In ruminants, symbiotic relationship is established with rumen microorganisms, by which the animal provides the nutrients and optimal environmental conditions to allow fermentation of their feed by the rumen microorganisms. Instead, microorganisms degrade fiber and synthesize microbial protein to supply energy and protein needs of the host animal (Van Soest, 1982). However, this symbiotic relationship is somewhat inefficient through loss of energy (methane) and protein (N-NH3) (Van Nevel and Demeyer, 1988). These losses not only reduce production performance, but they also contribute to release of environmental pollutants (Tamminga, 1996). Ruminant nutritionists have long been interested in modulating competition among different microbial populations with the objective of improving the efficiency of energy and protein utilization in the rumen. This has been achieved through optimized diet formulations and use of feed additives that modify fermentation in the rumen and enhance or inhibit specific microbial populations (Calsamiglia et al. 2006). Antibiotic ionophores have been very successful in reducing energy and protein losses in the rumen (Van Nevel and Demeyer, 1988). However, the use of antibiotics in animal feed is becoming less socially acceptable because of residue and production of resistant strains of bacteria. The use of antibiotics in animal feed has been banned in the European Union since January 2006 (Directive 1831/2003/CEE, European Commission, 2003). For this reason, scientists have become interested in developing alternative ways to modulate rumen fermentation such as application of yeasts, organic acids, plant extracts, probiotics and antibodies (Calsamiglia et al. 2006). Essential oils (EO) are complex mixtures of secondary metabolites and volatile compounds extracted from plants by methods of distillation that seem to have no direct function in their growth and development (Balandrin and Klocke, 1985; Benchaar et al. 2008). However, they are responsible odor and color in plants (Guenther, 1948). Application of EO can be a useful strategy to improve efficiency of nutrient utilization by ruminants (Benchaar et al. 2008; Tajodini et al. 2014). EO have an important role in plants, for example protection against invasive insects, bacteria, viruses and fungi, as well as in attracting pollinators (Bakkali et al. 2008). EO have demonstrated antimicrobial activity against a variety of microorganisms, including gram positive and gram-negative bacteria, protozoa, yeast and fungi (Helander et al. 1998; Greathead, 2003; Reichling et al. 2009). The mechanism by which EO are thought to exert their antimicrobial activity is by disrupting the cell wall structure, affecting electron transport, ion gradients, protein translocation, phosphorylation steps and other enzyme-dependent reactions (Ultee et al. 1999; Dorman and Deans, 2000). The antimicrobial activity of plant extract is attributed to a number of secondary plant metabolites, including saponins, terpenoids and phenylpropanoids present in the EO fraction of many plants (Dorman and Deans, 2000). A number of recent in vitro studies have evaluated the effects of various EO on ruminal microorganisms and ruminal metabolism and have reported varied results. Many studies have reported the stimulatory and inhibitory effects of EO on a large variety of microorganisms under in vitro and in vivo conditions (Newbold et al. 2004; Benchaar et al. 2007; Nanon et al. 2014; Khorrami et al. 2015). Fernandez et al. (1997) showed that a commercial product of a blended of EO compounds inhibits protein degradation in the rumen, thus has the potential to increase protein supply to the post-ruminal tract. McIntosh et al. (2003), reported that EO can cause a decrease in the ruminal fungal population. The objective of this study was to evaluate the effects of various medicinal plant EO on in vitro ruminal fermentation of alfalfa hay, sugar beet pulp and barley grain and in vitro ruminal fungal population.
MATERIALS AND METHODS
Substrates and EO preparation
The tested feed samples were as follows; alfalfa hay (AH), barley grain (BG) and sugar beet pulp (BP); these were ground to pass through a 1mm screen (AOAC, 1990). Treatments were feed samples without EO (as controls) or substrates plus 200 or 400 μL/g DM dose of EO of cinnamon, black pepper, cumin, fennel or garlic oil. Monensin was also used as a positive control in the medium at 5 µmol. These values were selected based on our previous experiments. The EO content was obtained for each plant with hydro-distillation of ground samples using Clevenger apparatus (Jahani-Azizabadi et al. 2014). The EO samples were stored in a refrigerator (4 ˚C) until they were used in the experiment.
