Influence of seaweed species and harvest season on in vitro rumen fermentation and methane production with a pasture-based substrate
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Abstract
This study aimed to evaluate the effects of Chilean brown and red seaweeds on in vitro rumen fermentation, methane (CH4) production, and bacterial community structure using a pasture-based substrate. Seven treatments were tested in an in vitro fermentation system using rumen fluid collected from Holstein-Friesian cows under a randomised block design, including three seaweed species (brown: Lessonia spicata [Ls] and Macrocystis pyrifera [Mp]; red: Gracilaria chilensis [GCh]), two harvest seasons (winter [WIN] and summer [SUM]), and a control treatment consisting of freeze-dried permanent pasture and concentrate mixed at an 80:20 ratio. Seaweeds were included at 10% of dietary dry matter, replacing part of the concentrate fraction of the substrate. Gas pressure was measured at 2, 4, 6, 8, 10, 12, 24, 36, and 48 h; CH4 was measured at 4, 24, and 48 h; and ammonia (NH3) and short-chain fatty acids (SCFA) were analysed at 4 and 48 h. Contrasts were used to evaluate the control treatment vs. seaweed treatments (CTR-SW), comparisons between brown seaweed species (L. spicata vs. M. pyrifera; LS-MP), comparison between the red seaweed GCh and brown seaweed Mp (G. chilensis vs. M. pyrifera; GCH-MP), and winter vs. summer harvest seasons (WIN-SUM), where sentence-case abbreviations (e.g., Mp, Ls, and GCh) refer to seaweed species and uppercase abbreviations denote the planned contrasts. Compared with the control treatment (CTR-SW), seaweed treatments reduced NH3 concentration at 48 h (P = 0.01) and CH4 production at 24 and 48 h (P < 0.01). Among the brown seaweeds (LS-MP), Mp resulted in 19% and 13% lower NH3 than Ls and GCh, respectively, and the lowest total gas and CH4 production (P < 0.01). Relative to GCh (GCH-MP), Mp increased propionate concentration and reduced the acetate:propionate ratio by 12.9% (P < 0.01). Although the control treatment (CTR-SW) showed higher total SCFA and nutrient digestibility (P < 0.01), harvest season (WIN-SUM) did not affect fermentation or CH4 production (expressed as a percentage of total gas and mL g OM-1). Denaturing Gradient Gel Electrophoresis Analysis revealed distinct bacterial clustering for Mp (winter) and Ls (both seasons). Overall, Mp reduced in vitro ruminal CH4 and NH3 concentrations; however, these effects were accompanied by a decrease in digestibility, indicating a trade-off between mitigation and fermentation efficiency. Under the evaluated conditions, none of the seaweeds reduced CH4 or NH3 without negatively affecting fermentation, thereby limiting their applicability as mitigation strategies.
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References
Almassri, N., Trujillo, F. J., Klieve, A. V., Bell, R., Ying, D., & Shiferaw Terefe, N. (2025). Microencapsulation of β-Glucosidase in Alginate Beads for Post-Rumen Release in Ruminant Gut. Bioengineering, 12(12), 1341. https://doi.org/10.3390/bioengineering12121341
AOAC. (1996). Official methods of analysis of AOAC International. AOAC International.
