Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening.

To increase intramuscular fat accumulation, Japanese Black cattle are commonly fed a high-grain diet from 10 to 30 months of age although it can result in the abnormal accumulation of organic acids in the rumen. We explored the effect of long-term high-concentrate diet feeding on ruminal pH and fermentation, and its effect on the rumen bacterial community in Japanese Black beef cattle during a 20-month fattening period. Nine castrated and fistulated Japanese Black beef cattle were housed with free access to food and water throughout the study period (10-30 months of age). The fattening stages included Early, Middle, and Late stages (10-14, 15-22, and 23-30 months of age, respectively). Cattle were fed high-concentrate diets for the experimental cattle during fattening. The body weight of the cattle was 439 ± 7.6, 561 ± 11.6, and 712 ± 18.5 kg (mean ± SE) during the Early, Middle, and Late stages, respectively. Ruminal pH was measured continuously during the final 7 days of each stage, and rumen fluid and blood samples were collected on day 4 (fourth day during the final 7 days of the pH measurements). The 24-h mean ruminal pH during the Late stage was significantly lower than that during the Early stage. Total volatile fatty acid (VFA) during the Late stage was significantly lower than during the Early and Middle stages, but no changes were noted in individual VFA components. The lactic acid concentration during the Late stage was significantly higher than that during the Early and Middle stages. The bacterial richness indices decreased significantly during the Late stage in accordance with the 24-h mean ruminal pH. Among the 35 bacterial operational taxonomic units (OTUs) shared by all samples, the relative abundances of OTU8 (Family Ruminococcaceae) and OTU26 (Genus Butyrivibrio) were positively correlated with the 24-h mean ruminal pH. Total VFA concentration was negatively correlated with OTU167 (Genus Intestinimonas), and lactic acid concentration was correlated positively with OTU167 and OTU238 (Family Lachnospiraceae). These results suggested that long-term high-grain diet feeding gradually lowers ruminal pH and total VFA production during the Late fattening stage. However, the ruminal bacterial community adapted to feeding management and the lower pH during the Late stage by preserving their diversity or altering their richness, composition, and function, to enhance lactic acid production in Japanese Black beef cattle.

Introduction

A high-grain based diet is essential for beef and dairy cattle, to maximize growth, productivity, and high-quality meat or milk. However, highly fermentable carbohydrate feeding can result in the accumulation of organic acids in the rumen, such as volatile fatty acids (VFAs) and lactic acid [1, 2]. As a result, ruminal pH decreases; subacute ruminal acidosis (SARA) and ruminal acidosis (RA) are defined by ruminal pHs values of ≤ 5.6 and below, respectively [2]. The production of organic acids by microbes, and their removal or neutralization by the gastrointestinal tract, constitutes a well-balanced regulatory system in the rumen [1]. The ruminal bacterial community and ruminal pH can adapt to and influence each other [3], and the effects of short- (days) and mid-term (weeks) SARA and RA challenges have been explored previously [3, 4, 5, 6]. In general, the ruminal bacterial community has similar proportions between the phyla Firmicutes and Bacteroidetes under a high-forage diet with higher ruminal pH [3, 7, 8]. However, grain-based SARA challenge induces death or lysis of Gram-negative bacteria, such as Bacteroidetes and Proteobacteria, and eventually the proportion of Firmicutes increases with severely low ruminal pH [3, 7, 8].

Japanese Black cattle are characterized by the ability to deposit a large amount of intramuscular fat [9]. The fattening of Japanese Black cattle typically begins at about 10 months and is completed by about 30 months of age. Although hypovitaminosis A may be related to the occurrence of hepatic disorders, the fattening cattle are generally fed high-grain, low vitamin A-containing diets to induce greater intramuscular fat deposition, leading to highly marbled meat during the fattening period [10]. However, limited information is available on the effects of a rumen environment on Japanese Black cattle fattening, in terms of the ruminal pH, fermentation, and bacterial communities.

Therefore, we explored the effects of the ruminal pH, bacterial community and fermentation characteristics on different ages of Japanese Black beef cattle fed a long-term (20-month) high-grain diet. In addition, these findings increase our understanding of the ruminal pH, fermentation, and bacterial community as an adaptation to long-term high-grain diet, thereby contributing to the understanding of their relationships in Japanese Black beef cattle.

Materials and methods

An experimental protocol was approved by the Iwate University Laboratory Animal Care and Use Committee (A201720; Morioka, Japan), and all animal experiments were conducted following the animal experiment policy of Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries (Hyogo Prefecture, Japan).

Animals and experimental design

A total of nine castrated (at age 5–6 months) and subsequently fistulated (at age 12 months under local anesthesia) Japanese Black beef cattle were housed with free access to food and water throughout the study period (10–30 months of age). The fattening stages included Early, Middle, and Late stages (10–14, 15–22, and 23–30 months of age, respectively) according to general agreement in Japan [10, 11]. The concentrate diet and rice straw were given a calculated amount for daily gain of 0.8 kg/day during the Early stage and ad libitum during the Middle and Late fattening stages (Table 1). The concentrate diet was composed of barely, steam-flaked corn, wheat bran, and soybean meal and contains 71.2% total digestible nutrient (TDN) and 15.7% crude protein (CP), 72.2% TDN and 13.9% CP, and 72.8% TDN and 12.0% CP during the Early, Middle, and Late stage, respectively. Feed refusal rate of concentrate and forage diet were 12.6 and 12.2%, respectively, during the Early stage. The forage-to-concentrate ratio was 26:74, 13:87, and 14:86 during the Early, Middle, and Late stages, respectively. The mean ± SE body weight of the cattle was 335 ± 4.4, 439 ± 7.6, 562 ± 11.6, and 712 ± 18.5 kg on prior to the experiment (10 months of age), and Early (14 months of age), Middle (21 months of age), and Late (29 months of age) fattening stage sampling days, respectively. The forage diet was supplied daily in two equal portions at 0930 and 1530 h, and concentrate diet was supplied 1 h after a forage diet feeding to maximize forage diet intake and to prevent excessive consumption of concentrate diet during the Early stage. Abnormalities of body condition (body temperature, appetite, hydration, and defecation) were observed daily throughout the study period. Daily intake amounts of concentrate and forage were recorded daily during the final 7 days (days 1–7) of the Early, Middle, and Late fattening stages. The body weight, intake amount, and chemical composition of the Early, Middle, and Late fattening stage diets are shown in Table 1. Chemical composition of the diets was analyzed according to the official method analysis of the Association of Official Analytical Chemists (AOAC) that registered in the Official Method Feed Analysis of Japan [12]. The adequacy rate of diet was calculated based on the nutrient requirement of Japanese Feeding Standard for Beef cattle [13].

Table 1

Body weight, dietary composition, and chemical analysis of diets in Japanese Black beef cattle during the Early, Middle, and Late fattening stages.

Items	Stage1			SEM
	Early	Middle	Late
Body weight (kg)	439.1a	561.8b	712.4c	12.6
Daily intake amount2 (kg)
Concentrate3	6.0a	7.6b	6.1a	0.32
Rice straw	2.1a	1.1b	1.0b	0.13
Nutrient adequacy rate4 (%)
DM5	88.7a	96.1a	75.4b	3.48
TDN6	91.2a	102.4a	74.2b	3.85
NDF7	43.9a	36.8b	31.5c	0.54

Mean within a row, different superscripts significantly differ (P < 0.05)

1The age of cattle in the Early, Middle, and Late stages were 14, 21, and 29 months, respectively.

2Organic matter basis

3The concentrate diet composed of barely, steam-flaked corn, wheat bran, and soybean meal and contains 71.2% total digestible nutrient (TDN) and 15.7% crude protein (CP), 72.2% TDN and 13.9% CP, and 72.8% TDN and 12.0% CP during the Early, Middle, and Late stage, respectively.

4Nutrient adequacy rate was based on the nutrient requirement of Japanese Feeding Standard for Beef Cattle [13], with an expected daily weight gain of 0.8, 0.65, and 0.7 kg during the Early, Middle, and Late stages, respectively.

5DM = dry matter

6TDN = total digestible nutrients

7NDF = neutral detergent fiber.

Sampling and measurements

Ruminal pH was measured continuously every 10 minutes during the final 7 days (days 1–7) of the Early, Middle, and Late fattening stages using a radio transmission system (YCOW-S; DKK-TOA, Yamagata, Japan), as described previously [14]. A pH sensor was placed in the ventral sac of the rumen through the rumen fistula. Calibration was performed at standard pH values of 4–7, before and after obtaining data in each fattening stage; no change in pH was observed during calibration. Rumen fluid samples were collected on day 4 (fourth day during the final 7 days of the pH measurements) during the Early, Middle, and Late stages, for analysis of the bacterial community, total VFA, individual VFAs, lactic acid concentration, and lipopolysaccharide (LPS) activity. The collected samples were immediately filtered through two layers of cheesecloth and stored at –80°C until use.

For the VFA analyses, 1 mL of 25% HO3P in 3 N H2SO4 was added to 5 mL of rumen fluid. Total VFA and individual VFAs (i.e., acetic acid, propionic acid, and butyric acid) were separated and quantified by gas chromatography (GC-2014; Shimadzu, Kyoto, Japan) using a packed glass column (Thermon-3000; 3%) with a Shimalite TPA 60–80 mesh support (Shinwa Chemical Industries Ltd., Kyoto, Japan). For lactic acid analyses, fluid samples were centrifuged at 2,000 × g for 15 min at 4°C, and the concentration of lactic acid in the supernatant was determined using a commercially available kit (F-kit [d-lactate/l-lactate]; J.K. International Co., Tokyo, Japan). To measure the NH3-N level, fluid samples were analyzed using the steam distillation method with an NH3-N analyzer (Kjeltec Auto Sampler System 1035 Analyzer; Tecator Inc., Höganäs, Sweden). To measure ruminal LPS activity, rumen fluid samples were centrifuged at 11,000 × g for 15 min at 4°C, and supernatant LPS activity was assayed using a kinetic Limulus amebocyte lysate assay (Pyrochrome with Glucashield; Seikagaku Corporation, Tokyo, Japan). Details of the sample preparation and method validation procedures have been described previously [15].

DNA isolation

Total bacterial DNA was extracted from rumen fluid samples, as described previously [16]. Briefly, samples were incubated with 750 μg/mL lysozyme (Sigma-Aldrich Co., St. Louis, MO, USA) at 37°C for 90 min. This was followed by the addition of 10 μL of purified achromopeptidase (Wako Pure Chemical Industries Ltd., Osaka, Japan) at a concentration of 10,000 U/mL, and the resulting mixture was incubated at 37°C for 30 min. This suspension was treated with 60 μL of 1% sodium dodecyl sulfate and 1 mg/mL proteinase K (Merck Japan Ltd., Tokyo, Japan) and incubated at 55°C for 5 min. Lysate was treated three times with phenol/chloroform/isoamyl alcohol (25:24:1) (Wako Pure Chemical Industries Ltd.) and chloroform (Life Technologies Japan Ltd., Tokyo, Japan). DNA was precipitated with 5 M NaCl and 100% ethanol, followed by centrifugation at 21,900 × g for 15 min at 4°C. The DNA pellet was rinsed with 70% ethanol, dried, and dissolved in Tris-hydrochloride buffer. Purified DNA was quantified using a Biospec-nano spectrophotometer (Shimadzu Biotech, Kyoto, Japan) and stored at –80°C until further analyses.

