Screening compounds of the phenylpropanoid pathway and commercial products to mitigate methane emissions using batch culture in vitro incubations

Screening compounds of the phenylpropanoid pathway and commercial products to mitigate methane emissions using batch culture in vitro incubations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Screening compounds of the phenylpropanoid pathway and commercial products to mitigate methane emissions using batch culture in vitro incubations Monique Hodgson, Alexandre Vieira Chaves This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8999036/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Enteric methane (CH₄) produced during ruminal fermentation is a major contributor to agricultural greenhouse gas emissions and represents an energy loss to the animal. Plant-derived phenolic compounds have been proposed as dietary strategies to mitigate methane formation; however, their efficacy depends on compound structure and inclusion level. This study evaluated phenylpropanoid-derived phenolics identified in Brassica rapa subsp. chinensis and selected commercial additives for their ability to reduce methane without impairing ruminal fermentation. Lucerne pellets (500 mg dry matter) were incubated for 24h in an anaerobic in vitro batch culture system using rumen fluid from three pasture-fed fistulated cows (n = 3). Treatments included p-coumaric acid (5, 10, 15 mM), trans-cinnamic acid (5, 10, 15 mM), their combination (2.5, 5, 7.5 mM each), gallic acid (5, 10, 15 mM), tannic acid (0.41, 0.81, 1.22, 4.05 mM), N-carbamyl-L-glutamic acid (5, 10, 15 mM), and two commercial formulations (4.5 and 9% of substrate dry matter). Total gas production, CH₄ concentration and yield, in vitro dry matter disappearance (IVDMD), fermentation pH, and volatile fatty acid (VFA) profiles were measured. p-Coumaric acid reduced (P < 0.01) CH₄ concentration and yield in a dose-dependent manner, with reductions up to 43% at 15 mM; however, IVDMD was reduced (P < 0.01) at ≥ 10 mM. Trans-Cinnamic acid reduced (P < 0.01) CH₄ yield only at 15 mM. The combination of p-coumaric acid and trans-cinnamic acid reduced (P < 0.01) CH₄ production by up to 41% while maintaining IVDMD at 2.5–5 mM. Gallic acid did not affect CH₄ (P ≥ 0.16). Tannic acid produced the greatest CH₄ suppression (up to 85%; P < 0.01) but reduced gas production, IVDMD, and total VFA concentration (P < 0.01) at higher doses. N-carbamyl-L-glutamic acid and the two commercial formulations did not reduce CH₄ under these conditions (P ≥ 0.05). Moderate inclusion of p-coumaric acid (5 mM) alone or combined with trans-cinnamic acid (2.5 mM each) reduced methane without measurable adverse effects on fermentation, supporting further in vivo validation and dose optimisation for practical methane mitigation strategies. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Microbiology Plant secondary metabolites phenolic compounds enteric fermentation Introduction Agriculture is a cornerstone of Australia’s economy and food security, yet it contributes approximately 19% of national greenhouse gas (GHG) emissions, with enteric methane (CH₄) from ruminant livestock accounting for nearly three-quarters of agricultural emissions 1 . Projections indicate that, without mitigation, enteric emissions could increase substantially in response to expanding red meat demand 2,3 . Beyond its environmental impact, enteric CH₄ represents a 3–12% loss of gross dietary energy, reducing feed efficiency and production performance 4 . Mitigation strategies must therefore balance environmental responsibility with agricultural productivity. Effective solutions need to be economically viable, safe for animals and ecosystems, compatible with diverse feeding systems, and readily adoptable by producers. Identifying practical approaches that reduce CH₄ emissions without compromising animal health or performance is essential to sustaining both the environmental and economic resilience of Australia’s ruminant industries. Dietary additives such as plant secondary metabolites (PSM) represent a promising avenue for reducing enteric CH 4 emissions in agricultural production. The shikimate and phenylpropanoid metabolic pathways in plants produce over 8,000 aromatic PSMs, including l-phenylalanine-derived phenylpropanoids, more specifically phenolic compounds, with roles in plant growth, defense, and stress tolerance 5 . Secondary metabolites of these central anabolic pathways have been investigated as dietary additives in ruminants for their potential to manipulate the rumen microbiota, showing reductions in CH 4 production of up to 50% 4 . Phenolic PSMs possess a hydroxyl group, enabling high antimicrobial activity 6,7 . They can act directly, by interfering with the growth rate of rumen microbes via disruption of cytoplasmic membranes, and ultimately reducing populations of cellulolytic bacteria, such as Ruminococcus flavefaciens, Fibrobacter succinogenes , and methanogenic archaea 4 . Indirect mechanisms of PSMs in the rumen include promoting alternative H 2 sink mechanisms, such as utilising excess (free) hydrogen ions in volatile fatty acids (VFA) formation, for example, propionic acid production 8,9 . Forage brassicas are increasingly used in beef production systems to fill seasonal feed gaps due to their rapid growth in cool conditions, heat tolerance, and cost-effectiveness 10 . Forage brassicas have been characterised by high nutritional quality, including 70% dry matter (DM) of total digestible nutrients, 25% DM crude protein, with up to 89% DM digestibility and high metabolisable energy levels of 12-14MJ/kg DM 10,11 . Brassica diets can depress voluntary feed intake caused by fermentation by-product, disulphide. However, young ruminants fed forage brassicas have exhibited high growth rates (173–225 g/d), suggesting the effect of disulphide is not clinically significant 10 . Brassica rapa subsp. chinensis (bok choy) contains high levels of phenolic PSMs that show evidence for the capacity to reduce enteric CH 4 production 12 . The objective of this study was to evaluate the effect of phenolic compounds that are plant secondary metabolites (PSMs) derived from the phenylpropanoid pathway in Brassica rapa subsp. chinensis . The study also included other commercially available plant-derived compounds. These compounds were tested as potential dietary additives for ruminant species. Their effects on enteric methane production and fermentation profiles were assessed after 24 hours of in vitro batch culture incubation. It was hypothesised that phenolic compounds: p-coumaric acid (PCA), trans-cinnamic acid (TCA), gallic acid (GA), and commercial products: tannic acid (TA), N-carbamyl-L-glutamic acid (NCG), ‘Yi Chang Bao’ (YCB), and ‘Liu Wei Suan 600’ (LWS600) would elicit a inhibitory effect on methane production compared to a control treatment without negative effects on the overall fermentation. Results Table 3 presents the effects of plant secondary metabolites derived from the phenylpropanoid pathway on in vitro rumen fermentation characteristics after 24h of incubation. Total gas production was not affected by treatment (P = 0.32), ranging from 86.4 to 100.9 mL g ⁻1 DM compared with 99.0 mL g ⁻1 DM in the control. Relative to the control (14.6%), p-coumaric acid reduced (P < 0.01) methane concentration by 20.1% at 5 mM, 35.7% at 10 mM, and 38.4% at 15 mM. Trans-cinnamic acid reduced (P < 0.01) methane concentration by 12.7% at 5 mM, 16.2% at 10 mM, and 27.1% at 15 mM. The combination of p-coumaric acid and trans-cinnamic acid reduced (P < 0.01) methane concentration by 16.8% at 2.5 mM, 23.9% at 5 mM, and 35.7% at 7.5 mM. Gallic acid did not affect methane concentration. Table 3 . Effects of increasing concentrations of plant secondary metabolites derived from the phenylpropanoid pathway in Brassica rapa subsp. chinensis on in vitro batch culture fermentation characteristics after 24 h of incubation. Variables measured include total gas production (mL g⁻ 1 DM incubated), methane concentration (%), methane production (mL g⁻ 1 DM incubated), in vitro dry matter disappearance (IVDMD, %), fermentation pH, total volatile fatty acid (VFA) concentration and individual VFA percentages (% of total VFA), net VFA production (24 h – 0 h; mM), and acetate-to-propionate ratio (A:P). mM Control p -coumaric acid (PCA) Trans-cinnamic acid (TCA) PCA + TCA Gallic acid 0 5 10 15 5 10 15 2.5 5 7.5 5 10 15 SEM P-Value Gas, mL g −1 DM 99.0 96.8 89.7 92.0 100.5 100.4 99.4 86.9 86.4 91.5 100.9 96.6 94.0 5.09 0.32 CH 4 , % 14.60 11.66* 9.39* 8.99* 12.74* 12.24* 10.65* 12.15* 11.11* 9.39* 14.18 13.97 14.06 0.612 < 0.01 CH 4 , mL g −1 DM 14.57 11.19* 8.38* 8.26* 12.83 12.28 10.57* 10.61* 9.73* 8.54* 14.31 13.59 13.27 0.832 < 0.01 IVDMD, % 61.8 60.1 55.5* 52.8* 60.0 60.7 56.8 60.5 58.7 55.2* 62.0 60.6 60.5 2.92 < 0.01 pH 6.10 6.06 6.08 6.06 6.07 6.03 6.03 6.04 6.03 6.04 6.06 6.07 6.05 0.029 0.55 Total VFA, mM 96.1 96.7 94.5 93.6 100.4 102.9 96.8 99.5 94.3 90.7 101.4 99.8 101.6 4.07 0.52 Percentages of individual VFA of total VFA, % Acetate (A) 60.7 64.5 64.6 63.8 63.5 63.3 64.7 62.8 64.4 65.0 62.0 63.2 62.7 1.13 0.16 Propionate (P) 24.4 22.8 23.0 22.2 22.5 22.6 22.5 23.4 23.2 21.7 23.4 22.2 21.8 0.83 0.25 Butyrate 9.72 9.88 8.89 8.46 9.22 9.04 8.86 8.98 8.95 9.11 9.44 9.40 9.56 0.399 0.37 BCVFA 3.29 2.08 2.27 3.28 2.96 3.02 2.51 2.94 2.29 2.58 3.18 3.22 3.54 0.412 0.28 Ratio A:P 2.49 2.83 2.81 2.88 2.83 2.81 2.88 2.68 2.78 3.00 2.65 2.85 2.88 0.144 0.86 Net VFA production (24h-0h), mM Total VFA 70.0 70.5 68.4 67.4 74.2 76.7 70.7 73.4 68.2 64.6 75.3 73.7 75.4 4.82 0.52 Acetate (A) 42.6 46.1 44.9 43.6 47.5 48.9 46.3 46.3 44.5 42.7 46.9 46.9 47.5 3.22 0.84 Propionate (P) 16.4 15.5 15.0 13.9 15.8 16.4 15.0 16.5 15.1 12.9 16.9 15.4 15.4 1.56 0.64 Butyrate 6.77 6.93 6.01 5.49* 6.85 6.90 6.17 6.54 6.03 5.85† 7.07 6.97 7.31 0.28 < 0.01 BCVFA 2.50 1.39 1.50 2.44 2.35 2.49 1.83 2.32 1.57 1.75 2.59 2.61 2.99 0.38 0.10 CH 4 , methane; BCVFA, branched chain volatile fatty acids (iso-butyrate + iso-valerate); SEM, standard error of the means. *Indicates differences (P ≤ 0.05) from control treatments, † Indicates tendency from control treatment (P ≤ 0.10). Compared with the control (14.57 mL g ⁻1 DM), p-coumaric acid reduced (P < 0.01) methane yield by 23.2% at 5 mM, 42.5% at 10 mM, and 43.3% at 15 mM. Trans-cinnamic acid reduced (P < 0.01) methane yield by 27.5% at 15 mM, whereas 5 and 10 mM had no detectable effect. The combination of p-coumaric acid and trans-cinnamic acid reduced methane yield by 27.2% at 2.5 mM, 33.2% at 5 mM, and 41.4% at 7.5 mM. Gallic acid did not affect methane production. In vitro dry matter disappearance (IVDMD) was affected by treatment (P < 0.01). Relative to the control (61.8%), p-coumaric acid reduced digestibility by 10.2% at 10 mM and 14.6% at 15 mM, whereas 5 mM had no detectable effect. The combination treatment reduced digestibility by 10.7% at 7.5 mM only. Trans-cinnamic acid and gallic acid did not affect