In vitro evaluation of Rhus succedanea extracts for ruminants

Objective This study was conducted to evaluate the effects of Rhus succedanea extract addition on in vitro ruminal fermentation and microbial growth. Methods Two ruminally-fistulated steers consuming 600 g/kg timothy- and 400 g/kg cracked corn-based concentrate with free access to water and mineral block were used as rumen fluid donors. In vitro batch fermentation, with timothy as a substrate, was conducted for up to 72 h, with Rhus succedanea extracts added to achieve final concentrations of 0, 10, 30, 50, 70, and 90 mg/L. Results Effective dry matter (DM) degradability rate linearly decreased (p = 0.046) depending on extract dosing levels. Total gas production after 24 to 72 h incubation tended to decrease following extract addition, beginning with 50 mg/L starting dose (significance of quadratic effects: p = 0.006, p<0.001, and p = 0.008 for 24, 48, and 72 h, respectively). Methane production decreased depending on dosing levels following 24 h (p<0.05) and 48 h (p<0.005) incubations and was the lowest with the 50 mg/L dose. The Rhus succedanea extracts increased the abundance of Fibrobacter succinogenes (p<0.05) and Ruminococcus flavefaciens (p = 0.0597) and decreased the abundance of methanogenic archaea (p<0.05) following 24 h incubation. Conclusion Rhus succedanea was shown to reduce methane production and increase cellulolytic bacteria without any signs of toxic effects and with a minor effect on DM degradability.


INTRODUCTION
The efficiency of ruminal fermentation can be facilitated by modifying the feeding regime of ruminants using natural feed additives. A number of methanogenic inhibitors have been developed to improve feed conversion efficiency of ruminant feeds, which are claimed to be effective in suppressing methanogens or overall bacterial activities [1]. However, some compounds are toxic or may not be economically feasible, or an adaptive response may occur in some bioactive compounds after supplementation.
In recent years, there has been a global trend toward the use of natural plants as medicinal and functional food additives. Medicinal plants have various characteristics, such as anti microbial, antiviral, and immune system stimulating activities, which can be beneficial to animal health and production. Rhus succedanea, the wax tree, is a flowering plant species found in Asia. Rhus succedanea (formerly Toxicodendron succedaneum) has been used in indigenous medicine for quite a long time in the treatment of asthma, cough, and colicky pains [2], and has antirumor, antioxidation, hangover cure, and gastritis suppression effects [3]. Bioactive constituents from R. succedanea have been isolated and characterized. These mostly include urushiol, flavonoids, and phenols [3].
Although a number of studies using plant extracts have been conducted to apply them as feed additives, R. succedanea extracts have rarely been applied to ruminant animals. Kim et al [4] reported that dietary Rhus verniciflua supplementa tion of Hanwoo cattle feed was effective in increasing meat color stability, waterholding capacity, and unsaturated fatty acid content, as well as retarding lipid oxidation. However, information on the effects of application of R. succedanea on ruminal fermentation is limited. Therefore, this study was conducted to evaluate the effects of R. succedanea extracts on in vitro ruminal metabolites, gas production, and micro bial growth.

Sample preparation
Rhus succedanea extracts were obtained from the Plant Extract Bank (KRIBB, Daejeon, Korea). R. succedanea was cut into small pieces and dried naturally under shade. Extraction from the dried pieces (100 g each) was then performed with 99.9% methyl alcohol (1,000 mL) using an ultrasonic cleaner (Branson Ultrasonics Corporation, Danbury, CT, USA) at room temper ature for three days. After extraction, the solutions were filtered and the solvents were evaporated under vacuum conditions. Stock solution (20 mg/mL) of the extracts was dissolved in dimethyl sulfoxide (SigmaAldrich Chemical Co., St. Louis, MO, USA) and diluted using culture medium immediately before in vitro incubation. R. succedanea extracts were pre pared to achieve final dosing concentrations of 0, 10, 30, 50, 70, and 90 mg/L.

