Introduction
In recent years, the use of antibiotics as growth promoters in animal nutrition has been limited in the European Union (EU) because of its relation to the increase in the number of antibiotic-resistant bacteria to drugs which are used by humans (Cancho et al., Reference Cancho, García and Simal2000). Consequently, new commercial additives are required that offer more safety, but also have the property of manipulating rumen fermentation. Thus, additives of vegetal origin, considered to be natural products, have been proposed to livestock producers as possible replacers of growth promoting antibiotics. The antimicrobial capacity of several natural extracts, mainly essential oils, has been demonstrated (Cowan, Reference Cowan1999). The antimicrobial properties of essential oils could be used to modulate rumen microbial activities (McIntosh et al., Reference McIntosh, Newbold, Losa, Williams and Wallace2000). Some effects could be related to a decrease in the number of proteolytic rumen bacteria and to a reduction in the rate of deamination in the rumen (Losa et al., Reference Losa, Frehner, Newbold and Wallace2002), or to other mechanisms such as the control of ruminal methanogenesis (Broudiscou et al., Reference Broudiscou, Papon and Broudiscou2000; Evans and Martin, Reference Evans and Martin2000). Currently, it is necessary to evaluate the effects of active components from plant extracts on ruminal parameters, in order to confirm their usefulness as an alternative to the use of growth-promoting antibiotics in ruminants.
Carvacrol and thymol, two phenolic derivatives, are considered to be the main active components in the essential oil fraction of oregano and thyme (Sivropoulou et al., Reference Sivropoulou, Papanikolaou, Nikolaou, Kokkini, Lanaras and Arsenakis1996), and to exhibit antimicrobial activities (Helander et al., Reference Helander, Alakomi, Latva-Kala, Mattila-Sandholm, Pol, Smid, Gorris and Wright1998). Thus, the aim of this study was to assess the influence of carvacrol on the in vitro ruminal degradability of dry matter (DM), crude protein (CP) and neutral-detergent fibre (NDF) and on end-products of fermentation as volatile fatty acids (VFA), in comparison with monensin.
Material and methods
Substrate
The substrate used for the in vitro procedure was composed of barley seed (Hordeum vulgare L.) and alfalfa hay (Medicago sativa L.) with a concentrate to forage ratio of 70:30 on a DM basis. The substrate was ground through a 1-mm screen. DM was determined by drying at 60°C for 48 h. CP (Kjeldahl N × 6.25) and ash were analysed according to the Association of Official Analytical Chemists (1990). NDF (using method with sodium sulphite and heat-stable α-amylase), acid-detergent fibre (ADF) and acid-detergent lignin (ADL) were determined as described by Van Soest et al. (Reference Van Soest, Robertson and Lewis1991). Table 1 shows the chemical composition of the incubated ingredients.
Table 1 Chemical composition of incubated ingredients†
† DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral-detergent fibre; ADF = acid-detergent fibre; ADL = acid-detergent lignin.
Collection of ruminal fluid
Two goats of Murciano-Granadina breed were fed alfalfa hay ad libitum for 15 days, sacrificed 4 h after feeding and rumens from both animals removed immediately. The content of the rumens was squeezed through four layers of cheesecloth. A volume of 1600 ml of ruminal fluid, resulting from the mixture of both ruminal contents, was purged with deoxygenated CO2, and placed at 39.5°C just before its use as ruminal inoculum in the in vitro procedure.
In vitro degradability
The in vitro procedure was conducted in a commercial incubator (Daisy II200/220 incubator, ANKOM Technology Corp., Fairport, NY) to determine the degradability of the substrate. The incubator consists of four independent digestion jars, and this incubation instrument allows keeping the fermentation medium in continuous agitation and at a specific temperature (39.5 ± 0.5°C). Each jar was filled with the digestion medium that contained: 1584 ml of buffer solution pre-warmed at 39°C, 400 ml of the ruminal inoculum and 16 ml of a solution with the test additive diluted in ethanol or ethanol alone in the negative control group. The buffer solution was made from two other buffer solutions (A and B) in a ratio of 1:5 to obtain a final pH of 6.8 at 39°C. The A buffer solution contained: KH2PO4 (10 g/l), MgSO4.7H2O (0.5 g/l), NaCl (0.5 g/l), CaCl2.2H2O (0.1 g/l) and urea (0.5 g/l); and the B buffer solution contained: Na2CO3 (15.0 g/l) and Na2S.9H2O (1.0 g/l). Four treatment groups were established (i.e. one per digestion jar): negative control, without additive (C); positive control, 7.5 mg/l monensin (M); 250 mg/l carvacrol (C250) and 500 mg/l carvacrol (C500). The doses of the evaluated additives were set to meet or exceed the minimum inhibitory concentrations (MICs) provoking antimicrobial activity based on former studies (monensin, Domescik and Martin (Reference Domescik and Martin1999); Wang et al. (Reference Wang, Alexander and McAllister2004) and carvacrol, Juliano et al. (Reference Juliano, Mattana and Usai2000)). The reactants used were: monensin sodium salt (C36H61NaO11, Fluka BioChemika, Steinheim, Germany) and 5-isopropyl-2-methylphenol (carvacrol) (C10H14O, Panreac, Barcelona, Spain).
