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Volume 15
Issue 12
10.3390/metabo15120762
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Rumen Fluid Metabolomics and Microbiome Profiling of Dairy Cows Fed Combinations of Prebiotics, Essential Oil Blend, and Onion Peel Using the RUSITEC System

by
Joel O. Alabi
1,
Ahmed E. Kholif
1,2,
Kelechi A. Ike
1,
Deborah O. Okedoyin
1,
Oludotun O. Adelusi
1,
Michael Wuaku
1,
Chika C. Anotaenwere
1,
James M. Enikuomehin
1,
Olatunde A. Oderinwale
1,
John O. Adebayo
1,
Andrea R. Gentry-Apple
1 and
Uchenna Y. Anele
1,*
1
Department of Animal Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC 27411, USA
2
Dairy Science Department, National Research Centre, 33 Bohouth St. Dokki, Giza 12622, Egypt
*
Author to whom correspondence should be addressed.
Metabolites 2025, 15(12), 762; https://doi.org/10.3390/metabo15120762
Submission received: 9 October 2025 / Revised: 19 November 2025 / Accepted: 20 November 2025 / Published: 25 November 2025
(This article belongs to the Special Issue Dysbiosis and Metabolic Disorders of the Microbiota)
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Abstract

Background and Objectives: Dairy products provide vital energy, high-quality protein, and micronutrients for over six billion people worldwide, with dairy cows contributing nearly 81% of global milk production. Sustainable strategies to enhance productivity are therefore critical. Feed additives such as essential oil blends (EOB), onion peel (OPE), and prebiotics including mannan oligosaccharides (MOS) and galacto-oligosaccharides (GOS) have been proposed to improve rumen fermentation, modulate microbial ecology, and mitigate greenhouse gas emissions. This study evaluated the combined effects of EOB, OPE, MOS, and GOS on rumen metabolism using the rumen simulation technique (RUSITEC). Materials and Methods: Rumen inoculum from three cannulated Holstein Friesian cows was incubated across 16 vessels (four treatments × four replicates) for nine days. Treatments included a control (CON; TMR only), GEO (TMR + GOS + EOB + OPE), MEO (TMR + MOS + EOB + OPE), and OLEO (TMR + a 1:1 mixture of GOS and MOS + EOB + OPE). Additives were included at 3 µL/g TMR for EOB and 30 mg/g TMR (3% w/w) for OPE, GOS, MOS, or OLG. Rumen effluents were collected for untargeted metabolomic profiling by liquid chromatography–mass spectrometry, identifying 661 metabolites. Results: Partial least squares-discriminant analysis revealed clear separation between CON and additive groups, confirming distinct metabolic shifts. GEO primarily enhanced tryptophan, tyrosine, and purine metabolism; MEO stimulated phosphonate and pyrimidine pathways and bile acid biosynthesis; OLEO promoted phosphonate, nicotinamide, and taurine metabolism. Microbial analysis showed enrichment of taxa such as Lachnospira, Succinivibrionaceae, Macellibacteroides, Lysinibacillus, and Christensenellaceae, indicating complementary effects on fermentation and microbial stability. Conclusions: These results demonstrate that dietary supplementation with GEO, MEO, or OLEO modulates rumen metabolism and microbial ecology without impairing fermentation, supporting improved nutrient utilization, antioxidant defenses, and metabolic resilience in dairy cows, with potential benefits for productivity and sustainability.
Keywords: dairy cows; dysbiosis; feed additives; metabolome; microbiota; RUSITEC dairy cows; dysbiosis; feed additives; metabolome; microbiota; RUSITEC
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MDPI and ACS Style

Alabi, J.O.; Kholif, A.E.; Ike, K.A.; Okedoyin, D.O.; Adelusi, O.O.; Wuaku, M.; Anotaenwere, C.C.; Enikuomehin, J.M.; Oderinwale, O.A.; Adebayo, J.O.; et al. Rumen Fluid Metabolomics and Microbiome Profiling of Dairy Cows Fed Combinations of Prebiotics, Essential Oil Blend, and Onion Peel Using the RUSITEC System. Metabolites 2025, 15, 762. https://doi.org/10.3390/metabo15120762

AMA Style

Alabi JO, Kholif AE, Ike KA, Okedoyin DO, Adelusi OO, Wuaku M, Anotaenwere CC, Enikuomehin JM, Oderinwale OA, Adebayo JO, et al. Rumen Fluid Metabolomics and Microbiome Profiling of Dairy Cows Fed Combinations of Prebiotics, Essential Oil Blend, and Onion Peel Using the RUSITEC System. Metabolites. 2025; 15(12):762. https://doi.org/10.3390/metabo15120762

Chicago/Turabian Style

Alabi, Joel O., Ahmed E. Kholif, Kelechi A. Ike, Deborah O. Okedoyin, Oludotun O. Adelusi, Michael Wuaku, Chika C. Anotaenwere, James M. Enikuomehin, Olatunde A. Oderinwale, John O. Adebayo, and et al. 2025. "Rumen Fluid Metabolomics and Microbiome Profiling of Dairy Cows Fed Combinations of Prebiotics, Essential Oil Blend, and Onion Peel Using the RUSITEC System" Metabolites 15, no. 12: 762. https://doi.org/10.3390/metabo15120762

APA Style

Alabi, J. O., Kholif, A. E., Ike, K. A., Okedoyin, D. O., Adelusi, O. O., Wuaku, M., Anotaenwere, C. C., Enikuomehin, J. M., Oderinwale, O. A., Adebayo, J. O., Gentry-Apple, A. R., & Anele, U. Y. (2025). Rumen Fluid Metabolomics and Microbiome Profiling of Dairy Cows Fed Combinations of Prebiotics, Essential Oil Blend, and Onion Peel Using the RUSITEC System. Metabolites, 15(12), 762. https://doi.org/10.3390/metabo15120762

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