Effects of ruminal lipopolysaccharide exposure on primary bovine ruminal epithelial cells.

The objective of this study was to investigate the immunopotential of ruminal lipopolysaccharides (LPS) on cultured primary bovine rumen epithelial cells (REC). Primary bovine REC were isolated from 6 yearling steers and grown in culture for 3 experiments. Experiment 1 aimed to determine the immunopotential of ruminal LPS, experiment 2 aimed to assess tolerance to chronic LPS exposure, and experiment 3 aimed to evaluate antagonistic interactions between ruminal and Escherichia coli LPS. In experiments 1 and 2, REC were exposed to nonpyrogenic water, 20 µg/mL E. coli LPS (EC20), 10 µg/mL ruminal LPS, 20 µg/mL ruminal LPS, and 40 µg/mL ruminal LPS, either continuously or intermittently. For the continuous exposure, REC underwent a 6 h exposure, whereas for the intermittent exposure, the procedure was: (1) a 12 h continuous exposure to treatments followed by LPS removal for 24 h and then another 12 h of exposure (RPT), and (2) a 12 h continuous exposure to treatments followed by LPS removal and a recovery period of 36 h (RCV). In experiment 3, REC were exposed to nonpyrogenic water, 1 µg/mL E. coli LPS, 1 µg/mL ruminal LPS to 1 µg/mL E. coli LPS, 10 µg/mL ruminal LPS to 1 µg/mL E. coli LPS, and 50 µg/mL ruminal LPS to 1 µg/mL E. coli LPS. Each experiment was done as a complete randomized block design with 6 REC donors. The REC-donor was used as blocking factor. Each treatment had 2 technical replicates, and treatment responses for all data were analyzed with the MIXED procedure of SAS. For all experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine the relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF, IL1B, and IL6), chemokines (CXCL2 and CXCL8), growth factor-like cytokines (CSF2 and TGFB1), and a lipid mediator (PTGS2). In experiment 1, the targeted genes were upregulated by EC20, whereas all ruminal LPS treatments resulted in a lower transcript abundance. Regarding RPT, and RCV condition, in experiment 2, the expression of targeted genes was not affected or was at a lower abundance to EC20 when compared with ruminal LPS treatments. Lastly, in experiment 3, all targeted genes resulted in lower or similar transcript abundance on all ruminal LPS ratios. Overall, our results indicate that ruminal LPS have a limited capacity to activate the TLR4/NF-kB pathway and to induce the expression of inflammatory genes.

Effects of amino resin-treated and heat-treated soybean meal on ruminal fermentation, nutrient digestion, and nitrogen partitioning in continuous culture.

The objective of this study was to evaluate the effects of amino resin-treated soybean meal (SBM) on ruminal fermentation, nutrient digestion, and N partitioning. The treatments were (1) untreated solvent-extracted SBM, (2) amino resin-treated SBM (AR-SBM), and (3) heat-treated SBM (HT-SBM). The experimental design was arranged as a replicated 3 × 3 Latin square with 6 fermentors in a dual-flow continuous culture system. Treatments were randomly assigned to fermentors within a Latin square for each period. Each fermentor was fed 106 g/d of diet DM equally distributed in 2 feeding times daily at 0800 and 1800 h. Diets were formulated to contain 16% CP, 30% NDF, and 30% starch across treatments. The experiment consisted of 3 experimental periods, each lasting for 10 d. The first 7 d of each period were considered adaptation, and the last 3 d were used for sampling and data collection. On d 8 and 9, samples were collected for analysis of diurnal variation in concentrations of NH3-N, pH, and VFA during the first 8 h after feeding. On d 8, 9, and 10, samples were collected from the liquid and solid effluents accumulated over 24 h for analysis of daily averages of NH3-N and VFA pools, and true ruminal digestibility estimates. Data were analyzed using the MIXED procedure of SAS, and significance was declared when P ≤ 0.05. The model included the fixed effect of treatment and random effects of square, period, and fermentor within square, whereas time and interaction treatment × time were included for analyses of diurnal variation, with time as repeated measures. Compared with SBM, the cultured ruminal contents of AR-SBM and HT-SBM had lower NH3-N concentrations, indicating lower microbial fermentation of protein. Molar proportions of isovalerate and isobutyrate were greater in SBM than AR-SBM and HT-SBM, with greater molar proportion of isobutyrate for SBM, particularly during the first 2 h after feeding. The flow of NH3-N was greater for SBM compared with AR-SBM and HT-SBM, whereas NAN flow, bacterial N flow, and N efficiency were greater for AR-SBM and HT-SBM compared with SBM. Our results indicate that both the amino resin and heat treatments of SBM allow for similar decreases in microbial degradation of CP without limiting microbial protein synthesis in diets with 16% CP. Amino resin treatment may be effective in reducing microbial fermentation of protein in the rumen without adverse effects on digestibility or fermentation parameters as compared with SBM.

