The Ecacy of Mootral Supplementation on Methane Production and Rumen Fermentation Characteristics in Ruminants Fed Different Styles

Background: Using natural feed supplements to mitigate methane emissions from ruminants is a promising strategy. Many antimethanogenic compounds have been used to alter rumen fermentation, yet their potential to reduce methane production effectively is not consistent across different kinds of feeding styles (forage:concentrate ratios). Therefore, this study was conducted to investigate the impacts of Mootral (MT), a natural combination of garlic powder and bitter orange extract, on methane production, rumen fermentation, and digestibility in different feeding models commonly used for ruminants. The dietary treatments were 1000 g grass/kg ration (10 GRS), 8 GRS + 200 g concentrate/kg ration (2CON), 6GRS + 4CON, 4GRS + 6CON, and 2GRS + 8CON. MT was supplemented at 200 g/kg of the feed. Each group consisted of 6 replicates. The experiment was performed as a batch culture for 24 h at 39 °C. This procedure was repeated in 3 consecutive runs. Results: The results of this experiment showed that supplementation with MT strongly reduced methane production in all kinds of feeding models (P<0.001). Its ecacy in reducing methane/digestible dry matter was 44% in the 10GRS diet, and this reductive power increased with the inclusion of CON up to a 69.5% reduction with the 2GRS + 8CON diet. MT application signicantly increased gas and carbon dioxide production and the concentration of ammonia-nitrogen, but decreased the pH (P<0.001). In contrast, it did not interfere with organic matter and ber digestibility. Supplementation with MT was effective in altering rumen fermentation toward less acetate and more propionate and butyrate. Additionally, it improved the production of total volatile fatty acids in all feeding models (P<0.001). Conclusions: The MT combination showed effective methane reduction by improving rumen fermentation characteristics without exhibiting adverse effects on ber digestibility. Thus, MT could be used with all kinds of feeding models to effectively mitigate methane emissions from ruminants. dry matter, crude protein, NDF: neutral detergent ber, ADF: acid detergent ber, ADL: acid detergent lignin, IVDMD: in vitro dry matter digestibility, IVOMD: in vitro organic matter digestibility, IVNDFD: in vitro neutral detergent ber digestibility, IVADFD: In vitro acid detergent ber digestibility, TVFA: total volatile fatty acids,

90% garlic granules and 10% citrus extract powder. The garlic powder used for MT preparation was sourced from cultivated and carefully processed and dried non-GMO garlic of Chinese origin. The dried garlic granules were standardized to contain 1% (w/w) Allicin potential (S-Prop-2-en-1-yl prop-2-ene-1sul nothioate). Allicin concentration was determined by High Performance Liquid Chromatography (HPLC) as described in details by Eger et al. [15]. The citrus components for the MT mixture (Naringin, Naringenin, Neohesperidin, Rhoifolin, and Neoeriocitrin) were developed from commercially available citrus extracts (Khush Ingredients, Oxford, United Kingdom) mainly extracted from bitter oranges (Citrus aurantium). The total polyphenol content of the citrus extract was standardized to 45% (w/w) by the Folin-Ciocalteau method [17]. Flavonoid concentrations were analyzed by HPLC using standards from (Sigma-Aldrich Ltd., Dorset, United Kingdom). Further information on the MT preparation was described in details by Eger et al. [15]. This mixture was provided by a Swiss company (Mootral SA, Rolle, Switzerland).
The experimental diets were as follows: 1-1000 g grass/kg ration (10GRS), 2-10GRS + 200 g MT/kg of substrate (2MT), 3-8GRS + 200 g concentrate/kg ration (2CON), 4-8GRS + 2CON + 2MT, 5-6GRS + 4CON, 6-6GRS + 4CON + 2MT, 7-4GRS + 6CON, 8-4GRS + 6CON + 2MT, 9-2GRS + 8CON, and 10-2GRS + 8CON + 2MT. Five hundred milligrams of each of the experimental substrates (GRS and CON) was added to preweighed ANKOM lter bags (F57, ANKOM Technology, Macedon, NY, USA), which were heat-sealed and placed in 120 mL glass bottles, while the MT feed supplement was added directly to the bottles one day before incubation. The MT dose used in the current study was based on the most effective dose in our previous study [16]. The chemical composition of the substrates and the MT are described in Table 1. The procedure of in vitro batch culture was performed as described by Menke and Steingass [18]. In the laboratory, the collected rumen uids from the two cows were mixed together in one beaker under a constant stream of CO 2 . Forty milliliters of fresh buffer solution at a pH of 6.8 prepared according to McDougall [19] with twenty mL of rumen uid was added to each 120 mL bottle under continuous CO 2 ushing to maintain anaerobic conditions. Thereafter, the fermentation bottles were ushed with CO 2 before sealing with butyl rubber stoppers and aluminum caps (Maruemu Co., Ltd, Osaka, Japan). All bottles were incubated for 24 h at 39 °C. This batch culture procedure was repeated in three consecutive runs on three different days. In each run, two blanks without substrate (empty lter bag plus 60 mL of buffered rumen uid) were included to be used for digestibility and gas production correction. In total, 180 bottles plus 6 blank bottles were examined in this study.

