated microorganisms and the potential of applying hydrogen-utilizing bacteria for mitigation.
      ). For example, it has been suggested to shift [H] from CH4 to propionate, which may be a way to increase the available metabolizable energy of animals (
      • Martin S.A.
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      Effects of monensin, pyromellitic diimide, and 2-bromoethanesulfonic acid on rumen fermentation in vitro.
      ). Propionate is a short chain fatty acid formed by the fermentation of carbohydrates in the diet, and is absorbed and used by animals and participates in the process of gluconeogenesis in the body (
      • Reichardt N.
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      ;
      • Morrison D.J.
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      Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism.
      ). As the main precursor of glucose in cows, propionate accounts for 60% of the amount of glucose released by the liver through gluconeogenesis (
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      ;
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      ). Furthermore, in lactating cows, approximately 80% of the glucose supply comes from gluconeogenesis (
      • Galindo C.E.
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      ). Three pathways of propionate production are known: the succinate pathway, the propylene glycol pathway, and the acrylate pathway. Of which the succinate pathway is the most predominant one (
      • Louis P.
      • Flint H.J.
      Formation of propionate and butyrate by the human colonic microbiota.
      ). Propionate production in the succinate pathway involves propionyl-CoA transferase reaction. Vitamin B12 is a necessary factor for propionyl-CoA transferase, a key enzyme in the formation of propionate in the succinate pathway (
      • Louis P.
      • Flint H.J.
      Formation of propionate and butyrate by the human colonic microbiota.
      ), and accumulation of succinate was observed in vitamin B12 deficient cultures of rumen Prevotella ruminicola (
      • Strobel H.J.
      Vitamin B12-dependent propionate production by the ruminal bacterium Prevotella ruminicola 23.
      ).
      In view of the role of vitamin B12 in propionic acid metabolism, it should play an important role in regulating [H] utilization pathway. Therefore, the objectives of this study were to determine the effects of vitamin B12 on rumen propionate and CH4 production in dairy cows by using in vitro fermentation methods, and to explore whether its combined application with 3-NOP in vitro would have synergistic effects on increasing rumen propionate and reducing CH4 production in dairy cows.

      MATERIALS AND METHODS

      The experiments were conducted at the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China), and the animal procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Agricultural Sciences (IAS2021–100; Beijing, China).

      Experimental Design

      Performed in vitro culture experiment to assess the combined effect of 3-NOP and vitamin B12, the treatments were laid out as a 2 × 3-factorial arrangement in randomized complete block design: two 3-NOP levels (0 or 2 mg/g DM) and 2 vitamin B12 levels (0 or 2 mg/g DM). The compound 3-NOP levels were selected based on the highest 3-NOP dose used in a previous in vitro experiment (
      • Romero-Pérez A.
      • Okine E.K.
      • Guan L.L.
      • Duval S.M.
      • Kindermann M.
      • Beauchemin K.A.
      Effects of 3-nitrooxypropanol and monensin on methane production using a forage-based diet in Rusitec fermenters.
      ). The selection of vitamin B12 level is based on the addition dose selected by our in vitro experiment of adding vitamin B12 alone. The experiment consisted of three 24-h in vitro cultures. Each run consisted of 20 samples: 4 treatments × 4 replicates as well as 4 blanks containing only inoculum. The 4 treatments were: control (CON), vitamin B12, 3-NOP, 3-NOP + vitamin B12. Reagents were added accurately to the fermentation flasks and the volume of liquid in all fermentation flasks was equalized with distilled water. The compound 3-NOP powder (>98%) was purchased from Shanghai Aladdin Biochemical Technology Co. Ltd.; vitamin B12 powder (>98%) was purchased from Solarbio Life Science.

      In Vitro Incubation

      Three lactating multiparous Holstein cows with permanent ruminal fistulas served as donors of ruminal fluid (3 ± 1 of parity; 618 ± 100 kg of BW; 23 ± 2.8 kg of milk yield/d). Donor cows received a TMR diet, which consisted (DM basis) of corn silage (25.7%), alfalfa hay (18.6%), steam flaked corn (26.0%), soybean meal (7.4%), cottonseed meal (7.4%), beet meal (5.6%), distillers dried grains with solubles (7.4%), and minerals and vitamins (1.9%). The rumen fluid was collected about 2 h after morning feeding. And the rumen fluid from the 3 donor cows was mixed together in equal volumes and placed in a pre-heated thermos, and then brought back to the laboratory within 30 min. The rumen fluid was then filtered through 2 layers of gauze to remove impurities and then diluted with warm (39°C) buffer (each liter of solution containing 8.75 g of NaHCO3, 1.00 g of NH4HCO3, 1.43 g of Na2HPO4, 1.55 g of KH2PO4, 0.15 g of MgSO4·7H2O, 0.52 g of Na2S, 0.017 g of CaCl·2H2O, 0.015 g of MnCl2·4H2O, 0.002 g of CoCl·6H2O, 0.012 g of FeCl3·6H2O, and 1.25 mg of resazurin) as inoculum. The inoculum was placed in a water bath at 39°C with constant agitation to ensure adequate mixing of the rumen fluid and buffer (1:2, vol/vol). The whole process was performed under continuous flushing with CO2. The fermentation substrate was the same TMR offered to the cows, which was dried at 55°C for 48 h and passed through a 1-mm screen with a Wiley mill (Arthur H Thomas Co). Under anaerobic conditions of continuous washing with CO2, an accurately weighed quantity of the fermentation substrate (0.5 g) and 75 mL of inoculum were added to the fermentation flask (120 mL). The compound 3-NOP and vitamin B12 solutions (both 2 mg/mL of water) were prepared, and 1 mL of 3-NOP, 1 mL of vitamin B12, and 1 mL of 3-NOP + 1 mL of vitamin B12 solutions were accurately injected into the fermentation flasks of vitamin B12, 3-NOP, and 3-NOP + vitamin B12, respectively. The flasks were then sealed by capping with butyl glue and wrapping aluminum foil. A gas bag was connected with the fermentation flask for gas collection after evacuation, and then placed into a thermostatic incubator at 39°C for 24 h at a frequency of 60 rpm. The fermentation flasks were shaken gently every 2 h during the whole cultivation process so that the fermentation substrate and inoculum were mixed thoroughly. When the incubation time reached 24 h, all the fermentation flasks were taken out and put into ice water to terminate the fermentation. The samples were then collected and preserved for subsequent analysis.

      Sample Collection and Analysis

      Total gas production from each air bag was measured using a calibrated glass syringe (100 mL, Häberle Labortechnik). A portable pH meter (Seven Go portable pH meter, Mettler-Toledo) was used to measure the pH values of the in vitro incubations. The liquid sample was collected after it had been completely cooled. Liquid samples (2.5 mL × 3) were collected from each fermentation flask, respectively for the analysis of VFA and microorganisms. The liquid collected from each bottle was centrifuged at 15,000 × g for 10 min at 4°C. Then the supernatant was acidified with 0.15 mL of 25% metaphosphate and stored at −20°C for VFA analysis. Samples for microbial DNA extraction and 16S rRNA gene sequencing and analysis were immediately frozen in liquid nitrogen and then transferred to a −80°C freezer. All the biomass materials in each bottle were separately filtered through a pre-weighed nylon bag (8 cm × 12 cm, 42 µm). The nylon bags were repeatedly rinsed with tap water until the effluent became clear and then oven-dried at 55°C for 48 h for measuring the dry matter degradation (DMD). Methane production was calculated by CH4 concentration and total gas volume. The concentrations of CH4 and VFA were determined using an Agilent 7890B gas chromatograph (7890B, Agilent Technologies).

