Effect of essential oils on the production of methane in the in vitro fermentation of Koronivia grass

a Universidad Autónoma de Guerrero. Facultad de Medicina Veterinaria y Zootecnia No. 2, Carretera Acapulco-Pinotepa Nacional, kilómetro 197, Cuajinicuilapa, 41940, Guerrero, México.

The objective was to evaluate the increasing use of garlic, sesame, and cinnamon oil in the production of CH4 in 60 d in vitro regrowth of Koronivia grass. The addition of 0, 2.5, 5.0, 5.0, 7.5, and 10 % garlic, cinnamon, or sesame oil was evaluated in an in vitro fermentation using a 60-d regrowth of Koronivia grass as substrate. The variables evaluated were cumulative CH4 production at 12, 24, 36, 36, 48, and 72 h; dry matter degradation (DMD), and CH4 production kinetics estimators (A= CH4 production potential, b= CH4 production rate constant, and k= lag time). The CH4 production and the DMD were analyzed with a completely randomized experimental design and orthogonal contrast. The estimators were subjected to a descriptive analysis. An increase of garlic oil and cinnamon linearly reduced CH4 production at 12, 24, 36, 48, and 72 h. The DMD decreased linearly with the use of any of the three oils (P<0.05). The highest value of A was obtained with 2.5 % garlic oil, and the highest value of k and b, with 10 % cinnamon oil. In conclusion, the use of garlic and cinnamon oils resulted in a linear decrease of Koronivia grass CH4 and Koronivia grass DMD under in vitro conditions.

Keywords: Garlic oil, Cinnamon oil, Sesame seed oil, Dry matter degradation, In vitro.

Livestock production generates 37 % of global greenhouse gas (GHG) emissions and is expected to increase to 40 % by 2050; 80.7 % of these GHGs come from the enteric fermentation of ruminants(1,2). The main GHGs produced by ruminants are enteric and manure methane (CH4), carbon dioxide (CO2), and nitrous oxide (N2O)(3). CH4 is a GHG that contributes to the formation of tropospheric ozone; it also has a short life span (9 to 12 yr), but is 25 times more harmful than CO2, so its reduction helps mitigate the adverse effects of climate change(1,2).

GHG mitigation strategies are aimed at not affecting animal performance, reducing environmental impact, and enhancing the productivity and profitability of production systems(1). Because of this, additives should be able to modify rumen fermentation to improve energy use efficiency while decreasing rumen methanogenesis(4). Today, approximately 200,000 plant secondary metabolites have been identified as potential modulators of the rumen microbiota, specifically in the reduction of energy los

Essential oil (EO) is a complex mixture of lipophilic, plant-specific volatile or aromatic chemicals; its constituents are terpenoids, phenylpropanoids, monoterpenes, sesquiterpenes, and alcohols, aldehydes, ethers, esters, ketones, and phenols(1,5). These oils possess antioxidant properties and induce changes in the ruminal microbiome, resulting in a reduction of CH4, increase in propionate or ruminal by-pass protein(1,4,6), antimicrobial effect of bacteria, fungi and protozoa, as well as decreased dry matter (DM) digestibility and ruminal fermentation(4), inhibition of amino acid deamination, ammonia nitrogen and acetate reduction(5), and they influence electron pathways affecting the integrity of cell membranes(1,2). The decrease in enteric CH4 production with the use of EO is due to the reduction of hydrogen production (alternative sinks), direct inhibition of archaea, and interruption of the symbiosis between protozoa and archaea(1).

Garlic (Allium sativa) oil has a broad spectrum of antibacterial activity against gram-negative and gram-positive bacteria(7); its bioactive compounds are organic sulfides, saponins, phenolic compounds, and polysaccharides, allicin, S-allyl cysteine, diallyl disulfide, diallyl trisulfide, diallyl sulfide, and ajoene(8). Cinnamon (Cinnamomum verum) oil has an antimicrobial effect due to its transcinnamaldehyde content and antioxidant activity derived from its phenolic and polyphenolic compounds(9). For its part, sesame (Sesamum indicum) oil contains oleic and linoleic acid, tocopherol, sesamin, sesamolin, polyphenols, phytosterols, flavonoids, and lignans, which have anti-inflammatory and antimutagenic effects(10).

