ANIMAL SCIENCE

Effect of cinnamaldehyde on in vitro ruminal degradability and volatile fatty acids production.

P. Catalá-Gregori^{1  *}  , V. García¹  , F. Hernández¹  , J. Madrid¹  , M. D. Megías¹  , J. Orengo¹ 

¹Department of Animal Production, University of Murcia, Murcia 30071, Spain

ABSTRACT

The aim of this research was to study the effect of cinnamaldehyde on nutrients ruminal degradation and total production and molar proportion of volatile fatty acids in a ruminal in vitro system (DaisyII Ankom Technology, USA). The diet was composed of a mixture of barley seed: alfalfa hay (70:30) which was incubated with 4 treatments: without additive or negative control (NC), with monensin at 7.5 ppm or positive control (CP) and with cinnamaldehyde at 250 (C250) and 500 ppm (C500). The inhibitory effect of cinnamaldehyde on disappearance of DM, NDF and ADF (from 48 to 72 h incubation time, P < 0.01) was similar to CP treatment. All supplemented treatments tended to decrease the potential degradability of DM, CP, NDF, ADF, as the effective degradability of DM, NDF and ADF. However, contrary to monensin, cinnamaldehyde increased total VFA production and did not affect molar proportion of VFA. The two doses of cinnamaldehyde evaluated showed the same effect. Results indicate that cinnamaldehyde modified in vitro ruminal fermentation.

Key words: goat; cinnamon; monensin; ruminal fermentation; artificial incubator

INTRODUCTION

Ruminant animals are unable to produce fiber-degrading enzymes. They have developed a symbiotic relationship with ruminal microorganisms (bacteria, fungi and protozoa) that provide them protein, vitamins and volatile fatty acids in exchange of a suitable habitat for growth. Contrary to wild ruminants, domestic ruminants are often fed an abundance of grain and little fiber. When ruminants are fed fiber-deficient rations, physiological mechanisms of homeostasis are disrupted, ruminal pH declines, microbial ecology is altered, and the animal becomes more susceptible to metabolic disorders and, in some cases, infectious diseases. Some disorders can be counteracted by feed additives as ionophores (^{Russell and Rychlik 2001}).

One of the additives commonly used in domestic ruminants is monensin, because of its positive effect on improvement of energy and nitrogen metabolism, and its preventing effect against digestive disorders resulting from abnormal rumen fermentation (^{Castillo et al. 2004}). However, the use of antibiotics as growth promoters in livestock has been limited in the European Union because of their probable implication in the development of microbial antibiotic resistance. In order to face this prohibition, alternatives must be proposed to keep animal health, productivity and microbial food safety.

Plants produce several secondary compounds with antimicrobial activity (^{Cowan 1999}). Cinnamaldehyde is the main compound of cinnamon’s essential oil and its in vitro antimicrobial effect has been widely demonstrated (^{Hili et al. 1997}, ^{Helander et al. 1998} and ^{Valero and Salmerón 2003}). It has been reported that cinnamaldehyde could be considered as potential alternative to monensin to modify rumen fermentation in beef cattle (^{Khorrami et al. 2015}). Cinnamaldehyde had a limited potential to improve feed efficiency and growth in lambs fed concentrate-based diets (^{Chaves et al. 2011}). A diet-effect was reported in sheep when using cinnamaldehyde to modify rumen fermentation (^{Mateos et al. 2013}). No data of cinnamaldehyde used as additive are available for Murciano-Granadina goats. Additionally, essential oils have been shown to be promising feed additives in mitigating methane and ammonia emissions (^{Cobellis et al. 2016}). However, the mode of action and activities of essential oils on rumen microbiome remain poorly understood (^{Cobellis et al. 2016}).

The effect of cinnamaldehyde on goat rumen in vitro fermentation was evaluated. To achieve this, degradability of dry matter, protein and fiber were measured in an artificial incubator. Volatile fatty acids (acetic, propionic and butyric acids) and cinnamaldehyde concentrations in ruminal fluid were also determined.

