The effect of Macleaya cordata extract on in vitro ruminal fermentation and methanogenesis

Abstract Methane production is the main gas energy loss in ruminants and generates a powerful greenhouse gas that contributes to climate changes. Macleaya cordata is a plant commonly utilized additive in livestock diet as it contains various chemical compounds with beneficial health effects. This study aimed to explore the influence of M. cordata extract on in vitro methanogenesis and rumen fermentation. Three cannulated Jingjiang cattle were used as rumen fluid donors. The mixture of rumen fluid and a buffer (60 ml, 1:2 volume ratio) was treated with 6 different concentrations of M. cordata extract (0.01%, 0.11%, 0.21%, 0.31%, 0.41%, and 0.51%) and incubated for 12 and 24 hr. The control sample, with no addition of plant extract, was also analyzed. At each time point, pH, total gas, methane, dry matter (DM) digestibility, neutral detergent fiber (NDF), acid detergent fiber (ADF), ammonia nitrogen (NH3‐N), microbial protein (MCP), and volatile fatty acids (VFA) concentrations were determined. Total gas production decreased with increasing the amount of M. cordata extract at all time points. Methane production also decreased dose‐dependently with M. cordata extract after 3, 6, 9, and 12 hr of incubation, but increased after 24 hr. M. cordata extract decreased the concentration of NH3‐N and VFA, and the amount of acetic, propionic, and butyric acid. M. cordata extract decreased the MCP concentration after 12 hr, but its level returned to the initial value after 24 hr. Supplementation with 0.01, 0.11, and 0.21% of M. cordata extract did not affect the DM digestibility. However, supplementation with 0.31, 0.41, and 0.51% of M. cordata extract significantly decreased the DM digestibility. Supplementation with 0.11% of M. cordata extract effectively reduced methane production without affecting the DM digestion in vitro. However, its effect on in vivo methane production, rumen fermentation, and ruminant production requires further investigation.


| INTRODUC TI ON
The growing amount of greenhouse gases such as methane in the atmosphere poses a huge risk to global warming, as its warming potential is 25-fold higher than carbon dioxide (Gill et al., 2010). Therefore, there is an urgent need for the reduction in methane in the atmosphere. The unique digestion metabolism of ruminants produces methane, and this process represents an energy loss as 2%-15% of dietary intake is utilized in methanogenesis (Ellis et al., 2007;Johnson & Johnson, 1995). Therefore, the reduction in methanogenesis in ruminants is an important goal for environment protection and the development of more efficient farming technologies.
However, few studies focused on in vitro effect of M. cordata on ruminal fermentation metabolism and gas production. Therefore, this study aimed to explore the influence of M. cordata extract added in the livestock diet on in vitro fermentation parameters such as total gas production, methane production, dry matter (DM) disappearance, neutral detergent fiber (NDF) content, acid detergent fiber (ADF) content, ammonia nitrogen (NH 3 -N) concentration, microbial protein (MCP) concentration, and volatile fatty acids (VFA) composition.

| Animal care
All experiments conducted in this study were done following the national legislation and good scientific practices, and the procedures were approved by the Animal Ethics Committee of the Jiangxi Academy of Sciences.

| Macleaya cordata extract
Macleaya cordata extract was obtained from Hunan Micolta Bioresource Inc. in China. The M. cordata extract is a solid powder with 40% sanguinarine and 20% chelerythrine and is stored in a dark and dry place.

| In vitro fermentation design
The rumen fluid was taken from three cannulated Jingjiang cattle (250 ± 15 kg) that were provided with ad libitum sources of water, minerals, and vitamins. The cattle were fed with ryegrass and concentrated in the ratio of 60:40 twice daily at 09:00 and 17:00, equivalent to 2% of body weight. The rumen fluid was collected before morning feeding. The rumen fluid was stored in a bottle, previously kept warm and filled with O 2 -free CO 2 gas, carried to the laboratory, and filtered through four layers of cheesecloth before mixing with artificial saliva (1:2) maintained at 39℃ and under O 2 -free N 2 gas.
The following six dietary regimens were applied: control (CON), basal diet without M. cordata extract; treatment 1 (TM1), basal diet with 0.01% M. cordata extract; treatment 2 (TM2), basal diet with 0.11% M. cordata extract; treatment 3 (TM3), basal diet with 0.21% M. cordata extract; treatment 4 (TM4), basal diet with 0.31% M. cordata extract; treatment 5 (TM5), basal diet with 0.41% M. cordata extract; and treatment 6 (TM6), basal diet with 0.51% M. cordata extract (amount of M. cordata extract was relative to substrate). The rumen fluid-buffer mixture (60 ml) was transferred anaerobically into a 150ml serum bottle containing 0.5 g of substrate (Table 1) and M. cordata extract, filled with O 2 -free N 2 gas, and then capped with a rubber stopper. The serum bottles were then incubated with shaking (100 rpm/ min) at 39℃ for 12 hr and 24 hr. The in vitro fermentation experiment was a completely randomized block design with three runs and five replicates per run. Each run was conducted on separate weeks.