Library preparation and DNA sequencing

Sequencing libraries preparation was performed according to the Illumina 16S Metagenomic Sequencing Library preparation guide (2013) [17]. Bacterial 16S rRNA gene was amplified using barcoded universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) spanning the V3–V4 hyper variable region [18]. Polymerase chain reaction (PCR) was performed on a 25 μL mixture containing 12.5 μL of 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems Ltd., UK), 5 μL of each primer (1 μM), and 2.5 μL of template DNA (10 ng/μL). The thermal cycling conditions were 95°C for 3 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. Amplicons were purified using AMPure XP beads (Beckman Coulter, High Wycombe, UK) according to the manufacturer’s instructions. Libraries were constructed by ligating sequencing adapters and indices onto purified PCR products using the Nextera XT Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Paired-end sequencing (2 × 150 bp) was conducted on the Illumina MiSeq platform according to standard protocols. The sequence data were deposited into the Sequence Read Archive of the National Center for Biotechnology Information and can be accessed via SRA accession number PRJNA548210 (https://submit.ncbi.nlm.nih.gov/subs/sra/).

Sequencing data analyses

All sequencing reads were processed using the MOTHUR program (version 1.41.1; University of Michigan; http://www.mothur.org/wiki/; [19]), following the standard operating procedure for MiSeq (https://mothur.org/wiki/MiSeq_SOP; [20]) with minor modifications. To obtain a non-redundant set of sequences, unique sequences were identified and aligned against the SILVA reference database (SSURef release 128; [21]); then, candidate sequences were screened and filtered, unique sequences were determined, candidate sequences were pre-clustered to eliminate outliers, chimeras were removed using the “chimera.vsearch” command, and sequence comparisons were performed using the Mothur Ribosomal Database Project (RDP) training set (version 16). Sequences identified as being of eukaryotic origin were removed and a distance matrix was generated from the remaining sequences. Sequences were clustered and classified into operational taxonomic units (OTUs) using a cutoff value of 97% similarity. All samples were standardized by random subsampling (6,741 sequences/sample) using the “sub.sample” command, resulting in the elimination of two samples from the Middle and Late stages, which were then subjected to further analysis. The OTU values and rarefaction curves for each group were analyzed using the “rarefaction.single” command according to the 97% similarity cutoff. The “summary.single” command was used to analyze the OTU, Chao1, abundance-based coverage estimator (ACE) richness indices and Shannon, Simpson, and Heip diversity indices.

Representative sequences for each OTU were determined using the “get.oturep” command, and sequence comparisons were performed using the BLASTn program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against a 16S ribosomal RNA sequence database (Bacteria and Archaea; May 2019). Representative sequences and tabulated raw count data were submitted to the piphillin website (http://piphillin.secondgenome.com/; [22]). For the analysis of functional categories, a sequence identity cutoff of 97% was applied, and metagenomic functions were assigned using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (October 2018).

Blood sampling and plasma metabolite profiles

Blood samples were collected on day 4 (fourth day during the final 7 days of the pH measurements), with rumen fluid samples from the jugular vein collected into 10 mL evacuated serum-separating tubes and tubes containing heparin (BD Vacutainer, Franklin Lakes, NJ, USA). Samples were immediately centrifuged (1,500 × g, 15 min, 4°C) to separate the serum and plasma, and then preserved at –80°C until analyses. Total protein (TP), blood urea nitrogen (BUN), total cholesterol (T-CHO), aspartate transaminase (AST), γ-glutamyl transpeptidase (GGT), and calcium (Ca) were measured using an automated biochemistry analyzer (Accute, Toshiba Ltd., Tokyo, Japan). Concentrations of retinol (vitamin A), α-tocopherol (vitamin E), and β-carotene were analyzed by high-performance liquid chromatography according to Katamoto et al. [23]. The plasma concentration of lipopolysaccharide-binding protein (LBP) was measured using a commercially available kit (HK503; HyCult Biotechnology, Uden, The Netherlands) according to Takemura et al. [24].

Statistical analyses

The normality of the data distribution was assessed using the Shapiro-Wilk test. Significant differences in ruminal pH, duration of time where pH < 5.6 and < 5.8, area under curve (AUC) values for pH < 5.6 and < 5.8, VFAs, lactic acid concentration, LPS activity, and blood metabolites among the Early, Middle, and Late stages were evaluated using paired t-test for normal variables and the Wilcoxon rank sum test for non-normal variables. Significant differences in the relative abundances of bacterial phyla, genera, OTUs, and bacterial richness and diversity indices among the Early, Middle, and Late stages were evaluated using the unpaired t-test for normal variables and Mann–Whitney U test for non-normal variables. Principal component analysis (PCA) plots were constructed using the R package ggbiplot (R software version 3.3.2; R Foundation for Statistical Computing, Vienna, Austria), including the 24-h mean ruminal pH, duration of time where pH < 5.6 and < 5.8, and AUC values for pH < 5.6 and < 5.8, and non-metric multidimensional scaling (NMDS) plots were constructed using the R package ggplot, including the OTUs and KEGG pathway categories. Pearson’s correlation coefficients (r) were calculated between the rumen parameters (24-h mean, minimum, and maximum ruminal pH, duration of time where pH < 5.6 and < 5.8, total VFA and lactic acid concentrations, proportions of individual VFAs, LPS activity, and peripheral blood LBP concentration) and OTUs. A heatmap was constructed using Prism software (version 8.10; GraphPad Software Inc., La Jolla, CA, USA) based on the Pearson correlation data. All numerical data were also analyzed using Prism. A P-value < 0.05 was considered to indicate a significant difference, while P < 0.10 was taken as a trend towards significance.

Results

Body weight and dietary intake

No adverse health condition throughout the study period and effects of ruminal cannulation after surgery were observed for any of the cattle. The body weight of the Japanese Black cattle increased gradually but significantly across the Early, Middle, and Late fattening stages (P < 0.05; Table 1). The concentrate diet intake during the Middle stage was significantly higher than that during the Early and Late stages (P < 0.05), and forage consumption during the Early stage was significantly higher than that during the Middle and Late stages (P < 0.05). Nutrient adequacy rates of dry matter (DM) and TDN, calculated based on the total consumption amounts of concentrate diet and forage, during the Late stage were significantly lower than during the Early and Middle stages (P < 0.05), while that of neutral detergent fiber (NDF) was significantly higher during the Middle stage compared with the Early stage, and during the Late stage versus the Early and Middle stages (P < 0.05; Table 1).

Ruminal pH, VFAs, and blood metabolites

The 24-h ruminal pH data were summarized as minimum, mean, and maximum pH values, duration of time where pH < 5.6 and < 5.8, and AUC values for pH < 5.6 and < 5.8 (Table 2). The minimum and mean ruminal pH during the Late stage were significantly lower than those during the Early stage (P < 0.05). In accordance with the 24-h mean ruminal pH, the duration of time where pH < 5.6 was significantly longer, and the AUC value for pH < 5.6 was significantly higher, during the Late stage than the Early stage (p < 0.05). In addition, the duration of time where pH < 5.8 during the Late stage was also significantly longer than during the Early and Middle stages (P < 0.05). Diurnal changes in the 10-minute mean ruminal pH were shown for the Early, Middle, and Late stages (Fig 1), and a gradual decrease in ruminal pH during the latter fattening stages was seen.

Table 2

The 24-h mean ruminal pH, duration of time, and area under curve (for pH <5.6 and 5.8) in Japanese Black beef cattle during the Early, Middle, and Late fattening stages.

Item	Stage			SEM
	Early	Middle	Late
24-h mean ruminal pH
Minimum	5.43a	5.30ab	4.98b	0.10
Mean	6.22a	6.06ab	5.73b	0.03
Maximum	6.79	6.76	6.69	0.09
Duration of ruminal pH (min/d)
pH <5.6	139a	287a	688b	105
pH <5.8	226a	460ab	802b	110
Area under curve (pH × min/d)
pH <5.6	4.29a	7.68ab	13.7b	5.17
pH <5.8	5.24a	12.7ab	24.0b	1.51

Mean within a row, different superscripts significantly differ (P < 0.05)

Fig 1

Diurnal changes in the 10-minute mean ruminal pH in Japanese Black beef cattle.

Days 1–7 correspond to observations made during the final 7 days of each fattening stage. Arrows indicate the sample collection time (1300 h).

Total VFA concentration during the Late stage was significantly lower than during the Early and Middle stages (P < 0.05; Table 3). The ruminal acetic acid-to-propionic acid ratio during the Early stage was significantly higher than during the Middle stage (P < 0.05). The lactic acid concentration during the Late stage was significantly higher than during the Early and Middle stages (P < 0.05), and that during the Middle stage was significantly lower than during the Early stage (P < 0.05). Ruminal LPS activity during the Early stage was significantly lower than during the Middle and Late stages (P < 0.05; Table 3).

Table 3

Total VFA, individual VFA proportions, acetic acid to propionic acid (A/P) ratio, lactic acid concentrations, and LPS activity in Japanese Black beef cattle during the Early, Middle, and Late fattening stages.

Item	Stage			SEM
	Early	Middle	Late
Total VFA (mmol/dL)	13.1a	12.3a	9.77b	0.72
Acetic acid (%)	62.5	57.1	58.6	1.69
Propionic acid (%)	21.4	27.1	27.1	2.08
Butyric acid (%)	11.9	12.8	11.2	0.92
Other (%)	4.20	3.04	3.08	0.53
A/P ratio	3.06a	2.24b	2.34ab	0.24
Lactic acid (mmol/dL)	0.75a	0.28b	1.57c	0.10
LPS (×104 EU/mL)	1.34a	4.29b	6.62b	1.49

Mean within a row, different superscripts significantly differ (P < 0.05)

Serum AST activity during the Late stage was significantly higher than during the Early and Middle stages (P < 0.05; Table 4). During the Late fattening stage, the vitamin A level was significantly higher (P < 0.05), and β-carotene and vitamin E levels were significantly lower (P < 0.05) than during the Early and Middle stages.

Table 4

Peripheral blood metabolite analysis in Japanese Black beef cattle during the Early, Middle, and Late fattening stages.