MATERIALS AND METHODS

Materials and chemicals. The substrate used for incubation was a mixture (dry matter basis) of 700 g barley grain (Hordeum vulgare) and 300 g alfalfa hay (Medicago sativa) Barley grain and alfalfa hay were milled through 1 mm screen (Hammer mill, Culatti, Italy). Cinnamaldehyde, monensin (95%) and propionic acid were supplied by Fluka Chemika (Switzerland). Acetic acid was purchased from Riedel-de Haen (Germany) and butyric and 4-methylvaleric acid from Sigma Aldrich (USA).

Rumen fluid. Ruminal fluid was collected from two fistulated Murciano-Granadina goats fed alfalfa hay ad libitum. Rumen fluids were pooled and transported to the laboratory in a sealed thermos. Then, it was immediately strained through four layers of cheesecloth and mantained with CO₂ flow to keep anaerobic conditions.

In vitro incubations and treatments. In vitro incubations were performed with the DaisyII (Ankom Technology, USA) incubator (^{Mandebvu et al. 2001}). Incubator is composed of four digestion jars (2 L capacity) maintained at 39.5 ºC in constant rotation and under a CO₂ saturated atmosphere. Each digestion jar was filled with prewarmed (39.5 ºC) buffer solution (containing 1317 mL solution A: KH₂PO₄, 10 g/L; MgSO₄.7H₂O, 0.5 g/L; NaCl, 0.5 g/L; CaCl₂.2H₂O, 0.1 g/L and CO (NH₂)₂, 0.5 g/L and 267 mL solution B: Na₂CO₃, 15 g/L and Na₂S.9H₂O, 1 g/L, fitted pH to 6.8); 400 mL of rumen fluid and 16 mL of additive solution.

Four treatments were tested, one per each in vitro jar: Negative control (NC), positive control (CP) with monensin 7.5 ppm and cinnamaldehyde at 250 ppm (C250) and 500 ppm (C500). Additives (monensin and cinnamaldehyde) were initially dissolved in 16 mL of ethanol. So as to preclude all confusion due to main effects of ethanol, this product was also added (16 mL) to negative control treatment (NC). The selected concentrations of monensin and cinnamaldehyde were based on published data, that they had showed in vitro antimicrobial properties, for monensin ^{Dennis et al. (1981)}, ^{Domescik and Martin (1999)} and ^{Wang et al. (2004)} and for cinnamaldehyde ^{Hili et al. (1997)}; ^{Chang et al. (2001)} and ^{Valero and Salmerón (2003)}.

Twenty-eight sample were weighed (0.5 g) per jar into ANKOM F57 filter bags (ANKOM Technology, USA). The bags (size 5.5 cm x 5 cm) had a pore size of 25 μm and were heat sealed. Four bags per treatment were removed at 0, 4, 8, 12, 24, 48 and 72h incubations times. The bags were immediately washed under running tap water until the water was clear, oven-dried at 60 °C for 48 h and weighed. The content of bags was frozen until posterior analysis.

Chemical analyses. Dry matter was determined by drying the samples at 60ºC during 48h; crude protein (CP) by Kjeldahl method (^{AOAC, 1990}); neutral detergent fiber (NDF) and acid detergent fiber (ADF) by ^{Van Soest and Roberston (1991)}.

Samples preparations for GC analysis. At 48 h of incubation two samples of 50 mL of rumen fluid from each jar were collected. The samples were centrifuged at 5,000 g for 20 min and the supernatant was acidified with 1 mL of 50% sulphuric acid. The supernatant was stored at -20 ºC. For GC analysis 5 mL of acidified ruminal samples, 5 mL of deionized water and 0.5 mL of an internal standard solution (4 methyl-n-valeric acid 30 mM) were mixed and centrifuged (7 min at 7,500 g).