      DNA Extraction and 16SrRNA Genes Sequencing and Analysis

      Rumen microbial DNA was extracted using cetyltrimethylammonium bromide (CTAB) plus bead beating (
      • Jin D.
      • Zhao S.G.
      • Zheng N.
      • Bu D.P.
      • Beckers Y.
      • Denman S.E.
      • McSweeney C.S.
      • Wang J.Q.
      Differences in ureolytic bacterial composition between the rumen digesta and rumen wall based on ureC gene classification.
      ). The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific). The hypervariable region V3–V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by an ABI Gene Amp 9700 PCR thermocycler (ABI). The V4–V5 region of the archaeal 16S rRNA gene was amplified by primers 524F10extF (5′-TGYCAGCCGCCGCGGTAA-3′) and Arch958RmodR (5′-YCCGGCGTTGA VTCCAA TT-3′). The PCR amplification of 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles (bacteria) or 33 cycles (archaea) of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s, and single extension at 72°C for 10 min, and end at 4°C. The PCR mixtures contained 5 × TransStart FastPfu buffer (4 μL), 2.5 mM dNTP (2 μL), forward primer [(5 μM) 0.8 μL], reverse primer [(5 μM) 0.8 μL], TransStart FastPfu DNA Polymerase (0.4 μL), template DNA (10 ng), and finally double-distilled H2O up to 20 μL. The PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) according to the manufacturer's instructions and quantified using a Quantus Fluorometer (Promega). Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform (Illumina) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd.
      The data were analyzed on the free online platform of Majorbio Cloud Platform (www.majorbio.com). Different samples were identified based on unique barcodes. The 16S rRNA sequences were perfectly matched to different samples, quality filtered by fastp (version 0.20.0), and merged by FLASH (version 1.2.7;
      • Magoč T.
      • Salzberg S.L.
      FLASH: Fast length adjustment of short reads to improve genome assemblies.
      ): (1) filtered the bases with a tail quality value below 20 of the reads, and set a 50 bp window. If the average quality value in the window was lower than 20, the back-end bases would be truncated from the window, filtered the reads with the quality control value below 50 bp, and removed the reads containing N bases; (2) according to the overlap relationship between PE reads, paired reads were spliced into a sequence with a minimum overlap length of 10 bp. Then the primers and barcodes were removed, and chimeras were filtered to obtain valid reads. After filtration, the valid reads of bacterial and archaeal communities were 2,491,440 and 2,990,098, respectively, and the average lengths of valid reads were 418 and 428 bp, respectively. Valid reads were clustered with operational taxonomic units (OTU) at a similarity cutoff of 97% using UPARSE (version 7.1;
      • Edgar R.C.
      • Haas B.J.
      • Clemente J.C.
      • Quince C.
      • Knight R.
      UCHIME improves sensitivity and speed of chimera detection.
      ). Classification analysis with a confidence threshold of 0.8 and the SILVA database (version 138) was performed using the RDP classifier (version 2.11;
      • Quast C.
      • Pruesse E.
      • Yilmaz P.
      • Gerken J.
      • Schweer T.
      • Yarza P.
      • Peplies J.
      • Glockner F.O.
      The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools.
      ). Alpha diversity was analyzed using the QIIME 2 package, and the indices Simpson, Shannon, Chao1, coverage, and ACE were calculated. Principal coordinate analysis was performed based on weighted UniFrac distances (
      • Lozupone C.A.
      • Hamady M.
      • Kelley S.T.
      • Knight R.
      Quantitative and qualitative beta diversity measures lead to different insights into factors that structure microbial communities.
      ). Differences in bacterial and archaeal relative abundances were visualized by extended error bar plots with bioinformatics software (STAMP). Welch's 2-sided test was used, with a Welch's inversion test of 0.95 (
      • Parks D.H.
      • Tyson G.W.
      • Hugenholtz P.
      • Beiko R.G.
      STAMP: Statistical analysis of taxonomic and functional profiles.
      ).
      The raw reads were deposited into the National Center for Biotechnology Information Sequence Read Archive database (Accession Number: SRP327717).

      Statistical Analysis

      All data presented were the average of the 3 experiments. The analysis of in vitro fermentation parameters (pH, DMD, total gas, CH4, and VFA), and abundance and diversity of bacteria and archaea were performed using the MIXED procedure of SAS 9.4 version (SAS Institute Inc.). The data were tested for normality using the UNIVARIATE procedure of SAS. The model used for data analysis was as follows:
      Yij = μ + Pi + Sj + PSij + eij,


      where Yij is the observed value, μ is the overall mean, Pi is the fixed effect of treatment with 3-NOP, Sj is the fixed effect of treatment with vitamin B12, PSij is the interaction effect of treatment with 3-NOP + vitamin B12, and eij is the random error. The difference was significant when P ≤ 0.05, and showed a trend when 0.05 < P ≤ 0.10.

      RESULTS

      Methane Production and Fermentation Characteristics

      The effects of 3-NOP, vitamin B12, and their interactions on fermentation characteristics and CH4 production are shown in Table 1. The addition of 3-NOP reduced the total gas production (P = 0.02), whereas vitamin B12 had no effect on the total gas production (P = 0.30). The addition of 3-NOP and vitamin B12 both respectively decreased the CH4 production by 9% (P = 0.30). The compound 3-NOP in combination with vitamin B12 decreased CH4 production by 12% (P = 0.26). Both 3-NOP (P < 0.01) and vitamin B12 (P = 0.08) increased the pH of the fermentation liquid. All treatments had no effect on DMD. Corresponding to the increased pH, both 3-NOP (P < 0.01) and vitamin B12 (P = 0.03) significantly decreased the concentration of total VFA. The compound 3-NOP significantly decreased the concentrations of acetate (P < 0.01) and valerate (P < 0.01), significantly increased the concentration of propionate (P = 0.02), and tended to decrease the concentration of isobutyrate (P = 0.06). Vitamin B12 significantly decreased the concentration of acetate (P < 0.01) and increased the concentration of propionate (P = 0.04), whereas it had no effect on other VFA (P > 0.1). Both 3-NOP (P < 0.01) and vitamin B12 (P < 0.01) significantly reduced the ratio of acetate to propionate. The combination of 3-NOP and vitamin B12 significantly increased the concentration of propionate and decreased the concentration of acetate and the ratio of acetate to propionate.
      Table 1Effects of the combination of 3-NOP and vitamin B12 on CH4 production, VFA profiles, and other fermentation parameters in vitro
      n = 15.
      Item0NOP
      Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      P-value
      NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.
      0VB0VBNOPVBNPV
      pH6.556.566.576.580.010.010.94
      DMD
      DMD = apparent disappearance of dry matter.
      0.720.710.710.700.460.370.86
      Gas production (mL)143.3139.5135.9134.60.020.300.61
      CH4 (mL)8.157.417.417.170.030.030.26
      Concentration (mM)
       Total VFA107.19106.11105.14104.690.010.030.40
       Acetate67.6366.7065.8765.360.010.010.31
       Propionate21.6022.0822.1522.430.020.040.64
       Isobutyrate1.111.091.081.060.060.210.73
       Butyrate13.0412.5012.3912.210.120.220.54
       Isovalerate2.021.981.961.940.210.400.91
       Valerate1.801.781.721.690.010.170.83
       Acetate or propionate3.133.022.982.920.010.010.35
      1 n = 15.
      2 Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      3 NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.
      4 DMD = apparent disappearance of dry matter.

