Assessing milk response to different combinations of branched-chain volatile fatty acids and valerate in Jersey cows.

Some cellulolytic bacteria require 1 or more branched-chain volatile fatty acids (BCVFA) for the synthesis of branched-chain AA and branched-chain long-chain fatty acids because they are not able to uptake branched-chain AA or lack 1 or more enzymes to synthesize branched-chain AA de novo. Supplemental BCVFA and valerate were included previously as a feed additive that was later removed from the market; these older studies and more current studies have noted improvements in neutral detergent fiber digestibility and milk efficiency. However, most studies provided a single BCVFA or else isobutyrate (IB), 2-methylbutyrate (MB), isovalerate, and valerate altogether without exploring optimal combinations. Our objective was to determine a combination of isoacids that is optimal for milk production. Sixty (28 primiparous and 32 multiparous) lactating Jersey cows (106 ± 54 days in milk) were blocked and assigned randomly to either a control (CON) treatment without any isoacids, MB [12.3 mmol/kg dry matter (DM)], MB + IB (7.7 and 12.6 mmol/kg DM of MB and IB, respectively), or all 4 isoacids (6.2, 7.3, 4.2, and 5.1 mmol/kg DM of MB, IB, isovalerate, and valerate, respectively). Cattle were fed the CON treatment for a 2-wk period, then were assigned randomly within a block to treatments for 8 wk (n = 15). There was a trend for an interaction of supplement and parity for milk components. There were no differences in components for primiparous cows, whereas MB + IB tended to increase protein concentration by 0.04 and 0.08 percentage units in multiparous cows compared with the CON and MB treatments, respectively. Feeding MB + IB increased fat concentration by 0.23 to 0.31 percentage units compared with all other treatments in multiparous cows. Milk yield and dry matter intake (DMI) did not change with treatment. Treatment interacted with week for milk net energy for lactation/DMI; MB + IB tended to increase milk net energy of lactation/DMI by 0.10 Mcal/kg compared with MB and approached a trend for CON, mainly during the early weeks of the treatment period, whereas differences decreased during the last 2 wk of the treatment period. Cows fed MB had the highest 15:0 anteiso fatty acids in the total milk fatty acid profile, which was greater than that for CON or MB + IB cows, but not cows supplemented with isoacids. Cows fed MB alone had the numerically lowest milk net energy for lactation/DMI. The combination of MB + IB appeared optimal for increasing feed efficiency in our study and was not at the expense of average daily gain. Further research is needed for evaluating how potential changes in supplemental isoacid dosage should vary under differing dietary conditions.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. III: Relation with solid passage rate and pH on prokaryotic fatty acid profile and community in continuous culture.

Our objectives were to evaluate potential interactions in culture conditions that influence how exogenously dosed branched-chain VFA (BCVFA) would be recovered as elongated fatty acids (FA) or would affect bacterial populations. A 2 × 2 × 2 factorial arrangement of treatments evaluated 3 factors: (1) without versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate; each dose was partially substituted with 13C-enriched tracers before and during the collection period); (2) high versus low pH (ranging diurnally from 6.3 to 6.8 vs. 5.7 to 6.2); and (3) low versus high particulate-phase passage rate (kp; 2.5 vs. 5.0%/h) in continuous cultures administered a 50:50 forage:concentrate diet twice daily. Samples of effluent were collected and composited before harvesting bacteria from which FA and DNA were extracted. Profiles and enrichments of FA in bacteria were evaluated by gas chromatography and isotope-ratio mass spectrometry. The 13C enrichment in bacterial FA was calculated as percentage recovery of dosed 13C-labeled BCVFA. Dosing BCVFA increased the even-chain iso-FA, preventing the reduced concentration at higher kp and potentially as a physiological response to decreased pH. However, decreasing pH decreased recovery of 13C in these even-chain FA, suggesting greater reliance on isobutyrate produced from degradation of dietary valine. The iso-FA were decreased, whereas anteiso-FA and 16:0 increased with decreasing pH. Thus, 2-methylbutyrate still appeared to be important as a precursor for anteiso-FA to counter the increased rigidity of bacterial membranes that had more saturated straight-chain FA when pH decreased. Provision of BCVFA stimulated the relative sequence abundance of Fibrobacter and Treponema, both of which require isobutyrate and 2-methylbutyrate. Numerous bacterial community members were shifted by low pH, including increased Prevotella and genera within the phylum Proteobacteria, at the expense of members within phylum Firmicutes. Because of relatively few interactions with pH and kp, supplementation of BCVFA can stimulate neutral detergent fiber degradability via key fibrolytic bacteria across a range of conditions. Decreasing pH shifted bacterial populations and their FA composition, suggesting that further research is needed to distinguish pH from dietary changes.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. II: Relation with solid passage rate and pH on neutral detergent fiber degradation and microbial function in continuous culture.

