Ability of three dairy feed evaluation systems to predict postruminal outflows of amino acids in dairy cows: A meta-analysis.

Adequate prediction of postruminal outflows of essential AA (EAA) is the starting point of balancing rations for EAA in dairy cows. The objective of this meta-analysis was to compare the performance of 3 dairy feed evaluation systems (National Research Council [NRC], Cornell Net Protein and Carbohydrate System version 6.5.5 [CNCPS], and National Academies of Sciences, Engineering and Medicine [NASEM]) to predict EAA outflows (Trp was not tested). The data set included a total of 354 treatment means from 70 duodenal and 24 omasal studies. To avoid Type I error, mean and linear biases were considered of concern if statistically significant and representing >5.0% of the observed mean. Analyses were conducted on raw observed values and on observations adjusted for the random effect of study. The analysis on raw data indicates the ability of the feed evaluation system to predict absolute values whereas the analysis on adjusted values indicates its ability to predict responses of EAA outflows to dietary changes. For the prediction of absolute values (based on raw data), NRC underpredicted outflows of all EAA, from 5.3% to 8.6% of the observed mean (%obs.mean) except for Leu, Lys, and Val; NASEM overpredicted Lys (10.8%obs.mean); and CNCPS overpredicted Arg, His, Lys, Met, and Val (5.2 to 26.0%obs.mean). No EAA had a linear bias of concern with NASEM, followed by NRC for His (6.8%obs.mean), and CNCPS for all EAA (5.6 to 12.2%obs.mean) except Leu, Phe, and Thr. In contrast, for the prediction of responses to dietary changes (based on adjusted data), NRC had 2 EAA presenting a linear bias of concern, followed by NASEM and CNCPS with 4 and 6 EAA, respectively. Predictions of His showed a linear bias of concern (5.3 to 9.6%obs.mean) with the 3 feed evaluation systems. Measured chemistry of crude protein and EAA were reported for 1 or more feed ingredients of the ration in 36% of the studies, and resulted in decreased linear biases in the 3 feed evaluation systems. The difference in mean biases of Met outflows was systematically positive when comparing omasal versus duodenal studies. Predictions of Met outflows with NRC had a higher concordance correlation coefficient in duodenal (used to develop NRC equations) versus omasal studies, whereas the opposite was observed with CNCPS, the latter showing the lowest mean bias for Met in omasal sampling studies. The 30% difference in Met mean biases between sampling sites appeared related to a similar difference found for observed Met versus nonammonia nitrogen outflows between duodenal and omasal studies, which is independent of predictions. In conclusion, NRC and NASEM yielded accurate predictions of EAA outflows, with a small superiority of NASEM to predict absolute values, and slight superiority of NRC to predict the responses to dietary changes. In comparison, CNCPS may present mean and linear biases of concern for many EAA. Moreover, it remains to determine which sampling site is more representative of the true supply of EAA to the cows.

Ability of three dairy feed evaluation systems to predict postruminal outflows of nitrogenous compounds in dairy cows: A meta-analysis.

