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.

Replacing soybean meal with microalgae biomass in diets with contrasting carbohydrate profiles can reduce in vitro methane production and improve short-chain fatty acid production.

The objective of this study was to evaluate the interaction of dietary carbohydrate profile and soybean meal (SBM) replacement with either Chlorella pyrenoidosa (CHL) or Spirulina platensis (SPI) on in vitro fermentation. This experiment was conducted as a randomized complete block design, with fermentation run (3 runs) considered as blocks. The treatments were arranged in a 2 × 5 factorial design, where the first factor was the carbohydrate profile, which was composed of diets containing 42.5% neutral detergent fiber (NDF) and 26.8% starch (HF-LS) or 26.8% NDF and 40.6% starch (LF-HS), and the second factor was the protein source, in which a control diet (100% SBM), partial replacement of SBM with CHL (1/2CHL) or SPI (1/2SPI), or total replacement of SBM with CHL or SPI were used. All experimental diets were formulated to have 17% crude protein. The ruminal fluid was collected from 2 lactating Holstein cows, buffered with Van Soest medium at a ratio of 1:2 and added to serum bottles containing 0.50 g of the experimental diets. Bottles were incubated at 39°C for 24 and 48 h in triplicate; headspace pressure was measured, along with gas collection for methane (CH4) quantification at 0, 2, 4, 8, 16, 24, 36, and 48 h after incubation. The final medium was used to measure pH, ammonia, and VFA. After incubation, feed bags were recovered and used for estimation of dry matter (DM), NDF, and organic matter (OM) degradability. Statistical analysis was carried out using the MIXED procedure of SAS, with carbohydrate profile, protein source, assay, and their interactions as fixed effects, with run and bottle as random effects. Orthogonal contrasts were used to compare carbohydrate profile, algae species, carbohydrate profile × algae interaction, and linear and quadratic effects of SBM replacement with CHL or SPI. There was no interaction effect between carbohydrate profile and algae source. The LF-HS improved gas production, degradability of nutrients, and VFA, mainly increasing the production of butyrate and propionate. When compared with CHL, SPI had a greater degradability of nutrients and branched VFA, along with reduction in total gas production and tended to reduce total CH4 yield. The replacement of SBM with algae linearly reduced the degradability of nutrients, along with a linear reduction in gas production. When replacement of SBM with only SPI was evaluated, SPI slightly reduced the degradability of nutrients; however, it promoted a linear reduction in CH4 yield, as well as reduction in CH4 yield by unit of degraded DM, NDF, and OM. In summary, there was no interaction of carbohydrate profile and protein source, which means that SBM replacement had a similar effect, regardless of dietary carbohydrate profile. Spirulina may be a more suitable algae source than Chlorella due to the potential to reduce CH4.

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.

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.

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.

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.

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.

Combinations of bacterial cultures, exogenous enzymes, and yeast-based feed additives and their impact on ruminal microbiome.

Our objective was to evaluate the effects of bacteria (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus lichenformis, Bacillus subtilis, and Enterococcus faecium), enzymes (amylase, hemicellulose, and xylanase), and yeast as additives on the ruminal microbiome. We hypothesized that inclusion of bacteria, enzymes, and yeast would impact butyric bacterial populations. Eight fermenters were arranged in a duplicated 4 × 4 Latin square with the following treatments: 1) control without additives (CTRL); 2) bacterial culture and enzyme blend (EB); 3) bacterial culture and enzyme blend with a live yeast and yeast culture blend (EBY); and 4) double dose of bacterial culture and enzyme blend and the yeast products blend (2X). We conducted four fermentation periods of 10 d each, with the last 3 d for collection of samples. Overall, 64 solid and liquid samples were analyzed by amplification of the V4 region of bacterial 16S rRNA. Data were analyzed with R and SAS. The following orthogonal contrasts were used: 1) ADD-the control compared to all treatments with additives (CTRL vs. EB, EBY, and 2X); 2) YEAST-treatment without yeast compared to those with yeast (EB vs. EBY and 2X); and 3) DOSE-the single dose of enzymes, bacteria, and yeast compared to the doubled dose (EBY vs. 2X). Family Prevotellaceae was more abundant when additives were added (ADD). Additives (ADD) also increased relative abundance of Prevotellaceae Ga6A1 and YAB2003 in solid fraction, and of Prevotellaceae Ga6A1 and two members of Lachnospiracea family in liquid fraction. Yeast (YEAST) decreased relative abundance of Succinivibrionaceae UCG-001 and increased abundance of Ruminococcus and Prevotellaceae UCG-003 in solid fraction. Doubling the dose of enzymes and microbial additives (DOSE) decreased the abundance of Succiniclasticum in solid fraction and Selenomonadaceae in the liquid. Molar proportion of butyrate was highly correlated with abundance of Prevotellaceae Ga6A1 in solid (r = 0.68) and liquid fraction (r = 0.79), and with Unclassified Lachnospiraceae in liquid (r = 0.70). Our results demonstrate that YEAST decreases abundance of succinate synthesizing bacteria, while DOSE decreases abundance of bacteria that metabolize succinate into propionate. Combined bacteria, enzymes, and yeast increase the relative abundance of specific genera primarily within the Prevotellaceae family, which may explain the increase in butyrate molar proportion observed with ADD.

