Megasphaera elsdenii and Saccharomyces cerevisiae as direct fed microbials and their impact on ruminal microbiome during an acute acidosis challenge in continuous culture.

Our objective was to evaluate the effects of combinations of Saccharomyces cerevisiae and Megasphaera elsdenii as direct-fed microbials (DFM) on ruminal microbiome during an acute acidosis challenge in a continuous culture system. Treatments provided a DFM dose of 1 × 108 colony-forming unit (CFU)/mL, as follows: 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 strains 1 and 2). We conducted four experimental periods of 11 d, which consisted of non-acidotic days (1 to 8) and acidotic challenge days (9 to 11) to establish acute ruminal acidosis conditions with a common basal diet containing 12% neutral detergent fiber and 58% starch. Treatments were applied from days 8 to 11, and samples of liquid and solid-associated bacteria were collected on days 9 to 11. Overall, 128 samples were analyzed by amplification of the V4 region of bacterial 16S rRNA, and data were analyzed with R and SAS for alpha and beta diversity, taxa relative abundance, and correlation of taxa abundance with propionate molar proportion. We observed a lower bacterial diversity (Shannon index, P = 0.02) when YM1 was added to the diet in comparison to the three other treatments. Moreover, compared to control, addition of YM1 to the diet increased relative abundance of phylum Proteobacteria (P = 0.05) and family Succinivibrioceae (P = 0.05) in the solid fraction and tended to increase abundance of family Succinivibrioceae (P = 0.10) and genus Succinivibrio (P = 0.09) in the liquid fraction. Correlation analysis indicated a positive association between propionate molar proportion and relative abundance of Proteobacteria (r = 0.36, P = 0.04) and Succinivibrioceae (r = 0.36, P = 0.05) in the solid fraction. The inclusion of YM1 in high-grain diets with a high starch content resulted in greater abundance of bacteria involved in succinate synthesis which may have provided the substrate for the greater propionate synthesis observed.

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 partially replacing dietary corn with sugars in a dual-flow continuous culture system on the ruminal microbiome.

The objective of this study was to evaluate the effects of feeding sugars as a replacement for starch on the ruminal microbiome using a dual-flow continuous culture system. Four periods of 10 days each were conducted with 8 fermenters in a 4 × 4 replicated Latin square design. Treatments included: 1) control with corn-CON, 2) molasses-MOL, 3) untreated condensed whey permeate-CWP, and 4) CWP treated with a caustic agent-TCWP as a partial substitute for corn. Sugars were defined as the water-soluble carbohydrates (WSC) concentration. Diets were formulated by replacing 4% of the diet DM in the form of starch from corn with the sugars in byproducts. Microbial samples for DNA analysis were collected from the solid and liquid effluent containers at 3, 6, and 9 h after feeding. Bacterial community composition was analyzed with sequencing the V4 region of the 16S rRNA gene using Illumina MiSeq platform. Data were analyzed with R 4.1.3 packages vegan, lmer, and ggplot to determine the effects of treatment on the relative abundance of taxa in the solid and liquid fractions, as well as the correlation of Acetate: Propionate ratio and pH to taxa relative abundance. Treatments did not affect alpha or beta diversity. At the phylum level the relative abundance of Proteobacteria was increased in CON compared to sugars in the solid fraction. In the liquid fraction, Firmicutes had greater relative abundance in sugar treatments while Bacteroidota and Spirochaetota were present in lower relative abundance in CWP. For solid and liquid samples, the family Lachnospiraceae had greater relative abundance in sugar treatments compared to CON. The decreased relative abundance of Christensenellaceae and Rikenellaceae paired with the greater relative abundance of Selenomonadaceae in CWP could help explain greater propionate molar proportion and decreased ruminal pH previously observed for this treatment. The genera Olsenella a lactic acid-producing bacterium, had the greatest relative abundance in MOL. Incorporating TCWP or MOL as a partial replacement for starch was more conservative of fibrolytic bacterial taxa compared to CWP. Additionally, TCWP did not increase bacterial taxa associated with synthesis of lactate as compared to MOL. Overall, replacing starch with sugars is mostly conservative of the ruminal microbiome; however, changes observed coincide with differences observed in acetate and propionate proportions and ruminal pH.

The Effects of Incremental Doses of Aflatoxin B1 on In Vitro Ruminal Nutrient Digestibility and Fermentation Profile of a Lactating Dairy Cow Diet in a Dual-Flow Continuous Culture System.