digestibility. Fermentation pH, total VFA concentration, the percentages of acetate, propionate, butyrate and BCVFA of total volatile fatty acids were not (P ≥ 0.16) affected by treatment. The acetate-to-propionate ratio remained unchanged (P = 0.86). Net total volatile fatty acids, net acetate and net propionate production (24h − 0h) were not affected (P ≥ 0.52). Net butyrate production was affected by treatment (P < 0.01), with p-coumaric acid at 15 mM reducing net butyrate by 18.9% relative to the control (6.77 mM). A tendency was observed for the combination treatment at 7.5 mM, which reduced net butyrate by 13.6% (P ≤ 0.10). Net branched-chain volatile fatty acid production showed a tendency for treatment effects (P = 0.10), although pairwise differences from the control were not detected at P ≤ 0.05. Table 4 presents the effects of commercial products on in vitro rumen fermentation characteristics after 24h of incubation. Total gas production was affected by tannic acid, with 4.05 mM reducing (P < 0.01) gas production by 38.1% relative to the control (99.0 mL g ⁻1 DM). Methane concentration was reduced (P < 0.01) by tannic acid by 21.5% at 0.81 mM, 28.6% at 1.22 mM, and 75.4% at 4.05 mM, while methane yield declined by 32.4%, 43.1%, and 85.0% at the same respective doses compared with the control (14.57 mL g ⁻1 DM). In vitro dry matter disappearance decreased (P < 0.01) with tannic acid by 8.1%, 12.3%, and 25.9% at 0.81, 1.22, and 4.05 mM, respectively, and Yi Chang Bao at 9% reduced digestibility by 9.2%. Fermentation pH was reduced (P < 0.01) by N-carbamyl-L-glutamic acid at 15 mM by 3.6%. Total volatile fatty acid concentration declined (P < 0.01) by 32.8% with tannic acid at 4.05 mM. The percentage of acetate increased (P = 0.02) with tannic acid at 0.41 mM and 1.22 mM by 7.1% and 10.2%, respectively, while propionate increased (P < 0.01) by 32.0% at 4.05 mM tannic acid. Butyrate percentage decreased (P < 0.01) by 56.6% at 1.22 mM and 96.9% at 4.05 mM tannic acid, whereas Yi Chang Bao at 9% increased butyrate by 31.6%. Branched-chain volatile fatty acids were reduced (P < 0.01) by up to 86.8% at 4.05 mM tannic acid, and the acetate-to-propionate ratio declined (P < 0.01) by 18.1% at this concentration. Net total volatile fatty acid production was reduced (P < 0.01) by 41.9% at 4.05 mM tannic acid, with net acetate reduced by 38.3% and net branched-chain volatile fatty acids reduced by up to 86.8%, whereas net propionate was not affected (P = 0.15). Table 4 Effects of increasing concentrations of commercial compounds on in vitro batch culture fermentation characteristics after 24 h of incubation. Variables measured include total gas production (mL g⁻ 1 DM incubated), methane concentration (%), methane production (mL g⁻ 1 DM incubated), in vitro dry matter disappearance (IVDMD, %), fermentation pH, total volatile fatty acid (VFA) concentration and individual VFA concentrations (% of total VFA), net VFA production (24 h – 0 h; mM), and acetate-to-propionate ratio (A:P). Control Tannic acid, mM N-carbamyl-L-glutamic acid, mM Yi Chang Bao, % of substrate DM Liu Wei Suan 600, % of substrate DM 0 0.41 0.81 1.22 4.05 5 10 15 4.5 9 4.5 9 SEM P-Value Gas, mL g − 1 DM 99.0 89.0 86.8 80.1 61.3* 93.4 100.9 101.8 85.4 85.7 95.8 98.8 7.53 < 0.01 CH 4 , % 14.60 13.28 11.46* 10.42* 3.59* 14.55 14.56 14.30 15.71 15.97 15.06 14.74 0.862 < 0.01 CH 4 , mL g − 1 DM 14.57 11.75 9.85* 8.29* 2.19* 13.45 14.65 14.48 13.78 13.61 14.32 14.51 0.863 < 0.01 IVDMD, % 61.8 58.0 56.8* 54.2* 45.8* 61.1 61.6 62.2 59.0 56.1* 60.7 61.4 2.97 < 0.01 pH 6.10 6.07 6.05 6.04 6.07 6.01 5.98 5.88* 6.08 6.11 6.08 6.07 0.055 < 0.01 Total VFA, mM 96.1 96.3 94.2 88.4 64.6* 94.6 91.6 92.8 90.6 88.5 101.5 103.1 3.89 < 0.01 Percentages of individual VFA of total VFA, % Acetate (A) 60.7 65.0* 64.7† 66.9* 65.8* 64.8* 64.4† 64.6† 63.8 63.1 64.4† 63.8 1.18 0.02 Propionate (P) 24.4 22.7 25.5 27.3† 32.2* 22.6 22.8 23.2 21.8 21.4† 22.3 22.5 1.47 < 0.01 Butyrate 9.72 8.62 7.79 4.22* 0.30* 8.67 9.45 9.41 11.06 12.79* 9.63 9.90 0.608 < 0.01 BCVFA 3.29 2.48 1.55* 1.28* 1.43* 2.55 2.32 2.05 2.17 1.80 2.42 2.55† 0.529 0.05 Ratio A:P 2.49 2.87 2.54 2.45 2.04* 2.87 2.82 2.79 2.93 2.95 2.89 2.84 0.217 < 0.01 Net VFA production (24h-0h), mM Total VFA 70.0 70.2 68.0 63.3 40.7* 68.7 65.5 66.6 64.4 62.4 75.4 77.0 5.19 < 0.01 Acetate (A) 42.6 46.3 44.7 42.8 26.3* 44.9 42.8 43.6 41.6 39.7 49.1 49.5 3.22 < 0.01 Propionate (P) 16.4 15.1 17.3 17.3 14.1 14.6 14.2 14.8 13.0 12.1 16.0 16.3 2.33 0.15 Butyrate 6.77 5.90 4.89* 4.77* - 6.91 6.25 6.31 7.60 8.95* 7.35 7.80 0.352 < 0.01 BCVFA 2.50 1.80 0.84* 0.52* 0.33* 1.82 1.52 1.29 1.36 1.00* 1.84 2.07 0.429 < 0.01 **CH4, methane; BCVFA, branched chain volatile fatty acids (iso-butyrate + iso-valerate); SEM, standard error mean. * Indicates differences (P ≤ 0.05) from control treatments, † indicates tendency from control treatment (P ≤ 0.10). Discussion In vitro 24h batch culture incubations provide a practical screening approach for antimethanogenic additives, but they do not reproduce digesta passage, absorption and host-mediated hydrogen sinks, or longer-term microbial adaptation; therefore, responses observed here should be interpreted as short-term rumen microbial effects under controlled conditions 19 . Among phenylpropanoid-derived metabolites, p-coumaric acid consistently reduced methane concentration and yield in a dose-dependent manner, while maintaining total gas production and VFA profiles at the lowest dose tested, indicating a selective inhibition of methanogenesis rather than a broad suppression of fermentation 13,14,20 . The decline in digestibility at ≥ 10 mM suggests that higher antimicrobial pressure can extend beyond methanogens to fibrolytic populations, consistent with the known membrane-disruptive activity of phenolic compounds 6,7 . Trans-Cinnamic acid produced a weaker response, reducing methane yield only at 15 mM, which may reflect structural differences that influence antimicrobial potency 20 . The p-coumaric acid + trans-cinnamic acid combination reduced methane across all inclusion levels, with digestibility maintained at 2.5–5 mM, supporting an additive effect at moderate doses 23 . Gallic acid did not reduce methane under these conditions, despite reports of antimethanogenic effects in other in vitro systems, highlighting the importance of compound structure, dose and experimental context 24–26 . Within the commercial additives, tannic acid produced the greatest methane suppression but was accompanied by clear depression of overall fermentation at higher doses, including reduced gas production, digestibility and total VFA, indicating that methane inhibition arose largely from broad microbial inhibition rather than a targeted shift in hydrogen utilisation 22,27–30 . Shifts in VFA percentages at the highest tannic acid dose occurred alongside reduced total VFA, reinforcing that fermentation was constrained rather than redirected 31 . N-carbamyl-L-glutamic acid did not affect methane or major fermentation endpoints in vitro, which is consistent with evidence that responses to this compound are more variable in vivo and may be mediated through systemic nitrogen metabolism rather than direct rumen microbial inhibition 32–34 . Yi Chang Bao and Liu Wei Suan 600 did not reduce methane in the batch culture system; medium-chain fatty acids and organic acids can influence rumen fermentation in other contexts, but microencapsulation and release characteristics may have limited immediate ruminal exposure within 24h 35–37 . The present study evaluated whether selected phenolic compounds and commercial additives could reduce methane production without impairing overall ruminal fermentation. The results demonstrate that p-coumaric acid, trans-cinnamic acid, and their combination consistently reduced methane concentration and yield in a dose-dependent manner, with reductions of up to 43% in methane yield, while maintaining total gas production, total volatile fatty acid concentration, acetate-to-propionate ratio, and overall fermentation profile at lower inclusion levels. Importantly, p-coumaric acid at 5 mM reduced methane without affecting in vitro dry matter disappearance, indicating that methane mitigation can be achieved without compromising substrate degradation when applied at appropriate doses. In contrast, higher inclusion levels of p-coumaric acid and the combined treatment reduced digestibility, suggesting that excessive concentrations may impair fermentative efficiency. Gallic acid did not influence methane production or fermentation characteristics under the conditions evaluated. Among the commercial products, tannic acid produced the greatest methane suppression, reducing methane yield by up to 85%; however, this response was accompanied by marked reductions in gas production, digestibility, total volatile fatty acids, and shifts in fermentation end-products, indicating substantial inhibition of overall rumen activity at higher doses. N-carbamyl-L-glutamic acid, Yi Chang Bao, and Lui Wei Suan 600 showed limited or inconsistent effects on methane production and did not provide meaningful mitigation without affecting other fermentation parameters. Collectively, these findings partially support the initial hypothesis. Certain phenolic compounds, particularly p-coumaric acid at moderate inclusion levels, reduced methane production without detrimental effects on overall fermentation, thereby meeting the criteria for effective mitigation. However, the hypothesis must be rejected for tannic acid at higher concentrations and for the remaining commercial products, as methane suppression was either accompanied by impaired fermentation or was not observed. These results highlight that effective methane mitigation requires a balance between inhibitory activity against methanogenesis and preservation of rumen fermentative function, with compound type and dose being critical determinants of this balance. Methods The in vitro study was conducted at the University of Sydney (Sydney, NSW, Australia). Protocols and animals used in this study are cared for under the approval and guidance of The University of Sydney Animal Ethics Committee (Approved Protocol Number 2022/AE002180). Information provided in the manuscript complies with the essential recommendations for reporting of the ARRIVE guidelines. Substrate preparation Substrate was prepared by grinding lucerne pellets using a 1.0-mm particle-size sieve feed mill (Retsch ZM 200; Haan, NW, Germany). For each in vitro incubation, 500 mg DM of lucerne substrate was weighed into an ANKOM® bag (model F57) with three replicates per treatment and sealed. Each bag was placed into a 50 mL amber serum bottle. The lucerne pellets were sent to an accredited laboratory for analysis of chemical composition using AOAC methods. Results are presented in Table 1 . Table 1 Dry matter content, chemical composition and dry matter digestibility of lucerne pellet used as the basal diet for in vitro batch culture incubations. Dry matter – as fed, % Crude Protein (CP) Neutral Detergent Fibre (NDF) Acid Detergent Fibre (ADF) Crude Fat (CF) Inorganic Ash Non-fibrous carbohydrates (NFC*) Dry Matter digestibility (DMD) Lucerne pellet 91.25 20.9 35.4 26.9 1.8 9.3 32.6 58.8 *NFC = 100 – (CP + NDF+CF + Ash) Treatments Eight treatments were tested, including three PSMs found abundantly in Brassica rapa subsp. chinensis . 