Item1	Stage			SEM
	Early	Middle	Late
TP (g/dL)	6.68	6.89	6.96	0.18
BUN (mg/dL)	12.1	14.5	11.3	0.98
TCHO (mg/dL)	106	109	93.0	7.31
AST (IU/L)	69.2a	71.3a	100b	10.1
GGT (IU/L)	27.7	26.9	25.5	3.18
Ca (mg/dL)	9.97	9.84	9.72	0.12
Vitamin A (IU/dL)	40.2a	35.5a	62.4b	3.67
β-carotene (μg/dL)	0.48a	0.33a	0.11b	0.06
Vitamin E (μg/dL)	122a	171a	92.6b	12.5
LBP (ng/mL)	283	493	389	67.3

Mean within a row, different superscripts differ (P < 0.05)

1TP = total protein; BUN = blood urea nitrogen; TCHO = total cholesterol; AST = aspartate transaminase; GGT = γ-glutamyl transpeptidase; Ca = calcium; LBP = lipopolysaccharide binding protein.

Principal component analyses of rumen parameters and peripheral blood metabolites

The 24-h mean and minimum ruminal pH were the dominant factors influencing ruminal pH parameters in the Early stage, and the duration of time where pH < 5.6 and 5.8, and AUC values for pH < 5.6 and 5.8, were the most influential variables during the Late stage (principal components 1 + 2, explaining 82.2% of the variance; Fig 2A). The proportions of acetic and butyric acids were the most dominant factors influencing rumen fermentation parameters in the Early stage, and lactic acid concentration and ruminal LPS activity were the most influential factors during the Late stage (principal components 1 + 2, explaining 57.7% of the variance; Fig 2B). In the peripheral blood metabolites, PCA plots showed that the Early and Middle stage were most influenced by T-CHO, vitamin E, β-carotene, BUN, GGT, and Ca, and the Late stage was most affected by vitamin A and AST (principal components 1 + 2, explaining 50.7% of the variance; Fig 2C).

Fig 2

Principal component analysis (PCA) plots for Japanese Black beef cattle.

PCA plots were generated for ruminal pH parameters (A), rumen fermentation parameters (B), and peripheral blood metabolites (C). PC1 and PC2 represent principal components 1 and 2, respectively.

Bacterial richness and diversity analysis

Bacterial richness indices (OTU, Chao1, and ACE) showed a gradual decrease from the Early to Late stages, and bacterial richness during the Late stage was significantly lower than during the Early stage (P < 0.05; Fig 3). However, bacterial diversity indices (Shannon, Simpson, and Heip) did not differ among the Early, Middle, and Late stages.

Fig 3

Column scatter plots of bacterial richness and diversity indices.

The Mothur program (version 1.41.1; University of Michigan; http://www.mothur.org/wiki/; Schloss et al., 2009) was used to analyze the bacterial richness (operational taxonomic unit; OTU, Chao1, and abundance-based coverage estimator; ACE) and diversity (Shannon, Simpson, and Heip) indices. *significant difference at P < 0.05.

Non-metric multidimensional scaling analyses of OTUs and KEGG pathway categories

The NMDS plots of OTUs during the Early stage showed a relatively clustered appearance compared with those during the Middle and Late stages, while those of OTUs during the Middle stage showed the most dispersed distribution (Fig 4A). Similarly, the NMDS plots of KEGG pathway categories during the Middle stage also showed a more scattered distribution compared with the Early and Late stage plots (Fig 4B). The stress of NMDS analysis was 0.10 for the OTU-based ordination and 0.14 for the KEGG pathway-based ordination.

Fig 4

Non-metric multidimensional scaling (NMDS) plots for Japanese Black beef cattle.

NMDS plots were generated for the bacterial OTUs (A) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway categories (B). The stress of NMDS analysis was 0.10 and 0.14 for the OTU- and KEGG pathway-based ordinations, respectively.

Relative abundances of core bacterial OTUs

OTU1 (unclassified Ruminococcaceae) and OTU2 (unclassified Lachnospiraceae) were the most abundant (% of total sequence data) OTUs in the bacterial community. The relative abundances of OTU5 (unclassified Firmicutes), OTU8 (unclassified Ruminococcaceae), OTU26 (Butyrivibrio), OTU30 (unclassified Firmicutes), OTU37 (Unclassified Clostridiales Incertae Sedis XIII), OTU110 (Unclassified Clostridiales), OTU189 (Unclassified Firmicutes), and OTU238 (Unclassified Lachnospiraceae) during the Early stage were significantly higher (P < 0.05), and those of OTU43 (unclassified Lachnospiraceae), OTU55 (Unclassified Firmicutes), and OTU62 (Ruminococcus) during the Early stage were significantly lower (P < 0.05), than those during the Middle stage (Table 5). The relative abundances of OTU26, OTU30, OTU37, OTU43, and OTU199 (unclassified Lachnospiraceae) during the Early stage were significantly higher (P < 0.05), and those of OTU55 and OTU167 (Intestinimonas) during the Early stage were significantly lower (P < 0.05), than during the Late stage. The relative abundances of OTU30, OTU167, and OTU238 during the Middle stage were significantly lower (P < 0.05), and those of OTU62 and OTU199 during the Middle stage was significantly higher (P < 0.05), than during the Late stage.

Table 5

Relative abundances and taxonomic classification of core operational taxonomic units (OTU; shared by all samples) in Japanese Black beef cattle during the Early, Middle, and Late fattening stages.

OTU	Stage			SEM
	Early	Middle	Late		RDP1 classification (genus level)	BLASTn2 classification	Percent to BLASTn identity3	Accession no.
OTU1	11.7	6.73	6.84	3.05	Unclassified Ruminococcaceae	Ruminococcus bromii strain ATCC 27255	97.8	NR_025930.1
OTU2	6.85	6.47	6.46	2.31	Unclassified Lachnospiraceae	Faecalimonas umbilicata strain EGH7	95.2	NR_156907.1
OTU4	2.89	3.46	2.86	1.42	Succiniclasticum	Succiniclasticum ruminis strain SE10	95.9	NR_026205.1
OTU5	5.14a	1.85b	2.14ab	0.77	Unclassified Firmicutes	Thermotalea metallivorans strain B2-1	88.6	NR_044503.1
OTU6	2.31	3.16	1.40	0.84	Prevotella	Prevotella ruminicola strain Bryant 23	98.9	NR_102887.1
OTU8	4.62a	1.37b	1.91b	0.45	Unclassified Ruminococcaceae	Pseudoflavonifractor phocaeensis strain Marseille-P3064	92.2	NR_147370.1
OTU10	2.10	2.04	0.80	0.57	Unclassified Ruminococcaceae	Ruminococcus bromii strain ATCC 27255	95.6	NR_025930.1
OTU13	1.66	2.01	1.48	0.42	Mogibacterium	Mogibacterium neglectum strain P9a-h	94.8	NR_027203.1
OTU15	1.77	0.69	2.26	0.66	Unclassified Ruminococcaceae	Ruminococcus bromii strain ATCC 27255	94.5	NR_025930.1
OTU24	0.98	0.53	0.72	0.27	Unclassified Lachnospiraceae	Faecalicatena orotica strain JCM 1429	93.3	NR_114392.1
OTU26	0.97a	0.17b	0.20b	0.14	Butyrivibrio	Butyrivibrio proteoclasticus strain B316	99.3	NR_102893.1
OTU30	1.13a	0.13b	0.35c	0.11	Unclassified Firmicutes	Novibacillus thermophilus strain SG-1	87.4	NR_136797.1
OTU34	0.65	0.28	0.71	0.17	Unclassified Firmicutes	Monoglobus pectinilyticus strain 14	90.0	NR_159227.1
OTU35	0.56	0.62	0.77	0.29	Olsenella	Olsenella profusa DSM 13989	98.5	NR_116938.1
OTU37	0.70a	0.14b	0.34b	0.10	Unclassified Clostridiales Incertae Sedis XIII	Emergencia timonensis strain SN18	93.3	NR_144737.1
OTU43	0.38a	0.48b	0.20b	0.13	Unclassified Lachnospiraceae	Acetatifactor muris strain CT-m2	91.5	NR_117905.1
OTU54	0.26	0.38	0.19	0.10	Schwartzia	Schwartzia succinivorans strain S1-1	99.3	NR_029325.1
OTU55	0.12a	0.59b	0.57b	0.17	Unclassified Firmicutes	Salinithrix halophila strain R4S8	87.0	NR_134171.1
OTU58	0.20	0.46	0.21	0.12	Unclassified Firmicutes	Gracilibacter thermotolerans JW/YJL-S1	89.6	NR_115693.1
OTU62	0.09a	0.45b	0.12a	0.06	Ruminococcus	Ruminococcus flavefaciens strain C94	98.9	NR_025931.1
OTU64	0.19	0.41	0.36	0.15	Unclassified Planctomycetaceae	Thermostilla marina strain SVX8	84.8	NR_148598.1
OTU68	0.23	0.24	0.89	0.19	Unclassified Lachnospiraceae	Blautia glucerasea strain JCM 17039	93.0	NR_113231.1
OTU79	0.21	0.27	0.30	0.09	Unclassified Clostridiales	Ihubacter massiliensis strain Marseille-P2843	93.7	NR_144749.1
OTU80	0.24	0.28	0.12	0.07	Unclassified Firmicutes	Geosporobacter ferrireducens strain IRF9	88.6	NR_148302.1
OTU86	0.14	0.21	0.23	0.08	Unclassified Clostridiales	Vallitalea pronyensis strain FatNI3	90.4	NR_125677.1
OTU90	0.27	0.13	0.24	0.07	Atopobium	Atopobium parvulum DSM 20469	95.5	NR_102936.1
OTU103	0.14	0.12	0.22	0.07	Unclassified Ruminococcaceae	Sporobacter termitidis strain SYR	93.0	NR_044972.1
OTU110	0.23a	0.04b	0.10ab	0.05	Unclassified Clostridiales	Anaerobacterium chartisolvens strain T-1-35	89.3	NR_125464.1
OTU125	0.09	0.12	0.23	0.05	Unclassified Ruminococcaceae	Ruminococcus flavefaciens strain C94	94.1	NR_025931.1
OTU167	0.08a	0.06a	0.17b	0.02	Intestinimonas	Intestinimonas butyriciproducens strain SRB-521-5-I	96.7	NR_118554.1
OTU183	0.09	0.04	0.07	0.02	Unclassified Clostridiales	Eubacterium nodatum ATCC 33099	91.8	NR_118781.1
OTU184	0.03	0.09	0.08	0.02	Ruminococcus	Ruminococcus albus 7 = DSM 20455	98.5	NR_074399.1
OTU189	0.09a	0.04b	0.09ab	0.02	Unclassified Firmicutes	Caloramator fervidus strain RT4. B1	89.6	NR_025899.1
OTU199	0.07a	0.06a	0.02b	0.01	Unclassified Lachnospiraceae	[Clostridium] aminophilum strain F	93.3	NR_118651.1
OTU238	0.07a	0.02b	0.08a	0.02	Unclassified Lachnospiraceae	Merdimonas faecis strain BR31	91.1	NR_157642.1

