Abstract

Rumen fluid plays a crucial role in in vitro studies for evaluating ruminant feed. Maintaining microbial activity in rumen fluid can serve as a breakthrough approach to reducing dependence on fresh rumen fluid collection by utilizing sequential culture techniques. This study aimed to assess the effectiveness of rumen microbial inoculants through sequential cultures with a 48-hour incubation period. A completely randomized design was applied with four treatments: K1 = Culture 1 (inoculant derived from fresh rumen fluid), K2 = Culture 2 (inoculant derived from Culture 1), K3 = Culture 3 (inoculant derived from Culture 2), and K4 = Culture 4 (inoculant derived from Culture 3). The test substrates included dwarf elephant grass and Indigofera zollingeriana leaves using analysis in vitro sequential cultures adapted from Tilley and Terry (1963) and the Consecutive Batch Culture (CBC) method. Parameters measured included rumen fermentation characteristics such as pH, ammonia nitrogen (N-NH₃) concentration, total volatile fatty acid (VFA) production, and dry matter digestibility. Data were analyzed using analysis of variance (ANOVA) followed by Tukey’s HSD (Honest Significant Difference) test. The results showed that the sequential culture process significantly affected in vitro rumen fermentation characteristics. The pH remained stable within the optimal range (6.67–6.78). Increased culture sequences enhanced N-NH₃ concentration, total VFA production, and dry matter digestibility. It can be concluded that rumen microbial inoculants remain effective up to the fourth sequential culture for in vitro evaluation of ruminant feeds.