1) p-coumaric acid; 2) trans-cinnamic acid; and 3) Gallic acid. The combination of p-coumaric acid + trans-cinnamic acid (TCA + PCA) was also tested to verify additional (synergistic, additive or antagonistic) effect of these compounds. Additionally, four commercially available compounds,1) Tannic acid, 2) N-carbamyl-L-glutamic acid; 3) Yi Chang Bao; and 4) Liu Wei Suan 600 were tested. Treatments were prepared at varying concentrations to evaluate dose-dependent effects on ruminal fermentation, making a total of twenty-three treatments and a lucerne-only control. The complete list of treatments and their respective concentrations tested are listed on Table 2 . Table 2 Inclusion levels of plant secondary metabolites derived from Brassica rapa subsp. chinensis and commercial compounds evaluated in the in vitro batch culture incubation. Category Treatment Concentration Control Lucerne substrate only 0 mM Plant secondary metabolites p-Coumaric acid (PCA) 5, 10, 15 mM trans-Cinnamic acid (TCA) 5, 10, 15 mM PCA + TCA (1:1 combination) 2.5 + 2.5, 5 + 5, 7.5 + 7.5 mM Gallic acid (GA) 5, 10, 15 mM Commercial compounds Tannic acid (TA, 81% purity*) 0.41, 0.81, 1.22, 4.05 mM N-carbamyl-L-glutamic acid (NCG) 5, 10, 15 mM Yi Chang Bao 4.5, 9% of substrate DM Liu Wei Suan 600 4.5, 9% of substrate DM *Inclusion levels of tannic acid were corrected for 81% purity; values shown represent actual molar concentrations. Yi Chang Bao (Guangdong Ruisheng Technology Group Co., Ltd., Guangdong, China) is a commercial medium-chain fatty acid (MCFA) blend containing caproic (C6), caprylic (C8), capric (C10), and lauric (C12) acids. The product is manufactured using microencapsulation technology to stabilise the fatty acids and allow gradual release in the gastrointestinal tract. Liu Wei Suan 600 (Guangdong Ruisheng Technology Group Co., Ltd., Guangdong, China) is a microencapsulated organic acid blend composed primarily of benzoic, fumaric, citric, formic, propionic, and lactic acids, together with plant essential oils. Both products are supplied as powder formulations. In the present study, Yi Chang Bao and Liu Wei Suan 600 were incorporated at 4.5% and 9% of the total substrate (500 mg DM) to evaluate dose-dependent effects on ruminal fermentation. Inclusion levels of PSM and N-carbamyl-L-glutamic acid were selected based on doses used in previous studies demonstrating effective CH 4 reduction 13–15 . All treatments were fine-powder additives and kept at the particle size commercially provided. Treatments were weighed using an analytical scale (AB54-S, Mettler Toledo, Greifensee, Switzerland) and transferred into the pre-prepared amber serum bottles containing substrate. Each treatment concentration was tested in triplicate (e.g., 3 serum bottles per treatment per incubation run). Inoculum and incubation Inoculum for the in vitro incubations was obtained from three ruminally fistulated cows (n = 3) grazing pasture (15% crude protein; 64% neutral detergent fibre). Each cow was treated as an individual experimental unit. Rumen fluid was collected separately from each animal 2–3 h post-feeding, filtered through a single layer of nylon–elastane fabric to remove particulate matter, and immediately transferred into individual 1 L pre-warmed insulated thermos flasks for transport to the laboratory. Inoculum was prepared by mixing rumen fluid with a rumen solution, reducing agent, and resazurin, as described by Menke et al. 16 . Each 50 mL amber serum bottle was gassed with CO 2 to create an anaerobic environment, 25 mL of inoculum was transferred into the bottle, and the bottle was immediately fitted with a rubber stopper to contain the gases produced during fermentation. Each serum bottle was then placed into a shaking incubator set at 39 o C and 120 oscillations per minute for 24h. The entire incubation was repeated three times. Measurement of total gas, in vitro dry matter disappearance and pH At 24h of incubation, 25 mL of headspace was sampled from each serum bottle with a syringe and immediately transferred into a 12 mL evacuated exetainer to ensure positive pressure (Labco Ltd., High Wycombe, United Kingdom) for CH 4 analysis. The remaining gas in each serum bottle was measured using a water-displacement apparatus 17 . Total gas production for each serum bottle was calculated as the sum of the volume measured using the water displacement apparatus and the 25 mL of headspace gas previously removed and expressed as mL gas g − 1 DM incubated. After 24h of incubation, and after gas was sampled and the volume measured, the fermentation serum bottles were opened and placed on ice to stop fermentation. The pH of each culture was measured using a pH probe inserted into the serum bottles (Activon Model 209, Gladesville, NSW, Australia). The pH probe was washed with distilled water between each serum bottle, using a wash bottle (LDPE Wash Bottle, ThermoFisher, Brisbane, NSW, Australia). The ANKOM® bags were then removed from the serum bottles and washed thoroughly using a top load washing machine for 10 minutes, twice, then dried at 55 o C for 48h using forced air circulated oven (BMT Venticell 111, Cambridge Scientific, Watertown, MA, USA) and weighed to calculate in vitro dry matter disappearance (IVDMD). Determination of volatile fatty acids (VFA) The liquid fraction of the fermentation at 0 and 24h incubation was subsampled from each serum bottle. 1.5 mL subsamples were transferred into 2 mL microcentrifuge tubes. Samples were centrifuged at 4,500 × g for 15 min at 5 o C to precipitate particulate matter and protein. A 1.2 mL sample of supernatant was transferred into 2 mL micro-centrifuge tubes containing 0.3 mL of metaphosphoric acid (0.25; w/v) and frozen at -20 o C until analysed for VFA concentrations. The VFA analysis was performed as described by Forwood et al. 18 . The 0h samples were analysed for VFA to calculate the net total VFA production [24h – 0h, mM]. The concentration of total VFA is expressed in mM, and individual VFA is expressed as % of total VFA. Determination of CH Methane (CH₄) concentration was determined from the headspace gas collected in 12 mL evacuated exetainers. For each sample, 3 mL of exetainer gas was withdrawn using a gas-tight syringe and transferred into a 2 mL evacuated autosampler vial to maintain positive pressure and avoid atmospheric contamination. The autosampler vials were then loaded onto the gas chromatograph for analysis of CH₄ by GC–FID. The Agilent model 7890a gas chromatograph with a flame ionisation detector (FID) calibrated to 250°C, airflow 300 mL min − 1 , H 2 fuel flow 30 mL min − 1 , makeup flow (N 2 ) 30 mL min − 1 installed with a capillary column (Restek Rt-Q-Bond, 30 m × 0.53 mm ID × 20 µm). The Split-Splitless Inlet was heated to 60°C, 9.526 PSI, with a helium total flow of 33 mL min − 1 , septum purge flow of 3 mL min − 1 , split ratio of 5:1, split flow of 25 mL min − 1 and an oven temperature of 60°C. Quantification was performed using an external standard calibration curve prepared with CH₄ concentrations of 0, 5, 10 and 20% (v/v), yielding a linear response (R 2 ≥ 0.98). Methane measurements are expressed as concentration (%), and production (mL CH 4 g − 1 DM incubated). Statistical analysis Data from the in vitro fermentations were analysed as a completely randomised design using PROC MIXED in SAS (version 9.4; SAS Institute Inc., Cary, NC, USA). Plant secondary metabolites and commercial products were analysed in separate models to avoid confounding between treatment categories. Within each model, treatment (dose) was included as a fixed effect, and animal (rumen fluid donor; n = 3) was included as a random effect. Incubation run (i.e., individual rumen fluid collection from each cow) served as the experimental unit and error term for assessing treatment effects on CH₄ production, fermentation parameters, and IVDMD. Treatment means were compared with the control using least-squares means with Dunnett adjustment. Statistical significance was declared at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. Data availability The data and the model are available from the corresponding author upon reasonable request. Declarations Competing interests The authors declare no conflict of interests. Funding Not applicable. Author contributions MH: investigation, writing original draft, review & editing, visualisation, lab analysis. AVC: investigation, writing second draft, review & editing, visualisation, formal analysis, validation, methodology, conceptualisation, supervision. References DCCEEW. Australia’s emissions projections 2024 . Australian Government (2024). O’Mara, F. P. The significance of livestock as a contributor to global greenhouse gas emissions today and in the near future. Anim. Feed Sci. Technol. 166–167 , 7–15 (2011). CIE. Australian Agricultural Emissions Projections to 2025 . The Centre for International Economics (2013). Ku-Vera, J. C. et al. Role of secondary plant metabolites on enteric methane mitigation in ruminants. Front. Vet. Sci. 7 , 584 (2020). Dong, N.-Q. & Lin, H.-X. Contribution of phenylpropanoid metabolism to plant development and plant–environment interactions. J. Integr. Plant Biol. 63 , 180–209 (2021). Lobiuc, A. et al. Future antimicrobials: natural and functionalized phenolics. Molecules 28 , 3 (2023). Dorman, H. J. D. & Deans, S. G. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88 , 308–316 (2000). Kamra, D. N., Pawar, M. & Singh, B. Effect of plant secondary metabolites on rumen methanogens and methane emissions by ruminants. In Dietary Phytochemicals and Microbes (ed. Patra, A. K.) 351–370 (Springer, Dordrecht, 2012). Cardoso-Gutierrez, E. et al. Effect of tannins from tropical plants on methane production from ruminants: a systematic review. Vet. Anim. Sci. 14 , 100214 (2021). Barry, T. N. The feeding value of forage brassica plants for grazing ruminant livestock. Anim. Feed Sci. Technol. 181 , 15–25 (2013). Dillard, S. L. et al. Assessment of forage brassica species for dairy and beef-cattle fall grazing systems. Appl. Anim. Sci. 36 , 157–166 (2020). Yeo, H. J. et al. Metabolomic analysis reveals the interaction of primary and secondary metabolism in white, pale green, and green pak choi ( Brassica rapa subsp. chinensis ). Appl. Biol. Chem. 64 , 3 (2021). Jayanegara, A. Ruminal methane production on simple phenolic acids addition in in vitro gas production method. Media Peternak. 32 , 1–8 (2009). Giuburuncă, M., Criste, A. & Vioara, M. Effects of p-coumaric acid on ruminal fermentation parameters in in vitro ruminal cultures. Bull. UASVM Anim. Sci. Biotechnol. 