60f93627d9c4ab319347fa45dc25f4d2 animals-15-02105-v2.pdf 5923d39dc86e43af1dec1c372a7e27d58118a950 animals-15-02105-v2.pdf ab35e7e886dff2e00fc4c32ac3f23afe2e94e5d566944278ba4aa05f5995135b animals-15-02105-v2.pdf Title: Optimizing Essential Oil Mixtures: Synergistic Effects on Cattle Rumen Fermentation and Methane Emission Subject: Ruminant livestock contribute significantly to methane emissions, necessitating sustainable mitigation strategies. Essential oils (EOs) show promise for modulating ruminal fermentation, but their synergistic effects remain underexplored. Two 24 h in vitro experiments evaluated the synergistic effects of EO blends on rumen microbial fermentation. Exp. 1 screened five oils using two triad combinations. Triad 1 tested 10 combinations of thyme (THY), peppermint (PPM), and cinnamon leaf (CIN) oils. Triad 2 tested 10 combinations of anise (ANI), clove leaf (CLO), and peppermint (PPM) oils. Each blend was tested at 400 mg/L, using batch culture methods measuring: pH, ammonia-N (NH3-N), and volatile fatty acids (VFAs). The two most effective blends, designated as T1 and T2, were selected for Exp. 2 to assess total gas and methane (CH4) production using pressure transducer methods. All treatments were incubated in a rumen fluid–buffer mix with a 50:50 forage-to-concentrate substrate (pH 6.6). In Exp. 1, data were analyzed according to the Simplex Centroid Design using R-Studio. In Exp. 2, an analysis was conducted using the MIXED procedure in SAS. Mean comparisons were assessed through Tukey’s test. The results from Exp. 1 identified CIN+PPM (80:20) and ANI+CLO (80:20) as optimal combinations, both increasing total VFAs while reducing acetate/propionate ratios and NH3-N concentrations. In Exp. 2, both combinations significantly reduced total gas and CH4 productions compared to the control, with CIN+PPM achieving the greatest methane reduction (similar to monensin, the positive control). Specific essential oil combinations demonstrated synergistic effects in modulating rumen fermentation and reducing methane emissions, offering potential for sustainable livestock production. Further in vivo validation is required to optimize dosing and assess long-term effects on animal performance. Keywords: essential oils; feed additives; methane mitigation; rumen fermentation; synergistic effects Author: Memoona Nasir, María Rodríguez-Prado, Marica Simoni, Susana M. Martín-Orúe, José Francisco Pérez and Sergio Calsamiglia Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Thu Jul 17 10:25:51 2025 CEST ModDate: Thu Jul 17 10:28:13 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 27 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2151707 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DAYYKT+URWPalladioL-Ital Type 1 Custom yes yes yes 155 0 FAVYLD+URWPalladioL-Bold Type 1 Custom yes yes yes 156 0 MIKRSI+VnURWPalladioL Type 1 Custom yes yes yes 157 0 PXGSIS+URWPalladioL-Roma Type 1 Custom yes yes yes 158 0 OSPFSW+CMSY10 Type 1 Builtin yes yes yes 277 0 GIFGLH+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 270 0 GIFGKF+PalatinoLinotype-Bold CID TrueType Identity-H yes 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application/pdf PDFMetadata: Objects: 876 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Experimental Design and Ethics Statement Item: Title: Experiment 1: Optimizing Fermentation Characteristics Through Targeted EO Synergy Analysis Children: Item: Title: Experimental Treatments and Incubation Substrate Item: Title: In Vitro System Item: Title: Rumen Fluid Collection Item: Title: The in Vitro Procedure for Evaluating Rumen Microbial Fermentation Item: Title: Sample Collection Item: Title: Experiment 2 (Optimizing Total Gas and CH4 Output Using Targeted EO Combinations) Children: Item: Title: Experimental Treatments and Incubation Substrate Item: Title: The in Vitro Procedure for Evaluating Total Gas and CH4 Production and Kinetics Item: Title: Gas Measurements and Analysis Item: Title: Statistical Analysis Item: Title: Results Children: Item: Title: Experiment 1 (Screening the Best Combination for an Optimum Rumen Fermentation) Children: Item: Title: Combination Triad 1 (Different Proportions of THY, PPM, and CIN EOs) Item: Title: Combination Triad 2 (Different Proportions of ANI, CLO, and PPM EOs) Item: Title: Experiment 2 (Total Gas and Methane Production) Item: Title: Discussion Children: Item: Title: Experiment 1 (Rumen Microbial Fermentation) Children: Item: Title: Individual Efficacy: Baseline Performance of Essential Oils Item: Title: Enhanced Efficacy: Synergistic Outcomes of Essential Oil Combinations Item: Title: Experiment 2 (Total Gas and Methane Production) Item: Title: Conclusions Item: Title: References Info: Title: Optimizing Essential Oil Mixtures: Synergistic Effects on Cattle Rumen Fermentation and Methane Emission Author: Memoona Nasir, María Rodríguez-Prado, Marica Simoni, Susana M. Martín-Orúe, José Francisco Pérez and Sergio Calsamiglia Subject: Ruminant livestock contribute significantly to methane emissions, necessitating sustainable mitigation strategies. Essential oils (EOs) show promise for modulating ruminal fermentation, but their synergistic effects remain underexplored. Two 24 h in vitro experiments evaluated the synergistic effects of EO blends on rumen microbial fermentation. Exp. 1 screened five oils using two triad combinations. Triad 1 tested 10 combinations of thyme (THY), peppermint (PPM), and cinnamon leaf (CIN) oils. Triad 2 tested 10 combinations of anise (ANI), clove leaf (CLO), and peppermint (PPM) oils. Each blend was tested at 400 mg/L, using batch culture methods measuring: pH, ammonia-N (NH3-N), and volatile fatty acids (VFAs). The two most effective blends, designated as T1 and T2, were selected for Exp. 2 to assess total gas and methane (CH4) production using pressure transducer methods. All treatments were incubated in a rumen fluid–buffer mix with a 50:50 forage-to-concentrate substrate (pH 6.6). In Exp. 1, data were analyzed according to the Simplex Centroid Design using R-Studio. In Exp. 2, an analysis was conducted using the MIXED procedure in SAS. Mean comparisons were assessed through Tukey’s test. The results from Exp. 1 identified CIN+PPM (80:20) and ANI+CLO (80:20) as optimal combinations, both increasing total VFAs while reducing acetate/propionate ratios and NH3-N concentrations. In Exp. 2, both combinations significantly reduced total gas and CH4 productions compared to the control, with CIN+PPM achieving the greatest methane reduction (similar to monensin, the positive control). Specific essential oil combinations demonstrated synergistic effects in modulating rumen fermentation and reducing methane emissions, offering potential for sustainable livestock production. Further in vivo validation is required to optimize dosing and assess long-term effects on animal performance. 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213, 399, 251, 410 Annotation: Subtype: Link Rect: 224, 361, 262, 371 Annotation: Subtype: Link Rect: 59, 323, 96, 333 Checksum: 5f8b997a Type: CRC32 Checksum: 60f93627d9c4ab319347fa45dc25f4d2 Type: MD5 Checksum: 5923d39dc86e43af1dec1c372a7e27d58118a950 Type: SHA-1 Checksum: ab35e7e886dff2e00fc4c32ac3f23afe2e94e5d566944278ba4aa05f5995135b Type: SHA-256