Beltran, I. E., Gregorini, P., Daza, J., Balocchi, O. A., Morales, A., & Pulido, R. G. (2019). Diurnal Concentration of Urinary Nitrogen and Rumen Ammonia Are Modified by Timing and Mass of Herbage Allocation. Animals, 9(11), 961. https://doi.org/10.3390/ani9110961
Camus, C., Hernández-González, M. d. C., & Buschmann, A. H. (2019). The seaweed resources of Chile over the period 2006–2016: moving from gatherers to cultivators. Botanica Marina, 62(3), 237-247. https://doi.org/10.1515/bot-2018-0030
De Bhowmick, G., & Hayes, M. (2023). Potential of Seaweeds to Mitigate Production of Greenhouse Gases during Production of Ruminant Proteins. Global Challenges, 7(5), 2200145. https://doi.org/10.1002/gch2.202200145
de la Moneda, A., Carro, M. D., Weisbjerg, M. R., Roleda, M. Y., Lind, V., Novoa-Garrido, M., & Molina-Alcaide, E. (2019). Variability and Potential of Seaweeds as Ingredients of Ruminant Diets: An In Vitro Study. Animals, 9(10), 851. https://doi.org/10.3390/ani9100851
Durmic, Z., Duin, E. C., Bannink, A., Belanche, A., Carbone, V., Carro, M. D., Crüsemann, M., Fievez, V., Garcia, F., Hristov, A., Joch, M., Martinez-Fernandez, G., Muetzel, S., Ungerfeld, E. M., Wang, M., Yáñez-Ruiz, D. R. (2025). Feed additives for methane mitigation: Recommendations for identification and selection of bioactive compounds to develop antimethanogenic feed additives. Journal of Dairy Science, 108(1), 302-321. https://doi.org/10.3168/jds.2024-25045
Ferreres, F., Lopes, G., Gil-Izquierdo, A., Andrade, P. B., Sousa, C., Mouga, T., & Valentão, P. (2012). Phlorotannin Extracts from Fucales Characterized by HPLC-DAD-ESI-MSn: Approaches to Hyaluronidase Inhibitory Capacity and Antioxidant Properties. Marine Drugs, 10(12), 2766-2781. https://doi.org/10.3390/md10122766
France, J., Dijkstra, J., Dhanoa, M. S., Lopez, S., & Bannink, A. (2007). Estimating the extent of degradation of ruminant feeds from a description of their gas production profiles observed in vitro:derivation of models and other mathematical considerations. British Journal of Nutrition, 83(2), 143-150. https://doi.org/10.1017/S0007114500000180
Garrido, O., & Mann, E. (1981). Composición Química, Digestibilidad y Valor Energético de una pradera permanente de pastoreo a través del año [Unpublished undergraduate thesis]. Universidad Austral de Chile, Valdivia, Chile.
Goering, H. K., & Van Soest, P. J. (1970). Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). U.S. Agricultural Research Service.
Grossi, G., Goglio, P., Vitali, A., & Williams, A. G. (2018). Livestock and climate change: impact of livestock on climate and mitigation strategies. Animal Frontiers, 9(1), 69-76. https://doi.org/10.1093/af/vfy034
Jerez-Timaure, N., Pulido, R., Heinsohn, F., Mendoza, J., Fuentes, J., Brintrup, M., & Berkhoff, M. (2023). Effects of the inclusion of brown seaweed (Macrocystis pyrifera) additive in the diet of grass-fed steers on carcass performance, meat quality, and nutrient composition. Austral Journal of Veterinary Sciences, 55, 95-103. https://doi.org/10.4067/S0719-81322023000200095
Kaplan, A. (1969). The Determination of Urea, Ammonia, and Urease. In Methods of Biochemical Analysis (pp. 311-324). https://doi.org/10.1002/9780470110355.ch7
Kim, Y. R., Park, K. Y., Ghassemi Nejad, J., Yoon, W. J., Kim, S. C., Lee, J. S., & Lee, H. G. (2022). Rumen methane abatement by phlorotannin derivatives (phlorofucofuroeckol-A, dieckol, and 8,8′-bieckol) and its relationship with the hydroxyl group and ether linkage. Animal Feed Science and Technology, 293, 115468. https://doi.org/10.1016/j.anifeedsci.2022.115468
Klieve, A. V., O'Leary, M. N., McMillen, L., & Ouwerkerk, D. (2007). Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet. Journal of Applied Microbiology, 103(6), 2065-2073. https://doi.org/10.1111/j.1365-2672.2007.03492.x
