cjasCuban Journal of Agricultural ScienceCuban J. Agric. Sci.2079-3480Ediciones ICA00003ANIMAL SCIENCEEffect of cinnamaldehyde on in vitro ruminal degradability and volatile fatty acids production.Catalá-GregoriP.1*GarcíaV.1HernándezF.1MadridJ.1MegíasM. D.1OrengoJ.1Department of Animal Production, University of Murcia, Murcia 30071, SpainUniversidad de MurciaDepartment of Animal ProductionUniversity of MurciaMurciaSpain
Email: direccion@cecav.es051220191220195343613721701201905052019This is an open-access article distributed under the terms of the Creative Commons Attribution LicenseABSTRACT
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.
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 ԑijk 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.
Chemical composition of incubated mixed diet (as DM basis) <sup>a</sup>
Composition (g/kg DM)
Mixed diet (700 g barley grain + 300 g alfalfa hay)
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).
Effect of treatments on dry matter degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B30">Ørskov and McDonald (1979)</xref> model
Treatmenta
Incubation time (h)
0
4
8
12
24
48
72
SEM
P-value
Negative control
44.5
50.8bc
62.6
64.7
72.6
82.3c
83.0c
0.5
0.001
Monensin 7.5ppm
46.3
54.8c
60.5
63.2
72.4
76.8b
80.0b
0.5
0.001
Cinnamaldehyde 250ppm
45.5
47.9b
60.2
66.3
73.1
75.9b
79.2b
0.4
0.001
Cinnamaldehyde 500ppm
44.2
48.1b
59.8
65.9
73.7
76.1b
79.7b
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
R2
RSDf
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).
Effect of treatments on crude protein degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B30">Ørskov and McDonald (1979)</xref> model
Treatmenta
Incubation time (h)
0
4
8
12
24
48
72
SEM
P-value
Negative control
79.0
81.2b
88.0
89.1
94.1
98.1d
98.4
0.3
0.001
Monensin 7.5ppm
81.0
84.9c
87.3
88.4
94.5
94.9c
98.2
0.4
0.001
Cinnamaldehyde 250ppm
81.5
80.5b
87.5
90.8
93.8
94.9c
97.6
0.4
0.001
Cinnamaldehyde 500ppm
79.2
84.8c
89.6
90.7
95.1
93.3b
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
R2
RSDg
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).
Effect of treatments on neutral detergent fiber degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B30">Ørskov and McDonald (1979)</xref> or <xref ref-type="bibr" rid="B25">McDonald (1981)</xref> model
Treatmenta
Incubation time (h)
0
4
8
12
24
48
72
SEM
P-value
Negative control
6.0d
14.9d
19.7c
24.6d
29.8c
44.3c
49.5d
0.3
0.001
Monensin 7.5ppm
5.2d
14.5d
18.8c
21.0c
25.1b
31.5b
37.9c
0.3
0.001
Cinnamaldehyde 250ppm
2.3c
9.9c
16.2c
23.3cd
26.3b
27.8b
34.6bc
0.4
0.001
Cinnamaldehyde 500ppm
0.0b
0.0b
0.0b
17.4b
25.6b
31.5b
31.7b
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
R2
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 RSD = Residual standard deviation.
Effect of treatments on acid detergent fiber degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B25">McDonald (1981)</xref> model.
Treatmenta
Incubation time (h)
12
24
48
72
SEM
P-value
Negative control
5.0d
11.0c
28.3c
37.7c
0.4
0.001
Monensin 7.5ppm
0.0b
2.2b
12.6b
22.1b
0.2
0.001
Cinnamaldehyde 250ppm
5.2d
9.1c
11.3b
20.7b
0.5
0.001
Cinnamaldehyde 500ppm
2.8c
9.1c
15.4b
18.0b
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
R2
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.
Effect of treatments on volatile fatty acids production at 48 h of <italic>in vitro</italic> 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.
