Abstract
Background:
Tropical seaweeds show promise as methane-reducing feed additives; however, optimal levels of supplementation remain undefined.

Aim: This study aimed to determine the optimal dose of three tropical seaweed species (brown, green, and red) for suppressing the production of enteric methane. Furthermore, the study evaluated their impact on nutrient digestibility and rumen fermentation characteristics using in vitro methods.

Methods:
The experiment followed a 3 × 5 factorial, completely randomized design (CRD). Factor A consisted of the following seaweed species: brown (Sargassum binderi), green (Kappaphycus striatum), and red (Gracilaria sp.). Factor B represents the supplementation levels: 0%, 2.5%, 5%, 7.5%, and 10% of the substrate. Parameters measured included nutrient digestibility, total gas and methane production, and rumen fermentation profiles (pH, NH₃, microbial protein synthesis, protozoa populations, and VFA profiles).

Results:
In vitro supplementation with tropical seaweeds significantly reduces methane production, although the optimal dose is species-specific. At a 5% inclusion rate, brown seaweed emerged as the most effective candidate for methane mitigation, reducing methane production to 11.21 mL/g DM (a 30.1% decrease) while maintaining the highest organic matter digestibility (74.44%). Conversely, low doses of green seaweed (2.5%–5%) increased methane (+35.7% to +41.2%); however, a 7.5% dose optimized mitigation, lowering methane to 10.72 mL/g DM (a 33.1% decrease) while preserving peak crude protein digestibility (74.09%). Red seaweed at 7.5% reduced methane production (13.51 mL/g DM, a 15.7% decrease) and enhanced microbial protein synthesis by 41.7% and total VFA production by 86.5%. All optimal doses maintained the rumen pH within physiological ranges (7.02–7.29).

Conclusion:
The optimal inclusion levels for methane mitigation are species-dependent: Sargassum binderi (5%), Kappaphycus striatum (7.5%), and Gracilaria sp. (7.5%). These findings provide a scientific basis for developing local seaweed-based feed supplements. However, further in vivo validation is required to confirm their efficacy and safety for sustainable, low-emission dairy production.

Key words: Feed supplementation; In vitro; Methane mitigation; Tropical seaweed.