Kocherginskaya, S. A., Aminov, R. I., & White, B. A. (2001). Analysis of the Rumen Bacterial Diversity under two Different Diet Conditions using Denaturing Gradient Gel Electrophoresis, Random Sequencing, and Statistical Ecology Approaches. Anaerobe, 7(3), 119-134. https://doi.org/10.1006/anae.2001.0378
Leyton, A., Pezoa-Conte, R., Barriga, A., Buschmann, A. H., Mäki-Arvela, P., Mikkola, J. P., & Lienqueo, M. E. (2016). Identification and efficient extraction method of phlorotannins from the brown seaweed Macrocystis pyrifera using an orthogonal experimental design. Algal Research, 16, 201-208. https://doi.org/10.1016/j.algal.2016.03.019
Manoni, M., Gschwend, F., Amelchanka, S., Terranova, M., Pinotti, L., Widmer, F., Sliacci, P., & Tretola, M. (2024). Gallic and Ellagic Acids Differentially Affect Microbial Community Structures and Methane Emission When Using a Rumen Simulation Technique. Journal of Agricultural and Food Chemistry, 72(49), 27163-27176. https://doi.org/10.1021/acs.jafc.4c06214
Martínez, E. D., Turnbull, K. E., Quigley, S. P., Streeter, S. J., Swain, A., Klieve, A. V., Ouwerkerk, D., & Poppi, D. P. (2012). Liquid-phase denaturant gradient gel electrophoresis profiles of rumen bacteria from Brahman cross steers selected into two groups on the basis of post-weaning liveweight gain on low crude protein pasture. Animal Production Science, 52(7), 647-652. https://doi.org/10.1071/AN11234
McSweeney, C., Kang, S., Gagen, E., Davis, C., Morrison, M., & Denman, S. (2009). Recent developments in nucleic acid based techniques for use in rumen manipulation. Revista Brasileira de Zootecnia, 38. https://doi.org/10.1590/S1516-35982009001300034
Mihaila, A. A., Glasson, C. R. K., Lawton, R., Muetzel, S., Molano, G., & Magnusson, M. (2022). New temperate seaweed targets for mitigation of ruminant methane emissions: an in vitro assessment. Applied Phycology, 3(1), 274-284. https://doi.org/10.1080/26388081.2022.2059700
Mora, B., Calabrò, S., Cutrignelli, M. I., Barbier, E., Haroutounian, S., Yanza, Y. R., Jayanegara, A., Torrent, A., Vastolo, A., Hoste, H., & Niderkorn, V. (2025). Screening of mediterranean agro-industrial by-products rich in phenolic compounds for their ability to modulate in vitro ruminal fermentation. Italian Journal of Animal Science, 24(1), 1478-1488. https://doi.org/10.1080/1828051X.2025.2527717
Muyzer, G., de Waal, E. C., & Uitterlinden, A. G. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology, 59(3), 695-700. https://doi.org/10.1128/aem.59.3.695-700.1993
Olate-Gallegos, C., Barriga, A., Vergara, C., Fredes, C., García, P., Giménez, B., & Robert, P. (2019). Identification of Polyphenols from Chilean Brown Seaweeds Extracts by LC-DAD-ESI-MS/MS. Journal of Aquatic Food Product Technology, 28(4), 375-391. https://doi.org/10.1080/10498850.2019.1594483
Pacheco, D., & Waghorn, G. C. (2008). Dietary nitrogen - definitions, digestion, excretion and consequences of excess for grazing ruminants. NZ Grassland association, 70. https://doi.org/10.33584/jnzg.2008.70.2738
Pandey, D., Hansen, H. H., Dhakal, R., Aryal, N., Rai, S. P., Sapkota, R., Nielsen, M. O., Novoa-Garrido, M., & Khanal, P. (2022). Interspecies and seasonal variations in macroalgae from the Nordic region: Chemical composition and impacts on rumen fermentation and microbiome assembly. Journal of Cleaner Production, 363, 132456. https://doi.org/10.1016/j.jclepro.2022.132456
Park, K. Y., Jo, Y. H., Ghassemi Nejad, J., Lee, J. C., & Lee, H. G. (2022). Evaluation of nutritional value of Ulva sp. and Sargassum horneri as potential eco-friendly ruminants feed. Algal Research, 65, 102706. https://doi.org/10.1016/j.algal.2022.102706