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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Email: direccion@cecav.esRESUMEN
El objetivo de esta investigación fue estudiar el efecto del cinamaldehído en la degradabilidad ruminal de los nutrientes y la producción total y la proporción molar de ácidos grasos volátiles en un sistema ruminal in vitro (DaisyII Ankom Technology,USA). La dieta estaba compuesta por una mezcla de semilla de cebada: heno de alfalfa (70:30) que se incubó con 4 tratamientos: sin control aditivo o negativo (CN), con monensina a 7,5 ppm o control positivo (CP) y con cinamaldehído a 250 (C250) y 500 ppm (C500). El efecto inhibidor de cinnamaldehyde en la desaparición de MS, FDN y FDA (de 48 a 72 h de tiempo de incubación, P <0.01) fue similar al tratamiento con CP. Todos los tratamientos suplementados tendieron a disminuir la degradabilidad potencial de MS, PB, FDN, FDA, como la degradabilidad efectiva de la MS, FDN y FDA. Sin embargo, a diferencia de la monensina, el cinamaldehído aumentó la producción total de AGV y no afectó la proporción molar de AGV. Las dos dosis evaluadas de cinamaldehído mostraron el mismo efecto. Los resultados indicaron que cinnamaldehyde modificó la fermentación ruminal in vitro.
Los animales rumiantes son incapaces de producir enzimas degradadoras de la fibra. Han desarrollado una relación simbiótica con microorganismos ruminales (bacterias, hongos y protozoos) que les proporcionan proteínas, vitaminas y ácidos grasos volátiles a cambio de un hábitat adecuado para el crecimiento. Al contrario de los rumiantes salvajes, los rumiantes domésticos a menudo se alimentan de gran cantidad de grano y poca fibra. Cuando los rumiantes se alimentan con raciones deficientes en fibra, se interrumpen los mecanismos fisiológicos de la homeostasis, disminuye el pH ruminal, se altera la ecología microbiana y el animal se vuelve más susceptible a los trastornos metabólicos y, en algunos casos, a las enfermedades infecciosas. Algunos trastornos pueden ser contrarrestados por los aditivos alimenticios como ionóforos (Russell y Rychlik, 2001).
Uno de los aditivos comúnmente utilizados en los rumiantes domésticos es la monensina, debido a su efecto positivo en la mejora del metabolismo de la energía y el nitrógeno, y su efecto preventivo contra los trastornos digestivos derivados de la fermentación anormal del rumen (Castillo et al. 2004). Sin embargo, el uso de antibióticos como promotores del crecimiento en el ganado ha sido limitado en la Unión Europea debido a su probable implicación en el desarrollo de la resistencia a los antibióticos microbianos. Para enfrentar esta prohibición, se deben proponer alternativas para mantener la salud animal, la productividad y la seguridad microbiana de los alimentos.
Las plantas producen varios compuestos secundarios con actividad antimicrobiana (Cowan 1999). El cinamaldehído es el principal compuesto del aceite esencial de canela y su efecto antimicrobiano in vitro ha sido ampliamente demostrado (Hili et al. 1997, Helander et al. 1998 y Valero y Salmerón 2003). Se ha reportado que el cinamaldehído podría considerarse una alternativa potencial a la monensina para modificar la fermentación del rumen en el ganado de carne (Khorrami et al. 2015). El cinamaldehído tenía un potencial limitado para mejorar la eficiencia alimentaria y el crecimiento en corderos alimentados con dietas basadas en concentrados (Chaves et al. 2011). Se informó un efecto de la dieta en ovejas cuando se usó cinamaldehído para modificar la fermentación del rumen (Mateos et al. 2013).No se dispone de datos del cinamaldehído utilizado como aditivo para las cabras Murciano-Granadina. Además, se ha demostrado que los aceites esenciales son aditivos alimentarios prometedores para mitigar las emisiones de metano y amoníaco (Cobellis et al. 2016). Sin embargo, el modo de acción y los aditivos de los aceites esenciales en el microbioma del rumen siguen siendo poco conocidos (Cobellis et al. 2016).
Se evaluó el efecto del cinamaldehído en la fermentación in vitro del rumen de cabra. Para lograr esto, se midió la degradabilidad de la materia seca, las proteínas y la fibra en una incubadora artificial. También se determinaron las concentraciones de ácidos grasos volátiles (ácidos acético, propiónico y butírico) y cinamaldehído en el fluido ruminal.