Effects of cashew nut shell extract and monensin on in vitro ruminal fermentation, methane production, and ruminal bacterial community.

The objective of this study was to evaluate the effects of cashew nut shell extract (CNSE) and monensin on ruminal in vitro fermentation, CH4 production, and ruminal bacterial community structure. Treatments were as follows: control (CON, basal diet without additives); 2.5 µM monensin (MON); 0.1 mg CNSE granule/g DM (CNSE100); and 0.2 mg CNSE granule/g DM (CNSE200). Each treatment was incubated with 52 mL of buffered ruminal content and 500 mg of total mixed ration for 24 h using serum vials. The experiment was performed as a complete randomized block design with 3 runs. Run was used as a blocking factor. Each treatment had 5 replicates, in which 2 were used to determine nutrient degradability, and 3 were used to determine pH, NH3-N, volatile fatty acids, lactate, total gas, CH4 production, and bacterial community composition. Treatment responses for all data, excluding bacterial abundance, were analyzed with the GLIMMIX procedure of SAS v9.4. Treatment responses for bacterial community structure were analyzed with a PERMANOVA test run with the R package vegan. Orthogonal contrasts were used to test the effects of (1) additive inclusion (ADD: CON vs. MON, CNSE100, and CNSE200); (2) additive type (MCN: MON vs. CNSE100 and CNSE200); and (3) CNSE dose (DOS: CNSE100 vs. CNSE200). We observed that pH, acetate, and acetate:propionate ratio in the CNSE100 treatment were lower compared with CNSE200, and propionate in the CNSE100 treatment was greater compared with CNSE200. Compared with MON, CNSE treatments tended to decrease total lactate concentration. Total gas production of CON was greater by 2.63% compared with all treatments, and total CH4 production was reduced by 10.64% in both CNSE treatments compared with MON. Also, compared with MON, in vitro dry matter degradabilities in CNSE treatments were lower. No effects were observed for NH3-N or in vitro neutral detergent fiber degradability. Finally, the relative abundances of Prevotella, Treponema, and Schwartzia were lower, whereas the relative abundances of Butyrivibrio and Succinivibrio were greater in all treatments compared with CON. Overall, the inclusion of CNSE decreased CH4 production compared with MON, making CNSE a possible CH4 mitigation additive in dairy cattle diets.

Partial replacement of soybean meal with microalgae biomass on in vitro ruminal fermentation may reduce ruminal protein degradation.

