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.     

Active dry Saccharomyces cerevisiae can alleviate the effect of subacute ruminal acidosis in lactating dairy cows

The objective of the study was to determine the effect of active dry Saccharomyces cerevisiae (ADSC) supplementation on dry matter intake, milk yield, milk components, ruminal pH, and microbial community during a dietary regimen that leads to subacute ruminal acidosis (SARA). Sixteen multiparous, rumen-cannulated lactating Holstein cows were randomly assigned to 1 of 2 dietary treatments that included ADSC (Biomate; AB Vista, Marlborough, UK; 8 × 10¹⁰ cfu/head per day) or control. During wk 1 to 6, all cows received a high-forage (HF) diet (77:23, forage:concentrate). Cows were then abruptly switched during wk 7 to a high-grain (HG) diet (49:51, forage:concentrate) and remained on the HG until the end of wk 10. Feed intake and milk yields were recorded daily. Ruminal pH was recorded continuously using an indwelling system for 1 to 2 d per week during the pre-experimental phase, and wk 6, 7, and 10. Ruminal digesta samples were collected at the end of the experiment and analyzed for relative change in microbial communities using real-time quantitative PCR. Cows were considered to have SARA if the duration below pH 5.6 was ≥300 min/d. Ruminal pH during wk 6 (HF plateau) was not different across treatments (15 ± 46 min/d at pH <5.6). The dietary regimen successfully induced SARA during wk 7 (transition from HF to HG diet), and ruminal pH (551 ± 46 min/d at pH <5.6) was not different across treatments. However, cows receiving ADSC had an improved ruminal pH (122 ± 57 vs. 321 ± 53 min/d at pH <5.6) during wk 10 (HG plateau) compared with control. Additionally, cows receiving ADSC had a better dry matter intake (23.3 ± 0.66 vs. 21.6 ± 0.61 kg/d) and 4% fat-corrected milk yield (29.6 ± 1.2 vs. 26.5 ± 1.2 kg/d) than control cows during the HG phase (wk 8 to 10). During HG feeding, cows receiving ADSC had greater total volatile fatty acid and propionate concentrations (175 ± 7.5 vs. 154 ± 7.5 and 117 ± 6.1 vs. 94 ± 5.7 mM for ADSC and control, respectively) and lower acetate:propionate ratio (0.26 ± 0.5 vs. 0.36 ± 0.05 for ADSC and control, respectively). Microbial analyses conducted on samples collected during wk 10 showed that cows supplemented with S. cerevisiae had a 9-fold, 2-fold, 6-fold, 1.3-fold, and 8-fold increase in S. cerevisiae, Fibrobacter succinogenes, Anaerovibrio lipolytica, Ruminococcus albus, and anaerobic fungi, respectively, which suggested an increase in cellulolytic microbes within the rumen. Cows supplemented with ADSC had 2.2-fold reduction in Prevotella albensis, which is a gram-negative bacterium predominant during SARA. Prevotella spp. are suggested to be an important source of lipopolysaccharide responsible for inflammation within the rumen. Cows supplemented with ADSC had a 2.3-fold increase in Streptococcus bovis and a 12-fold reduction in Megasphaera elsdenii. The reduction in M. elsdenii may reflect lower concentration of lactic acid within the rumen for ADSC cows. In conclusion, ADSC supplementation to dairy cows was demonstrated to alleviate the condition of SARA caused by abrupt dietary changes from HF to HG, and can potentially improve rumen function, as indicated by greater numbers of cellulolytic microorganisms within the rumen.     

Evidence for the inhibition of the terminal step of ruminal α-linolenic acid biohydrogenation by condensed tannins

Effects of condensed tannins (CT), either via extract or plant-bound, and saponin extract on ruminal biohydrogenation of α-linolenic acid (ALA) were investigated in vitro. Grass-clover hay served as basal diet (control). The control hay was supplemented with extracts contributing either CT from Acacia mearnsii [7.9% of dietary dry matter (DM)] or saponins from Yucca schidigera (1.1% of DM). The fourth treatment consisted of dried sainfoin (Onobrychis viciifolia), a CT-containing forage legume, in an amount also providing 7.9% CT in dietary DM. All diets were supplemented with linseed oil at a level contributing 60% of total dietary ALA in all treatments. Diets were incubated for 10 d (n = 4) in the rumen simulation technique system, using the last 5 d for statistical evaluation. Fatty acids were analyzed in feed, feed residues, incubation fluid, and its effluent. Data were subjected to ANOVA considering diet and experimental run as main effects. Both CT treatments reduced ruminal fiber and crude protein degradation, and lowered incubation fluid ammonia concentration. Only the CT extract suppressed methane formation and shifted microbial populations toward bacteria at cost of protozoa. The saponin extract remained without clear effects on fermentation characteristics except for increased protozoal counts. The extent of ALA biohydrogenation was 20% less with the CT plant, but this probably resulted from reduced organic matter degradability rather than from an inhibition of biohydrogenation. After incubation analysis of incubation fluid effluent and feed residues showed a considerable proportion of the 3 biohydrogenation intermediates, cis-9, trans-11, cis-15 C18:3, trans-11, cis-15 C18:2, and trans-11 C18:1, which did not occur in the initial feeds. Only the CT-extract diet led to a different profile in the effluent compared with the control diet with trans-11 C18:1 being considerably increased at cost of C18:0. This could have been achieved by suppressing protozoa and enhancing the bacterial population, thus removing potential microbes involved in biohydrogenation and increasing competition between bacteria involved in biohydrogenation and others. The elevation of trans-11 C18:1 as the precursor of cis-9, trans-11 conjugated linoleic acid formed in body tissue and mammary gland is probably favorable from a human health point of view.     