In vitro batch fermentation
Ruminal contents were collected from two ruminallyfistulated steers (with mean body weight±standard error of 450±30 kg), which had been consuming 600 g/kg body weight timothy and 400 g/kg cracked cornbased concentrate (crude protein, 120 g/kg; ether extracts, 15 g/kg; crude fiber, 150 g/kg; crude ash, 120 g/kg; Ca, 7.5 g/kg; P, 9.0 g/kg; total digestible nutrients, 690 g/kg dry matter [DM] basis) with free access to water and mineral block (supplied per kilogram of diet: vitamin [Vit] A, 3,800 IU; Vit E, 400 IU; Vit E, 500 IU; Fe, 7 mg; Cu, 2.4 mg; Mn, 30 mg; Zn, 6.0 mg; I, 1.5 mg; Se and Co, 1.5 mg). The diet was fed at 3% of body weight of the steers in two equal portions at 07h00 and 17h00 daily. The steers were acclimated to the diet for a minimum of 14 days.
On the day of fermentation testing, approximately 2 kg of ruminal contents were collected from the dorsal, ventral, and caudal rumen of each steer 2 h after the morning feeding, and collected into an insulated container for transport to the labo ratory, usually within 10 min after collection. Ruminal contents were processed with a Waring blender under a CO 2 atmo sphere and strained through four layers of cheesecloth and glass wool prior to combining with McDougall buffer [5]. The McDougall buffer (1,000 mL) and ruminal inoculums (500 mL) were combined, and 25 mL of this mixture was then added to 60mL fermentation vessels (serum bottles) containing 0 or 300 mg (based on DM) of substrate. The substrate was sup plied from the timothy that was fed to the steers, and was used after being ground up in a Wiley mill until it could pass through a 2 mm screen. The fermentation vessels were closed with butyl rubber stoppers under the anaerobic gassing sys tems while being connected to a source of oxygenfree gas, and then were sealed with aluminum caps and placed in an incubator at 39°C for 3, 6, 9, 12, 24, 48, and 72 h without shak ing. Fermentation was conducted in a completely randomized order and in duplicate for each sample, and was then repli cated on three separate days (n = 3 for each dose in statistical analyses).
Total gas and methane production, ammonia-nitrogen, and volatile fatty acid content At the end of each incubation, a detachable pressure trans ducer and a digital readout voltmeter (Laurel Electronics, Inc., Costa Mesa, CA, USA) were used to measure the headspace gas pressure in each vessel. Gas samples for methane analysis were drawn from each vessel into sampling syringes and trans ferred into a vacuum test tube (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA). Gas samples were analyzed for me thane concentrations by gas chromatography (Agilent Technologies HP 5890, Santa Clara, CA, USA) conducted using a thermal conductivity detector with a Column Car boxen 1006PLOT capillary column, measuring 30 m×0.53 mm (Supelco, Bellefonte, PA, USA), as described by Zafarian and Manafi [6].
