Identification of Bioactive Phytochemicals from Six Plants: Mechanistic Insights into the Inhibition of Rumen Protozoa, Ammoniagenesis, and α-Glucosidase

Rumen protozoa prey on feed-degrading bacteria synthesizing microbial protein, lowering nitrogen utilization efficiency in ruminants. In this in vitro study, we evaluated six plants (Adansonia digitata, Flemingia macrophylla, Kalimeris indica,Brassica rapa subsp. chinensis, Portulaca oleracea, and Calotropis gigantea) for their potential to inhibit rumen protozoa and identified the phytochemicals potentially responsible for protozoa inhibition. Rumen protozoa were anaerobically cultured in vitro in the presence of each plant at four doses. All of the tested plants reduced total rumen protozoa (p ≤ 0.05), but C. gigantea and B. rapa were the most inhibitory, inhibiting rumen protozoa by 45.6 and 65.7%, respectively, at the dose of 1.1 mg/mL. Scanning electron microscopy revealed a disruption of the extracellular structure of protozoa cells. Only C. gigantea also decreased the wasteful ammoniagenesis (p ≤ 0.05). Moreover, the A. digitata extract inhibited α-glucosidase activity by about 70% at 100 µg/mL. Reversed-phase high-performance liquid chromatography analysis detected quercetin, anthraquinone, 3-hydroxybenzoic acid, astragaloside, and myricetin in the tested plant leaves. These plants may hold potential as feed additives to reduce rumen protozoa and α- glucosidase activity. Future research is needed to identify the specific anti-protozoal compound(s), the effects on the rumen microbiome, and its fermentation characteristics.

Identification of Bioactive Phytochemicals from Six Forest Plants: Insights into the Mechanistic Inhibition of Rumen Protozoa, Ammoniagenesis, and Α-Glucosidase

Rumen protozoa have a little contribution to the feed digestibility but Entodinium, the most predominant genus, is unfortunately culprit of the nitrogen utilization inefficiency. To bridge the gap, antibiotics have been used to inhibit the rumen protozoa but unfortunately, due to the health concern, this could not be really applied at the farm level, especially in the organic farms where the use of natural plants is an utmost priority. Therefore, our study aimed at investigating the nutritional and functional properties of six forest plants for their potential as feed additives in animal husbandry. The plants were analyzed for major phytochemicals using reversed phase-HPLC analysis and then evaluated for their in-vitro suppressing effect on rumen protozoa, ammoniagenesis, and microbial α-glucosidase activity. For each plant, four doses (0, 0.7, 0.9, and 1.1 mg/mL culture fluid) displayed in a complete randomized design were used. Quercetin, anthraquinone, 3-hydroxybenzoic acid, astragaloside, and myricetin were found to different extent (P ≤ 0.05) in the plant leaves. All the tested plants reduced total rumen protozoa counts but C. gigantea and B. rapa had the most inhibitory effect (P ≤ 0.05), inhibiting the rumen protozoa by 45.6 and 65.7%, respectively, at the dose of 1.1 mg/mL. Moreover, the scanning electron microscopy revealed the mechanistic disruption of the extracellular membrane of the protozoa, indicating their metabolic death pathway. Only C. gigantea inhibited the rumen protozoa in a proportion that also led to the reduction of the wasteful ammonia production (P ≤ 0.05). Besides, A. digitata and F. macrophylla have the higher inhibition rate (70%) of the microbial α-glucosidase activity at 100 µg/mL of crude extract. Overall, the plants showed promising results as functional feed additives although future research on bio-guided fractionation is needed to accurately identify the pure anti-protozoal bioactive compound(s).

Diet and breed alter community structures of rumen protozoa in cattle subjected to different feeding systems

The objective were to identify and quantify the rumen protozoa from two genetic groups of beef cattle (Nellore and crossbred European beef cattle) subjected to three feeding systems (pasture, pasture supplement, and confined). Samples of rumen contents were obtained from the center of the ruminal mass after slaughter. Quantification and identification of rumen protozoa were performed with a Sedgewick-Rafter counting chamber by optic microscopy. Data were analyzed using general linear analysis methodology with a Poisson distribution (1 %). In addition, the data underwent of Analisys Principal Component. A effect on ruminal protozoa was observed for the diets and breeds analyzed. Was identified 14 genera, among which Entodinium was predominant in all animals analyzed. Ciliates belonging to Entodiniomorphida, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium, and Ostracodinium showed highest density in Nellore animals when compared with the crossbreed, Nellore × European. Larger numbers of protozoa from the family Isotrichidae (Dasytricha and Isotricha) were observed in animals maintained by pasture or pasture supplement feeding. According to the kind of feeding, those fed exclusively with pasture had a greater diversity of ciliates in comparison to those restricted to, or supplemented with pasture. In one animal, the occurrence of ciliates belonging to the genus Buetschlia, was observed (prevalence of 1.66 %), which is one of only a few records of this genus in ruminants.