Increasing Buffering Capacity Alters Rumen Microbiota Composition and Enhances Rumen Fermentation Characteristics of High-Concentrate Fed Hanwoo Steers

Increasing Buffering Capacity Alters Rumen Microbiota Composition and Enhances Rumen Fermentation Characteristics of High-Concentrate Fed Hanwoo Steers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Increasing Buffering Capacity Alters Rumen Microbiota Composition and Enhances Rumen Fermentation Characteristics of High-Concentrate Fed Hanwoo Steers Sonny Ramos, Seon Ho Kim, Chang Dae Jeong, Lovelia L. Mamuad, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-354029/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Rumen bacterial community is mainly affected by the type of diet consumed by the host animals. High concentrate diet increases the abundance of lactic acid producers and utilizers due to high level of non-structural carbohydrates thus reducing the number of fiber-degrading bacteria because of drastic decrease in pH. Dietary buffers are essential in regulating rumen pH through the compounds responsible in resisting drastic decrease in pH once cattle were fed with high-concentrate diet. However, no study has evaluated the effects of buffering capacity and efficiency in alleviating chronic acidosis in rumen. Ruminal metataxonomic and fermentation characteristics analyses were conducted to evaluate the effect of different buffering capacities on in vitro and in vivo experiments in high-concentrate fed Hanwoo steers. Results: Results revealed that BC 0.9% and BC 0.5% had similar and significant effect ( P < 0.05) on in vitro ruminal fermentation at 3 to 24 h incubation. Both BC 0.9% and BC 0.5% had significantly highest ( P < 0.05) buffering capacity, pH, and ammonia-nitrogen (NH 3 -N) than BC 0.3% and CON at 24 h of incubation. Individual and total volatile fatty acids (VFA) were significantly lowest in CON. Increasing buffering capacity concentration showed linear effect on pH at 6 to 24 h while total gas and NH 3 -N at 3 and 12 h. Phylum Bacteroidetes dominated all treatments but a higher abundance of Firmicutes in BC 0.5% than others. Ruminoccocus bromii and Succiniclasticum ruminis were dominant in BC 0.5% and Bacteroides massiliensis in BC 0.3% . The normalized data of relative abundance of observed OTUs’ representative families have grouped the CON with BC 0.3% in the same cluster, whereas BC 0.5% and BC 0.9% were clustered separately which indicates the effect of varying buffering capacity of buffer agents. Principal coordinate analysis (PCoA) on unweighted UniFrac distances revealed close similarity of bacterial community structures within and between treatments and control, in which BC 0.9% and BC 0.3% groups showed dispersed community distribution. Conclusion: Our findings showed that increasing buffering capacity enhances rumen fermentation parameters and affects rumen microbiome by altering bacterial community through distinct structure between high and low buffering capacity, thus an important factor contributed to the prevention of ruminal acidosis during a high-concentrate diet. General Microbiology Applied & Industrial Microbiology Buffering capacity Hanwoo steers Microbiome Ruminal metataxonomic Fermentation characteristic Acidosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Energy and essential nutrients are obtained by ruminants through a complicated symbiotic relationship with the rumen microbiome [ 1 ] and bacterial community alterations can affect the productivity and health of the host animal [ 2 ]. A high forage diet is usually switched to a high concentrate diet to improve the productivity of the ruminants; however, it alters the rumen ecosystem due to high non-structural carbohydrates level [ 1 ]. Meanwhile, the core ruminal bacterial community is dominated by the phyla Bacteroidetes, Firmicutes, and Proteobacteria regardless of diet composition [ 3 ]. However, a high-concentrate diet induces death and cell lysis [ 4 ], thus decrease in abundance of Firmicutes in the rumen [ 3 ]. It also enhances the growth of lactic acid utilizers like Megasphaera elsdenii , Selenomonas ruminantium , and Veillonella parvula resulting to a drastic reduction of fiber-degrading bacteria such as Fibrobacter succinogenes and Ruminococcus spp [ 1 ]. Feeding of highly fermentable diets is the current practices in high producing beef to increase growth rates, but it causes microbial disturbances resulting to digestive disorders such as ruminal acidosis [ 5 ]. The rapid fermentation of non-structural carbohydrates resulted in the accumulation of volatile fatty acid and lactic acid in the rumen causing a drastic decrease in pH [ 6 ]. Hence, the use of buffer could be useful to resist changes in rumen pH whenever cattle are being fed with high concentrate, low forage, fermented and fine-chopped forage [ 7 ]. Compounds that increase the buffering capacity of ruminal fluid help maintain a more stable ruminal pH and direct neutralization of VFA especially during a diet or fermentation-related acid challenge [ 5 , 6 ]. Rumen buffering could avert the sudden decrease in pH, thus could enhance rumen microbial growth, activity and diversity, microbial protein synthesis, and fermentation end product [ 10 ]. Buffering capacity (BC) is then referred to as the number of moles of H + that should be added to a 1L solution to decrease pH by 1 unit [ 11 ]. Weak acids and bases are known to provide better buffering in comparison to strong acids and bases because of the equilibrium establishment between the acid and the conjugate base [ 12 ]. Various studies have reported that adding a buffer solution, such as sodium bicarbonate (NaHCO 3 ) with magnesium oxide (MgO) increased dry matter intake when corn silage was the sole or major source of forage in the diet [ 13 ]. NaHCO 3 is commonly used in preventing ruminal acidosis because it provides a natural buffer; however, its high solubility limits the buffering activity against acidic conditions [ 14 ]. Le Ruyet and Tucker [ 15 ] proved that NaHCO 3 had high BC in an in vitro study. It contained 26% more actively buffering the CO 3 portion of the molecule that is important to neutralize the acid. MgO, on the other hand, appears to work efficiently in combination with NaHCO 3 [ 14 ]. Shaver et al. [ 16 ] stated that supplementing NaHCO 3 and MgO in a 3:1 ratio is the recommended level of dietary buffer for the best response. The efficiency and mechanisms of buffer responsible for alleviating chronic acidosis are variable and often inconsistent [ 9 ]. Research on different level of buffering capacity in enhancing rumen fermentation parameters and microbiome during high concentrate diet has not yet been investigated. In the present work, ruminal metataxonomic and fermentation characteristics analyses were conducted using rumen fluid samples to evaluate the effect of different buffering capacities on in vitro and in vivo trials in high-concentrate fed Hanwoo steers. Results Effect of different buffering capacities on in vitro rumen fermentation parameters The buffering capacity of BC 0.9% and BC 0.5% were significantly greatest ( P < 0.05) after 24 h incubation compared to BC 0.3% and CON (Table 1). Both BC 0.9% and BC 0.5% exhibited significantly highest ( P < 0.05) buffering capacity value of 106.00 meq/L, hence had a similar effect on in vitro after 24 h. The ruminal pH obtained from BC 0.9% and BC 0.5% showed similar effects and were consistently higher ( P < 0.05) than the other treatments throughout the incubation period. In gas production, BC 0.9% , BC 0.5% , and BC 0.3% had significantly higher ( P < 0.05) gas produced than CON and showed similar effects at 3 and 12 h incubation. Ruminal NH 3 -N concentration was significantly higher ( P < 0.05) in BC 0.9% and BC 0.5% , thus, it also had a similar effect on this parameter. However, at 6 and 12 h, no effect observed on treatments except that BC 0.5% tended to increase ( P = 0.073) NH 3 -N concentration followed by BC 0.9% and the rest treatments. Significantly higher concentrations ( P < 0.05) of acetate were observed in BC 0.9% at 12 h; however, BC 0.5% and BC 0.3% obtained the highest value ( P < 0.05) after 24 h (Table 2). Propionate and butyrate concentrations were both highest ( P < 0.05) in BC 0.3% and BC 0.9% at 6 h. Subsequently, distinct effects of BC 0.3% , BC 0.5% , and BC 0.9% were observed at 24 h which had significantly higher ( P < 0.05) propionate concentrations than CON. A similar pattern was noticeable with butyrate at 12 h such that BC 0.3% , BC 0.5% , and BC 0.9% obtained the highest concentration ( P < 0.05) compared with CON. During this period, a similar effect can be seen between the 3 treatments; however, no significant effect was observed after 24 h. Total volatile fatty acid contents were greater ( P < 0.05) in BC 0.3% , BC 0.5% and BC 0.9% at 12 h but had a slight change after 24 h. At this time point, treatments BC 0.3% and BC 0.5% were highest ( P < 0.05) compared to BC 0.9% and CON. Furthermore, there were no treatment effects on acetate to propionate ratio after 24 h incubation. Consequently, increasing the concentration of buffering capacity showed linear effects ( P < 0.05) on pH, total gas production, NH 3 -N, and at some certain time point of individual VFA. Table 1 Effect of different buffering capacity concentrations on in vitro rumen fermentation parameters at 3, 6, 12 and 24 h Parameters Time (h) Treatment e SEM P -value CON BC 0.3% BC 0.5% BC 0.9% All Linear Buffering capacity (meq/L) 3 76.44 d,z 83.89 c,y 87.56 b,x 90.11 a 0.398 <0.001 <0.001 6 76.45 c,z 84.17 b,y 92.89 a,x 92.89 a 0.246 <0.001 <0.001 12 85.33 c,y 87.22 c,y 96.67 b,x 99.22 a 0.469 <0.001 <0.001 24 100.22 c,z 102.56 b,y 106.00 a,x 106.00 a 0.462 <0.001 <0.001 pH 3 6.00 c 6.03 bc 6.08 ab 6.13 a 0.013 0.012 0.052 6 5.68 c,z 5.80 b,y 5.91 a,x 5.93 a 0.014 <0.001 <0.001 12 5.42 c,z 5.54 b,y 5.60 a,x 5.62 a 0.014 <0.001 <0.001 24 5.14 b,y 5.15 b,y 5.22 a,x 5.24 a 0.009 0.001 0.003 Total gas (mL) 3 74.67 b,y 82.00 a,x 81.67 a,x 82.00 a 1.287 0.016 0.004 6 122.33 124.33 123.00 123.67 1.353 0.777 0.488 12 169.00 b,y 187.00 a,x 179.00 a,x 182.67 a 2.492 0.007 0.010 24 251.67 269.00 256.00 256.67 3.877 0.078 0.195 NH 3 -N (mg/dL) 3 11.26 c,y 12.24 b,x 13.05 a,x 13.39 a 0.205 0.001 0.003 6 13.35 13.60 16.09 14.74 0.569 0.178 0.095 12 13.90 y 14.95 xy 16.23 x 15.84 0.482 0.073 0.010 24 19.90 c 20.39 bc 21.35 ab 22.43 a 0.378 0.012 0.052 a-d Means with different superscripts in a row differ significantly ( P < 0.05) e CON (no buffer added); BC 0.3% (0.3% buffer); BC 0.5% (0.5% buffer); BC 0.9% (0.9% buffer) x,y,z Means within a row indicate linear effect among CON, BC 0.3% , and BC 0.5% ( P < 0.05) Table 2 Volatile fatty acid production during in vitro rumen fermentation incubated at 3, 6, 12, and 24 h Parameters Time (h) Treatment e SEM P- value CON BC 0.3% BC 0.5% BC 0.9% All Linear Acetate (mmol/L) 3 100.13 94.81 93.41 90.36 2.505 0.265 0.237 6 100.03 100.31 99.79 100.39 0.592 0.901 0.775 12 102.52 b 104.34 b 103.36 b 107.76 a 0.291 0.001 0.412 24 103.71 c,y 112.05 ab,xy 117.27 a,x 108.47 bc 2.182 0.018 0.009 Propionate (mmol/L) 3 27.44 26.51 24.02 25.59 1.395 0.598 0.280 6 27.81 b 28.67 a 27.66 b 28.37 a 0.085 0.001 0.421 12 32.19 32.65 30.59 33.85 0.756 0.227 0.350 24 35.24 b,y 40.32 a,xy 42.31 a,x 38.09 ab 1.203 0.045 0.020 Butyrate (mmol/L) 3 17.60 20.15 19.60 19.42 0.501 0.088 0.098 6 22.23 a,x 22.78 a,x 20.95 b,y 22.81 a 0.276 0.009 0.006 12 27.46 b,y 40.52 a,x 39.69 a,x 40.08 a 1.660 0.007 <0.001 24 53.85 54.58 56.43 54.94 0.783 0.327 0.125 Total VFA (mmol/L) 3 145.16 141.47 137.02 135.37 3.381 0.430 0.285 6 150.07 151.76 148.40 151.57 0.803 0.077 0.196 12 162.18 b,y 177.51 a,x 173.63 a,x 181.69 a 2.424 0.005 0.005 24 192.80 c,y 206.95 ab,xy 216.01 a,x 201.51 bc 3.447 0.013 0.008 A:P ratio 3 3.74 3.57 3.89 3.53 0.134 0.618 0.670 6 3.60 ab 3.50 c 3.61 a 3.54 bc 0.018 0.014 0.719 12 3.19 3.20 3.41 3.18 0.084 0.465 0.266 24 2.94 2.78 2.78 2.85 0.069 0.434 0.178 a-c Means with different superscripts in a row differ significantly ( P < 0.05) e CON (no buffer added); BC 0.3% (0.3% buffer); BC 0.5% (0.5% buffer); BC 0.9% (0.9% buffer) x,y Means within a row indicate linear effect among CON, BC 0.3% , and BC 0.5% ( P < 0.05) Effect of different buffering capacities on rumen fermentation characteristics in Hanwoo steers The effect of different buffering capacity concentrations on rumen fermentation characteristics of Hanwoo steers in four treatments are presented in Table 3. Average pH had no significant effects among CON and treatments. However, BC 0.3% , BC 0.5% , and BC 0.9% had significantly higher ( P < 0.05) buffering capacity value than CON, and showed linearly significant effect ( P < 0.05). Ammonia-nitrogen, acetate to propionate ratio, individual and total VFA concentrations of rumen fluid from steers under all treatments were not significant and showed similar effects after the in vivo experiment. Table 3 Effect of different buffering capacity concentrations on rumen fermentation characteristics in Hanwoo steers Parameters Treatment c SEM P- value CON BC 0.3% BC 0.5% BC 0.9% All Linear Average pH 6.28 6.56 6.77 6.48 0.146 0.226 0.528 Buffering capacity (meq/L) 83.47 b,y 99.53 a,x 101.00 a,x 94.80 a 3.058 0.015 0.004 NH 3 -N (mg/dL) 4.84 4.26 5.40 4.59 1.260 0.945 0.794 Total VFA (mmol/L) 47.24 45.29 54.16 55.87 5.234 0.632 0.563 Acetate (mmol/L) 29.67 27.28 30.70 35.56 2.421 0.532 0.890 Propionate (mmol/L) 11.30 9.26 14.37 12.75 2.190 0.412 0.380 Butyrate (mmol/L) 6.28 8.76 9.08 7.56 1.556 0.652 0.299 A:P ratio 2.81 3.10 2.13 2.91 0.451 0.431 0.370 a-b Means with different superscripts in a row differ significantly ( P < 0.05) c CON (no buffer added); BC 0.3% (0.3% buffer); BC 0.5% (0.5% buffer); BC 0.9% (0.9% buffer) x,y Means within a row indicate linear effect among CON, BC 0.3% , and BC 0.5% ( P < 0.05) Bacterial diversity of the rumen contents of Hanwoo steers The boxplot representation of alpha diversity indices is shown in Figure 1. Alpha diversity indices are composite indices that reflect abundance and consistency. Chao1 which reflect the OTU abundance in the samples showed that BC 0.9% was the highest among treatments followed by BC 0.5% and the rest of the treatments (Figure 1a). Shannon index which reflects the diversity of the OTU in samples presented BC 0.9% as the most diverse among treatments and BC 0.3% being the least (Figure 1b). Moreover, Figure 1c showed the boxplot of OTUs of observed species from the samples. The number of OTUs in BC 0.9% was higher followed by BC 0.5% and the rest of the treatments. The diversity index is used to analyze the temporal and spatial changes in species composition which reflects whether bacterial communities between groups have differences. Our results showed that the rumen bacterial composition of BC 0.5% and BC 0.9% had overall higher alpha diversity than other treatment groups, although no significant difference was observed after statistical analysis. Effect of treatments on bacterial community composition of Hanwoo steers rumen contents Bacterial taxonomic compositions at the phylum, genera, and species level are shown in Figure 2. Results at the phylum level revealed that 15 bacterial phyla were identified in the rumen digesta samples of Hanwoo steers (Figure 2a). The majority of the sequences obtained from all treatments belonged to Bacteroidetes followed by Firmicutes. It was noticeable that BC 0.3% had the highest abundance of Bacteroidetes (71.90%) and lowest Firmicutes (22.13%). On the contrary, BC 0.9% had the lowest abundance of Bacteroidetes (54.19%) among treatments; however, BC 0.5% had the highest Firmicutes (33.84%) relative abundance. Furthermore, Proteobacteria was dominant upon incorporating BC 0.9% in the diet resulting in declining its abundance from that of BC 0.3% and BC 0.5% . Spirochaetes increased its abundance when animals received supplementation of BC 0.9% , whereas there was a sudden decrease once the steers received BC 0.5% in their diet. At the genus level (Figure 2b), Prevotella was the predominant among genera in all treatments followed by Bacteroides (10.36%) and Ruminococcus (8.93%) in BC 0.3% and BC 0.5% , respectively. Furthermore, Succiniclasticum was dominant among genera after supplementing BC 