Mean within a row, different superscripts significantly differ (P < 0.05)

1Ribosomal Database Project tools training set version 16 in the MiSeq standard operating procedure (MiSeq SOP) in Mothur (https://mothur.org/wiki/MiSeq_SOP; [19])

2Basic Local Alignment Search Tool

3Percent identity

Pearson correlation analyses of rumen parameters and core bacterial OTUs

Among the OTUs that were significantly correlated with rumen fermentation parameters (P < 0.05), the relative abundances of OTU1 (unclassified Ruminococcaceae), OTU8 (unclassified Ruminococcaceae), and OTU26 (Butyrivibrio) were positively correlated with the 24-h mean ruminal pH (r = 0.416, 0.427, and 0.476, respectively) and 24-h minimum ruminal pH (r = 0.458, 0.454, and 0.435, respectively), while they were negatively correlated with the duration of time where pH < 5.6 (r = -0.476, -0.472, and -0.432, respectively) and < 5.8 (r = -0.509, -0.476, and -0.473, respectively) (Fig 5). In contrast, the relative abundance of OTU68 (unclassified Lachnospiraceae) was negatively correlated with the 24-h mean (r = -0.530), minimum (r = -0.531), and maximum (r = -0.499) ruminal pH, and positively correlated with the duration of time where pH < 5.6 (r = 0.453) and < 5.8 (r = 0.417). Total VFA concentration was negatively correlated with OTU167 (r = -0.412, P < 0.05). Lactic acid concentration was positively correlated with OTU34 (unclassified Firmicutes; r = 0.521), OTU68 (r = 0.503), OTU167 (r = 0.556), and OTU238 (r = 0.587) and negatively correlated with OTU6 (r = -0.449; Prevotella) and OTU62 (r = -0.453; all P < 0.05). The ruminal LPS activity was positively correlated with OTU125 (r = 0.519; unclassified Ruminococcaceae) and negatively correlated with OTU5 (r = -0.413), OTU8 (r = -0.440), OTU13 (r = -0.414; Mogibacterium), and OTU37 (r = -0.470; all P < 0.05).

Fig 5

Correlation analyses between the core OTUs (shared by all samples) and rumen parameters.

Cells are colored based on Pearson correlation analyses. Blue represents a negative correlation and red represents a positive correlation. *significant correlation between OTUs and rumen parameters at P < 0.05. Mean = 24-h mean ruminal pH; Minimum = 24-h minimum ruminal pH; Maximum = 24-h maximum ruminal pH; Time pH < 5.6 = duration of time where pH < 5.6; Time pH < 5.8 = duration of time where pH < 5.8; LPS = lipopolysaccharide; LBP = lipopolysaccharide-binding protein.

Discussion

Japanese Black beef cattle are raised on a high-grain diet for about 20 months, i.e., between 10 and 30 months of age, to increase intramuscular fat accumulation (marbling score), and the fattening period consists of three fattening stages. We collected samples over the entire fattening period and explored long-term changes in ruminal pH and fermentation, as well as their consequences with respect to the rumen bacterial community.

The occurrence of SARA may cause various health problems in cattle, such as feed intake depression, reduced fiber digestion, milk fat depression, diarrhea, laminitis, liver abscesses, increased production of bacterial endotoxins, and inflammation [25]. In the present study, however, the cattle showed no clinical sign of abnormal body condition, such as high body temperature, acute feed intake, dehydration, and diarrhea, throughout the study period, and the body weight of them showed a gradual but significant increase across the three fattening stages. Dietary intake amounts were highest during the Middle stage and lowest during the Late stage as an adaptation to long-term high-grain diet feeding or response to significantly lowered ruminal pH during the latter fattening stage. However, growth performance during the Late stage was not impaired, and changes in the 24-h mean ruminal pH were not consistent with dietary intake or rates of DM and TDN. The 24-h mean ruminal pH gradually decreased toward the end of the fattening period. Although the 24-h mean pH value during the Early and Middle stages (6.22 and 6.06, respectively) were similar level compared with the SARA challenge model for 2 days (5.94 and 5.81; [26]), for 1 week (6.10; [3]) and mid-term for 6 weeks (5.97; [4]) in Holstein cattle studies, Japanese Black cattle presented more severe depression of ruminal pH (5.73) during the Late stage in the present study. Furthermore, the total VFA concentration, where VFAs are the most abundant organic acids in the rumen [3], is correlated negatively with ruminal pH (r = -0681, P > 0.05; [27]). However, total VFA concentration did not accord with the changes in ruminal pH parameters (r = 0.258, P > 0.05), and gradually decreased toward the end of the fattening period. The significantly higher lactic acid concentration during the Late stage may play a role in the lowered ruminal pH during the same stage, suggesting that different mechanisms underlie low ruminal pH values occurring in the Late versus Middle and Early stages due to 10 times less protonated lactic acid property than VFA (pKa 4.9 vs. 3.9) [3]. Furthermore, decrease and increase in the proportions of acetic and propionic acids, respectively, were consistent with general feature of high-grain diet feeding in Holstein cattle [3, 4]. The PCA plots showed that lactic acid and LPS were the most influential variables during the Late stage (Fig 3B). The Japanese Black beef cattle suffered from SARA due to higher lactic acid levels during the Late stage, suggestive of a partial transition from VFA production to lactic acid production in the rumen or enhanced absorption of VFA by rumen epithelium transporters (i.e. sodium hydrogen exchanger isoform 3; [28], and monocarboxylate transporter isoform 4; [29]). However, lower ruminal pH during the Late stage did not disrupt the gastrointestinal barrier or induce higher LBP levels in the peripheral blood, despite inducing significantly higher LPS levels in the rumen [30, 31].

In the present study, the cattle were typically healthy, with no recent antibiotic use during fistulation surgery (performed at 12 months of age) and no signs of obvious illness during the experimental period; thus, there was no indication of negative effects of antibiotics or fistulation surgery on the rumen bacterial community. Regarding the relationships of ruminal pH, bacterial diversity and richness indices, correlations of pH parameters with bacterial diversity and richness were generally positive; low ruminal pH leads to low bacterial diversity and richness [3, 4, 6, 7]. In the present study, the decrease in ruminal pH was consistent with the decrease in bacterial richness (OTU, Chao1, and ACE), but not with the Shannon, Simpson, and Heip bacterial diversity indices. The mean bacterial diversity and richness indices during the Early stage were similar to those in a short-term SARA challenge model of Holstein cattle with a similar sampling size (4,623 sequences/sample; [3]). This suggests that ruminal pH during the Early stage was low (SARA “challenge level”) and gradually decreased during the latter fattening period, thus reducing ruminal bacterial richness but not bacterial diversity. To best our knowledge, this is the first study demonstrating the relationship between the long-term high-grain diet feeding and bacterial diversity or richness, and suggests that long-term high-grain diet consumption results in the preservation of bacterial diversity to protect against dysbiosis of the entire rumen bacterial community in Japanese Black beef cattle.

NMDS plots showed the taxonomic and genetic structures of the rumen bacterial communities based on OTUs and KEGG pathway categories. Previously, short-term high-grain diet feeding of Holstein cattle was associated with dispersed principal coordinate analysis plot data, with lower ruminal pH and higher VFA concentrations compared with control diet [26, 32, 33]. In the present study, NMDS plots for the Middle fattening stage showed a scattered appearance for both OTU and KEGG data. Therefore, we suggest that the bacterial community during the Middle stage was in the process of adapting to long-term high-grain diet feeding, before producing more lactic acid in the rumen of Japanese Black beef cattle during the Late fattening stage. Further studies are required to fully explore the relationships of KEGG categories (predicted functional pathway) and ruminal pH or fermentation parameters.

The present study showed that a total of 35 OTUs (core microbiota) were shared by all samples and all fattening stages. In addition, taxonomic classification was performed against the RDP training set; further classification was performed using the GenBank database, to assign OTUs to a specific taxonomic level. In the present study, unclassified Ruminococcaceae, unclassified Lachnospiraceae, and Prevotella were the most abundant genera in Japanese Black cattle, which is not consistent with previous studies showing that the genus Prevotella was generally the most predominant in the rumen bacterial community of Holstein cattle [3, 7, 16, 34]. This is because different breeds of cattle may have different feed passage rate through the digestive tract due to different eating and rumination behaviors [35], and Holstein cows are fed high-grain based diet to maximize productivity, but considering health and reproductivity [36], compared with those fed to maximize productivity and to produce beef with quality [11]. Furthermore, the relative abundances of several OTUs showed significant changes across the fattening stages. For example, changes in the proportion of OTUs were consistent with changes in the ruminal pH and fermentation parameters based on Pearson correlation analysis; the relative abundances of OTU8 (Family Ruminococcaceae) and OTU26 (Genus Butyrivibrio) were positively correlated with the 24-h mean ruminal pH. The unclassified Ruminococcaceae and unclassified Lachnospiraceae have been associated with the maintenance of gut health and play a role as active plant degraders [37], where the Ruminococcus may contribute to starch fermentation [3] and Butytivibrio is dominant in the rumen due to fermentation of a range of substrates [38]. In addition, both OTU167 and OTU238 proportions were positively correlated with the lactic acid concentration. OTU238 (Family Lachnospiraceae; Merdimonas faecis strain BR31) may be associated with the production of lactic acid in the rumen [39], and increased lactic acid concentrations may be exploited by OTU167 (Genus Intestinimonas; Intestinimonas butyriciproducens strain SRB-521-5-I) as its primary energy and carbon source [40]. Collectively, changes in bacterial richness, structure, and composition during the fattening period may represent an adaptation to long-term high-grain diet feeding, and was shown to affect or be affected by changes in ruminal pH and fermentation. Furthermore, the core microbiota in the present study may have arisen as a consequence of adaptation to and endurance of a long-term, harsh ruminal environment, to protect against dysbiosis of the entire rumen bacterial community, resulting in unique and distinct microbiota in Japanese Black beef cattle.

Conclusions

Long-term high-grain feeding (from 10 to 30 months of age) in Japanese Black cattle induced a gradual decrease in ruminal pH and total VFA production during the latter fattening period. In contrast, the lactic acid concentration during the Late stage increased significantly compared with the earlier stages, suggestive of a different underlying mechanism of SARA. Regarding the rumen bacterial composition, the unclassified Ruminococcaceae and unclassified Lachnospiraceae were the most abundant bacterial genera, while family Lachnospiraceae (OTU238) and genus Intestinimonas (OTU167) may be associated with lactic acid production and utilization, respectively, during the Late stage. Taken together, the specialized fattening technique applied herein to Japanese Black beef cattle resulted in unique changes in the rumen fermentation characteristics and bacterial community composition, as adaptations to long-term high-grain diet feeding.

