Animals and diets
Four non-lactating, rumen-fistulated Holstein cows were used as rumen fluid donors in Experiments 1 and 2. The average body weight was 748 ± 59 kg. Throughout the trials, the cows had free access to water and were fed twice a day (0800 and 1900 h). The diet was composed of 31.0% Chinese wildrye, 27.0% silage corn, 8.0% soybean hull, and 34.0% concentrate.
Eight mid-lactating, rumen-fistulated Holstein cows were used in Experiment 3. The study was conducted from early April to early May 2019 at an experimental farm near Nanjing, China. All the cows were housed in individual tie-stalls during the 4-week trial period. The cows were 186 ± 16 day in milk, with a mean milk yield of 17.2 ± 2.42 kg/d, and the average body weight was 617 ± 45 kg at the beginning of the experiment. The diet was formulated to satisfy the energy requirement of a 620 kg Holstein cow yielding 25 kg/d of milk containing 4.0% milk fat and 3.5% milk protein according to NRC (2001). Approximately 5% feed residual was allowed to minimize sorting. The cows were fed twice in 24 h, at 0800 and 1900 h, and milked twice daily. All the cows had free access to fresh water throughout the experiment.
Experimental design and sampling
Two in vitro and one in vivo experiment were conducted. In Experiment 1, the effect of methanogen activity inhibition on the TMA utilization by rumen microbiota was investigated by rumen in vitro fermentation over 48 h. In Experiment 2, the metabolic pathway from choline to TMA to methane was investigated by rumen in vitro fermentation over 72 h. In Experiment 3, the effect of supplementary choline chloride on the TMA concentration in the rumen fluid and TMAO concentration in the blood and milk of dairy cows was studied over 4 weeks.
Experiment 1. Four incubation runs were carried out with 4 bottles per treatment. The treatments were as follows: control (no additive treatment), nitroglycerin (NG, 300 μmol/L), TMA (0.69 mg/mL), and NG (300 μmol/L) + TMA (0.69 mg/mL). NG is an inhibitor of rumen methanogens . It was purchased from Beijing Yimin Pharmaceutical Co., Ltd. (Beijing, China). TMA was supplemented in the form of trimethylamine hydrochloride (Sigma-Aldrich, St. Louis, USA). The fermentation substrate was rice straw, which was milled to 1 mm before being weighed into the bottles. The experimental procedure was based on that reported by Martínez-Fernández et al. . The substrate (600 mg, dry matter basis) was weighed into 100 mL serum bottles. Ruminal fluid was obtained from the four cows before the morning feeding, pooled, and strained through four layers of cheesecloth into an insulated flask under anaerobic conditions. The filtered rumen fluid was mixed with the buffer solution devised by Menke and Steingass  at a ratio of 1:9 (vol:vol) at 39 ℃ under anaerobic conditions, and 60 mL was inoculated in each bottle. NG and TMA were directly added to the bottles before inoculation with the buffered rumen fluid. The bottles were sealed with rubber stoppers and aluminum caps, incubated for 48 h with shaking at 80 rpm at 39 ℃. The supernatant (2 mL) of the fermentation fluid was sampled for TMA measurement at the beginning and end of the fermentation. Gas production (hydrogen and methane) was measured at the end of the fermentation. The pH value was measured by a pH meter (Ecoscan pH 5, Singapore). The bottles were then immersed in an ice bath to stop the fermentation. The content in each bottle was homogenized and sampled for DNA extraction.
Experiment 2. Four incubation runs were carried out with 4 bottles per treatment. The treatments were as follows: control (no additive treatment), NG (300 μmol/L), choline (choline chloride, 1.0 g/L), and NG (300 μmol/L) + choline (choline chloride 1.0 g/L). Choline was added in the form of choline chloride (Shanghai Ryon Biological Technology CO., Ltd, Shanghai, China). The procedure for this experiment was identical to that of Experiment 1. A serum bottle (180 mL) containing 1.0 g of milled rice straw was filled with 100 mL buffered rumen fluid and incubated for 72 h. The gas production (methane and hydrogen) and TMA were measured at 0, 24, 48, and 72 h. The content in each bottle was homogenized and sampled for DNA extraction at the end of the fermentation.