Adequate prediction of postruminal outflow of protein fractions is the starting point for the determination of metabolizable protein supply in dairy cows. The objective of this meta-analysis was to compare the performance of 3 dairy feed evaluation systems (National Research Council [NRC], Cornell Net Protein and Carbohydrate System [CNCPS], and National Academies of Sciences, Engineering and Medicine [NASEM]) to predict outflows (g/d) of nonammonia nitrogren (NAN), microbial N (MiN), and nonammonia nonmicrobial N (NANMN). Predictions of rumen degradabilities (% of nutrient) of protein (RDP), NDF, and starch were also evaluated. The data set included 1,294 treatment means from 312 digesta flow studies. The 3 feed evaluation systems were compared using the concordance correlation coefficient (CCC), the ratio of root mean square prediction error (RMSPE) on standard deviation of observed values (RSR), and the slope between observed and predicted values. Mean and linear biases were deemed biologically relevant and are discussed if higher than a threshold of 5% of the mean of observed values. The comparisons were done on observed values adjusted or not for the study effect; the adjustment had a small effect on the mean bias but the linear bias reflected a response to a dietary change rather than absolute predictions. For the absolute predictions of NAN and MiN, CNCPS had the best-fit statistics (8% greater CCC; 6% lower RMSPE) without any bias; NRC and NASEM underpredicted NAN and MiN, and NASEM had an additional linear bias indicating that the underprediction of MiN increased at increased predictions. For NANMN, fit statistics were similar among the 3 feed evaluation systems with no mean bias; however, the linear bias with NRC and CNCPS indicated underprediction at low predictions and overprediction at elevated predictions. On average, the CCC were smaller and RSR ratios were greater for MiN versus NAN indicating increased prediction errors for MiN. For NAN responses to a dietary change, CNCPS also had the best predictions, although the mean bias with NASEM was not biologically relevant and the 3 feed evaluation systems did not present a linear bias. However, CNCPS, but not the 2 other feed evaluation systems, presented a linear bias for MiN, with responses being overpredicted at increased predictions. For NANMN, responses were overpredicted at increased predictions for the 3 feed evaluation systems, but to a lesser extent with NASEM. The site of sampling had an effect on the mean bias of MiN and NANMN in the 3 feed evaluation systems. The mean bias of MiN was higher in omasal than duodenal studies in the 3 feed evaluation systems (from 55 to 61 g/d) and this mean bias was twice as large when 15N labeling was used as a microbial marker compared with purines. Such a difference was not observed for duodenal studies. The reasons underlying these systematic differences are not clear as the type of measurements used in the current meta-analysis does not allow to delineate if one site or one microbial marker is yielding the "true" postruminal N outflows. Rumen degradabilities of protein was underpredicted with CNCPS, and RDP responses to a dietary change was underpredicted by the 3 feed evaluation systems with increased RDP predictions. Rumen degradability of NDF was underpredicted and had poor fit statistics for NASEM compared with CNCPS. Fit statistics were similar between CNCPS and NASEM for rumen degradability of starch, but with an underprediction of the response with NASEM and absolute values being overpredicted with CNCPS. Multivariate regression analyses showed that diet characteristics were correlated with prediction errors of N outflows in each feed evaluation system. Globally, compared with NAN and NANMN, residuals of MiN were correlated with several moderators in the 3 feed evaluation systems reflecting the complexity to measure and model this outflow. In addition, residuals of NANMN were correlated positively with RDP suggesting an overestimation of this parameter. In conclusion, although progress is still to be made to improve equations predicting postruminal N outflows, the current feed evaluation systems provide sufficient precision and accuracy to predict postruminal outflows of N fractions.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. I: Digestibility, microbial protein, and prokaryotic community structure.

Branched-chain amino acids are deaminated by amylolytic bacteria to branched-chain volatile fatty acids (BCVFA), which are growth factors for cellulolytic bacteria. Our objective was to determine the dietary conditions that would increase the uptake of BCVFA by rumen bacteria. We hypothesized that increased forage would increase cellulolytic bacterial abundance and incorporation of BCVFA into their structure. Supplemental polyunsaturated fatty acids, supplied via corn oil (CO), should inhibit cellulolytic bacteria growth, but we hypothesized that additional BCVFA would alleviate that inhibition. Further, supplemental BCVFA should increase neutral detergent fiber degradation and efficiency of bacterial protein synthesis more with the high forage and low polyunsaturated fatty acid dietary combination. The study was an incomplete block design with 8 dual-flow continuous cultures used in 4 periods with 8 treatments (n = 4 per treatment) arranged as a 2 × 2 × 2 factorial. The factors were: high forage (HF) or low forage (LF; 67 or 33%), without or with supplemental CO (3% dry matter), and without or with 2.15 mmol/d (which included 5 mg/d of 13C each of BCVFA isovalerate, isobutyrate, and 2-methylbutyrate). The isonitrogenous diets consisted of 33:67 alfalfa:orchardgrass pellet, and was replaced with a concentrate pellet that mainly consisted of ground corn, soybean meal, and soybean hulls for the LF diet. The main effect of supplementing BCVFA increased neutral detergent fiber (NDF) degradability by 7.6%, and CO increased NDF degradability only in LF diets. Supplemental BCVFA increased bacterial N by 1.5 g/kg organic matter truly degraded (6.6%) and 0.05 g/g truly degraded N (6.5%). The relative sequence abundance decreased with LF for Fibrobacter succinogenes, Ruminococcus flavefaciens, and genus Butyrivibrio compared with HF. Recovery of the total 13C dose in bacterial pellets decreased from 144 µg/ mg with HF to 98.9 µg/ mg with LF. Although isotope recovery in bacteria was greater with HF, BCVFA supplementation increased NDF degradability and efficiency of microbial protein synthesis under all dietary conditions. Therefore, supplemental BCVFA has potential to improve feed efficiency in dairy cows even with dietary conditions that might otherwise inhibit cellulolytic bacteria.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. II: Biohydrogenation and incorporation into bacterial lipids.