After determination of gas production, the vessels were uncapped, the pH of fermentation media was then measured, and a 5mL aliquot of the fermentation medium was com bined with 0.5 mL of 2ethylbutyrate (85 mM) as an internal standard and 0.5 mL of 50 g/kg metaphosphoric acid for analy sis of volatile fatty acid (VFA) concentrations. These samples were centrifuged at 39,000×g at 23°C for 15 min, transferred to vials, capped, and analyzed for VFA concentrations by gas chromatography (model GC14B, Shimadzu Co. Ltd., Tokyo, Japan) using a Thermon3000 5% Shincarbon A columm (1.6 m×3.2 mm i.d., 60 to 80 mesh, Shinwakako, Kyoto, Japan) and flameionization detector (column temperature = 130°C, injector and detector temperature = 200°C). The carrier gas (N 2 ) flow rate was 50 mL/min. A 5mL aliquot of the fer mentation medium was combined with 0.5 mL of 25 g/kg metaphosphoric acid for analysis of NH 3 N concentration using a spectrophotometer (Model 680, BioRad Laboratories, Hercules, CA, USA) and methods based on glutamate de hydrogenase. Soil kit (Machereynagel, Düren, Germany). This was accom plished by taking a 1.0mL aliquot from the culture medium using a widebore pipette to ensure collection of a homoge neous sample, and then centrifugating the aliquot at 39,000×g. Nucleic acid concentrations were measured using a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Relative quantification of specific ruminal microbes
Polymerase chain reaction (PCR) primer sets were then used in this study to detect and amplify DNA from Fibrobacter succinogenes (forward primer: GTT CGG AAT TAC TGG GCG TAA A; reverse primer: CGC CTG CCC CTG AAC TAT C), Ruminococcus flavefaciens (forward primer: CGA ACG GAG ATA ATT TGA GTT TAC TTA GG, reverse primer: CGG TCT CTG TAT GTT ATG AGG TAT TAC C), and Ruminococcus albus (forward primer: CCC TAA AAG CAG TCT TAG TTC G; reverse primer: CCT CCT TGC GGT TAG AAC A), and the primers used were the same as those referenced by Denman and McSweeney [7], Koike and Kobayashi [8], and Skillman et al [9], respectively. A total bacteria primer set (forward: CGG CAA CGA GCG CAA CCC; reverse: CCA TTG TAG CAC GTG TGT AGC C) was used as the internal standard [7].
Realtime PCR (RTPCR) assays for enumeration of mi crobes were performed according to the methods described by Denman and McSweeney [7] and Denman et al [10] on a realtime PCR Machine (CFX96 RealTime system, BioRad, USA) using the SYBR Green Supermix (QPK201, Toyobo Co., Ltd., Tokyo, Japan). The values of the cycle threshold (Ct) after PCR reactions were used to determine fold change (num ber of fold difference) of different microbial populations relative to the control without additives. Abundance of these microbes was expressed by the equation: relative quantifica tion = 2 -∆Ct(Target) -∆Ct(Control) , where Ct represents the threshold cycle. All RTPCR reaction mixtures (final volume of 25 μL) contained forward and reverse primers (10 pmol each), the SYBR Green Supermix (QPK201, Toyobo Co., Ltd., Japan), and DNA templates ranging from 10 to 100 ng. A negative control without template DNA was used in every RTPCR assay for each primer. The amplification of the target DNA was performed as described by Denman and McSweeney [7], Koike and Kobayashi [8], and Skillman et al [9].

Calculations and statistical analysis
To give a more precise estimate of gas production throughout fermentation, the following calculation was used to analyze the kinetic data, as described by Ørskov and McDonald [11]: G P = a+b(1-exp -c×time ), where G P is gas production (mL/g DM of substrate) at time t; a, b, and c are the scaling factors for the Yaxis intercept (mL/g of DM), potential gas production (mL/g of DM), and the rate constant for gas production per h, re spectively. Gas production rate was fitted to the model by using the NLIN procedure of SAS (version 9.1, SAS Inst. Inc., Cary, NC, USA) employing Marquadt's algorithm, while varying a, b, and c. Effective gas production (EG P , i.e. substrate availability) from the culture was estimated as EG P = a+b(k d /[k d +k p ]), where k d is a gas production rate constant, and k p is a passage rate constant assumed to be 0.05/h [12].