A 0.5-g sample of substrate was directly weighed into a filter bag (F57 Filter Bags, ANKOM Technology), placing 28 bags with substrate and seven blanks (without substrate, empty bags) inside each digestion jar. Once all bags were placed into each digestion jar, which was already filled with the fermentation medium, the corresponding bags per treatment were removed for 0 h of incubation. The selected incubation times were: 0, 4, 8, 12, 24, 48 and 72 h. At each time, five filter bags per treatment were randomly removed (i.e. four bags with substrate and one blank); then the jar was purged with CO2 and sealed. The removed bags were washed with tap water until the rinsing water was clean. Washed bags were dried at 60°C for 48 h and weighed to determine DM residues. CP, by the Kjeldahl method (N × 6.25) and NDF, using the method described by Van Soest et al. (Reference Van Soest, Robertson and Lewis1991), were determined on DM residues. In vitro DM, CP and NDF disappearance data were calculated from the concentration of each nutrient in the residues and the original samples after correction for amounts of DM, CP and NDF in blank bags.
Volatile fatty acids (VFA)
After 48 h of incubation, a duplicate of 50 ml of the fermentation medium was removed per treatment, and centrifuged at 3000 × g for 20 min. Supernatants were transferred into plastic containers, 1 ml of 50% sulphuric acid was added and they were frozen at − 20°C until VFA determination. The VFA concentration was measured by capillary gas chromatography as described by Madrid et al. (Reference Madrid, Megías and Hernández1999). The gas chromatograph (TRACE GC Ultra, Thermo Finnigan Italia SpA, Milan, Italy) was equipped with a flame ionisation detector. The capillary column was fused silica, 30 m × 0.25 mm × 0.25 μm ID and coated with FFAP-TR as the stationary phase (Teknokroma, Spain). Standard solutions of acetic, propionic and butyric acids were prepared for calibration, using 4-methyl-n-valeric acid (30 mmol/l) as internal standard. Original supernatant solutions were diluted twice and internal standard was added in ratio 10:1 (diluted supernatant/internal standard). The solution was mixed and centrifuged at 7000 × g for 7 min at 4°C. Then, 1 μl aliquot of this solution was injected onto the column.
Kinetic of degradability and statistical analyses
In vitro nutrient disappearance data were used to estimate degradation parameters using non-linear equations. The model used was: p = a+b (1 – e− ct), described by Ørskov and McDonald (Reference Ørskov and McDonald1979), where p is the in vitro disappearance (%) at time t, a is the soluble fraction (%), b is the slowly degradable fraction (%), c is the fractional rate at which the b fraction is degraded (per h) and a+b is the potential degradability. Effective degradabilities (ED) were estimated using the equation: ED = a+ bc/(c+k), where k is the rumen outflow rate, taken as 0.06 per h (Sauvant et al., Reference Sauvant, Perez and Tran2003).
The effects of supplements on in vitro DM, CP and NDF degradability for each time, the results of VFAs at 48 h of incubation and kinetic parameters of degradability were subjected to one-way analysis of variance (Steel et al., Reference Steel, Torrie and Dickey1996), using SPSS software (Statistical Packages for the Social Sciences, 1997). The comparison between means was analysed by LSD test.
Results
In vitro degradation
Approximately 50% of the total DM loss occurred after 12 h of incubation in all the evaluated treatments; except for C500 that had a lower DM loss ( ≈ 40%; Table 2). All supplements reduced (P < 0.001) potential DM degradation. This is mainly due to a reduction (P < 0.001) in the slowly degradable fraction (b), but fractional degradation rates were higher for M and C250 (P < 0.05). Consequently, effective DM degradation only has been reduced (P < 0.001) by C500.