3 Sep 2019

PONE-D-19-20947

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening

PLOS ONE

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Reviewer #1: General comments

The paper intitled ‘Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening’ described rumen characteristics, such as pH, VFA, and lactic acid, blood metabolites, and rumen microbial abundance and diversity of Japanese Black beef cattle during Early, Mid and Late fattening stages.

The manuscript is well-prepared

The novelty about the work lies on the evaluation of rumen characteristics of cattle which are fed high levels of concentrate from 10 to 30 months, and how animals can cope the diet.

The study has one major flaw which should be addressed. According to experimental design described, animals were on considered on Early fattening stage from 10 to 14 months. However, animals were fistulated when they were 12 months old. Thus, considering at least on month for recovery in the best case scenario, and another 3 weeks for diet adaption, different fattening stages should be considered.

Also, minor considerations should be the forage-to-concentrate ratio that was modified throughout the experiment, being 26:74 during Early stage, 13:87 during the Middle stage, and 14:86 during Late stage, which might have affected the rumen environment and pH values. Also, the diet composition should be presented, and the methods used for chemical analysis. Vitamin A concentration should also be included in the Table 1, as you discussed hypovitaminosis in the Introduction section.

Finally, the lack of mechanical lysis of rumen content is troublesome and data interpretation and extrapolation should be made carefully. Several studies have shown that disruption of bacteria with tough cell walls is more efficient with a mechanical approach than by an enzyme-based protocol. Furthermore, extraction methods have important implications on the results, and studies using different extraction procedures should not be compared. For that, the conclusion needs to be re-worked.

Despite these considerations, the paper is well-prepared. Studies exploring the rumen microbiome and metabolic disorders are needed.

If necessary, I would be available to look at the revised version.

Specific comments

Please, verify financial disclosure guidelines and amend if appropriate. According to PLOS ONE guidelines, Funded studies should have statements with the following details: Initials of the authors who received each award; Grant numbers awarded to each author; The full name of each funder; URL of each funder website; and whether the sponsors or funders play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

P02L26: what ‘specialized high-concentrate diets’ means?

P02L29: the term ‘were collected on day 4 of pH measurement’ is confusing.

P02L45-46: I respectfully disagree with the first sentence of the Introduction section, as pasture based diet can also promote growth, productivity, and high-quality meat or milk.

P04L48-50: there are parts of the manuscript which are confusing, such as the phrase ‘As a result, ruminal pH decreases; subacute ruminal acidosis (SARA) and ruminal acidosis (RA) are defined by ruminal pHs of ≤ 5.6 and below, respectively (Nagaraja and Titgemeyer, 2007).’. For example, I would suggest modifying to ‘ruminal pHs values of ≤ 5.6’.

P05L69-71: this phrase should be re-worked as you did not evaluate the ‘effect of ruminal pH, bacterial community and fermentation on the fattening of 10-month-old Japanese Black beef cattle’. Rather, this work characterized ruminal pH, bacterial community and fermentation characteristics on different ages of Japanese Black beef cattle.

P05L71: ‘this findings’ is incorrect.

P06L82: what percentage of refusals?

P06L83: why 10–14, 15–22, and 23–30 months of age were selected as different stages? It seems arbitrary. This should be addressed.

P06L89: the rationale to feed concentrate after 1 hour of forage should be addressed. Also, how concentrate availability was ensured?

P06L93: the term ‘sufficient rate’ is dubious. Use other term.

P06L93: please, refer the Japanese feeding standard.

P08L104: the phrase ‘Rumen fluid samples were collected on day 4 of the pH measurements during the Early…’ is confusing and should be re-worked.

P09L122: there are two references of Kim et al. 2016, which should be differentiated according to PLOS ONE manuscript preparation guidelines.

P09L128: what was the ratio of phenol/chloroform/isoamyl alcohols?

P09L134: authors need to clarify whether pyrosequencing was the used approach. Furthermore, this should be addressed throughout the manuscript.

P11L175: the phrase ‘Rumen fluid samples were collected on day 4 of the pH measurements during the Early…’ is confusing and should be re-worked.

Table 2: please, use ‘ab’ to indicate similarities between stages. For example, the minimum pH values should be presented as ‘5.43a, 5.30ab, and 4.98b’ for Early, Middle, and Late stages, respectively. This should be considered for other tables.

Table 3: data on acetic, propionic and butyric acids should be discussed in the text.

P26L349: the effect of feed intake reduction in the Late stage should be discussed. Did animals decrease feed intake, and reduced concentrate intake by 20% to mitigate health problems?

P27L385: the discussion on reduction of ruminal bacterial richness but not bacterial diversity, should not be limited to one study. Please expand this discussion using other studies that corroborate with you study, other which does not, as it is central to your work. I would also consider bacterial and sequencing limitations, as bead beating was not used.

P28L401-405: the different core microbiota observed in your study compared to the literature was expected. A diet enriched in concentrate was used, having greater levels compared to dairy cattle diets. Furthermore, dairy cattle is anatomically different to beef cattle, which has implication of passage rate for example, and passage rate has huge influence on the rumen microbiota. Finally, generally beef cattle is fed to maximize productivity and to produce beef with quality. On the other hand, dairy cattle are fed to maximize productivity, but considering health and reproductivity. Thus, nutrient requirements and managements are different.

Reviewer #2: Line Comment

29 It this total VFA concentration?

72 Perhaps “understand” could be changed to “the understanding of”.

81, 374 It is noted that the cattle were rumen-fistulated at 12-mnths of age, during the Early stage of the trial. Please speculate whether this would have influenced the results obtained during that phase. It was also noted that fistualtion was said to be done at 10-months of age on line 374. If cattle were fistulated at 10 months, no discussion of affects on the animals is necessary.

85, 88, 90, etc. Suggest using either “roughage” or “forage” for that portion of the diet.

93, Table 1 What is “sufficient rate”? Is this a requirement? If it is a requirement, why are the units in “%”? Are the dairy intakes in kg of DM? Please clarify. Also, please supply a citation for the Japanese feeding standard.

117 Was the supernatant or the pellet analyzed for LPS activity? I ask because the LPS is presumably associated with the microbes and likely would be largely with the 11,000 x g pellet.

213-217 Again, please clarify what is meant by “rates”.

Table 2 Why are there no superscripts on several of the values for the Middle treatment (as is seen in Table 4)? Normally, one would expect that, if those values were not different from Early and/or Late, they would share the superscript with the Early and/or Late. Sometimes the Middle values are intermediate, or even greater than, either Early and Late.

Table 3 Why are there no superscripts for A/P ration for the Late treatment?

383-385 Do you mean to say that the risk of SARA during the Early stage was low, because pH was actually higher during this stage?

Additional comment Normally, one would expect to see a table of diet composition, showing the feedstuffs used to construct the 3 diets fed in this trial. The authors might consider adding a table containing this information to the paper.

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17 Sep 2019

PONE-D-19-20947

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening

Dear Editor and Reviewers

Authors would like to thank Editor and Reviewers for their helpful comments and suggestions. We have done our best to address all the issues raised by Reviewers very carefully in this first revision, which we believe has improved the quality of the paper further. To facilitate the reviewing process, we have highlighted all changes done by Authors (Yellow) in the revised manuscript. We have responded to every comment done by the Reviewers below, and also have indicated the changes made with respective new lines.

Before proceeding the present revision, we would like to declare that we have two corresponding authors who contributed equally to this work. The relevant changes are declared in the cover letter and title page of the manuscript.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments

The paper intitled ‘Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening’ described rumen characteristics, such as pH, VFA, and lactic acid, blood metabolites, and rumen microbial abundance and diversity of Japanese Black beef cattle during Early, Mid and Late fattening stages. The manuscript is well-prepared. The novelty about the work lies on the evaluation of rumen characteristics of cattle which are fed high levels of concentrate from 10 to 30 months, and how animals can cope the diet.

AU: Authors would like to thank Reviewer 1 for your helpful comments and suggestions. We have done our best to address all the issues raised by Reviewer 1 very carefully in this new revision, which we believe has improved the quality of the paper further.

The study has one major flaw which should be addressed. According to experimental design described, animals were on considered on Early fattening stage from 10 to 14 months. However, animals were fistulated when they were 12 months old. Thus, considering at least on month for recovery in the best case scenario, and another 3 weeks for diet adaption, different fattening stages should be considered.

AU: We would like to thank you for your comment. As you mentioned, animals were fistulated when they were 12 months old. After the surgery by skilled veterinarian, calves suffered from temporal loss of appetite, but no longer than 2 or 3 days, and no apparent adverse effects of cannulation were observed as similar to previous report (Kristensen et al., 2010; Technical note: Ruminal cannulation technique in young Holstein calves: Effects of cannulation on feed intake, body weight gain, and ruminal development at six weeks of age) although there is differences in age (6 weeks vs. 12 months of age) and breed (Holstein vs. Japanese Black cattle). Furthermore, no sign of obvious illness and antibiotic use was observed during the experimental period as mentioned in the original manuscript L363-365, which also are added in the Result section L220-221. Therefore, the effect of surgery was minimized in the present study, and no additional dietary adaptation was needed because Early fattening stage diet was fed before 2 months before surgery (10 months of age). Even if considering at least one month for recovery in the best case scenario and another 3 weeks for diet adaption, we considered that calves were fully adapted to Early stage diet during 13 to 14 months of age.

Also, minor considerations should be the forage-to-concentrate ratio that was modified throughout the experiment, being 26:74 during Early stage, 13:87 during the Middle stage, and 14:86 during Late stage, which might have affected the rumen environment and pH values. Also, the diet composition should be presented, and the methods used for chemical analysis. Vitamin A concentration should also be included in the Table 1, as you discussed hypovitaminosis in the Introduction section.

AU: We would like to appreciate for your comment. As you mentioned, the diet composition was modified throughout the experiment period, and it affected the rumen environment and pH values. During the fattening period, dietary modification according to different fattening stage is a common practice in Japanese Black cattle (original manuscript L343-345), and we followed general agreement in Japan (research and commercial farm) as introduced previously (Oka et al., 1998; Ogata et al., 2019). Furthermore, the present experiment presupposed dietary modification during the entire fattening stages to explore long-term changes in ruminal pH and fermentation, as well as their consequences with respect to the rumen bacterial community (original manuscript L345-347). However, we would like to apology for our mistake that the forage-to-concentrate ratio in the original manuscript was based on the intake amount, and it is revised accordingly in the revised manuscript L89-90 as “The forage-to-concentrate ratio was gradually decreased from 38:62 to 22:78, 13:87 to 10:90 and 8:92 to 7:93 during the Early, Middle, and Late stages, respectively”. In addition, chemical composition of the diets was analyzed according to the official method analysis of the Association of Official Analytical Chemists (AOAC) that registered in the Official Method Feed Analysis of Japan (MAFF, 2008) as we mentioned in the revised manuscript L98-101. Regarding the vitamin A concentration in the diet, we agree with your comment that it should be included in the Table 1. However, because vitamin A concentration is not directly related to our experiment, we did not measure dietary vitamin A concentration, and it can be predicted by peripheral blood concentration.