      Changes of Microbial Community

      Effects on Bacterial Community.

      The α diversity of bacterial community is shown in Table 2. The coverage of sample OTU was more than 99% and there was no significant difference among treatments, indicating that the accuracy and repeatability of sequencing results were ideal for analysis. Alpha diversity indices such as Shannon, Simpson, ACE, and Chao1 did not differ significantly among treatments (P > 0.05), indicating that the addition of 3-NOP and vitamin B12 did not affect the richness and diversity of the bacterial community. Propionyl-CoA analysis based on the weighted UniFrac distance algorithm showed no obvious separation of each treatment group from CON (Figure 1). Similarity analysis (ANOSIM) revealed no significant difference in bacterial community structure between each treatment group and CON (r = 0.01, P = 0.58).
      Table 2Alpha diversity indices of archaea and bacteria among treatments in vitro
      n = 15.
      Item0NOP
      Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      P-value
      NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.
      0VB0VBVBNOPNPV
      Bacteria
       ACE
      ACE = abundance-based coverage estimator.
      1,604.71,568.51,609.31,587.60.110.790.52
       Chao1,634.31,579.81,616.61,612.90.060.400.48
       Coverage0.990.990.990.990.500.550.29
       Shannon5.335.315.315.280.750.630.29
       Simpson0.020.020.020.020.790.860.72
      Archaea
       ACE241.0227.7179.7284.10.790.290.44
       Chao219.3198.7161.8244.90.640.290.60
       Coverage1.001.001.001.000.610.260.67
       Shannon1.751.661.701.710.030.310.44
       Simpson0.330.350.340.340.060.430.33
      1 n = 15.
      2 Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      3 NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.
      4 ACE = abundance-based coverage estimator.
      Figure thumbnail gr1
      Figure 1Principal coordinate (PC) analysis of the rumen bacterial community among treatments in vitro. CON = control group, a basic TMR; VB = CON plus vitamin B12 (2 mg/g DM); NOP = CON plus 3-nitrooxypropanol (2 mg/g DM); NPV = 3-nitrooxypropanol (2 mg/g DM) plus vitamin B12 (2 mg/g DM).
      A total of 18 phyla of bacteria were identified in rumen samples. Among them, Bacteroidota (48.7%) and Firmicutes (47.5%) had higher relative abundances and were the 2 dominant phyla (Figure 2A). A total of 18 dominant genera were identified which accounted for more than 1% of the total sequence. These genera were: Rikenellaceae_RC9_gut_group (18.0%), Prevotella (10.9%), Succiniclasticum (9.2%), NK4A214_group (7.9%), Christensenellaceae_R-7_group (6.4%), norank_f__F082 (6.3%), norank_f__UCG-011 (5.4%),norank_f__Bacteroidales_RF16_group (3.8%), Prevotellaceae_UCG-003 (3.3%), norank_f__Muribaculaceae (2.5%), Selenomonas (1.6%), Lachnospiraceae_NK3A20_group (1.6%), unclassified_f__Prevotellaceae (1.5%), Ruminococcus (1.4%), Acetitomaculum (1.3%), Ruminococcus_gauvreauii_group (1.3%), Butyrivibrio (1.0%), and Veillonellaceae_UCG-001 (0.9%; Figure 2B). The experimental treatments had no significant effect on the relative abundance of bacterial phyla and most genera. The changes in the relative abundance of bacteria at the phylum and genus level are shown in Table 3. At the phylum level, we observed no significant difference in the relative abundance of Bacteroidota and Firmicutes among the treatment groups. The compound 3-NOP significantly increased the relative abundances of Christensenellaceae_R-7_group (P < 0.05) and Lachnospiraceae_NK3A20_group (P < 0.05). Vitamin B12 tended to decrease the relative abundances of Rikenellaceae_RC9_gut_group (P = 0.07) and Lachnospiraceae_NK3A20_group (P = 0.06). Vitamin B12 significantly increased the relative abundance of unclassified_f__Prevotellaceae (P < 0.05) and tended to increase the relative abundance of Prevotellaceae_UCG-003 (P = 0.06).
      Figure thumbnail gr2
      Figure 2Composition of the predominant bacterial community among treatments in vitro. (A) phyla level; (B) genera level. CON = control group, a basic TMR; VB = CON plus vitamin B12 (2 mg/g DM); NOP = CON plus 3-nitrooxypropanol (2 mg/g DM); NPV = 3-nitrooxypropanol (2 mg/g DM) plus vitamin B12 (2 mg/g DM).
      Table 3Difference in the relative abundances of bacterial community among treatments in vitro
      n = 15.
      Item0NOP
      Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      P-value
      NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.
      0VB0VBNOPVBNPV
      Phylum level abundance (%)
      Bacteroidota48.6848.6346.3847.080.210.820.81
      Firmicutes47.4546.7249.3848.630.190.610.99
      Actinobacteriota1.091.150.991.150.620.310.64
      Proteobacteria0.801.111.121.060.480.550.38
      Genus level abundance (%)
      Rikenellaceae_RC9_gut_group17.9516.3117.7816.240.890.070.96
      Prevotella10.8912.018.7510.740.190.230.74
      Succiniclasticum9.1610.848.759.570.460.270.70
      Christensenellaceae_R-7_group6.395.876.957.290.020.820.30
      Prevotellaceae_UCG-0033.334.202.963.710.310.060.89
      Unclassified_f__Prevotellaceae1.532.031.401.940.620.020.92
      Ruminococcus1.571.551.731.630.420.490.76
      Lachnospiraceae_NK3A20_group1.421.391.511.430.520.060.84
      1 n = 15.
      2 Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      3 NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.

      Effects on Archaeal Community.

