Flavonoids and their aromatic derivatives in Piper betle powder promote in vitro methane mitigation in a variety of diets

At present, there is little information regarding whether supplementation with Piper betle powder (PBP) and sunflower oil (SFO) has a synergistic effect on lowering methane emissions without negatively impacting ruminal fermentation. This study investigated the effects of PBP, supplemented either with or without SFO, on biogas release, fermentation end-products, and microorganisms in the rumen of lactating goats. The treatments were run in a completely randomized 3 × 5 factorial arrangement, whereby 0, 15, and 30 mg SFO were combined with 0, 15, 30, 45, and 60 mg PBP on a dry matter basis. The outcomes were assessed in vitro . PBP was obtained from the perennial plant Piper betle L., which is an abundant source of flavonoids and their aromatic derivatives. SFO, which reduces dietary methane emissions, was supplemented to confirm whether it interacted with other nutrients in the ruminant diet. SFO × PBP significantly (p < 0.05) decreased methane production, enhanced total volatile fatty acid concentrations, and decreased the number of rumen protozoa. We found that 15–30 mg, but not 45–60 mg, PBP combined with 0, 15, and 30 mg SFO increased (p < 0.05) total gas production (including CO 2 ) from fermentation. However, our results suggested that at least 45 mg PBP, either alone or combined with SFO, was required to reduce ammonia-N (p < 0.05). Not all treatments affected rumen pH. In conclusion, supplementing PBP (< 30 mg), either alone or combined with SFO, has a suppressing effect on methane production while preserving an optimum rate of rumen fermentation.


INTRODUCTION
Methane accounts for approximately 14% of greenhouse gases worldwide; this gas is emitted by ruminants under anaerobic conditions during ruminal fermentation (Sejian et al., 2011). As a result, there has been global interest in suppressing methane emissions without deleterious consequences on feed utilization (Yanza et al., 2018). Providing feed materials such as plant polyphenols and vegetable oils was expected to alter rumen inhabitants, resulting in improved rumen fermentation characteristics while reducing methane production since these supplementations have well-documented antimicrobial effects (Hook;Wright;McBride, 2010). Despite the considerable benefits of supplementing plant polyphenols and vegetable oils from a fermentation perspective, these rumen modifiers could modulate other nutrients in the ruminant diet (Lourenço et al., 2014;Elghandour et al., 2017;Purba;Paengkoum, 2020). Notably, sunflower oil (SFO) has been suggested as a cost-effective way to alleviate methane production and provide concurrent nutrients such as fat inclusions (Vargas et al., 2017).
Piper betle L. is a part of the Piperaceae family and has been traditionally used raw as a natural antiseptic for humans, particularly for cleaning their mouths. P. betle leaves have protein, fat, carbohydrate, mineral, and other contents of about 3-3.5%, 0.4-1.0%, 0.5-6.10%, 2.3-3.3%, and 86.5-93.5% respectively, of total nutrients in dry matter (Guha, 2006). Further, these leaves contain a host of polyphenols, especially flavonoids and essential oils (Purba;. Polyphenols originating from an extracted assay or a whole plant could be considered methane inhibitors as they have been shown to decrease enteric methane production (Jayanegara;Leiber;Kreuzer, 2012;Vasta;Daghio;Cappucci, 2019;Purba;Yuangklang;Paengkoum, 2020). However, there were adverse responses to feed digestion and ruminal fermentation when plant polyphenols were dosed at exceedingly high thresholds to obtain optimal methane mitigation Yu, 2013;Vasta;Daghio;Cappucci, 2019). n detrimental cases, animals experienced toxicity resulting in impaired ruminant productivity and health (Waghorn;McNabb, 2003). In contrast, the above disastrous effects can be avoided at a lower threshold, but the methane mitigation would be decreased as well Yu, 2015). A combination of a low amount of methane inhibitors, such nitrate and saponin, has been reported to decrease enteric methane production while maintaining feed digestion or substrate fermentation Yu, 2013). At present, however, it is unknown whether supplementation with Piper betle powder (PBP) and SFO has a synergistic effect and can boost animal performance. We postulated that PBP could alter the ruminal fermentation pathway. Therefore, the objective of this study was to investigate the effects of PBP at five different doses, either with or without SFO, on biogas production, fermentation end-products, and microbial composition in rumen fluids from lactating goats, as estimated by in vitro techniques.