Finally, the lack of mechanical lysis of rumen content is troublesome and data interpretation and extrapolation should be made carefully. Several studies have shown that disruption of bacteria with tough cell walls is more efficient with a mechanical approach than by an enzyme-based protocol. Furthermore, extraction methods have important implications on the results, and studies using different extraction procedures should not be compared. For that, the conclusion needs to be re-worked.

AU: We would like to appreciate for your comment, and partly agree with your comment about mechanical lysis of rumen content. As you mentioned, DNA extraction methods and sampling techniques may affect the rumen microbial community structure (Henderson et al., 2013; Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities). In our study, total bacterial DNA was extracted as described previously Morita et al. (2007; An improved DNA isolation method for metagenomics analysis of the microbial flora of the human intestine) with minor modifications. Morita et al. (2007) reported that the improved DNA extraction method using lysozyme, proteinase K, and achromopeptidase gave stable and high-level lysis (>90%) for all the human fecal samples compared to the reference method (13.3-84.6%) and QIAamp DNA stool mini kit (38.8-69.2%). In addition, our study aimed to explore changes in the rumen fluid bacterial community after filtering through two layers of cheesecloth, and nearly no rumen contents are included in the fluid sample, possibly suggesting mechanical lysis of rumen contents has not much meaning in the present study. Unfortunately, to the best of our knowledge, we could not find studies that used enzymatic DNA extraction method in our field. Therefore, our DNA extraction method may reflect nearly true genomic information in the rumen microbial flora, while there are not many citable references in this kind of study.

Despite these considerations, the paper is well-prepared. Studies exploring the rumen microbiome and metabolic disorders are needed. If necessary, I would be available to look at the revised version.

AU: Authors would like to gratefully appreciate Reviewer 1 for your helpful comments and suggestions. Regarding studies exploring the rumen microbiome and metabolic disorder, a sentence about health problems induced by SARA is added in the revised manuscript L360-362 as “The occurrence of SARA may cause various health problems in cattle, such as feed intake depression, reduced fiber digestion, milk fat depression, diarrhea, laminitis, liver abscesses, increased production of bacterial endotoxins, and inflammation (Plaizier et al., 2008)”.

Specific comments

Please, verify financial disclosure guidelines and amend if appropriate. According to PLOS ONE guidelines, Funded studies should have statements with the following details: Initials of the authors who received each award; Grant numbers awarded to each author; The full name of each funder; URL of each funder website; and whether the sponsors or funders play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AU: We would like to appreciate for your comment. However, we do not have any grant information in this study. Therefore, we did not declare grant information in the manuscript.

P02L26: what ‘specialized high-concentrate diets’ means?

AU: In the present study, high-concentrate diets were not commercially available merchandise, but specially designed for the experimental cattle based on the Japanese Feeding Standard for Beef cattle. The relevant description is revised accordingly as “Specially designed high-concentrate diet” in the revised manuscript L26 and L86.

P02L29: the term ‘were collected on day 4 of pH measurement’ is confusing.

AU: In the present study, ruminal pH was measured during the final 7 days (days 1-7) of each fattening stage as described in the manuscript L101. The sentence “were collected on day 4 of pH measurement” is revised more in detail as “were collected on day 4 (fourth day during the final 7 days of pH measurement) during the ~” in the revised manuscript L29-30, L113-114, and L186-187.

P02L45-46: I respectfully disagree with the first sentence of the Introduction section, as pasture based diet can also promote growth, productivity, and high-quality meat or milk.

AU: We would like to appreciate for your comment on our thought. We completely agree with your comment, and people, including us, easily overlook the importance of forage and the relevant things that the ruminants live on pasture. Recently, however, high-concentrate based diet is well known for high-energy property and used broadly because of a human’s purpose in the world. Therefore, the sentence is revised accordingly as “A high-grain based diet is essential for beef and dairy cattle, to maximize growth, productivity, and high-quality meat or milk.” in the revised manuscript L46-47.

P04L48-50: there are parts of the manuscript which are confusing, such as the phrase ‘As a result, ruminal pH decreases; subacute ruminal acidosis (SARA) and ruminal acidosis (RA) are defined by ruminal pHs of ≤ 5.6 and below, respectively (Nagaraja and Titgemeyer, 2007).’. For example, I would suggest modifying to ‘ruminal pHs values of ≤ 5.6’.

AU: We would like to thank you for your comment. The relevant sentence is revised according to your comment in the revised manuscript L49-51.

P05L69-71: this phrase should be re-worked as you did not evaluate the ‘effect of ruminal pH, bacterial community and fermentation on the fattening of 10-month-old Japanese Black beef cattle’. Rather, this work characterized ruminal pH, bacterial community and fermentation characteristics on different ages of Japanese Black beef cattle.

AU: We would like to appreciate for your comment. The relevant sentence is revised accordingly in the revised manuscript L70-71 as “we explored the effects of the ruminal pH, bacterial community and fermentation characteristics on different ages of Japanese Black beef cattle”.

P05L71: ‘this findings’ is incorrect.

AU: We would like to apology for our careless mistake. This sentence is revised accordingly in the revised manuscript L72 as “these findings”.

P06L82: what percentage of refusals?

AU: We would like to thank you for your comment. During the Early stage, a fixed amount of concentrate and forage diets were offered, and feed refusal rate of concentrate and forage diets were 12.6% and 12.2%, respectively as revised in the revised manuscript L88-89. However, diets were fed ad libitum during the Middle and Late stages, and the percentage of refusal was not accessed during these stages.

P06L83: why 10–14, 15–22, and 23–30 months of age were selected as different stages? It seems arbitrary. This should be addressed.

AU: As we mentioned above, there is general agreement in Japanese Black cattle fattening stage, which included Early, Middle, and Late stages (10–14, 15–22, and 23–30 months of age, respectively). Although specific months of age is not documented, many research groups and commercial farms followed this guideline as reported previously (Oka et al., 1998; Ogata et al., 2019; Maeda et al., 2019; Effect of feeding wood kraft pulp on the growth performance, feeding digestibility, blood components, and rumen fermentation in Japanese Black fattening steers). Therefore, we followed general agreement in Japan as introduced previously (Oka et al., 1998; Ogata et al., 2019), and this information is added in the revised manuscript L84-86 as “The fattening stages included Early, Middle, and Late stages (10–14, 15–22, and 23–30 months of age, respectively) according to general agreement in Japan (Oka et al., 1998; Ogata et al., 2019)”.

P06L89: the rationale to feed concentrate after 1 hour of forage should be addressed. Also, how concentrate availability was ensured?

AU: Generally, cattle consumes concentrate diet first, and then, access to forage diet latter. Under our high-grain diet feeding condition, the cattle may consume excessive amount of concentrate diet and suffer from severe depression of ruminal pH, such as acute ruminal acidosis. Therefore, to prevent possible disorder, we decided to offer forage diet in advance to concentrate diet. The relevant information is added in the revised manuscript L94-96 as “concentrate diet was supplied 1 h after a forage diet feeding to maximize forage diet intake and to prevent excessive consumption of concentrate diet during the Early stage”. In addition, diets were offered individually to each cattle, and daily intake amounts of concentrate and forage were also recorded individually. Therefore, there was no problem in concentrate diet availability during the entire study period.

P06L93: the term ‘sufficient rate’ is dubious. Use other term.

AU: We would like to apology for our dubious term. At first, we intended to describe how much offered feed can cover an expected daily weight gain in the experimental cattle. However, we choose inappropriate term in the relevant description as “sufficient rate”, and thus, we revise this description accordingly as “Nutrient adequacy rate” in the revised Table 1. Furthermore, supplementary description is added in the footnote as “Nutrient adequacy rate was based on the nutrient requirement of Japanese Feeding Standard for Beef Cattle (National Agriculture and Food Research Organization (NARO), 2009), with an expected daily weight gain of 0.8, 0.65, and 0.7 kg during the Early, Middle, and Late stages, respectively”.

P06L93: please, refer the Japanese feeding standard.

AU: We would like to apology for our careless mistake. The reference is added accordingly in the revised manuscript L101-102 as “The adequacy rate of diet was calculated based on the nutrient requirement of Japanese Feeding Standard for Beef cattle (NARO, 2009)”

P08L104: the phrase ‘Rumen fluid samples were collected on day 4 of the pH measurements during the Early…’ is confusing and should be re-worked.

AU: As we mentioned above, this sentence “were collected on day 4 of pH measurement” is also revised more in detail as “were collected on day 4 (fourth day during the final 7 days of pH measurement) during the ~” in the revised manuscript L113-114.

P09L122: there are two references of Kim et al. 2016, which should be differentiated according to PLOS ONE manuscript preparation guidelines.

AU: We would like to apology for our careless mistake. The references are distinguished accordingly (Kim et al., 2016a; Front Microbiol, and Kim et al., 2016b; Physiol Genomics) in the revised manuscript.

P09L128: what was the ratio of phenol/chloroform/isoamyl alcohols?

AU: The ratio of phenol/chloroform/isoamyl alcohols was 25:24:1, and the relevant information is added in the revised manuscript L137-138 as “with phenol/chloroform/isoamyl alcohol (25:24:1) (Wako Pure Chemical Industries Ltd.)”.

P09L134: authors need to clarify whether pyrosequencing was the used approach. Furthermore, this should be addressed throughout the manuscript.

AU: We would like to appreciate for your comment. In the present study, pyrosequencing approach was performed followed by manufacturer’s instruction (16S Metagenomic Sequencing Library Preparation; Preparing 16S Robosimal RNA Gene Amplicons for the Illumina Miseq System). Therefore, the information is added in the revised manuscript L144-145 as “Sequencing libraries preparation was performed according to the Illumina 16S Metagenomic Sequencing Library preparation guide (2013)”.

P11L175: the phrase ‘Rumen fluid samples were collected on day 4 of the pH measurements during the Early…’ is confusing and should be re-worked.

AU: As we mentioned above, this sentence “were collected on day 4 of pH measurement” is also revised more in detail as “were collected on day 4 (fourth day during the final 7 days of pH measurement) during the ~” in the revised manuscript L186-187.

Table 2: please, use ‘ab’ to indicate similarities between stages. For example, the minimum pH values should be presented as ‘5.43a, 5.30ab, and 4.98b’ for Early, Middle, and Late stages, respectively. This should be considered for other tables.

AU: We would like to appreciate for your helpful comment, and to apology for our careless mistake. As you mentioned, superscript ‘ab’ is added throughout the tables.

Table 3: data on acetic, propionic and butyric acids should be discussed in the text.