Effects of ruminal protozoa on methane emissions in ruminants-A meta-analysis.

The effects of different ruminal protozoa (RP) on CH4 emissions from ruminants were evaluated in a meta-analysis, using 64 publications reporting data from 79 in vivo experiments. Experiments included in the database reported CH4 emissions (g/d) and total RP (TRP, log10 cells/mL) from the same group of animals. The relationship between CH4 emissions and RP (TRP, entodiniomorphids, and isotrichids), and TRP-, entodiniomorphid-, and isotrichid-based CH4 emission prediction models, were evaluated as mixed models with experiment as a random effect and weighted by the reciprocal of the standard error of the mean and centered around one. Positive associations existed between TRP and isotrichids with CH4 emissions but not between entodiniomorphids and CH4 emissions. A reduction in CH4 emissions was observed, averaging 7.96 and 4.25 g/d, per log unit reduction in TRP and isotrichid concentrations, respectively. Total RP and isotrichids were important variables in predicting CH4 emissions from ruminants. Isotrichid CH4 prediction model was more robust than the TRP, evidenciated by lower predicted sigma hat study (%), and error (%), and with higher concordance correlation coefficient. Both TRP and isotrichid models can accurately predict CH4 emissions across different ruminant types, as shown by the low square root of the mean square prediction error, with 6.59 and 4.08% of the mean of root of the mean square prediction error in the TRP and isotrichid models, respectively. Our results confirm that isotrichids are more important than entodiniomorphids in methanogenesis. Distinguishing these 2 populations yielded a more robust CH4 prediction model than combining them as total protozoa.

Effects of sucrose and lactose as partial replacement to corn in lactating dairy cow diets: a review.

Carbohydrates are one of the three macronutrients that provides energy in diets and are classified by their structures. Starch is a nonstructural carbohydrate and polysaccharide made of glucose monomers used for storage in plant cells. When starch makes up greater than 30% of the DM in diets there can be adverse effects on NDF digestibility due to decreases in ruminal pH. Sugars are water soluble carbohydrates that consist of monosaccharide and disaccharide units. Sugars ferment faster than starch because microorganisms in the rumen can ferment carbohydrates at different rates depending on their structure; however, this has not been shown to have negative effects on the ruminal pH. Sources of sugars such as molasses (sucrose) or whey (lactose) can be included in the diet as a partial replacement for starch in dairy cow diets. The purpose of replacing starch with sugars in a diet would be to add differing sources of carbohydrates in the diet to allow for continual fermentation of carbohydrates by the microorganisms in the rumen. It has been seen in studies and previous literature that the partial replacement of starch with sugars has the potential to maintain the ruminal environment and milk yield and composition in dairy cows without reducing NDF digestibility. The objective of this review is to evaluate the effects of partially replacing starch with sugars in dairy diets and its implication on ruminal fermentation, nutrient utilization, milk production, and feeding replacement strategy.

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.

Effects of choline chloride on the ruminal microbiome at 2 dietary neutral detergent fiber concentrations in continuous culture.