To maintain membrane homeostasis, ruminal bacteria synthesize branched-chain fatty acids (BCFA) or their derivatives (vinyl ethers) that are recovered during methylation procedures as branched-chain aldehydes (BCALD). Many strains of cellulolytic bacteria require 1 or more branched-chain volatile fatty acid (BCVFA). Therefore, the objective of this study was to investigate BCVFA incorporation into bacterial lipids under different dietary conditions. The study was an incomplete block design with 8 continuous culture fermenters used in 4 periods with treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were high (HF) or low forage (LF, 67 or 33% forage, 33:67 alfalfa:orchardgrass), without or with supplemental corn oil (CO; 3% dry matter, 1.5% linoleic fatty acid), and without or with 2.15 mmol/d (5 mg/d 13C each of isovalerate, isobutyrate, and 2-methylbutyrate). After methylation of bacterial pellets collected from each fermenter's effluent, fatty acids and fatty aldehydes were separated before analysis by gas chromatography and isotope ratio mass spectrometry. Supplementation of BCVFA did not influence biohydrogenation extent. Label was only recovered in branched-chain lipids. Lower forage inclusion decreased BCFA in bacterial fatty acid profile from 9.45% with HF to 7.06% with LF and decreased BCALD in bacterial aldehyde profile from 55.4% with HF to 51.4% with LF. Supplemental CO tended to decrease iso even-chain BCFA and decreased iso even-chain BCALD in their bacterial lipid profiles. The main 18:1 isomer was cis-9 18:1, which increased (P < 0.01) by 25% from CO (data not shown). Dose recovery in bacterial lipids was 43.3% lower with LF than HF. Supplemental CO decreased recovery in the HF diet but increased recovery with LF (diet × CO interaction). Recovery from anteiso odd-chain BCFA and BCALD was the greatest; therefore, 2-methylbutyrate was the BCVFA primer most used for branched-chain lipid synthesis. Recovery in iso odd-chain fatty acids (isovalerate as primer) was greater than label recovery in iso even-chain fatty acids (isobutyrate as primer). Fatty aldehydes were less than 6% of total bacterial lipids, but 26.0% of 13C recovered in lipids were recovered in BCALD because greater than 50% of aldehydes were branched-chain. Because BCFA and BCALD are important in the function and growth of bacteria, especially cellulolytics, BCVFA supplementation can support the rumen microbial consortium, increasing fiber degradation and efficiency of microbial protein synthesis.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. III: Protein metabolism and incorporation into bacterial protein.

