Background and Aim: Ipomoea aquatica (Water Spinach) is the most potential for livestock growth performance, including chickens, pigs, cattle, and goats, especially in a tropical country like Cambodia. It is not only an alternative feed source but also one kind of supplemented feed for goat raising. Supplementation with Water Spinach in the utilization of low-quality tree foliage results in an increase in dry matter intake in goat production. This study aimed to identify the effectiveness of supplementation of Water Spinach in the utilization of Mimosa pigra and Leucaena leucocephala leaf in in vitro fermentation.

Materials and Methods: The study was designed according to a 2 × 2 factorial arrangement in randomized design of seven treatments with different ratios consisted of different three types of dietary treatments, including M. pigra, L. leucocephala, and Water Spinach. The treatments were arranged according to a completely randomized design and were as follow: T1 = M. pigra leaf (100%); T2 = L. leucocephala leaf (100%); T3 = M. pigra leaf and L. leucocephala leaf (50% and 50%); T4 = M. pigra leaf and Water Spinach (99.5% and 0.5%); T5 = L. leucocephala leaf and Water Spinach (99.5% and 0.5%); T6 = M. pigra leaf and Water Spinach (99% and 1%); and T7 = L. leucocephala leaf and Water Spinach (99% and 1%). A total of 200 mg (dry matter) of dietary treatments were prepared in a 60 mL syringe. Each treatment was replicated 3 time. Gas recording of each treatment lasted for 3 days. In vitro was performed for 72 h, was followed by Makkar method. Gas production was recorded at 2, 4, 8, 12, 24, 36, 48, and 72 h of incubation by using strict anaerobic technique. A mixture of rumen fluid and dietary treatments were carried out under continuous flushing with CO₂ in sharking incubator at 39°C. After incubating for 72 h, the ammonia concentration (NH₃-N) was measured and recorded to identify pH, nutrient digestibility, and ammonia concentration (NH₃-N).

Results: Nutrient digestibility of the treatment with Water Spinach supplement in the utilization of L. leucocephala was obtained at a higher digestibility than treatment with M. pigra (p < 0.05). Gas production was different between groups (p < 0.05). Treatment with only M. pigra leaf had the highest gas production (A), while treatment with Water Spinach supplementation had the lowest gas production (A). At 0–24 h, the treatment with L. leucocephala leaf and Water Spinach 0.5% had the highest gas production, but after 24 h, M. pigra leaf and Water Spinach 1% and L. leucocephala leaf and Water Spinach 0.5% produced more gas compared to the other treatments (p < 0.05).

Conclusion: The supplementation of Water Spinach 1% in treatment with M. pigra and L. leucocephala leaf resulted in increased degradability, gas production, and NH₃-N concentration without a change in the pH value rumen condition. Based on these results, it is recommended that the level of Water Spinach supplementation should be 1% of dietary intake. Future studies should consider investigating the rumen ecology associated with Water Spinach supplementation. Feeding with Water Spinach remains a good supplement for ruminant performance; therefore, further studies should be conducted using Water Spinach in ruminant feeding in both metabolic and feeding trials.

Keywords: In vitro fermentation, Leucaena leucocephala leaf, Mimosa pigra, Nutrient digestibility, Supplementation, Utilization, Water Spinach (Ipomoea Aquatica).