MATERIALES Y MÉTODOS
Materiales y productos químicos. El sustrato utilizado para la incubación fue la mezcla de (base de materia seca) 700 g de grano de cebada (Hordeum vulgare) y 300 g de heno de alfalfa (Medicago sativa). El grano de cebada y el heno de alfalfa se molieron a través de un tamiz de 1 mm (Molino de martillo, Culatti, Italia). El cinamaldehído, la monensina (95 %) y el ácido propiónico se suministraron por Fluka Chemika (Switzerland). El ácido acético se adquirió en Riedel-de Haen (Alemania) y el ácido butírico y 4-metilvalérico en Sigma Aldrich (USA).
Líquido ruminal. El líquido ruminal se extrajo de dos cabras Murciano-Granadina fistuladas, alimentadas con heno de alfalfa ad libitum. Los fluidos del rumen se agruparon y se transportaron al laboratorio en un termo sellado. Luego, se filtró inmediatamente a través de cuatro capas de estopilla y se purgó con CO2 para mantener las condiciones anaeróbicas.
Incubaciones y tratamientos in vitro. Las incubaciones in vitro se realizaron en la incubadora DaisyII (Ankom Technology,USA.) (Mandebvu et al. 2001). La incubadora está compuesta por cuatro frascos de digestión (2 L de capacidad) mantenidos a 39.5 ºC en rotación constante y bajo atmósfera saturada de CO2. Cada frasco de digestión se llenó con solución tampón precalentada (39.5ºC) (que contenía 1317 mL de solución A: KH2PO4, 10 g/L; MgSO4,.7H2O 0.5 g/L; NaCl, 0.5 g/L, CaCl2, 2H2O, 0.1 g/L y CO (NH2)2, 0.5 g/L y 267 mL de solución B: Na2CO3, 15 g/L y Na2S.9H2O, 1 g/L, pH ajustado a 6,8); 400 mL de líquido ruminal y 16 mL de solución aditiva.
Se probaron cuatro tratamientos, uno por cada frasco in vitro: control negativo (CN), control positivo (CP) con monensina 7,5 ppm y cinnamaldehído a 250 ppm (C250) y 500 ppm (C500). Los aditivos (monensina y cinamaldehído) se disolvieron inicialmente en 16 mL de etanol. Con el fin de evitar toda confusión debido a los efectos principales del etanol, este producto también se añadió (16 mL) al tratamiento de control negativo (CN). Las concentraciones seleccionadas de monensina y cinamaldehído se basaron en datos publicados, que habían mostrado propiedades antimicrobianas in vitro, para la monensina Dennis et al. (1981), Domescik y Martin (1999); Wang et al. (2004) y para cinnamaldehyde Hili et al. (1997) y Chang et al. (2001) y Valero y Salmerón (2003).
Se pesaron veintiocho muestras (0,5 g) por frascos en bolsas de filtro ANKOM F57 (ANKOM Technology, USA). Las bolsas (tamaño 5,5 cm x 5 cm) tenían un tamaño de poro de 25 µm y se sellaron térmicamente. Se retiraron cuatro bolsas por tratamiento a 0, 4, 8, 12, 24, 48 y 72 horas de incubación. Las bolsas se lavaron inmediatamente con agua corriente hasta que el agua estuvo clara, se secaron en estufa a 60 °C durante 48 h y se pesaron. El contenido de las bolsas se congeló hasta su posterior análisis.
Análisis químico. La materia seca se determinó secando las muestras a 60ºC durante 48 h; la proteína cruda (PC) por el método Kjeldahl (AOAC, 1990); la fibra detergente neutra (FDN) y la fibra detergente ácida (FDA) por el método Van Soest y Roberston (1991).