MATERIAL AND METHODS
All experimental procedures were approved and carried out in accordance with the Rules of Animal Welfare and all research on animals was conducted according to the Institutional Committee on Animal Use (SUT 4/2558).

Animal, feed and Piper betle L. powder
Four multiparous Saanen goats, Capra aegagrus hircus, (43 ± 1.29 kg body weight) were assigned as rumen inoculum donors. All goats received a total mixed ration (TMR) consisting of a 50:50 mix of Pangola hay (particle size > 4 cm) and concentrate, and had free access to drinking water. The TMR was offered in two portions (60% at 09:00 and 40% at 17:00) and this feed was dedicated as a basal substrate in the later in vitro incubation. The formulation and chemical analysis of the feed (substrate) are presented in Table 1.
P. betle leaves were purchased from a local market in the Prachinburi area of east Thailand. Fresh biomass of leafy plant material was pooled, rinsed, and kept overnight at 4 °C. The leaves were air-dried using an oven set at 40 °C for 2 days, turned into a powder (hereafter referred to as P. betle powder, PBP), loaded in sealed plastic, and kept in a desiccator until used. Flavonoid and aromatic contents were extracted and assayed in water, methanol, and hexane. The quantification procedure was performed using a highperformance liquid chromatography machine with a diodearray detector (HPLC-DAD Agilent Technologies 1260 Infinity, USA and Canada) (Purba; Paengkoum, 2019). Briefly, 5 g of PBP were extracted using 20 mL solvents individually and then run in a Soxhlet apparatus for 3-4 hours. The extracts were finally filtered through 0.45 µm polyvinyl difluoride syringe filters and subsequently diluted with mobile phase solution (1:9, HPLC-grade acetonitrile:1% acetic acid). We injected 20 µL of diluted extract into the HPLC-DAD machine, in which the extract was set in a 10-mm flow cell with 100 loops of automatic sample injection and four solvent delivery system quaternary pumps. Separation was achieved using a reversed-phase Zorbax SB-C18 column (3.5 µm particle size, i.d. 4.6 × 250 mm) (Agilent Technologies). A standard stock solution was prepared using commercial quercetin and eugenol (Sigma-Aldrich, St. Louis, MO). Data collection was performed using OpenLAB CDS v1.8.1 (Agilent Technologies). All measurements were performed in triplicate and chemical standards were included in each analytical run as appropriate (Table 1).

