Sustainability is interwoven with consumer expectations of organic production systems, yet there are few nutritional options for organic dairy systems to approach methane (CH4) mitigation. The objective of the current study was to compare 3 feed additives for CH4 mitigation. We hypothesized that each additive would decrease CH4 production in continuous culture when compared with a control diet. Using dual-flow continuous culture fermenters fitted for CH4 and hydrogen (H2) sampling, 4 treatments were arranged in a 4 × 4 Latin square design. Treatments were a negative control (CON, 60:40 concentrate:orchardgrass pellet mix, 17.1% CP, 33.0% NDF, 20.1% ADF, and 27.1% starch) fed twice daily a total 80 g/d DM (CON) and comparing one of the 3 additives: kelp powder (KP) at 1.7 g/d, essential oils (EO) at 3 mg/d, or pistachio-based biochar (BC) at 1.6 g/d. All dosages were calculated based on previous data and supplier recommendations scaled to dual-flow continuous culture functional volume. Experimental periods included 7 d of adaptation and 4 d of sampling (11 d total), and buffer and solids dilution rates were maintained at 7%/h and 5%/h, respectively. The main statistical model included fixed effect of treatment and random effects of fermenter and period. Gas production data were measured by feeding; thus, analysis included a repeated effect of feeding and hourly VFA samples a repeated effect of hour. Only EO decreased CH4 production compared with CON (43.1 vs. 47.4 mmol/feeding, SD: 3.96). There was no effect of treatment on H2 emission, nor H2 or CH4 in the aqueous phase. There was also no effect of treatment on nutrient digestibility despite large numerical differences in fiber digestibility, nor the production of primary VFA. However, KP decreased production of isobutyrate and isovalerate compared with CON. While the present data illustrate efficacy of an essential oil product to decrease CH4 emission, decreases in CH4 were not supported by statistically significant gains in VFA that could translate to increased milk production in the dairy cow.

Purpose This literature review explains current understanding of the mechanisms by which branched-chain volatile fatty acids (BCVFA), historically termed isoacids, can improve fiber digestibility, microbial protein production, and either milk production or production efficiency. Sources Peer-reviewed literature and ADSA abstracts were the primary course of information reviewed. Synthesis Although traditionally included in isoacid supplements with the 3 BCVFA, the straight-chain valerate does not warrant being included. Because of the high concentration of its precursor leucine in corn protein, isovalerate is less likely to be needed than 2-methylbutyrate and isobutyrate. If conditions are not amenable for BCVFA assimilation into microbes, particularly if ruminal ammonia is deficient, more supplemental BCVFA are available for postruminal metabolism. Isovalerate is likely metabolized primarily in the rumen epithelium, whereas isobutyrate and 2-methylbutyrate are likely metabolized in the liver or peripheral tissues. If BCVFA improve fiber degradability, typically by 3 to 5 percentage units, a more balanced consortium of ruminal microbes should improve efficiency of microbial protein production so long as ruminal nitrogenous precursors are adequate. Conclusions and Applications Increased acetate production from improved fiber digestibility is thought to increase milk fat production, particularly in multiparous cows. In primiparous cows, the acetate from improved fiber degradability might be diverted to body weight gain. Milk production efficiency responses of 5% to 10% have been recorded with optimum doses of BCVFA and adequate rumen-degraded protein supply.

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.

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.

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.

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.

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.

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 sup-press 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 summa-rized 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 opti-mize fiber digestibility in the rumen. Our developments of mechanisms of action for various rumen-active modi-fiers should help nutrition advisors anticipate when a benefit in field conditions is more likely.

Previous research revealed a potential effect of dietary trace mineral source on both ruminal and fecal microbiota. However, the effect of Zn source, specifically, has not previously been considered. Based on reported postruminal solubility, we hypothesized that Zn hydroxychloride would decrease Treponema spp. fecal excretion relative to cows fed Zn sulfate. To test this hypothesis, lactating Holstein cows (n = 24; 685 ± 9 kg of body weight; 159 ± 8 d in milk; parity 3 ± 0.2) were randomly assigned to 1 of 2 dietary treatments: control (75 mg/kg Zn from ZnSO4) or Zn hydroxychloride (HYD; 75 mg/kg IntelliBond Z; Micronutrients USA LLC). Single fecal grab samples were collected on d 1 before dietary treatments and on d 27 after dietary treatments were applied. Fecal microbial DNA was extracted and sequenced to establish taxonomy using a universal primer for the 16S rRNA gene. Supplementation of HYD decreased the relative abundance of Treponema 2 by 3-fold (14.7% vs. 4.9%). Poor sequencing resolution at the species level limited inference of Treponema spp. toward management or gut health implications of HYD supplementation. However, the inclusion of pathogenic species among Treponema spp. indicates a potential implication of HYD feeding to reduce environmental exposure of the dairy cow to Treponema spp.

Previous research revealed a potential effect of dietary trace mineral source on both ruminal and fecal microbiota. However, the effect of Zn source, specifically, has not previously been considered. Based on reported postruminal solubility, we hypothesized that Zn hydroxychloride would decrease Treponema spp. fecal excretion relative to cows fed Zn sulfate. To test this hypothesis, lactating Holstein cows (n = 24; 685 +/- 9 kg of body weight; 159 +/- 8 d in milk; parity 3 +/- 0.2) were randomly assigned to 1 of 2 dietary treatments: control (75 mg/kg Zn from ZnSO 4 ) or Zn hydroxychloride (HYD; 75 mg/kg IntelliBond Z; Micronutrients USA LLC). Single fecal grab samples were collected on d 1 before dietary treatments and on d 27 after dietary treatments were applied. Fecal microbial DNA was extracted and sequenced to establish taxonomy using a universal primer for the 16S rRNA gene. Supplementation of HYD decreased the relative abundance of Treponema 2 by 3-fold (14.7% vs. 4.9%). Poor sequencing resolution at the species level limited inference of Treponema spp. toward management or gut health implications of HYD supplementation. However, the inclusion of pathogenic species among Treponema spp. indicates a potential implication of HYD feeding to reduce environmental exposure of the dairy cow to Treponema spp.

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.

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.

Objective: Our objective was to determine whether preweaned steer and heifer calves given a phytogenic starter supplement (either one containing caraway, licorice, oak bark, and vanilla that was soluble in milk, PM, or one containing caraway, licorice, oak bark, and vanilla, PC) or no phytogenic supplement, control (CTR), at a commercial calf ranch would have increased starter intake, weight gain, or rumen development. Materials and Methods: Holstein calves (124) were randomly assigned to 1 of 3 treatments, CTR, PM, or PC, at 2 d of age. Supplements PM or PC were added to individual feed buckets at each feeding at the rate of 0.25 g/kg of starter at a.m. and p.m. feedings. Calves were weighed at enrollment and at weaning, and blood samples were collected from a subset of 13 calves per treatment to assess rumen development. Weekly average DMI, milk intake, Glu, and BHB were analyzed using the Mixed procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC) Results and Discussion: Weekly average DMI (P < 0.01), BHB (P < 0.01), and Glu (P < 0.01) were different by week but not by treatment. Product PC group was numerically greater in total DMI, gain, and ADG and had faster rumen development indicated by overall greater BHB values but were not different among treatments. Implications and Applications: Addition of PM or PC to starter feed did not increase DMI, rumen development, or BW gain. But, the dose may have been too small or not fed long enough to elicit a response during the preweaning period.

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 β-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.