Sample collection
After 24 h of incubation, the total gas production was measured, and gas samples (3 mL) were collected from the headspace of the glass bottles into vacutainer tubes (BD Vacutainer®, Becton Drive, USA). The tubes were stored at room temperature until CH 4 and CO 2 determination. Thereafter, the bottles' caps were removed, and the pH of each tube was recorded using a pH meter (LAQUA F-72, HORIBA Scienti c, Kyoto, Japan). Then, aliquots of the culture uid were transferred into 1.5 ml Eppendorf tubes and centrifuged at 16,000×g and 4 °C for 5 minutes. The supernatant was collected and transferred into a new Eppendorf tube® (Eppendorf AG, Hamburg, Germany), which was stored at − 20 °C until use for volatile fatty acid (VFA) and ammonia nitrogen (NH 3 -N) analysis. The bags were removed from the bottles, washed under running tap water until the draining uid became clear, and then dried at 60 °C for 48 h to determine the in vitro dry matter digestibility (IVDMD). After IVDMD determination, the bags were used for the determination of in vitro organic matter digestibility (IVOMD), in vitro neutral detergent ber digestibility (IVNDFD), and in vitro acid detergent ber digestibility (IVADFD). The residues in the fermentation bottles were discarded.

Chemical analysis
The chemical composition of the GRS, CON, MT and remaining substrate in the bags was determined following the standard procedure of AOAC [20]. DM content was measured by drying the samples in an air-forced oven at 135 °C for 2 h (930.15). OM and ash were measured by placing the samples into a mu e furnace at 500 °C for 3 h (942.05). Nitrogen (N) was measured according to the method of Kjeldahl (984.13) using an electrical heating digester (DK 20, VELP Scienti ca, Usmate (MB), Italy) and an automatic distillation apparatus (UDK 129 VELP Scienti ca, Usmate (MB), Italy), and then CP was estimated as N The concentrations of CH 4 and CO 2 in the gas samples were determined by injection of 1 mL using a Hamilton gastight syringe (Hamilton Company, Reno, Nevada, USA) into a gas chromatograph (GC-8A, Shimadzu Corp., Kyoto, Japan). The carrier gas was helium. The temperatures of the infuser port, column, and detector were 70 °C, 150 °C, and 150 °C, respectively. The identi cation of CH 4 and CO 2 was based on the retention time.

Volatile fatty acids and NH 3 -N analysis
The concentration of VFA was determined using high-performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) after diluting the supernatant 3 times with distilled water. Brie y, the analytical speci cations were as follows: column, Shim-pak SCR-102H (7 mm, i.d. 8.0 mm×300 mm, Shimadzu Corp., Kyoto, Japan); eluent ow rate and mobile phase for organic acid analysis (Shimadzu Corp., Kyoto, Japan) at 0.8 mL/min; column temperature, 40 °C; reaction reagent and ow rate, pH buffer for organic acid analysis (Shimadzu Corp., Kyoto, Japan) at 0.8 mL/min; conductivity detector (CDD-10AVP, Shimadzu Corp., Kyoto, Japan). Quanti cation of the VFA concentration was performed using an external standard quantitation method [16].
The NH 3 -N concentration was measured by diluting samples 50 times with 0.1 M phosphate buffer (pH 5.5) and then they were analyzed following the procedure of the modi ed Fujii-Okuda method [21] using an NH 3 kit (FUJIFILM Wako Pure Chemical Corp, Osaka, Japan). The plate was read by a microplate reader (SH-1000 Lab, Corona Electric Co., Ltd., Japan) at an optical density of 630 nm.