Gas production technique
Two sheep (45±2 kg, body weight) fitted with rumen cannulae were used as donors for rumen fluid. They were fed 1.5 kg DM alfalfa hay and 0.4 kg DM concentrates (165 g CP/kg DM) per head per day. Rumen content was collected before feeding in the morning. Rumen fluid was strained through 4 layers of cheesecloth and incubated at 39 ˚C. The gas production method of Menke and Steingass (1988) was used. Rumen fluid was immediately strained through four layers of cheesecloth and mixed in a 2:1 with buffer, then, 40 mL of diluted fluid was added to the syringes. Each syringe was gassed with CO2 then incubated at 38.6 ˚C. The volume of gas produced was determined at 2, 4, 8, 12, 24, 36, 72 and 96 h after incubation. Gas production data were fitted using an exponential equation of
b: volume of gas produced.
c: fractional rate constant of gas production (/h).
t: incubation time (h).
P: volume of gas produced at time t.
Ruminal quantification of fungi using batch culture
Feed samples were alfalfa hay (AH), barley grain (BG) and sugar beet pulp (BP). Treatments were substrates without EO (as control) and substrates plus 200 μL/g DM of EO of cinnamon, black pepper, cumin and fennel. Samples were incubated in a medium prepared as described in Arroquy et al. (2005). The fermentation medium was pre-reduced anaerobically sterilized ruminal fluid medium consisting 150 mL/L of mineral mixtures I (3 g/L K2HPO4), 150 mL/L mineral mixture II (3 g/L KH2PO4, 6 g/L (NH4)2SO4, 6 g/L NaCl, 0.6 g/L MgSO4_7H2O, 0.6 g/L CaCl2), 0.05 g/L of cellobiose, 400 mL/L of cell-free ruminal fluid, 1 mL/L resazurin, 300 mL/L of distilled water, 4 g/L NaHCO3 and 0.5 g/L of cysteine-HCL. The fermentation medium was autoclaved for about 20 minutes, cooled and then 4 g/L sodium bicarbonate was added. An amount of 45 ml of medium was supplied to a 100 mL bottle that contained 0.45 g of a feed sample (4 replicates per each sample). Then, each bottle was inoculated under carbon dioxide with 5 mL of mixed rumen microbes. Rumen fluid was obtained from three sheep (49.5±2.5 kg) each fitted with a rumen fistulae before the morning feed, and immediately strained through four layers of cheesecloth. The animals were fed with 1.5 kg of DM alfalfa hay and 0.4 kg DM concentrates (165 g CP/kg of DM) per head per day. The bottles were incubated for 120 h at 39 ˚C. Then, each bottle content was filtered through a 42 µm filter, and unfiltered residue was dried using a forced-air oven at 60 ˚C for 48 h, weighted and analyzed for DM and NDF content. Evaluations for ammonia nitrogen (N-NH3) concentration, medium pH and dry matter (DM) disappearance were carried out after 48 h of incubation. At the end of the incubation period, 2 mL of medium content (liquid and solid phase) were taken and stored at -20 ˚C until the next analysis.
After thawing, samples were shaken and transferred to 1.5 ml micro tubes containing glass beads and vortexed twice for 2 min with incubation on ice between shakings. Tubes were centrifuged at 200 × g for 5 min at 4 ˚C for the sedimentation of feeds particles. The supernatants (200 μL) were transferred to a fresh 1.5 mL micro tubes and DNA extraction was performed using a genomic DNA Extraction Kit (AccuPrepTM, Bioneer Corporation) following the manufacturer′s instructions.