Some cellulolytic bacteria cannot transport branched-chain AA (BCAA) and do not express complete synthesis pathways, thus depending on cross-feeding for branched-chain volatile fatty acid (BCVFA) precursors for membrane lipids or for reductive carboxylation to BCAA. Our objective was to assess BCVFA uptake for BCAA synthesis in continuous cultures administered high forage (HF) and low forage (LF) diets without or with corn oil (CO). We hypothesized that BCVFA would be used for BCAA synthesis more in the HF than in LF diets. To help overcome bacterial inhibition by polyunsaturated fatty acids in CO, BCVFA usage for bacterial BCAA synthesis was hypothesized to decrease when CO was added to HF diets. The study was an incomplete block design with 8 dual-flow fermenters used in 4 periods with 8 treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were: HF or LF (67 or 33% forage, 33:67 alfalfa:orchardgrass pellets), without or with supplemental CO (3% of dry matter), and without or with 2.15 mmol/d (5 mg/d 13C) each of isovalerate, isobutyrate, and 2-methylbutyrate for one combined BCVFA treatment. The flow of bacterial BCAA increased by 10.7% by supplementing BCVFA and 9.14% with LF versus HF; similarly, dosing BCVFA versus without BCVFA increased BCAA by 1.98% in total bacterial AA, whereas LF increased BCAA by 1.92% versus HF. Additionally, BCVFA supplementation increased bacterial AA flow by 16.6% when supplemented in HF - CO and 12.4% in LF + CO diets, but not in the HF + CO (-1.5%) or LF - CO (+6.7%) diets (Diet × CO × BCVFA interaction). The recovery of 13C in bacterial AA flow was 31% lower with LF than with HF. Of the total 13C recovered in bacteria, 13.8, 17.3, and 30.2% were recovered in Val, Ile, and Leu, respectively; negligible 13C was recovered in other AA. When fermenters were dosed with BCVFA, nonbacterial and total effluent flows of AA, particularly of alanine and proline, suggest decreased peptidolysis. Increased ruminal outflow of bacterial AA, especially BCAA, but also nonbacterial AA could potentially support postabsorptive responses from BCVFA supplementation to dairy cattle.

Effects of corn silage and grain expressing α-amylase on ruminal nutrient digestibility, microbial protein synthesis, and enteric methane emissions in lactating cows.

Increasing ruminal starch digestibility has the potential to improve microbial protein synthesis (MPS), milk production, and feed efficiency. Enogen corn (Syngenta Seeds LLC) expresses high α-amylase activity, and we evaluated effects of Enogen corn silage (CS) and grain (CG) on ruminal starch digestibility, MPS, and milk production in lactating dairy cows. Fifteen Holstein cows (6 ruminally cannulated and 9 noncannulated; average ± standard deviation at the beginning of the trial: 170 ± 40 d in milk; milk yield, 37.2 ± 7.73 kg/d; body weight, 714 ± 37 kg) were used in a replicated 3 × 3 Latin square design (28 d per period) with 3 treatments: a diet containing isoline CS and CG (control, CON); a diet with Enogen CS and isoline CG (ECS); and a diet with Enogen CS and CG (ECSCG). Dry matter (DM; 30%), starch (35% of DM), and particle size distribution of the isoline and Enogen CS were similar. However, the mean particle size of Enogen CG was larger (1.05 vs. 0.65 mm) than that of the isoline CG. Cannulated cows were used for digestibility and nutrient flow measurements, noncannulated cows were used for enteric CH4 measurements, and all cows were used for production evaluation. Dry matter intake (DMI) and milk yield were greater for ECS and ECSCG compared with CON (26.7 and 26.6 vs. 25.1 kg/d and 36.5 and 34.1 vs. 33.1 kg/d, respectively) without a difference between ECS and ECSCG. Milk protein yield was greater (1.27 vs. 1.14 and 1.17 kg/d) for ECS compared with CON and ECSCG. Milk fat content was greater (3.79 vs. 3.32%) for ECSCG compared with ECS. Milk fat yield and energy-corrected milk did not differ among treatments. Ruminal digestibilities of DM, organic matter, starch, and neutral detergent fiber were not different among treatments. However, ruminal digestibility of nonammonia, nonmicrobial N was greater (85 vs. 75%) for ECS compared with ECSCG. Total-tract apparent starch digestibility was lower (97.6 and 97.1 vs. 98.3%) for ECS and ECSCG compared with CON, respectively, and tended to be lower (97.1 vs. 98.3%) for ECSCG compared with ECS. Ruminal outflows of bacterial OM and nonammonia N tended to be greater for ECS than for ECSCG. Efficiency of MPS tended to be greater (34.1 vs. 30.6 g of N/kg of organic matter truly digested) for ECS versus ECSCG. Ruminal pH and total and individual short-chain fatty acid concentrations did not differ among treatments. Concentration of ruminal NH3 for ECS and ECSCG was lower (10.4 and 12.4 vs. 13.4 mmol/L, respectively) compared with CON. Methane per unit of DMI decreased for ECS and ECSCG compared with CON (11.4 and 12.2 vs. 13.5 g/kg of DMI, respectively) without a difference between ECS and ECSCG. In conclusion, ECS and ECSCG did not increase ruminal or total-tract starch digestibility. However, the positive effects of ECS and ECSCG on milk protein yield, milk yield, and CH4 per unit of DMI may show potential benefits of feeding Enogen corn. Effects of ECSCG were not apparent when compared with ECS, partly due to larger particle size of Enogen CG compared with its isoline counterpart.