In vitro experiment
After a 15-day adaptation period, rumen fluids were suctioned from the rumen using an oral lavage solution and a suction pump (CV-SF18, Hitachi, Tokyo, Japan) before morning feeding time (Tian et al., 2018). The pH of the rumen fluids were strictly checked to avoid contamination from goat saliva (Menke;Steingass, 1988). Rumen fluid was moved to the laboratory (14°52'36''N, 102°00'54''E; elevation above 200 m) in a pre-warmed thermal flask, then strained using a nylon membrane (400 µm; Fisher Scientific S.L., Madrid, Spain) while bubbled with CO 2 . The artificial solution was prepared per an earlier protocol (Menke;Steingass, 1988) and mixed with strained rumen liquors (2:1, volume: volume) under continuous CO 2 at 39 °C. Every hundred glass syringes containing the earlier SFO and PBP as treatment were combined with 500 mg of basal substrate. For instance, the control treatment contained 500 mg of basal substrate, 0 mg of SFO, and 0 mg of PBP. We then added 30 mL of rumen mixture as a final preparation prior to incubation. Once the glass syringes were locked with 3-way stopcocks and capped by glass plungers, the glass syringes were subsequently shaken and placed in a water bath set at 39 °C. The incubation was run for 72 h, including shaking once per hour. To get representative results, all incubations were completed in 10 replications over three runs on separate days and gas production was corrected with blanks (three glass syringes that only contained rumen mixture were included in each run).
using a forced-air oven at 105 °C for 4 h (AOAC, 2005). Organic matter (OM) content was calculated as a hundred percent minus ash percentage, which was obtained after incineration in a muffle furnace at 550 °C for 5 h (#942.05) (AOAC, 2005). Total N was measured using the Kjeldahl method and crude protein concentration was calculated as total N × 6.25 (#984.13), (AOAC, 2005). Ether extract concentration was measured by extraction with petroleum ether (#920.39) (AOAC, 2005) and fatty acid concentration was calculated from methylation using a gas chromatography machine (Agilent 7890A GC, Agilent Technologies) with external FAME standards (Supelco 37-Component FAME Mix, Supelco Inc., Bellefonte, PA) (Lourenço et al., 2014). Concentrations of acid detergent fiber and neutral detergent fiber were measured by sequential analysis without amylase (we used sodium sulfite instead) and expressed with residual ash included (Van Soest; Robertson; Lewis, 1991). The gross energy was determined using a bomb calorimeter with an O 2 carrier (Parr 6200 bomb calorimeter, Parr Instruments Co., Moline, IL) according to the manufacturer's instructions. All measurements were performed in triplicate and chemical standards were included in each analytical run as appropriate (Table 1). Gas production was read, recorded, and measured at 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, and 72 h by adapting a prior gas pressure technique (Theodorou et al., 1994). To calculate the cumulative volume of gas production (Equaton 1), the numerical measurement was fitted to the model of Orskov and McDonald (1970) as: where a (mL g -1 DM) is the gas production from the soluble fraction, b (mL g -1 DM) is the gas production from the insoluble fraction, c (h -1 ) is the gas production rate constant for the insoluble fraction (b), t (h) is the incubation time, (a + b) (mL g -1 DM) is the potential extent of gas production, and y (mL g -1 DM) is the gas produced at time 't'.
At 24 and 72 h, 10 mL of gas was transferred into a disposal syringe for directly injecting into the gas chromatography machine (Agilent 7890A, Agilent Technologies) to measure methane and carbon dioxide levels. To obtain the acceptable separation, internal standards were applied into the peak rate for methane (FIDI A, front signal, 0.900-0.999 min) and carbon dioxide (TCD2 B, back signal, 0.924-0950 min). The methane, carbon dioxide, and cumulative gas production were expressed as mL/g of OM disappeared after 24 and 72 h of incubation (Ribeiro Junior et al., 2014).
After 24 h of incubation, rumen fluids were then filtered through four layers of cheesecloth. Once the glass syringes were unplugged, pH was immediately measured using a pH meter (Oakton 700, Cole-Parmer, Vernon Hills, IL). Samples were divided into 2 portions; the first portion was centrifuged at 6,000 × g for 15 min at 4 °C and the supernatant was stored at -20 °C before NH 3 -N analysis using the micro-Kjeldahl methods (Kjeltec 8100, Hilleroed, Denmark) (AOAC, 2005) and volatile fatty acid (VFA) detection by gas chromatography (Agilent 6890 GC, Agilent Technologies) (Erwin;Marco;Emery, 1961). The second portion was fixed with 10% formalin solution in a sterilized 0.9% saline solution for calculating total bacteria, protozoa, and fungal zoospores using a Neubauer counting chamber. Subsequently, the fixed solution was diluted with autoclaved deionized water as a medium 100, 10, and 10 times and counted using 10 × 40, 10 × 40, and 10 × 10 ocular × objective for total bacteria, fungal zoospores, and protozoa, respectively (Galyean, 1989).
In vitro degradability was determined after 24 and 72 h of incubation following an earlier protocol (Tilley;Terry, 1963). The filtered contents were collected through pre-weighed Gooch crucibles and residual dry matter was calculated. The percent loss in weight was calculated and dedicated as the in vitro dry matter degradability. The dried feed sample and residue left from the above process was incinerated in a furnace at 550 °C for 5 h for determination of the in vitro organic matter degradability.

Statistical analysis
The data were analyzed in a 3 × 5 factorial arrangement in a completely randomized design using the PROC GLM procedure in SAS 9.4. Since the data from the three consecutive runs of incubations were comparable, the data were averaged and analyzed using the one-way analysis of variance. Multiple comparisons among SFO supplementations, PBP treatments, and interactions were performed using Tukey's honestly significant difference test (Kaps;Lamberson, 2004). Differences among means at p < 0.05 were accepted as representing statistically significant differences.

