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Identification of the core rumen bacterial taxa and their population dynamics during the fattening period in Japanese Black cattle

Abstract The rumen microbiota comprises a vast range of bacterial taxa, which may affect the production of high‐quality meat in Japanese Black cattle. The aim of this study was to identify core rumen microbiota in rumen fluid samples collected from 74 Japanese Black cattle raised under different dietary conditions using 16S rRNA gene amplicon sequencing. In the rumen of fattening Japanese Black cattle, 10 bacterial taxa, showing >1% average relative abundance and >95% prevalence, irrespective of the dietary conditions and the fattening periods, were identified as the core rumen bacterial taxa, which accounted for approximately 80% of the rumen microbiota in Japanese Black cattle. Additionally, population dynamics of the core rumen bacterial taxa revealed two distinct patterns: Prevotella spp. and unclassified Bacteroidales decreased in the mid‐fattening period, whereas unclassified Clostridiales, unclassified Ruminococcaceae, Ruminococcus spp., and unclassified Christensenellaceae increased during the same period. Therefore, the present study reports the wide distribution of the core rumen bacterial taxa in Japanese Black cattle, and the complementary nature of the population dynamics of these core taxa, which may ensure stable rumen fermentation during the fattening period.


| INTRODUCTION
Ruminant animals harbor dense and diverse microbial populations in their rumen. The rumen microbes ferment feed and produce volatile fatty acids (VFAs), the main energy source for the host. Of the ruminal microbes, including bacteria, protozoa, and fungi, bacteria are considered to play vital roles in nutrient acquisition for the host animals; they constitute the largest biomass pool among the rumen microbes (Allison, 1993), and a majority (>90%) of the microbial genes expressed in the rumen are bacterial in origin (Brulc et al., 2009). Furthermore, comprehensive analysis of 16S rRNA genes has demonstrated the presence of more than 5,000 bacterial species in the rumen (Kim et al., 2011). A study from the Global Rumen Census project (Henderson et al., 2015) identified abundant bacterial groups, including Prevotella spp., unclassified Clostridiales, unclassified Bacteroidales, unclassified Ruminococcaceae, unclassified Lachnospiraceae, Ruminococcus spp., and Butyrivibrio spp., in the rumen as the core members of the rumen microbiota, irrespective of the ruminant species.
It is well known that the population sizes of particular bacterial species or groups in the rumen (the composition of the rumen microbiota) are affected by several factors such as host species (Henderson et al., 2015;Iqbal et al., 2017), dietary conditions (Tajima et al., 2001), and even cattle breeds. Paz et al. (2016) compared the compositions of rumen microbiota of Holstein and Jersey dairy cows under the same feeding management and demonstrated that the relative abundances of Prevotellaceae and Lachnospiraceae were significantly different between the breeds. Although members of the core rumen microbiota have been reported in a previous study, their distribution in different cattle breeds needs further investigation to extend our understanding of the key players in stable rumen fermentation.
Japanese Black cattle are the most popular beef breed, accounting for 67.2% of the beef cattle in Japan (MAFF, 2019). This breed is known to produce high-quality meat in terms of taste, tenderness, and marbling (Gotoh et al., 2009). To produce highly marbled meat, Japanese Black cattle are fed approximately five metric tons of concentrate diet during the fattening period, which is generally about 20 months long starting from the age of 10 months, and the proportion of concentrate diet is gradually increased up to approximately 90% (as fed) of the total diet. A high-concentrate diet can cause a drop in the ruminal pH, increasing the risk of digestive disorders such as subacute ruminal acidosis (Khafipour et al., 2009) and leading to loss of appetite, thereby resulting in low meat quality (Abdela, 2016).
Consequently, stable rumen fermentation during the fattening period, wherein animals are given a high-concentrate diet, is one of the most important aspects of high-quality meat production in Japanese Black cattle. Therefore, it is necessary to identify the core members of the rumen microbiota in this breed and their relation to fermentation to achieve stable rumen fermentation through appropriate feeding management.
Recently, Ogata et al. (2019Ogata et al. ( , 2020 studied the rumen microbiota of Japanese Black cattle using nine animals and showed high relative abundances of unclassified Ruminococcaceae, unclassified Lachnospiraceae, and Prevotella spp. during the fattening period. The abundance of these bacterial groups supported a previous report (Henderson et al., 2015) in which the core members of the rumen microbiota were determined using 742 samples from 32 animal species of 35 countries. However, further investigation with a larger sample size is necessary to identify the core members of the rumen microbiota in Japanese Black cattle. Therefore, in the present study, the findings of the previous studies on the rumen microbiota of Japanese Black cattle were validated using a larger sample size (n = 74). In addition, the rumen fluid samples of Japanese Black cattle that were fed different diets on different farms were subjected to rumen microbiota analysis. We hypothesized that if bacterial groups showed similar distribution and population dynamics under different conditions (locations and diets), it could be assumed that those bacterial groups were widely distributed in the rumen of Japanese Black cattle. Therefore, the objective of the present study was to generalize the core rumen microbiota that contributes to stable rumen fermentation in Japanese Black cattle.