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.

Invited review: Rumen modifiers in today's dairy rations.

Our aim was to review feed additives that have a potential ruminal mechanism of action when fed to dairy cattle. We discuss how additives can influence ruminal fermentation stoichiometry through electron transfer mechanisms, particularly the production and usage of dihydrogen. Lactate accumulation should be avoided, especially when acidogenic conditions suppress ruminal neutral detergent fiber digestibility or lead to subclinical acidosis. Yeast products and other probiotics are purported to influence lactate uptake, but growing evidence also supports that yeast products influence expression of gut epithelial genes promoting barrier function and resulting inflammatory responses by the host to various stresses. We also have summarized methane-suppressing additives for potential usage in dairy rations. We focused on those with potential to decrease methane production without decreasing fiber digestibility or milk production. We identified some mitigating factors that need to be addressed more fully in future research. Growth factors such as branched-chain volatile fatty acids also are part of crucial cross-feeding among groups of microbes, particularly to optimize fiber digestibility in the rumen. Our developments of mechanisms of action for various rumen-active modifiers should help nutrition advisors anticipate when a benefit in field conditions is more likely.

Effects of acetate, propionate, and pH on volatile fatty acid thermodynamics in continuous cultures of ruminal contents.

To investigate the effects of acetate, propionate, and pH on thermodynamics of volatile fatty acids (VFA) in the rumen, a dual-flow continuous culture study was conducted to quantify production of major VFA, interconversions among the VFA, and H2 and CH4 emissions in a 4 × 4 Latin square design. The 4 treatments were (1) control: pH buffered to an average of 6.75; (2) control plus 20 mmol/d of infused acetate (InfAc); (3) control plus 7 mmol/d of infused propionate (InfPr); and (4) a 0.5-unit decline in pH elicited by adjustment of the buffer (LowpH). All fermentors were fed 40 g of a pelleted diet containing whole alfalfa pellets and concentrate mix pellets (50:50) once daily. After 7 d of treatment, sequential, continuous infusions of [2-13C] sodium acetate (3.5 mmol/d), [U-13C] sodium propionate (2.9 mmol/d), and [1-13C] sodium butyrate (0.22 mmol/d) were carried out from 12 h before feeding for 36 h. Filtered liquid effluent (4 mL) was sampled at 0, 2, 4, 6, 8, 12, 16, and 22 h after feeding, and assessed for VFA concentrations, with another filtered sample (20 mL) used to quantify aqueous concentrations of CH4 and H2. Headspace CH4 and H2 gases were monitored continuously. Ruminal microbes were isolated from the mixed effluent samples, and the microbial community structure was analyzed using the 16S rRNA amplicon sequencing technique. The digestibility of neutral detergent fiber, acid detergent fiber, and starch and microbial C sequestrated from VFA were not affected by treatments. The LowpH treatment increased net propionate production and decreased H2 and CH4 headspace emissions, primarily due to shifts in metabolic pathways of VFA formation, likely due to the observed changes in bacterial community structure. Significant interconversions occurred between acetate and butyrate, whereas interconversions of other VFA with propionate were relatively small. The InfAc and InfPr treatments increased net acetate and propionate production, respectively; however, interconversions among VFA were not affected by pH, acetate, or propionate treatments, suggesting that thermodynamics might not be a primary influencer of metabolic pathways used for VFA formation.