RESULTS AND DISCUSSION
The PBP used in this study contained roughly 52.24 g/kg DM and 11.51 g/kg DM of secondary metabolites, which accounted for flavonoids and aromatic/ essential oils, respectively (Table 1). Among the present secondary metabolites, quercetin and eugenol were major compounds. In the present study, we tested whether these two active plant metabolites interacted with SFO supplementations to modulate rumen fermentation and its biogas production. All outcomes were expected to reduce methane production and enhance the fermentation endproduct. Therefore, we evaluated the effect of different levels of SFO supplementation with different doses of PBP.

Ruminal biogas and degradability
Increased PBP levels in different substrates (0, 15, 30 mg of SFO) did not show a reduction in gas production after 72 h of incubation (Figure 1). Adding PBP at 15-30 mg, either with or without SFO, increased both total gas production (including CO 2 ) from fermentation and organic matter degradability (Table 2). SFO did not affect those parameters. Exceedingly high doses of PBP (45-50 mg) were shown to have an opposite effect. SFO × PBP significantly (p < 0.05) affected methane production after 24 and 72 h of incubation. Methane production decreased when PBP was combined with 0 mg of SFO (up to 37%), 15 mg of SFO (up to 20%), and 30 mg of SFO (up to 16%).
The gas production trend of substrates treated by Piper betle L. powder (PBP). S1, control or no PBP content; S2, 15 mg PBP; S3, 30 mg PBP; S4, 45 mg PBP; and S5, 60 mg PBP. It has been previously reported that gases such as hydrogen (H 2 ), methane (CH 4 ), and carbon dioxide (CO 2 ) are produced as a result of substrate fermentation in the rumen (Wolin;1979). Carbohydrates, proteins, and lipids of substrates are degraded by rumen microorganisms and shifted into microbial cells. In the present study, the lack of SFO effect on total gas production (including CO 2 ) from fermentation and degradability was consistent with a previous study by Vargas et al. (2017), who reported comparable results for estimated total gas production and degradability rate maintained in semi-continuous flow fermenters (RUSITEC) in rumen fluid from sheep. This notion was substantiated by the fact that increasing SFO supplementations in the substrate tended to maintain total gas production (including CO 2 ). Thus, it appears that SFO is a relatively unfermentable nutrient source. However, in the present study, increasing PBP, which contained flavonoids and their aromatic derivatives, enhanced the fermentable organic degradability in substrate fermentation, resulting in a greater total gas production (including CO 2 ). Notably, PBP is an abundant carbohydrate source (Table 1). It has been reported that plant polyphenols  richer in flavonoids could be degraded in nutrient fermentation, especially by carbohydrate-degrading bacteria (McSweeney et al., 2001). Given this information, the presence of PBP has been shown to increase ruminal biogases whereby the degraded PBP made a predominant contribution to additional fermentable nutrient sources, such as carbohydrates (Orskov;McDonald, 1970). The carbohydrate content of the feed substrate was identical at the onset of each treatment in the present study; thus, the surge in ruminal biogases and degradability rates were expected from modulated carbohydrates and other glucose fermentations degraded by rumen microorganisms during substrate incubation. However, exceedingly high levels of PBP (> 10%) supplementation have shown an adverse effect that might increase toxicity for rumen inhabitants (Yanza et al., 2018). Furthermore, the supplementation of SFO and PBP modulated ruminal fermentation and effectively reduced methane production. The extent of methane mitigation by SFO and PBP was equal to that reported by previous studies (Vargas et al., 2017;Yanza et al., 2018). This methane reduction might have occurred due to the flavonoids and essential oils of PBP. Previous references reported similar results that flavonoids and essential oils had a well-documented antimicrobial effect in rumen methanogenesis through interfering with the outer membrane of gram-positive bacteria Saxena, 2010;Canova et al., 2015;Purba;Yuangklang;Paengkoum, 2020). This motion caused bacteria to lose their control over ion gradients, electron mobilization, phosphorylation cascades, protein translocation, and other enzymatic reactions (Ultee;Bennik;Moezelaar, 2002). In the present study, the combination of SFO and PBP tended to cause a reduction in CH 4 . To our knowledge, the present study is the first investigation of the effects of a combination of SFO and polyphenol on enteric methane production. However, a previous study combining SFO and microalgae could be assessed due to the polyphenolic properties ascertained in microalgae (Haoujar;Cacciola;Abrini, 2019). Schizochytrium microalgae and SFO appeared to alleviate ruminal methane production in rumen fluid of steers and goats (Elghandour et al., 2017). It is worth highlighting that the varied responses to SFO and microalgae inclusions in goat rumen were expected due to the type and dose of the basal diet and lipid inclusion (Hook;Wright;McBride, 2010). Our results elaborated that the combination of SFO and PBP attenuated bacteria activity in the rumen, especially the methanogen group, in which flavonoids of PBP may compete with SFO to possess the target sites through their hydrophobic interaction. Antimicrobial properties of flavonoids and oils have been observed to understand the efficacy of their activity against varied bacteria (Mandalari et al., 2007). This notion was substantiated by the fact that increasing SFO supplementations with PBP levels led to augmented rumen methanogenesis activity.