72 , 1–6 (2015). Aboagye, I. A. & Beauchemin, K. A. Potential of molecular weight and structure of tannins to reduce methane emissions from ruminants: a review. Animals 9 , 856 (2019). Menke, K. H. et al. The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from gas production when incubated with rumen liquor in vitro. J. Agric. Sci. 93 , 217–222 (1979). Fedorah, P. M. & Hrudey, S. E. A simple apparatus for measuring gas production by methanogenic cultures in serum bottles. Environ. Technol. Lett. 4 , 425–432 (1983). Forwood, D. L. et al. Crop sorghum ensiled with unsalable vegetables increases silage microbial diversity. Front. Microbiol. 10 , 2599 (2019). Dijkstra, J., Mills, J. A. N. & France, J. The role of dynamic modelling in understanding the microbial contribution to rumen fermentation. Nutr. Res. Rev. 15 , 67–90 (2002). Peña-Torres, E. F. et al. Hydroxycinnamic acids in animal production: pharmacokinetics, pharmacodynamics and growth promoting effects. Rev. Mex. Cienc. Pecu. 10 , 391–415 (2019). El-Zaiat, H. M. et al. Assessment of different phytogenic-based additives on in vitro rumen fermentation profile and methane emissions. Front. Vet. Sci. 12 , 1591700 (2025). Magnani, E. et al. Tannin-based product in feedlot diet as a strategy to reduce enteric methane emissions of Nellore cattle finished under tropical conditions. Transl. Anim. Sci. 7 , txad048 (2023). Huang, R. et al. Evaluating the effect of phenolic compounds as hydrogen acceptors when ruminal methanogenesis is inhibited in vitro. Part 1: Dairy cows. Animal 17 , 100788 (2023). Zhu, W. et al. Effects of gallic acid on in vitro ruminal fermentation, methane emission, microbial composition, and metabolic functions. Animals 15 , 1959 (2025). Getachew, G. et al. The influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis. Anim. Feed Sci. Technol. 140 , 444–461 (2008). Nørskov, N. P. et al. Methane reduction by quercetin, tannic and salicylic acids: influence of molecular structures on methane formation and fermentation in vitro. Sci. Rep. 13 , 16023 (2023). Min, B. R. & Solaiman, S. Comparative aspects of plant tannins on digestive physiology, nutrition and microbial community changes in sheep and goats: a review. J. Anim. Physiol. Anim. Nutr. 102 , 1181–1193 (2018). Frutos, P., Hervás, G., Giráldez, F. J. & Mantecón, A. R. An in vitro study on the ability of polyethylene glycol to inhibit the effect of quebracho tannins and tannic acid on rumen fermentation in sheep, goats, cows, and deer. Aust. J. Agric. Res. 55 , 1125–1132 (2004). Yang, K. et al. Effects of dietary supplementing tannic acid in the ration of beef cattle on rumen fermentation, methane emission, microbial flora and nutrient digestibility. J. Anim. Physiol. Anim. Nutr. 101 , 302–310 (2017). Zhao, R. et al. Effects of dietary tannic acid and tea polyphenol supplementation on rumen fermentation, methane emissions, milk protein synthesis and microbiota in cows. Microorganisms 13 , 8 (2025). Newbold, C. J. et al. Propionate precursors and other metabolic intermediates as possible alternative electron acceptors to methanogenesis in ruminal fermentation in vitro. Br. J. Nutr. 94 , 27–35 (2005). Sun, X. et al. Effects of dietary addition of N-carbamylglutamate on growth performance, rumen fermentation, rumen microflora and methane emission of Holstein bulls. J. Anim. Sci. 98 , 2020 (2020). (Add article number if available.) Li, Y. et al. Microbiome-metabolome responses in ruminal content and feces of lactating dairy cows with N-carbamylglutamate supplementation under heat stress. Front. Vet. Sci. 9 , 902001 (2022). Liu, Z. et al. N-Carbamoylglutamate supplementation on digestibility, rumen fermentation, milk quality, antioxidant parameters and metabolites of Jersey cattle in high-altitude areas. Front. Vet. Sci. 9 , 848912 (2022). Machmüller, A. Medium-chain fatty acids and their potential to reduce methanogenesis in domestic ruminants. Agric. Ecosyst. Environ. 112 , 107–114 (2006). Rajaraman, B. et al. Effects of caprylic acid and β-cyclodextrin complexes on digestibility, energy balance and methane production in Korean Hanwoo heifers. Anim. Feed Sci. Technol. 234 , 72–77 (2017). Castillo, C. et al. Organic acids as a substitute for monensin in diets for beef cattle. Anim. Feed Sci. Technol. 115 , 101–116 (2004). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8999036","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":607570017,"identity":"ab06cb01-51c1-4567-9d54-db1e9cac4c7e","order_by":0,"name":"Monique Hodgson","email":"","orcid":"","institution":"University of Sydney","correspondingAuthor":false,"prefix":"","firstName":"Monique","middleName":"","lastName":"Hodgson","suffix":""},{"id":607570018,"identity":"c891c1ff-f4fd-44bb-a61c-a5746daeca7d","order_by":1,"name":"Alexandre Vieira Chaves","email":"data:image/png;base64,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","orcid":"","institution":"University of Sydney","correspondingAuthor":true,"prefix":"","firstName":"Alexandre","middleName":"Vieira","lastName":"Chaves","suffix":""}],"badges":[],"createdAt":"2026-03-01 03:53:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8999036/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8999036/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105034652,"identity":"e672bc3b-9dc3-48d8-978f-e143a815f8db","added_by":"auto","created_at":"2026-03-20 07:23:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":992980,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8999036/v1/17621d30-216a-4cb0-855f-6381fe957e5a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening compounds of the phenylpropanoid pathway and commercial products to mitigate methane emissions using batch culture in vitro incubations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAgriculture is a cornerstone of Australia\u0026rsquo;s economy and food security, yet it contributes approximately 19% of national greenhouse gas (GHG) emissions, with enteric methane (CH₄) from ruminant livestock accounting for nearly three-quarters of agricultural emissions\u003csup\u003e1\u003c/sup\u003e. Projections indicate that, without mitigation, enteric emissions could increase substantially in response to expanding red meat demand\u003csup\u003e2,3\u003c/sup\u003e. Beyond its environmental impact, enteric CH₄ represents a 3\u0026ndash;12% loss of gross dietary energy, reducing feed efficiency and production performance\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitigation strategies must therefore balance environmental responsibility with agricultural productivity. Effective solutions need to be economically viable, safe for animals and ecosystems, compatible with diverse feeding systems, and readily adoptable by producers. Identifying practical approaches that reduce CH₄ emissions without compromising animal health or performance is essential to sustaining both the environmental and economic resilience of Australia\u0026rsquo;s ruminant industries.\u003c/p\u003e \u003cp\u003eDietary additives such as plant secondary metabolites (PSM) represent a promising avenue for reducing enteric CH\u003csub\u003e4\u003c/sub\u003e emissions in agricultural production. The shikimate and phenylpropanoid metabolic pathways in plants produce over 8,000 aromatic PSMs, including l-phenylalanine-derived phenylpropanoids, more specifically phenolic compounds, with roles in plant growth, defense, and stress tolerance\u003csup\u003e5\u003c/sup\u003e. Secondary metabolites of these central anabolic pathways have been investigated as dietary additives in ruminants for their potential to manipulate the rumen microbiota, showing reductions in CH\u003csub\u003e4\u003c/sub\u003e production of up to 50%\u003csup\u003e4\u003c/sup\u003e. Phenolic PSMs possess a hydroxyl group, enabling high antimicrobial activity\u003csup\u003e6,7\u003c/sup\u003e. They can act directly, by interfering with the growth rate of rumen microbes via disruption of cytoplasmic membranes, and ultimately reducing populations of cellulolytic bacteria, such as \u003cem\u003eRuminococcus flavefaciens, Fibrobacter succinogenes\u003c/em\u003e, and methanogenic archaea\u003csup\u003e4\u003c/sup\u003e. Indirect mechanisms of PSMs in the rumen include promoting alternative H\u003csub\u003e2\u003c/sub\u003e sink mechanisms, such as utilising excess (free) hydrogen ions in volatile fatty acids (VFA) formation, for example, propionic acid production\u003csup\u003e8,9\u003c/sup\u003e. Forage brassicas are increasingly used in beef production systems to fill seasonal feed gaps due to their rapid growth in cool conditions, heat tolerance, and cost-effectiveness\u003csup\u003e10\u003c/sup\u003e. Forage brassicas have been characterised by high nutritional quality, including 70% dry matter (DM) of total digestible nutrients, 25% DM crude protein, with up to 89% DM digestibility and high metabolisable energy levels of 12-14MJ/kg DM\u003csup\u003e10,11\u003c/sup\u003e. Brassica diets can depress voluntary feed intake caused by fermentation by-product, disulphide. However, young ruminants fed forage brassicas have exhibited high growth rates (173\u0026ndash;225 g/d), suggesting the effect of disulphide is not clinically significant\u003csup\u003e10\u003c/sup\u003e. \u003cem\u003eBrassica rapa subsp. chinensis\u003c/em\u003e (bok choy) contains high levels of phenolic PSMs that show evidence for the capacity to reduce enteric CH\u003csub\u003e4\u003c/sub\u003e production\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe objective of this study was to evaluate the effect of phenolic compounds that are plant secondary metabolites (PSMs) derived from the phenylpropanoid pathway in \u003cem\u003eBrassica rapa subsp. chinensis\u003c/em\u003e. The study also included other commercially available plant-derived compounds. These compounds were tested as potential dietary additives for ruminant species. Their effects on enteric methane production and fermentation profiles were assessed after 24 hours of in vitro batch culture incubation. It was hypothesised that phenolic compounds: p-coumaric acid (PCA), trans-cinnamic acid (TCA), gallic acid (GA), and commercial products: tannic acid (TA), N-carbamyl-L-glutamic acid (NCG), \u0026lsquo;Yi Chang Bao\u0026rsquo; (YCB), and \u0026lsquo;Liu Wei Suan 600\u0026rsquo; (LWS600) would elicit a inhibitory effect on methane production compared to a control treatment without negative effects on the overall fermentation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the effects of plant secondary metabolites derived from the phenylpropanoid pathway on in vitro rumen fermentation characteristics after 24h of incubation. Total gas production was not affected by treatment (P\u0026thinsp;=\u0026thinsp;0.32), ranging from 86.4 to 100.9 mL g\u003csup\u003e⁻1\u003c/sup\u003e DM compared with 99.0 mL g\u003csup\u003e⁻1\u003c/sup\u003e DM in the control. Relative to the control (14.6%), p-coumaric acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) methane concentration by 20.1% at 5 mM, 35.7% at 10 mM, and 38.4% at 15 mM. Trans-cinnamic acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) methane concentration by 12.7% at 5 mM, 16.2% at 10 mM, and 27.1% at 15 mM. The combination of p-coumaric acid and trans-cinnamic acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) methane concentration by 16.8% at 2.5 mM, 23.9% at 5 mM, and 35.7% at 7.5 mM. Gallic acid did not affect methane concentration.