Preparación de muestras para análisis de CG. A las 48 h de la incubación, se recogieron dos muestras de 50 mL de líquido ruminal de cada frasco. Las muestras se centrifugaron a 5.000 g durante 20 minutos y el sobrenadante se acidificó con 1 mL de ácido sulfúrico al 50%. El sobrenadante se almacenó a -20ºC. Para el análisis de CG, se mezclaron 5 mL de muestras ruminales acidificadas, 5 mL de agua desionizada y 0,5 mL de una solución estándar interna (4 ácido metil-n-valérico 30 mM) y se centrifugaron (7 minutos a 7.500 g).
Condiciones de análisis de la CG. La determinación de los ácidos grasos volátiles y el cinamaldehído en el líquido ruminal se realizó utilizando un cromatógrafo de gases Thermo Finnigan Trace (Thermo, Italia), equipado con un detector de ionización de llama y columna capilar de sílice fundida TR-FFAP de 30 m × 0,25 mm i.d., 0,25 µm (Teknokroma, España). La temperatura de la estufa se mantuvo a 90ºC durante 1 minuto y aumentó de 90 a 125ºC a una velocidad de 2,6ºC/ min y de 125 a 180ºC a 10 ºC/min. Las temperaturas del inyector y del detector fueron de 220 y 260ºC, respectivamente. El flujo del gas portador fue de 2.0 mL/min y el volumen de inyección fue de 1 μL. El flujo de aire del FID fue de 350 mL/min, mientras que el flujo de H2 fue de 35 mL/min (Madrid et al. 1999). El procesamiento de datos se realizó a través del Chrom-Card Data System (Finnigan, Italia). Todos los productos se determinaron en comparación con el área del pico del estándar interno (ácido 4 metil-n-valérico).
Cálculos y análisis estadístico. Los valores de desaparición de MS, PB, FDN y FDA de las bolsas de filtro en cada tiempo de incubación se ajustaron a la ecuación exponencial descrita por Ørskov y McDonald (1979) y = a + b (1-e-ct), donde y es el la pérdida del componente de alimentación analizado de la bolsa de filtro en el tiempo t, a es la fracción soluble, b es la fracción insoluble potencialmente degradable y c es la tasa fraccional de degradación b. Cuando el valor de a es negativo o el valor de b es mayor que 100, se empleó la ecuación descrita por McDonald (1981) y = b (1-e-c (tL)) donde L indica el tiempo que falta para comenzar la degradación de la fracción b. La degradabilidad potencial (DP) es la suma de las fracciones a y b. La degradabilidad efectiva (DE) se calculó por la ecuación descrita por Ørskov y McDonald (1979) DE = a + ((bc)/(c + r)) o por la ecuación descrita por McDonald (1981) DE = ((bc)/(c + r))(- (c + r) L) asumiendo un valor constante de la tasa fraccional de paso (r) de 0.06 h-1 (Sauvant et al. 2004).
Los datos de fermentación en la incubadora DAISYII se analizaron por ANOVA. Para la degradación de nutrientes, el modelo utilizado fue Y = µ + A + B + AB + ԑ, donde µ es la media, A y B son los efectos del tipo de aditivo y el tiempo de incubación, respectivamente, AB es la interacción tipo de aditivo x tiempo de incubación y ԑijk es el error. Los datos de los AGV se analizaron por ANOVA. Las diferencias entre las medias de tratamiento se determinaron mediante la prueba Least Significant Difference (LSD). La significancia estadística se estableció cuando P <0.05. Todos los cálculos se realizaron utilizando SPSS (1997).
RESULTADOS Y DISCUSIÓN
La composición química de la dieta incubada se presenta en la tabla 1. La dieta fue 12.87 % PB, 28.54 % FDN y 12.84 % FDA.