The objective of this study was to evaluate the effects of partially replacing soybean meal (SBM) with algal sources on in vitro ruminal fermentation. Using 6 fermenters in a 3 × 3 replicated Latin square with 3 periods of 10 d each, we tested 3 treatments: a control diet (CRT) with SBM at 17.8% of the diet dry matter (DM); and 50% SBM biomass replacement with either Chlorella pyrenoidosa (CHL); or Spirulina platensis (SPI). The basal diet was formulated to meet the requirements of a 680-kg Holstein dairy cow producing 45 kg/d of milk with 3.5% fat and 3% protein. All diets had a similar nutritional composition (16.0% CP; 34.9% NDF; 31.0% starch, DM basis) and fermenters were provided with 106 g DM/d split into 2 portions. After 7 d of adaptation, samples were collected for 3 d of each period for analyses of ruminal fermentation at 0, 1, 2, 4, 6, and 8 h after morning feeding for evaluation of the ruminal fermentation kinetics. For the evaluation of the daily production of total metabolites and for the evaluation of nutrient degradability, samples from the effluent containers were collected daily. Statistical analysis was performed with the MIXED procedure of SAS with treatment, time, and their interactions considered as fixed effects; day, square, and fermenter were considered as random effects. Orthogonal contrasts (CRT vs. algae; and CHL vs. SPI) were used to depict the treatment effect, and significance was declared when P ≤ 0.05. Fermenters that received algae-based diets had a greater propionate molar concentration and molar proportion when compared with the fermenters fed CRT diets. In addition, those algae-fed fermenters had lower branched short-chain fatty acids (BSCFA) and isoacids (IA), which are biomarkers of ruminal protein degradation, along with lower ammonia (NH3-N) concentration and greater nonammonia nitrogen (NAN). When contrasting with fermenters fed SPI-diets, fermenters fed based CHL-diets had a lower molar concentration of BSCFA and IA, along with lower NH3-N concentration and flow, and greater NAN, bacterial nitrogen flow, and efficiency of nitrogen utilization. Those results indicate that CHL protein may be more resistant to ruminal degradation, which would increase efficiency of nitrogen utilization. In summary, partially replacing SBM with algae biomass, especially with CHL, is a promising strategy to improve the efficiency of nitrogen utilization, due to the fact that fermenters fed CHL-based diets resulted in a reduction in BSCFA and IA, which are markers of protein degradation, and it would improve the efficiency of nitrogen utilization. However, further validation using in vivo models are required.

Effects of cashew nutshell extract and monensin on microbial fermentation in a dual-flow continuous culture.

The objective of this study was to compare cashew nutshell extract (CNSE) to monensin and evaluate changes in in vitro mixed ruminal microorganism fermentation, nutrient digestibility, and microbial nitrogen outflow. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square design with 4 experimental periods of 10 d (7 d for diet adaptation and 3 d for sample collection). Basal diets contained 43.5:56.5 forage: concentrate ratio and each fermenter was fed 106 g of DM/d divided equally between 2 feeding times. Treatments were control (CON, basal diet without additives), 2.5 µM monensin (MON), 0.1 mg CNSE granule/g DM (CNSE100), and 0.2 mg CNSE granule/g DM (CNSE200). On d 8 to10, samples were collected for pH, lactate, NH3-N, volatile fatty acids (VFA), mixed protozoa counts, organic matter (OM), and neutral detergent fiber (NDF) digestibility. Data were analyzed with the GLIMMIX procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). We observed that butyrate concentration in all treatments was lower compared with CON and the concentration for MON was lower compared with CNSE treatments. Protozoal population in all treatments was lower compared with CON. No effects were observed for pH, lactate, NH3-N, total VFA, OM, or N utilization. Within the 24-h pool, protozoal generation time, tended to be lower, while NDF digestibility tended to be greater in response to all additives. Furthermore, the microbial N flow, and the efficiency of N use tended to be lower for the monensin treatment compared with CNSE treatments. Overall, our results showed that both monensin and CNSE decreased butyrate synthesis and protozoal populations, while not affecting OM digestibility and tended to increase NDF digestibility; however, such effects are greater with monensin than CNSE nutshell.

Production, physiological response, and calcium and magnesium balance of lactating Holstein cows fed different sources of supplemental magnesium with or without ruminal buffer.

The objective of this study was to evaluate the effects of dietary replacement of magnesium oxide (MgO) with calcium-magnesium hydroxide [CaMg(OH)2] and its interaction with ruminal buffer (sodium sesquicarbonate) supplementation on production, Ca and Mg balance, and overall physiological response of mid-lactation Holstein dairy cows. Sixty cows averaging 40.5 ± 7.0 kg of milk/d were used. Treatments were assigned following a 2 × 2 factorial arrangement: (1) MgO, (2) MgO + buffer, (3) CaMg(OH)2, or (4) CaMg(OH)2 + buffer. Diets were formulated to have 16.5% of crude protein, 1.82 Mcal/kg of net energy for lactation, 0.67% Ca, 0.39% P, and 0.25% Mg, all on a dry matter (DM) basis. Treatments were individually top dressed. Milk production, composition, and DM intake were evaluated. A subsample of 20 cows were randomly selected for the evaluation of Ca and Mg balance, blood gases, and electrolytes. Ruminal fluid was also collected for evaluation of pH and Ca and Mg solubility. Effects of Mg source, buffer, and the interaction Mg source × buffer were analyzed through orthogonal contrasts. An interaction of Mg source × buffer was found for DM intake and feed efficiency, in which cows fed CaMg(OH)2 had a similar feed efficiency regardless of ruminal buffer inclusion; however, when cows were fed MgO, the inclusion of buffer reduced feed efficiency. No effects on body weight and milk yield were observed. Buffer addition tended to increase the concentrations of fat, protein, and solids-not-fat, without affecting the yields of these milk components. Magnesium source and buffer did not affect ruminal fluid, blood, urine, or fecal pH; however, buffer supplementation increased urinary pH. Treatment with CaMg(OH)2 increased blood concentration of HCO3-, total CO2, and base excess compared with cows fed MgO. No differences were observed in the ruminal solubility of Ca and Mg or on milk or urinary Ca and Mg excretion. Greater plasma Mg concentration was observed for animals fed MgO compared with cows fed CaMg(OH)2; however, both sources were above the threshold recommended in the literature for dairy cows. Also, a reduction in fecal Mg excretion was observed in animals fed CaMg(OH)2. In summary, we provide evidence that CaMg(OH)2 could replace MgO without affecting performance, overall physiological response, or Ca and Mg balance of mid-lactating dairy Holstein cows.

