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

Abstract

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 °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.

Metabolite Profile, Ruminal Methane Reduction, and Microbiome Modulating Potential of Seeds of Pharbitis nil

“…The fluid was then diluted with O 2 -free buffer ( McDougall, 1948 ; adjusted to pH 7.0) at a ratio of 1:2 (v/v) and placed in a water bath pre-heated to 39°C with continuous CO 2 flushing. The in vitro trial was performed as described in Bharanidharan et al (2021a) to test the effect of ground PA. Briefly, incubation was carried out with three replicates with each comprising 30 mL of rumen fluid mixture in 60-mL serum bottles containing 210 mg DM of substrate.…”

“…After 24 h of incubation, the total gas production was measured using water displacement apparatus ( Fedorah and Hrudey, 1983 ). CH 4 concentration in the headspace gas and VFAs in the medium were determined using the Agilent 7890B GC system (Agilent Technologies, Santa Clara, CA, USA) with a flame ionization detector using methods described by Bharanidharan et al (2021a) . The pH was measured using a pH meter (model AG 8603; Seven Easy pH, Mettler-Toledo, Schwerzenbach, Switzerland) and the NH 3 -N concentration was determined using a modified colorimetric method ( Chaney and Marbach, 1962 ).…”

“…Samples of rumen head space gas were taken at 1, 2, and 3 h after morning feeding by passing a 20-mL graduated syringe connected to a two-way stopcock (KOVAX, Seoul, South Korea) into the rumen through the cannula stopper. Six samples (three each during rumen contraction and relaxation) per sampling period were collected and immediately transferred to a vacuum tube (ref 364979, BD Vacutainer, Becton Dickinson, NJ, United States) for CH 4 analysis as described previously ( Bharanidharan et al, 2021a ). Ruminal fluid samples were collected on days −1, 3, 11, and 21 at 3.5 h after morning feeding.…”

“…Our previous in vitro screening study involving 152 plant extracts reported that ethanolic extracts from the seeds of Pharbitis nil (Pharbitis semen; PA) can decrease CH 4 by up to 37% ( Bharanidharan et al, 2021a ). Pharbitis nil (Convolvulaceae) is an annual climbing herb widely distributed throughout Korea, Japan, and China, and PA is used as a purgative agent and in treating digestive disorders ( Kim et al, 2020 ).…”

“…Previous phytochemical investigations have reported the isolation of bioactive chemical constituents such as resin glycosides ( Ono, 2017 ), monoterpenoids ( Lee et al, 2017 ), diterpenoids ( Ki et al, 2009 ; Woo et al, 2017 ), triterpenoid saponins ( Jung et al, 2008 ), flavonoids, chlorogenic acid derivatives ( Saito et al, 1994 ), and phenolic compounds ( Kim et al, 2011 ) from different parts of P. nil . Similarly, we reported the enrichment of PUFAs in PA using gas chromatography-mass spectroscopy (GC-MS) without derivatization ( Bharanidharan et al, 2021a ). However, a complete secondary metabolite and fatty acid (FA) profile of PA is needed to understand their effects on methanogenesis and fermentation.…”