The efficiency of rumen fermentation is leading to the search for natural alternatives to mitigate GHG emissions without compromising livestock productivity; the concentration of atmospheric CH4 continues to increase, so strategies are needed to help reduce its production. The hypothesis was that the addition of garlic, sesame, and cinnamon oils decreases CH4 production during in-vitro ruminal fermentation of Koronivia grass substrate with 60 d of regrowth. Thus, the objective of this research was to evaluate the use of increasing doses of garlic, sesame, and cinnamon oil in the in vitro ruminal fermentation of 60-d-old Koronivia grass as a substrate for methane production and dry matter degradation.

The study was conducted in the animal nutrition laboratory of the Faculty of Veterinary Medicine and Animal Husbandry No. 2, located in the municipal seat of Cuajinicuilapa, Guerrero, Mexico.

The essential oils utilized were garlic (Yerbatex), sesame (Yerbatex), and cinnamon (Yerbatex). The proportions of oil evaluated were 0, 2.5, 5.0, 7.5, and 10.0 % of oil. The Koronivia grass (Brachiaria dictyoneura) was harvested 60 d after regrowth. The grass was dehydrated at 60 °C for 48 h in an oven (FELISA® FE-293A, Mexico) and ground to 1 mm size in a Thomas-Wiley Mill (Thomas Scientific®, Swedesboro, NJ, USA). The bromatological composition of the grass was 22.4 % dry matter (DM), 3.4 % crude protein (CP), 71.1 % neutral detergent fiber (NDF), 42.1 % acid detergent fiber (ADF), and 8.7 % ash (Ash).

The culture medium for the in vitro tests consisted of two-thirds reduced buffer-mineral solution and one-third fresh rumen fluid(11). The reduced buffer-mineral solution contained: 150 mL of mineral solution I [6 g K2HPO4 (Sigma) in 1,000 mL of distilled H2O], 150 mL of mineral solution II [6 g K2HPO4 (Sigma) in 1,000 mL of distilled H2O], 150 mL of mineral solution II [6 g KH2PO4 (Sigma) + 6 g (NH4)2SO4 (Merck) + 12 g NaCl (Sigma-Aldrich) + 2.45 g MgSO4 (Sigma) + 1.6 g CaCl-2H2O (Sigma) in 1 000 ml of distilled H2O], 100 mL of 8 % solution of Na2CO3 (Merck), 100 mL of reducing solution [0.1 g L-cysteine (Sigma) + 0.1 g Na2S-9H2O (Meyer) + 2 mL NaOH (2N; Meyer) in 100 mL distilled H2O] and 2 mL resazurin at 0.1% (Sigma-Aldrich). Fresh rumen fluid was obtained from a bovine with a rumen cannula grazing on pasture with pangola grass and filtered with a sky blanket to remove macroparticles of organic matter. The cattle were handled by the internal bioethics and welfare regulations of the Autonomous University of Guerrero, based on the official norms NOM-062-ZOO-1999 and NOM-051-ZOO-1995.

Subsequently, the following ratios of oil and ground plainer grass were each placed directly in a (120 mL) serological vial: 0 % (1 g grass), 2.5 % (0.025 g oil and 0.975 g grass), 5 % (0.05 g oil and 0.95 g grass), 7.5 % (0.075 g oil and 0.925 g grass), and 10 % (0.1 g oil and 0.9 g grass). 50 mL of culture medium was added to each vial, under a continuous flow of CO2, to maintain anaerobic conditions. The vials were closed with a neoprene cap and aluminum ring with a removable center and were considered biodigesters. The biodigesters were incubated in a double boiler at 39 °C for 72 h.

Methane (CH4) production was determined using a Taygon® hose (2.38 mm inner Ø and 45 cm length) with hypodermic needles (20 G x 32 mm) at the ends. The needles were used to couple a biodigester with a vial trap containing NaOH (2N); this was placed inversely in a modified test tube that was used to collect the NaOH solution (2N) displaced by the CH4 produced during incubation through the hypodermic needle placed as an outlet valve. CH4 production was measured at 0, 12, 24, 36, 48, and 72 h(12,13). The main reason for using this technique is that the main gases produced by the final products of in vitro microbial fermentation are CO2 and CH4, given that the rest of the gases produced in in vitro techniques are trace gases(14). Likewise, NaOH can capture CO2, as its reaction generates HCO3(15), so the CH4 production is measured as the displaced milliliters of NaOH solution.

