Abstract
Background:
Indonesia, a tropical country with rich biodiversity, is home to numerous native plant species, including the betel leaf. Several varieties of betel leaf are found across the country, such as green betel (Piper betle Linn.), red betel (Piper ornatum) and yellow betel (Piper betle Linn. var aureum). Despite their widespread use in traditional practices, little research has examined the bioactive compounds in each type of betel leaf. Studies exploring their potential as feed additives for ruminants also remain limited.

Aim:
This study aimed to determine the optimal inclusion rate and to characterize the bioactive compound profiles of green, red and yellow betel leaves when used as feed additives for ruminant livestock.

Methods:
An in vitro experiment was carried out using a completely randomized 3 × 5 factorial design with three replicates. The first factor was betel leaf type (green, red and yellow) and the second factor was the inclusion dose (0%, 2.5%, 5%, 7.5% and 10%). The basal diet consisted of a 60:40 ratio of forage and concentrate. Parameters measured included total gas and methane gas production, rumen protozoa population, protein digestibility, microbial protein synthesis (MPS), NH₃ concentration, partial VFA profile (acetate, propionate, n-butyrate, iso butyrate, isovaleric acid, and n-valerate) and the bioactive compound composition of the betel leaves.

Results:
The supplementation of green, red or yellow betel leaves significantly influenced total gas and methane gas production, rumen protozoa population, protein digestibility, MPS, NH₃ concentration and partial VFA (P < 0.01). Morphological differences among the three betel leaf types were associated with variations in secondary metabolite composition. These differences resulted in different optimal inclusion doses.

Conclusion:
The findings indicate optimal inclusion doses of 7.5% for green betel leaf, 5% for red betel leaf and 2.5% for yellow betel leaf. The optimal dose is based on the most favorable balance between methane reduction and maintenance of rumen fermentation performance. MPS, protein digestibility, NH3 and partial VFA indicated this.

Key words: Betel leaf; Methane; Microbial protein; Partial VFA; Secondary metabolite.