Experiment 3. Eight cows were randomly assigned to one of two groups: control (n = 4) and choline (n = 4). The cows in the choline group were gradually supplemented with choline chloride as follows: 100, 150, 200, and 250 g/(cow·d) in weeks 1, 2, 3, and 4, respectively. Dry matter intake (DMI) was recorded daily. Rumen fluid was sampled from the rumen cannula before morning feeding and 4 h after the morning feeding to measure TMA, pH, and volatile fatty acids (VFAs). The rumen digesta were mixed and homogenized for DNA extraction. Blood samples were collected from the tail vein with evacuated tubes containing sodium heparin before the morning feeding at day 28. Upon collection, the blood samples were placed on ice and then centrifuged at 1125×g for 15 min at room temperature for plasma separation within 30 min. The separated plasma was collected and stored in liquid nitrogen to measure the TMAO. Milk samples were collected and stored in liquid nitrogen at day 28. The milk samples from the two time points were mixed in the ratio of 1:1 before measuring the TMAO.
Gas production was measured by a pressure transducer and a calibrated syringe . Hydrogen and methane were measured by a gas chromatograph (GC) (Agilent 7890B, Agilent, California, USA) according to Jin et al. . The gases were separated on packed GC columns (Porapak Q and MolSieve 5A packings, Agilent, California, USA) at a column temperature of 80 ℃. The injection temperature was 200 ℃. The temperature of the thermal conductivity detector was 200 ℃. The carrier gas was nitrogen.
The VFAs were measured by the aforementioned GC system according to Jin et al. . The samples were separated on a fused silica capillary column (Supelco, Bellefonte, USA) with a column temperature procedure of 110 ℃ for 3 min followed by 110–150 ℃ (40 ℃/min). The injection temperature was 200 ℃. The temperature of the flame ionization detector (FID) was 220 ℃. Nitrogen was used as the carrier gas.
The TMA contents of the rumen fluid and rumen in vitro fermentation fluid were analyzed by the GC system. A sample of 1.0 mL was centrifuged at 15000× g for 5 min at 4 ℃. An aliquot containing 200 μL of supernatant was thoroughly mixed with 50 μL potassium hydroxide (10 mol/L) and then centrifuged at 15000× g for 2 min at 4 ℃. The supernatant was filtered with a 0.22 μm filter. An aliquot of 1 μL filtrate was separated on a WEL-PEG20M capillary column (Welch Technology, Shanghai, China) using a column temperature procedure of 60 ℃ for 3 min followed by 60–150 ℃ at 45 ℃/min and 150 ℃ for 2 min. The injection temperature was 220 ℃. The temperature of the FID was 220 ℃. Nitrogen served as the carrier gas.
The concentrations of the TMAO in the plasma and milk were determined by liquid chromatography–mass spectrometry (LC–MS). The exact molecular weight of the TMAO adduct ion ([M + H]+) is 76.07569 g/mol. The sample (100 μL) was added to a 1.5 mL microcentrifuge tube, following which 200 μL of acetonitrile was added to precipitate the protein. The mixture was mechanically vortexed for 2 min and centrifuged at 14000× g for 10 min at 4 °C. The supernatant was transferred to vials for the LC-MS. Sample supernatant (2 μL) was injected into a U3000 dual-gradient LC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The analytes were separated on a Waters BEH Amide column (100 mm × 2.1 mm, 1.8 μm particle size) at 35 °C. The mobile phase consisted of 90% methanol containing 0.1% acetic acid (solvent A) and water containing 0.1% acetic acid (solvent B) at a flow rate of 0.3 mL/min. The following gradient procedure was utilized: 90% solvent A and 10% solvent B for 0–2 min, followed by a linear gradient to 90% solvent B from 1–15 min, 90% solvent B for 5 min, and the initial conditions from 20–25 min. The compounds were ionized in the electrospray ionization ion source of the MS operated in the positive mode. The ionizing voltage was +3000 V, and the ion source temperature was 350 °C. The sheath gas, auxiliary gas, and capillary temperatures were 30, 10, and 320 °C, respectively. The data were collected by Xcalibur and analyzed using TraceFinder 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA) with the external standard method.