Statistical analysis
All variables were analyzed using PROC MIXED by SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). The model included the treatment (diet) effect, MT effect, and their interaction as xed effects, while the experimental runs were considered random effects. Least square means and standard error (SEM) were calculated, and the differences of means were estimated by pairwise t-tests (PDIFF option of PROC MIXED). Signi cance was declared at P < 0.05, and a tendency toward signi cance was declared when the P value was between 0.05 and 0.10.

Results
Effect of Mootral supplementation on in vitro pH, gas production, and gas composition Supplementation of MT to all feeding models reduced the pH (P < 0.001) when compared with its corresponding group without MT supplementation in the same feeding model (Table 2). Moreover, the inclusion of MT increased the absolute total gas production when correlated with DM and digestible DM in all feeding styles (P < 0.001, Table 2).  Adding MT to all feeding styles decreased the proportion of CH 4 but increased the proportion of CO 2 in the produced gas (P < 0.001, Fig. 1A Fig. 1B). Furthermore, the CH 4 /CO 2 ratio in the produced gas (ml/ml) decreased in all feeding models due to MT's effect (P < 0.001, Fig. 1D).
MT inclusion was effective with all diets in reducing the production of CH 4 /DM (ml/g) (P < 0.001, Fig. 2A) Fig. 2C). In contrast, the production of CO 2 /DM and CO 2 /digestible DM (ml/g) increased (P < 0.001) due to the effect of MT in all feeding styles ( Fig. 2B, 2D, respectively).
Effect of Mootral supplementation on in vitro nutrient digestibility and ammonia-nitrogen concentration MT supplementation did not affect the IVDMD in the different experimental diets except in the 8GRS + 2CON and 6GRS + 4CON diets, where adding MT to these styles reduced the IVDMD (P < 0.01, Table 3). However, IVOMD, IVNDFD, and IVADFD did not show any differences when MT was added as compared with their corresponding groups without MT supplementation (P > 0.05, Table 3). MT inclusion increased the NH 3 -N concentration (P < 0.01) in 10GRS and 6GRS + 4CON and it tended to increase in 4GRS + 6CON (P = 0.088), while there was a non-signi cant numerical increase in 8GRS + 2CON (P = 0.106) and 2GRS + 8CON (P = 0.32) ( Table 3).