Fermentation parameter
There were significant interactions (p < 0.05) between SFO and PBP for total VFA and the ratio of acetic to propionic acid and its acid fractions, except iso-acids (Table 3). Increased PBP levels in different substrates (0, 15, 30 mg of SFO) increased acetic acid without altering pH. Supplementing at least 45 mg of PBP, either alone or in combination with SFO, decreased ammonia-N (p < 0.05).
It has been previously reported that adding SFO (ca. 2% of total feed substrate) containing unsaturated fatty acids increased VFA concentration (Vargas et al., 2017). Furthermore, polyphenol utilization by adding doses of commercial quercetin and eugenol at 250-500 mg L -1 has shown inconsistent results (Lourenço et al., 2014). Quercetin, but not eugenol, resulted in a greater concentration of VFA. Given those reports, the increased total VFA in the present study might be due to the content of unsaturated fatty acids and carbohydrate sources in SFO and PBP, respectively. However, a previous meta-analysis reported that additional oil in a diet did not modulate rumen NDF degradability and displayed a negative effect, e.g. reducing dry matter intake (Weld;Armentano, 2017). It is worth noting that constituent nutrients of PBP may be more easily degraded than SFO by rumen inhabitants, thereafter boosting fermentation to produce more VFA (McSweeney et al., 2001). However, taken altogether, the present results were similar to prior studies both observed in vitro (Elghandour et al., 2017;Vargas et al., 2017) and in vivo (Atikah et al., 2018). Presumably, SFO might still affect the total volatile fermentation by modulating fiberdegrading bacteria in the rumen.
In the present study, SFO increased propionic acid, which was in agreement with a prior study (Vargas et al., 2017). However, PBP seemed to have the reverse effect on the propionate shift by systematically altering ruminal metabolism. This suggested that active plant compounds of PBP might have a broad effect on the breakdown in the activity of propionate-producing bacteria during nutrient metabolism via a propionate-generating pathway intermediate. Consequently, this deterioration amounted to less degradation of fermentable cellulose, which was concomitant of a reduced propionic fraction. A lower propionic number may be linked to a lower concentration of valeric acid as well (Andries et al., 1987).
Furthermore, our results addressed the greater concentrations of acetic and butyric acids that may have occurred because of the quercetin and eugenol in PBP. However, these findings contradicted prior studies (Castillejos;Calsamiglia;Ferret, 2006;Oskoueian;Abdullah;Oskoueian, 2013). A possible reason for these different outcomes could be to the interaction between the basal substrate used and the available plant secondary metabolite. For example, Oskoueian, Abdullah and Oskoueian (2013) and Castillejos, Calsamiglia and Ferret (2006) fed the fermenters a 60:40 forage: concentrate diet, whereas in our study, a 50:50 Pangola hay: concentrate diet was used. In addition, those reports used a commercial extract to obtain quercetin and eugenol. These differences in concentrations of acetic and butyric acids could be due to the varied molecular weight content of the plant secondary metabolite used. Hence, response-dependent purity has been suggested to affect substrate fermentation (Petlum;Liang, 2019). In the present study, organic compounds such as quercetin and eugenol might still bind other natural compounds such as phenolic acids (Purba; Paengkoum 2019). Therefore, quercetin and eugenol still allowed ruminal microorganisms to produce a wide extent of acetic and butyric fractions.
Supplementation of PBP decreased ammonia deposition in the present study. PBP seemed to affect the deamination process through an ammonia-inhibiting feature exhibiting slow affinity due to high amounts of PBP (at least 45-50 mg) supplementations. This result was consistent with other studies involving dietary quercetin (Oskoueian;Abdullah;Oskoueian, 2013) and eugenol (Castillejos;Calsamiglia;Ferret, 2006). Given those references, the reduction in ammonia might be due to eugenol. Eugenol seemed to have stronger antimicrobial properties compared to the other secondary metabolites in inhibiting protein degradability in the rumen (Castillejos;Calsamiglia;Ferret, 2006). Eugenol supplementation dosed to at least 50 mg L -1 in the fermenter decreased the ammonia concentration without changing the pH (Castillejos;Calsamiglia;Ferret, 2006). In the present study, the ranges of ammonia concentration and pH were 15.7-18.0 mg 100 mL -1 and 6.8-6.9, respectively, which represented the acceptable conditions for ruminal microorganisms to survive for modulating microbial growth and fermentation efficiency (Ørskov;MacLeod, 1982).

