Effects of Bacillus subtilis on in vitro ruminal fermentation and methane production.

The objective of this study was to evaluate the effect of a proprietary strain of a Bacillus subtilis on in vitro ruminal fermentation and methane production in batch culture serum bottles. One hundred forty-nine batch culture bottles were used in a complete randomized block design. The arrangement of treatments was a 3 × 3 × 4 factorial to evaluate the effects of inoculum, time, diet, and their respective interactions. There were three experimental runs total, where the run was used as block. Inoculum treatments were 1.85 mg/mL of microcrystalline cellulose (CON); 10 billion B. subtilis plus microcrystalline cellulose (A1); and 60 billion B. subtilis plus microcrystalline cellulose (A2). Diet treatments were 0.50 g of early lactation diet (E, 30% starch), mid-lactation diet (M, 25% starch), or dry cow diet (D, 18% starch). The combination resulted in total of nine treatments. Each treatment had five replicates, two of which were used to determine nutrient degradability at 24 and 48 h after inoculation, and three were used to determine pH, ammonia nitrogen (NH3-N), volatile fatty acids, lactate, total gas, and methane production at 3, 6, 24, and 48 h after inoculation. Fixed effects of inoculum, diet, and their interaction were tested using the GLIMMIX procedure of SAS. Significance was declared at P ≤ 0.05. We observed that, compared to control, the supplementation of B. subtilis, decreased the production of acetate and propionate, while increasing the production of butyrate, iso-butyrate, valerate, iso-valerate, and caproate within each respective diet. Additionally, the total methane production exhibited mixed responses depending on the diet type. Overall, the inclusion of B. subtilis under in vitro conditions shows the potential to reduce ruminal methane production when supplemented with a mid-lactation diet, constituting a possible methane mitigation additive for dairy cattle diets.

Effects of monensin and cashew nut-shell extract on bacterial community composition in a dual-flow continuous culture system.

The objective of this study was to evaluate the effects of including monensin and two doses of CNSE in a high producing dairy cow diet on ruminal bacterial communities. 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 d (17% crude protein and 27% starch). There were four experimental treatments: the basal diet without any feed additive (CON), 2.5 µM monensin (MON), 100 ppm CNSE granule (CNSE100), and 200 ppm CNSE granule (CNSE200). Samples were collected from the fluid and solid effluents at 3, 6, and 9 h after feeding; a composite of all time points was made for each fermenter within their respective fractions. Bacterial community composition was analyzed by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Treatment responses for bacterial community structure were analyzed with the PERMANOVA test run with the R Vegan package. Treatment responses for correlations were analyzed with the CORR 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). Significance was declared at P ≤ 0.05. We observed that the relative abundance of Sharpea (P < 0.01), Mailhella (P = 0.05), Ruminococcus (P = 0.03), Eubacterium (P = 0.01), and Coprococcus (P < 0.01) from the liquid fraction and the relative abundance of Ruminococcus (P = 0.03) and Catonella (P = 0.02) from the solid fraction decreased, while the relative abundance of Syntrophococcus (P = 0.02) increased in response to MON when compared to CNSE treatments. Our results demonstrate that CNSE and monensin have similar effects on the major ruminal bacterial genera, while some differences were observed in some minor genera. Overall, the tested additives would affect the ruminal fermentation in a similar pattern.

In vitro evaluation of microencapsulated organic acids and pure botanicals as a supplement in lactating dairy cows diet on in vitro ruminal fermentation.

The utilization of microencapsulated organic acids and pure botanicals (mOAPB) is widely used in the monogastric livestock industry as an alternative to antibiotics; in addition, it can have gut immunomodulatory functions. More recently, an interest in applying those compounds in the ruminant industry has increased; thus, we evaluated the effects of mOAPB on ruminal fermentation kinetics and metabolite production in an in vitro dual-flow continuous-culture system. For this study, two ruminal cannulated lactating dairy Holstein cows were used as ruminal content donors, and the inoculum was incubated in eight fermenters arranged in a 4 × 4 Latin square design. The basal diet was formulated to meet the nutritional requirements of a 680-kg Holstein dairy cow producing 45 kg/d of milk and supplemented with increasing levels of mOAPB (0; 0.12; 0.24; or 0.36% of dry matter [DM]), which contained 55.6% hydrogenated and refined palm oil, 25% citric acid, 16.7% sorbic acid, 1.7% thymol, and 1% vanillin. Diet had 16.1 CP, 30.9 neutral detergent fiber (NDF), and 32.0 starch, % of DM basis, and fermenters were fed 106 g/d split into two feedings. After a 7 d adaptation, samples were collected for 3 d in each period. Samples of the ruminal content from the fermenters were collected at 0, 1, 2, 4, 6, and 8 h postmorning 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 at days 8 to 10. The statistical analysis was conducted using MIXED procedure of SAS and treatment, time, and its interactions were considered as fixed effects and day, Latin square, and fermenter as random effects. To depict the treatment effects, orthogonal contrasts were used (linear and quadratic). The supplementation of mOAPB had no major effects on the ruminal fermentation, metabolite production, and degradability of nutrients. The lack of statistical differences between control and supplemented fermenters indicates effective ruminal protection and minor ruminal effects of the active compounds. This could be attributed to the range of daily variation of pH, which ranged from 5.98 to 6.45. The pH can play a major role in the solubilization of lipid coat. It can be concluded that mOAPB did not affect the ruminal fermentation, metabolite production, and degradability of dietary nutrients using an in vitro rumen simulator.