Our objective was to evaluate the effects of unprotected choline chloride (Cho) on the ruminal microbiome at 2 dietary neutral detergent fiber (NDF) concentrations. We hypothesized that the effects of Cho on ruminal bacterial populations would depend on NDF. Eight dual-flow continuous-culture fermentors were arranged in a duplicated 4 × 4 Latin square as a 2 × 2 factorial with the following treatments: (1) 30% NDF-control (30% NDF diet, no supplemental choline); (2) 30% NDF-Cho (30% NDF diet plus 1.9 g of choline ion per kg of dry matter); (3) 40% NDF-control (40% NDF diet, no supplemental choline); and (4) 40% NDF-Cho (40% NDF diet plus 1.9 g of choline ion per kg of dry matter). We did 4 fermentation periods of 10 d each and used the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 32 solid and 32 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R (R Project for Statistical Computing) and SAS (SAS Institute Inc.) to determine effects of Cho, NDF, and NDF × Cho on taxa relative abundance. The correlation of propionate molar proportion with taxa relative abundance was also analyzed. At the phylum level, relative abundance of Firmicutes in the liquid fraction tended to be greater when Cho was supplemented with a 30% NDF diet. At the order level, Cho increased Coriobacteriales in solid fraction and decreased Fibrobacterales in liquid fraction. Moreover, Cho decreased abundance of Clostridiales and increased Selenomonadales in the solid fraction, only with the 30% NDF diet. For genera, lower abundance of Pseudobutyrivibrio resulted from Cho in solid and liquid fractions. Greater abundance of Succinivibrio in solid and Selenomonas and Selenomonas 1 in liquid resulted from Cho with the 30% NDF diet. Propionate molar proportion was positively correlated with relative abundance of order Selenomonadales in solid and liquid fractions, and with genus Succinivibrio in solid and genera Selenomonas and Selenomonas 1 in liquid. Our results indicate that Cho primarily decreases abundance of bacteria involved in fiber degradation and increases abundance of bacteria mainly involved in nonstructural carbohydrate degradation and synthesis of propionate, particularly when a diet with 30% NDF is provided.

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 lactic acid-producing bacteria as direct-fed microbials on the ruminal microbiome.

The objective of this study was to evaluate ruminal microbiome changes associated with feeding Lactobacillus plantarum GB-LP1 as direct-fed microbials (DFM) in high-producing dairy cow diets. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin square design. A basal diet was formulated to meet the requirements of a cow producing 45 kg of milk per day (16% crude protein and 28% starch). There were 4 experimental treatments: the basal diet without any DFM (CTRL); a mixture of Lactobacillus acidophilus, 1 × 109 cfu/g, and Propionibacterium freudenreichii, 2 × 109 cfu/g [MLP = 0.01% of diet dry matter (DM)]; and 2 different levels of L. plantarum, 1.35 × 109 cfu/g (L1 = 0.05% and L2 = 0.10% of diet DM). Bacterial samples were collected from the fluid and particulate effluents before feeding and at 2, 4, 6, and 8 h after feeding; a composite of all time points was made for each fermentor within their respective fractionations. Bacterial community composition was analyzed through sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequenced data were analyzed on DADA2, and statistical analyses were performed in R (RStudio 3.0.1, https://www.r-project.org/) and SAS 9.4 (SAS Institute Inc.); orthogonal contrasts were used to compare treatments. Different than in other fermentation scenarios (e.g., silage or beef cattle high-grain diets), treatments did not affect pH or lactic acid concentration. Effects were mainly from overall DFM inclusion, and they were mostly observed in the fluid phase. The relative abundance of the phylum Firmicutes, family Lachnospiraceae, and 6 genera decreased with DFM inclusion, with emphasis on Butyrivibrio_2, Saccharofermentans, and Ruminococcus_1 that are fibrolytic and may display peptidase activity during fermentation. Lachnospiraceae_AC2044_group and Lachnospiraceae_XPB1014_group also decreased in the fluid phase, and their relative abundances were positively correlated with NH3-N daily outflow from the fermentors. Specific effects of MLP and L. plantarum were mostly in specific bacteria associated with proteolytic and fibrolytic functions in the rumen. These findings help to explain why, in the previous results from this study, DFM inclusion decreased NH3-N concentration without altering pH and lactic acid concentration.

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.

Enzymatic effects of Pleurotus ostreatus spent substrate on whole-plant corn silage and performance of lactating goats.

