Skip to main content Skip to main navigation menu Skip to site footer
Original article
Austral J. Vet. Sci.
Vol 58, e5809 (2026)
DOI: 10.4206/ajvs.58.09

Influence of seaweed species and harvest season on in vitro rumen fermentation and methane production with a pasture-based substrate

1 Instituto de Investigaciones Agropecuarias, INIA Remehue, Osorno, Chile.
2 Instituto de Producción Animal, Facultad de Ciencias Agrarias y Alimentarias, Universidad Austral de Chile, Valdivia, Chile.
3 Centro i-mar, Núcleo Milenio MASH & CeBiB, Universidad de Los Lagos, Puerto Montt, Chile.
4 Facultad de Ciencias Veterinarias, Instituto de Ciencia Animal, Universidad Austral de Chile, Valdivia, Chile.
5 Instituto de Investigaciones Agropecuarias, INIA Tamel Aike, Coyhaique, Chile.
6 Facultad de Zootecnia y Ecología, Universidad Autónoma de Chihuahua, Chihuahua, México.
Keywords: ammonia enteric methane greenhouse gas macroalgae phlorotannins rumen microbiota

Submitted: 2025-12-16

Accepted: 2026-06-08

Published: 2026-07-15

*Corresponding author:
ignacio.beltran@inia.cl

How to Cite

Beltran, I., Keim, J. P., Buschmann, A., Pereda, S., Salazar, F., Martinez, E., Saldivia, M., Pulido, R., Daza, J., & Vargas-Bello-Pérez, E. (2026). Influence of seaweed species and harvest season on in vitro rumen fermentation and methane production with a pasture-based substrate. Austral Journal of Veterinary Sciences, 58, e5809. https://doi.org/10.4206/ajvs.58.09

License

Copyright (c) 2026 Ignacio Beltran, Juan Pablo Keim, Alejandro Buschmann, Sandra Pereda, Francisco Salazar, Emilio Martinez, Marcelo Saldivia, Ruben Pulido, Jose Daza, Einar Vargas-Bello-Pérez

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

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.

Downloads

Download data is not yet available.

References

  1. 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
  2. AOAC. (1996). Official methods of analysis of AOAC International. AOAC International.
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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.
  11. Goering, H. K., & Van Soest, P. J. (1970). Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). U.S. Agricultural Research Service.
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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
  23. 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
  24. 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
  25. 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
  26. 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
  27. 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
  28. 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
  29. 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
  30. 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
  31. 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
  32. 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
  33. 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
  34. 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
  35. 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
  36. 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
  37. 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
  38. 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
  39. 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
  40. 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
  41. 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
  42. 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

Make a Submission

Make a Submission

Journal Metrics (2025) & Ranking

JOURNAL METRICS & RANKING (2025)

Impact Factor (2025) 0.5
5 years Impact Factor 1.0
JCR Quartile Q4
JIF Rank 146/170 (Veterinary Sciences)
SJR (2025) 0.208

Indexed in

Publisher


This journal is published by the Facultad de Ciencias Veterinarias, Universidad Austral de Chile

Keywords