Can dietary magnesium sources and buffer change the ruminal microbiota composition and fermentation of lactating dairy cows?

Magnesium oxide (MgO) is one of the most used Mg supplements in livestock. However, to avoid relying upon only one Mg source, it is important to have alternative Mg sources. Therefore, the objective of this study was to evaluate the effects of the interaction of two Mg sources with buffer use on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. Twenty lactating Holstein cows were blocked by parity and days in milk into five blocks with four cows each, in a 2 × 2 factorial design. Within blocks, cows were assigned to one of four treatments: 1) MgO; 2) MgO + Na sesquicarbonate (MgO+); 3) calcium-magnesium hydroxide (CaMgOH); 4) CaMgOH + Na sesquicarbonate (CaMgOH+). For 60 d, cows were individually fed a corn silage-based diet, and treatments were top-dressed. Ruminal fluid was collected via an orogastric tube, for analyses of the microbiota composition, volatile fatty acids (VFA), lactate, and ammonia nitrogen (NH3-N). The microbiota composition was analyzed using V4/16S rRNA gene sequencing, and taxonomy was assigned using the Silva database. Statistical analysis was carried out following the procedures of block design analysis, where block and cow were considered random variables. Effects of Mg source, buffer, and the interaction between Mg Source × Buffer were analyzed through orthogonal contrasts. There was no interaction effect of the two factors evaluated. There was a greater concentration of NH3-N, lactate, and butyrate in the ruminal fluid of cows fed with CaMg(OH)2, regardless of the buffer use. The increase in these fermentation intermediates/ end-products can be explained by an increase in abundance of micro-organisms of the genus Prevotella, Lactobacillus, and Butyrivibrio, which are micro-organisms mainly responsible for proteolysis, lactate-production, and butyrate-production in the rumen, respectively. Also, dietary buffer use did not affect the ruminal fermentation metabolites and pH; however, an improvement of the apparent total tract digestibility of dry matter (DM), organic matter (OM), neutral fiber detergent (NDF), and acid fiber detergent (ADF) were found for animals fed with dietary buffer. In summary, there was no interaction effect of buffer use and Mg source, whereas buffer improved total tract apparent digestibility of DM and OM through an increase in NDF and ADF digestibility and CaMg(OH)2 increased ruminal concentration of butyrate and abundance of butyrate-producing bacteria.

Magnesium oxide (MgO) is extensively used as a dietary magnesium (Mg) source in dairy cow diets. However, dairy operations can benefit from other Mg sources. Thus, we evaluated the replacement of dietary MgO with calcium­magnesium hydroxide (CaMg(OH)2) in diets with and without ruminal buffer and their effects on the ruminal microbiota composition, ruminal fermentation, and nutrient digestibility in lactating dairy cows. The study used 20 lactating Holstein cows that were blocked in groups of four and randomly assigned to one of the four treatments. The ruminal content, feed, feces, and urine were collected for analysis of the microbiota composition, ruminal fermentation, nitrogen metabolism, and apparent nutrient digestibility. There was no interaction effect of dietary buffer use and Mg source, while buffer improved total tract apparent digestibility of the dry matter and fiber components; CaMg(OH)2 increased the ruminal concentration of butyrate and the abundance of butyrate-producing bacteria. In summary, we conclude that using CaMg(OH)2 can improve ruminal fermentation regardless of buffer use, which indicates that we can take advantage of the mineral formulation in the diet to modulate the ruminal microbiota composition.

Effects of ruminal lipopolysaccharides on growth and fermentation end products of pure cultured bacteria.