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003e.\u003c/b\u003eEffects of increasing concentrations of plant secondary metabolites derived from the phenylpropanoid pathway in Brassica rapa subsp. chinensis on in vitro batch culture fermentation characteristics after 24 h of incubation. Variables measured include total gas production (mL g⁻\u003csup\u003e1\u003c/sup\u003e DM incubated), methane concentration (%), methane production (mL g⁻\u003csup\u003e1\u003c/sup\u003e DM incubated), in vitro dry matter disappearance (IVDMD, %), fermentation pH, total volatile fatty acid (VFA) concentration and individual VFA percentages (% of total VFA), net VFA production (24 h \u0026ndash; 0 h; mM), and acetate-to-propionate ratio (A:P).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"16\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003emM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-coumaric acid (PCA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eTrans-cinnamic acid (TCA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e \u003cp\u003ePCA\u0026thinsp;+\u0026thinsp;TCA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c14\" namest=\"c12\"\u003e \u003cp\u003eGallic acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGas, mL g\u003csup\u003e\u0026minus;1\u003c/sup\u003eDM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e86.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e86.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e91.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e96.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e94.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e5.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.66*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.39*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.99*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.74*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.24*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.65*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12.15*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e11.11*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9.39*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e13.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e14.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.612\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e, mL g\u003csup\u003e\u0026minus;1\u003c/sup\u003eDM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.19*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.38*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.26*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.57*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10.61*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9.73*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e8.54*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e13.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e13.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIVDMD, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.5*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e60.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e58.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e55.2*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e62.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e60.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e60.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e2.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e6.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal VFA, mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e93.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e102.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e96.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e99.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e94.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e90.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e101.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e99.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e101.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e4.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e \u003cp\u003ePercentages of individual VFA of total VFA, %\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetate (A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e63.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e64.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e62.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e65.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e62.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e63.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e62.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionate (P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e23.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e23.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e21.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e23.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e22.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e21.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e8.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e9.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e9.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e9.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCVFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e3.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRatio A:P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e2.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e \u003cp\u003eNet VFA production (24h-0h), mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal VFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e74.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e70.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e73.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e68.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e64.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e75.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e73.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e75.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e4.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetate (A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e48.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e46.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e46.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e44.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e42.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e46.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e46.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e47.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionate (P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e12.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.49*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5.85\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e7.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e6.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e7.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCVFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e2.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"16\"\u003eCH\u003csub\u003e4\u003c/sub\u003e, methane; BCVFA, branched chain volatile fatty acids (iso-butyrate\u0026thinsp;+\u0026thinsp;iso-valerate); SEM, standard error of the means.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"16\"\u003e*Indicates differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) from control treatments, \u0026dagger; Indicates tendency from control treatment (P\u0026thinsp;\u0026le;\u0026thinsp;0.10).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCompared with the control (14.57 mL g\u003csup\u003e⁻1\u003c/sup\u003e DM), p-coumaric acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) methane yield by 23.2% at 5 mM, 42.5% at 10 mM, and 43.3% at 15 mM. Trans-cinnamic acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) methane yield by 27.5% at 15 mM, whereas 5 and 10 mM had no detectable effect. The combination of p-coumaric acid and trans-cinnamic acid reduced methane yield by 27.2% at 2.5 mM, 33.2% at 5 mM, and 41.4% at 7.5 mM. Gallic acid did not affect methane production. In vitro dry matter disappearance (IVDMD) was affected by treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Relative to the control (61.8%), p-coumaric acid reduced digestibility by 10.2% at 10 mM and 14.6% at 15 mM, whereas 5 mM had no detectable effect. The combination treatment reduced digestibility by 10.7% at 7.5 mM only. Trans-cinnamic acid and gallic acid did not affect digestibility. Fermentation pH, total VFA concentration, the percentages of acetate, propionate, butyrate and BCVFA of total volatile fatty acids were not (P\u0026thinsp;\u0026ge;\u0026thinsp;0.16) affected by treatment. The acetate-to-propionate ratio remained unchanged (P\u0026thinsp;=\u0026thinsp;0.86).\u003c/p\u003e \u003cp\u003eNet total volatile fatty acids, net acetate and net propionate production (24h \u0026minus;\u0026thinsp;0h) were not affected (P\u0026thinsp;\u0026ge;\u0026thinsp;0.52). Net butyrate production was affected by treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with p-coumaric acid at 15 mM reducing net butyrate by 18.9% relative to the control (6.77 mM). A tendency was observed for the combination treatment at 7.5 mM, which reduced net butyrate by 13.6% (P\u0026thinsp;\u0026le;\u0026thinsp;0.10). Net branched-chain volatile fatty acid production showed a tendency for treatment effects (P\u0026thinsp;=\u0026thinsp;0.10), although pairwise differences from the control were not detected at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the effects of commercial products on in vitro rumen fermentation characteristics after 24h of incubation. Total gas production was affected by tannic acid, with 4.05 mM reducing (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) gas production by 38.1% relative to the control (99.0 mL g\u003csup\u003e⁻1\u003c/sup\u003e DM). Methane concentration was reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by tannic acid by 21.5% at 0.81 mM, 28.6% at 1.22 mM, and 75.4% at 4.05 mM, while methane yield declined by 32.4%, 43.1%, and 85.0% at the same respective doses compared with the control (14.57 mL g\u003csup\u003e⁻1\u003c/sup\u003e DM). In vitro dry matter disappearance decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) with tannic acid by 8.1%, 12.3%, and 25.9% at 0.81, 1.22, and 4.05 mM, respectively, and Yi Chang Bao at 9% reduced digestibility by 9.2%. Fermentation pH was reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by N-carbamyl-L-glutamic acid at 15 mM by 3.6%. Total volatile fatty acid concentration declined (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by 32.8% with tannic acid at 4.05 mM. The percentage of acetate increased (P\u0026thinsp;=\u0026thinsp;0.02) with tannic acid at 0.41 mM and 1.22 mM by 7.1% and 10.2%, respectively, while propionate increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by 32.0% at 4.05 mM tannic acid. Butyrate percentage decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by 56.6% at 1.22 mM and 96.9% at 4.05 mM tannic acid, whereas Yi Chang Bao at 9% increased butyrate by 31.6%. Branched-chain volatile fatty acids were reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by up to 86.8% at 4.05 mM tannic acid, and the acetate-to-propionate ratio declined (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by 18.1% at this concentration. Net total volatile fatty acid production was reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by 41.9% at 4.05 mM tannic acid, with net acetate reduced by 38.3% and net branched-chain volatile fatty acids reduced by up to 86.8%, whereas net propionate was not affected (P\u0026thinsp;=\u0026thinsp;0.15).