Pleurotus ostreatus (oyster mushroom) synthesizes enzymes that degrade lignin, cellulose, and hemicellulose. The objectives of this study were to evaluate the effect of Pleurotus ostreatus spent substrate (POSS) on whole-plant corn silage (WPCS) chemical composition, antioxidant capacity, lignin monomers, and in vitro digestibility, as well as the performance of lactating goats fed corn silage treated with different levels of POSS. In experiment 1, 4 levels of lignocellulolytic enzymes were tested in a complete randomized design: 0, 10, 20, and 30 mg of lignocellulosic enzymes per kilogram of fresh matter, 4 replicates per treatment (vacuum-sealed bags). The bags were opened 60 d after ensiling. In experiment 2, corn silage treated with 3 enzyme levels (0, 10, or 30 mg/kg of fresh matter) was fed to lactating goats as part of the total mixed ration. Nine lactating Saanen goats (62.68 ± 7.62 kg BW; 44 ± 8 d in milk; 2.91 ± 0.81 kg of milk/day, mean ± SD) were assigned to three 3 × 3 Latin squares. Data were analyzed using the GLIMMIX procedure of SAS (version 9.4, SAS Institute Inc.), and means were compared by linear and quadratic orthogonal contrast. In experiment 1, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, and cellulose quadratically decreased in the WPCS treated with POSS. At the nadir point, POSS decreased NDF by 14.1%, ADF by 19.5%, lignin by 9.07%, and cellulose by 22.1% compared with the untreated silage. Therefore, POSS led to a quadratic increase in in vitro dry matter digestibility of WPCS (+8.88% at the vertex) compared with the untreated silage. In experiment 2, POSS quadratically increased the in vivo total-tract ADF digestibility. Also, the concentration of polyphenols in the milk of goats linearly increased with the addition of POSS, and no differences were observed among treatments for milk yield and composition. In summary, adding 10 mg of lignocellulolytic enzymes from POSS per kilogram of fresh matter of whole-plant corn at ensiling had a more evident reduction in lignin and cellulose concentration, leading to greater in vitro digestibility, as well as greater in vivo ADF digestibility; however, milk yield was not different among treatments.

Shrink, weighing accuracy, and weighing precision of mineral supplement in five commercial dairies in the Western United States.

Shrink, accuracy, and precision of ingredient weighing are critical factors of efficiency in TMR-fed dairy systems. Those factors have been evaluated for major feeds; however, we are not aware of any reports for mineral supplement. Farms commonly mix mineral supplement with other low-inclusion ingredients into a premix which is used later as a single ingredient for TMR formulation. Our objectives were to evaluate shrink, weighing accuracy, and weighing precision of mineral supplement during premix formulation, and variation in concentration of minerals in mineral supplement and TMR, in five large dairies in the Western United States. We used the automated weight-tracking system at each farm to account for all the mineral supplement loaded into the mixing-wagon and collected samples of mineral supplement and TMR from time of mineral supplement delivery at the farm until 100% of it was consumed. Mean, standard deviation and coefficient of variation (CV) for each variable were calculated with SAS 9.4. Average shrink was estimated at 2.0% for mineral supplement during storage and loading, ranging from 0.37% to 3.25%. Mineral supplement weighing deviation from the targeted amount was 1.54% on average for the five dairies with a 95% CV. Mineral composition of mineral supplements averaged 11.3%, 0.27%, and 3.16% for Ca, P, and Mg, and 215, 881, and 1533 ppm for Cu, Mn, and Zn, respectively. Mineral compositions in TMR averaged 0.84%, 0.41%, and 0.37% for Ca, P, and Mg, respectively; and 15.1, 71, and 94.5 ppm for Cu, Mn, and Zn, respectively. The CV of all minerals except Ca, were larger for mineral supplement than TMR, and with the exception of P in mineral supplement, CV of trace minerals were larger than CV values for macro minerals. Our shrink estimates for mineral supplement represent an initial approximation to this issue. Results of our weighing deviation analysis suggest some room for improvement on the precision of weighing mineral supplement at the time when premix is prepared at the farm, which could improve consistency in chemical composition of the premix and consequently reduce the variation (CV values) of mineral concentrations in TMR.

Effects of lactic acid bacteria in a silage inoculant on ruminal nutrient digestibility, nitrogen metabolism, and lactation performance of high-producing dairy cows.