Fungi rDNA concentrations were measured using real time PCR relative to total bacteria amplification (ΔΔCt) and the SYBR Green PCR Master Mix Kit (SYBR Green I qPCR Master Mix, Syntol, Russia). The 16s rRNA gene-targeted primer sets used in the present study are described in Table 1. Templates (1 μL) were added to amplification reactions (25 μL) containing 0.6 μL of primer mixture containing 10 pmol of each primer, 11.5 μL of SYBR Green I qPCR Master Mix (Syntol) and 12 μL of deionized water. SYBR Green I qPCR. Master Mix contained KCl, Tris-HCL (pH 8.8), 6.25 mM MgCl2, dNTP, Taq DNA polymerase, Tween, and SYBR Green I. A no-template (sterile distilled water) negative control was loaded on each plate run to screen for contamination and dimmer formation and to set the background fluorescence for plate normalization. Amplification and detection were performed using an applied biosystems (ABI) 7300 sequence detection system under the following conditions: initial denaturation at 95 ˚C for 5 min was followed by 40 cycles of denaturation at 95 ˚C for 15 s, annealing at 61 ˚C for 15 s, extension at 72 ˚C for 30 s, and then by the melting curve program (60-95 ˚C with a heating rate of 0.2 ˚C per second and a continuous fluorescence measurement). For total bacteria the threshold cycle of each standard dilution was determined during the exponential phase of amplification and regressed against the logarithm of known total bacterial. Total bacteria population size is reported as nano gram (ng) per μL of extracted DNA. The copy number of total bacteria 16 S ribosomal RNA gene was determined as: Log10 copy number= Ct-(y-intercept/efficiency), where the formula parameters were derived from a standard curve of total bacteria. The population of fungi were expressed relative to the estimated abundance of total bacterial 16 S ribosomal RNA gene.
The chemical composition of the diet is reported in Table 2. DM content determined for each substrate by drying samples for 48 h in a 65 ˚C forced air oven (AOAC, 1990). Dry samples of the feed were maintained overnight at 550 ˚C in a furnace and organic matter (OM) was subsequently calculated as 100 minus the percentage ash (AOAC, 1990). Total nitrogen of each diet was determined by the kjeldahl method (AOAC, 1990), crud protein was calculated as N × 6.25. Ether extract was determined following the AOAC (1990). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) of the feed samples were analyzed by the detergent system using the sequential procedure of Van Soest et al. (1991). For N-NH3 concentration determination, a 5 mL of each sample was acidified with 5 mL of 0.2 N HCL and was measured by the kjeldahl method. The medium pH was measured at the end of incubation with a pH meter (Metrohm744, Switzerland). The disappearance of DM was calculated as original weight of the DM minus weight of dry residue (after incubation) divided by weight of the original sample.
Table 1 PCR primers utilized for amplifying the target bacteria
Table 2 Dry matter and chemical composition of alfalfa hay, sugar beet pulp and barley grain
Statistical analysis of data from in vitro gas production test were made using SAS (1999) with the following model:
Yijk= μ + Ai + βj + Tk + eijk
Y: depended variable.
μ: overall mean.
Ai: effect of EO.
Bj: effect of feed sample.
Tk: effect of concentration.
eijk: residual error, were conducted in a 3 × 6 × 3 factorial design.
Statistical analysis of rumen fermentation characteristics were conducted as a randomized complete design using SAS program with the following model:
Yij= μ + Ai + eij
Y: depended variable.
μ: overall mean.
Ai: effect of EO.
eij: residual error.
Evaluations of significance of difference between means of treatments and controls were determined at (P<0.05) using the Dunnett test.