GC analysis conditions. The determination of the volatile fatty acids and cinnamaldehyde in rumen fluid was performed by using a Thermo Finnigan Trace gas chromatograph (Thermo, Italy), equipped with a flame ionization detector and 30 m × 0.25 mm i.d., 0.25 μm TR-FFAP fused silica capillary column (Teknokroma, Spain). The oven temperature was held at 90 ºC for 1 min and increased from 90 to 125 ºC at the rate of 2.6 ºC/min and from 125 to 180 ºC at 10 ºC/min. The temperatures of the injector and detector were 220 and 260 ºC, respectively. The flow rate of carrier gas was 2.0 mL/min and injection volume was 1 μL. FID air flow was 350 mL/min, while the H2 flow was 35 mL/min (^{Madrid et al. 1999}). Data processing was performed by with Chrom-Card Data System (Finnigan, Italy). All the products were determined in compared to the peak area of the internal standard (4 methyl-n-valeric acid).

Calculations and statistical analysis. The disappearance values of DM, CP, NDF and ADF from the filter bags at each incubation time were fitted to the exponential equation described by ^{Ørskov and McDonald (1979)} y=a +b (1-e^-ct), where y is the loss of the analysed feed component from the filter bag at time t, a is the soluble fraction, b is the insoluble potentially degradable fraction, and c is the fractional rate of b degradation. When a value is negative or b value is more than 100, we made use of the equation described by ^{McDonald (1981)} y=b (1-e^{-c (t-L)}) where L indicates the time that remains to begin the degradation of b fraction. Potential degradability (PD) is the sum of a and b fraction. Effective degradability (ED) was calculated for the equation described by ^{Ørskov and McDonald (1979)} ED =a + ((bc)/(c + r)) or for the equation described by ^{McDonald (1981)} ED =((bc)/(c + r)) ^(-(c+r)L) assuming a constant value of the fractional rate of passage (r) of 0.06 h^-1 (^{Sauvant et al. 2004}).

Data of fermentation in DAISYII incubator were analysed by ANOVA. For nutrient degradation the model used was Y= µ +A+B+AB+ԑ, where µ is the mean, A and B are the effects of additive type and incubation time, respectively, ABis the interaction additive type x incubation time and ԑ_ijk is the error. Data of VFA were analyzed by one-way ANOVA. Differences between treatment means were established with a Least Significant Difference (LSD) test. Statistical significance was stated when P < 0.05. All calculations were done using ^{SPSS (1997)}.

RESULTS AND DISCUSSION

The chemical composition of incubated diet is presented in table 1. The diet was 12.87% CP, 28.54% NDF and 12.84% ADF.

Table 1 Chemical composition of incubated mixed diet (as DM basis) ^a 

Composition (g/kg DM)	Mixed diet (700 g barley grain + 300 g alfalfa hay)
OM	941.6
Ash	58.1
CP	128.7
NDF	285.4
ADF	128.4
ADL	19.6

^a OM = organic matter; CP = crude protein; NDF = neutral-detergent fibre; ADF = acid-detergent fibre; ADL = acid-detergent lignin.

Dry matter degradability. Dry matter disappearance according to treatment type and incubation time, as kinetic of DM degradability are shown in table 2. There was a significative interaction between incubation time and treatment (P = 0.007). Additives (P < 0.05) influenced DM disappearance. At 48h and 72h incubation times, PC, C250 and C500 treatments reduced (P < 0.001) DM disappearance in comparison with NC treatment. On the other hand, DM disappearance (P < 0.001) increased when incubation time advanced. All additives reduced DM potential degradability (table 2) in comparison to NC treatment (79.4, 78.5, 78.6 vs 83.6 %). The decrease of PD corresponded with a reduction of potentially degradable fraction b. In vitro studies (^{Jalc et al. 1992} and ^{Wang et al. 2004}) have demonstrated a reduction of DM disappearance when monensin was added at 2.5 and 15 ppm. The same effect for monensin has been reported in vivo (^{Rogers et al. 1997} and ^{Wang et al. 2004}) studies. For an essential oils blend (thymol (5-methyl-2-(1-methylethyl) phenol), guaiacol (2-methoxyphenol), limonene (1-methyl-4-(1-methylethenyl) cyclohexene)) has been reported a reduction of in situ DM disappearance for concentrate feedstuff as peas (Pisum sativum) (^{Molero et al. 2004}) or soybean meal (Glycine max) (^{Newbold et al. 2004}). These essential oils blend inhibited the growth of the most pure cultures of ruminal bacteria at concentrations of less than 100 ppm (^{McIntosh et al. 2003}).