      The α diversity of archaeal community is shown in Table 2. A total of 1,041 OTU were identified in the 4 treatments. The coverage of sample OTU over 99% was close to 100%, and we observed no significant difference among treatments, indicating that the accuracy and repeatability of sequencing results were ideal for analysis. Alpha diversity indices such as Shannon, Simpson, ACE and Chao1 did not differ significantly among treatments (P > 0.05), indicating that the addition of 3-NOP and vitamin B12 did not affect the richness and diversity of the archaeal community. Propionyl-CoA analysis based on the weighted UniFrac distance algorithm showed no obvious separation of each treatment group from CON (Figure 3). ANOSIM revealed no significant difference in archaeal community structure between each treatment group and CON (r = 0.03, P = 0.20).
      Figure thumbnail gr3
      Figure 3Principal coordinate (PC) analysis of the rumen archaeal community among treatments in vitro. CON = control group, a basic TMR; VB = CON plus vitamin B12 (2 mg/g DM); NOP = CON plus 3-nitrooxypropanol (2 mg/g DM); NPV = 3-nitrooxypropanol (2 mg/g DM) plus vitamin B12 (2 mg/g DM).
      At the phylum level, Euryarchaeota and unclassified_ k__ norank_ d__ Archaea represented the 2 main archaeal communities, of which Euryarchaeota accounted for an average of 98.5% (Figure 4A). At the genus level, genera that accounted for ≥1% of the total sequences were selected for analysis, and 3 dominant genera were identified: Methanobrevibacter (95.3%), Methanosphaera (3.2%), and unclassified_ k__ norank_ d__ Archaea (1.2%; Figure 4B). No effect of 3-NOP or vitamin B12 on archaeal microbial community was observed at the phylum and genus levels. The species and OTU levels of archaeal community were further analyzed to investigate changes in the archaeal microbial community (Table 4). At the species level, vitamin B12 tended to decrease the relative abundance of uncultured_methanogenic_archaeon_g_ _Methanobrevibacter (P = 0.07). The compound 3-NOP tended to decrease the relative abundances of unclassified_g__Methanobrevibacter (P = 0.07) and uncultured_methanogenic_archaeon_g_ _Methanobrevibacter (P = 0.06), whereas tended to increase the relative abundance of Methanobrevibacter _sp._AbM4 (P = 0.09). Combined supplementation of 3-NOP and vitamin B12 had a significant interaction effect on increasing the relative abundance of Methanobrevibacter_sp._AbM4 (P < 0.05) and decreasing the relative abundance of uncultured_methanogenic_archaeon_g__Methanobrevibacter (P < 0.05), and we observed a trend of interaction on decreasing the relative abundance of unclassified_g__Methanobrevibacter (P = 0.07). At the OTU level, vitamin B12 significantly reduced the relative abundance of OTU55 (P < 0.05). The compound 3-NOP significantly decreased the relative abundances of OTU55 (P < 0.05) and OTU1147 (P < 0.05), and had a trend of decreasing the relative abundance of OTU1056 (P = 0.08). The compound 3-NOP significantly increased the relative abundances of OTU1125 (P < 0.05) and OTU95 (P < 0.05). Combined supplementation of 3-NOP and vitamin B12 had a significant interaction effect on increasing the relative abundances of OTU1125 and OTU95 and decreasing the relative abundances of OTU1056, OTU55, and OTU1147 (P < 0.05).
      Figure thumbnail gr4
      Figure 4Composition of the predominant archaeal community among treatments in vitro. (A) phyla level; (B) genera level. CON = control group, a basic TMR; VB = CON plus vitamin B12 (2 mg/g DM); NOP = CON plus 3-nitrooxypropanol (2 mg/g DM); NPV = 3-nitrooxypropanol (2 mg/g DM) plus vitamin B12 (2 mg/g DM).
      Table 4Difference in the relative abundances of archaeal community among treatments in vitro
      n = 15.
      Item0NOP
      Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      P-value
      NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.
      0VB0VBNOPVBNPV
      Species level abundance (%)
      Uncultured_archaeon_g_Methanobrevibacter62.6265.0664.1865.040.220.100.24
      Unclassified_g_Methanobrevibacter13.3912.4612.1211.620.070.190.07
      Methanobrevibacter_sp._AbM46.756.507.547.630.090.420.02
      Uncultured_rumen_-methanogen_g_Methanobrevibacter7.036.736.816.730.310.260.24
      Uncultured_methanogenic_archaeon_g_Methanobrevibacter6.996.306.276.020.060.070.04
      Unclassified_g_Methanosphaera2.782.592.722.610.750.180.40
      OTU level abundance (%)
       OTU89355.6458.0356.4057.070.440.100.36
       OTU10569.058.238.027.640.080.240.02
       OTU11256.977.037.787.970.010.930.01
       OTU956.756.507.547.630.090.420.02
       OTU554.684.233.983.840.010.020.01
       OTU11473.723.483.293.170.040.260.04
      1 n = 15.
      2 Data were analyzed using two 3-nitrooxypropanol levels (0 or 2.0 mg/g DM) and 2 vitamin B12 levels (0 or 2.0 mg/g DM).
      3 NOP = 3-nitrooxypropanol; VB = vitamin B12; NPV = the interaction between 3-nitrooxypropanol and vitamin B12.