Lipid oxidation products of heated soybeans as a possible cause of protection from ruminal biohydrogenation

Heating oilseeds has been shown to improve the milk fatty acid profile when given to dairy cows, compared to raw oilseeds. However, results from published studies are conflicting. The conditions of heating and storage of the oilseeds could be responsible for these differences, probably partly through their effects on lipid oxidation, the products of which could act on ruminal biohydrogenation (BH). Thus, 15 different treatments were applied to ground soybeans: three levels of heating (no heating, 30 min at 110 or 150°C) × 5 ambient storage durations (0, 1, 2, 4, or 6 months). Soybeans were incubated in vitro with ruminal fluid for 6 h. Triacylglycerol (TAG) polymers, hydroperoxides and hydroxyacids (HOA), aldehydes, and fatty acids were assayed in soybeans and ruminal culture. No TAG polymer was detected in any treatment. Soybeans stored for a long time had a high content of HOA, whereas those heated at 150°C, whatever the storage duration, had high aldehyde contents. The percentage disappearance of cis‐9,cis‐12 18:2 and cis‐9,cis‐12,cis‐15 18:3 in incubates decreased significantly in cultures with heated soybeans, especially at 150°C, suggesting that this partial protection of polyunsaturated fatty acids (PUFA) from BH was at least in part linked to the aldehyde content of the heated soybeans. Practical applications: Oilseeds given to ruminants are often heated, and heat treatment is known to generate oxidation products. Knowing what oxidation products influence ruminal biohydrogenation of unsaturated fatty acids could result in technological processes allowing a better transfer of unsaturated fatty acids from oilseeds to ruminant products.     

A single mild episode of subacute ruminal acidosis does not affect ruminal barrier function in the short term

Twenty-four German Merino sheep (72.3 ± 10.1 kg of body weight) were fed an all-hay diet and assigned to either the subacute ruminal acidosis (SARA) treatment (n = 17) or sham treatment (n = 7). The SARA sheep were orally dosed with a 2.2 M glucose solution to supply 5 g of glucose/kg of body weight, whereas sham sheep received an equal volume of water. Ruminal pH was measured for 48 h before and 3 h after the oral dose. Sheep were then killed and ruminal epithelia from the ventral sac were mounted in Ussing chambers. The serosal-to-mucosal flux rate of partially ³H-labeled mannitol (J_mannitol-SM), an indicator of barrier function, was measured while epithelia were exposed to 3 sequential in vitro measurement periods lasting 1 h each. The measurement periods consisted of baseline, challenge, and recovery periods and were interspersed by 30-min periods for treatment equilibration. Baseline conditions were pH 6.1 (mucosal solution) and pH 7.4 (serosal solution) with a bilateral osmolarity of 293 mOsm/L. During the challenge period, the mucosal side of the epithelia was exposed to either an acidotic challenge (pH 5.2, osmolarity 293 mOsm/L) or an osmotic challenge (pH 6.1, osmolarity 450 mOsm/L); a third group served as control (pH 6.1, osmolarity 293 mOsm/L). The mucosal buffer solution was replaced for the recovery period. In vivo, sheep on the SARA treatment had lower mean (5.77 vs. 6.67) and nadir (5.48 vs. 6.47) ruminal pH for the 3 h following the oral drench compared with sham sheep, indicating the successful induction of SARA with the oral glucose dose. Despite the marked reduction in pH in vivo, induction of SARA had no detectable effects on the baseline measurements of J_mannitol-SM, tissue conductance (G_t), and short-circuit current (I_sc) in vitro. However, reducing mucosal pH to 5.2 in vitro had negative effects on epithelial barrier function in the recovery period, including increased J_mannitol-SM, increased G_t, and decreased I_sc. The osmotic challenge increased J_mannitol-SM and G_t and decreased I_sc during the challenge period, which was reversible in the recovery period except for slight reduction in I_sc. Interactions between the in vitro treatment and measurement period were detected for J_mannitol-SM, G_t, and I_sc. These data indicate that a mild episode of SARA (nadir pH, 5.48; duration ruminal pH <5.8, 111 min relative to the 180-min measurement period) does not affect ruminal epithelial barrier function immediately after the episode but that a rapid and more severe acidification (pH 5.2) in vitro increases epithelial permeability following the insult.     