Table 2 Effect of treatments on dry matter degradability. Disappearance kinetic’s according to ^{Ørskov and McDonald (1979)} model 

Treatment^a	Incubation time (h)
	0	4	8	12	24	48	72	SEM	P-value
Negative control	44.5	50.8^bc	62.6	64.7	72.6	82.3^c	83.0^c	0.5	0.001
Monensin 7.5ppm	46.3	54.8^c	60.5	63.2	72.4	76.8^b	80.0^b	0.5	0.001
Cinnamaldehyde 250ppm	45.5	47.9^b	60.2	66.3	73.1	75.9^b	79.2^b	0.4	0.001
Cinnamaldehyde 500ppm	44.2	48.1^b	59.8	65.9	73.7	76.1^b	79.7^b	0.6	0.001
SEM	1.1	0.7	0.8	0.5	0.3	0.4	0.3
P-value	0.895	0.015	0.635	0.110	0.314	0.001	0.005
Exponential equation: y=a+b(1-e ^-ct )
	a (%)	b(%)	c( h^-1)	PD (%)^d	ED (%)^e	R^2	RSD^f
Negative control	44.4	39.2	0.061	83.6	64.2	95.6	3.0
Monensin 7.5ppm	46.9	32.5	0.062	79.4	63.4	95.6	2.4
Cinnamaldehyde 250ppm	43.4	35.1	0.075	78.5	62.9	94.5	3.0
Cinnamaldehyde 500ppm	42.8	35.8	0.077	78.6	62.9	93.6	3.3

^a Incubation time x treatment interaction, P = 0.007.

^b,c Means with different superscript within a column are significantly different (P<0.05).

^d PD = a + b.

^e ED = a + [( bc)/(c+r)], assuming a constant value of r of 0.06 h^-1.

^f RSD = Residual standard deviation.

Crude protein degradability. Crude protein disappearance according to treatment type and incubation time, as kinetic of CP degradability are shown in table 3. There was a significative interaction between incubation time and treatment (P = 0.030). The effects of the tested additives on CP degradation were not so noticeable, as a very high CP soluble fraction (a) was found. Monensin and cinnamaldehyde did not affect CP disappearance (P > 0.05), though a significant (P < 0.05) interaction between treatment type and incubation time was noticed (table 3). Moreover, at 48 h of incubation, CP disappearance was smaller (P < 0.001) in supplemented treatments than in treatment NC (94.9, 94.9 and 93.3% vs 98.1% for PC, C250, C500 and NC treatments respectively). The potentially degradable fraction (b) decreases lightly when additives were included, a similar trend in the PD was observed. It is known that monensin decreases ruminal CP degradation (^{Van Nevel and Demeyer 1977} and ^{Hillaire et al. 1989}). ^{Ghorbani et al. (2010)} showed that monensin could decrease the amount of ammonia in rumen liquid. This effect could be due to the fact that momensin negatively affects the gram-positive bacteria population that have high activity of ammonia production (^{Duffield et al. 2002}). However, the ruminal microbial synthesis does not seem to be affected by monensin inclusion (^{Ghorbani et al. 2010}). On the other hand, some works did not find any effect of monensin on CP degradation (^{Vanhaecke et al. 1985}) while others showed an increase of CP degradation (^{Benchaar et al. 2006}), probably due to several factors, as monensin concentration or the type of diet used. In situ CP degradation can also be reduced by antimicrobial plants products. A blend of thymol (5-methyl-2-(1-methylethyl)phenol), guaiacol (2-methoxyphenol) and limonene (1-methyl-4-(1-methylethenyl)cyclohexene) reduced the soybean meal CP degradation in sheeps (^{Newbold et al., 2004}) and lupin seeds (Lupinus angustifolius), green peas (Pisum sativum) and sunflower meal (Helianthus annuus) CP degradation in growing heifers fed with a high concentrate diet (^{Molero et al. 2004}). ^{Tager and Krause (2010)} showed that crude protein digestibility was depressed with cinnamaldehyde and eugenol (500mg/L/d).