Aflatoxin B1 (AFB1) is a mycotoxin known to impair human and animal health. It is also believed to have a deleterious effect on ruminal nutrient digestibility under in vitro batch culture systems. The objective of this study was to evaluate the effects of increasing the dose of AFB1 on ruminal dry matter and nutrient digestibility, fermentation profile, and N flows using a dual-flow continuous culture system fed a diet formulated for lactating dairy cows. Eight fermenter vessels were used in a replicated 4 × 4 Latin square design with 10 d periods (7 d adaptation and 3 d sample collection). Treatments were randomly applied to fermenters on diet DM basis: (1) 0 µg of AFB1/kg of DM (Control); (2) 50 µg of AFB1/kg of DM (AF50); (3) 100 µg of AFB1/kg of DM (AF100); and (4) 150 µg of AFB1/kg of DM (AF150). Treatments did not affect nutrient digestibility, fermentation, and N flows. Aflatoxin B1 concentration in ruminal fluid increased with dose but decreased to undetectable levels after 4 h post-dosing. In conclusion, adding incremental doses of AFB1 did not affect ruminal fermentation, digestibility of nutrients, and N flows in a dual-flow continuous culture system fed diets formulated for lactating dairy cows.

Effects of calcium-magnesium carbonate and calcium-magnesium hydroxide as supplemental sources of magnesium on ruminal microbiome.

Our objective was to evaluate the inclusion of calcium-magnesium carbonate [CaMg(CO3)2] and calcium-magnesium hydroxide [CaMg(OH)4] in corn silage-based diets and their impact on ruminal microbiome. Our previous work showed a lower pH and molar proportion of butyrate from diets supplemented with [CaMg(CO3)2] compared to [CaMg(OH)4]; therefore, we hypothesized that ruminal microbiome would be affected by Mg source. Four continuous culture fermenters were arranged in a 4 × 4 Latin square with the following treatments defined by the supplemental source of Mg: 1) Control (100% MgO, plus sodium sesquicarbonate as a buffer); 2) CO 3 [100% CaMg(CO3)2]; 3) OH [100% CaMg(OH)4]; and 4) CO 3 /OH [50% Mg from CaMg(CO3)2, 50% Mg from CaMg(OH)4]. Diet nutrient concentration was held constant across treatments (16% CP, 30% NDF, 1.66 MCal NEl/kg, 0.67% Ca, and 0.25% Mg). We conducted four fermentation periods of 10 d, with the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 16 solid and 16 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R and SAS to determine treatment effects on taxa relative abundance of liquid and solid fractions. Correlation of butyrate molar proportion with taxa relative abundance was also analyzed. Treatments did not affect alpha and beta diversities or relative abundance of phylum, class and order in either liquid or solid fractions. At the family level, relative abundance of Lachnospiraceae in solid fraction was lower for CO3 and CO3/OH compared to OH and Control (P < 0.01). For genera, abundance of Butyrivibrio (P = 0.01) and Lachnospiraceae ND3007 (P < 0.01) (both from Lachnospiraceae family) was lower and unclassified Ruminococcaceae (P = 0.03) was greater in CO3 than Control and OH in solid fraction; while abundance of Pseudobutyrivibrio (P = 0.10) and Lachnospiraceae FD2005 (P = 0.09) (both from Lachnospiraceae family) and Ruminobacter (P = 0.09) tended to decrease in CO3 compared to Control in liquid fraction. Butyrate molar proportion was negatively correlated to Ruminococcaceae (r = -0.55) in solid fraction and positively correlated to Pseudobutyrivibrio (r = 0.61) and Lachnospiraceae FD2005 (r = 0.61) in liquid. Our results indicate that source of Mg has an impact on bacterial taxa associated with ruminal butyrate synthesis, which is important for epithelial health and fatty acid synthesis.

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.

Effects of neutral detergent fiber digestibility estimation method on calculated energy concentration of canola meals from 12 Canadian processing plants.