Chemical composition of incubated mixed diet (as DM basis) <sup>a</sup>
Composition (g/kg DM)
Mixed diet (700 g barley grain + 300 g alfalfa hay)
Degradabilidad de la materia seca. La desaparición de la materia seca según el tipo de tratamiento y el tiempo de incubación, como la cinética de la degradabilidad de la MS se muestran en la tabla 2. Hubo interacción significativa entre el tiempo de incubación y el tratamiento (P = 0,007). Los aditivos (P <0.05) influyeron en la desaparición de la MS. En los tiempos de incubación de 48 y 72 horas, los tratamientos con CP, C250 y C500 redujeron (P <0,001) la desaparición de la MS en comparación con el tratamiento con CN. Por otro lado, la desaparición de la MS (P <0.001) aumentó cuando el tiempo de incubación avanzó. Todos los aditivos redujeron la degradabilidad potencial de la MS (tabla 2) en comparación con el tratamiento con CN (79.4, 78.5, 78.6 vs 83.6 %). La disminución de la DP se correspondió con una reducción de la fracción degradable b. Los estudios in vitro (Jalc et al. 1992 y Wang et al. 2004) demostraron una reducción de la desaparición de la MS cuando se agregó monensina a 2,5 y 15 ppm. El mismo efecto para la monensina se informó en estudios in vivo (Rogers et al. 1997 y Wang et al. 2004). Para una mezcla de aceites esenciales (timol (5-metil-2- (1-metiletil) fenol), guayacol (2-metoxifenol), limoneno (1-metil-4-(1-metiletenil) ciclohexeno) se reportó una reducción de la desaparición de MS in situ para alimentos concentrados como guisantes (Pisum sativum) (Molero et al. 2004) o harina de soja (Glycine max) (Newbold et al. 2004). La mezcla de estos aceites esenciales inhibió el crecimiento de los cultivos más puros de bacterias ruminales en concentraciones de menos de 100 ppm (McIntosh et al. 2003).
Effect of treatments on dry matter degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B30">Ørskov and McDonald (1979)</xref> model
Treatmenta
Incubation time (h)
0
4
8
12
24
48
72
SEM
P-value
Negative control
44.5
50.8bc
62.6
64.7
72.6
82.3c
83.0c
0.5
0.001
Monensin 7.5ppm
46.3
54.8c
60.5
63.2
72.4
76.8b
80.0b
0.5
0.001
Cinnamaldehyde 250ppm
45.5
47.9b
60.2
66.3
73.1
75.9b
79.2b
0.4
0.001
Cinnamaldehyde 500ppm
44.2
48.1b
59.8
65.9
73.7
76.1b
79.7b
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
R2
RSDf
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.
Degradabilidad de la proteína bruta. La desaparición de la proteína bruta según el tipo de tratamiento y el tiempo de incubación, como la cinética de la degradabilidad de la PB se muestran en la tabla 3. Hubo interacción significativa entre el tiempo de incubación y el tratamiento (P = 0.030). Los efectos de los aditivos probados en la degradación de la PB no fueron tan notables, ya que se encontró una fracción soluble (a) de PB muy alta. La monensina y el cinamaldehído no afectaron la desaparición de la PB (P> 0.05), aunque se observó interacción significativa (P <0.05) entre el tipo de tratamiento y el tiempo de incubación (tabla 3). Además, a las 48 h de incubación, la desaparición de la PB fue menor (P <0,001) en los tratamientos suplementados que en el tratamiento CN (94.9, 94.9 y 93.3 % vs 98.1 % para los tratamientos con PB, C250, C500 y CN respectivamente). La fracción potencialmente degradable (b) disminuye ligeramente cuando se incluyen los aditivos, se observó una tendencia similar en la DP. Es sabido que la monensina disminuye la degradación ruminal de la PB (Van Nevel y Demeyer 1977 y Hillaire et al. 1989). Ghorbani et al. (2010) demostraron que la monensina podría disminuir la cantidad de amoníaco en el líquido ruminal. Este efecto podría deberse al hecho de que la momensina afecta negativamente a la población de bacterias grampositivas que tienen una alta actividad de producción de amoníaco (Duffield et al. 2002). Sin embargo, la síntesis microbiana ruminal no parece verse afectada por la inclusión de monensina (Ghorbani et al. 2010). Por otro lado, algunos estudios no encontraron ningún efecto de la monensina en la degradación de la PB (Vanhaecke et al. 1985), mientras que otros mostraron aumento de la degradación de la PB (Benchaar et al. 2006), probablemente debido a varios factores, como la concentración de monensina o el tipo de dieta utilizada. La degradación in situ de CB también puede reducirse por productos de plantas antimicrobianas. Una mezcla de timol (5-metil-2-(1-metiletil)fenol), guaiacol (2-metoxifenol) y limoneno (1-metil-4-(1-metiletenil) ciclohexeno) redujo la degradación de la PB de la harina de soya en las ovejas( Newbold et al. 2004) y las semillas de lupino (Lupinus angustifolius), guisantes verdes (Pisum sativum) y harina de girasol (Helianthus annuus) disminuyeron la degradación de la PB en las novillas en crecimiento alimentadas con una dieta altamente concentrada (Molero et al. 2004). Tager y Krause (2010) demostraron que la digestibilidad de la proteína bruta fue baja con cinamaldehído y eugenol (500 mg/L/d).
