Simple SummaryDespite studies conducted with other seaweed species on in vitro gas production, until now, no studies have been reported on the use of pelagicSargassumas potential ruminant feed. Thus, the objective of the present study was to determine the effect ofSargassuminclusion, using tropical grass as substrate, on in vitro gas production kinetics. Additionally, heavy metals and macro- and microminerals were determined inSargassum. For that, in vitro incubations were performed with different levels ofSargassuminclusion on a basal substrate (Stargrass hay). In vitro results showed that up to 30% pelagicSargassumcould be included in hay-based substrates from tropical grasses.AbstractThis study determined the effect of pelagicSargassumon in vitro dry matter and organic matter degradation, total gas production (TGP), and protozoa population. The treatments were different levels ofSargassuminclusion on a basal substrate (Stargrass hay;Cynodon nlemfuensis) as follows: T0 (control treatment based on Stargrass hay), T10 (90% Stargrass hay + 10%Sargassum), T20 (80% Stargrass hay + 20%Sargassum), and T30 (70% Stargrass hay + 30%Sargassum). Ruminal fermentation kinetics and protozoa population were determined during 72 h of in vitro incubations. Compared to control, dry matter degradability at 48 and 72 h and organic matter degradability at 24 and 48 h were higher inSargassumtreatments. TGP was lower with T20 at 48 h. The total population of protozoa and the concentration ofEntodiniumspp. were lower at T20 at 48 h and T30 at 72 h. Cl, S, Ca, K, and Zn (103, 5.97, 88.73, 285.70 g/kg, and 15,900 mg/kg) were high inSargassum, reaching twice or even nine times higher than the contents in Stargrass (11.37, 1.60, 43.53, 87.73 g/kg, and 866.67 mg/kg). Overall, up to 30% pelagicSargassumcould be included in hay-based substrates from tropical grasses without negative effects on in vitro dry matter and organic matter degradability.