| Animals and sampling
The animal experimental procedures used in the present study were approved by the Animal Care Committee National Livestock Breeding Center (Authorized Number: H20-53) and performed in accordance with the principles and guidelines for animal use set by the National Livestock Breeding Center.
In the present study, 74 Japanese Black cattle that were fed one of the four diets on five farms were sampled (Table 1). Thirty-seven animals were raised under the standard dietary conditions for Japanese Black fattening cattle following the conventional feeding program in the respective farms and were assigned to the Standard group (Table 1). Twenty-one animals in the Rice group were fed the same diet as the Standard group, except that the concentrate was partially replaced by steamed unhulled rice. The cattle in the corn silage (CS) group (n = 8) were offered CS-based total mixed ration (TMR), whereas 40% of the concentrate in the TMR was replaced with rice for the corn silage-rice (CSR) group (n = 8) (  (Table 1). In the Rice group, feeding regimens were the same as the Standard group except that 10%-15% of the concentrate was replaced by steamed unhulled rice (7.5% CP and 76.2% starch on a DM basis). In the CS group, cattle were fed TMR in which corn silage (8.4% CP, 25.7% NDF, and 25.7% starch on a DM basis) and concentrate (15.8% CP and 43.3% starch on a DM basis) were mixed in a ratio of 50:50 (DM basis) throughout the fattening period. In the CSR group, feeding regimens were the same as the CS group except that 40% of the concentrate was replaced by unhulled rice (7.5% CP and 76.2% starch on a DM basis). The animals were fed twice daily at 08:30 and 16:00.
Rumen fluid was collected from individual cattle using a stomach tube, 4 h after feeding in the morning. The sampling time points were as follows: 14, 22, and 26 months of age (considering as early, mid-, and late fattening periods, respectively) for the farms in Akita, Fukushima, Iwate, and Miyagi, and 17, 21, and 26 months of age (considering as early, mid-, and late fattening periods, respectively) for the farm in Hokkaido. Collected rumen fluid samples were stored at À80 C without separating the solid and liquid fractions until use.
Rumen fluid collection from one animal in the Standard group raised at the farm in Iwate was not conducted during the mid-and late fattening periods due to a metabolic disorder.

| Measurement of fermentation parameters
The rumen fluid samples were subjected to pH measurement using a pH electrode (LAQUAtwin B-712; Horiba, Kyoto, Japan) and VFA measurement using a gas chromatograph (GC-14B; Shimadzu, Kyoto, Japan), as described previously (Oh et al., 2017). NH 3 -N levels in the rumen samples were measured using the phenol-hypochlorite method (Weatherburn, 1967

| Bioinformatics analysis
Data obtained from amplicon sequencing using the MiSeq platform were analyzed using the QIIME2 version 2019.4 (Bolyen et al., 2019). Paired-end reads were filtered, dereplicated, merged, and chimera-filtered using q2-dada2 plugin (Callahan et al., 2016) to generate amplicon sequence variants (ASVs). The taxonomic classification of ASVs was performed at the genus level using the recently updated Greengenes database (v.13_8). ASVs classified as archaea, mitochondria, and chloroplasts were removed from the analysis.
Three samples (one from the Standard group in the mid-fattening period and two from the Rice group in the early fattening period) with less than 1,500 reads remaining after this step were excluded from further analysis. Filtered ASV count    To study the population dynamics patterns of the core rumen bacterial taxa during the fattening periods, correlation analysis was performed. Figure 3a represents the correlation matrix based on the abundances of the core rumen bacterial taxa in the Standard group during the fattening period. The abundance of Prevotella spp. was negatively correlated (p < 0.05) with that of the other core members, except for the unclassified Bacteroidales. Similar to Prevotella spp., unclassified Bacteroidales showed a negative correlation (p < 0.05) with many of the core members (Figure 3a).