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.

Relationship between urinary energy and urinary nitrogen or carbon excretion in lactating Jersey cows.

Measurement of urinary energy (UE) excretion is essential to determine metabolizable energy (ME) supply. Our objectives were to evaluate the accuracy of using urinary N (UN) or C (UC) to estimate UE and ultimately improve the accuracy of estimating ME. Individual animal data (n = 433) were used from 11 studies with Jersey cows at the University of Nebraska-Lincoln, where samples were analyzed after drying (n = 299) or on an as-is basis (n = 134). Dried samples resulted in greater estimated error variance compared with as-is samples, and thus only as-is samples were used for final models. The as-is data set included a range (min to max) in dry matter intake (11.6-24.6 kg/d), N intake (282-642 g/d), UE excretion (1,390-3,160 kcal/d), UN excretion (85-220 g/d or 20.6-59.5% of N intake), and UC excretion (130-273 g/d). As indicated by a bias in residuals between observed and predicted ME as dietary crude protein (CP; range of 14.9-19.1%) increased, the National Research Council dairy model did not accurately predict ME of diets, as dietary CP varied. The relationship between UE (kcal/d) and UN (g/d) excretion was linear and had an intercept of 880 ± 140 kcal. Because an intercept of 880 is biologically unlikely, the intercept was forced through 0, resulting in linear and quadratic relationships. The regressions of UE (kcal/d) on UN (g/d) excretion were UE = 14.6 ± 0.32 × UN, and UE = 20.9 ± 1.0 × UN - 0.0357 ± 0.0056 × UN2. In the quadratic regression, UE increased, but at a diminishing rate as UN excretion increased. As UC increased, UE linearly and quadratically increased. However, error variance was greater for regression with UC compared with UN as explanatory variables (8.42 vs. 7.42% of mean UE). The use of the quadratic regression between UN and UE excretion to predict ME resulted in a slope bias in ME predictions as dietary CP increased. The linear regression between UE and UN excretion removed slope bias between predicted ME and CP, and thus may be more appropriate for predicting UE across a wider range of dietary CP. Using equations to predict UE from UN should improve our ability to predict diet ME in Jersey cows compared with calculating ME directly from digestible energy.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 2. Ruminal fermentation, fatty acid, and bacterial community structure.

The experiment was conducted to understand ruminal effects of diet modification during moderate milk fat depression (MFD) and ruminal effects of 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa) and isoacids on alleviating MFD. Five ruminally cannulated cows were used in a 5 × 5 Latin square design with the following 5 dietary treatments (dry matter basis): a high-forage and low-starch control diet with 1.5% safflower oil (HF-C); a low-forage and high-starch control diet with 1.5% safflower oil (LF-C); the LF-C diet supplemented with HMTBa (0.11%; 28 g/d; LF-HMTBa); the LF-C diet supplemented with isoacids [(IA) 0.24%; 60 g/d; LF-IA]; and the LF-C diet supplemented with HMTBa and IA (LF-COMB). The experiment consisted of 5 periods with 21 d per period (14-d diet adaptation and 7-d sampling). Ruminal samples were collected to determine fermentation characteristics (0, 1, 3, and 6 h after feeding), long-chain fatty acid (FA) profile (6 h after feeding), and bacterial community structure by analyzing 16S gene amplicon sequences (3 h after feeding). Data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) in a Latin square design. Preplanned comparisons between HF-C and LF-C were conducted, and the main effects of HMTBa and IA and their interaction within the LF diets were examined. The LF-C diet decreased ruminal pH and the ratio of acetate to propionate, with no major changes detected in ruminal FA profile compared with HF-C. The α-diversity for LF-C was lower compared with HF-C, and ß-diversity also differed between LF-C and HF-C. The relative abundance of bacterial phyla and genera associated indirectly with fiber degradation was influenced by LF-C versus HF-C. As the main effect of HMTBa within the LF diets, HMTBa increased the ratio of acetate to propionate and butyrate molar proportion. Ruminal saturated FA were increased and unsaturated FA concentration were decreased by HMTBa, with minimal changes detected in ruminal bacterial diversity and community. As the main effect of IA, IA supplementation increased ruminal concentration of all branched-chain volatile FA and valerate and increased the percentage of trans-10 C18 isomers in total FA. In addition, α-diversity and the number of functional features were increased for IA. Changes in the abundances of bacterial phyla and genera were minimal for IA. Interactions between HMTBa and IA were observed for ruminal variables and some bacterial taxa abundances. In conclusion, increasing diet fermentability (LF-C vs. HF-C) influenced rumen fermentation and bacterial community structure without major changes in FA profile. Supplementation of HMTBa increased biohydrogenation capacity, and supplemental IA increased bacterial diversity, possibly alleviating MFD. The combination of HMTBa and IA had no associative effects in the rumen and need further studies to understand the interactive mechanism.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 1. Production, milk fatty acid profile, and nutrient digestibility.