      DISCUSSION

      Effects on Methanogenesis

      In the current study, the addition of 3-NOP alone or vitamin B12 alone reduced the CH4 emission by 9%. Similar to other previous studies on cows, 3-NOP significantly reduced CH4 emission (
      • Haisan J.
      • Sun Y.
      • Guan L.L.
      • Beauchemin K.A.
      • Iwaasa A.
      • Duval S.
      • Barreda D.R.
      • Oba M.
      The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation.
      ;
      • Reynolds C.K.
      • Humphries D.J.
      • Kirton P.
      • Kindermann M.
      • Duval S.
      • Steinberg W.
      Effects of 3-nitrooxypropanol on methane emission, digestion, and energy and nitrogen balance of lactating dairy cows.
      ;
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.W.
      • Harper M.T.
      • Weeks H.L.
      • Branco A.F.
      • Moate P.J.
      • Deighton M.H.
      • Williams S.R.
      • Kindermann M.
      • Duval S.
      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      ;
      • Lopes J.C.
      • de Matos L.F.
      • Harper M.T.
      • Giallongo F.
      • Oh J.
      • Gruen D.
      • Ono S.
      • Kindermann M.
      • Duval S.
      • Hristov A.N.
      Effect of 3-nitrooxypropanol on methane and hydrogen emissions, methane isotopic signature, and ruminal fermentation in dairy cows.
      ). The observed effectiveness of inhibiting CH4 emission in studies of 3-NOP ranges from 7 to 60% (
      • Haisan J.
      • Sun Y.
      • Guan L.L.
      • Beauchemin K.A.
      • Iwaasa A.
      • Duval S.
      • Barreda D.R.
      • Oba M.
      The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation.
      ;
      • Reynolds C.K.
      • Humphries D.J.
      • Kirton P.
      • Kindermann M.
      • Duval S.
      • Steinberg W.
      Effects of 3-nitrooxypropanol on methane emission, digestion, and energy and nitrogen balance of lactating dairy cows.
      ), but in most studies, 3-NOP reduces CH4 emission by an average of 30% in cases where it is provided to cows by way of mixing with TMR for ad libitum feeding (
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.W.
      • Harper M.T.
      • Weeks H.L.
      • Branco A.F.
      • Moate P.J.
      • Deighton M.H.
      • Williams S.R.
      • Kindermann M.
      • Duval S.
      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      ;
      • Lopes J.C.
      • de Matos L.F.
      • Harper M.T.
      • Giallongo F.
      • Oh J.
      • Gruen D.
      • Ono S.
      • Kindermann M.
      • Duval S.
      • Hristov A.N.
      Effect of 3-nitrooxypropanol on methane and hydrogen emissions, methane isotopic signature, and ruminal fermentation in dairy cows.
      ). In addition, in 2 in vitro experiments, the use of 3-NOP at a dose similar to that in our study reduced CH4 emission by 70–80% (
      • Romero-Pérez A.
      • Okine E.K.
      • Guan L.L.
      • Duval S.M.
      • Kindermann M.
      • Beauchemin K.A.
      Effects of 3-nitrooxypropanol on methane production using the rumen simulation technique (Rusitec).
      ,
      • Romero-Pérez A.
      • Okine E.K.
      • Guan L.L.
      • Duval S.M.
      • Kindermann M.
      • Beauchemin K.A.
      Rapid communication: Evaluation of methane inhibitor 3-nitrooxypropanol and monensin in a high-grain diet using the rumen simulation technique (Rusitec).
      ). However, different with our study, these 2 in vitro studies used high-grain diets as the fermentation substrates, whereas the forage-based diet was used in our study. The use of different dietary types as fermentation substrates may induce potential differences in the effects on inhibiting CH4 emission. The compound 3-NOP was more effective in CH4 alleviation when it was added to the high-grain diet. For example, the same supplementation of 200 mg of 3-NOP/kg of DM reduced CH4 emission by 38% in beef cattle consuming a forage-based diet and by 84% in beef cattle consuming a high-grain diet (
      • Vyas D.
      • McGinn S.M.
      • Duval S.M.
      • Kindermann M.
      • Beauchemin K.A.
      Effects of sustained reduction of enteric methane emissions with dietary supplementation of 3-nitrooxypropanol on growth performance of growing and finishing beef cattle.
      ). Another reason for the lower efficacy of 3-NOP in inhibiting CH4 emission observed in the current study may be caused by the difference methods of administration of 3-NOP between in vitro addition and directly mixing into cow feed.
      • Ungerfeld E.M.
      Inhibition of rumen methanogenesis and ruminant productivity: A meta-analysis.
      reported that the methods of administration were an important factor affecting the efficacy of 3-NOP in inhibiting CH4 emission. The compound 3-NOP is volatile and metabolized rapidly in rumen fluid due to the increase of temperature after entering the rumen (
      • Romero-Pérez A.
      • Okine E.K.
      • McGinn S.M.
      • Guan L.L.
      • Oba M.
      • Duval S.M.
      • Kindermann M.
      • Beauchemin K.A.
      Sustained reduction in methane production from long-term addition of 3-nitrooxypropanol to a beef cattle diet.
      ;
      • Van Wesemael D.
      • Vandaele L.
      • Ampe B.
      • Cattrysse H.
      • Duval S.
      • Kindermann M.
      • Fievez V.
      • De Campeneere S.
      • Peiren N.
      Reducing enteric methane emissions from dairy cattle: Two ways to supplement 3-nitrooxypropanol.
      ). Mixing 3-NOP into TMR can create synchronization between the gradual entry of 3-NOP into the rumen along with the fermentation of the feed, and this synchrony may elevate the inhibitory potential of 3-NOP on CH4 emission (
      • Romero-Pérez A.
      • Okine E.K.
      • McGinn S.M.
      • Guan L.L.
      • Oba M.
      • Duval S.M.
      • Kindermann M.
      • Beauchemin K.A.
      Sustained reduction in methane production from long-term addition of 3-nitrooxypropanol to a beef cattle diet.
      ). Thus, 3-NOP addition after mixing with feed is a key measure for the intake of 3-NOP to be evenly distributed throughout the day. Similar to the current findings, a study by
      • Reynolds C.K.
      • Humphries D.J.
      • Kirton P.
      • Kindermann M.
      • Duval S.
      • Steinberg W.
      Effects of 3-nitrooxypropanol on methane emission, digestion, and energy and nitrogen balance of lactating dairy cows.
      adding 3-NOP directly to the rumen via a rumen fistula reduced CH4 discharge by 9.8%. To our knowledge, the current study was the first to report the effect of vitamin B12 in suppressing CH4 emission, and the combination of 3-NOP with vitamin B12 elevated the effect of suppression compared with either alone (9 to 12%).