0.5% ; however, a decreasing abundance of Paludibacter was noticeable as BC 0.5% is incorporated into the diet, which had a reverse effect as did CON. Species-level analyses revealed that Prevotella ruminicola predominated the treatments CON, BC 0.3% , BC 0.5% and BC 0.9% with the relative abundance of 24.85%, 32.16%, 26.73%, and 23.17%, respectively (Figure 2c). The comparison of single species analyzed through statistical analysis showed a significant effect of the treatments only in the case of Prevotella brevis. This species was more abundant ( P = 0.015) in the CON and as steers received a diet supplemented with BC 0.9% , BC 0.5% and BC 0.3% its abundance decreased. Owing to the BC 0.5% supplemented in the diet, a decreasing abundance of Paludibacter propionicigenes was observed; however, it increased in CON. Incorporation of BC 0.5% increased the microbial population of Ruminococcus bromii and Succiniclasticum ruminis. Moreover, the smaller percentage of BC 0.3% resulted in a higher abundance of Bacteroides massiliensis which led to a sudden decrease in its population as the concentration of treatments increases. Supplementing buffers of different buffering capacity concentration may affect the rumen microbiota through the relative abundance of bacterial species. The core, shared and unique bacterial community of observed species of the rumen microbiome after treatment of buffer agents with varying level of buffering capacity is presented in Figure 3 as Venn diagram. A total of 211 (59.6%) observed species can be found across all the samples (core), 79 (22.32%) for shared by 2 or 3 samples, and 64 (18.08%) are specific and are distributed to the four samples. The normalized data presented in Figure 4 shows the clustering based on the similarity of relative abundance between representative families of OTUs (row), and treatments (column). The analysis divided the representative families into two major clusters distinguishing families which represents low relative abundance on all treatments (upper cluster in red), and families that have varying relative abundance between treatments (lower cluster, colored from peach to blue). On the cluster presenting varying abundance between treatments, two sub-clusters were also distinguishable; (1) families which represent variation from very low (red) to average (peach) abundance, and (2) cluster representing families which have average to high (blue) abundance. The cluster in the bottom of the heatmap (labelled) contains the families that represent the above average relative abundance. On this cluster, family Prevotellaceae had branched out because it presents the highest abundance with very small variations between treatments ( p = 0.092). Family Ruminicoccaceae were also found in all treatments, but varying relative abundance was observed, with BC 0.5% presenting the highest. Families Acidaminococcaceae and Lachnospiraceae were significantly highest ( P < 0.05) in BC 0.3% and BC 0.5% , respectively. Also, the Unclassified Clostridiales had significantly highest ( P < 0.05) relative abundance in BC 0.5% . A certain unclassified family under order Bacteroidales also showed major abundance especially in BC 0.3% , while families Vibrionaceae and Spirochaetaceae were highest in BC 0.9% . Meanwhile, the normalized data of relative abundance of representative families of observed OTUs have grouped the control sample together with BC 0.3% in a single cluster, while BC 0.5% and BC 0.9% are on their own cluster, which could indicate the effect of varying buffering capacity of buffer agents. The comparison of the bacterial communities by principal coordinate analysis (PCoA) is presented in Figure 5. The PCoA plots showed close similarity within and between treatments and control, whereas those under BC 0.9% and BC 0.3% groups showed dispersed distribution of bacterial communities. The PCoA plot showed dissimilarity of bacterial community and revealed a distinct structure between high buffering capacity and low buffering capacity. Monitoring of acidosis The changes in the 24 h mean ruminal pH monitored for 30 d is presented in Table 4. During this period of the feeding challenges, mean pH values were >5.8. Minimum pH was lowest in CON, whereas it was highest in BC 0.5% . Additionally, BC 0.9% had a low minimum pH value second to that of CON. It was noticeable that BC 0.3% and BC 0.5% had higher minimum and mean pH values compared to BC 0.9% and CON. Obtained results indicated that the duration of time where pH was <5.8 and 5.8 < 6.0 was longer in CON followed by BC 0.9% and BC 0.3% . Meanwhile, BC 0.3% also exhibited good results in the duration of time where pH was approximately 6.0 and above; however, BC 0.5% had even better effects and did not show any signs of acidosis in the rumen. Based on the data gathered, BC 0.5% stabilized the pH of rumen preventing it from becoming acidotic. Table 4. Changes in the 24 h mean ruminal pH, duration of time where pH was <5.8 monitored for 30 d challenge diet Item Treatment a CON BC 0.3% BC 0.5% BC 0.9% 24 h mean ruminal pH Minimum 5.28 5.72 5.83 5.40 Mean 6.36 6.56 6.47 6.22 Duration of ruminal pH pH <5.8, min/d 66.29 1.45 0.00 13.06 pH 5.8 < 6.0, min/d 90.97 21.77 10.16 106.45 pH 6.0 and above, min/d 1214.52 1296.77 1202.42 999.68 a CON (no buffer added); BC 0.3% (0.3% buffer); BC 0.5% (0.5% buffer); BC 0.9% (0.9% buffer) Discussion Currently, one of the major health issues in dairy farming is the sudden decline of ruminal pH which causes a reduction of feed intake, problems with digestion, and production losses. Cattle health mainly suffers and additional costs in management increase due to its prevalence. Sodium bicarbonate is widely used for the prevention of rumen acidosis because it serves as a natural buffer in the rumen. Despite its buffering ability, it only functions for a short period of time and because of the high solubility, it is rapidly used by the ruminants. Most studies have suggested that magnesium oxide act either as a neutralizer or buffer in rumen or intestine [ 22 ]. It also increases starch digestion in the intestine of animals fed with a high-concentrate diet. This may result in an increase of pH in the small intestines allowing starch-digesting enzymes to become more active [ 16 ]. Mao et al. [ 23 ] reported that supplementation of the bicarbonate group had higher pH, total gas production, and total VFA concentration although ammonia-nitrogen concentrations remained unaltered. Addition of combined buffers in high concentrate rations altered rumen pH, liquid turnover, and patterns of rumen fermentation [ 24 ]. Consequently, commercial buffer agent (CBA) is developed as a buffer premix and considered as more powerful alternative to sodium bicarbonate. This premix is a mixture of various raw materials, differing in acid-binding capacity and solubility that contained live yeast, which promoted the conversion of lactate to propionate; thus, improving rumen conditions. Research data have shown its efficiency in maintaining the stability of ruminal pH, thus preventing the stimulation of subacute ruminal acidosis (Provimi™, Rotterdam, Netherlands). Meanwhile, the results of the present study are in accordance with their experimental output. The result of the present study showed that BC 0.9% , as well as the BC 0.5% , had similar effects on rumen content. Both treatments had significant effects on pH, buffering capacity, and ammonia-nitrogen concentration relative to that of the negative control. An increase in ruminal pH upon supplementation of sodium bicarbonate is a result of dissociation of sodium (Na + ) and bicarbonate (HCO 3 − ) [ 11 ]. Meanwhile, the results on gas production were supported by the claims of Rauch et al. [ 25 ] and Kang and Wanapat [ 10 ], who stated that supplementation with sodium bicarbonate enhanced gas production. The increase in gas production might be caused by the dissociation of sodium bicarbonate resulting to increase gas volume because of CO 2 liberation [ 25 ]. Also, it might be due to the conversion of some bicarbonate to carbonic acid which soon released as carbon dioxide [ 11 ]. Moreover, obtained data from the present study is in accordance with the results of Le Ruyet and Tucker [ 15 ] on the temporal effects of ruminal buffers in terms of buffering capacity and pH of ruminal fluid from cows fed a high concentration diet. Buffering compounds increased the ruminal fluid buffering value index and were beneficial in preventing postprandial increases in ruminal fluid hydrogen ion concentration. Shaver et al. [ 16 ] also stated that magnesium oxide and sodium bicarbonate were the best rumen buffers, which increased the acetate: propionate ratio and prevented declines in pH. The effect of buffers on VFA in this study was the same as the data obtained by Kang and Wanapat [ 10 ] wherein supplementation with buffering agents increased the total VFA. High ruminal VFA concentration is caused by increased carbohydrate fermentation in the rumen [ 26 ]. Although the present study did not show a significant effect on molar concentration of VFA, the noticeable increasing numerical values were observed in buffer-supplemented treatments. Subsequently, the metagenomic survey of bacterial community composition was identified in the rumen digesta samples of Hanwoo steers. Obtained results at the phylum level were in accordance with the data gathered by Nagata et al. [ 27 ] wherein the relative abundance of Bacteroidetes was higher during the high-concentrate period of the experimental animals. Additionally, Zhao et al. [ 28 ] stated that the microbial community of beef cattle was dominated by Bacteroidetes and Firmicutes at the phylum level regardless of group. An increase in the phylum Bacteroidetes resulted in increased Prevotella and repressed Firmicutes, which was attributed to decreasing Ruminococcaceae. Dodd et al. [ 29 ] and Naas et al. [ 30 ] indicated that the Bacteroidetes in the rumen represented another numerically dominating phylum that was not associated with cellulose degradation, rather its saccharolytic status is based on limited case studies of noncellulolytic Prevotella rumen isolates. Because of the ability of Prevotella to use a variety of substrates, it tends to dominate in the rumen under a range of diets [ 31 ]. In the present study, Prevotella ruminicola appeared to be the predominant species among all treatments. This species constitutes one of the most numerous groups recovered from the rumen and plays important roles in the utilization of polysaccharides of plant origin [ 32 – 34 ] and the metabolism of peptides and proteins [ 35 – 39 ]. Moreover, the low-relative abundance of Ruminococcus (8.93%) in this study was in contrast with the findings obtained by Klieve et al. [ 40 ], who used a high-grain diet (75% barley) for the animals, although this genus was identified and largely comprised the cellulolytic bacteria. High propionate concentration of BC 0.5% might be caused by the high relative abundance of Succiniclasticum ruminis. This result is in accordance with the study of Van Gylswyk [ 41 ], who stated that this species specializes in fermenting and converting succinate to propionate, which is an important precursor of glucose in ruminants. Ueki et al. [ 42 ] described Bacteroides massiliensis as a producer of acetate, propionate, and succinate which can explain the increase in molar concentrations of VFA on in vivo study. The abundance of Paludibacter propionicigenes might be due to its description as a sugars utilizer and a producer of acetate and propionate, an end product of fermentation [ 43 ]. Acidosis was defined as impaired ruminal health accompanied by a reversible ruminal pH depression [ 40 , 44 – 47 ]. Ruminal microbes convert carbohydrates to short-chain fatty acids at a rate that exceeds the rumen’s absorptive, buffering, and outflow capacity causing a rapid decrease in ruminal pH [ 48 ]. Data gathered in this experiment agreed with the results obtained by Tucker et al. [ 8 ] that the addition of a buffer, especially sodium bicarbonate, was effective in reducing ruminal fluid acidity and retards the drop in pH that normally occurs from 6 to 12 h post-feeding. Also, Zamarreño et al. [ 9 ] stated that the use of sodium bicarbonate and magnesium oxide or even mixed antacids were recommended for satisfactory results. They concluded that the increase in buffering capacity and increase in acid consuming capacity contributed to the correction of animal acidosis. Conclusion Different BC concentrations were evaluated and results showed that BC 0.9% and BC 0.5% had a similar effect on an in vitro ruminal fermentation. Increasing the concentration of BC showed a linear effect on pH, NH 3 -N, and some incubation time of individual VFA. Metagenomics survey on bacterial abundance revealed that phylum Bacteroidetes dominated all the treatments. A higher abundance of Firmicutes was observed in BC 0.5% ; however, gradually decreased as the BC concentration decreases. Meanwhile, as BC increases, the relative abundance of Proteobacteria also increases. Prevotella ruminicola dominated all treatments and had the highest abundance in BC 0.3% . Supplementing BC 0.5% in the diet increased the abundance of Ruminoccocus bromii and Succiniclasticum ruminis while a drastic increase in the population of Bacteroides massiliensis in BC 0.3% . Overall, data gathered from the present study showed that increasing buffering capacity enhances rumen fermentation and alters rumen microbiome which is an important factor contributed positively to the correction of animal acidosis during a high-concentrate diet. Methods Animals, rumen fluid collection and in vitro rumen fermentation Three ruminally cannulated Hanwoo steers (500 ± 47 kg body weight; 20 mos. of age) were used to provide rumina fluid for in vitro rumen fermentation. The animals were fed twice daily with concentrate feed and kleingrass. Ruminal contents were collected before morning feeding. Samples were squeezed and strained through four layers of surgical gauze and pooled in an amber bottle with an oxygen-free headspace, which was subsequently capped after collection. Collected samples were immediately transported to the laboratory while being maintaining at a temperature of 39 °C [49]. Seventy milliliters of rumen fluid were dispensed into serum bottles containing each treatment and 2.5 g dry matter of ground corn grain served as substrate, mixed, and flushed with CO 2 [50]. Samples were in triplicate and incubated at 39 °C for 3, 6, 12, and 24 h while shaking horizontally at 100 rpm, as described by Hattori and Matsui [51]. The buffer used in treatments is composed of calcium carbonate, magnesium oxide, sodium carbonate, and calcified seaweed (Rupromin Balance™, Rotterdam, Netherlands). Treatments consisted of CON (negative control, no buffer added), BC 0.3% (low buffering capacity, 0.3% buffer), BC 0.5% (medium buffering capacity, 0.5% buffer), and BC 0.9% (high buffering capacity, 0.9% buffer). The buffer and the concentrate given to experimental animals were supplied by Purina ® Cargill, Korea. The ingredients and chemical composition of the experimental concentrate offered are presented in Table 5. Treatments were initially tested for determining their neutralizing (NC) and buffering capacity (BC) through titration using 2N acetic acid from its initial pH to 6.50, and 5.50, respectively (Table 6). The buffering agents used in every treatment are in powdered form. Table 5 Ingredients and chemical composition of experimental concentrate Ingredients a Percentage (%) Corn fine 31.17 Corn gluten feed 21.00 Soy hulls 13.00 Wheat fine 10.00 Rice bran 5.00 Wheat flour 5.32 DDGS 3.40 Molasses 3.00 Palm kernel meal 2.14 Limestone fine 1.90 Palm kernel meal (Solvent) 1.80 CMS 1.50 Brown rice 0.45 Salt 0.17 Mineral/Vitamin premix 1) 0.15 Total 100.00 Calculated nutrients, as fed Crude Protein 13.03 Crude Fat 3.84 Ash 5.77 NDF 23.44 Moisture 11.50 Crude Fiber 8.65 Calcium 1.00 Phosphorus 0.48 Ca/P 2.09 Sulfur 0.20 Potassium 0.84 Magnesium 0.24 Sodium 0.21 TDN 76.44 a DDGS, dried distillers’ grains with solubles; CMS, condensed molasses solubles; NDF, neutral detergent fiber; TDN, total digestible nutrients. 