Effect of Mootral supplementation on in vitro volatile fatty acids
MT supplementation did not show any effect on the acetate concentration in all feeding models; however, the interaction between MT and treatment showed a difference for the MT supplemented groups to be increased in 10GRS (P < 0.001) and to be decreased in 2GRS + 8CON (P < 0.05) compared with its corresponding treatment without MT inclusion (Table 4). In contrast, the acetate ratio decreased in all feeding models due to MT supplementation (P < 0.01), while the interaction between MT and treatment did not have a signi cant effect (P > 0.05, Table 4). The concentration and the ratio of propionate and butyrate increased (P < 0.001) by adding MT in all feeding models. Additionally, the concentration of total volatile fatty acids (TVFA) showed the same nding (Table 4). The acetate/propionate (A/P) ratio decreased (P < 0.001) with MT supplementation in all feeding styles (Table 4). Discussion CH 4 emissions from ruminants are not only a serious environmental issue but also a signi cant source of energy loss to the animals. Different kinds of antimethanogenic compounds have already been studied to investigate their potential to reduce CH 4 production; however, there are limitations to their use due to their negative impacts on rumen fermentation characteristics [22], and they exhibited inconsistent e ciency with different feeding styles [23]. Therefore, sustainable and immediate CH 4 mitigation strategies for the livestock industry are in high demand. MT, a natural plant-based combination of garlic and citrus extracts, showed promising results when used as a feed supplement for methane mitigation from ruminants [15,24]. Therefore, this study was performed to evaluate the e cacy of MT with different kinds of feeding styles in ruminants.
Similar to the ndings of the current study, MT increased gas production when used as a feed supplement with rumen uid collected from sheep, which may re ect a stimulating effect of MT on rumen microbes [16]. This nding has been reported previously from a 48 h in vitro gas production study conducted by Hansen and Nielsen [25]. Furthermore, MT increased the concentration of ruminal NH 3 -N, which might be due to the role of MT in enhancing the proteolysis process. This nitrogen source can be used by rumen microorganisms to build their own protein, which in turn would be used as a protein source for the animal [26]. A similar effect has been reported when MT was used as a feed supplement with a 70 forage:30 concentrate diet in the RUSITEC system [27]. The same nding has also been observed with garlic oil with a 50 forage:50 concentrate diet for 24 h incubation by Busquet et al. [28].
MT supplementation did not interfere with ber degradability in all feeding models, which was similar to the ndings of García-González et al. [29], who reported that inclusion of garlic bulbs in the substrate in an in vitro trial did not affect IVNDFD, and Wanapat et al. [30], who declared that adding garlic powder to concentrates did not change the NDF and ADF digestibility through an in vivo trial using steers. Rumen microbiome analysis in upcoming studies would provide a better understanding of MT's effect on nutrient digestibility and proteolytic bacteria.
The synergism between the organosulfur compounds and avonoids in the MT mixture was effective in decreasing CH 4 production in all feeding models. The reduction in CH 4 may be due to the direct inhibitory effect of MT on methanogenic archaea. Eger et al. [15] and Ahmed et al. [16] reported a lower abundance of the family Methanobacteriaceae, which is the major CH 4 producer in the rumen, in MT supplemented treatment. This was attributed to the toxicity of organosulfur compounds of garlic, such as diallyl sul de and allicin, to inhibit certain sulfhydryl-containing enzymes essential for the metabolic activities of methanogenic archaea [31,32]. It has been established that ruminal ciliated protozoa could enhance methanogenesis, as they are major H 2 producers in the rumen and are in symbiotic relationships with methanogens [33]. Although the impact of MT on protozoa has not yet been investigated, allicin and avonoids have shown toxic effects on protozoa [34,35]. Any effect of MT on protozoa has to be con rmed in additional studies.
It is well established that CH 4 formation has been positively associated with more acetate production and negatively associated with increased propionate production [36]. MT was able to shift rumen fermentation toward less acetate and more propionate and butyrate. This increase in propionate may be due to the role of MT in increasing the abundance of the Prevotellaceae and Veillonellaceae families, which was con rmed by Ahmed et al. [16]. Prevotellaceae is one of the dominant families in rumen uid, and it is well known to produce propionate by utilizing hydrogen (H 2 ) produced during the fermentation of carbohydrates [37]. This pathway is the main pathway for H 2 consumption and it represents a competitive and alternative pathway to methanogenesis [38,39]. Moreover, the family Veillonellaceae showed high relative abundance due to the effect of avonoids extracted from citrus [34], and it was associated with propionate production [40]. Supplementation of steers with garlic powder reduced the A/P ratio [30]. Similarly, the current study showed the same nding. An increase in butyrate was also associated with a reduction in CH 4 production when the basal diet of ewes was supplemented with garlic extract [41].
Reports about the effects of garlic and avonoid components on TVFAs are inconsistent. Some studies reported that they had no effect on TVFA [8,29,42,43], while others reported an adverse effect [28,34,44] using an in vitro batch culture system. In contrast, in the current study, the MT formulation improved the production of TVFA. This may be attributed to the role of MT in stimulating the metabolic activity of some rumen microbes, which may be proven by increasing the production of total gas and CO 2 . This nding has also been observed previously in studies using in vitro batch culture [16] and the RUSITEC system [15].

Conclusion
In the present study, we investigated the e ciency of the MT mixture on CH 4 production, rumen fermentation and digestibility in different feeding styles commonly applied in ruminant farms. This study has con rmed the potential of MT to effectively reduce CH 4 production with all feeding styles. MT showed a high reducing power up to 70% when the amount of CON comprised up to 800 g/kg of the ration. Moreover, MT supplementation improved the production of TVFA by shifting the fermentation pro le toward less acetate and more propionate and butyrate. Additionally, MT did not impair ber digestibility. Therefore, MT could be used as a feed supplement with all feeding styles to e ciently reduce CH 4 production by ruminants.  Effect of Mootral (MT) supplementation on the proportion of in vitro gas composition in different feeding styles. A-Proportion of methane (CH4) in the produced gas, B-Proportion of CH4/g digestible dry matter (DM), C-Proportion of carbon dioxide (CO2) in the produced gas, D-CH4/CO2 ratio (ml/ml).
Asterisks between groups mean signi cant difference; *** (P<0.001). Data are represented as the mean and standard error (n = 18).