Effects of exogenous amylolytic or fibrolytic enzymes inclusion on in vitro fermentation of lactating dairy cow diets in a dual-flow continuous-culture system.

The objective of this study was to determine the effects of including exogenous amylolytic or fibrolytic enzymes in a diet for high-producing dairy cows on in vitro ruminal fermentation. Eight dual-flow continuous-culture fermentors were used in a replicated 4 × 4 Latin square. The treatments were control (CON), a xylanase and glucanase mixture (T1), an α-amylase mixture (T2), or a xylanase, glucanase, and α-amylase mixture (T3). Treatments were included at a rate of 0.008% of diet dry matter (DM) for T1 and T2 and at 0.02% for T3. All treatments replaced the equivalent amount of soybean meal in the diet compared with CON. All diets were balanced to have the same nutrient composition [30.2% neutral detergent fiber (NDF), 16.1% crude protein (CP), and 30% starch; DM basis], and fermentors were fed 106 g/d divided into 2 feedings. At each feeding, T2 was pipetted into the respective fermentor and an equivalent amount of deionized water was added to each fermentor to eliminate potential variation. Experimental periods were 10 d (7 d for adaptation and 3 d for sample collection). Composite samples of daily effluent were collected and analyzed for volatile fatty acids (VFA), NH3-N, and lactate concentrations, degradability of DM, organic matter, NDF, CP, and starch, and flow and metabolism of N. Samples of fermentor contents were collected from each fermentor at 0, 1, 2, 4, 6, and 8 h after feeding to determine kinetics of pH, NH3-N, lactate, and VFA concentrations over time. All data were analyzed using PROC GLIMMIX of SAS (SAS Institute Inc.), and the repeated variable of time was included for kinetics measurements. Treatment did not affect mean pH, degradability, N flow and metabolism, or the concentrations of VFA, NH3-N, or lactate in the effluent samples. Treatment did not affect pH, acetate:propionate ratio, or the concentrations of lactate, NH3-N, total VFA, acetate, propionate, butyrate, isobutyrate, valerate, or caproate. However, the concentration of total VFA tended to change at each time point depending upon the treatment, and T2 tended to have a greater proportion of 2-methylbutyrate and isovalerate than CON, T1, or T3. As 2-methylbutyrate and isovalerate are branched-chain VFA that are synthesized from branched-chain amino acids, T2 may have an increased fermentation of branched-chain amino acids or decreased uptake by fibrolytic microorganisms. Although we did not observe changes in N metabolism due to the enzymes, there could be changes in microbial populations that utilize branched-chain VFA. Overall, the tested enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows.

Effects of partially replacing dietary corn with molasses, condensed whey permeate, or treated condensed whey permeate on ruminal microbial fermentation.