1) Mineral & vitamin premix contained vit. A 2,650,000 IU, vit. D 3 530,000 IU, vit. E 1,050 IU, niacin 10,000 mg, Mn 4,400 mg, Zn 4,400 mg, Fe 13,200 mg, Cu 2,200 mg, iodine 440 mg, and Co, 440 mg/kg of Grobic-DC provided from Bayer Health Care (Leverkusen, Germany) Table 6 Titration results to determine the neutralizing and buffering capacity of sample buffers used in treatments Parameters Sample buffers d SEM P- value BC 0.3% BC 0.5% BC 0.9% All Linear Initial pH 6.94 a 7.09 b 7.42 c 0.007 <0.001 <0.001 Neutralizing capacity (mmol/L) 0.03 a 0.06 b 0.16 c 0.001 <0.001 <0.001 Buffering capacity (mmol/L) 0.16 a 0.30 b 0.43 c 0.003 <0.001 <0.001 a,b,c Within row indicate linear effect ( P < 0.05) d BC 0.3% (0.3% buffer); BC 0.5% (0.5% buffer); BC 0.9% (0.9% buffer) Analyses of in vitro rumen fermentation parameters and buffering capacity Ruminal fermentation parameters were monitored at the end of each incubation time period. Total gas production was measured from each serum bottle after the incubation time using a pressure meter (Laurel Electronics, Inc., Costa Mesa, Calif., USA). Consequently, a needle channel connected to the machine was extended into the sealed fermentation bottle for measuring positive pressure created by the gas build up inside the bottle. A gas flow regulator was then opened to allow gas flow inside a syringe barrel and the plunger was subsequently pulled gradually until the pressure reading on the machine display was zero. The volume of gas trapped inside the barrel was recorded as the total gas produced [49, 52]. The pH value was determined using a pH meter (Metler Toledo, Germany) after uncapping each serum bottle. Samples of fermenta were also collected into two 1.5 ml microcentrifuge tubes and stored at -80 °C prior to ammonia-nitrogen and VFA analyses. Frozen samples were thawed at room temperature; after which, they were centrifuged for 10 min at 13,000 rpm at 4 °C using a Micro 17TR centrifuge (Hanil Science Industrial, Korea). The resulting supernatant was used for ammonia-nitrogen and VFA concentration analyses. Ammonia-nitrogen concentration was measured according to the colorimetric method developed by Chaney and Marbach [53] using a Libra S22 spectrophotometer (Biochrom Ltd., CB40FJ, England) at an absorbance of 630 nm. NH 3 -N is the vital source of nitrogen for microbial protein synthesis in the rumen [54]. Analysis of volatile fatty acid concentration was done using high-performance liquid chromatography (Agilent Technologies 1200 series, Tokyo, Japan) with a UV detector set at 210 nm and 220 nm. Samples were isocratically eluted with 0.0085N H 2 SO 4 at a flow rate of 0.6 mL/min and a column temperature of 35 °C. Ruminal fluid pH was recorded following 1 min of equilibration. Buffering capacity, defined as the resistance to change in pH from pH 7 to 5, was determined by titrating a 30 ml aliquot of ruminal fluid with continuous stirring from its initial pH to pH 5 with 1N HCl and titrating an additional 30 ml aliquot from its initial pH to a pH of 7 with 1N NaOH. If the initial pH was higher than 7, only the volume of acid required to reduce the pH from 7 to 5 was recorded. Buffering capacity was converted to milliequivalents per liter as follows: BC = [(milliliters of 1N HCl) + (milliliters of 1N NaOH)] × 10 3 /30 [15]. Analysis of rumen fermentation characteristics in Hanwoo steers In vivo experiment was conducted using four Hanwoo steers (765 ± 60 kg body weight; 24 mos. of age) in a 4 × 4 Latin square design to assess the effects of treatments on rumen fermentation characteristics and ruminal bacterial composition and diversity of the experimental animals for four months. The feeding trial was conducted with 4 treatments comprised of CON which served as the negative control, BC 0.3% , BC 0.5% , and BC 0.9% . The Hanwoo steers were fed daily of 2:8 forage and concentrate ratio in 2 equal portions at 0900 and 1600 h. Animals in all treatments received the same vaccinations, medications, and were under the same management programs unless otherwise stated. Steers were confined in free-stall barns and had free access to water and exercise lots. Rumen fluid samples were collected before morning feeding using an oral stomach tube on the 30 th day right before transitioning to the next feeding trial for the analysis of ruminal fermentation parameters. These parameters were all evaluated using the same protocol as used in the in vitro experiment. However, rumen pH change in every experimental period of about 30 days was monitored using eCow (hathor.ecow.co.uk). It was done basically to monitor the occurrence of acidosis through a pH value of <5.8 for several hours a day. 16S rRNA amplicon sequencing and metataxonomic analyses Samples obtained from each treatment were sent to Macrogen, Korea for DNA extraction, 16S rRNA sequencing and microbiome analysis. In brief, DNA was extracted using DNeasy Power Soil Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was quantified using Quant-IT PicoGreen (Invitrogen). The sequencing libraries were prepared according to the Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 region. The input gDNA was PCR amplified with 1 × reaction buffer, 1 nM of dNTP mix, 500 nM each of the universal F/R PCR primer, and 2.5 U of Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA). The cycle condition for 1st PCR was 3 min at 95 °C for heat activation, and 25 cycles of 30 sec at 95 °C, 30 sec at 55 °C and 30 sec at 72 °C, followed by a 5-min final extension at 72 °C. The universal primer pair with Illumina adapter overhang sequences used for the first amplification was V3-F (5’-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3’) and V4-R (5’- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3’). The 1st PCR product was purified with AMPure beads (Agencourt Bioscience, Beverly, MA). Following purification, the 2 uL of 1st PCR product was PCR amplified for final library construction containing the index using NexteraXT Indexed Primer. The cycle condition for 2nd PCR was the same as the 1st PCR condition except for 10 cycles. The PCR product was purified with AMPure beads. The final purified product is then quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Sequencing was done using the Illumina Miseq (Illumina Inc., San Diego, CA, USA) platform. The raw data files (fastq) containing the sequenced paired-end (PE) reads were obtained using the bcls2fastq package (Illumina Inc., San Diego, CA, USA) from the base call binary data produced by real-time analysis. The PE raw reads were filtered from adapter sequences using Scythe (v0.994) [55] and Sickle [56] programs then assembled using Fast Length Adjustment of Short Reads (FLASH 1.2.11) [57]. Assembled reads were quality filtered and trimmed for short and extra-long reads, and duplicate reads were removed, then clustered at 100% identity using CD-HIT-OTU [58]. Chimeric reads were identified and the initial clusters were recruited to primary clusters. Then, noise filtering was done and the remaining non-chimeric clusters were binned to operational taxonomic units (OTU) following a greedy algorithm with a cut-off value of 97% species level identity using CD-HIT-OTU [58]. Representative sequences from the clustered OTU were taxonomically assigned using Quantitative Insights Into Microbial Ecology (QIIME Version 1) [18] from the NCBI 16S rRNA database, and the taxonomy composition was generated using QIIME-UCLUST [59]. The produced bacterial taxonomy and composition data were used to generate a biological information matrix (BIOM) [60] in Mothur [61]. The generated BIOM file were used to visualize the alpha and beta diversity indices, and the bacterial composition using programs utilized by Metagenomics Core Microbiome Exploration Tool (MetaCOMET) [17]. Statistical analysis Data analysis was performed using Statistical Analysis Systems (SAS) version 9.1 (SAS Institute Inc., Cary, NC). The data of rumen fermentation, alpha diversity indices and relative abundance of individual taxa of rumen microbiota were statistically evaluated using Proc general linear model (GLM) for a completely randomized design. All treatments in the in vitro experiment were conducted in triplicate and Duncan’s Multiple Range Test (DMRT) was used to identify differences between specific treatments. The linear effects of different buffering capacity concentrations were analyzed using orthogonal polynomial coefficients to describe the functional relationships among the control and treatment groups. A P < 0.05 was considered indicative of significant differences. Abbreviations BC: Buffering capacity; NaHCO 3 : Sodium bicarbonate; MgO: Magnesium oxide; DDGS: dried distillers’ grains with solubles; CMS: condensed molasses solubles; NDF: neutral detergent fiber; TDN: total digestible nutrients; SEM: standard error of the mean; NH 3 -N: Ammonia nitrogen; VFA: Volatile fatty acid; DNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction; FLASH: Fast Length Adjustment of Short Reads; OTU: Operational taxonomic units; QIIME: Quantitative Insights Into Microbial Ecology; BIOM: Biological information matrix; MetaCOMET: Metagenomics Core Microbiome Exploration Tool; SAS: Statistical Analysis Systems; GLM: General linear model; DMRT: Duncan’s Multiple Range Test; PCoA: Principal Coordinate Analysis Declarations Acknowledgment This research was supported and funded by Provimi Singapore Pte Ltd, 138 Market Street, #17-01 CapitaGreen, Singapore 048946. Authors’ contributions Conceptualization: SSL., CDJ, TGK, JSL, KKC. Supervision: SSL. Experiment: SCR, CDJ. Data curation: SCR, CDJ. Formal analysis: SCR, CDJ, SSL. Methodology: SCR, CDJ, SSL. Software: SHKim, ARS, YIC, Sung Sill Lee (SSL). Validation: SSL. Investigation: SCR, CDJ, SSL. Writing – original draft: SSL, CDJ, SCR, TGK, JSL. Writing – review and editing: SCR, LLM, SHKang, SSL. All authors have read and agreed to the published version of the manuscript. Funding This research is funded by Provimi Singapore Pte Ltd, 138 Market Street, #17-01 CapitaGreen, Singapore 048946. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Animals used in this experiment and all experimental protocols were reviewed and approved by the Sunchon National University Animal Research Ethics Committee (SCNU IACUC, approval number: SCNU IACUC-2018-01). All experiments were performed in accordance with the guidelines and regulation set by the governing body. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University, 413 Jungangno, Jeonnam 57922, Suncheon, Republic of Korea. 2 The University of Queensland Diamantina Institute, Faculty of Medicine, Brisbane, Australia. 3 Animal Disease and Diagnostic Laboratory, Department of Animal Science and Technology, Sunchon National University, 413 Jungangno, Jeonnam 57922, Suncheon, Republic of Korea. 4 Rupromin Balance™, 5th. Bonsol Blg. 445, Teheran-ro, Gangnam-gu, Seoul 06158, Republic of Korea. 5 Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea. References Lee M, Jeong S, Seo J, Seo S. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-354029","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":18353632,"identity":"2323b1af-f01b-4acf-b31c-2c2c89ee6e36","order_by":0,"name":"Sonny Ramos","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sonny","middleName":"","lastName":"Ramos","suffix":""},{"id":18353633,"identity":"4d9ff1aa-01c3-4fde-aef5-2e4acd7f5af1","order_by":1,"name":"Seon Ho Kim","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seon","middleName":"Ho","lastName":"Kim","suffix":""},{"id":18353634,"identity":"49536b9d-9ffb-4593-952c-3e02a2dcc638","order_by":2,"name":"Chang Dae Jeong","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chang","middleName":"Dae","lastName":"Jeong","suffix":""},{"id":18353635,"identity":"6e02c7c0-f675-47d2-802d-ae8ef4ecb8ee","order_by":3,"name":"Lovelia L. Mamuad","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lovelia","middleName":"L.","lastName":"Mamuad","suffix":""},{"id":18353636,"identity":"1b4e8b34-0f2c-40bf-a7a9-eb1addc5491e","order_by":4,"name":"A-rang Son","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"A-rang","middleName":"","lastName":"Son","suffix":""},{"id":18353638,"identity":"2812d3e9-07bb-41f2-b52f-fb20d563f8bc","order_by":5,"name":"Seung Ha Kang","email":"","orcid":"","institution":"The University of Queensland Diamantina Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Ha","lastName":"Kang","suffix":""},{"id":18353639,"identity":"b00013d6-c6a6-4d3c-aa41-a023951e41fc","order_by":6,"name":"Yong Il Cho","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Il","lastName":"Cho","suffix":""},{"id":18353640,"identity":"105785d7-d08a-427b-8280-e8739f8b4347","order_by":7,"name":"Tae Gyu Kim","email":"","orcid":"","institution":"Rupromin Balance™","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tae","middleName":"Gyu","lastName":"Kim","suffix":""},{"id":18353642,"identity":"b53e33e7-9c98-4be2-9068-643870262321","order_by":8,"name":"Jin Sung Lee","email":"","orcid":"","institution":"Rupromin Balance™","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jin","middleName":"Sung","lastName":"Lee","suffix":""},{"id":18353645,"identity":"d62d0f4d-1679-49aa-98a0-d2de99cdeba1","order_by":9,"name":"Kwang Keun Cho","email":"","orcid":"","institution":"Gyeongnam National University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kwang","middleName":"Keun","lastName":"Cho","suffix":""},{"id":18353646,"identity":"52a3e73b-1e59-482d-bc00-19884fcd97b0","order_by":10,"name":"Sung Sill Lee","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung","middleName":"Sill","lastName":"Lee","suffix":""},{"id":18353647,"identity":"25107674-cc9c-4d17-baf2-ccd23f8299cf","order_by":11,"name":"Sang Suk Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYNCCCjgrgVgtZ0jWwthGihbzGdmJjwvn1cmbSyQwfvjBkJZPUIvMjdzNxjO3HTbcOSOBWbKHIceygZAWCYncbdK82w4kGNxIYJAGBoQBQVuAWrb/5p1TB9LC/JtYLduYeRuYQVrYgLbkEKGF5+1maZ5jhw03nHnYZtljkEaEFvbcjZ95aurkDY4nH77xoyKZsBYkwNjAwECShlEwCkbBKBgFOAEArns0Z55lvNcAAAAASUVORK5CYII=","orcid":"","institution":"Sunchon National University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sang","middleName":"Suk","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2021-03-23 04:29:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-354029/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-354029/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":7401420,"identity":"db18cbe4-f395-4db1-b59b-5a5530244870","added_by":"auto","created_at":"2021-03-26 21:55:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42519,"visible":true,"origin":"","legend":"Boxplot representation of alpha diversity indices: (a) chao1, (b) Shannon, and (c) observed OTUs, between treatment groups. Alpha-diversity metrics visualization were done in MetaCOMET [17] and computed using QIIME [18]. CON (no buffer added); BC0.3% (0.3% buffer); BC0.5% (0.5% buffer); BC0.9% (0.9% buffer)","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-354029/v1/d7e8f307e5963baa789eedbc.png"},{"id":7401422,"identity":"3bd756cc-1edb-405c-8865-a8fa7a82e909","added_by":"auto","created_at":"2021-03-26 21:55:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":194951,"visible":true,"origin":"","legend":"Relative abundance of the observed (a) phyla, (b) genera, and (c) species from the four different treatments. Relative abundance was computed using QIIME [18]. CON (no buffer added); BC0.3% (0.3% buffer); BC0.5% (0.5% buffer); BC0.9% (0.9% buffer); asterisk (*): represents significant differences (P \u003c 0.05)","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-354029/v1/3c6163a66d15275fefdbdfdf.png"},{"id":7401421,"identity":"00161def-b153-44c7-9014-0a8b32a0b9ba","added_by":"auto","created_at":"2021-03-26 21:55:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":88332,"visible":true,"origin":"","legend":"Membership-based representation of unique, shared and core bacterial community of rumen microbiome after treatment supplementation with varying level of buffering capacity, and the total size of observed species per treatment. Venn diagram was generated in MetaCOMET [17] using jvenn [19]. CON (no buffer added); BC0.3% (0.3% buffer); BC0.5% (0.5% buffer); BC0.9% (0.9% buffer)","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-354029/v1/4db8b3f4cd14a7713bb43a36.png"},{"id":7401236,"identity":"dc6eee75-90e9-40b9-9042-2a80384fabab","added_by":"auto","created_at":"2021-03-26 