To support improving genetic potential for increased milk production, intake of digestible carbohydrate must also increase to provide digestible energy and microbial protein synthesis. We hypothesized that the provision of exogenous branched-chain volatile fatty acids (BCVFA) would improve both neutral detergent fiber (NDF) degradability and efficiency of microbial protein synthesis. However, BCVFA should be more beneficial with increasing efficiency of bacterial protein synthesis associated with increasing passage rate (kp). We also hypothesized that decreasing pH would increase the need for isobutyrate over 2-methylbutyrate. To study these effects independent from other sources of variation in vivo, we evaluated continuous cultures without (control) versus with BCVFA (0 vs. 2 mmol/d each of isobutyrate, isovalerate, and 2-methylbutyrate), low versus high kp of the particulate phase (2.5 vs. 5.0%/h), and high versus low pH (ranging from 6.3 to 6.8 diurnally vs. 5.7 to 6.2) in a 2 × 2 × 2 factorial arrangement of treatments. Diets were 50% forage pellets and 50% grain pellets administered twice daily. Without an interaction, NDF degradability tended to increase from 29.7 to 35.0% for main effects of control compared with BCVFA treatments. Provision of BCVFA increased methanogenesis, presumably resulting from improved NDF degradability. Decreasing pH decreased methane production. Total volatile fatty acid (VFA) and acetate production were decreased with increasing kp, even though true organic matter degradability and bacterial nitrogen flow were not affected by treatments. Decreasing pH decreased acetate but increased propionate and valerate production, probably resulting from a shift in bacterial taxa and associated VFA stoichiometry. Decreasing pH decreased isobutyrate and isovalerate production while increasing 2-methylbutyrate production on a net basis (subtracting doses). Supplementing BCVFA improved NDF degradability in continuous cultures administered moderate (15.4%) crude protein diets (excluding urea in buffer) without major interactions with culture pH and kp.

Conditions stimulating neutral detergent fiber degradation by dosing branched-chain volatile fatty acids. I: Comparison with branched-chain amino acids and forage source in ruminal batch cultures.

Three experiments assessed branched-chain volatile fatty acid (BCVFA) stimulation of neutral detergent fiber (NDF) disappearance after 24 h of incubation in batch cultures derived from ruminal fluid inocula that were enriched with particulate-phase bacteria. In experiment 1, a control was compared with 3 treatments with isomolar doses of all 3 BCVFA (plus valerate), all 3 branched-chain AA (BCAA), or half of each BCVFA and BCAA mix with either alfalfa or grass hays (50%) and ground corn grain (50%). A portion of the BCAA and BCVFA doses were enriched with 13C, and valerate (also enriched with 13C) was added with BCVFA. Although BCAA yielded a similar production of BCVFA compared with dosing BCVFA, equimolar substitution of BCVFA for BCAA decreased the percentage of N in bacterial pellets when alfalfa hay was fed but increased N when grass hay was fed. Substituting BCVFA for BCAA increased total fatty acid (FA) concentration with alfalfa hay. Dosing of BCAA or BCVFA did not affect total branched-chain FA, iso-FA, or anteiso-FA percentages in bacterial total FA, whereas numerous individual FA isomers and their 13C enrichments were affected by these treatments. Increasing recovery of the 13C dose from respective labeled BCVFA primers indicated facilitated BCVFA uptake and incorporation into FA compared with BCAA, whereas increased recovery of 13C from labeled BCAA in the bacteria pellet but not in the FA fraction suggested direct assimilation into bacterial protein. The BCVFA and valerate were dosed in varying combinations that either summed to 4 mM (experiment 2) or had only 1 mM no matter what combination (experiment 3). In general, grass hay was more responsive to stimulation in NDF digestibility by BCVFA than was alfalfa hay, which was attributed to the higher degradable protein in the latter. The net production of the BCVFA (after subtracting dose) was affected by source and combination of BCVFA. Isovalerate dosing tended to increase its own net production; in contrast, isobutyrate seemed to be used more when it was added alone, but 2-methylbutyrate seemed to be preferred over isobutyrate when 2-methylbutyrate was added. Results supported potential interactions, including potential feedback in production from feed BCAA or increased concentration-dependent competition for dosed BCVFA into cellular products. Under our conditions, the BCVFA appear to be more readily available than BCAA, probably because of regulated BCAA transport and metabolism. Valerate consistently provided no benefit. Using nonparametric ranking, all 3 BCVFA or either isovalerate or isobutyrate (both yielding iso-FA) should be combined with 2-methylbutyrate (yielding anteiso-FA) as a potential opportunity to improve NDF digestibility when rumen-degraded BCAA are limited in diets to decrease environmental impact from N in waste.