The pH, ammonia nitrogen (N-NH3), and dry matter degradation (DMD) were determined after 72 h of incubation. A potentiometer (Hanna® HI2211, Italy; calibration pH 7 and 4) was utilized to estimate the pH of the culture medium. To measure The ammonia nitrogen (N-NH3) was measured by taking 1 mL of the medium contained in the biodigester and mixing it with 0.25 mL of 25 % metaphosphoric acid (Meyer®; 4:1 ratio) in a 2 mL Eppendorf tube (Neptune®, Mexico). The tube sample was centrifuged for 25 min at 3,500 xg, and the supernatant was recovered in 2 mL vials. A volume of 20 μL of this supernatant was mixed in a volumetric vial with 1 ml of phenol solution [10 mg of Na2(NO) Fe(CN)5.H2O (Meyer®) + 10 g of phenol crystals (Meyer®) in 1,000 mL of distilled water] and 1 mL of hypochlorite solution [7.5 g of NaOH (Reasol®) + 21.3 g of Na2HPO4 (Meyer®) + 15 mL of hypochlorite (5 %; Reasol®) in 1,000 ml of distilled water]. The mixture was incubated for 30 min at 37 °C in a (Shel Lab® 1227, USA) double boiler. Subsequently, 5 mL of distilled water was added for dilution and vortexed (Genie 2 G-560, USA). The absorbance was measured at 630 nm in a UV-VIS spectrophotometer (Jenway® 6850, USA), calibrating with a nitrogen concentration method (r2= 0.9994)(16). The DMD was quantified by filtering the residual solid sample from the biodigester using ANKOM® bags previously dried to constant weight. The sample bags were dried at 60 °C for 24 h in an oven. In vitro dry matter degradation (DMD) was calculated using the formula DMD %= (initial sample - residual sample / initial sample) * 100(17).

The cumulative CH4 production values were used to estimate the kinetics of CH4 production using the Gompertz model(18). The estimators A, b and k were estimated by nonlinear regression analysis, using the PROC NLMIXED procedure of the SAS statistical package(19). The model used was:

b= constant CH4 production rate of the potentially degradable material (ml h-1);

k= time lag (h), microbial efficiency constant factor, defined as the intercept of the time axis of the tangent line at the point of inflection;

The cumulative methane production at 12, 24, 36, 48 and 72 h, as well as the in vitro fermentative characteristics of each oil (garlic, sesame, and cinnamon) were analyzed using a completely randomized design with the GLM procedure of SAS(19). Mean values were compared with Tukey's test (P<0.05). The response to the growing increase in oil was calculated using linear and quadratic orthogonal contrasts. It should be noted that a descriptive analysis of the CH4 production kinetics estimators was performed.

CH4 production decreased linearly at 12, 24, 36, 36, 48, and 72 h of Koronivia grass fermentation (P<0.05) as the amaount of added garlic (Table 1) and cinnamon oil increased (Table 2). Sesame oil did not exhibit a linear or quadratic contrast in CH4 production after 12, 24, 36, 48, and 72 h of grass fermentation (P>0.05) as more of it was added (Table 3). This indicates that garlic and cinnamon oil reduce methane production in in vitro tests. However, in the case of garlic oil, the trend in the decrease and difference between inclusion levels became evident only with 7.5 % or more (Table 1). While, in the case of cinnamon oil, the effect on the decrease could be observed even with as little as 2.5 % (Table 2).

Table 1: Effect of garlic oil level on CH4 production and in vitro fermentative characteristics of Koronivia grass at 60 days of regrowth