21:52:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":63436,"visible":true,"origin":"","legend":"Heatmap presentation of relative abundance of representative families of observed OTU’s. Treatments (columns) and families (rows) are clustered using Bray-Curtis dissimilarity test and Ward linkage. Normalized relative abundance are plotted from low (red), mid (peach), and high (blue). Heatmap clustering was generated in MetaCOMET [17] utilizing the InCHlib application [20]. CON (no buffer added); BC0.3% (0.3% buffer); BC0.5% (0.5% buffer); BC0.9% (0.9% buffer); asterisk (*): represents significant differences (P \u003c 0.05)","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-354029/v1/fab7616ee3bc272c13d6081d.png"},{"id":7401232,"identity":"4cb32eb3-aa29-4e88-b72d-ca55f81536c3","added_by":"auto","created_at":"2021-03-26 21:52:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31149,"visible":true,"origin":"","legend":"Principal Coordinate Analysis (PCoA) of all samples using Bray-Curtis distance derived from the subset of identified OTUs. PCoA plot was generated using EMPeror [21]. CON (no buffer added); BC0.3% (0.3% buffer); BC0.5% (0.5% buffer); BC0.9% (0.9% buffer)","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-354029/v1/e71412f9e9ae54b0e13b6052.png"},{"id":13682520,"identity":"50a5b3ae-e0a5-46d6-9cd3-83a11cc43376","added_by":"auto","created_at":"2021-09-17 11:57:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":964854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-354029/v1/67ec2fcd-d236-47a9-bcb2-e279a35272f2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIncreasing Buffering Capacity Alters Rumen Microbiota Composition and Enhances Rumen Fermentation Characteristics of High-Concentrate Fed Hanwoo Steers\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eEnergy and essential nutrients are obtained by ruminants through a complicated symbiotic relationship with the rumen microbiome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and bacterial community alterations can affect the productivity and health of the host animal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A high forage diet is usually switched to a high concentrate diet to improve the productivity of the ruminants; however, it alters the rumen ecosystem due to high non-structural carbohydrates level [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Meanwhile, the core ruminal bacterial community is dominated by the phyla Bacteroidetes, Firmicutes, and Proteobacteria regardless of diet composition [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, a high-concentrate diet induces death and cell lysis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], thus decrease in abundance of Firmicutes in the rumen [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It also enhances the growth of lactic acid utilizers like \u003cem\u003eMegasphaera elsdenii\u003c/em\u003e, \u003cem\u003eSelenomonas ruminantium\u003c/em\u003e, and \u003cem\u003eVeillonella parvula\u003c/em\u003e resulting to a drastic reduction of fiber-degrading bacteria such as \u003cem\u003eFibrobacter succinogenes\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e spp [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Feeding of highly fermentable diets is the current practices in high producing beef to increase growth rates, but it causes microbial disturbances resulting to digestive disorders such as ruminal acidosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The rapid fermentation of non-structural carbohydrates resulted in the accumulation of volatile fatty acid and lactic acid in the rumen causing a drastic decrease in pH [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hence, the use of buffer could be useful to resist changes in rumen pH whenever cattle are being fed with high concentrate, low forage, fermented and fine-chopped forage [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Compounds that increase the buffering capacity of ruminal fluid help maintain a more stable ruminal pH and direct neutralization of VFA especially during a diet or fermentation-related acid challenge [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Rumen buffering could avert the sudden decrease in pH, thus could enhance rumen microbial growth, activity and diversity, microbial protein synthesis, and fermentation end product [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Buffering capacity (BC) is then referred to as the number of moles of H\u003csup\u003e+\u003c/sup\u003e that should be added to a 1L solution to decrease pH by 1 unit [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Weak acids and bases are known to provide better buffering in comparison to strong acids and bases because of the equilibrium establishment between the acid and the conjugate base [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious studies have reported that adding a buffer solution, such as sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e) with magnesium oxide (MgO) increased dry matter intake when corn silage was the sole or major source of forage in the diet [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. NaHCO\u003csub\u003e3\u003c/sub\u003e is commonly used in preventing ruminal acidosis because it provides a natural buffer; however, its high solubility limits the buffering activity against acidic conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Le Ruyet and Tucker [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] proved that NaHCO\u003csub\u003e3\u003c/sub\u003e had high BC in an \u003cem\u003ein vitro\u003c/em\u003e study. It contained 26% more actively buffering the CO\u003csub\u003e3\u003c/sub\u003e portion of the molecule that is important to neutralize the acid. MgO, on the other hand, appears to work efficiently in combination with NaHCO\u003csub\u003e3\u003c/sub\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Shaver et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] stated that supplementing NaHCO\u003csub\u003e3\u003c/sub\u003e and MgO in a 3:1 ratio is the recommended level of dietary buffer for the best response. The efficiency and mechanisms of buffer responsible for alleviating chronic acidosis are variable and often inconsistent [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Research on different level of buffering capacity in enhancing rumen fermentation parameters and microbiome during high concentrate diet has not yet been investigated. In the present work, ruminal metataxonomic and fermentation characteristics analyses were conducted using rumen fluid samples to evaluate the effect of different buffering capacities on \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e trials in high-concentrate fed Hanwoo steers.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of different buffering capacities on \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe buffering capacity of BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e were significantly greatest (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) after 24 h incubation compared to BC\u003csub\u003e0.3%\u003c/sub\u003e and CON (Table 1). Both BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e exhibited significantly highest (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) buffering capacity value of 106.00 meq/L, hence had a similar effect on \u003cem\u003ein vitro \u003c/em\u003eafter 24 h. The ruminal pH obtained from BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e showed similar effects and were consistently higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) than the other treatments throughout the incubation period. In gas production, BC\u003csub\u003e0.9%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e, and BC\u003csub\u003e0.3%\u003c/sub\u003e had significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) gas produced than CON and showed similar effects at 3 and 12 h incubation. Ruminal NH\u003csub\u003e3\u003c/sub\u003e-N concentration was significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e, thus, it also had a similar effect on this parameter. However, at 6 and 12 h, no effect observed on treatments except that BC\u003csub\u003e0.5%\u003c/sub\u003e tended to increase (\u003cem\u003eP\u003c/em\u003e = 0.073) NH\u003csub\u003e3\u003c/sub\u003e-N concentration followed by BC\u003csub\u003e0.9%\u003c/sub\u003e and the rest treatments.\u003c/p\u003e\n\u003cp\u003eSignificantly higher concentrations (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) of acetate were observed in BC\u003csub\u003e0.9%\u003c/sub\u003e at 12 h; however, BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.3%\u003c/sub\u003e obtained the highest value (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) after 24 h (Table 2). Propionate and butyrate concentrations were both highest (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in BC\u003csub\u003e0.3%\u003c/sub\u003e and BC\u003csub\u003e0.9%\u003c/sub\u003e at 6 h. Subsequently, distinct effects of BC\u003csub\u003e0.3%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e, and BC\u003csub\u003e0.9%\u003c/sub\u003e were observed at 24 h which had significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) propionate concentrations than CON. A similar pattern was noticeable with butyrate at 12 h such that BC\u003csub\u003e0.3%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e, and BC\u003csub\u003e0.9%\u003c/sub\u003e obtained the highest concentration (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) compared with CON. During this period, a similar effect can be seen between the 3 treatments; however, no significant effect was observed after 24 h. Total volatile fatty acid contents were greater (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) in BC\u003csub\u003e0.3%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.9%\u003c/sub\u003e at 12 h but had a slight change after 24 h. At this time point, treatments BC\u003csub\u003e0.3%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e were highest (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) compared to BC\u003csub\u003e0.9%\u003c/sub\u003e and CON. Furthermore, there were no treatment effects on acetate to propionate ratio after 24 h incubation. Consequently, increasing the concentration of buffering capacity showed linear effects (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) on pH, total gas production, NH\u003csub\u003e3\u003c/sub\u003e-N, and at some certain time point of individual VFA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Effect of different buffering capacity concentrations on \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation parameters at 3, 6, 12 and 24 h\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e\u003cstrong\u003eTime (h)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"273\"\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment \u003csup\u003ee\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"118\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e\u003cstrong\u003eCON\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.3%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.5%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.9%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u003cstrong\u003eAll\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"92\"\u003e\n\u003cp\u003eBuffering capacity (meq/L)\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e76.44\u003csup\u003ed,z\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e83.89\u003csup\u003ec,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e87.56\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e90.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.398\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e76.45\u003csup\u003ec,z\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e84.17\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e92.89\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e92.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.246\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e85.33\u003csup\u003ec,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e87.22\u003csup\u003ec,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e96.67\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e99.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.469\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e100.22\u003csup\u003ec,z\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e102.56\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e106.00\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e106.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.462\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"92\"\u003e\n\u003cp\u003epH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e6.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e6.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e6.08\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e6.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.052\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e5.68\u003csup\u003ec,z\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e5.80\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e5.91\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e5.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e5.42\u003csup\u003ec,z\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e5.54\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e5.60\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e5.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e5.14\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e5.15\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e5.22\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e5.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.003\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"92\"\u003e\n\u003cp\u003eTotal gas (mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e74.67\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e82.00\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e81.67\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e82.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e1.287\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.004\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e122.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e124.33\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e123.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e123.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e1.353\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.777\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.488\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e169.00\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e187.00\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e179.00\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e182.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e2.492\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.010\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e251.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e269.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e256.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e256.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3.877\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.078\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.195\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"92\"\u003e\n\u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N (mg/dL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e11.26\u003csup\u003ec,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e12.24\u003csup\u003eb,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e13.05\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e13.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.205\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.003\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e13.35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e13.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e16.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e14.