Rumen microbial responses to supplemental nitrate. I. Yeast growth and protozoal chemotaxis in vitro as affected by nitrate and nitrite concentrations.

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH4 emissions. In the NO3- reduction process, NO2- can accumulate, which could directly inhibit methanogens and some bacteria. However, little information is available on eukaryotic microbes in the rumen. Protozoa were hypothesized to enhance nitrate reductase but also have more circling swimming behavior, and the yeast Saccharomyces cerevisiae was hypothesized to lessen NO2- accumulation. In the first experiment, a culture of S. cerevisiae strain 1026 was evaluated under 3 growth phases: aerobic, anoxic, or transition to anoxic culture. Each phase was evaluated with a control or 1 of 3 isonitrogenous doses, including NO3-, NO2-, or NH4+ replacing peptone in the medium. Gas head phase, NO3-, or NH4+ did not influence culture growth, but increasing NO2- concentration increasingly inhibited yeast growth. In experiment 2, rumen fluid was harvested and incubated for 3 h in 2 concentrations of NO3-, NO2-, or sodium nitroprusside before assessing chemotaxis of protozoa toward glucose or peptides. Increasing NO2- concentration decreased chemotaxis by isotrichids toward glucose or peptides and decreased chemotaxis by entodiniomorphids but only toward peptides. Live yeast culture was inhibited dose-responsively by NO2- and does not seem to be a viable mechanism to prevent NO2- accumulation in the rumen, whereas a role for protozoal nitrate reductase and NO2- influencing signal transduction requires further research.

Rumen microbial responses to supplemental nitrate. II. Potential interactions with live yeast culture on the prokaryotic community and methanogenesis in continuous culture.

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH4 emissions. In the NO3- reduction process, NO2- can accumulate, which could directly inhibit methanogens and possibly other microbes in the rumen. Saccharomyces cerevisiae yeast was hypothesized to decrease NO2- through direct reduction or indirectly by stimulating the bacterium Selenomonas ruminantium, which is among the ruminal bacteria most well characterized to reduce both NO3- and NO2-. Ruminal fluid was incubated in continuous cultures fed diets without or with NaNO3 (1.5% of diet dry matter; i.e., 1.09% NO3-) and without or with live yeast culture (LYC) fed at a recommended 0.010 g/d (scaled from cattle to fermentor intakes) in a 2 × 2 factorial arrangement of treatments. Treatments with LYC had increased NDF digestibility and acetate:propionate by increasing acetate molar proportion but tended to decrease total VFA production. The main effect of NO3- increased acetate:propionate by increasing acetate molar proportion; NO3- also decreased molar proportions of isobutyrate and butyrate. Both NO3- and LYC shifted bacterial community composition (based on relative sequence abundance of 16S rRNA genes). An interaction occurred such that NO3- decreased valerate molar proportion only when no LYC was added. Nitrate decreased daily CH4 emissions by 29%. However, treatment × time interactions were present for both CH4 and H2 emission from the headspace; CH4 was decreased by the main effect of NO3- until 6 h postfeeding, but NO3- and LYC decreased H2 emission up to 4 h postfeeding. As expected, NO3- decreased methane emissions in continuous cultures; however, contrary to expectations, LYC did not attenuate NO2- accumulation.

Evaluation of the National Research Council (2001) dairy model and derivation of new prediction equations. 1. Digestibility of fiber, fat, protein, and nonfiber carbohydrate.