      Effects on Rumen Fluid Incubation

      In the current study, neither 3-NOP nor vitamin B12 affected DMD. This result was similar to the previous studies that supplementation of 3-NOP to cows did not affect DMI (
      • Haisan J.
      • Sun Y.
      • Guan L.L.
      • Beauchemin K.A.
      • Iwaasa A.
      • Duval S.
      • Barreda D.R.
      • Oba M.
      The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation.
      ;
      • Reynolds C.K.
      • Humphries D.J.
      • Kirton P.
      • Kindermann M.
      • Duval S.
      • Steinberg W.
      Effects of 3-nitrooxypropanol on methane emission, digestion, and energy and nitrogen balance of lactating dairy cows.
      ;
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.W.
      • Harper M.T.
      • Weeks H.L.
      • Branco A.F.
      • Moate P.J.
      • Deighton M.H.
      • Williams S.R.
      • Kindermann M.
      • Duval S.
      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      ;
      • Lopes J.C.
      • de Matos L.F.
      • Harper M.T.
      • Giallongo F.
      • Oh J.
      • Gruen D.
      • Ono S.
      • Kindermann M.
      • Duval S.
      • Hristov A.N.
      Effect of 3-nitrooxypropanol on methane and hydrogen emissions, methane isotopic signature, and ruminal fermentation in dairy cows.
      ;
      • Haisan J.
      • Sun Y.
      • Guan L.
      • Beauchemin K.A.
      • Iwaasa A.
      • Duval S.
      • Kindermann M.
      • Barreda D.R.
      • Oba M.
      The effects of feeding 3-nitrooxypropanol at two doses on milk production, rumen fermentation, plasma metabolites, nutrient digestibility, and methane emissions in lactating Holstein cows.
      ). Consistent with previous studies, supplementation with 3-NOP was generally accompanied by an increase in pH and a decrease in total VFA while inhibiting methanogenesis (
      • Guyader J.
      • Ungerfeld E.M.
      • Beauchemin K.A.
      Redirection of metabolic hydrogen by inhibiting methanogenesis in the rumen simulation technique (RUSITEC).
      ;
      • Ungerfeld E.M.
      Inhibition of rumen methanogenesis and ruminant productivity: A meta-analysis.
      ). Meanwhile, as we hypothesized, vitamin B12 had a significant effect on fermentation patterns and methanogenesis, resulting in a decreased acetate to propionate ratio and CH4 production. Vitamin B12 plays an important role in the production of propionate, and the pathway to propionate formation provides an alternative sink for the use of metabolic hydrogen compared with acetate, and an inverse correlation between methanogenesis and propionate formation has been reported (
      • Janssen P.H.
      Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics.
      ). This may explain that the addition of vitamin B12 resulted in a decreased acetate to propionate ratio and lower CH4 production in the current study. Supplementation of 3-NOP also decreased the concentration of acetate, increased that of propionate, and decreased the acetate to propionate ratio. It is worth mentioning that these changes in VFA concentrations were most significant when 3-NOP was combined with vitamin B12. In addition, the decrease of isobutyrate and valerate concentrations was also observed in the 3 treatment groups. Therefore, the decrease of total VFA concentration in the current study may be due to the reduced concentrations of acetate, isobutyrate and valerate. Cows supplemented with 3-NOP typically observed reduced concentration of acetate. This might be related to the accumulation of [H] due to the inhibition of CH4 production, because the increase of [H] concentration in rumen will reduce the production of acetate. (
      • van Lingen H.J.
      • Plugge C.M.
      • Fadel J.G.
      • Kebreab E.
      • Bannink A.
      • Dijkstra J.
      Thermodynamic driving force of hydrogen on rumen microbial metabolism: A theoretical investigation.
      ;
      • Melgar A.
      • Harper M.T.
      • Oh J.
      • Giallongo F.
      • Young M.E.
      • Ott T.L.
      • Duval S.
      • Hristov A.N.
      Effects of 3-nitrooxypropanol on rumen fermentation, lactational performance, and resumption of ovarian cyclicity in dairy cows.
      ). Although the emission of H2 was not measured in this study, multiple previous studies have observed that 3-NOP suppresses CH4 emission while resulting in a dramatic increase in H2 emission. The H2 that is excreted as gas is only a fraction of the excess H2 that is produced as a result of inhibited CH4 synthesis. So, there is still a significant amount of [H] diverted to other metabolic pathways (
      • Hristov A.N.
      • Oh J.
      • Giallongo F.
      • Frederick T.W.
      • Harper M.T.
      • Weeks H.L.
      • Branco A.F.
      • Moate P.J.
      • Deighton M.H.
      • Williams S.R.
      • Kindermann M.
      • Duval S.
      An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production.
      ;
      • van Gastelen S.
      • Dijkstra J.
      • Binnendijk G.
      • Duval S.M.
      • Heck J.M.L.
      • Kindermann M.
      • Zandstra T.
      • Bannink A.
      3-Nitrooxypropanol decreases methane emissions and increases hydrogen emissions of early lactation dairy cows, with associated changes in nutrient digestibility and energy metabolism.
      ;
      • Zhang X.M.
      • Gruninger R.J.
      • Alemu A.W.
      • Wang M.
      • Tan Z.L.
      • Kindermann M.
      • Beauchemin K.A.
      3-Nitrooxypropanol supplementation had little effect on fiber degradation and microbial colonization of forage particles when evaluated using the in situ ruminal incubation technique.
      ;
      • Melgar A.
      • Lage C.F.A.
      • Nedelkov K.
      • Raisanen S.E.
      • Stefenoni H.
      • Fetter M.E.
      • Chen X.
      • Oh J.
      • Duval S.
      • Kindermann M.
      • Walker N.D.
      • Hristov A.N.
      Enteric methane emission, milk production, and composition of dairy cows fed 3-nitrooxypropanol.
      ). Propionate generation is the thermodynamically most favorable pathway in the case of [H] accumulation (
      • Ellis J.L.
      • Dijkstra J.
      • Kebreab E.
      • Bannink A.
      • Odongo N.E.
      • McBride B.W.
      • France J.
      Aspects of rumen microbiology central to mechanistic modelling of methane production in cattle.
      ,
      • Janssen P.H.
      Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics.
      ). When CH4 generation is inhibited, propionate generation becomes an alternative pathway that uses [H] (
      • Mcallister T.A.
      • Newbold C.J.J.A.
      Redirecting rumen fermentation to reduce methanogenesis.
      ), which might explain the increased propionate concentration in the current study. However, the increased production of propionate by 3-NOP does not fully explain the metabolic fate of [H]. Vitamin B12 is an important coenzyme of the propionate production pathway and can become deficient when propionate production increases (
      • Frobish R.A.
      • Davis C.L.J.
      Theory involving propionate and Vitamin B12 in the low-milk fat syndrome.
      ). This may explain the increase in propionate with the addition of vitamin B12 in the current study. Moreover, when 3-NOP was combined with vitamin B12, the magnitude of the increase in the concentration of propionate was greater compared with the addition of 3-NOP alone, which may to some extent alleviate the problem of increased H2 emission caused by 3-NOP addition.