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.569\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.178\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.095\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e13.90\u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e14.95\u003csup\u003exy\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e16.23\u003csup\u003ex\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e15.84\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.482\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.073\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.010\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e19.90\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e20.39\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e21.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"62\"\u003e\n\u003cp\u003e22.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e0.378\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.052\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea-d\u003c/sup\u003e Means with different superscripts in a row differ significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ee\u003c/sup\u003e CON (no buffer added); BC\u003csub\u003e0.3%\u003c/sub\u003e (0.3% buffer); BC\u003csub\u003e0.5%\u003c/sub\u003e (0.5% buffer); BC\u003csub\u003e0.9%\u003c/sub\u003e (0.9% buffer)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ex,y,z\u003c/sup\u003e Means within a row indicate linear effect among CON, BC\u003csub\u003e0.3%\u003c/sub\u003e, and BC\u003csub\u003e0.5%\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 \u003c/strong\u003eVolatile fatty acid production during \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation incubated at 3, 6, 12, and 24 h\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"104\"\u003e\n\u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"49\"\u003e\n\u003cp\u003e\u003cstrong\u003eTime (h)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"289\"\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment \u003csup\u003ee\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"64\"\u003e\n\u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"112\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e\u003cstrong\u003eCON\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.3%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.5%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.9%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e\u003cstrong\u003eAll\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"104\"\u003e\n\u003cp\u003eAcetate (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e100.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e94.81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e93.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e90.36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e2.505\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.265\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.237\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e100.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e100.31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e99.79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e100.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.592\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.901\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.775\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e102.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e104.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e103.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e107.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.291\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.412\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e103.71\u003csup\u003ec,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e112.05\u003csup\u003eab,xy\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e117.27\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e108.47\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e2.182\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.018\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"104\"\u003e\n\u003cp\u003ePropionate (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e27.44\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e26.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e24.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e25.59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e1.395\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.598\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.280\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e27.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e28.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e27.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e28.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.085\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.421\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e32.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e32.65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e30.59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e33.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.756\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.227\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.350\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e35.24\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e40.32\u003csup\u003ea,xy\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e42.31\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e38.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e1.203\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"104\"\u003e\n\u003cp\u003eButyrate (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e17.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e20.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e19.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e19.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.501\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.088\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.098\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e22.23\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e22.78\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e20.95\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e22.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.276\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.006\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e27.46\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e40.52\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e39.69\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e40.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e1.660\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e53.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e54.58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e56.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e54.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.783\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.327\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.125\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"104\"\u003e\n\u003cp\u003eTotal VFA (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e145.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e141.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e137.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e135.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e3.381\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.430\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.285\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e150.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e151.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e148.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e151.57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.803\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.077\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.196\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e162.18\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e177.51\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e173.63\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e181.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e2.424\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e192.80\u003csup\u003ec,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e206.95\u003csup\u003eab,xy\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e216.01\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e201.51\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e3.447\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"4\" width=\"104\"\u003e\n\u003cp\u003eA:P ratio\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e3.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e3.57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e3.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e3.53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.134\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.618\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.670\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e3.60\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e3.50\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e3.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e3.54\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.018\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.719\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e3.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e3.20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e3.41\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e3.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.084\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.465\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.266\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"49\"\u003e\n\u003cp\u003e24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"71\"\u003e\n\u003cp\u003e2.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e2.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e2.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e2.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e0.069\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\n\u003cp\u003e0.434\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.178\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea-c\u003c/sup\u003e Means with different superscripts in a row differ significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ee\u003c/sup\u003e CON (no buffer added); BC\u003csub\u003e0.3%\u003c/sub\u003e (0.3% buffer); BC\u003csub\u003e0.5%\u003c/sub\u003e (0.5% buffer); BC\u003csub\u003e0.9%\u003c/sub\u003e (0.9% buffer)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ex,y\u003c/sup\u003e Means within a row indicate linear effect among CON, BC\u003csub\u003e0.3%\u003c/sub\u003e, and BC\u003csub\u003e0.5%\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of different buffering capacities on rumen fermentation characteristics in Hanwoo steers \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of different buffering capacity concentrations on rumen fermentation characteristics of Hanwoo steers in four treatments are presented in Table 3. Average pH had no significant effects among CON and treatments. However, BC\u003csub\u003e0.3%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e, and BC\u003csub\u003e0.9%\u003c/sub\u003e had significantly higher (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) buffering capacity value than CON, and showed linearly significant effect (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Ammonia-nitrogen, acetate to propionate ratio, individual and total VFA concentrations of rumen fluid from steers under all treatments were not significant and showed similar effects after the \u003cem\u003ein vivo\u003c/em\u003e experiment.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 \u003c/strong\u003eEffect of different buffering capacity concentrations on rumen fermentation characteristics in Hanwoo steers\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"182\"\u003e\n\u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"273\"\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment \u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e\u003cstrong\u003eCON\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.3%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.5%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.9%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e\u003cstrong\u003eAll\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eAverage pH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e6.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e6.56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e6.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e6.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e0.146\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.226\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.528\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eBuffering capacity (meq/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e83.47\u003csup\u003eb,y\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e99.53\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e101.00\u003csup\u003ea,x\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e94.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e3.058\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.004\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N (mg/dL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e4.84\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e4.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e5.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e4.59\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e1.260\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.945\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.794\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eTotal VFA (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e47.24\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e45.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e54.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e55.87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e5.234\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.632\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.563\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eAcetate (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e29.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e27.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e30.70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e35.56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e2.421\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.532\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.890\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003ePropionate (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e11.30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e9.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e14.37\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e12.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e2.190\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.412\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.380\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eButyrate (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e6.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e8.76\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e9.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e7.56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e1.556\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.652\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.299\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"182\"\u003e\n\u003cp\u003eA:P ratio\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"63\"\u003e\n\u003cp\u003e2.81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"67\"\u003e\n\u003cp\u003e3.