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

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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.