Variable	Inclusion of garlic oil					MSE	Tukey test	Linear	Square
Variable	0 %	2.5 %	5.0 %	7.5 %	10 %	MSE	Tukey test	Linear	Square
Me12	11.49a	11.61a	11.15a	8.64b	9.62ab	0.35	0.0027	0.0013	0.8488
Me24	24.99ab	28.01a	25.43ab	21.96b	23.31b	0.62	0.0027	0.0058	0.1887
Me36	34.48ab	37.24a	33.73ab	28.81c	31.08bc	0.84	0.0007	0.0007	0.5140
Me48	38.95b	43.73a	37.11bc	34.21c	36.63bc	0.88	<0.0001	<0.0001	0.7042
Me72	43.98b	48.86a	41.53bc	39.61c	40.33c	0.92	<0.0001	<0.0001	0.2223
pH	6.01c	6.10b	6.11ab	6.14ab	6.17a	0.02	<0.0001	<0.0001	0.0912
DMD	59.48	59.37	58.31	55.31	53.74	0.93	0.1815	0.0396	0.9975
N-NH3	9.75	9.75	14.12	11.83	11.00	0.92	0.5416	0.5451	0.3324

Me12= methane production at 12 h of fermentation (mL g-1 DM), Me24= methane production at 24 h of fermentation, Me36= methane production at 36 h of fermentation, Me48= methane production after 48 h of fermentation, Me72= methane production after 72 h of fermentation, pH= hydrogen ion potential, DMD= dry matter degradation percentage, N-NH3= mg dL-1 of ammonia nitrogen, MSE= mean standard error.

a,b,c Average values with different letters in the same row are different (P<0.05).

Table 2: Effect of cinnamon oil level on CH4 production and in vitro fermentative characteristics of Koronivia grass at 60 days of regrowth

Variable	Inclusion of cinnamon oil					MSE	Tukey test	Linear	Square
Variable	0 %	2.5 %	5.0 %	7.5 %	10 %	MSE	Tukey test	Linear	Square
Me12	11.66a	10.25ab	9.11bc	8.28c	8.14c	0.37	<0.0001	<0.0001	0.0374
Me24	25.32a	23.91ab	22.79bc	20.88c	23.68ab	0.42	0.0004	0.0010	0.0009
Me36	34.65a	31.77b	30.15bc	27.73c	31.45b	0.64	<0.0001	0.0001	0.0001
Me48	38.64a	36.56ab	34.71bc	33.13c	36.27ab	0.54	0.0004	0.0009	0.0004
Me72	43.31a	39.29b	38.92b	38.53b	40.71b	0.51	0.0007	0.0066	0.0001
pH	6.01c	6.11b	6.20a	6.16ab	6.22a	0.02	<0.0001	<0.0001	0.0035
DMD	59.48a	58.36a	56.88ab	54.58b	55.49b	0.53	0.0007	<0.0001	0.1621
N-NH3	9.75a	7.25a	8.50a	8.50a	8.91a	0.33	0.2040	0.8484	0.0983

a,b,c Average values with different letters in the same row are different (P<0.05).

Table 3: Effect of sesame oil level on CH4 production and in vitro fermentative characteristics of Koronivia grass at 60 days of regrowth

Variable	Inclusion of sesame oil					EEM	Tukey test	Lineal	Cuadratic
Variable	0 %	2.5 %	5.0 %	7.5 %	10 %	EEM	Tukey test	Lineal	Cuadratic
Me12	11.66	10.59	10.51	10.80	9.99	0.22	0.1939	0.0524	0.6139
Me24	25.32	25.28	23.84	24.48	24.42	0.30	0.5145	0.2516	0.4369
Me36	34.65	32.11	32.60	33.12	32.93	0.31	0.0737	0.1977	0.0461
Me48	38.64	37.57	37.86	38.52	37.74	0.21	0.4146	0.5729	0.5973
Me72	43.31	42.70	43.12	42.49	43.29	0.24	0.8175	0.9001	0.4485
pH	6.01c	6.13b	6.14b	6.17ab	6.22a	0.02	<0.0001	<0.0001	0.0085
DMS	59.48a	57.88ab	56.12b	53.33c	53.29c	0.68	<0.0001	<0.0001	0.2876
N-NH3	9.75	9.75	8.91	9.75	8.91	0.35	0.8964	0.5684	1.000

Mane productzion at 36 h of fermentation, Me48= methane production after 48 h of fermentation, Me72= methane production after 72 h of fermentation, pH= hydrogen ion potential, DMD= dry matter degradation percentage, N-NH3= mg dL-1 of ammonia nitrogen, MSE= mean standard error.

a,b,c Average values with different letters in the same row are different (P<0.05).