Silage treated with lactic acid bacteria inoculants has been reported to increase ruminal microbial biomass when tested in vitro. Therefore, we tested if alfalfa silage inoculated with Lactobacillus plantarum MTD-1 would improve ruminal N metabolism and increase milk production in high-producing dairy cows. Twenty-eight early lactation Holstein cows (8 ruminally cannulated) were blocked by DIM and milk production; animals were used in a double crossover design consisting of four 28-d periods. Animals in each block were randomly assigned to 2 treatments: a diet containing uninoculated alfalfa silage (control) and a diet containing alfalfa silage inoculated with L. plantarum MTD-1 (LP). Diets were formulated to contain 50% of alfalfa silage, 16% crude protein, and 25% neutral detergent fiber (dry matter basis). Milk production and dry matter intake were recorded in the last 14 d of each period. Milk samples were collected twice at both daily milkings on d 20, 21, 27, and 28 of each period. On d 22, omasal samples were collected from the cannulated animals over a period of 3 d to quantify ruminal digestibility and nutrient flows. Data were analyzed using mixed models of SAS 9.4 (SAS Institute). Compared to the control, cows receiving the LP treatment had greater milk production (40.4 vs. 39.6 kg/d) and lower milk urea nitrogen concentration (11.6 vs. 12.7 mg/dL), despite minor changes in energy-corrected milk. Milk lactose concentration was greater in the milk produced by cows fed the LP treatment, which reflected a tendency for increased milk lactose yield. Although milk true protein concentration was lower for cows in the LP treatment, milk true protein yield was the same on both control and LP treatments. Improvements in milk production of animals under the LP treatment were associated with greater organic matter truly digested in the rumen, especially ruminal neutral detergent fiber digestion. Minor changes were observed in total omasal microbial nonammonia N flow in cows receiving the LP treatment. Therefore, alfalfa silage treated with L. plantarum MTD-1 may improve ruminal fermentation and milk production; however, because of a lack of response in ruminal N metabolism, these changes did not result in greater energy-corrected milk in high-producing dairy cows.

Effects of bacterial cultures, enzymes, and yeast-based feed additive combinations on ruminal fermentation in a dual-flow continuous culture system.

Bacterial cultures, enzymes, and yeast-derived feed additives are often included in commercial dairy rations due to their effects on ruminal fermentation. However, the effects of these additives when fed together are not well understood. The objective of this study was to evaluate the changes in ruminal fermentation when a dairy ration is supplemented with combinations of bacterial probiotics, enzymes and yeast. Our hypotheses were that ruminal fermentation would be altered, indicated through changes in volatile fatty acid profile and nutrient digestibility, with the inclusion of (1) an additive, (2) yeast, and (3) increasing additive doses. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square with four 10 d experimental periods, consisting of 7 d for diet adaptation and 3 d for sample collection. Basal diets contained 52:48 forage:concentrate and fermenters were fed 106 g of dry matter per day divided equally between two feeding times. Treatments were: control (CTRL, without additives); bacterial culture/enzyme blend (EB, 1.7 mg/d); bacterial culture/enzyme blend with a blend of live yeast and yeast culture (EBY, 49.76 mg/d); and a double dose of the EBY treatment (2×, 99.53 mg/d). The bacterial culture/enzyme blend contained five strains of probiotics (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus lichenformis, Bacillus subtilis, and Enterococcus faecium) and three enzymes (amylase, hemicellulase, and xylanase). On d 8-10, samples were collected for pH, redox, volatile fatty acids, lactate, ammonia N, and digestibility measurements. Statistical analysis was performed using the GLIMMIX procedure of SAS. Repeated measures were used for pH, redox, VFA, NH3-N, and lactate kinetics data. Orthogonal contrasts were used to test the effect of (1) additives, ADD (CTRL vs. EB, EBY, and 2X); (2) yeast, YEAST (EB vs. EBY, and 2X); and (3) dose, DOSE (EBY vs. 2X). No effects (P > 0.05) were observed for pH, redox, NH3-N, acetate, isobutyrate, valerate, total VFA, acetate:propionate, nutrient digestibility or N utilization. Within the 24 h pool, the molar proportion of butyrate increased (P = 0.03) with the inclusion of additives when compared to the control while the molar proportion of propionate tended to decrease (P = 0.07). In conclusion, the inclusion of bacterial cultures, enzymes and yeast in the diet increased butyrate concentration; but did not result in major changes in ruminal fermentation.