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of increasing concentrations of commercial compounds on in vitro batch culture fermentation characteristics after 24 h of incubation. Variables measured include total gas production (mL g⁻\u003csup\u003e1\u003c/sup\u003e DM incubated), methane concentration (%), methane production (mL g⁻\u003csup\u003e1\u003c/sup\u003e DM incubated), in vitro dry matter disappearance (IVDMD, %), fermentation pH, total volatile fatty acid (VFA) concentration and individual VFA concentrations (% of total VFA), net VFA production (24 h \u0026ndash; 0 h; mM), and acetate-to-propionate ratio (A:P).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"15\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eTannic acid, mM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eN-carbamyl-L-glutamic acid, mM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eYi Chang Bao, % of substrate DM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c13\" namest=\"c12\"\u003e \u003cp\u003eLiu Wei Suan 600, % of substrate DM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.05\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGas, mL g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61.3*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e101.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e85.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e85.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e95.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e98.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.46*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.42*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.59*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e15.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e15.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e15.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e14.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e, mL g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.85*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.29*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.19*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e13.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e14.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIVDMD, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.2*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e62.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e59.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e56.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e60.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e61.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.88*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e6.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal VFA, mM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64.6*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e94.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e91.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e92.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e90.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e88.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e101.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e103.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e3.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"15\" nameend=\"c15\" namest=\"c1\"\u003e \u003cp\u003ePercentages of individual VFA of total VFA, %\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetate (A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.0*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.7\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.8*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e64.4\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64.6\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e63.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e63.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e64.4\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e63.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionate (P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.3\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32.2*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e23.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e21.4\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e22.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e22.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.22*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.30*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e11.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e12.79*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e9.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e9.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCVFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.55*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.28*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.43*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.55\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRatio A:P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.04*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"15\" nameend=\"c15\" namest=\"c1\"\u003e \u003cp\u003eNet VFA production (24h-0h), mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal VFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e65.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e66.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e62.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e75.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e77.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e5.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetate (A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.3*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e44.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e42.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e43.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e41.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e39.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e49.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e49.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionate (P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e14.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e16.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.89*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.77*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e8.95*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e7.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e7.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCVFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.84*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.52*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.33*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"15\"\u003e**CH4, methane; BCVFA, branched chain volatile fatty acids (iso-butyrate\u0026thinsp;+\u0026thinsp;iso-valerate); SEM, standard error mean.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"15\"\u003e* Indicates differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) from control treatments, \u0026dagger; indicates tendency from control treatment (P\u0026thinsp;\u0026le;\u0026thinsp;0.10).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn vitro 24h batch culture incubations provide a practical screening approach for antimethanogenic additives, but they do not reproduce digesta passage, absorption and host-mediated hydrogen sinks, or longer-term microbial adaptation; therefore, responses observed here should be interpreted as short-term rumen microbial effects under controlled conditions\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong phenylpropanoid-derived metabolites, p-coumaric acid consistently reduced methane concentration and yield in a dose-dependent manner, while maintaining total gas production and VFA profiles at the lowest dose tested, indicating a selective inhibition of methanogenesis rather than a broad suppression of fermentation\u003csup\u003e13,14,20\u003c/sup\u003e. The decline in digestibility at \u0026ge;\u0026thinsp;10 mM suggests that higher antimicrobial pressure can extend beyond methanogens to fibrolytic populations, consistent with the known membrane-disruptive activity of phenolic compounds\u003csup\u003e6,7\u003c/sup\u003e. Trans-Cinnamic acid produced a weaker response, reducing methane yield only at 15 mM, which may reflect structural differences that influence antimicrobial potency\u003csup\u003e20\u003c/sup\u003e. The p-coumaric acid\u0026thinsp;+\u0026thinsp;trans-cinnamic acid combination reduced methane across all inclusion levels, with digestibility maintained at 2.5\u0026ndash;5 mM, supporting an additive effect at moderate doses\u003csup\u003e23\u003c/sup\u003e. Gallic acid did not reduce methane under these conditions, despite reports of antimethanogenic effects in other in vitro systems, highlighting the importance of compound structure, dose and experimental context\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWithin the commercial additives, tannic acid produced the greatest methane suppression but was accompanied by clear depression of overall fermentation at higher doses, including reduced gas production, digestibility and total VFA, indicating that methane inhibition arose largely from broad microbial inhibition rather than a targeted shift in hydrogen utilisation\u003csup\u003e22,27\u0026ndash;30\u003c/sup\u003e. Shifts in VFA