Corn is a feedstuff commonly fed to dairy cows as a source of energy. The objective of this study was to evaluate whether partially replacing dietary corn with molasses or condensed whey permeate, in lactating dairy cow diets in a dual-flow continuous culture system, can maintain nutrient digestibility by ruminal microorganisms. Furthermore, this study evaluated whether treating condensed whey permeate before feeding could aid the fermentation of the condensed whey permeate in the rumen. Eight fermentors were used in a 4 × 4 replicated Latin square with 4 periods of 10 d each. The control diet (CON) was formulated with corn grain, and the other diets were formulated by replacing corn grain with either sugarcane molasses (MOL), condensed whey permeate (CWP), or treated condensed whey permeate (TCWP). Diets were formulated by replacing 4% of the diet dry matter (DM) in the form of starch from corn with sugars from the byproducts. Sugars were defined as water-soluble carbohydrates (WSC) in the rations. The fermentors were fed 52 g of DM twice daily of diets containing 17% crude protein, 28% neutral detergent fiber, and 45% nonfiber carbohydrates. Liquid treatments were pipetted into each fermentor. After 7 d of adaptation, samples were collected for analyses of volatile fatty acids (VFA), lactate, and ammonia, and fermentors' pH were measured at time points after the morning feeding for 3 d. Pooled samples from effluent containers were collected for similar analyses, nutrient flow, and N metabolism. Data were statistically analyzed using Proc MIXED of SAS version 9.4 (SAS Institute Inc.); fixed effects included treatment and time, and random effects included fermentor, period, and square. The interaction of treatment and time was included for the kinetics samples. The TCWP and MOL treatments maintained greater fermentor pH compared with CWP. Total VFA concentration was increased in CWP compared with MOL. The acetate:propionate ratio was increased in TCWP compared with CON, due to tendencies of increased acetate molar proportion and decreased propionate molar proportion in TCWP. Lactate concentration was increased in MOL. Digestibility of WSC was increased in the diets that replaced corn with byproducts. The partial replacement of 4% of DM from corn starch with the sugars in byproducts had minimal effects on ruminal microbial fermentation and increased pH. Treated CWP had similar effects to molasses.

Effects of replacing magnesium oxide with calcium-magnesium carbonate with or without sodium bicarbonate on ruminal fermentation and nutrient flow in vitro.

The objective of this study was to evaluate the effects of replacing magnesium oxide (MgO) with calcium-magnesium carbonate [CaMg(CO3)2] on ruminal fermentation with or without the addition of sodium bicarbonate (NaHCO3). Eight fermentors of a dual-flow continuous-culture system were distributed in a replicated (2) 4 × 4 Latin square design in a 2 × 2 factorial arrangement of treatments (magnesium sources × NaHCO3). The treatments tested were 0.21% MgO [MgO; dry matter (DM) basis; 144.8 mEq of dietary cation-anion difference (DCAD)]; 0.21% MgO + 0.50% NaHCO3 (MgO+NaHCO3; DM basis; 205.6 mEq of DCAD); 1.00% CaMg(CO3)2 [CaMg(CO3)2; DM basis; 144.8 mEq of DCAD]; and 1.00% CaMg(CO3)2 + 0.50% NaHCO3 [CaMg(CO3)2+NaHCO3; DM basis; 205.6 mEq of DCAD]. Diets were formulated to have a total of 0.28% of Mg (DM basis). The experiment consisted of 40 d, which was divided into 4 periods of 10 d each, where 7 d were used for adaptation and 3 d for sampling to determine pH, volatile fatty acids (VFA), ammonia (NH3-N), lactate, mineral solubility, N metabolism, and nutrient digestibility. The effects of Mg source [MgO vs. CaMg(CO3)2], NaHCO3 (with vs. without), and the interaction were tested with the MIXED procedure of SAS version 9.4 (SAS Institute). There was no Mg source × NaHCO3 interaction in the pH variables and mineral solubility, and Mg sources evaluated did not affect the variables related to ruminal pH and solubility of Mg. On the other hand, the inclusion of NaHCO3 increased the pH daily average, independent of Mg source, which led to a reduced time that pH was below 5.8 and decreased area under the curve. Total VFA and lactate concentration were similar among treatments regardless of NaHCO3 and Mg source; however, the molar proportion of isobutyrate and NH3-N concentration were lower in diets with CaMg(CO3)2 compared with MgO. Moreover, NaHCO3 inclusion increased NH3-N, total daily NH3-N flow, isobutyrate concentration, and acid detergent fiber digestibility. Our results showed that CaMg(CO3)2 leads to a lower NH3-N concentration and isobutyrate proportion. Therefore, because most of the tested variables were not significantly different between MgO and CaMg(CO3)2 when combined or not with NaHCO3, CaMg(CO3)2 can be a viable alternative source to replace MgO in dairy cow diets without affecting mineral solubility, ruminal pH, nutrient digestibility, total VFA, and the main ruminal VFA. Although Mg sources are known to have an alkalizing effect, NaHCO3 inclusion in diets with Mg supplementation allowed an increase in ruminal pH, as well as an increase in isobutyrate and NH3-N flow.