Evaluation of ration balancing systems such as the National Research Council (NRC) Nutrient Requirements series is important for improving predictions of animal nutrient requirements and advancing feeding strategies. This work used a literature data set (n = 550) to evaluate predictions of total-tract digested neutral detergent fiber (NDF), fatty acid (FA), crude protein (CP), and nonfiber carbohydrate (NFC) estimated by the NRC (2001) dairy model. Mean biases suggested that the NRC (2001) lactating cow model overestimated true FA and CP digestibility by 26 and 7%, respectively, and under-predicted NDF digestibility by 16%. All NRC (2001) estimates had notable mean and slope biases and large root mean squared prediction error (RMSPE), and concordance (CCC) ranged from poor to good. Predicting NDF digestibility with independent equations for legumes, corn silage, other forages, and nonforage feeds improved CCC (0.85 vs. 0.76) compared with the re-derived NRC (2001) equation form (NRC equation with parameter estimates re-derived against this data set). Separate FA digestion coefficients were derived for different fat supplements (animal fats, oils, and other fat types) and for the basal diet. This equation returned improved (from 0.76 to 0.94) CCC compared with the re-derived NRC (2001) equation form. Unique CP digestibility equations were derived for forages, animal protein feeds, plant protein feeds, and other feeds, which improved CCC compared with the re-derived NRC (2001) equation form (0.74 to 0.85). New NFC digestibility coefficients were derived for grain-specific starch digestibilities, with residual organic matter assumed to be 98% digestible. A Monte Carlo cross-validation was performed to evaluate repeatability of model fit. In this procedure, data were randomly subsetted 500 times into derivation (60%) and evaluation (40%) data sets, and equations were derived using the derivation data and then evaluated against the independent evaluation data. Models derived with random study effects demonstrated poor repeatability of fit in independent evaluation. Similar equations derived without random study effects showed improved fit against independent data and little evidence of biased parameter estimates associated with failure to include study effects. The equations derived in this analysis provide interesting insight into how NDF, starch, FA, and CP digestibilities are affected by intake, feed type, and diet composition.

Evaluation of the National Research Council (2001) dairy model and derivation of new prediction equations. 2. Rumen degradable and undegradable protein.

This work evaluated the National Research Council (NRC) dairy model (2001) predictions of rumen undegradable (RUP) and degradable (RDP) protein compared with measured postruminal non-ammonia, nonmicrobial (NANMN) and microbial N flows. Models were evaluated using the root mean squared prediction error (RMSPE) as a percent of the observed mean, mean and slope biases as percentages of mean squared prediction error (MSPE), and concordance correlation coefficient (CCC). The NRC (2001) over-estimated NANMN by 18% and under-estimated microbial N by 14%. Both responses had large mean biases (19% and 20% of MSPE, respectively), and NANMN had a slope bias (22% of MSPE). The NRC NANMN estimate had high RMSPE (46% of observed mean) and low CCC (0.37); updating feed library A, B, and C protein fractions and degradation rate (Kd) estimates with newer literature only marginally improved fit. The re-fit NRC models for NANMN and microbial N had CCC of 0.89 and 0.94, respectively. When compared with a prediction of NANMN as a static mean fraction of N intake, the re-derived NRC approach did not have improved fit. A protein system of intermediate complexity was derived in an attempt to estimate NANMN with improved fit compared with the static mean NANMN model. In this system, postruminal appearance of A, B, and C protein fractions were predicted in a feed-type specific manner rather than from estimated passage and degradation rates. In a comparison to independent data achieved through cross-validation, the new protein system improved RMSPE (34 vs. 36% of observed mean) and CCC (0.42 vs. 0.30) compared with the static mean NANMN model. When the NRC microbial N equation was re-derived, the RDP term dropped from the model. Consequently, 2 new microbial protein equations were formulated, both used a saturating (increasing at a decreasing rate) form: one saturated with respect to TDN and the other saturated over increasing intakes of rumen degraded starch and NDF. Both equations expressed maximal microbial N production as a linear function of RDP intake. The function relating microbial N to intake of rumen degradable carbohydrate improved RMSPE (24 vs. 28% of the observed mean) and CCC (0.63 vs 0.30) compared with the re-derived NRC model. The newly derived equations showed modest improvements in model fit and improved capacity to account for known biological effects; however, substantial variability in NANMN and microbial N estimates remained unexplained.