Table 3 Effect of treatments on crude protein degradability. Disappearance kinetic’s according to ^{Ørskov and McDonald (1979)} model 

Treatment^a	Incubation time (h)
	0	4	8	12	24	48	72	SEM	P-value
Negative control	79.0	81.2^b	88.0	89.1	94.1	98.1^d	98.4	0.3	0.001
Monensin 7.5ppm	81.0	84.9^c	87.3	88.4	94.5	94.9^c	98.2	0.4	0.001
Cinnamaldehyde 250ppm	81.5	80.5^b	87.5	90.8	93.8	94.9^c	97.6	0.4	0.001
Cinnamaldehyde 500ppm	79.2	84.8^c	89.6	90.7	95.1	93.3^b	97.4	0.5	0.001
SEM	0.9	0.3	0.9	0.5	0.2	0.2	0.2
P-value	0.714	0.013	0.798	0.293	0.303	0.001	0.401
Exponential equation: : y=a+b(1-e ^-ct )
	a (%)	b (%)	c ( h^-1)	PD (%)^e	ED (%)^f	R^2	RSD^g
Negative control	78.4	20.5	0.063	98.9	88.9	96.3	1.4
Monensin 7.5ppm	81.2	16.4	0.056	97.6	89.1	93.8	1.5
Cinnamaldehyde 250ppm	79.9	17.0	0.066	96.9	88.8	88.7	2.2
Cinnamaldehyde 500ppm	80.2	14.2	0.090	94.4	88.7	83.0	2.6

^a Incubation time x treatment interaction, P = 0.030.

^b,c,d Means with different superscript within a column are significantly different (P<0.05).

^e PD = a + b.

^f ED = a + [( bc)/(c+r)], assuming a constant value of r of 0.06 h^-1.

^g RSD = Residual standard deviation

Fiber degradability. NDF and ADF disappearance according to treatment type and incubation time, as kinetic of NDF and ADF degradability are shown in table 4 and 5. There was a significative interaction between incubation time and treatment (P = 0.001) for both NDF and ADF. NDF and ADF degradations were reduced (P < 0.001) with additive supplementation. In addition, the effect of cinnamaldehyde at 250 ppm on fiber degradation was similar to that observed with monensin. Fiber disappearance for C500 treatment did not begin until 8 h of incubation for NDF degradation and until 12 h of incubation in all treatments for ADF degradation. C500 treatment showed a time "lag" from which NDF degradation started. A time “lag” was necessary for ADF fraction in all treatments used. This result is consistent with the fact that ADF does not have soluble fraction (^{Van Soest, 1994}). PC, C250 and C500 treatments tended to reduce PD and ED of the fiber fraction, in comparison with NC treatment. Monensin inhibitory effect on fiber degradation is well documented by other authors in in vitro and in situ trials (^{Jalc et al. 1992}). The effect of plants extracts on fiber degradation is depending on their chemical structure. This way, tannins (^{Hervás et al. 2003}) and saponins (^{Wina et al. 2005}) could to reduce in situ NDF degradation, while Achillea millefolium extract containing flavonoids, increased NDF and ADF degradabilities (^{Broudiscou et al. 2002}).