Effects of supplemental source of magnesium and inclusion of buffer on ruminal microbial fermentation in continuous culture.

Magnesium oxide (MgO) is the most common supplemental source of Mg for dairy cows and a proven ruminal alkalizer when supplemented above NRC (2001) recommendations. However, overfeeding MgO may increase feeding costs, whereas the effects of alternative sources of Mg on ruminal fermentation are not well known. Moreover, it is still unclear if Mg supplementation influences the effects of bicarbonate-based buffers on ruminal fermentation. We aimed to evaluate the effect of Mg source on ruminal fermentation with diets formulated to a final concentration of 0.25% Mg, and to determine if the effect of sodium sesquicarbonate as a buffer varies with the source of Mg. We used 8 fermentors in a duplicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments, by combining 2 factors: (1) Mg source: using either MgO or an alternative source consisting of a blend of CaMg(OH)4 and CaMg(CO3)2 (BLN) and (2) sodium sesquicarbonate buffer inclusion, at 0 or 0.6% of dry matter intake. Based on preliminary tests of reactivity, we hypothesized that BLN plus buffer would allow for greater ruminal pH, acetate molar proportion, and NDF digestibility than diets with MgO or without buffer. Four 10-d periods were completed, where the last 3 d were used for pH measurements and collection of samples for volatile fatty acids (VFA), ammonia (NH3-N), Mg solubility, N metabolism, and nutrient digestibility. Effects of Mg source (source), sodium sesquicarbonate inclusion (buffer), and their interaction (source × buffer) were tested with the MIXED procedure of SAS (SAS Institute Inc.). We did not find an effect of Mg source on ruminal fermentation variables; however, concentration of soluble Mg in ruminal fluid was greater for MgO compared with BLN. On the other hand, buffer supplementation increased average ruminal pH, acetate molar proportion, and branched-chain VFA molar proportion; tended to increase NDF digestibility; and decreased both area under the curve and time below pH 6.0. An interaction of source × buffer was found for propionate, butyrate, and NH3-N, the first one decreasing and the 2 others increasing only when buffer was supplemented to the BLN diet. Our results indicate that supplementing Mg with either MgO or BLN promotes similar ruminal fermentation in diets with total concentration of 0.25% Mg. Further evaluations are needed to assess Mg availability and animal performance in dairy cows fed BLN.

Ruminal bacteria lipopolysaccharides: an immunological and microbial outlook.

Lipopolysaccharides (LPS) are outer membrane components of Gram-negative bacteria made of three regions: the O-antigen; the core oligosaccharide; and a glucosamine disaccharide linked to hydroxy fatty acids, which is named lipid A. The number phosphate groups, and hydroxy fatty acid chains is associated with the immunopotency and the immunomodulatory activity of LPS, where six-acyl chain lipid A with two phosphate groups is found in virulent strains and five- or four-acyl chain lipid A with one phosphate group are found in non-virulent bacteria strains. Ruminal bacteria are predominantly Gram-negative and their LPS have not been thoroughly investigated. In the rumen, LPS is comprised of mixed ruminal LPS. Drawing upon a body of theoretical and applied work, this paper aims to critically review the scientific literature regarding single-species and mixed ruminal bacteria LPS, highlighting the importance of ruminal LPS to the host. Lastly, future research directions are suggested in order to further our understanding of the roles of LPS in the rumen. Possible suggestions for further understanding ruminal LPS include (1) in silico evaluation of major bacteria contributing to ruminal LPS, (2) structural characterization of LPS from prominent ruminal bacteria species, such as ruminal selenomonads and Megasphaera elsdenii, and, (3) ruminal epithelial tissue immune response evaluation from single-species and mixed ruminal LPS. In conclusion, this review identifies numerous areas for future research, including setting the basis for future modeling and simulation of host microbiome interactions in ruminants.