Our aim was to determine whether the method used to estimate truly digestible neutral detergent fiber (tdNDF) affects calculated concentrations of total digestible nutrients (TDN1x) and net energy of lactation (NEL3x) of canola meal (CM). Samples were collected from 12 CM processing plants in Canada over 4 yr (2011 to 2014, n = 47) and analyzed for dry matter (DM), crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin (ADL), and neutral detergent insoluble CP (NDICP). Ruminal in situ incubation of CM samples was performed at 0, 24, 48, 96, and 288 h to determine NDF fractions (A, B, and C), effective ruminal NDF digestibility (ERNDFD), and indigestible NDF (iNDF) of CM. Three tdNDF-estimation methods were evaluated: 1) National Research Council (NRC) = 0.75 × (NDF - NDICP - ADL) × {1- [ADL/ (NDF - NDICP)]0.667}; 2) iNDF = 0.75 × (NDF - NDICP - NDF remaining after 288 h in situ); and 3) ERNDFD estimated from in situ NDF digestion kinetics. Resulting tdNDF values were used for calculation of TDN1x and NEL3x according to NRC (2001) equations. Data were analyzed with MIXED procedure of SAS 9.4 to determine the effect of processing plant on chemical composition, NDF degradation kinetics and NEL3x of CM. Effect of tdNDF estimation method on calculated TDN1x and NEL3x of CM was also evaluated. Model for analysis of processing plant included the fixed effect of plant and the random effect of year (plant) as replication, while analysis of tdNDF methods included the fixed effect of tdNDF estimation method and the random effects of processing plant and of year(plant) as replication. There was an effect of processing plant on DM (P = 0.03), CP (P < 0.01), EE (P < 0.01), and NDF (P < 0.01) of CM. Processing plant also had an effect on NDF fractions A (P < 0.01) and B (P = 0.02) but did not affect fraction C and ERNDFD. The tdNDF estimation method had an effect on tdNDF (P < 0.01), TDN1x (P < 0.01), and NEL3x (P < 0.01) of CM, yielding average NEL3x values of 1.72, 1.87, and 2.07 Mcal/kg for NRC, iNDF, and ERNDFD, respectively. Our results indicate that calculated energy concentration of CM according to NRC (2001) equations varies depending on the method used for estimation of tdNDF. Further research will be needed to determine the most accurate estimation method.

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.

Evaluation of performance and carcass traits for a five-cohort All Heifer, No Cow beef production system demonstration herd.

The All Heifer, No Cow (AHNC) beef production system is an alternative to conventional cow/calf production that involves insemination of nulliparous heifers with sexed semen to produce female calves that are early weaned at 3 mo of age. Dams are finished on a high-concentrate diet and harvested before reaching 30 mo of age. Objectives of this research were to document reproductive, feedyard, calf, and carcass performance of an AHNC herd; evaluate effects of carcass maturity on carcass quality; and determine if performance of initial cohorts (i.e., cohorts 1 and 2) differed from sustaining cohorts (i.e., cohorts 3-5). A total of 272 heifers were enrolled in the AHNC system via five annual cohorts. The system was initiated with 51 yearling, Angus-based heifers, and a replicate set (n = 56) was started 12 mo after. Heifers in cohorts 3 (n = 53), 4 (n = 56), and 5 (n = 56) were primarily offspring of prior cohorts (i.e., cohort 3 heifers born to cohort 1 females), but some were purchased to maintain inventory. Angus replacement heifers were purchased in cohorts 3 (n = 26), 4 (n = 26), and 5 (n = 28). Mean (±standard deviation) pregnancy rate at 30 d after fixed-time artificial insemination (AI) with sexed semen was 50.8% ± 9.4%, and 140-d pregnancy rate was 93.0% ± 1.5%. With AHNC, 61.0% ± 6.5% of females replaced themselves with a heifer. During finishing, average daily gain (ADG) was 1.9 ± 0.4 kg • d-1 and dry matter intake (DMI) was 14.9 ± 1.9 kg • d-1. Hot carcass weight (HCW) was 367 ± 35 kg. The USDA grading system classified 20.5% of all carcasses (n = 220) as C maturity (A00 = 100, B00 = 200, etc.), 62.4% ± 29.1% of carcasses as USDA Choice. USDA yield grade (YG) was 2.6 ± 0.7. Based on cohorts 1 and 2, there were no differences (P = 0.96) in Warner-Bratzler shear force values between A and B maturity vs. C maturity carcasses. Across all cohorts, there were no differences in USDA YG, marbling score (MA), and lean maturity between A and B maturity vs. C maturity carcasses; there were differences in age (P < 0.001), bone maturity (P < 0.001), and overall maturity (P <0.001). A comparison of initial vs. sustaining cohorts showed that initial cohorts had lower (P < 0.001) DMI, heavier (P < 0.001) HCW, and more advanced (P < 0.05) bone maturity. However, there were no differences for 30- and 140-d pregnancy rates, ADG, USDA YG, and MA between initial and sustaining cohorts. The AHNC beef production system can effectively produce female calves and quality carcasses for harvest.