Table 4 Effect of treatments on neutral detergent fiber degradability. Disappearance kinetic’s according to ^{Ørskov and McDonald (1979)} or ^{McDonald (1981)} model 

Treatment^a	Incubation time (h)
	0	4	8	12	24	48	72	SEM	P-value
Negative control	6.0^d	14.9^d	19.7^c	24.6^d	29.8^c	44.3^c	49.5^d	0.3	0.001
Monensin 7.5ppm	5.2^d	14.5^d	18.8^c	21.0^c	25.1^b	31.5^b	37.9^c	0.3	0.001
Cinnamaldehyde 250ppm	2.3^c	9.9^c	16.2^c	23.3^cd	26.3^b	27.8^b	34.6^bc	0.4	0.001
Cinnamaldehyde 500ppm	0.0^b	0.0^b	0.0^b	17.4^b	25.6^b	31.5^b	31.7^b	0.3	0.001
SEM	0.3	0.1	0.6	0.3	0.4	0.5	0.5
P-value	0.008	0.001	0.001	0.005	0.050	0.001	0.001
Exponential equation: y=a+b(1-e ^-ct ) or y= b(1-e ^-c(t-L) )
	a (%)	b (%)	c (h^-1)	L (h)	PD (%)^e	ED (%)^f	R^2	RSD ^g
Negative control	8.0	45.3	0.033		53.3	24.1	98.1	2.1
Monensin 7.5ppm	7.7	29.0	0.047		36.7	20.4	94.2	2.5
Cinnamaldehyde 250ppm	2.1	29.3	0.085		31.4	19.3	94.2	2.6
Cinnamaldehyde 500ppm		34.2	0.048	2.38	34.6	11.8	88.6	4.9

^a Incubation time x treatment interaction, P = 0.001.

^b,c,d Means with different superscript within a column are significantly different (P<0.05).

^e PD = a + b.

^f ED = a + [( bc)/(c+r)] or [( bc)/(c+r)]^(-(c+r)L), assuming a constant value of r of 0.06 h^-1.

^{g R}SD = Residual standard deviation.

Table 5 Effect of treatments on acid detergent fiber degradability. Disappearance kinetic’s according to ^{McDonald (1981)} model. 

Treatment^a	Incubation time (h)
	12	24	48	72	SEM	P-value
Negative control	5.0^d	11.0^c	28.3^c	37.7^c	0.4	0.001
Monensin 7.5ppm	0.0^b	2.2^b	12.6^b	22.1^b	0.2	0.001
Cinnamaldehyde 250ppm	5.2^d	9.1^c	11.3^b	20.7^b	0.5	0.001
Cinnamaldehyde 500ppm	2.8^c	9.1^c	15.4^b	18.0^b	0.3	0.001
SEM	0.2	0.7	0.6	0.7
P-value	0.003	0.040	0.001	0.002
Exponential equation y= b(1-e ^-c(t-L) )
	b (%)	c (h^-1)	L (h)	PD (%)^e	ED (%)^f	R^2	RSD ^g
Negative control	39.6	0.025	4.35	39.6	8.0	92.1	4.2
Monensin 7.5ppm	23.5	0.019	6.18	23.5	3.5	82.9	3.5
Cinnamaldehyde 250ppm	19.8	0.027	3.11	19.8	4.7	86.1	2.8
Cinnamaldehyde 500ppm	19.6	0.029	3.71	19.6	5.6	93.5	1.9

^a Incubation time x treatment interaction, P = 0.001.

^b,c,d Means with different superscript within a column are significantly different (P<0.05).

^e PD = a + b.

^f ED = [( bc)/(c+r)]^(-(c+r)L), assuming a constant value of r of 0.06 h^-1.

^g RSD = Residual standard deviation.