AU: We would like to thank you for your suggestion. In the present study, the proportions of these acids were not different throughout the fattening period, while they showed general feature of high-grain diet feeding in Holstein cattle as reported elsewhere (Khafipour et al., 2009; Nagata et al., 2018). Therefore, the data on acetic, propionic, and butyric acids are discussed in the Discussion section L383-385 as “Furthermore, decrease and increase in the proportions of acetic and propionic acids, respectively, were consistent with general feature of high-grain diet feeding in Holstein cattle (Khafipour et al., 2009; Nagata et al., 2018)”.

P26L349: the effect of feed intake reduction in the Late stage should be discussed. Did animals decrease feed intake, and reduced concentrate intake by 20% to mitigate health problems?

AU: Throughout the study period, animals did not show any health problems, such as high-temperature, dehydration, and diarrhea. In addition, there was no abnormal or critical (within physiological range) changes in blood metabolites during the fattening stages, and thus, we could not suggest any plausible answer to the reason. In the revised manuscript, these information are clarified in the Result (L220-221) and Discussion (L363-365) sections, and decreased feed intake is discussed in the Discussion (L367-368).

P27L385: the discussion on reduction of ruminal bacterial richness but not bacterial diversity, should not be limited to one study. Please expand this discussion using other studies that corroborate with you study, other which does not, as it is central to your work. I would also consider bacterial and sequencing limitations, as bead beating was not used.

AU: We would like to appreciate for your helpful comment. We also agree with your comment that discussion should be expanded using other studies that corroborate with our study. In the original manuscript of the Discussion section L396-399, we mentioned that “bacterial diversity and richness indices, correlations of pH parameters with bacterial diversity and richness were generally positive; low ruminal pH leads to low bacterial diversity and richness (Khafipour et al., 2009; Mao et al., 2013; Plaizier et al., 2017; Nagata et al., 2018)”. However, to our best knowledge, this is the first study demonstrating the relationship between the long-term high-grain diet feeding and bacterial diversity or richness. Therefore, we clarify this in the revised manuscript L405-407 as “To best our knowledge, this is the first study demonstrating the relationship between the long-term high-grain diet feeding and bacterial diversity or richness, and suggests that ~”.

P28L401-405: the different core microbiota observed in your study compared to the literature was expected. A diet enriched in concentrate was used, having greater levels compared to dairy cattle diets. Furthermore, dairy cattle is anatomically different to beef cattle, which has implication of passage rate for example, and passage rate has huge influence on the rumen microbiota. Finally, generally beef cattle is fed to maximize productivity and to produce beef with quality. On the other hand, dairy cattle are fed to maximize productivity, but considering health and reproductivity. Thus, nutrient requirements and managements are different.

AU: We would like to appreciate for your comment. Regarding the relevant differences in core microbiota in our study to Holstein cattle, we also agree with your comment. Therefore, the manuscript is revised accordingly to include your comment as “~ the genus Prevotella was generally the most predominant in the rumen bacterial community of Holstein cattle (Mao et al., 2013; Golder et al., 2014; Kim et al., 2016a; Nagata et al., 2018). This is because different breeds of cattle may have different feed passage rate through the digestive tract due to different eating and rumination behaviors (Aikman et al., 2008), and Holstein cows are fed high-grain based diet to maximize productivity, but considering health and reproductivity (Roche, 2006), compared with those fed to maximize productivity and to produce beef with quality (Ogata et al., 2019).” in the revised manuscript L425-431.

Reviewer #2: Line Comment

AU: Authors would like to thank Reviewer 2 for your helpful comments and suggestions. We have done our best to address all the issues raised by Reviewer 2 very carefully in this new revision, which we believe has improved the quality of the paper further.

29 It this total VFA concentration?

AU: We would like to apology for our ambiguous description. The description is revised accordingly as “total VFA production” in the revised manuscript L41.

72 Perhaps “understand” could be changed to “the understanding of”.

AU: We would like to appreciate for your suggestion. We revise the expression according to your suggestion as “the understanding of” in the revised manuscript L73-74.

81, 374 It is noted that the cattle were rumen-fistulated at 12-mnths of age, during the Early stage of the trial. Please speculate whether this would have influenced the results obtained during that phase. It was also noted that fistualtion was said to be done at 10-months of age on line 374. If cattle were fistulated at 10 months, no discussion of affects on the animals is necessary.

AU: We would like to thank you for your comment. As you mentioned as a response to Reviewer 1, no apparent adverse effects of cannulation were observed in the present study. After the surgery at 12 months of age by skilled veterinarian, calves suffered from temporal loss of appetite, but no longer than 2 or 3 days, and no apparent adverse effects of cannulation were observed as similar to previous report (Kristensen et al., 2010; Technical note: Ruminal cannulation technique in young Holstein calves: Effects of cannulation on feed intake, body weight gain, and ruminal development at six weeks of age) although there is differences in age (6 weeks vs. 12 months of age) and breed (Holstein vs. Japanese Black cattle). Furthermore, no sign of obvious illness and antibiotic use was observed during the experimental period as mentioned in the original manuscript L363-365, which also are added in the Result section L220-221. Therefore, we believe that the effect of surgery was minimized in the present study.

85, 88, 90, etc. Suggest using either “roughage” or “forage” for that portion of the diet.

AU: We would like to appreciate for your suggestion. “roughage” is revised to “forage” throughout the revised manuscript.

93, Table 1 What is “sufficient rate”? Is this a requirement? If it is a requirement, why are the units in “%”? Are the dairy intakes in kg of DM? Please clarify. Also, please supply a citation for the Japanese feeding standard.

AU: We would like to apology for our dubious term. As we responded to Reviewer 1, we intended to describe how much offered feed can cover an expected daily weight gain in the experimental cattle. However, we choose inappropriate term in the relevant description as “sufficient rate”, and thus, we revise this description accordingly as “Nutrient adequacy rate” in the revised Table 1. Furthermore, supplementary description is added in the footnote as “Nutrient adequacy rate was based on the nutrient requirement of Japanese Feeding Standard for Beef Cattle (NARO, 2009), with an expected daily weight gain of 0.8, 0.65, and 0.7 kg during the Early, Middle, and Late stages, respectively”. Regarding the Japanese Feeding standard, reference is added accordingly in the revised manuscript L101-102 as “The adequacy rate of diet was calculated based on the nutrient requirement of Japanese Feeding Standard for Beef cattle (NARO, 2009).”

117 Was the supernatant or the pellet analyzed for LPS activity? I ask because the LPS is presumably associated with the microbes and likely would be largely with the 11,000 x g pellet.

AU: We would like to thank you for your comment. In the present study, rumen fluid samples were centrifuged and supernatant and pellet were separated. Then, LPS activity was assayed using supernatant to minimize interruption of gram-negative bacterial cell membrane. Therefore, we clarify that supernatant LPS activity was assayed using a kinetic Limulus amebocyte lysate assay as “~ and supernatant LPS activity was assayed using a kinetic Limulus amebocyte lysate assay ~” in the revised manuscript L126-127.

213-217 Again, please clarify what is meant by “rates”.

AU: We would like to apology for our undescriptive description. As we mentioned above, “sufficient rate” is revised to “nutrient adequacy rate” in the Table 1, and thus, the relevant description “rate” is revised in detail as “Nutrient adequacy rates” in the revised manuscript L225.

Table 2 Why are there no superscripts on several of the values for the Middle treatment (as is seen in Table 4)? Normally, one would expect that, if those values were not different from Early and/or Late, they would share the superscript with the Early and/or Late. Sometimes the Middle values are intermediate, or even greater than, either Early and Late.

AU: We would like to appreciate for your helpful comment, and to apology for our careless mistake. As you and Reviewer 1 mentioned, superscript ‘ab’ is added throughout the tables.

Table 3 Why are there no superscripts for A/P ration for the Late treatment?

AU: We would like to appreciate for your helpful comment, and to apology for our careless mistake. As we mentioned above, superscript ‘ab’ is added throughout the tables.

383-385 Do you mean to say that the risk of SARA during the Early stage was low, because pH was actually higher during this stage?

AU: In the present study, ruminal pH during the Early fattening stage was higher than other stages. Although the duration of time where pH < 5.6 was not long to diagnose as SARA during the Early stage, ruminal pH value was already low to induce low bacterial diversity from the beginning of the fattening stage when compared with previous study (Nagata et al., 2018). Also, the depression was getting more severe during the latter period, resulting in reduced rumen bacterial richness but not bacterial diversity. Therefore, our conclusion was that the risk of SARA during the Early stage was “high”, because pH was actually “low” during the Early stage although we did not diagnose SARA based on the duration of time in the present study.

Additional comment

Normally, one would expect to see a table of diet composition, showing the feedstuffs used to construct the 3 diets fed in this trial. The authors might consider adding a table containing this information to the paper.

AU: We would like to thank you for your suggestion. During the three different fattening period, the cattle were fed simply two kind of diets, concentrate (specially designed for the experimental cattle) and rice straw. Therefore, forage in the Table 1 is revised in detail as “Rice straw” in the revised manuscript, and we consider that this can explain the feedstuffs used to construct the 3 diets in this study.

Submitted filename: #5 Authors Responses to Reviewers_Rev1-1 (JB, PLOS).docx

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23 Oct 2019

PONE-D-19-20947R1

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening

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I appreciate the explanations and revisions of the authors and believe, I can provide better comments and suggestions.

L83: please provide the body weight of animals prior to the experiment;

L86-91: the concentrate composition is still missing. There are several considerations about animal performance, and the reluctancy to include feed composition troubles me. I understand that authors might have another paper with performance and other data and if so, please discuss accordingly in this manuscript. I also understand the performance was not the objective of this manuscript. However, intake and performance are central to discuss rumen microbiome modulation. For example, according to the provided body weights, animals gained 122 kg from Early to Middle, with an average daily gain of 0.51, which is 21.79 % lower from the expected daily weight gain. Furthermore, animals had an average daily gain of 0.63 kg and the estimation was 0.7 kg, thus animals performed 10.12 % less than what was expected for the Late stage. Moreover, feed intake decreased by 18% (Table 1) from Middle to Late stages. Yet animals had a gain in average daily weight. What is the rationale of this phenomenon?

L85-88: please provide the concentrate composition rather the phrase ‘specially designed high-concentrate’;

L89: authors included new values for forage-to-concentrate ratio. My consideration about forage-to-concentrate ratio was that it was ‘modified throughout the experiment, being 26:74 during Early stage, 13:87 during the Middle stage, and 14:86 during Late stage, which might have affected the rumen environment and pH values’. Forage-to-concentrate ratio was properly presented. However, the increase percentage of concentrate in the diet and how it affected the rumen microbiome was not satisfactorily discussed in the previous version of the manuscript. I would suggest author to keep forage-to-concentrate ration as it was in the first version, or clarify if the new value is the recommend values according to practices in Japan;

L89-91: I would consider being more precise in the description of gradually decreased. Was it a weekly adjustment for example?