A representative 1 mL of the rumen digestor or rumen in vitro fermentation culture was used to extract microbial DNA. A FastPrep-24 Instrument (MP Biomedicals, South Florida, USA) set at 5 for 2 min and cetyltrimethylammonium bromide was used to break up the microbial cell walls . Further extraction was performed with phenol–chloroform–isopentanol . Finally, the DNA in the solution was precipitated with ethanol, and the pellet was suspended in 50 μL Tris–EDTA buffer. The concentration of the DNA was quantified by a NanoDrop ND-1000 Spectrophotometer (Nyxor Biotech, Paris, France).
Real-time PCR was performed using a QuantStudioTM 7 Flex System Real-Time PCR System (Applied Biosystems, California, USA) with a SYBR® Premix Ex Tag TM kit (TaKaRa, Dalian, China). The 16S rRNA genes of archaea and Mmc were quantified according to Jeyanathan et al. . The primers for the archaea were 915f 5’-AAG AAT TGG CGG GGG AGC AC and 1386r 5’-GCG GTG TGT GCA AGG AGC. The primers for Mmc were 762f 5’-GAC GAA GCC CTG GGT C and 1099r 5’-GAG GGT CTC GTT CGT TAT. The cutC genes were quantified with a pair of primers from Campo et al. . The primer sequences were 389-aa-f 5’-TTY GCI GGI TAY CAR CCN TT-3’ and 492-aa-r 5’-TGN GGR TCI ACY CAI CCC AT-3’. The reaction mixture (20 μL) was composed of 10 μL of SYBR® Premix Ex Tag TM, 0.2 μmol/L of each primer, and 2 μL of the template DNA.
The procedure for real-time PCR amplification of the archaea was initiated at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 59 °C for 30 s, and 72 °C for 30 s. The procedure for Mmc was initiated at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 56 °C for 30 s, and 72 °C for 30 s. The procedure for the cutC genes was initiated at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 30 s. Finally, each of the 3 procedures was followed by a melting curve at 95 °C for 15 s, 95 °C for 1 min, and 95°C for 15 s.
The genomic DNA of the rumen samples was amplified with the three pairs of primers. The amplicons were cloned into Escherichia coli JM109 using the pGEM-T Easy Vector (Promega, Madison, USA). The copy number of each standard plasmid was calculated based on its DNA concentration and molecular weight. The standard plasmids were used to construct the standard curves to estimate the 16S rRNA gene or cutC gene copy numbers. A 10-fold dilution series of the standard plasmids was run with the samples.
Methanogenic community analysis by Illumina MiSeq sequencing
The 16S rRNA genes of the methanogens were amplified according to Cersosimo et al. . The primer sequences were Met86F 5’-GCT CAG TAA CAC GTG G-3’ and Met471R 5’-GWR TTA CCG CGG CKG CTG-3’. The amplicons were sequenced on an Illumina MiSeq PE250 platform (Biozeron, Shanghai, China).
The raw data were processed using the QIIME software package (version 1.9.0) . Sequences were selected such that their lengths exceeded 250 bp, the vague base ‘N’ was absent, and the average base quality score was higher than 25. Open reference operational taxonomic unit picking was performed at a distance of 0.01 using UPARSE . Alpha and beta diversity analyses were conducted at a sequence depth of 20421. Taxonomic assignment was based on the RIM-DB database .
The data from Experiment 1 and the real-time PCR data from Experiment 2 were analyzed by one-way analysis of variance in SPSS (version 20, SPSS, Inc., IBM, Chicago, IL, USA). The DMI data from Experiment 3, and the hydrogen, methane, and TMA data from Experiment 2 were analyzed using a generalized linear model in SPSS. The model included fixed effects for treatment, the week, and their interaction. The data of real-time PCR, TMA, TMAO, pH, and VFAs in Experiment 3 were analyzed using the independent samples t-test procedure of SPSS. The nonparametric test in SPSS was used to compare the relative abundance of the methanogenic populations. Significance was declared at P values < 0.05.