In Vitro Screening of East Asian Plant Extracts for Potential Use in Reducing Ruminal Methane Production

Abstract

Simple Summary Methane from ruminants is a major contributor to total greenhouse gases. Therefore, ruminant nutritionists have proposed strategies to mitigate methane emissions, such as chemical inhibitors and ionophores. However, dietary manipulation including natural feed additives is more practical, considering consumer preferences. Therefore, the current experiment screened 137 plant species, indigenous to East Asian countries, to select novel anti-methanogenic candidates as natural feed additives. Among these species, an extract from the seeds of Pharbitis nil exhibited a maximum 37% reduction of methane in a conformation assay. Identification of active compounds present in the seeds of Pharbitis nil revealed enrichment of polyunsaturated fatty acids, which were dominated by linoleic acid (18:2). The extract had negative effects on the populations of ciliated protozoa and H-2-producing Ruminococcus flavefaciens, thereby increasing the proportion of propionate, similar to the effect of monensin. This is the first report to suggest that the seeds of P. nil could be a promising anti-methanogenic alternative to ionophores or oil seeds. Indiscriminate use of antibiotics can result in antibiotic residues in animal products; thus, plant compounds may be better alternative sources for mitigating methane (CH4) production. An in vitro screening experiment was conducted to evaluate the potential application of 152 dry methanolic or ethanolic extracts from 137 plant species distributed in East Asian countries as anti-methanogenic additives in ruminant feed. The experimental material consisted of 200 mg total mixed ration, 20 mg plant extract, and 30 mL diluted ruminal fluid-buffer mixture in 60 mL serum bottles that were sealed with rubber stoppers and incubated at 39 degrees C for 24 h. Among the tested extracts, eight extracts decreased CH4 production by >20%, compared to the corresponding controls: stems of Vitex negundo var. incisa, stems of Amelanchier asiatica, fruit of Reynoutria sachalinensis, seeds of Tribulus terrestris, seeds of Pharbitis nil, leaves of Alnus japonica, stem and bark of Carpinus tschonoskii, and stems of Acer truncatum. A confirmation assay of the eight plant extracts at a dosage of 10 mg with four replications repeated on 3 different days revealed that the extracts decreased CH4 concentration in the total gas (7-15%) and total CH4 production (17-37%), compared to the control. This is the first report to identify the anti-methanogenic activities of eight potential plant extracts. All extracts decreased ammonia (NH3-N) concentrations. Negative effects on total gas and volatile fatty acid (VFA) production were also noted for all extracts that were rich in hydrolysable tannins and total saponins or fatty acids. The underlying modes of action differed among plants: extracts from P. nil, V. negundo var. incisa, A. asiatica, and R. sachalinensis resulted in a decrease in total methanogen or the protozoan population (p < 0.05) but extracts from other plants did not. Furthermore, extracts from P. nil decreased the population of total protozoa and increased the proportion of propionate among VFAs (p < 0.05). Identifying bioactive compounds in seeds of P. nil by gas chromatography-mass spectrometry analysis revealed enrichment of linoleic acid (18:2). Overall, seeds of P. nil could be a possible alternative to ionophores or oil seeds to mitigate ruminal CH4 production.