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\n\u003cp\u003e2.13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"74\"\u003e\n\u003cp\u003e2.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e0.451\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"54\"\u003e\n\u003cp\u003e0.431\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e0.370\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea-b\u003c/sup\u003e Means with different superscripts in a row differ significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e CON (no buffer added); BC\u003csub\u003e0.3%\u003c/sub\u003e (0.3% buffer); BC\u003csub\u003e0.5%\u003c/sub\u003e (0.5% buffer); BC\u003csub\u003e0.9%\u003c/sub\u003e (0.9% buffer)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ex,y\u003c/sup\u003e Means within a row indicate linear effect among CON, BC\u003csub\u003e0.3%\u003c/sub\u003e, and BC\u003csub\u003e0.5%\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial diversity of the rumen contents of Hanwoo steers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe boxplot representation of alpha diversity indices is shown in Figure 1. Alpha diversity indices are composite indices that reflect abundance and consistency. Chao1 which reflect the OTU abundance in the samples showed that BC\u003csub\u003e0.9%\u003c/sub\u003e was the highest among treatments followed by BC\u003csub\u003e0.5%\u003c/sub\u003e and the rest of the treatments (Figure 1a). Shannon index which reflects the diversity of the OTU in samples presented BC\u003csub\u003e0.9%\u003c/sub\u003e as the most diverse among treatments and BC\u003csub\u003e0.3%\u003c/sub\u003e being the least (Figure 1b). Moreover, Figure 1c showed the boxplot of OTUs of observed species from the samples. The number of OTUs in BC\u003csub\u003e0.9%\u003c/sub\u003e was higher followed by BC\u003csub\u003e0.5%\u003c/sub\u003e and the rest of the treatments. The diversity index is used to analyze the temporal and spatial changes in species composition which reflects whether bacterial communities between groups have differences. Our results showed that the rumen bacterial composition of BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.9%\u003c/sub\u003e had overall higher alpha diversity than other treatment groups, although no significant difference was observed after statistical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of treatments on bacterial community composition of Hanwoo steers rumen contents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial taxonomic compositions at the phylum, genera, and species level are shown in Figure 2. Results at the phylum level revealed that 15 bacterial phyla were identified in the rumen digesta samples of Hanwoo steers (Figure 2a). The majority of the sequences obtained from all treatments belonged to Bacteroidetes followed by Firmicutes. It was noticeable that BC\u003csub\u003e0.3%\u003c/sub\u003e had the highest abundance of Bacteroidetes (71.90%) and lowest Firmicutes (22.13%). On the contrary, BC\u003csub\u003e0.9%\u003c/sub\u003e had the lowest abundance of Bacteroidetes (54.19%) among treatments; however, BC\u003csub\u003e0.5%\u003c/sub\u003e had the highest Firmicutes (33.84%) relative abundance. Furthermore, Proteobacteria was dominant upon incorporating BC\u003csub\u003e0.9%\u003c/sub\u003e in the diet resulting in declining its abundance from that of BC\u003csub\u003e0.3%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e. Spirochaetes increased its abundance when animals received supplementation of BC\u003csub\u003e0.9%\u003c/sub\u003e, whereas there was a sudden decrease once the steers received BC\u003csub\u003e0.5%\u003c/sub\u003e in their diet. At the genus level (Figure 2b), \u003cem\u003ePrevotella \u003c/em\u003ewas the predominant among genera in all treatments followed by \u003cem\u003eBacteroides\u003c/em\u003e (10.36%) and \u003cem\u003eRuminococcus \u003c/em\u003e(8.93%) in BC\u003csub\u003e0.3%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e, respectively. Furthermore,\u003cem\u003e Succiniclasticum \u003c/em\u003ewas dominant among genera after supplementing BC\u003csub\u003e0.5%\u003c/sub\u003e; however, a decreasing abundance of \u003cem\u003ePaludibacter \u003c/em\u003ewas noticeable as BC\u003csub\u003e0.5%\u003c/sub\u003e is incorporated into the diet, which had a reverse effect as did CON. Species-level analyses revealed that \u003cem\u003ePrevotella ruminicola \u003c/em\u003epredominated the treatments CON, BC\u003csub\u003e0.3%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.9%\u003c/sub\u003e with the relative abundance of 24.85%, 32.16%, 26.73%, and 23.17%, respectively (Figure 2c). The comparison of single species analyzed through statistical analysis showed a significant effect of the treatments only in the case of \u003cem\u003ePrevotella brevis.\u003c/em\u003e This species was more abundant (\u003cem\u003eP\u003c/em\u003e = 0.015) in the CON and as steers received a diet supplemented with BC\u003csub\u003e0.9%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.3%\u003c/sub\u003e its abundance decreased. Owing to the BC\u003csub\u003e0.5%\u003c/sub\u003e supplemented in the diet, a decreasing abundance of \u003cem\u003ePaludibacter propionicigenes \u003c/em\u003ewas observed; however, it increased in CON. Incorporation of BC\u003csub\u003e0.5%\u003c/sub\u003e increased the microbial population of \u003cem\u003eRuminococcus bromii \u003c/em\u003eand \u003cem\u003eSucciniclasticum ruminis. \u003c/em\u003eMoreover, the smaller percentage of BC\u003csub\u003e0.3%\u003c/sub\u003e resulted in a higher abundance of \u003cem\u003eBacteroides massiliensis \u003c/em\u003ewhich led to a sudden decrease in its population as the concentration of treatments increases. Supplementing buffers of different buffering capacity concentration may affect the rumen microbiota through the relative abundance of bacterial species.\u003c/p\u003e\n\u003cp\u003eThe core, shared and unique bacterial community of observed species of the rumen microbiome after treatment of buffer agents with varying level of buffering capacity is presented in Figure 3 as Venn diagram. A total of 211 (59.6%) observed species can be found across all the samples (core), 79 (22.32%) for shared by 2 or 3 samples, and 64 (18.08%) are specific and are distributed to the four samples.\u003c/p\u003e\n\u003cp\u003eThe normalized data presented in Figure 4 shows the clustering based on the similarity of relative abundance between representative families of OTUs (row), and treatments (column). The analysis divided the representative families into two major clusters distinguishing families which represents low relative abundance on all treatments (upper cluster in red), and families that have varying relative abundance between treatments (lower cluster, colored from peach to blue). On the cluster presenting varying abundance between treatments, two sub-clusters were also distinguishable; (1) families which represent variation from very low (red) to average (peach) abundance, and (2) cluster representing families which have average to high (blue) abundance. The cluster in the bottom of the heatmap (labelled) contains the families that represent the above average relative abundance. On this cluster, family Prevotellaceae had branched out because it presents the highest abundance with very small variations between treatments (\u003cem\u003ep \u003c/em\u003e= 0.092). Family Ruminicoccaceae were also found in all treatments, but varying relative abundance was observed, with BC\u003csub\u003e0.5%\u003c/sub\u003e presenting the highest. Families Acidaminococcaceae and Lachnospiraceae were significantly highest (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) in BC\u003csub\u003e0.3%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e, respectively. Also, the Unclassified Clostridiales had significantly highest (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) relative abundance in BC\u003csub\u003e0.5%\u003c/sub\u003e. A certain unclassified family under order Bacteroidales also showed major abundance especially in BC\u003csub\u003e0.3%\u003c/sub\u003e, while families Vibrionaceae and Spirochaetaceae were highest in BC\u003csub\u003e0.9%\u003c/sub\u003e. Meanwhile, the normalized data of relative abundance of representative families of observed OTUs have grouped the control sample together with BC\u003csub\u003e0.3%\u003c/sub\u003e in a single cluster, while BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.9%\u003c/sub\u003e are on their own cluster, which could indicate the effect of varying buffering capacity of buffer agents.\u003c/p\u003e\n\u003cp\u003eThe comparison of the bacterial communities by principal coordinate analysis (PCoA) is presented in Figure 5. The PCoA plots showed close similarity within and between treatments and control, whereas those under BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.3%\u003c/sub\u003e groups showed dispersed distribution of bacterial communities. The PCoA plot showed dissimilarity of bacterial community and revealed a distinct structure between high buffering capacity and low buffering capacity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMonitoring of acidosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe changes in the 24 h mean ruminal pH monitored for 30 d is presented in Table 4. During this period of the feeding challenges, mean pH values were \u0026gt;5.8. Minimum pH was lowest in CON, whereas it was highest in BC\u003csub\u003e0.5%\u003c/sub\u003e. Additionally, BC\u003csub\u003e0.9%\u003c/sub\u003e had a low minimum pH value second to that of CON. It was noticeable that BC\u003csub\u003e0.3%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e had higher minimum and mean pH values compared to BC\u003csub\u003e0.9%\u003c/sub\u003e and CON. Obtained results indicated that the duration of time where pH was \u0026lt;5.8 and 5.8 \u0026lt; 6.0 was longer in CON followed by BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.3%\u003c/sub\u003e. Meanwhile, BC\u003csub\u003e0.3%\u003c/sub\u003e also exhibited good results in the duration of time where pH was approximately 6.0 and above; however, BC\u003csub\u003e0.5%\u003c/sub\u003e had even better effects and did not show any signs of acidosis in the rumen. Based on the data gathered, BC\u003csub\u003e0.5%\u003c/sub\u003e stabilized the pH of rumen preventing it from becoming acidotic.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Changes in the 24 h mean ruminal pH, duration of time where pH was \u0026lt;5.8 monitored for 30 d challenge diet\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"244\"\u003e\n\u003cp\u003e\u003cstrong\u003eItem\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"333\"\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eCON\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.3%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.5%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.9%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003e\u003cem\u003e24 h mean ruminal pH\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003eMinimum\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e5.28\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e5.72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e5.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e5.40\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003eMean\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e6.36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e6.56\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e6.47\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e6.22\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003e\u003cem\u003eDuration of ruminal pH\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003epH \u0026lt;5.8, min/d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e66.29\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e1.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e13.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003epH 5.8 \u0026lt; 6.0, min/d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e90.97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e21.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e10.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e106.45\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"244\"\u003e\n\u003cp\u003epH 6.0 and above, min/d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e1214.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e1296.77\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"86\"\u003e\n\u003cp\u003e1202.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e999.68\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e CON (no buffer added); BC\u003csub\u003e0.3% \u003c/sub\u003e(0.3% buffer); BC\u003csub\u003e0.5% \u003c/sub\u003e(0.5% buffer); BC\u003csub\u003e0.9%\u003c/sub\u003e (0.9% buffer)\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eCurrently, one of the major health issues in dairy farming is the sudden decline of ruminal pH which causes a reduction of feed intake, problems with digestion, and production losses. Cattle health mainly suffers and additional costs in management increase due to its prevalence. Sodium bicarbonate is widely used for the prevention of rumen acidosis because it serves as a natural buffer in the rumen. Despite its buffering ability, it only functions for a short period of time and because of the high solubility, it is rapidly used by the ruminants. Most studies have suggested that magnesium oxide act either as a neutralizer or buffer in rumen or intestine [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It also increases starch digestion in the intestine of animals fed with a high-concentrate diet. This may result in an increase of pH in the small intestines allowing starch-digesting enzymes to become more active [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mao et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] reported that supplementation of the bicarbonate group had higher pH, total gas production, and total VFA concentration although ammonia-nitrogen concentrations remained unaltered. Addition of combined buffers in high concentrate rations altered rumen pH, liquid turnover, and patterns of rumen fermentation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, commercial buffer agent (CBA) is developed as a buffer premix and considered as more powerful alternative to sodium bicarbonate. This premix is a mixture of various raw materials, differing in acid-binding capacity and solubility that contained live yeast, which promoted the conversion of lactate to propionate; thus, improving rumen conditions. Research data have shown its efficiency in maintaining the stability of ruminal pH, thus preventing the stimulation of subacute ruminal acidosis (Provimi\u0026trade;, Rotterdam, Netherlands). Meanwhile, the results of the present study are in accordance with their experimental output.