Dry matter degradation (DMD) decreased linearly (P<0.05) as the inclusion of garlic (Table 1), cinnamon (Table 2), or sesame (Table 3) oil increased. This decrease was reflected in the pH value of the culture media; the pH augmented linearly (P<0.05) as the inclusion of garlic (Table 1), cinnamon (Table 2), and sesame (Table 3) oil increased.

The ammonia nitrogen content (N-NH3) did not exhibit (P>0.05) linear or quadratic effects, or differences between levels of inclusion of garlic oil (Table 1), cinnamon oil (Table 2), or sesame oil (Table 3), as their inclusion in the fermentation of Koronivia grass increased.

The kinetics of CH4 fermentation using garlic oil showed similar values in A and k when 5 and 7.5 %, respectively, were added, while in b they were lower with the addition of 7.5 and 10 %, compared to the values obtained without any added oil (control). Also, in relation to the control, the inclusion of 2.5 % of sesame oil resulted in lower values in the estimators A, k, and b. In contrast, in estimator A, all cinnamon oil inclusion levels exhibited lower values than the control; while in estimator b, the values decreased with the addition of 7.5 %, and in estimator k, with 5 and 7.5 % (Table 4).

Table 4: Average estimators of in vitro CH4 production kinetics of plainer grass with 60 days of regrowth supplemented with increasing levels of garlic, sesame or cinnamon oil

Oil	% of inclusion	A (ml g-1 of DM)	k (h)	b (ml h-1)
Control	0.0	42.54	3.55	0.077
Garlic oil	2.5	47.50	3.52	0.075
	5.0	42.53	3.47	0.075
	7.5	42.55	3.37	0.068
	10.0	43.77	3.35	0.067
Sesame oil	2.5	35.62	3.23	0.071
	5.0	44.72	3.91	0.076
	7.5	41.16	3.52	0.078
	10.0	43.45	3.42	0.071
Cinnamon oil	2.5	36.61	3.69	0.077
	5.0	39.66	3.49	0.074
	7.5	34.75	3.34	0.064
	10.0	38.98	4.17	0.082

A= total methane production potential, b= constant methane production rate, k= time lag.

The goal of ruminant microbiologists and nutritionists is to manipulate rumen microbial ecosystems to improve feed intake efficiency(7). Additives used as CH4 inhibitors act directly in the methanogenesis pathway, interrupting the process. Oils in the rumen environment exhibit toxic characteristics on methanogens and protozoa, hydrogenation of unsaturated fatty acids (alternative sink for hydrogen), and changes in propionic production leading to reduced CH4 production(20).

The decrease in accumulated CH4 production at different times measured by garlic and cinnamon oils are assumed to contain terpenoids and phenylpropanoids that interact in the cell membrane, as the hydrophobic nature of their cyclic hydrocarbons allows them to accumulate in the lipid bilayer, causing conformational changes in the membrane structure that result in loss of cell membrane stability(21).

Delgadillo-Ruiz et al(5) utilized nonlinear models for their estimates and reported yields of 183, 99, and 141 mM L-1 of CH4 when 0.1, 0.3, and 0.6 mL of cinnamon oil were added using 41.5 % alfalfa, 41.5 % wheat straw, and 17 % of a corn grain-based concentrate as substrate; these values differ from those obtained in the present study (Table 2) because they do not show a tendency to decrease CH4 as the addition of cinnamon oil increased. Cobellis et al(3) reported 3.67 mL CH4 g-1 of DM in a 24-h in vitro fermentation using alfalfa hay as substrate and adding 1.125 mL L-1 of cinnamon oil culture medium; these values are lower than those of the present study, even concerning the control treatment (Table 2). This is a consequence of the methodology used for measuring the CH4, substrate, inoculum source, etc.(13), all of which influence CH4 production.

Concentrations of up to 10 % were estimated in the present in vitro study; however, NRC(22) mentions that the added oils should not exceed 7 % of the dry matter of the diet, because higher contents of oil may hinder an adequate dry matter intake. For this reason, in vivo tests should be carried out to assess the effect of cinnamon oil on the reduction of greenhouse gases, as reductions were observed with the addition of at least 2.5 % of this oil, as well as with a minimum addition of 5 % of garlic oil.