Effect of treatments on crude protein degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B30">Ørskov and McDonald (1979)</xref> model
Treatmenta
Incubation time (h)
0
4
8
12
24
48
72
SEM
P-value
Negative control
79.0
81.2b
88.0
89.1
94.1
98.1d
98.4
0.3
0.001
Monensin 7.5ppm
81.0
84.9c
87.3
88.4
94.5
94.9c
98.2
0.4
0.001
Cinnamaldehyde 250ppm
81.5
80.5b
87.5
90.8
93.8
94.9c
97.6
0.4
0.001
Cinnamaldehyde 500ppm
79.2
84.8c
89.6
90.7
95.1
93.3b
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
R2
RSDg
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
Degradabilidad de la fibra. En la tabla 4 y 5 se muestran las desapariciones de la FDN y la FDA según el tipo de tratamiento y el tiempo de incubación, así como la cinética de la degradabilidad de la FDN y la FAD. Hubo interacción significativa entre el tiempo de incubación y el tratamiento (P = 0,001) tanto para la FDN como para la FDA. Las degradaciones de FDN y FDA se redujeron (P <0,001) con la suplementación de aditivos. Además, el efecto del cinamaldehído a 250 ppm en la degradación de la fibra fue similar al observado con la monensina. La desaparición de la fibra para el tratamiento con C500 no comenzó hasta las 8 h de incubación para la degradación de FDN y hasta las 12 h de incubación en todos los tratamientos para la degradación de FDA. El tratamiento con C500 mostró la fase de latencia a partir de la cual comenzó la degradación de la FDN. La fase de latencia fue necesaria para la fracción de FDA en todos los tratamientos utilizados. Este resultado es consistente con el hecho de la FDA no tiene fracción soluble (Van Soest, 1994). Los tratamientos con PB, C250 y C500 tendieron a reducir la DP y la DE de la fracción de fibra, en comparación con el tratamiento de CN. El efecto inhibitorio de la monensina en la degradación de la fibra está bien documentado por otros autores en ensayos in vitro e in situ (Jalc et al. 1992). El efecto de los extractos de plantas en la degradación de las fibras depende de su estructura química. De esta forma, los taninos (Hervás et al. 2003) y las saponinas (Wina et al. 2005) podrían reducir la degradación in situ de la FDN, mientras que el extracto de Achillea millefolium que contiene flavonoides, incrementa las degradabilidades de FDN y FDA (Broudiscou et al. 2002).
Effect of treatments on neutral detergent fiber degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B30">Ørskov and McDonald (1979)</xref> or <xref ref-type="bibr" rid="B25">McDonald (1981)</xref> model
Treatmenta
Incubation time (h)
0
4
8
12
24
48
72
SEM
P-value
Negative control
6.0d
14.9d
19.7c
24.6d
29.8c
44.3c
49.5d
0.3
0.001
Monensin 7.5ppm
5.2d
14.5d
18.8c
21.0c
25.1b
31.5b
37.9c
0.3
0.001
Cinnamaldehyde 250ppm
2.3c
9.9c
16.2c
23.3cd
26.3b
27.8b
34.6bc
0.4
0.001
Cinnamaldehyde 500ppm
0.0b
0.0b
0.0b
17.4b
25.6b
31.5b
31.7b
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
R2
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 RSD = Residual standard deviation.
Effect of treatments on acid detergent fiber degradability. Disappearance kinetic’s according to <xref ref-type="bibr" rid="B25">McDonald (1981)</xref> model.