\u003c/p\u003e \u003cp\u003eThe result of the present study showed that BC\u003csub\u003e0.9%\u003c/sub\u003e, as well as the BC\u003csub\u003e0.5%\u003c/sub\u003e, had similar effects on rumen content. Both treatments had significant effects on pH, buffering capacity, and ammonia-nitrogen concentration relative to that of the negative control. An increase in ruminal pH upon supplementation of sodium bicarbonate is a result of dissociation of sodium (Na\u003csup\u003e+\u003c/sup\u003e) and bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Meanwhile, the results on gas production were supported by the claims of Rauch et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Kang and Wanapat [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], who stated that supplementation with sodium bicarbonate enhanced gas production. The increase in gas production might be caused by the dissociation of sodium bicarbonate resulting to increase gas volume because of CO\u003csub\u003e2\u003c/sub\u003e liberation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Also, it might be due to the conversion of some bicarbonate to carbonic acid which soon released as carbon dioxide [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, obtained data from the present study is in accordance with the results of Le Ruyet and Tucker [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] on the temporal effects of ruminal buffers in terms of buffering capacity and pH of ruminal fluid from cows fed a high concentration diet. Buffering compounds increased the ruminal fluid buffering value index and were beneficial in preventing postprandial increases in ruminal fluid hydrogen ion concentration. Shaver et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] also stated that magnesium oxide and sodium bicarbonate were the best rumen buffers, which increased the acetate: propionate ratio and prevented declines in pH. The effect of buffers on VFA in this study was the same as the data obtained by Kang and Wanapat [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] wherein supplementation with buffering agents increased the total VFA. High ruminal VFA concentration is caused by increased carbohydrate fermentation in the rumen [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although the present study did not show a significant effect on molar concentration of VFA, the noticeable increasing numerical values were observed in buffer-supplemented treatments.\u003c/p\u003e \u003cp\u003eSubsequently, the metagenomic survey of bacterial community composition was identified in the rumen digesta samples of Hanwoo steers. Obtained results at the phylum level were in accordance with the data gathered by Nagata et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] wherein the relative abundance of Bacteroidetes was higher during the high-concentrate period of the experimental animals. Additionally, Zhao et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] stated that the microbial community of beef cattle was dominated by Bacteroidetes and Firmicutes at the phylum level regardless of group. An increase in the phylum Bacteroidetes resulted in increased \u003cem\u003ePrevotella\u003c/em\u003e and repressed Firmicutes, which was attributed to decreasing Ruminococcaceae. Dodd et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and Naas et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] indicated that the Bacteroidetes in the rumen represented another numerically dominating phylum that was not associated with cellulose degradation, rather its saccharolytic status is based on limited case studies of noncellulolytic \u003cem\u003ePrevotella\u003c/em\u003e rumen isolates. Because of the ability of \u003cem\u003ePrevotella\u003c/em\u003e to use a variety of substrates, it tends to dominate in the rumen under a range of diets [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the present study, \u003cem\u003ePrevotella ruminicola\u003c/em\u003e appeared to be the predominant species among all treatments. This species constitutes one of the most numerous groups recovered from the rumen and plays important roles in the utilization of polysaccharides of plant origin [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and the metabolism of peptides and proteins [\u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, the low-relative abundance of \u003cem\u003eRuminococcus\u003c/em\u003e (8.93%) in this study was in contrast with the findings obtained by Klieve et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], who used a high-grain diet (75% barley) for the animals, although this genus was identified and largely comprised the cellulolytic bacteria. High propionate concentration of BC\u003csub\u003e0.5%\u003c/sub\u003e might be caused by the high relative abundance of \u003cem\u003eSucciniclasticum ruminis.\u003c/em\u003e This result is in accordance with the study of Van Gylswyk [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], who stated that this species specializes in fermenting and converting succinate to propionate, which is an important precursor of glucose in ruminants. Ueki et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] described \u003cem\u003eBacteroides massiliensis\u003c/em\u003e as a producer of acetate, propionate, and succinate which can explain the increase in molar concentrations of VFA on \u003cem\u003ein vivo\u003c/em\u003e study. The abundance of \u003cem\u003ePaludibacter propionicigenes\u003c/em\u003e might be due to its description as a sugars utilizer and a producer of acetate and propionate, an end product of fermentation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcidosis was defined as impaired ruminal health accompanied by a reversible ruminal pH depression [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Ruminal microbes convert carbohydrates to short-chain fatty acids at a rate that exceeds the rumen\u0026rsquo;s absorptive, buffering, and outflow capacity causing a rapid decrease in ruminal pH [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Data gathered in this experiment agreed with the results obtained by Tucker et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] that the addition of a buffer, especially sodium bicarbonate, was effective in reducing ruminal fluid acidity and retards the drop in pH that normally occurs from 6 to 12 h post-feeding. Also, Zamarre\u0026ntilde;o et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] stated that the use of sodium bicarbonate and magnesium oxide or even mixed antacids were recommended for satisfactory results. They concluded that the increase in buffering capacity and increase in acid consuming capacity contributed to the correction of animal acidosis.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eDifferent BC concentrations were evaluated and results showed that BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e had a similar effect on an in vitro ruminal fermentation. Increasing the concentration of BC showed a linear effect on pH, NH\u003csub\u003e3\u003c/sub\u003e-N, and some incubation time of individual VFA. Metagenomics survey on bacterial abundance revealed that phylum Bacteroidetes dominated all the treatments. A higher abundance of Firmicutes was observed in BC\u003csub\u003e0.5%\u003c/sub\u003e; however, gradually decreased as the BC concentration decreases. Meanwhile, as BC increases, the relative abundance of Proteobacteria also increases. \u003cem\u003ePrevotella ruminicola\u003c/em\u003e dominated all treatments and had the highest abundance in BC\u003csub\u003e0.3%\u003c/sub\u003e. Supplementing BC\u003csub\u003e0.5%\u003c/sub\u003e in the diet increased the abundance of \u003cem\u003eRuminoccocus bromii\u003c/em\u003e and \u003cem\u003eSucciniclasticum ruminis\u003c/em\u003e while a drastic increase in the population of \u003cem\u003eBacteroides massiliensis\u003c/em\u003e in BC\u003csub\u003e0.3%\u003c/sub\u003e. Overall, data gathered from the present study showed that increasing buffering capacity enhances rumen fermentation and alters rumen microbiome which is an important factor contributed positively to the correction of animal acidosis during a high-concentrate diet.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals, rumen fluid collection and \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree ruminally cannulated Hanwoo steers (500 \u0026plusmn; 47 kg body weight; 20 mos. of age) were used to provide rumina fluid for \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation. The animals were fed twice daily with concentrate feed and kleingrass. Ruminal contents were collected before morning feeding. Samples were squeezed and strained through four layers of surgical gauze and pooled in an amber bottle with an oxygen-free headspace, which was subsequently capped after collection. Collected samples were immediately transported to the laboratory while being maintaining at a temperature of 39 \u0026deg;C [49].\u003c/p\u003e\n\u003cp\u003eSeventy milliliters of rumen fluid were dispensed into serum bottles containing each treatment and 2.5 g dry matter of ground corn grain served as substrate, mixed, and flushed with CO\u003csub\u003e2\u003c/sub\u003e [50]. Samples were in triplicate and incubated at 39 \u0026deg;C for 3, 6, 12, and 24 h while shaking horizontally at 100 rpm, as described by Hattori and Matsui [51]. The buffer used in treatments is composed of calcium carbonate, magnesium oxide, sodium carbonate, and calcified seaweed (Rupromin Balance\u0026trade;, Rotterdam, Netherlands). Treatments consisted of CON (negative control, no buffer added), BC\u003csub\u003e0.3%\u003c/sub\u003e (low buffering capacity, 0.3% buffer), BC\u003csub\u003e0.5%\u003c/sub\u003e (medium buffering capacity, 0.5% buffer), and BC\u003csub\u003e0.9%\u003c/sub\u003e (high buffering capacity, 0.9% buffer). The buffer and the concentrate given to experimental animals were supplied by Purina\u003csup\u003e\u0026reg;\u003c/sup\u003e Cargill, Korea. The ingredients and chemical composition of the experimental concentrate offered are presented in Table 5. Treatments were initially tested for determining their neutralizing (NC) and buffering capacity (BC) through titration using 2N acetic acid from its initial pH to 6.50, and 5.50, respectively (Table 6). The buffering agents used in every treatment are in powdered form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Ingredients and chemical composition of experimental concentrate\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003e\u003cstrong\u003eIngredients \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e\u003cstrong\u003ePercentage (%)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCorn fine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e31.17\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCorn gluten feed\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e21.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eSoy hulls\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e13.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eWheat fine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e10.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eRice bran\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e5.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eWheat flour\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e5.32\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eDDGS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e3.40\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eMolasses\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e3.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003ePalm kernel meal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e2.14\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eLimestone fine\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e1.90\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003ePalm kernel meal (Solvent)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e1.80\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCMS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e1.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eBrown rice\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.45\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eSalt\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.17\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eMineral/Vitamin premix\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.15\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eTotal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e100.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCalculated nutrients, as fed\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCrude Protein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e13.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCrude Fat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e3.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eAsh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e5.77\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eNDF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e23.44\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eMoisture\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e11.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCrude Fiber\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e8.65\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCalcium\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003ePhosphorus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eCa/P\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e2.09\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eSulfur\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.20\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003ePotassium\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eMagnesium\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.24\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eSodium\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e0.21\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"422\"\u003e\n\u003cp\u003eTDN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"166\"\u003e\n\u003cp\u003e76.44\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e DDGS, dried distillers\u0026rsquo; grains with solubles; CMS, condensed molasses solubles; NDF, neutral detergent fiber; TDN, total digestible nutrients.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1) \u003c/sup\u003eMineral \u0026amp; vitamin premix contained vit. A 2,650,000 IU, vit. D\u003csub\u003e3\u003c/sub\u003e 530,000 IU, vit. E 1,050 IU, niacin 10,000 mg, Mn 4,400 mg, Zn 4,400 mg, Fe 13,200 mg, Cu 2,200 mg, iodine 440 mg, and Co, 440 mg/kg of Grobic-DC provided from Bayer Health Care (Leverkusen, Germany)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u003c/strong\u003e Titration results to determine the neutralizing and buffering capacity of sample buffers used in treatments\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"223\"\u003e\n\u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" width=\"189\"\u003e\n\u003cp\u003e\u003cstrong\u003eSample buffers \u003csup\u003ed\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"68\"\u003e\n\u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"118\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.3%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.5%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e\u003cstrong\u003eBC\u003csub\u003e0.9%\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u003cstrong\u003eAll\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"223\"\u003e\n\u003cp\u003eInitial pH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e6.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e7.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e7.42\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e0.007\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"223\"\u003e\n\u003cp\u003eNeutralizing capacity (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e0.16\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"223\"\u003e\n\u003cp\u003eBuffering capacity (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"61\"\u003e\n\u003cp\u003e0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e0.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"59\"\u003e\n\u003cp\u003e0.43\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"68\"\u003e\n\u003cp\u003e0.003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"58\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"60\"\u003e\n\u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea,b,c\u003c/sup\u003e Within row indicate linear effect (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003e BC\u003csub\u003e0.3%\u003c/sub\u003e (0.3% buffer); BC\u003csub\u003e0.5%\u003c/sub\u003e (0.5% buffer); BC\u003csub\u003e0.9%\u003c/sub\u003e (0.9% buffer)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalyses of \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation parameters and buffering capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRuminal fermentation parameters were monitored at the end of each incubation time period. Total gas production was measured from each serum bottle after the incubation time using a pressure meter (Laurel Electronics, Inc., Costa Mesa, Calif., USA). Consequently, a needle channel connected to the machine was extended into the sealed fermentation bottle for measuring positive pressure created by the gas build up inside the bottle. A gas flow regulator was then opened to allow gas flow inside a syringe barrel and the plunger was subsequently pulled gradually until the pressure reading on the machine display was zero. The volume of gas trapped inside the barrel was recorded as the total gas produced [49, 52].