| DISCUSSION
Stable rumen fermentation is one of the most important factors for the production of high-quality meat in Japanese Black cattle. Therefore, identification of the core rumen bacterial taxa contributing to stable rumen fermentation is important to achieve high-quality meat production in this breed through appropriate feeding management. In  (Baek et al., 2020;Henderson et al., 2015;Lima et al., 2015). The possible roles of Pirellulaceae and unclassified F16, belonging to phylum TM7, in carbohydrate degradation have been reported in the animal gut (Kindaichi et al., 2016;Parata et al., 2020). Christensenellaceae has been recently recognized as an important hydrogen-producing bacterial group, which is utilized for methane production in the human gut and rumen (Greening et al., 2019;Waters & Ley, 2019 (Monteiro & Faciola, 2020). Gram-negative bacteria are generally more sensitive to low rumen pH compared with grampositive bacteria. In fact, the abundances of core members belonging to Bacteroidales and Clostridiales correlated positively and negatively, respectively, with ruminal pH (p < 0.05) ( Figure S2). Although the cattle used in the present study did not suffer from rumen acidosis, rumen pH was lower in the Standard and Rice groups during the mid-fattening period of the study. Therefore, a drop in pH due to an increase in the concentrate diet in the mid-fattening period could be a possible cause of the change in population dynamics of the core rumen bacterial taxa.
A negative correlation between the abundances of Prevotella spp.  (Henderson et al., 2015) and exhibit versatile functions, including the degradation and fermentation of both roughage and concentrate (Cotta, 1988;Flint, 1997). Ruminococcaceae includes Ruminococcus bromii, which is a major amylolytic bacterium in the rumen of cattle fed with a high-concentrate diet (Khongpradit et al., 2020;Klieve et al., 2007). Christensenellaceae, containing the Clostridiales R-7 group, was recently identified as one of the dominant bacterial groups and is possibly involved in xylan and xyloglucan degradation in the rumen (Seshadri et al., 2018). Therefore, these bacterial groups could play vital roles in rumen fermentation. The complementary population dynamics of core members results in maintaining the niche for all core rumen bacterial taxa, which ensures stable rumen fermentation during the fattening period in Japanese Black cattle.
A long-term fattening with high concentrate feeding in the production of Japanese Black cattle increases the risk of dysbiosis, leading to abnormal rumen fermentation, such as rumen acidosis.
Under high concentrate feeding, some lactic acid bacteria including Streptococcus bovis and Lactobacillus spp. exhibit rapid growth, and it is a trigger for rumen acidosis. Therefore, preventing the overgrowth of these lactic acid bacteria is important for normal rumen fermentation. It can be assumed that the rumen microbiota dominated by the core bacterial taxa in a complementary manner could increase the stability of the rumen ecosystem contributing to the prevention of the overgrowth of lactic acid bacteria in the rumen of Japanese Black cattle.
The complementary population dynamics in the core bacterial taxa during the fattening period were relatively smaller in TMR feeding groups (i.e., CS and CSR in the present study) than those in other dietary groups. This observation indicates the stability of rumen microbiota in TMR feeding groups and partly explains less fluctuation of rumen fermentation parameters in these groups. Although the complemental relationship among the core rumen bacterial taxa needs to be further validated, the negative correlation between Bacteroidetes and Firmicutes (the former contains Prevotella spp. and unclassified Bacteroidales, and the latter includes the families Ruminococcaceae and Christensenellaceae) is well recognized in the animal gut (Filippo et al., 2010). Besides, it is reported that Prevotella spp. showed a negative correlation with Butyrivibrio spp. and Shuttleworthia spp. belonging to Clostridiales in the rumen of Holstein dairy cows (Jami et al., 2014).
In conclusion, the present study identified the core rumen bacterial taxa that account for approximately 80% of the total bacteria in the rumen of Japanese Black cattle. The distribution of these core members was mainly affected by the increase in the concentrate diet during the fattening period. Additionally, the population dynamics of the core rumen bacterial members is likely to be complementary, resulting in their stable distribution during the fattening period. Therefore, the key findings of the present study provide an insight into the key players in stable rumen fermentation in Japanese Black cattle.