| Gas production measurement and ruminal fermentation profiles
Upon completion of incubation, total gas production was quantified according to the method of Theodorou et al. (1994) pressure transducer and a digital readout voltmeter were used to measure the headspace gas pressure of fermenting cultures after removing serum bottles from a shaking incubator. The transducer was connected to the inlet of a disposable Luer-lock three-way stopcock in order to quantify the total gas production in this system. Gas pressure in the headspace was read from the display unit after insertion of the hypodermic syringe needle through the rubber stopper above the culture medium. The amount of methane in a headspace gas above the culture medium was analyzed using gas chromatography (GC-2010, Shimadzu). The culture medium was analyzed for pH (FE-28, Mettle-Toledo), VFA concentration, NH 3 -N, and MCP concentration. The VFA analysis was performed with a gas chromatography (GC-2010, Shimadzu) as described method by Stewart and Duncan (Stewart & Duncan, 1985). The UV/Vis spectroscopy (Model 680, Bio-Rad laboratories) was applied for the quantification of NH 3 -N and MCP following the procedure described by Wang et al. (2008) and Wang (2003).
The rate of DM disappearance was determined using the nylon bag digestion method in vitro. After incubation, the nylon bags with substrate were rinsed with cold water until the water ran clear, dried at 60℃ until constant weight, weighed, and subsequently analyzed for DM digestibility.

| Statistical analysis
The data analysis was performed using one-way analysis of variance (ANOVA) using SPSS version 16.0 software. To test the effect of M. cordata extract on total gas production, methane production, pH, DM disappearance, and VFA profiles, the results were compared with control experiment, and the statistical significance between the results was obtained by applying Duncan's multiple comparison tests. A p < .05 was considered to be statistically significant.

| Gas production kinetics
The effects of M. cordata extract dose on total gas production and methane emission are shown in Table 2. Total gas production in samples supplemented with 0.11%, 0.21%, 0.31%, 0.41%, and 0.51% of M. cordata extract was lower than the value for CON (p < .05).

| In vitro ruminal fermentation characteristics
The effects of M. cordata extract dose on pH and DM disappearance are shown in Table 3. The pH of the fermentation medium at

| Volatile fatty acid profile and acetic acid/ propionic acid ratio
The outcome of supplementation with M. cordata extract on the content of VFA in rumen fluid such as acetic, propionic, butyric acid, and acetic acid/propionic acid ratio (A/P) is shown in Table 4.
The total VFA concentration, and the level of acetic, propionic, and butyric acid were significantly reduced upon 12-hr incubation with M. cordata extract (p < .05). In vitro experiments revealed a positive correlation between the gas production and digestibility of organic matter (Menke & Steingass, 1988 study. Moreover, the extracts enriched with alkaloids are recognized as a promising feed additive to reduce gas production during ruminal fermentation (Santos et al., 2013).

| D ISCUSS I ON
The addition of M. cordata extract to the diet significantly decreased methane production after 3, 6, 9, and 12 hr of incubation, but the gas production increased after prolonged incubation (24 hr).
This trend might be explained by the inhibitory effect of M. cordata extract on methanogens that was most pronounced at the beginning, and gradually disappeared in the next 12 hr, After 12 hr, the activity of methanogens increased rapidly, which resulted in the in-  VFA is the main source of energy in ruminants, and their content and composition directly reflect the level of metabolic activity in the rumen (Lee et al., 2018a(Lee et al., , 2018b. In the present study, the addition of M. cordata extract steadily decreased the total VFA, acetic acid, propionic acid and butyric acid concentration, and A/P ratio. This result is not consistent with the literature (Zhang et al., 2017), and the observed discrepancy could be ascribed to different dosage regimens used in two studies. Moreover, the results showed that M. cordata extract had a significant effect on pH. Nevertheless, the pH TA B L E 4 The effect of M. cordata extract on total volatile fatty acids (VFA), acetic acid, propionic acid, butyric acid, and acetic acid/ propionic acid ratio (A/P) according to incubation time value remains within the range 6.37-7.35, which is an appropriate pH range for cellulose digestion (6.0-6.8), protein synthesis (6.3-7.4), proteolytic activity (6.5-7.0), VFA production (6.0-6.6), and ruminal microbial activity (5.8-7.2; Hiltner & Dehority, 1983;McCullough et al., 1969). Compared with the control group, the DM disappearance did not change significantly when M. cordata extract was added at 0.01%, 0.11%, and 0.21%, while DM digestibility decreased when the dosage was 0.31%, 0.41%, and 0.51%. The results of our study suggest that the supplementation of the diet with more than 0.31% of M. cordata extract might reduce the rumen fermentation and DM digestion.

| CON CLUS ION
The present study shows that the addition of 0.11% M. cordata extract in diet decreases methane production, VFA, NH 3 -N, and MCP production, without affecting the DM digestibility. These results suggest that supplementation with 0.11% of M. cordata extract decreases methanogenesis without negative impact on ruminal fermentation and could be used as a potential additive for ruminants.
Additional research is required to evaluate the effects of M. cordata extract on in vivo ruminal methane production, fermentation, and microbiome, which is required for this extract to be used as a food additive in ruminant production.

CO N FLI C T O F I NTE R E S T
The authors confirm that there is no conflict of interests regarding this paper.

ETHICAL APPROVAL
The study protocol was approved by the Animal Ethics Committee of the Jiangxi Academy of Sciences.