      Effects on Microbial Community

      Bacteroidota and Firmicutes were the dominant phyla in the current study, which was consistent with several previous studies of in vitro fermentation in cows (
      • Wang K.
      • Nan X.
      • Chu K.
      • Tong J.
      • Yang L.
      • Zheng S.
      • Zhao G.
      • Jiang L.
      • Xiong B.
      Shifts of hydrogen metabolism from methanogenesis to propionate production in response to replacement of forage fiber with non-forage fiber sources in diets in vitro.
      ,
      • Wang K.
      • Nan X.M.
      • Zhao Y.G.
      • Tong J.J.
      • Jiang L.S.
      • Xiong B.H.
      Effects of propylene glycol on in vitro ruminal fermentation, methanogenesis, and microbial community structure.
      ;
      • Liu Z.
      • Wang K.
      • Nan X.
      • Cai M.
      • Yang L.
      • Xiong B.
      • Zhao Y.
      Synergistic effects of 3-nitrooxypropanol with fumarate in the regulation of propionate formation and methanogenesis in dairy cows in vitro.
      ). Bacteroidota and Firmicutes are widely recognized as the most abundant and common phyla in rumen (
      • Henderson G.
      • Cox F.
      • Ganesh S.
      • Jonker A.
      • Young W.
      • Janssen P.H.
      Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range.
      ;
      • Sun H.Z.
      • Xue M.
      • Guan L.L.
      • Liu J.
      A collection of rumen bacteriome data from 334 mid-lactation dairy cows.
      ). Firmicutes play an important role in fiber and cellulose breakdown and can produce H2. The main function of Bacteroidota is to degrade proteins and carbohydrates, and it is a utilizer of H2 (
      • Comtet-Marre S.
      • Parisot N.
      • Lepercq P.
      • Chaucheyras-Durand F.
      • Mosoni P.
      • Peyretaillade E.
      • Bayat A.R.
      • Shingfield K.J.
      • Peyret P.
      • Forano E.
      metatranscriptomics reveals the active bacterial and eukaryotic fibrolytic communities in the rumen of dairy cow fed a mixed diet.
      ;
      • Lan W.
      • Yang C.
      Ruminal methane production: Associated microorganisms and the potential of applying hydrogen-utilizing bacteria for mitigation.
      ). Prevotella is generally the predominant bacterial genus commonly found in the rumen and comprises multiple species (
      • Kim M.
      • Morrison M.
      • Yu Z.
      Status of the phylogenetic diversity census of ruminal microbiomes.
      ). Prevotella has a variety of extracellular degrading enzymes that degrade carbohydrates into short chain fatty acid, and different species produce final fermentation products formed differently by extracellular degrading enzymes, with acetate or propionate as the final fermentation product (
      • Stevenson D.M.
      • Weimer P.J.
      Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR.
      ;
      • Emerson E.L.
      • Weimer P.J.
      Fermentation of model hemicelluloses by Prevotella strains and Butyrivibrio fibrisolvens in pure culture and in ruminal enrichment cultures.
      ). Although we observed functional differences among species of Prevotella, propionate and succinate appear to be the major fermentation products of most Prevotella species (
      • de Menezes A.B.
      • Lewis E.
      • O’Donovan M.
      • O’Neill B.F.
      • Clipson N.
      • Doyle E.M.
      Microbiome analysis of dairy cows fed pasture or total mixed ration diets.
      ;
      • De Vadder F.
      • Kovatcheva-Datchary P.
      • Zitoun C.
      • Duchampt A.
      • Bäckhed F.
      • Mithieux G.
      Microbiota-produced succinate improves glucose homeostasis via intestinal gluconeogenesis.
      ). Furthermore, the abundance of some Prevotella species was generally positively associated with the low CH4-producing phenotype and high concentration of vitamin B12 in the rumen (
      • Kittelmann S.
      • Pinares-Patiño C.S.
      • Seedorf H.
      • Kirk M.R.
      • Ganesh S.
      • McEwan J.C.
      • Janssen P.H.
      Two different bacterial community types are linked with the low-methane emission trait in sheep.
      ;
      • Franco-Lopez J.
      • Duplessis M.
      • Bui A.
      • Reymond C.
      • Poisson W.
      • Blais L.
      • Chong J.
      • Gervais R.
      • Rico D.E.
      • Cue R.I.
      • Girard C.L.
      • Ronholm J.
      Correlations between the composition of the bovine microbiota and vitamin B12 abundance.
      ). In the current study, the addition of vitamin B12 increased the relative abundances of Prevotellaceae_UCG-003 and unclassified_f__Prevotellaceae.
      • Martinez-Fernandez G.
      • Denman S.E.
      • Yang C.L.
      • Cheung J.E.
      • Mitsumori M.
      • McSweeney C.S.
      Methane inhibition alters the microbial community, hydrogen flow, and fermentation response in the rumen of cattle.
      showed that Prevotella could increase propionate production when CH4 production was inhibited. Therefore, the increase of propionate concentration and the decrease of CH4 production in the current study were partly related to the change of the relative abundance of Prevotella. Lachnospiraceae_NK3A20_group is a fiber degrading bacterium, whose members can ferment various sugars to produce acetate, H2 and CO2 (
      • Xing D.
      • Ren N.
      • Li Q.
      • Lin M.
      • Wang A.
      • Zhao L.
      Ethanoligenens harbinense gen. nov., sp. nov., isolated from molasses wastewater.
      ). In the current study, vitamin B12 decreased the relative abundance of Lachnospiraceae_NK3A20_group, whereas 3-NOP increased the relative abundance of Lachnospiraceae_NK3A20_group. As mentioned above, many studies have shown that the addition of 3-NOP increased H2 emission. Therefore, the significant increase in the abundance of Lachnospiraceae_NK3A20_group after the addition of 3-NOP may lead to increased H2 production. In contrast, vitamin B12 supplementation may inhibit fiber fermentation and reduce acetate production by reducing the relative abundance of Lachnospiraceae_NK3A20_group. Christensenellaceae_R-7_ group is a member of Firmicutes, which is usually associated with good health and better digestive system function (
      • Morotomi M.
      • Nagai F.
      • Watanabe Y.
      Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposal of Christensenellaceae fam. nov.
      ;
      • Goodrich J.K.
      • Waters J.L.
      • Poole A.C.
      • Sutter J.L.
      • Koren O.
      • Blekhman R.
      • Beaumont M.
      • Van Treuren W.
      • Knight R.
      • Bell J.T.
      • Spector T.D.
      • Clark A.G.
      • Ley R.E.
      Human genetics shape the gut microbiome.
      ;
      • Chen R.
      • Li Z.
      • Feng J.
      • Zhao L.
      • Yu J.
      Effects of digestate recirculation ratios on biogas production and methane yield of continuous dry anaerobic digestion.
      ). The multiple metabolites secreted by the Christensenellaceae_R-7_ group are related to feed efficiency and may play an important role in promoting digestive tract health (
      • Xie J.
      • Yu R.
      • Qi J.
      • Zhang G.
      • Peng X.
      • Luo J.
      Pectin and inulin stimulated the mucus formation at a similar level: An omics-based comparative analysis.
      ). Several previous studies have shown that the abundance of the Christensenellaceae_R-7_ group appears reduced in individuals with intestinal inflammation (
      • Goodrich J.K.
      • Waters J.L.
      • Poole A.C.
      • Sutter J.L.
      • Koren O.
      • Blekhman R.
      • Beaumont M.
      • Van Treuren W.
      • Knight R.
      • Bell J.T.
      • Spector T.D.
      • Clark A.G.
      • Ley R.E.
      Human genetics shape the gut microbiome.
      ), and higher abundance of the Christensenellaceae_R-7_ group in healthy individuals compared with individuals with intestinal disease was observed (
      • Pittayanon R.
      • Lau J.T.
      • Leontiadis G.I.
      • Tse F.
      • Yuan Y.
      • Surette M.
      • Moayyedi P.
      Differences in gut microbiota in patients with vs without inflammatory bowel diseases: A systematic review.
      ). In the current study, 3-NOP addition significantly increased the relative abundance of Christensenellaceae_R-7_group, indicating that 3-NOP addition may also play a beneficial role in the health of the digestive tract of dairy cows.