Data obtained from the experiment were analyzed using the general linear model procedure of SAS (SAS Inst. Inc., USA) for a completely random design. The model included terms for dosing levels, time, and their interaction. Orthogo nal contrast was used to assess linear, quadratic, and cubic relationships between the dosing levels of R. succedanea ex tracts and the dependent variables. Orthogonal coefficients for unequally spaced dosing were acquired using the IML pro cedure (SAS Inst. Inc., USA). Table 1 shows the effects of different doses of R. succedanea extracts on DM degradability and their parameters after dif ferent incubation periods. The DM degradability was not affected by the dose of R. succedanea extracts, except after 24 and 72 h incubations, when it decreased by dose (significance of linear effect: p = 0.04; linear and cubic effects: p = 0.014 and p = 0.041, respectively). The DM degradability for 24 and 72 h incubations was decrease by 70 and 90 mg/L doses of R. succedanea extracts. Effective DM degradability rate (E DM ) decreased linearly with dose (p = 0.046). Cumulative gas pro duction rapidly increased from 12 to 72 h of incubation ( Table  2). The R. succedanea extracts increased total gas production depending on dosing level at 24 (linear, quadratic, and cubic effects: p = 0.005, 0.006, and 0.051, respectively), 48 (quadratic effect: p<0.001), and 72 h (quadratic and cubic effects: p = 0.008 and 0.031, respectively) incubations. In addition, the total gas production for 24 to 72 h incubation tended to de crease with 50 mg/L dosing of R. succedanea extract as a starting point (quadratic effects: p = 0.006, p<0.001, and p = 0.008 for 24, 48, and 72 h, respectively). The potential gas production (a+b) was decreased (cubic effect: p = 0.051) by dosing with 50 mg/L of R. succedanea extracts. The decrease in the poten tial gas production led to an increase in the k value. Effective gas production rate (E Gp ) was decreased (linear and quadratic effects: p = 0.003 and 0.069, respectively) depending on dos ing levels.

RESULTS
The pH was slightly increased after fermentation depend ing on the dose used at 24 h and 72 h, but remained within an optimal pH range of 6 to 7 (Table 3). AmmoniaN concen tration changed in a quadratic manner (p = 0.003) following 24 h incubation, which was increased by 50 mg/L of R. succedanea extracts. Total VFA concentration was decreased by 50 mg/L of R. succedanea extracts following 48 h (quadratic effect: p = 0.021) and by 70 and 90 mg/L of R. succedanea extracts following 72 h incubation (linear effect: p = 0.014). Total VFA concentration following 24 h of incubation de creased (p<0.05) and acetate concentration increased (p<0.05) relative to control with dosing of 70 mg/L of R. succedanea extracts (Figure 1). No differences were observed in propio nate and butyrate concentrations and acetate to propionate ratio. Methane production was decreased depending on dosing levels following 24 h (linear and quadratic effects: p<0.0001 and p = 0.002, respectively) and 48 h (linear and cubic effects: p<0.0001 and p<0.001, respectively; Table 4) incubations, and was the lowest at 50 mg/L doses of R. succedanea extract.
Realtime PCR analyses indicated that R. succedanea extracts affected the abundance of cellulolytic bacteria and methano genic archaea (Figure 2). The dose of R. succedanea extracts increased the abundance of Fibrobacter succinogenes (linear, quadratic, and cubic effects: p<0.0001, p = 0.0006, and p = 0.0078, respectively) and Ruminococcus flavefaciens (linear effect: p = 0.0597), and decreased the abundance of metha nogenic archaea (linear and quadratic effects: p<0.0001 and p = 0.0073, respectively) after 24 h of incubation.   1) Potential extent and rate of gas production were determined using the exponential model: G P = a+b(1-exp -c × time ), where G P is gas production (mL/g DM of substrate) at time t; a = gas production from the immediately soluble fraction; b = gas production from the insoluble fraction; c = the fractional rate of gas production per hour; a+b = potential extent of gas production; k = gas production rate constant for the insoluble fraction; E GP = effective gas production rate from the cultures, calculated as E GP = a+b[k d /(k d +k p )], where k d is a gas production rate constant, and kp is a passage rate constant assumed to be 0.05 h -1 .