The objectives of this experiment were to determine the effects of increased diet fermentability and polyunsaturated fatty acids (FA) with or without supplemental 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), isoacids (IA; isobutyrate, 2-methylbutyrate, isovalerate, and valerate) or the combination of these on milk fat depression (MFD). Ten Holstein cows (194 ± 58 DIM, 691 ± 69 kg BW, 28 ± 5 kg milk yield) were used in a replicated 5 × 5 Latin square design. Treatments included a high-forage control diet (HF-C), a low-forage control diet (LF-C) causing MFD by increasing starch and decreasing neutral detergent fiber (NDF), the LF-C diet supplemented with HMTBa at 0.11% (28 g/d), the LF-C diet supplemented with IA at 0.24% of dietary dry matter (60 g/d), and the LF-C diet supplemented with HMTBa and IA. Preplanned contrasts were used to compare HF-C versus LF-C and to examine the main effects of HMTBa or IA and their interactions within the LF diets. Dry matter intake was greater for LF-C versus HF-C, but milk yield remained unchanged. The LF-C diet decreased milk fat yield (0.87 vs. 0.98 kg/d) but increased protein yield compared with HF-C. As a result, energy-corrected milk was lower (28.5 vs. 29.6 kg/d) for LF-C versus HF-C. Although the concentration of total de novo synthesized FA in milk fat was not affected, some short- and medium-chain FA were lower for LF-C versus HF-C, but the concentrations of C18 trans-10 isomers were not different. Total-tract NDF apparent digestibility was numerically lower (42.4 vs. 45.6%) for LF-C versus HF-C. As the main effects, the decrease in milk fat yield observed in LF-C was alleviated by supplementation of HMTBa through increasing milk yield without altering milk fat content and by IA through increasing milk fat content without altering milk yield so that HMTBa or IA, as the main effects, increased milk fat yield within the LF diets. However, interactions for milk fat yield and ECM were observed between HMTBa and IA, suggesting no additive effect when used in combination. Minimal changes were found on milk FA profile when HMTBa was provided. However, de novo synthesized FA increased for IA supplementation. We detected no main effect of HMTBa, IA, and interaction between those on total-tract NDF digestibility. In conclusion, the addition of HMTBa and IA to a low-forage and high-starch diet alleviated moderate MFD. Although the mechanism by which MFD was alleviated was different between HMTBa and IA, no additive effects of the combination were observed on milk fat yield and ECM.

Inhibition of methanogenesis by nitrate, with or without defaunation, in continuous culture.