      Archaea uses H2 to produce CH4 in the rumen to maintain a lower H2 partial pressure. This low H2 partial pressure rumen environment is conducive to rumen microorganisms fermenting digestible fiber in the feed (
      • Ferry J.G.
      Physiological ecology of methanogens.
      ;
      • Zhou M.
      • Hernandez-Sanabria E.
      • Guan L.L.
      Characterization of variation in rumen methanogenic communities under different dietary and host feed efficiency conditions, as determined by PCR-denaturing gradient gel electrophoresis analysis.
      ). Euryarchaeota is the dominant archaea phylum and the only methanogenic microorganisms currently known in the rumen, which contains many different species (
      • Hook S.E.
      • Wright A.D.
      • McBride B.W.
      Methanogens: Methane producers of the rumen and mitigation strategies.
      ). In the current study, 98.5% of the total sequences of the archaeal community belonged to this dominant phylum. The species of methanogens are mainly divided into hydrogen-trophic, acetate-trophic and methyl-trophic types based on the difference of methanogenic substrates (H2, acetate, formate, and methylamine, respectively;
      • Lan W.
      • Yang C.
      Ruminal methane production: Associated microorganisms and the potential of applying hydrogen-utilizing bacteria for mitigation.
      ). At the genus level, similar to previous studies, Methanobacter was the dominant genus and was the most common methanogen (
      • Zhou M.
      • Hernandez-Sanabria E.
      • Guan L.L.
      Assessment of the microbial ecology of ruminal methanogens in cattle with different feed efficiencies.
      ;
      • Weimar M.R.
      • Cheung J.
      • Dey D.
      • McSweeney C.
      • Morrison M.
      • Kobayashi Y.
      • Whitman W.B.
      • Carbone V.
      • Schofield L.R.
      • Ronimus R.S.
      • Cook G.M.
      Development of multiwell-plate methods using pure cultures of methanogens to identify new inhibitors for suppressing ruminant methane emissions.
      ). At the phylum and genus level, we observed no effect of treatment on the archaeal community. Methane production may be related to the abundance of specific methanogens species rather than the total number of methanogens.
      • Shi W.
      • Moon C.D.
      • Leahy S.C.
      • Kang D.
      • Froula J.
      • Kittelmann S.
      • Fan C.
      • Deutsch S.
      • Gagic D.
      • Seedorf H.
      • Kelly W.J.
      • Atua R.
      • Sang C.
      • Soni P.
      • Li D.
      • Pinares-Patino C.S.
      • McEwan J.C.
      • Janssen P.H.
      • Chen F.
      • Visel A.
      • Wang Z.
      • Attwood G.T.
      • Rubin E.M.
      Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome.
      reported that if the total number of methanogens in the rumen did not differ, the composition of the methanogenic community may be an important factor in determining CH4 production. A similar conclusion was made in a study of the influence of nonforage fiber on rumen microbes in cows, where changes in archaeal community structure contributed to the reduction in CH4 production (
      • Wang K.
      • Nan X.
      • Chu K.
      • Tong J.
      • Yang L.
      • Zheng S.
      • Zhao G.
      • Jiang L.
      • Xiong B.
      Shifts of hydrogen metabolism from methanogenesis to propionate production in response to replacement of forage fiber with non-forage fiber sources in diets in vitro.
      ). At the species and OTU levels, 3-NOP + vitamin B12 increased the relative abundances of Methanobrevibacter_ sp._ Abm4, OTU1125, and OTU95 and decreased the relative abundances of uncultured_methanogenic_archaeon_g__Methanobrevibacter, OTU1147, OTU1056, and OTU55. Methanobrevibacter_ sp._ Abm4 is both a hydrogen-trophic and methyl-trophic methanogen (
      • Leahy S.C.
      • Kelly W.J.
      • Li D.
      • Li Y.
      • Altermann E.
      • Lambie S.C.
      • Cox F.
      • Attwood G.T.
      The complete genome sequence of Methanobrevibacter sp. AbM4.
      ). Methanobrevibacter_ sp._ Abm4 encodes only a few sticky proteins and can synthesize coenzyme M on its own. So, the external environment has little influence on its growth (
      • Kumar S.
      • Choudhury P.K.
      • Carro M.D.
      • Griffith G.W.
      • Dagar S.S.
      • Puniya M.
      • Calabro S.
      • Ravella S.R.
      • Dhewa T.
      • Upadhyay R.C.
      • Sirohi S.K.
      • Kundu S.S.
      • Wanapat M.
      • Puniya A.K.
      New aspects and strategies for methane mitigation from ruminants.
      ). The final step of all CH4 production reactions is catalyzed by coenzyme M (
      • Ermler U.
      • Grabarse W.
      • Shima S.
      • Goubeaud M.
      • Thauer R.K.
      Crystal structure of methyl-coenzyme M reductase: The key enzyme of biological methane formation.
      ). The molecular structure of 3-NOP is similar to coenzyme M, which can inactivate methyl coenzyme M reductase, block the combination of coenzyme M and coenzyme B, and inhibit the production of CH4 (
      • Duin E.C.
      • Wagner T.
      • Shima S.
      • Prakash D.
      • Cronin B.
      • Yanez-Ruiz D.R.
      • Duval S.
      • Rumbeli R.
      • Stemmler R.T.
      • Thauer R.K.
      • Kindermann M.
      Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol.
      ). Thus, the abundance of Methanobrevibacter_ sp._ Abm4 increased upon inhibition of CH4 production by 3-NOP. In addition, the methanogenic efficiency of different methanogens species is considered to be a more important factor than methanogens abundance in affecting CH4 production (
      • Shi W.
      • Moon C.D.
      • Leahy S.C.
      • Kang D.
      • Froula J.
      • Kittelmann S.
      • Fan C.
      • Deutsch S.
      • Gagic D.
      • Seedorf H.
      • Kelly W.J.
      • Atua R.
      • Sang C.
      • Soni P.
      • Li D.
      • Pinares-Patino C.S.
      • McEwan J.C.
      • Janssen P.H.
      • Chen F.
      • Visel A.
      • Wang Z.
      • Attwood G.T.
      • Rubin E.M.
      Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome.
      ). For example, Methanobrevibacter can use 1 mol of CO2 to produce 1 mol of CH4 (
      • Hook S.E.
      • Wright A.D.
      • McBride B.W.
      Methanogens: Methane producers of the rumen and mitigation strategies.
      ), whereas Methanosphaera consumes 4 moles of methanol to produce 3 moles of CH4 (
      • Fricke W.F.
      • Seedorf H.
      • Henne A.
      • Krüer M.
      • Liesegang H.
      • Hedderich R.
      • Gottschalk G.
      • Thauer R.K.
      The genome sequence of Methanosphaera stadtmanae reveals why this human intestinal archaeon is restricted to methanol and H2 for methane formation and ATP synthesis.
      ). Therefore, it is possible that the addition of 3-NOP and vitamin B12 may have altered the archaeal community structure by altering the abundance of methanogens, thereby inhibiting CH4 production.

      CONCLUSIONS

      In the current study, we studied the effects of 3-NOP and vitamin B12 on VFA, CH4 production, and microbial community structure of dairy cows by in vitro rumen fermentation. The combined addition of 3-NOP and vitamin B12 significantly decreased CH4 emission, increased propionate concentration, decreased acetate to propionate ratio, and changed rumen fermentation pattern. Moreover, compared with the addition of 3-NOP alone, the addition of 3-NOP in combination with vitamin B12 further enhanced the suppression effect on CH4 emission, with more [H] being transferred to the production of propionate. By analyzing the changes of bacterial and archaeal communities in the rumen, we demonstrated the effects of supplementation with 3-NOP + vitamin B12 on rumen fermentation and CH4 production, providing a basis for the application of 3-NOP + vitamin B12. In conclusion, 3-NOP added in combination with vitamin B12 may be a meaningful strategy to suppress CH4 emission in rumen. However, further studies are still needed to evaluate whether the same effect could be achieved in vivo in a longer period.

      ACKNOWLEDGMENTS

      This research was funded by National Key R&D Program of China (2019YFE0125600; Beijing) and National Key R&D Program of China (2021YFE2000804). The authors have not stated any conflicts of interest.

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