Volatile fatty acids. The effect of treatments on volatile fatty acids production is shown in table 6. The main effect of monensin on ruminal energy metabolism is to increase production of propionic acid and to reduce the production of acetic acid, resulting in a lower acetic: propionic ratio (^{Wang et al. 2004}). In our study, this effect was also found. Cinnamaldehyde increased the total VFA production (P < 0.01) compared with NC or PC, but did not affect the molar proportion of acetate, propionate or butyrate compared with the control treatments. When cinnamaldehyde was added at 2.2 mg/mL concentration in a continuous culture system, total VFA production and propionate molar proportion were not affected, but a lower molar proportion of acetate and a numerical increase in molar proportion of butyrate were noticed (^{Busquet et al. 2005}). In similar incubators, the addition of 0.22 mg/mL of an extract of cinnamon with a 59% of cinnamaldehyde increased the molar proportion of acetate and decreased the molar proportion of propionate and butyrate during the adaptation period. However, these effects disappeared after 6 days of fermentation (^{Cardozo et al. 2004}). An increase of total VFA production due to plant extracts has also been reported by other study (^{Wina et al. 2005}). Thus, the addition of 2 or 4 mg/mL of a methanol extract from Sapindus rarak containing saponins to glass syringes incubators in vitro, it increased the total VFA production at 48 h of incubation. In contrast, a dose of 540 mg/L of cinnamaldehyde reduced the total VFA production in a 16h incubation of mixed rumen microorganism with medium and high concentrate content diets, while lower doses (180, 60 and 20 mg/L) didn´t affect total VFA production in comparison to control group (^{Mateos et al. 2013}). This way, ^{Macheboeuf et al. (2008)} found no increase of total VFA production with cinnamaldehyde supplementation at doses of 132 or 264 mg/L, but total VFA production were reduced by cinnamaldehyde doses of 396 and 661 mg/L. ^{Blanch et al. (2016)} describes reduction in total VFA with a dose of 172 mg/L of cinnamaldehyde.

Table 6 Effect of treatments on volatile fatty acids production at 48 h of in vitro incubation 

Treatments	Total VFA (mM)	Molar proportion VFA (%)
		Acetic acid	Propionic acid	Butyric acid
Rumen fluid at 0 h of incubation	7.45	78.94	9.79	11.25
Rumen fluid at 48 h of incubation
Negative control	21.06 ^b	70.17	16.76 ^b	13.05 ^a
Monensin 7.5 ppm	16.65 ^b	66.16	23.74 ^a	10.09 ^b
Cinnamaldehyde 250 ppm	28.17 ^a	69.61	16.70 ^b	13.66 ^a
Cinnamaldehyde 500 ppm	28.91 ^a	69.85	17.92 ^b	12.18 ^ab
SEM	0.64	0.82	0.45	0.37
P-value	0.003	0.152	0.002	0.021

^a-b Means with different superscript within a column are significantly different (P<0.05)

When gas chromatography analyses were performed, a peak in ruminal samples with cinnamaldehyde treatments was found (Figure 1). This peak was identified using a standard solution of cinnamaldehyde. As consequence, the method developed could be used to determine both VFA and plant-derived compounds as cinnamaldehyde in ruminal fluid, method in which an apolar solvent extraction would be not needed as well.

Figure 1 Chromatogram of rumen fluid with cinnamaldehyde (500 ppm) at 48h of incubation. Peaks: 1= acetic acid, 2= propionic acid, 3= butyric acid, 4= 4 methyl n-valeric acid, 5= cinnamaldehyde. 

In conclusion, results indicated that cinnamaldehyde modified in vitro goat ruminal fermentation. This phenolic compound reduced DM and fiber degradation in the same fashion as monensin did. However, contrary to monensin, cinnamaldehyde increased total VFA production and did not affect molar proportion of VFA. The two doses of cinnamaldehyde used showed an identical effect and probably were too high. Further research with lower doses and in vivo study are required.

ACKNOWLEDGEMENTS

P. Catalá-Gregori was recipient of a research fellowship (AP2002-3340) from the Ministry of Education and Science of Spain.

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Received: January 17, 2019; Accepted: May 05, 2019

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