Prediction of ruminal pH from pasture-based diets

This study identified suitable predictors of ruminal pH and identified relationships between ruminal pH and animal measures for diets based on fresh pasture. Animal and dietary variables (121 treatment means from six countries) were collated from 23 studies of lactating dairy cows fed pasture. Mean daily ruminal pH ranged from 5.6 to 6.7 across studies. Within studies, a low ruminal pH was associated with higher (P < 0.05; r2 > 0.40) microbial N flow from the rumen, total and individual volatile fatty acid concentrations, milk and milk component yields, and dry matter intake, and with lower (P < 0.05; r2 > 0.30) concentrations of milk fat, fat:protein, and acetate:propionate. Large variation between studies meant that these ruminal and production variables could not be used to make reliable predictions of ruminal pH in future pasture-based studies or feeding scenarios. Ruminal pH was positively related (P < 0.05; r2 < 0.15) to forage neutral detergent fiber (NDF) and NDF content within study, and negatively related (P = 0.001; r2 = 0.14) to nonstructural carbohydrate across studies. No single dietary variable, or group of variables, could be used to make a reliable prediction of ruminal pH. Estimates of effective fiber for diets containing only pasture were made using the Cornell Net Carbohydrate and Protein System ruminal pH equation. Mean effectiveness of fiber in pasture was 43% of NDF, and ranged from 17 to 78% across studies. High flows of microbial nitrogen, milk, milk fat yield, and dry matter intake suggested that the performance of cows fed high quality pasture was not limited when mean ruminal pH decreased to 5.8.     

Short communication: Diurnal profiles of conjugated linoleic acids and trans fatty acids in ruminal fluid from cows fed a high concentrate diet supplemented with fish oil, linseed oil, or sunflower oil

Trans-18:1 and 18:2 isomer composition in ruminal fluid during the daily feeding cycle was examined in 3 cows fed a high concentrate diet (35:65) with 5% (DM basis) sunflower oil (SO), 5% linseed oil (LO), or 2.5% fish oil (FO) in a 3 x 3 Latin square with 3 4-wk periods. Grass hay and concentrate mixtures were fed at 0900, 1300, and 1700 h daily. Ruminal fluid was collected at 0900, 1100, 1300, 1500, 1700, 2000, and 0000 h. Feeding SO resulted in the greatest mean concentrations (% of total fatty acids) of trans10,cis12-18:2 and cis9,trans11-18:2. In particular, trans10,cis12-18:2 with SO was greater at 1500 (0.29%), 2000 (0.34%), and 0000 h (0.25%) relative to 0900 h (0.07%). Cis9,trans11-18:2 concentration increased from 0.47% at 0900 h to a peak of 2.06% at 1100 h; it remained greater than the percentage determined at 0900 h at 1300 (1.4%) through 0000 h (1.1%). Concentration of trans11,cis15-18:2 was greatest with LO, ranging from 3.3% (0900 h) to a peak of 11.4% at 2000 h. Mean trans10-18:1 concentration ranked by diet was SO > FO > LO. Peak trans10-18:1 with SO was observed at 1700 h (14.9%) compared with 0900 h (5.1%). Trans11-18:1 did not differ with diet or time. Stearic acid decreased over time with all diets reaching minimum concentrations at 1700 to 2000 h relative to 0900 h. Feeding FO, however, decreased mean 18:0 concentration 4-fold compared with LO or SO. The moderate effect on concentration of trans-18:1 coupled with accumulation of 18:2 intermediates and the decrease of 18:0 over time suggest that oils reduced the biohydrogenation of 18:2 isomers to trans-18:1.     

Short communication: Effects of subacute ruminal acidosis on free-choice intake of sodium bicarbonate in lactating dairy cows

The effect of inducing subacute ruminal acidosis (SARA) on the free-choice intake of sodium bicarbonate (SB) was investigated in four midlactation Holstein cows in a switchover experiment with four 1-wk periods. The SARA was induced by replacing 25% of the ad libitum intake of total mixed ration (TMR) with pellets containing 50% ground wheat and 50% ground barley and restricting access to TMR from 0700 to 1700 h. Control consisted of feeding TMR ad libitum. Powdered SB was provided for ad libitum consumption. Rumen pH was measured continuously using indwelling pH probes. Induction of SARA reduced (P < 0.05) the average daily rumen pH from 6.08 to 5.87, increased (P < 0.05) the average duration of rumen pH below 6 from 547 min x d(-1) to 916 min x d(-1), and increased (P < 0.05) the average duration of rumen pH below 5.6 from 132 min x d(-1) to 397 min x d(-1) (P < 0.05) but did not significantly affect SB intake. Average intake of SB was 26.8 g x d(-1) during SARA and 34.5 g x d(-1) during control. These low SB intakes must not have substantially affected rumen pH. Sodium bicarbonate intake differed significantly (P < 0.05) between cows. These data indicate that cows did not select SB in order to attenuate SARA.