\u003c/p\u003e\n\u003cp\u003eThe pH value was determined using a pH meter (Metler Toledo, Germany) after uncapping each serum bottle. Samples of fermenta were also collected into two 1.5 ml microcentrifuge tubes and stored at -80 \u0026deg;C prior to ammonia-nitrogen and VFA analyses. Frozen samples were thawed at room temperature; after which, they were centrifuged for 10 min at 13,000 rpm at 4 \u0026deg;C using a Micro 17TR centrifuge (Hanil Science Industrial, Korea). The resulting supernatant was used for ammonia-nitrogen and VFA concentration analyses. Ammonia-nitrogen concentration was measured according to the colorimetric method developed by Chaney and Marbach [53] using a Libra S22 spectrophotometer (Biochrom Ltd., CB40FJ, England) at an absorbance of 630 nm. NH\u003csub\u003e3\u003c/sub\u003e-N is the vital source of nitrogen for microbial protein synthesis in the rumen [54]. Analysis of volatile fatty acid concentration was done using high-performance liquid chromatography (Agilent Technologies 1200 series, Tokyo, Japan) with a UV detector set at 210 nm and 220 nm. Samples were isocratically eluted with 0.0085N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4 \u003c/sub\u003eat a flow rate of 0.6 mL/min and a column temperature of 35 \u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRuminal fluid pH was recorded following 1 min of equilibration. Buffering capacity, defined as the resistance to change in pH from pH 7 to 5, was determined by titrating a 30 ml aliquot of ruminal fluid with continuous stirring from its initial pH to pH 5 with 1N HCl and titrating an additional 30 ml aliquot from its initial pH to a pH of 7 with 1N NaOH. If the initial pH was higher than 7, only the volume of acid required to reduce the pH from 7 to 5 was recorded. Buffering capacity was converted to milliequivalents per liter as follows: BC = [(milliliters of 1N HCl) + (milliliters of 1N NaOH)] \u0026times; 10\u003csup\u003e3\u003c/sup\u003e/30 [15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of rumen fermentation characteristics in Hanwoo steers \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo \u003c/em\u003eexperiment was conducted using four Hanwoo steers (765 \u0026plusmn; 60 kg body weight; 24 mos. of age) in a 4 \u0026times; 4 Latin square design to assess the effects of treatments on rumen fermentation characteristics and ruminal bacterial composition and diversity of the experimental animals for four months. The feeding trial was conducted with 4 treatments comprised of CON which served as the negative control, BC\u003csub\u003e0.3%\u003c/sub\u003e, BC\u003csub\u003e0.5%\u003c/sub\u003e, and BC\u003csub\u003e0.9%\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eThe Hanwoo steers were fed daily of 2:8 forage and concentrate ratio in 2 equal portions at 0900 and 1600 h. Animals in all treatments received the same vaccinations, medications, and were under the same management programs unless otherwise stated. Steers were confined in free-stall barns and had free access to water and exercise lots.\u003c/p\u003e\n\u003cp\u003eRumen fluid samples were collected before morning feeding using an oral stomach tube on the 30\u003csup\u003eth\u003c/sup\u003e day right before transitioning to the next feeding trial for the analysis of ruminal fermentation parameters. These parameters were all evaluated using the same protocol as used in the \u003cem\u003ein vitro \u003c/em\u003eexperiment. However, rumen pH change in every experimental period of about 30 days was monitored using eCow (hathor.ecow.co.uk). It was done basically to monitor the occurrence of acidosis through a pH value of \u0026lt;5.8 for several hours a day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e16S rRNA amplicon sequencing and metataxonomic analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples obtained from each treatment were sent to Macrogen, Korea for DNA extraction, 16S rRNA sequencing and microbiome analysis. In brief, DNA was extracted using DNeasy Power Soil Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was quantified using Quant-IT PicoGreen (Invitrogen). The sequencing libraries were prepared according to the Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 region. The input gDNA was PCR amplified with 1 \u0026times; reaction buffer, 1 nM of dNTP mix, 500 nM each of the universal F/R PCR primer, and 2.5 U of Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA). The cycle condition for 1st PCR was 3 min at 95 \u0026deg;C for heat activation, and 25 cycles of 30 sec at 95 \u0026deg;C, 30 sec at 55 \u0026deg;C and 30 sec at 72 \u0026deg;C, followed by a 5-min final extension at 72 \u0026deg;C. The universal primer pair with Illumina adapter overhang sequences used for the first amplification was V3-F (5\u0026rsquo;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3\u0026rsquo;) and V4-R (5\u0026rsquo;- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3\u0026rsquo;). The 1st PCR product was purified with AMPure beads (Agencourt Bioscience, Beverly, MA). Following purification, the 2 uL of 1st PCR product was PCR amplified for final library construction containing the index using NexteraXT Indexed Primer. The cycle condition for 2nd PCR was the same as the 1st PCR condition except for 10 cycles. The PCR product was purified with AMPure beads. The final purified product is then quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany).\u003c/p\u003e\n\u003cp\u003eSequencing was done using the Illumina Miseq (Illumina Inc., San Diego, CA, USA) platform. The raw data files (fastq) containing the sequenced paired-end (PE) reads were obtained using the bcls2fastq package (Illumina Inc., San Diego, CA, USA) from the base call binary data produced by real-time analysis. The PE raw reads were filtered from adapter sequences using Scythe (v0.994) [55] and Sickle [56] programs then assembled using Fast Length Adjustment of Short Reads (FLASH 1.2.11) [57]. Assembled reads were quality filtered and trimmed for short and extra-long reads, and duplicate reads were removed, then clustered at 100% identity using CD-HIT-OTU [58]. Chimeric reads were identified and the initial clusters were recruited to primary clusters. Then, noise filtering was done and the remaining non-chimeric clusters were binned to operational taxonomic units (OTU) following a greedy algorithm with a cut-off value of 97% species level identity using CD-HIT-OTU [58]. Representative sequences from the clustered OTU were taxonomically assigned using Quantitative Insights Into Microbial Ecology (QIIME Version 1) [18] from the NCBI 16S rRNA database, and the taxonomy composition was generated using QIIME-UCLUST [59]. The produced bacterial taxonomy and composition data were used to generate a biological information matrix (BIOM) [60] in Mothur [61]. The generated BIOM file were used to visualize the alpha and beta diversity indices, and the bacterial composition using programs utilized by Metagenomics Core Microbiome Exploration Tool (MetaCOMET) [17].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analysis was performed using Statistical Analysis Systems (SAS) version 9.1 (SAS Institute Inc., Cary, NC). The data of rumen fermentation, alpha diversity indices and relative abundance of individual taxa of rumen microbiota were statistically evaluated using Proc general linear model (GLM) for a completely randomized design. All treatments in the \u003cem\u003ein vitro \u003c/em\u003eexperiment were conducted in triplicate and Duncan\u0026rsquo;s Multiple Range Test (DMRT) was used to identify differences between specific treatments. The linear effects of different buffering capacity concentrations were analyzed using orthogonal polynomial coefficients to describe the functional relationships among the control and treatment groups. A \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered indicative of significant differences.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBC: Buffering capacity; NaHCO\u003csub\u003e3\u003c/sub\u003e: Sodium bicarbonate; MgO: Magnesium oxide; DDGS: dried distillers\u0026rsquo; grains with solubles; CMS: condensed molasses solubles; NDF: neutral detergent fiber; TDN: total digestible nutrients; SEM: standard error of the mean; NH\u003csub\u003e3\u003c/sub\u003e-N: Ammonia nitrogen; VFA: Volatile fatty acid; DNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction; FLASH: Fast Length Adjustment of Short Reads; OTU: Operational taxonomic units; QIIME: Quantitative Insights Into Microbial Ecology; BIOM: Biological information matrix; MetaCOMET: Metagenomics Core Microbiome Exploration Tool; SAS: Statistical Analysis Systems; GLM: General linear model; DMRT: Duncan\u0026rsquo;s Multiple Range Test; PCoA: Principal Coordinate Analysis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported and funded by Provimi Singapore Pte Ltd, 138 Market Street, #17-01 CapitaGreen, Singapore 048946.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: SSL., CDJ, TGK, JSL, KKC. Supervision: SSL. Experiment: SCR, CDJ. Data curation: SCR, CDJ. Formal analysis: SCR, CDJ, SSL. Methodology: SCR, CDJ, SSL. Software: SHKim, ARS, YIC, Sung Sill Lee (SSL). Validation: SSL. Investigation: SCR, CDJ, SSL. Writing \u0026ndash; original draft: SSL, CDJ, SCR, TGK, JSL. Writing \u0026ndash; review and editing: SCR, LLM, SHKang, SSL. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is funded by Provimi Singapore Pte Ltd, 138 Market Street, #17-01 CapitaGreen, Singapore 048946.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals used in this experiment and all experimental protocols were reviewed and approved by the Sunchon National University Animal Research Ethics Committee (SCNU IACUC, approval number: SCNU IACUC-2018-01). All experiments were performed in accordance with the guidelines and regulation set by the governing body.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eRuminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University, 413 Jungangno, Jeonnam 57922, Suncheon, Republic of Korea. \u003csup\u003e2\u003c/sup\u003eThe University of Queensland Diamantina Institute, Faculty of Medicine, Brisbane, Australia. \u003csup\u003e3\u003c/sup\u003eAnimal Disease and Diagnostic Laboratory, Department of Animal Science and Technology, Sunchon National University, 413 Jungangno, Jeonnam 57922, Suncheon, Republic of Korea. \u003csup\u003e4\u003c/sup\u003eRupromin Balance\u0026trade;, 5th. Bonsol Blg. 445, Teheran-ro, Gangnam-gu, Seoul 06158, Republic of Korea. \u003csup\u003e5\u003c/sup\u003eDepartment of Animal Resources Technology, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLee M, Jeong S, Seo J, Seo S. Changes in the ruminal fermentation and bacterial community structure by a sudden change to a high-concentrate diet in Korean domestic ruminants. Asian-Australasian J Anim Sci. 2019;32:92\u0026ndash;102.\u003c/li\u003e\n\u003cli\u003eRussell JB. Rumen microbiology and its role in ruminant nutrition. Print book. Ithaca, NY: Ithaca, NY : Dept. of Microbiology, Cornell University, \u0026copy;2002.; 2002.\u003c/li\u003e\n\u003cli\u003eBi Y, Zeng S, Zhang R, Diao Q, Tu Y. Effects of dietary energy levels on rumen bacterial community composition in Holstein heifers under the same forage to concentrate ratio condition. 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Introducing mothur : Open-Source , Platform-Independent , Community-Supported Software for Describing and Comparing Microbial Communities. 2009;75:7537\u0026ndash;41.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Buffering capacity, Hanwoo steers, Microbiome, Ruminal metataxonomic, Fermentation characteristic, Acidosis","lastPublishedDoi":"10.21203/rs.3.rs-354029/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-354029/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eRumen bacterial community is mainly affected by the type of diet consumed by the host animals. High concentrate diet increases the abundance of lactic acid producers and utilizers due to high level of non-structural carbohydrates thus reducing the number of fiber-degrading bacteria because of drastic decrease in pH. Dietary buffers are essential in regulating rumen pH through the compounds responsible in resisting drastic decrease in pH once cattle were fed with high-concentrate diet. However, no study has evaluated the effects of buffering capacity and efficiency in alleviating chronic acidosis in rumen. Ruminal metataxonomic and fermentation characteristics analyses were conducted to evaluate the effect of different buffering capacities on \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments in high-concentrate fed Hanwoo steers. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Results revealed that BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5%\u003c/sub\u003e had similar and significant effect (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) on \u003cem\u003ein vitro\u003c/em\u003e ruminal fermentation at 3 to 24 h incubation. Both BC\u003csub\u003e0.9%\u003c/sub\u003e and BC\u003csub\u003e0.5% \u003c/sub\u003ehad significantly highest (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) buffering capacity, pH, and ammonia-nitrogen (NH\u003csub\u003e3\u003c/sub\u003e-N) than BC\u003csub\u003e0.3%\u003c/sub\u003e and CON at 24 h of incubation. Individual and total volatile fatty acids (VFA) were significantly lowest in CON. Increasing buffering capacity concentration showed linear effect on pH at 6 to 24 h while total gas and NH\u003csub\u003e3\u003c/sub\u003e-N at 3 and 12 h. Phylum Bacteroidetes dominated all treatments but a higher abundance of Firmicutes in BC\u003csub\u003e0.5%\u003c/sub\u003e than others. \u003cem\u003eRuminoccocus bromii\u003c/em\u003e and \u003cem\u003eSucciniclasticum ruminis\u003c/em\u003e were dominant in BC\u003csub\u003e0.5%\u003c/sub\u003e and \u003cem\u003eBacteroides massiliensis\u003c/em\u003e in BC\u003csub\u003e0.3%\u003c/sub\u003e. The normalized data of relative abundance of observed OTUs’ representative families have grouped the CON with BC\u003csub\u003e0.3% \u003c/sub\u003ein the same cluster, whereas BC\u003csub\u003e0.5%\u003c/sub\u003e and BC\u003csub\u003e0.9% \u003c/sub\u003ewere clustered separately which indicates the effect of varying buffering capacity of buffer agents. Principal coordinate analysis (PCoA) on unweighted UniFrac distances revealed close similarity of bacterial community structures within and between treatments and control, in which BC\u003csub\u003e0.9% \u003c/sub\u003eand BC\u003csub\u003e0.3%\u003c/sub\u003e groups showed dispersed community distribution. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our findings showed that increasing buffering capacity enhances rumen fermentation parameters and affects rumen microbiome by altering bacterial community through distinct structure between high and low buffering capacity, thus an important factor contributed to the prevention of ruminal acidosis during a high-concentrate diet.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Increasing Buffering Capacity Alters Rumen Microbiota Composition and Enhances Rumen Fermentation Characteristics of High-Concentrate Fed Hanwoo Steers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-26 21:52:01","doi":"10.21203/rs.3.rs-354029/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"736fbbf7-329d-4753-bda1-ec794072719a","owner":[],"postedDate":"March 26th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3230980,"name":"General Microbiology"},{"id":3230981,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2021-06-07T23:29:06+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-26 21:52:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-354029","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-354029","identity":"rs-354029","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}