Table 2 In vitro dry matter (DM) degradation of substrate without or with supplementation of monensin or carvacrol at two doses (C205, 250 mg/l; C500, 500 mg/l)
a-c Means in the same column with different superscript letters are significantly different.
† *P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant.
‡ ED = effective degradability values at 0.06 per h outflow rate.
Table 3 shows the effect of monensin and carvacrol on the in vitro CP degradability. Only C250 and C500 reduced (P < 0.05) CP degradation compared with the negative control after 72 h of incubation. M, C250 and C500 diminished the potential degradability of the protein by reducing the b fraction (P < 0.05). In addition, only C500 exerted a reduction of the CP effective degradability of approximately five points against the C treatment (P < 0.05).
Table 3 In vitro crude protein (CP) degradation of substrate without or with supplementation of monensin or carvacrol at two doses (C205, 250 mg/l; C500, 500 mg/l)
a-b Means in the same column with different superscript letters are significantly different.
† *P < 0.05; NS = not significant.
‡ ED = effective degradability values at 0.06 per h outflow rate.
A reduction in NDF degradability by all supplements from 24 to 72 h of incubation was found (Table 4). The highest NDF loss occurred at 72 h in C compared with the supplemented treatments (P < 0.001). The NDF potential degradation and effective degradability were also lower in the treated groups in comparison with the negative control (P < 0.01).
Table 4 In vitro neutral-detergent fibre (NDF) degradation of substrate without or with supplementation of monensin or carvacrol at two doses (C205, 250 mg/l; C500, 500 mg/l)
a-c Means in the same column with different superscript letters are significantly different.
† *P < 0.05; **P < 0.01; ***P < 0.001; NS = not significant.
‡ ED = effective degradability values at 0.06 per h outflow rate.
The kinetic model of DM and NDF degradability obtained a R 2 mean value of 0.942 and 0.954 respectively, whereas for the CP kinetic model the R 2 reached 0.750 only.
VFA production
The average concentration of VFAs and the molar proportion of each individual acid in the in vitro rumen medium at 0 and 48 h of incubation are presented in Table 5. The total VFA concentration after 48 h of incubation was influenced by supplements (P < 0.001). The VFA values in M and C250 were higher than in C after 48 h of incubation; whereas C500 showed the lowest VFA concentration. The molar proportion of acetate, propionate and butyrate was also affected (P < 0.001). C250 and M reduced acetate molar proportions; whereas C500 increased the acetate molar proportion. M increased propionate, diminishing acetate:propionate ratio, whereas the C250 treatment increased the molar proportion of butyrate.
Table 5 Volatile fatty acid (VFA) concentrations at 0 h and 48 h of incubation without or with supplementation of monensin or carvacrol at two doses (C205, 250 mg/l; C500, 500 mg/l)
a-d Means in the same row with different superscript letters are significantly different.
† ***P < 0.001.
Discussion
In the present study, carvacrol was shown to have the potential to modify in vitro ruminal fermentation and to increase total VFA concentration when supplemented at 250 mg/l. Despite C250 reduced DM degradability similar to M, a greater degradability reduction was obtained when C500 was included. The effect of M on DM degradability has been reported before by other authors (e.g. Wang et al., Reference Wang, Alexander and McAllister2004). On the other hand, in a study with heifers fed diets with different forage to concentrate ratios, Molero et al. (Reference Molero, Ibars, Casamiglia, Ferret and Losa2004) showed that the addition (700 mg/day) of a blend of essential oils, in which thymol (carvacrol is a naturally occurring isomer of thymol) was one of the major components, reduced in situ effective DM degradability of green peas compared with control diets; while in situ ruminal degradability of other feeds (e.g. maize-gluten feed, lupin seeds, fish, soya-bean and sunflower meal) was not affected. Moreover, Newbold et al. (Reference Newbold, McIntosh, Williams, Losa and Wallace2004) also observed a reduction of in situ DM degradation of soya-bean meal after 8 and 16 h of incubation when an essential oil (mixture of thymol, guajacol and limonene) was added to the diet of sheep. However, this mix had no effect on DM degradability of rapeseed meal and hay.
In our work, CP degradation had a tendency to decrease with the supplemented treatments after 72 h of incubation. Monensin has been reported before to reduce feed protein degradation in the rumen (Van Nevel and Demeyer, Reference Van Nevel and Demeyer1977). In the study of Molero et al. (Reference Molero, Ibars, Casamiglia, Ferret and Losa2004) the blend of the above-mentioned essential oil had little effect on protein degradation: a decreased CP degradation was only observed in three of the five vegetal protein sources in heifers fed a high concentrate diet. However, in an in vitro ruminal fermentation study, Busquet et al. (Reference Busquet, Calsamiglia, Ferret and Kamel2006) showed that carvacrol resulted in a 30 to 50% reduction in ammonia N concentration; but the carvacrol concentration used (3000 mg/l) was higher than both carvacrol levels used in our study.