Ramin, M., Franco, M., Roleda, M. Y., Aasen, I. M., Hetta, M., & Steinshamn, H. (2019). In vitro evaluation of utilisable crude protein and methane production for a diet in which grass silage was replaced by different levels and fractions of extracted seaweed proteins. Animal Feed Science and Technology, 255, 114225. https://doi.org/10.1016/j.anifeedsci.2019.114225
Robles-Jimenez, L. E., Ghavipanje, N., Ulloa, A., Rivero, A., Gallardo, P., & Gonzalez Ronquillo, M. (2024). Sub-Antarctic Macroalgae as Feed Ingredients for Sustainable Ruminant Production: In Vitro Total Gas and Methane Production. Methane, 3(3), 456-465. https://doi.org/10.3390/methane3030026
Stiger-Pouvreau, V., Bourgougnon, N., & Deslandes, E. (2016). Chapter 8 - Carbohydrates From Seaweeds. In J. Fleurence & I. Levine (Eds.), Seaweed in Health and Disease Prevention (pp. 223-274). Academic Press. https://doi.org/10.1016/B978-0-12-802772-1.00008-7
Tala, F., Velásquez, M., Mansilla, A., Macaya, E. C., & Thiel, M. (2016). Latitudinal and seasonal effects on short-term acclimation of floating kelp species from the South-East Pacific. Journal of Experimental Marine Biology and Ecology, 483, 31-41. https://doi.org/10.1016/j.jembe.2016.06.003
Tavendale, M. H., Meagher, L. P., Pacheco, D., Walker, N., Attwood, G. T., & Sivakumaran, S. (2005). Methane production from in vitro rumen incubations with Lotus pedunculatus and Medicago sativa, and effects of extractable condensed tannin fractions on methanogenesis. Animal Feed Science and Technology, 123-124, 403-419. https://doi.org/10.1016/j.anifeedsci.2005.04.037
Tilley, J. M. A., & Terry, R. A. (1963). A TWO-STAGE TECHNIQUE FOR THE IN VITRO DIGESTION OF FORAGE CROPS. Grass and Forage Science, 18(2), 104-111. https://doi.org/10.1111/j.1365-2494.1963.tb00335.x
Ungerfeld, E., Muñoz, C., Urrutia, N., Ávila, J., & Keim, J. P. (2025). Research on enteric methane mitigation in Chile: Ten years of scientific insights. Austral Journal of Veterinary Sciences, 57(1), e5710. https://doi.org/10.4206/ajvs.57.10
van der Weerden, T. J., Luo, J., de Klein, C. A. M., Hoogendoorn, C. J., Littlejohn, R. P., & Rys, G. J. (2011). Disaggregating nitrous oxide emission factors for ruminant urine and dung deposited onto pastoral soils. Agriculture, Ecosystems & Environment, 141(3), 426-436. https://doi.org/10.1016/j.agee.2011.04.007
Van Soest, P. J., Robertson, J. B., & Lewis, B. A. (1991). Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. Journal of Dairy Science, 74(10), 3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2
Véliz, K., Toledo, P., Araya, M., Gómez, M. F., Villalobos, V., & Tala, F. (2023). Chemical composition and heavy metal content of Chilean seaweeds: Potential applications of seaweed meal as food and feed ingredients. Food Chemistry, 398, 133866. https://doi.org/10.1016/j.foodchem.2022.133866
Villaseñor-Parada, C., Pauchard, A., Ramírez, M. E., & Macaya, E. C. (2018). Macroalgas exóticas en la costa de Chile: patrones espaciales y temporales en el proceso de invasión. Latin American Journal of Aquatic Research, 46, 147-165. https://doi.org/10.3856/vol46-issue1-fulltext-15
Vissers, A. M., Pellikaan, W. F., Bouwhuis, A., Vincken, J. P., Gruppen, H., & Hendriks, W. H. (2018). Laminaria digitata phlorotannins decrease protein degradation and methanogenesis during in vitro ruminal fermentation. Journal of the Science of Food and Agriculture, 98(10), 3644-3650. https://doi.org/10.1002/jsfa.8842
Wang, J., Chang, G., Liu, H., Yin, Z., Liu, P., Zhao, Y., Li, K., & Gao, T. (2024). Carbon balance analysis of agricultural production systems in oasis areas. Scientific Reports, 14(1), 16698. https://doi.org/10.1038/s41598-024-66972-4
Yu, Z., & Forster, R. J. (2005). Nucleic acid extraction, oligonucleotide probes and PCR methods. In H. P. S. Makkar & C. S. McSweeney (Eds.), Methods in Gut Microbial Ecology for Ruminants (pp. 81-104). Springer Netherlands. https://doi.org/10.1007/1-4020-3791-0_7
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