percentages at the highest tannic acid dose occurred alongside reduced total VFA, reinforcing that fermentation was constrained rather than redirected\u003csup\u003e31\u003c/sup\u003e. N-carbamyl-L-glutamic acid did not affect methane or major fermentation endpoints in vitro, which is consistent with evidence that responses to this compound are more variable in vivo and may be mediated through systemic nitrogen metabolism rather than direct rumen microbial inhibition\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e. Yi Chang Bao and Liu Wei Suan 600 did not reduce methane in the batch culture system; medium-chain fatty acids and organic acids can influence rumen fermentation in other contexts, but microencapsulation and release characteristics may have limited immediate ruminal exposure within 24h\u003csup\u003e35\u0026ndash;37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present study evaluated whether selected phenolic compounds and commercial additives could reduce methane production without impairing overall ruminal fermentation. The results demonstrate that p-coumaric acid, trans-cinnamic acid, and their combination consistently reduced methane concentration and yield in a dose-dependent manner, with reductions of up to 43% in methane yield, while maintaining total gas production, total volatile fatty acid concentration, acetate-to-propionate ratio, and overall fermentation profile at lower inclusion levels. Importantly, p-coumaric acid at 5 mM reduced methane without affecting in vitro dry matter disappearance, indicating that methane mitigation can be achieved without compromising substrate degradation when applied at appropriate doses. In contrast, higher inclusion levels of p-coumaric acid and the combined treatment reduced digestibility, suggesting that excessive concentrations may impair fermentative efficiency. Gallic acid did not influence methane production or fermentation characteristics under the conditions evaluated.\u003c/p\u003e \u003cp\u003eAmong the commercial products, tannic acid produced the greatest methane suppression, reducing methane yield by up to 85%; however, this response was accompanied by marked reductions in gas production, digestibility, total volatile fatty acids, and shifts in fermentation end-products, indicating substantial inhibition of overall rumen activity at higher doses. N-carbamyl-L-glutamic acid, Yi Chang Bao, and Lui Wei Suan 600 showed limited or inconsistent effects on methane production and did not provide meaningful mitigation without affecting other fermentation parameters.\u003c/p\u003e \u003cp\u003eCollectively, these findings partially support the initial hypothesis. Certain phenolic compounds, particularly p-coumaric acid at moderate inclusion levels, reduced methane production without detrimental effects on overall fermentation, thereby meeting the criteria for effective mitigation. However, the hypothesis must be rejected for tannic acid at higher concentrations and for the remaining commercial products, as methane suppression was either accompanied by impaired fermentation or was not observed. These results highlight that effective methane mitigation requires a balance between inhibitory activity against methanogenesis and preservation of rumen fermentative function, with compound type and dose being critical determinants of this balance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe in vitro study was conducted at the University of Sydney (Sydney, NSW, Australia). Protocols and animals used in this study are cared for under the approval and guidance of The University of Sydney Animal Ethics Committee (Approved Protocol Number 2022/AE002180). Information provided in the manuscript complies with the essential recommendations for reporting of the ARRIVE guidelines.\u003c/p\u003e\n\u003ch3\u003eSubstrate preparation\u003c/h3\u003e\n\u003cp\u003eSubstrate was prepared by grinding lucerne pellets using a 1.0-mm particle-size sieve feed mill (Retsch ZM 200; Haan, NW, Germany). For each in vitro incubation, 500 mg DM of lucerne substrate was weighed into an ANKOM\u0026reg; bag (model F57) with three replicates per treatment and sealed. Each bag was placed into a 50 mL amber serum bottle. The lucerne pellets were sent to an accredited laboratory for analysis of chemical composition using AOAC methods. Results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDry matter content, chemical composition and dry matter digestibility of lucerne pellet used as the basal diet for in vitro batch culture incubations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDry matter \u0026ndash; as fed, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCrude Protein (CP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNeutral Detergent Fibre (NDF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAcid Detergent Fibre (ADF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCrude Fat (CF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eInorganic Ash\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNon-fibrous carbohydrates (NFC*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDry Matter digestibility (DMD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLucerne pellet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e58.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e*NFC\u0026thinsp;=\u0026thinsp;100 \u0026ndash; (CP\u0026thinsp;+\u0026thinsp;NDF+CF\u0026thinsp;+\u0026thinsp;Ash)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eTreatments\u003c/h3\u003e\n\u003cp\u003eEight treatments were tested, including three PSMs found abundantly in \u003cem\u003eBrassica rapa subsp. chinensis\u003c/em\u003e. 1) p-coumaric acid; 2) trans-cinnamic acid; and 3) Gallic acid. The combination of p-coumaric acid\u0026thinsp;+\u0026thinsp;trans-cinnamic acid (TCA\u0026thinsp;+\u0026thinsp;PCA) was also tested to verify additional (synergistic, additive or antagonistic) effect of these compounds. Additionally, four commercially available compounds,1) Tannic acid, 2) N-carbamyl-L-glutamic acid; 3) Yi Chang Bao; and 4) Liu Wei Suan 600 were tested. Treatments were prepared at varying concentrations to evaluate dose-dependent effects on ruminal fermentation, making a total of twenty-three treatments and a lucerne-only control. The complete list of treatments and their respective concentrations tested are listed on Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInclusion levels of plant secondary metabolites derived from Brassica rapa subsp. chinensis and commercial compounds evaluated in the in vitro batch culture incubation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLucerne substrate only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant secondary metabolites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep-Coumaric acid (PCA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5, 10, 15 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etrans-Cinnamic acid (TCA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5, 10, 15 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePCA\u0026thinsp;+\u0026thinsp;TCA (1:1 combination)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;+\u0026thinsp;2.5, 5\u0026thinsp;+\u0026thinsp;5, 7.5\u0026thinsp;+\u0026thinsp;7.5 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGallic acid (GA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5, 10, 15 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCommercial compounds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTannic acid (TA, 81% purity*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.41, 0.81, 1.22, 4.05 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN-carbamyl-L-glutamic acid (NCG)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5, 10, 15 mM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYi Chang Bao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5, 9% of substrate DM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiu Wei Suan 600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.5, 9% of substrate DM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e*Inclusion levels of tannic acid were corrected for 81% purity; values shown represent actual molar concentrations.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eYi Chang Bao (Guangdong Ruisheng Technology Group Co., Ltd., Guangdong, China) is a commercial medium-chain fatty acid (MCFA) blend containing caproic (C6), caprylic (C8), capric (C10), and lauric (C12) acids. The product is manufactured using microencapsulation technology to stabilise the fatty acids and allow gradual release in the gastrointestinal tract. Liu Wei Suan 600 (Guangdong Ruisheng Technology Group Co., Ltd., Guangdong, China) is a microencapsulated organic acid blend composed primarily of benzoic, fumaric, citric, formic, propionic, and lactic acids, together with plant essential oils. Both products are supplied as powder formulations. In the present study, Yi Chang Bao and Liu Wei Suan 600 were incorporated at 4.5% and 9% of the total substrate (500 mg DM) to evaluate dose-dependent effects on ruminal fermentation. Inclusion levels of PSM and N-carbamyl-L-glutamic acid were selected based on doses used in previous studies demonstrating effective CH\u003csub\u003e4\u003c/sub\u003e reduction\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e. All treatments were fine-powder additives and kept at the particle size commercially provided. Treatments were weighed using an analytical scale (AB54-S, Mettler Toledo, Greifensee, Switzerland) and transferred into the pre-prepared amber serum bottles containing substrate. Each treatment concentration was tested in triplicate (e.g., 3 serum bottles per treatment per incubation run).\u003c/p\u003e\n\u003ch3\u003eInoculum and incubation\u003c/h3\u003e\n\u003cp\u003eInoculum for the in vitro incubations was obtained from three ruminally fistulated cows (n\u0026thinsp;=\u0026thinsp;3) grazing pasture (15% crude protein; 64% neutral detergent fibre). Each cow was treated as an individual experimental unit. Rumen fluid was collected separately from each animal 2\u0026ndash;3 h post-feeding, filtered through a single layer of nylon\u0026ndash;elastane fabric to remove particulate matter, and immediately transferred into individual 1 L pre-warmed insulated thermos flasks for transport to the laboratory. Inoculum was prepared by mixing rumen fluid with a rumen solution, reducing agent, and resazurin, as described by Menke et al.\u003csup\u003e16\u003c/sup\u003e. Each 50 mL amber serum bottle was gassed with CO\u003csub\u003e2\u003c/sub\u003e to create an anaerobic environment, 25 mL of inoculum was transferred into the bottle, and the bottle was immediately fitted with a rubber stopper to contain the gases produced during fermentation. Each serum bottle was then placed into a shaking incubator set at 39\u003csup\u003eo\u003c/sup\u003eC and 120 oscillations per minute for 24h. The entire incubation was repeated three times.