All additives decreased NDF degradability from 24 to 72 h of incubation. Ionophores, such as M, are reported selectively to inhibit gram-positive micro-organisms (Bergen and Bates, Reference Bergen and Bates1984), which include most of the cellulolytic bacteria, capable of hydrolysing fibre (e.g. Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens). In fact, in an in vitro degradability experiment, it has been demonstrated that M diminished NDF degradation (Jalc et al., Reference Jalc, Baran, Vendrak and Siroka1992). On the other hand, M also revealed to reduce lactic acid producing bacteria such as Streptococcus bovis (McGuffey et al., Reference McGuffey, Richardson and Wilkinson2001). In accordance with our results, Martínez et al. (Reference Martínez, Madrid, Hernández, Megías, Sotomayor and Jordán2006) reported that two essential oils of thyme at 1.35 μl/ml, one rich in carvacrol and the other in thymol, decreased the in vitro potential degradability of NDF (in our study the 250 and 500 mg/l concentrations were equivalent to 0.264 and 0.528 μl/ml of carvacrol).
It must be noted that the R 2 values of the CP models were rather low. This could be due to a bacterial contamination of the bag residue if the washing procedure of the filter bags applied here inappropriate to efficiently remove bacterial residues. As bacteria contain relatively high protein proportions and no NDF, disturbance through bacterial contamination might be of higher importance in CP compared with NDF models.
C500 showed the lowest total VFA concentration. It is likely that the use of such high doses of plant extracts and/or their secondary metabolites with antimicrobial activity decreased total microbial activity and diet fermentability (Cardozo et al., Reference Cardozo, Calsamiglia, Ferret and Kamel2004). Moreover, individual VFA proportions were affected by the additives. M changed the VFA profile as expected, increasing propionate proportions and diminishing the ratio acetate:propionate, which agrees with previous findings (Thornton and Owens, Reference Thornton and Owens1981). C250 decreased the molar proportion of acetate and propionate, but increased butyrate molar proportion. Evans and Martin (Reference Evans and Martin2000) also observed that the use of 400 mg/l of thymol decreased concentrations of CH4, acetate, propionate and lactate in a mixed ruminal micro-organism fermentation. In addition, Castillejos et al. (Reference Castillejos, Calsamiglia and Ferret2006) found that thymol at 5 mg/l tended to reduce the proportion of acetate and increased the proportion of butyrate. These authors also tested thymol at 50 and 500 mg/l, with the former concentration showing no effect on VFA profile, whereas total VFA production has been reduced at 500 mg/l. They concluded that the selection of the optimal dose may require further studies with doses between 5 to 50 mg/l and from 50 to 500 mg/l, to observe positive effects without affecting total VFA concentration.
Shifts in the fermentation pattern provoked through C250 supplementation clearly differed from M supplementation, suggesting that the mechanism of action of C250 is not the same as that of M. In fact, Helander et al. (Reference Helander, Alakomi, Latva-Kala, Mattila-Sandholm, Pol, Smid, Gorris and Wright1998) reported that monensin affects mainly some gram-positive bacteria, while essential oils and other vegetal secondary compounds inhibit both gram-positive and gram-negative bacteria.
Although gas production during the present work was not directly evaluated, the molar proportions of acetate, propionate and butyrate were used to predict rumen methanogenesis (mmol/mol VFA) according to an equation, derived from the stoichiometric relations as described by Demeyer and Fievez (Reference Demeyer and Fievez2000). These calculations suggest M supplementation reduced relative methane production, whereas higher methane production was associated with the supplementation of C250 and C500 (291, 241, 325 and 400 mmol CH4/mol VFA for C, M, C250 and C500, respectively). Therefore, carvacrol at the assayed doses seems to have a different effect compared with M on ruminal methanogenesis.
The action of M on rumen degradability, VFA pattern and rumen methanogenesis was as expected and reported before by other authors. C250 changed the VFA profile, but was different to the fermentation pattern observed with M supplementation. C500 reduced total VFA production. Future research, including in vivo studies, in order to understand the factors that contribute to that antimicrobial activity and the selection of the optimal dose is required.
