Preparation of hydrogen-rich water

Preparation of 800 ppb HRW: Distilled water (2 L) was added to a negative ion water generator (Model V8, Mrs. Li’s Electrical Appliance Co., Ltd., Zhongshan City) using a measuring cylinder. The apparatus was powered for 0.5 h to produce alkaline hydrogen-rich electrolyzed water. The resulting alkaline electrolyzed water had a pH of 8.69, an ORP of -554 mV, and a hydrogen gas concentration of 0.81 mg/L.

Preparation of 400 ppb: 1 L of HRW at a concentration of 800 ppb was mixed with 1 L of distilled water to obtain 2 L of HRW at a concentration of 400 ppb. The resulting alkaline electrolyzed water had a pH of 7.64, an ORP of -72 mV, and a hydrogen gas concentration of 0.44 mg/L.

Preparation of 200 ppb HRW: 1 L of HRW at a concentration of 400 ppb was mixed with 1 L of distilled water to obtain 2 L of HRW at a concentration of 200 ppb. The resulting alkaline electrolyzed water had a pH of 7.52, an ORP of -14 mV, and a hydrogen gas concentration of 0.22 mg/L.

Rumen fluid collection

Three Jinjiang cattle with permanent ruminal fistula installed (weight = 365.2 ± 27.4 kg) were taken as the rumen fluid donors for rumen content collection. The rumen content was obtained 1 h before morning feeding and then was filtered by four layers of gauze. All three collections from bulls were evenly mixed into a sterile bottle, which was finally used as the rumen fluid (culture medium) for the in vitro test. The rumen fluid pH of three cattle, measured immediately with a Rex PHBJ-260 m upon arrival at the laboratory using a Rex PHBJ-260 pH meter (Shanghai INESA Scientific Instrument Co., Ltd., Shanghai, China), averaged 6.82. The fermentation substrate was the total mixed ration for Jinjiang cattle, the ingredients and nutrient composition of the diet are listed in Table 1.

In vitro cultivation medium and experimental design

Mixing the following reagents in volume as cultivation medium: 520.2 mL of distilled water (treatment group using 200 ppb, 400ppb, and 800ppb HRW), 208.1 mL of buffer solution (4.0 g NH4HCO3 + 35 g NaHCO3 dissolved in distilled water and made up to 1000 mL), 208.1 mL of constant element solution (9.45 g Na2HPO4·12H2O + 6.2 g anhydrous KH2PO4 + 0.6 g MgSO4·7H2O dissolved in distilled water and made up to 1000 mL), 0.1 mL of trace element solution (13.2 g CaCl2·2H2O + 10.0 g MnCl2·4H2O + 1.0 g CoCl2·6H2O + 8.0 g FeCl3·6H2O dissolved in distilled water and made up to 1000 mL), and 62.4 mL of reducing solution (4.0 mL of 1 mol/L NaOH + 625 mg Na2S·9H2O + 625 mg cysteine hydrochloride + 95 mL distilled water), which was bubbled with CO2 until the solution turned colorless from light blue.

The prepared cultivation medium was warmed at 39 ℃. Proportionally prepared fermentation substrate (0.50 g) was placed in a glass bottle with a total volume of 100 mL, and then 40 mL of pre-warmed cultivation medium and 20 mL of rumen fluid were added to the above bottle and CO2 was injected to get rid of oxygen. The bottle was incubated in SHA-B oscillators (Guohua Enterprise, Changzhou, Jiangsu, China) for in vitro gastric fermentation experiments.

The experiment comprised 4 groups, including CON (control), 200 ppb, 400 ppb, and 800ppb HRW, with 10 replicates for each group (5 replicates were stopped at 12 h (hours) and the other 5 at 48 h), and the indicators were strictly measured according to the experimental steps and requirements. Rumen fermentation characteristics were determined at the incubation time of 12 h and 48 h.