DISCUSSION
Total gas production was closely related to the digestion of fermentation substrates, VFA production, and microbial ac tivity and growth [13]. Ruminal microbial activity was affected by the use of plant extracts and secondary plant metabolites [14].  [14] reported that at the highest concentrations of various plant extracts, most treatments showed decreased total VFA production, possibly reflecting decreased feed di gestion. Plant secondary metabolites are particularly attractive as rumen modifiers that are generally accepted to be environ mental friendly and safe to use in food production systems. Due to their potential to adversely affect feed intake and nu trient utilization, however, these should be administered at low concentrations to beneficially alter ruminal fermentation. The effect of R. succedanea extract dose on ammoniaN con centration was quadratic (p = 0.003) following 24 h and 48 h incubations, and this effect was increased for 50 mg/L extracts and decreased for 70 mg/L extracts. These results suggest that changes observed to be caused by R. succedanea extract on ruminal ammoniaN concentration may be contradictory depending on the dose used. The observed reduction in am  moniaN suggests that over 70 mg/L of R. succedanea extracts reduced amino acid deamination. Inhibition of amino acid deamination has practical implications because it may increase ruminal use of dietary protein and improve the efficiency of N use in the rumen [15]. Ruminal cellulolysis is conducted primarily by Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus [16], and their relative populations can potentially impact the ratios of VFA available to ruminants. In the present study, an increase in ruminal acetate concentration was observed to accompany the addition of 70 mg/L dose of R. succedanea extracts, and this was consistent with the observed increase in abundance of the gramnegative bacteria Fibrobacter succinogenes. This bacterium intensively degrades plant cell walls by an erosionlike mechanism, in which it burrows its way through the complex matrix of cellulose and hemicellulose in the cell wall resulting in the release of digestible and undi gested cell wall fragments [17]. Ruminococcus flavefaciens, a grampositive bacteria, was also observed to increase with addition of 70 mg/L dose of R. succedanea extract that corre sponded with the increase of acetate concentration. Production of methane and propionate are negatively correlated because both these processes compete for hydrogen [18]. However, the negative relationship between propionate concentration and methane output was not evident from our results. Forma tion of acetate and butyrate results in production of additional methanogenic substrates, which are formate and hydrogen, and propionate formation results in less hydrogen being avail able for methane production [18]. In addition, low methane production might be related to reduced fiber digestibility, thus, also influencing the energy input to the animal. In the present study DM degradability and gas production linearly decreased, whereas Fibrobacter succinogenes and Ruminococcus flavefaciens, which are considered to be primarily responsible for plant cell wall biodegradation, increased after 24 h of incu bation. Nevertheless, the decreasing methane production and abundance of methanogenic archaea observed with extract dosing were likely caused by the combined effects of decreased total VFA concentration and decreased DM fermentation. R. succedanea produces a cytotoxic biflavonoid, and it has been reported that the methanol extract of Rhus succedanea showed positive indications of the presence of phenols, steroids, al kaloids, flavonoids, tannins, glycosides, and carbohydrates [19]. It has been documented that alkaloids, flavonoids, tan nins, and phenols are plant secondary metabolites, which are wellknown for their antimicrobial activity in the rumen. The extract of stem bark of Rhus had antioxidant effects against hydroxyl radicals and antiproliferative activity against human cancer cell lines, and also augmented the activity of cellasso ciated detoxifying enzymes in hepatocytes [2022]. The sap of the wax tree (R. succedanea) is composed of urushiol, glyco protein, flavonoids, a gummy substance that contains laccase, stellacyanin, polysaccharides, peroxidase, and water [23]. Therefore, the observed antibacterial potency of methanol extracts from R. succedanea can be attributed to the nature of its biologically active components, which might be enhanced in the rumen. However, further work is needed to clarify the relationship between fibrolytic microbes and methanogens, and although suppression of methanogenic archaea by R. succedanea extracts was observed in this study, it must be noted that the longterm effects of the extracts might be different because adaptation of the rumen microbes might occur.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.