Microbial composition
Total bacteria were similar among the treatments (Table 4). Significant effects (p < 0.05) of SFO × PBP were observed for rumen protozoa and fungal zoospores (p < 0.01). It has been reported that a change of ruminal biogases and fermentation end-products in the rumen might indicate a shift in the ruminal microorganisms (Ungerfeld;Aedo;Martínez, 2019). In the present study, SFO and PBP disturbed ruminal protozoa activity and suppressed ruminal protozoa numbers. It has been previously reported that plants richer in polyphenols, especially tannins, flavonoids, and essential oils, reduced the protozoa community in rumens (Castillejos;Calsamiglia;Ferret, 2006;Oskoueian;Abdullah;Oskoueian, 2013;Cherdthong et al., 2019). In the present study, flavonoids and essential oils present in PBP, either with or without SFO, seemed to have a wider antimicrobial effect, leading to direct deteriorations of protozoa by undertaking cell wall synthesis or nucleic acid synthesis (Cherdthong et al., 2019). Moreover, the decrease in the protozoa did not affect the total number of rumen bacteria. Among flavonoids and essential oils in PBP, quercetin was the predominant active compound (Table 1). This might relate to an earlier report which indicated that supplementing flavonoids like quercetin did not vary the total bacteria in the rumen (Oskoueian;Ciência e Agrotecnologia, 44:e012420, 2020 Abdullah; Oskoueian, 2013). Quercetin has been suggested to have control in maintaining cellulolytic bacteria with lower protozoa and methanogen populations. Although, a possible reason for the escalated fungal zoospores may be related to the altered number of rumen protozoa. Newbold et al. (2015) reported that fungal zoospores slightly increased with a slightly decreased protozoal number, which reflected small competition between fungal zoospores and protozoa regarding consumption of the substrate during fermentation. To support this assessment, fungal zoospores have been reported to have a strong defense towards interruption by plant-containing polyphenols (Cherdthong et al., 2019). Therefore, fungal zoospores could be assessed as the remaining microorganisms that ingest the ruminal substrate during fermentation.

CONCLUSIONS
This study suggested that flavonoids and their aromatic derivatives obtained from the edible plant Piper betle L. dosed at 15-30 mg per incubation on a dry matter basis promotes in vitro methane mitigation while improving rumen fermentation. Incorporating Piper betle powder with sunflower oil as a dietary fat also showed a positive interaction. These findings might be useful for application in ruminant diets, as they are expected to reduce the existential greenhouse gas emissions from livestock. Furthermore, providing Piper betle powder could contribute to limiting agricultural waste. SFO, sunflower oil supplementation per incubation at no (0 mg); low (15 mg); high (30 mg); 2 PBP, Piper betle L. powder supplementation per incubation at S1 (0 mg); S2 (15 mg); S3 (30 mg); S4 (45 mg); S5 (60 mg); 3 dedicated as control treatment (only substrate); 4 SEM, standard error of mean. Within a parameter as column, means with different superscript (a, b, c) differ at p < 0.05 for the PBP effect in substrate; with different superscripts (x, y, z) at p < 0.05 for the SFO effect in substrate.