Rumen fermentation parameter determination

After 12 h and 48 h incubation, fermented contents were filtered with four layers of gauze to obtain supernatant samples. The pH value was measured by a pH meter (Testo 206-pH1, Desto Instrument Co., LTD, Shenzhen, China). These supernatant samples were stored at -80℃ to a determination of VFA, ammonia nitrogen (NH3–N), MCP, and rumen microorganisms. The NH3-N concentration was determined using the method of phenol-hypochlorite reaction as described in Broderick and Kang [40]. The Folin phenol method based on Lowry’s assay was taken to determine the concentration of microbial crude protein (MCP), as described by Makkar et al. [41]. The VFA measurements were determined according to the method of Qiu et al. [42]: using a gas chromatograph (GC-2014Shimadzu Corporation, Kyoto, Japan) equipped with a 30 m capillary column (Rtx-Wax, 0.25 mm ID × 0.25 μm film, Restek, Evry, France) to determine the contents of acetic acid, propionic acid, iso-butyric acid, butyric acid, iso-valeric acid and valeric acid. The sum of the six VFAs was defined as total VFA (TVFA), and the sum of iso-butyric acid and iso-valeric acid was defined as branched-chain VFA. The peak area method was used for identification and content conversion of each VFA based on relative retention time. The standard curve was prepared under the same conditions using the same method. The non-glucogenic to glucogenic acids ratio (NGR) and fermentation efficiency (FE) were calculated as follows:

$$} = }\left( + }2} \times } + }} \right)}/}\left( + }} \right)$$

$$\eqalign} = & \left( + 1.092 \times + 1.56 \times } \right) \cr & /\left( + + 2 \times } \right) \cr}$$

The culture medium was filtered through gauze, and the filter cake was transferred without damage into a nylon bag, which was then placed in a 65 °C drying oven to determine the solids content and calculate the degradation rate. The 12 h and 48 h solids were dried by reference to the method in GB/T6435-2006.

The in vitro solids degradation rate (V) = (W2-W3) / W1.

W1 = The weight of fermentation substrate (g).

W2 = The total weight of fermentation substrate and nylon bag (g).

W3 = The total weight of fermentation substrate and nylon bag after in vitro fermentation (g).

Net gas production rate and gas production parameters

The gas production was measured after incubating the culture for 3, 6, 9, 12, 18, 24, 27, 30, 36, and 48 h. The culture tubes were quickly removed from incubation and the piston displacement (mL) was immediately recorded. The net gas production for each period was calculated as:

Net gas production (mL) = Gas production at a time point (mL) - Gas production of blank at the same time point (mL).

Methane production (The CH4 production was estimated using the equation described by Moss et al. [43].

CH4 (mmol/L) = 0.45 × C2 − 0.275 × C3 + 0.40 × C4.

Note: C2 = Concentration of acetate (mmol/L), C3 = Concentration of propionate (mmol/L), C4 = Concentration of butyrate (mmol/L).

Bacterial community analysis

A total of twenty microbial community genomic DNA using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) were transported to the Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) or PCR amplification and MiSeq sequencing. 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, Wilmington, USA). 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 GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The amplification reaction system and program were the same as Mao et al. [27] report. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA). Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA1174395).

The raw 16 S rRNA gene sequencing reads were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of < 20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of the overlap region is 0.2. Reads that could not be assembled were discarded; (iii) Samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matching.

Operational taxonomic units (OTUs) with 97% similarity cut-off were clustered using UPARSE (version 7.1, http://drive5.com/uparse/), and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by the RDP Classifier (http://rdp.cme.msu.edu/) against the 16 S rRNA database (e.g. Silva v138) using a confidence threshold of 0.7. Correlations between rumen fermentation characteristics and rumen bacterial community were presented with a heat map, which was performed using SPSS (version 17.0, IBM, Armonk, NY, USA) and Origin (version 2018, Origin Software, Inc., Northampton, Massachusetts, USA).

Statistical analysis

The data analyses were statistically analyzed by one-way ANOVA with SPSS statistical software (Version 17.0, IBM, Armonk, NY, USA). The results are shown as the mean and standard error mean (SEM). Differences among means were determined using Tukey’s multiple range test was done when the interaction was significant. The level of statistical significance was set at P < 0.05.