L91: the body weight should be 562 instead of 561. According to data on table 1, the weight was 561.8, thus it should be rounded up accordingly;

L96-97: do authors collected feed intake for the whole trial, or only during the sampling weeks? If that was the case, please address possible limitations of this approach in the discussion section. For example, animals could have suffered acute feed intake by the end of the animal trial, which help explain intake and performance data;

Table 1: please indicate if data for daily intake amount is based on organic or dry matter.

L143;160;161: as I understand, you used the Illumina platform, which is based on reversible dye-terminator instead of pyrosequencing. Usually the pyrosequencing was employed using the Roche 454 sequencing platform. Please, address this accordingly;

L364-365: since body temperature, dehydration and diarrhea were considered clinical signs, please include how these were monitored in the Material and Methods sections;

L366-368: in the Late stage animals experienced rumen pH value under 5.6 for almost 11.5 hours, decreased VFA production, increased lactic acid, LPS, and aspartate transaminase concentration, which suggests SARA and decreased feed intake. However, check your data on feed intake and forage-to-concentrate ratio;

Table 5: Please, verify if differences between stages are correct for the OUT8. Should Late and Middle stage be similar?

Reviewer #2: (No Response)

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1 Nov 2019

PONE-D-19-20947R1

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening

Dear Editor and Reviewers

Authors would like to appreciate Editor and Reviewers for your helpful comments and suggestions. We have done our best to address all the issues raised by Reviewer #1 very carefully in this second revision, which we believe has improved the quality of the paper further. To facilitate the reviewing process, we have highlighted all changes done by Authors (Yellow) in the revised manuscript. We have responded to every comment done by the Reviewer #1 below, and also have indicated the changes made with respective new lines.

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to the Author

I appreciate the explanations and revisions of the authors and believe, I can provide better comments and suggestions.

AU: Authors would like to thank Reviewer #1 for your helpful comments and suggestions. We have done our best to address all the issues raised by you very carefully in this new revision, which we believe has improved the quality of the paper further.

L83: please provide the body weight of animals prior to the experiment;

AU: We would like to appreciate for your comment. The body weight of animals prior to the experiment (10 months of age) is added in the revised manuscript L92-94 as “The mean ± SE body weight of the cattle was 335 ± 4.4, 439 ± 7.6, 562 ± 11.6, and 712 ± 18.5 kg on prior to the experiment (10 months of age), and Early (14 months of age), Middle (21 months of age), and Late (29 months of age) fattening stage sampling days, respectively.”.

L86-91: the concentrate composition is still missing. There are several considerations about animal performance, and the reluctancy to include feed composition troubles me. I understand that authors might have another paper with performance and other data and if so, please discuss accordingly in this manuscript. I also understand the performance was not the objective of this manuscript. However, intake and performance are central to discuss rumen microbiome modulation. For example, according to the provided body weights, animals gained 122 kg from Early to Middle, with an average daily gain of 0.51, which is 21.79 % lower from the expected daily weight gain. Furthermore, animals had an average daily gain of 0.63 kg and the estimation was 0.7 kg, thus animals performed 10.12 % less than what was expected for the Late stage. Moreover, feed intake decreased by 18% (Table 1) from Middle to Late stages. Yet animals had a gain in average daily weight. What is the rationale of this phenomenon?

AU: We would like to apology for our missing data. The concentrate composition is added in the revised manuscript L87-90 as “The concentrate diet was composed of barely, steam-flaked corn, wheat bran, and soybean meal and contains 71.2% total digestible nutrient (TDN) and 15.7% crude protein (CP), 72.2% TDN and 13.9% CP, and 72.8% TDN and 12.0% CP during the Early, Middle, and Late stage, respectively.”

Regarding the animal performance, we partly agree with your opinion that intake and performance are central to discuss rumen microbiome modulation. In the present study, actual daily gain of cattle was observed only 2 weeks of period at each stage (from 1 week before to 1 week after sample collection day), and average daily gain was 0.75 (93.8% than calculated amount for daily gain), 0.69 (106.2%), and 0.46 (65.7%) kg/day during the Early, Middle, and Late stages, respectively. In addition, average daily gain during the entire experimental period was 0.63 kg/day in the present study. However, as you mentioned, the performance was not the main objective of the present study. Furthermore, the Japanese Feeding Standard for Beef cattle aimed to increase intramuscular fat accumulation (highly marbled meat) as discussed in the Introduction and Discussion sections, which may not fit the general farming object in other countries that maximize growth performance of beef cattle. We also agree with the fact that feed intake is undoubtedly important to meet energy requirement for production. However, other factors, such as peripheral hormones, environment, and genetic strain, also significantly affect the animal performance. As an example, peripheral blood vitamin A level is known for modulating intramuscular fat deposition, and the fattening cattle are generally fed high-grain, low vitamin A-containing diets to induce greater intramuscular fat deposition, leading to highly marbled meat during the fattening period (Oka et al., 1998) as introduced in the Introduction (L62-66) and Discussion (L355-357) sections. Therefore, we considered that descriptions in our manuscript is sufficient to concisely discuss and suggest our study aim. Although our description is somewhat unclear to elucidate the rationale of the phenomenon, we alternatively added our opinion on the dietary intake in the revised manuscript L365-367 as “Dietary intake amounts were highest during the Middle stage and lowest during the Late stage as an adaptation to long-term high-grain diet feeding or response to significantly lowered ruminal pH during the latter fattening stage”.

L85-88: please provide the concentrate composition rather the phrase ‘specially designed high-concentrate’;

AU: We would like to apology for our missing data. As we mentioned above, concentrate composition is added in the revised manuscript L87-90. Please see the revised manuscript.

L89: authors included new values for forage-to-concentrate ratio. My consideration about forage-to-concentrate ratio was that it was ‘modified throughout the experiment, being 26:74 during Early stage, 13:87 during the Middle stage, and 14:86 during Late stage, which might have affected the rumen environment and pH values’. Forage-to-concentrate ratio was properly presented. However, the increase percentage of concentrate in the diet and how it affected the rumen microbiome was not satisfactorily discussed in the previous version of the manuscript. I would suggest author to keep forage-to-concentrate ration as it was in the first version, or clarify if the new value is the recommend values according to practices in Japan;

AU: We would like to thank you for your valuable suggestion, and the relevant description about forage-to-concentrate ratio is reversed as it was in the original manuscript. Regarding the increase percentage of concentrate in the diet and how it affected the rumen microbiome, the dietary composition (e.g. forage-to-concentrate ratio) was changed monthly during the experiment period as revised in the previous revision despite it is reversed to the original description. However, rumen fluid sample was collected only once at the end of each fattening period, and sample collection is not accord with the dietary compositional changes (monthly dietary change vs. fattening periodic sample collection). As a result of feeding management and as we presented in the manuscript, total VFA concentration decreased, and lactic acid concentration increased during the Late stage as the adaptation or response to long-term high-grain diet feeding and long-term high-grain diet compositional change. Although we fully agree with your valuable suggestion on further discussion on the dietary change and rumen microbiome, we believe that our data connected long-term fattening diet feeding to ruminal pH, rumen fermentation, bacterial community composition and structure, blood metabolites, their relationships, and their consequences to leading enhanced lactic acid production are presented accordingly in the present study.

L89-91: I would consider being more precise in the description of gradually decreased. Was it a weekly adjustment for example?

AU: We would like to appreciate for your comment. However, as you mentioned above, the relevant description is reversed as it in the original manuscript. Please see the revised manuscript L91-92.

L91: the body weight should be 562 instead of 561. According to data on table 1, the weight was 561.8, thus it should be rounded up accordingly;

AU: We would like to apology for our careless mistake. The body weight is revised accordingly as 562 in the revised manuscript L92.

L96-97: do authors collected feed intake for the whole trial, or only during the sampling weeks? If that was the case, please address possible limitations of this approach in the discussion section. For example, animals could have suffered acute feed intake by the end of the animal trial, which help explain intake and performance data;

AU: We would like to appreciate for your helpful comment. In the present study, feed intake amount was collected daily throughout the experiment period. In addition, we checked that there was no acute increase or decrease in feed intake amount throughout the study period. Therefore, we added the information in the revised manuscript L364 as “no clinical sign of abnormal body condition, such as high body temperature, acute feed intake, dehydration ~”.

Table 1: please indicate if data for daily intake amount is based on organic or dry matter.

AU: We would like to apology for our undetailed description. In the Table 1, daily intake amount was based on the organic matter, and it is added in the footnote of the Table 1.

L143;160;161: as I understand, you used the Illumina platform, which is based on reversible dye-terminator instead of pyrosequencing. Usually the pyrosequencing was employed using the Roche 454 sequencing platform. Please, address this accordingly;

AU: We would like to appreciate for your comment. The description is revised accordingly in the revised manuscript L145 (Library preparation and DNA sequencing), 161 (Sequencing data analyses), and 162 (All sequencing reads were~).

L364-365: since body temperature, dehydration and diarrhea were considered clinical signs, please include how these were monitored in the Material and Methods sections;

AU: We would like to appreciate for your suggestion. The relevant information is added in the revised manuscript L97-98 as “Abnormalities of body condition (body temperature, appetite, hydration, and defecation) were observed daily throughout the study period”.

L366-368: in the Late stage animals experienced rumen pH value under 5.6 for almost 11.5 hours, decreased VFA production, increased lactic acid, LPS, and aspartate transaminase concentration, which suggests SARA and decreased feed intake. However, check your data on feed intake and forage-to-concentrate ratio;

AU: We would like to appreciate for your comment. In the original manuscript, we interpreted the relevant results with caution to avoid any overspeculation beyond our study result and aim. Therefore, as you mentioned above, we carefully suggest the reasons for decreased feed intake during the Late stage in the revised manuscript L365-367 as “Dietary intake amounts were highest during the Middle stage and lowest during the Late stage as an adaptation to long-term high-grain diet feeding or response to significantly lowered ruminal pH during the latter fattening stage”.

Table 5: Please, verify if differences between stages are correct for the OTU8. Should Late and Middle stage be similar?

AU: We would like to appreciate for your comment and apology for our careless mistake on the statistical analysis. As you mentioned, we checked all the validity of data, and found statistical mistakes only in the Table 5. Therefore, we revised the relevant descriptions in the Result (L311-324) and superscripts in the Table 5. However, these revisions have any influence on other part of the manuscript.

Submitted filename: #5 Authors Responses to Reviewers_Rev2-1 (JB, PLOS).docx

Click here for additional data file.

6 Nov 2019

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening

PONE-D-19-20947R2

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13 Nov 2019

PONE-D-19-20947R2

Long-term high-grain diet altered the ruminal pH, fermentation, and composition and functions of the rumen bacterial community, leading to enhanced lactic acid production in Japanese Black beef cattle during fattening

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