The addition of garlic oil did not exhibit differences in dry matter degradation (DMD) between oil inclusion levels; its tendency to reduce DMD value was observed with the addition of 5 % or more. In the case of sesame and cinnamon oil, differences (P<0.05) and a tendency to reduce DMD were observed with the addition of as little as 5 %. The decrease in DMD can be assumed to be a consequence of hydrogen accumulation that affected fiber degradation(20), and it may be inferred that the oils reduced protein and starch degradation in response to inhibition of the bacteria used in the inoculum(23). Also, unsaturated fatty acids are toxic to fiber-hydrolyzing bacteria; these acids adhere to the cell wall(24), thereby reducing the ability of the bacteria to attach to the grass and hydrolyze it.

In vitro fermentation of bermudagrass (Cynodon dactylon) produced a linear decrease in dry matter degradation with the increasing addition of thyme oil (50, 250 and 500 mL g-1)(25), a similar situation to that reported in the present study with the three oils. Likewise, Cobellis et al(3) reported lower values of DMD than the present study, as they assessed 55 % of DMD in an in vitro fermentation where they used alfalfa hay as substrate and added 1.125 mL L-1 of cinnamon oil culture medium.

The pH values in the present study are ascribed to the DMD, since volatile fatty acids, which decrease the pH, are a product of its degradation; however, when the DMD decreased as the concentration of the different oils evaluated increased, the production of volatile fatty acids diminished and did not affect the pH value. Busquet et al(7) reported a quadratic effect on the pH value of the culture media, with a tendency to increase as more garlic oil was added to a diet containing 50:50 forage:concentrate, a behavior similar to that reported in the present study with the three oils.

The concentration of N-NH3 in the present study is because oils do not inhibit the metabolism of ammonia nitrogen-producing bacteria(23). Researchers(3) reported 13.5 mg dL-1 of ammonia nitrogen in an in vitro fermentation of alfalfa with 1.125 mL L-1 of cinnamon oil; these values are higher than those obtained in the present study because alfalfa has a higher protein content than the Koronivia grass. Another study reported(7) that the N-NH3 content exhibited no differences concerning the treatment that did not contain garlic oil, a situation similar to that of the present study with the three oils.

The effects of these oils tend to be influenced by their components, which make it difficult to analyze their effect on ruminant nutrition. Therefore, further studies are required to identify the metabolites contained in each oil to establish their real effect on the fermentation of forages as the main producers of methane, given the fermentation stoichiometry.

The modified Gompertz equation is a common model for CH4 production by degradation of a simple organic substrate(26). The literature includes several studies that utilized this model to estimate CH4 production. He et al(27) applied a modified Gompertz model and a first-order kinetic model to evaluate CH4 production during the in vitro fermentation of wheat straw using bull and heifer fluid as inoculum; their results showed lower values in A (22 ml CH4 g-1) and k ( 0.945 h), as well as higher values in b (0.105 mL h-1). Another study(28) evaluated the CH4 potential and the CH4 production rate of stalk bark, stalk pith, and corn stubble leaves from batch anaerobic digestion, reporting higher values than those estimated in the present study in A (204.8 ml CH4 g-1) and lower values in k (0.1553 h). Zhang et al(26) reported higher values than the present study, as they published values of 94.38 mL CH4 g-1 for A, 12.38 h for k, and 2.46 ml h-1 for b in the fermentation of cow manure with corn stubble. With the differences in the estimators reported by other authors(26,27,28) used for comparison and those of the present study, it is assumed that whether essential oils with anti-metagenomic properties are added or not depends on the conditions under which the experiments were performed and the substrates used, since they directly influence the kinetics of methane production.

Therefore, the modeling of CH4 production under the conditions of the present study was important, because these serve for the design, construction and application of chemical or biochemical processes. Furthermore, they describe the characteristics of the process and allow its subsequent optimization(29).

The addition of garlic or cinnamon oil to the in vitro fermentation of Koronivia grass reduces methane production and dry matter degradation. Sesame oil does not exhibit anti-methanogenic activity under the conditions of the present study, but it reduces the in vitro degradation of dry matter.

The authors are grateful to the academic team of the "Sustainable Production of Ruminants in the Tropics" for financing this project, and to the student Adrián Medina Calvo for his support in the work done in the laboratory as part of his professional practices of the Veterinary Medicine and Animal Husbandry degree of the Autonomous University of Guerrero.