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of total gas, in vitro dry matter disappearance and pH\u003c/h2\u003e \u003cp\u003eAt 24h of incubation, 25 mL of headspace was sampled from each serum bottle with a syringe and immediately transferred into a 12 mL evacuated exetainer to ensure positive pressure (Labco Ltd., High Wycombe, United Kingdom) for CH\u003csub\u003e4\u003c/sub\u003e analysis. The remaining gas in each serum bottle was measured using a water-displacement apparatus\u003csup\u003e17\u003c/sup\u003e. Total gas production for each serum bottle was calculated as the sum of the volume measured using the water displacement apparatus and the 25 mL of headspace gas previously removed and expressed as mL gas g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM incubated. After 24h of incubation, and after gas was sampled and the volume measured, the fermentation serum bottles were opened and placed on ice to stop fermentation. The pH of each culture was measured using a pH probe inserted into the serum bottles (Activon Model 209, Gladesville, NSW, Australia). The pH probe was washed with distilled water between each serum bottle, using a wash bottle (LDPE Wash Bottle, ThermoFisher, Brisbane, NSW, Australia). The ANKOM\u0026reg; bags were then removed from the serum bottles and washed thoroughly using a top load washing machine for 10 minutes, twice, then dried at 55\u003csup\u003eo\u003c/sup\u003eC for 48h using forced air circulated oven (BMT Venticell 111, Cambridge Scientific, Watertown, MA, USA) and weighed to calculate in vitro dry matter disappearance (IVDMD).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of volatile fatty acids (VFA)\u003c/h3\u003e\n\u003cp\u003eThe liquid fraction of the fermentation at 0 and 24h incubation was subsampled from each serum bottle. 1.5 mL subsamples were transferred into 2 mL microcentrifuge tubes. Samples were centrifuged at 4,500 \u0026times; g for 15 min at 5\u003csup\u003eo\u003c/sup\u003eC to precipitate particulate matter and protein. A 1.2 mL sample of supernatant was transferred into 2 mL micro-centrifuge tubes containing 0.3 mL of metaphosphoric acid (0.25; w/v) and frozen at -20\u003csup\u003eo\u003c/sup\u003eC until analysed for VFA concentrations. The VFA analysis was performed as described by Forwood et al.\u003csup\u003e18\u003c/sup\u003e. The 0h samples were analysed for VFA to calculate the net total VFA production [24h \u0026ndash; 0h, mM]. The concentration of total VFA is expressed in mM, and individual VFA is expressed as % of total VFA.\u003c/p\u003e\n\u003ch3\u003eDetermination of CH\u003c/h3\u003e\n\u003cp\u003eMethane (CH₄) concentration was determined from the headspace gas collected in 12 mL evacuated exetainers. For each sample, 3 mL of exetainer gas was withdrawn using a gas-tight syringe and transferred into a 2 mL evacuated autosampler vial to maintain positive pressure and avoid atmospheric contamination. The autosampler vials were then loaded onto the gas chromatograph for analysis of CH₄ by GC\u0026ndash;FID. The Agilent model 7890a gas chromatograph with a flame ionisation detector (FID) calibrated to 250\u0026deg;C, airflow 300 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003e fuel flow 30 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, makeup flow (N\u003csub\u003e2\u003c/sub\u003e) 30 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e installed with a capillary column (Restek Rt-Q-Bond, 30 m \u0026times; 0.53 mm ID \u0026times; 20 \u0026micro;m). The Split-Splitless Inlet was heated to 60\u0026deg;C, 9.526 PSI, with a helium total flow of 33 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, septum purge flow of 3 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, split ratio of 5:1, split flow of 25 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and an oven temperature of 60\u0026deg;C. Quantification was performed using an external standard calibration curve prepared with CH₄ concentrations of 0, 5, 10 and 20% (v/v), yielding a linear response (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.98). Methane measurements are expressed as concentration (%), and production (mL CH\u003csub\u003e4\u003c/sub\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM incubated).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData from the in vitro fermentations were analysed as a completely randomised design using PROC MIXED in SAS (version 9.4; SAS Institute Inc., Cary, NC, USA). Plant secondary metabolites and commercial products were analysed in separate models to avoid confounding between treatment categories. Within each model, treatment (dose) was included as a fixed effect, and animal (rumen fluid donor; n\u0026thinsp;=\u0026thinsp;3) was included as a random effect. Incubation run (i.e., individual rumen fluid collection from each cow) served as the experimental unit and error term for assessing treatment effects on CH₄ production, fermentation parameters, and IVDMD. Treatment means were compared with the control using least-squares means with Dunnett adjustment. Statistical significance was declared at P\u0026thinsp;\u0026le;\u0026thinsp;0.05 and tendencies at 0.05\u0026thinsp;\u0026lt;\u0026thinsp;P\u0026thinsp;\u0026le;\u0026thinsp;0.10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data and the model are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMH: investigation, writing original draft, review \u0026amp; editing, visualisation, lab analysis. AVC: investigation, writing second draft, review \u0026amp; editing, visualisation, formal analysis, validation, methodology, conceptualisation, supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDCCEEW. \u003cem\u003eAustralia\u0026rsquo;s emissions projections 2024\u003c/em\u003e. Australian Government (2024).\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Mara, F. P. The significance of livestock as a contributor to global greenhouse gas emissions today and in the near future. \u003cem\u003eAnim. Feed Sci. Technol.\u003c/em\u003e \u003cstrong\u003e166\u0026ndash;167\u003c/strong\u003e, 7\u0026ndash;15 (2011).\u003c/li\u003e\n\u003cli\u003eCIE. \u003cem\u003eAustralian Agricultural Emissions Projections to 2025\u003c/em\u003e. The Centre for International Economics (2013).\u003c/li\u003e\n\u003cli\u003eKu-Vera, J. C. et al. Role of secondary plant metabolites on enteric methane mitigation in ruminants. \u003cem\u003eFront. Vet. 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Technol.\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 101\u0026ndash;116 (2004).\u003c/li\u003e\n\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plant secondary metabolites, phenolic compounds, enteric fermentation","lastPublishedDoi":"10.21203/rs.3.rs-8999036/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8999036/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnteric methane (CH₄) produced during ruminal fermentation is a major contributor to agricultural greenhouse gas emissions and represents an energy loss to the animal. Plant-derived phenolic compounds have been proposed as dietary strategies to mitigate methane formation; however, their efficacy depends on compound structure and inclusion level. This study evaluated phenylpropanoid-derived phenolics identified in \u003cem\u003eBrassica rapa\u003c/em\u003e subsp. \u003cem\u003echinensis\u003c/em\u003e and selected commercial additives for their ability to reduce methane without impairing ruminal fermentation. Lucerne pellets (500 mg dry matter) were incubated for 24h in an anaerobic in vitro batch culture system using rumen fluid from three pasture-fed fistulated cows (n\u0026thinsp;=\u0026thinsp;3). Treatments included p-coumaric acid (5, 10, 15 mM), trans-cinnamic acid (5, 10, 15 mM), their combination (2.5, 5, 7.5 mM each), gallic acid (5, 10, 15 mM), tannic acid (0.41, 0.81, 1.22, 4.05 mM), N-carbamyl-L-glutamic acid (5, 10, 15 mM), and two commercial formulations (4.5 and 9% of substrate dry matter). Total gas production, CH₄ concentration and yield, in vitro dry matter disappearance (IVDMD), fermentation pH, and volatile fatty acid (VFA) profiles were measured. p-Coumaric acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) CH₄ concentration and yield in a dose-dependent manner, with reductions up to 43% at 15 mM; however, IVDMD was reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) at \u0026ge;\u0026thinsp;10 mM. Trans-Cinnamic acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) CH₄ yield only at 15 mM. The combination of p-coumaric acid and trans-cinnamic acid reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) CH₄ production by up to 41% while maintaining IVDMD at 2.5\u0026ndash;5 mM. Gallic acid did not affect CH₄ (P\u0026thinsp;\u0026ge;\u0026thinsp;0.16). Tannic acid produced the greatest CH₄ suppression (up to 85%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but reduced gas production, IVDMD, and total VFA concentration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) at higher doses. N-carbamyl-L-glutamic acid and the two commercial formulations did not reduce CH₄ under these conditions (P\u0026thinsp;\u0026ge;\u0026thinsp;0.05). Moderate inclusion of p-coumaric acid (5 mM) alone or combined with trans-cinnamic acid (2.5 mM each) reduced methane without measurable adverse effects on fermentation, supporting further in vivo validation and dose optimisation for practical methane mitigation strategies.\u003c/p\u003e","manuscriptTitle":"Screening compounds of the phenylpropanoid pathway and commercial products to mitigate methane emissions using batch culture in vitro incubations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 22:27:09","doi":"10.21203/rs.3.rs-8999036/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T07:05:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T08:58:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T20:44:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210075261468868353371843972980296881167","date":"2026-03-20T05:21:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220323841885318337913537302067883574896","date":"2026-03-19T16:45:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-17T06:43:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-17T06:39:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-17T06:21:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T00:46:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-09T21:07:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a3f60ec-ac2b-48d6-a813-e2226fb52621","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64650401,"name":"Biological sciences/Biochemistry"},{"id":64650402,"name":"Biological sciences/Biotechnology"},{"id":64650403,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-23T07:10:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 22:27:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8999036","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8999036","identity":"rs-8999036","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}