Within the rumen, nitrate can serve as an alternative sink for aqueous hydrogen [H2(aq)] accumulating during fermentation, producing nitrite, which ideally is further reduced to ammonium but can accumulate under conditions not yet explained. Defaunation has also been associated with decreased methanogenesis in meta-analyses because protozoa contribute significantly to H2 production. In the present study, we applied a 2 × 2 factorial treatment arrangement in a 4 × 4 Latin square design to dual-flow continuous culture fermentors (n = 4). Treatments were control without nitrate (-NO3-) versus with nitrate (+NO3-; 1.5% of diet dry matter), factorialized with normal protozoa (faunated, FAUN) versus defaunation (DEF) by decreasing the temperature moderately and changing filters over the first 4 d of incubation. We detected no main effects of DEF or interaction of faunation status with +NO3-. The main effect of +NO3- increased H2(aq) by 11.0 µM (+117%) compared with -NO3-. The main effect of +NO3- also decreased daily CH4 production by 8.17 mmol CH4/d (31%) compared with -NO3-. Because there were no treatment effects on neutral detergent fiber digestibility, the main effect of +NO3- also decreased CH4 production by 1.43 mmol of CH4/g of neutral detergent fiber degraded compared with -NO3-. There were no effects of treatment on other nutrient digestibilities, N flow, or microbial N flow per gram of nutrient digested. The spike in H2(aq) after feeding NO3- provides evidence that methanogenesis is inhibited by substrate access rather than concentration, regardless of defaunation, or by direct inhibition of NO2-. Methanogens were not decreased by defaunation, suggesting a compensatory increase in non-protozoa-associated methanogens or an insignificant contribution of protozoa-associated methanogens. Despite adaptive reduction of NO3- to NH4+ and methane inhibition in continuous culture, practical considerations such as potential to depress dry matter intake and on-farm ration variability should be addressed before considering NO3- as an avenue for greater sustainability of greenhouse gas emissions in US dairy production.

Potential roles of nitrate and live yeast culture in suppressing methane emission and influencing ruminal fermentation, digestibility, and milk production in lactating Jersey cows.

Concern over the carbon footprint of the dairy industry has led to various dietary approaches to mitigate enteric CH4 production. One approach is feeding the electron acceptor NO3-, thus outcompeting methanogens for aqueous H2. We hypothesized that a live yeast culture (LYC; Saccharomyces cerevisiae from Yea-Sacc 1026, Alltech Inc., Nicholasville, KY) would stimulate the complete reduction of NO3- to NH3 by selenomonads, thus decreasing the quantity of CH4 emissions per unit of energy-corrected milk production while decreasing blood methemoglobin concentration resulting from the absorbed intermediate, NO2-. Twelve lactating Jersey cows (8 multiparous and noncannulated; 4 primiparous and ruminally cannulated) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Cattle were fed diets containing 1.5% NO3- (from calcium ammonium nitrate) or an isonitrogenous control diet (containing additional urea) and given a top-dress of ground corn without or with LYC, with the fourth week used for data collection. Noncannulated cows were spot measured for CH4 emission by mouth using GreenFeed (C-Lock Inc., Rapid City, SD). The main effect of NO3- decreased CH4 by 17% but decreased dry matter intake by 10% (from 19.8 to 17.8 kg/d) such that CH4:dry matter intake numerically decreased by 8% and CH4:milk net energy for lactation production was unaffected by treatment. Milk and milk fat production were not affected, but NO3- decreased milk protein from 758 to 689 g/d. Ruminal pH decreased more sharply after feeding for cows fed diets without NO3-. Acetate:propionate was greater for cows fed NO3-, particularly when combined with LYC (interaction effect). Blood methemoglobin was higher for cattle fed NO3- than for those fed the control diet but was low for both treatments (1.5 vs. 0.5%, respectively; only one measurement exceeded 5%), indicating minimal risk for NO2- accumulation at our feeding level of NO3-. Although neither apparent organic matter nor neutral detergent fiber digestibilities were affected, apparent N digestibility had an interaction for NO3- × LYC such that apparent N digestibility was numerically lowest for diets containing both NO3- and LYC compared with the other 3 diets. Under the conditions of this study, NO3- mitigated ruminal methanogenesis but also depressed dry matter intake and milk protein yield. Based on the fact that few interactions were detected, LYC had a minimal role in attenuating negative cow responses to NO3- supplementation.

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.

Draft Macronuclear Genome Sequence of the Ruminal Ciliate Entodinium caudatum.

The transcriptionally active macronucleus of a ruminal ciliate, Entodinium caudatum MZG-1, was sequenced using the Illumina MiSeq and Oxford Nanopore MinION platforms. This is the first draft macronuclear genome sequence of a ruminal protozoon, and the genomic information will provide useful insight into the metabolism, physiology, and ecology of ruminal ciliates.