Elevated levels of ruminal lipopolysaccharides (LPS) have been linked to ruminal acidosis; however, they result in reduced endotoxicity compared to LPS derived from species like Escherichia coli. Additionally, there is a knowledge gap on the potential effect of LPS derived from ruminal microbiome on ruminal bacteria species whose abundance is associated with ruminal acidosis. The objective of this study was to evaluate the effects of LPS-free anaerobic water (CTRL), E. coli-LPS (E. COLI), ruminal-LPS (RUM), and a 1:1 mixture of E. coli and ruminal-LPS (MIX) on the growth characteristics and fermentation end products of lactate-producing bacteria (Streptococcus bovis JB1, Selenomonas ruminantium HD4) and lactate-utilizing bacterium (Megasphaera elsdenii T81). The growth characteristics were predicted based on the logistic growth model, the ammonia concentration was determined by the phenolic acid/hypochlorite method and organic acids were analyzed with high performance liquid chromatography. Results indicate that, compared to the CTRL, the maximum specific growth rate of S. bovis JB1 decreased by approximately 19% and 23% when RUM and MIX were dosed, respectively. In addition, acetate and lactate concentrations in Se. ruminantium HD4 were reduced by approximately 30% and 18%; respectively, in response to MIX dosing. Compared to CTRL, lactate concentration from S. bovis JB1 was reduced approximately by 31% and 22% in response to RUM and MIX dosing; respectively. In summary, RUM decreased the growth and lactate production of some lactate-producing bacteria, potentially mitigating the development of subacute ruminal acidosis by restricting lactate availability to some lactate-utilizing bacteria that metabolize lactate into VFAs thus further contributing to the development of acidosis. Also, RUM did not affect Megasphaera elsdenii T81 growth.

Megasphaera elsdenii and Saccharomyces Cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge.

This study aimed to evaluate the effects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle finishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-flow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × 108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1-8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9-11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No differences in pH, D-, L-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment effects were observed on NH3-N concentration; however, efficiency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3-N concentration was reduced to approximately 2 mg/dL as the challenge progressed.

Adaptation of in vitro methodologies to estimate the intestinal digestion of lipids in ruminants.

The objective of this study was to adapt existing in vitro methodologies to determine the extent of intestinal digestion of corn oil (CO), canola oil (CA), and beef tallow (BT) via manipulation of incubation length and concentrations of lipase, bile, and calcium within a buffer solution. Unless otherwise stated, 0.5 g of each lipid source were incubated separately and in triplicate, with triplicate batch culture runs for each treatment in 40 mL of 0.5 M KH2PO4 (pH = 7.6) for 24 h with pancreatin (8 g/L), bovine bile (2.5 g/L), and CaCl2 (10 mM). Individually, concentrations of pancreatin, bile, and CaCl2, as well as incubation length were tested. To examine the use of this assay to estimate in vitro total tract digestion, a KH2PO4 solution with concentrated amounts to reach the same final concentrations of pancreatin, bile, and Ca were used as the third step in a three-step total tract digestibility procedure. Free glycerol and free fatty acid (FFA) concentrations were measured using colorimetric assays as indicators of digestion. Data wereanalyzed as a completely randomized block design (block = run), using the Glimmix procedure of SAS. For each lipid source, free glycerol increased with increasing pancreatin; however, FFA was lowest at 0 g/L pancreatin but was similar at 6, 8, and 10 g/L. Both glycerol and FFA were greater for 2.5 and 5 g/L of bile than for 0 g/L for each lipid source. Calcium concentration did not affect glycerol or FFA for either CO or CA; however, glycerol and FFA for BT were greater when calcium was included at 5 and 10 mM than at 0 mM. For all fat sources, free glycerol and FFA increased after 1 h until 12 h, but did not increase from 12 to 24 h. When a concentrated mixture was used following fermentation and acidification steps, digestibility using FFA concentration increased as compared to just adding buffer; however, free glycerol concentration was indeterminable. Thus, free glycerol and FFA can be used as indicators of lipid digestion when a lipid source is incubated for at least 12 h in a buffer solution containing 8 g/L pancreatin, 2.5 g/L bile, and 5 mM Ca when only estimating in vitro intestinal digestion; however, when utilizing this assay in a three-step in vitro total tract digestibility procedure, only FFA can be used.

Ruminal Lipopolysaccharides Analysis: Uncharted Waters with Promising Signs.

The objective of this review is to present the need for the development of a comprehensive ruminal lipopolysaccharide (LPS) extraction, purification and analysis protocol and state hypotheses that could contribute to planning novel strategies against ruminal acidosis. Lipopolysaccharide is an immunostimulatory molecule of Gram-negative bacterial outer membranes and has been reported to contribute to ruminal acidosis in cattle. Bacterial death and lysis are normal processes, and thus LPS is normally present in ruminal fluid. However, ruminal LPS concentration is much greater during subacute ruminal acidosis (SARA). Contrary to the widely known LPSs, ruminal LPS seems to be composed of a variety of LPS chemotypes that may interact with each other resulting in an LPS "mixture". Hypotheses regarding the influence of each specific ruminal bacterial specie to innate immunity during SARA, and the representativeness of the exclusive use of the Escherichia coli LPS to rumen epithelial tissue challenges, could expand our knowledge regarding SARA. In addition, possible correlation between the monomeric Toll-like Receptor 4 (TRL4) and the antagonistic penta-acylated lipid A of LPS could contribute to novel strategies to tackle this nutrition disorder.