Treatmenta
Incubation time (h)
12
24
48
72
SEM
P-value
Negative control
5.0d
11.0c
28.3c
37.7c
0.4
0.001
Monensin 7.5ppm
0.0b
2.2b
12.6b
22.1b
0.2
0.001
Cinnamaldehyde 250ppm
5.2d
9.1c
11.3b
20.7b
0.5
0.001
Cinnamaldehyde 500ppm
2.8c
9.1c
15.4b
18.0b
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
R2
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.
Ácidos grasos volátiles. El efecto de los tratamientos en la producción de ácidos grasos volátiles se muestra en la tabla 6. El efecto principal de la monensina en el metabolismo de la energía ruminal es aumentar la producción de ácido propiónico y reducir la producción de ácido acético, lo que resulta en menor relación acético: propiónico (Wang et al. 2004). En este estudio, este efecto también se encontró. El cinamaldehído aumentó la producción total de AGV (P <0.01) en comparación con CN o PB, pero no afectó la proporción molar de acetato, propionato o butirato en comparación con los tratamientos de control. Cuando se agregó cinamaldehído a la concentración de 2,2 mg/mL en un sistema de cultivo continuo, la producción total de AGV y la proporción molar de propionato no se afectaron, pero se notó una proporción molar inferior de acetato y un aumento numérico en la proporción molar de butirato (Busquet et al. 2005). En incubadoras similares, la adición de 0,22 mg/mL de un extracto de canela con 59 % de cinamaldehído aumentó la proporción molar de acetato y disminuyó la proporción molar de propionato y butirato durante el período de adaptación. Sin embargo, estos efectos desaparecieron después de 6 días de fermentación (Cardozo et al. 2004). Otro estudio (Wina et al. 2005) también informó aumento en la producción total de AGV debido a extractos de plantas. Por lo tanto, la adición de 2 o 4 mg/mL de extracto con metanol de Sapindus rarak que contiene saponinas a incubadoras in vitro de jeringas de vidrio, incrementó la producción total de AGV a las 48 h de incubación. A diferencia de, una dosis de 540 mg/L de cinamaldehído redujo la producción total de AGV en una incubación de 16 horas de mezcla de microorganismos de rumen mixto con dietas de contenido de concentrado medio y alto, mientras que las dosis más bajas (180, 60 y 20 mg/L) no afectaron la producción total de AGV en comparación con el grupo control (Mateos et al. 2013). De esta manera, Macheboeuf et al. (2008) no encontraron aumento de la producción total de AGV con suplementación de cinamaldehído a dosis de 132 o 264 mg/L, pero la producción total de AGV se redujo con dosis de cinamaldehído de 396 y 661 mg/L. Blanch et al. (2016) describen la reducción total de AGV con una dosis de 172 mg/L de cinamaldehído.
Effect of treatments on volatile fatty acids production at 48 h of <italic>in vitro</italic> 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)
Cuando se realizaron los análisis de cromatografía de gases, se encontró un pico en las muestras de rumen con tratamientos con cinamaldehído (figura 1). Este pico se identificó utilizando una solución estándar de cinamaldehído. Como consecuencia, el método desarrollado podría usarse para determinar tanto los AGV y los compuestos derivados de plantas como el cinamaldehído en el fluido ruminal, método en el que tampoco sería necesaria una extracción con solvente apolar.
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.
En conclusión, los resultados indicaron que el cinamaldehído modificó la fermentación ruminal in vitro de la cabra. Este compuesto fenólico redujo la MS y la degradación de la fibra de la misma manera que la monensina. Sin embargo, a diferencia de la monensina, el cinamaldehído aumentó la producción total de AGV y no afectó la proporción molar de AGV. Las dos dosis de cinamaldehído utilizadas mostraron efecto idéntico y probablemente fueron demasiado altas. Se requieren investigaciones futuras con dosis más bajas y estudios in vivo.
AGRADECIMIENTOS
P. Catalá-Gregori recibió una beca de investigación (AP2002-3340) del Ministry of Education and Science of Spain.