RESULTS AND DISCUSSION
The effect of treatments on in vitro gas production parameters
The effects of EO on in vitro gas production parameters are shown in Table 3. Addition EO to AH showed that cinnamon significantly decreased c parameter at 200 µL (P<0.05), but it did not produce gas at 400 µL. Also, supplementation of cumin significantly decreased gas production parameters and b parameter at 200 and 400 µL (P<0.05), respectively.Moreover, addition of garlic oil and fennel significantly increased c parameter at 200 µL (P<0.05) and black pepper significantly increased c parameter at both 200 and 400 µL (P<0.05) and monensin significantly increased both gas production parameters (P<0.05). In addition EO, BP showed that cinnamon and black pepper significantly decreased c parameter but increased b parameter at 200 µL, while, cinnamon did not produce gas at 400 µL. Moreover, cumin addition significantly decreased gas production parameters and b parameter at 200 and 400 µL (P<0.05), respectively. Also, fennel addition significantly decreased b parameter at both 200 and 400 µL (P<0.05), and garlic oil decreased b parameter at 200 µL (P<0.05), while, monensin significantly increased it. Addition evaluations for EO to BG showed that cinnamon and garlic oil additions significantly increased b parameter at 200 µL, while it did not produce gas at 400 µL. Also, black pepper addition and monensin significantly decreased c parameter but increased b parameter (P<0.05), while fennel decreased b parameter at 200 µL (P<0.05).
The effects of treatments on rumen fermentation characteristics on in vitro condition
The effects of treatments on disappearance of concentrations of DM, pH and N-NH3 are shown in Table 4 after 48 h incubation, respectively. Additions of all EO to AH significantly increased pH of culture (P<0.05), but decreased disappearance of DM except in fennel. Black pepper and cinnamon significantly decreased N-NH3 concentration (P<0.05). In addition, addition of cinnamon and cumin significantly decreased disappearance of DM and N-NH3 concentration of BP (P<0.05), while, cinnamon significantly increased pH of culture (P<0.05). Moreover, all treatments significantly decreased disappearance of DM of BG except fennel. Also, cinnamon and cumin significantly increased pH of culture but decreased N-NH3 concentration (P<0.05).
Table 3 Effect of treatments on gas production of alfalfa hay, sugar beet pulp and barley grain
SEM: standard error of the means.
1 Monensin used as 5 μmol in medium.
The effects of treatments on ruminal anaerobic fungal population
Effects of treatments on ruminal anaerobic fungal population are shown in Figure 1. Results of the present study demonstrate difference between treatments for evaluations of ruminal fungal population. All treatments significantly decreased (P<0.05) both fungal population of AH and fungal population of BG in rumen except cumin. In general, rumen microbial activity and gas production were affected by the use of EO. These results are consistent with those reported in Fraser et al. (2007) and Macheboeuf et al. (2008) in that cinnamon decreased gas production at incubation periods of 24 h and 16 h. Also, Jahani-Azizabadi et al. (2009) reported that addition of cumin powder (4% of incubated DM) on in vitro rumen microbial fermentation of alfalfa hay decreased production of gas and methane. On the other hand, results of the present study did not conform to those reported in Hodjatpanah‐Montazeri et al. (2015) in which cumin and cinnamon increased gas production 24 h post incubation. Other reports have demonstrated that garlic decreased gas production (Busquet et al. 2005; Kilic et al. 2011). Furthermore, it has been reported that garlic oil has antimicrobial activity and a high dose of garlic could have a detrimental effect on ruminal fermentation (Feldberg et al. 1988). Cinnamon is a monophenolic compound that binds proteins and disturbs energy metabolism in cells (Wendakoon and Sakaguchi, 1995). Oussalah et al. (2006) also reported that cinnamon had an inhibitory effect on pseudomonas putida. The chemical structure determines the activity and antimicrobial effect of an EO (Dorman and Deans, 2000).
Table 4 Effect of treatments on rumen fermentation characteristics in in vitro condition of alfalfa hay, sugar beet pulp and barley grain
SEM: standard error of the means.
Various results have been reported on the effects of EO on ruminal pH. The findings of the present study show that cinnamon and cumin caused a significant (P<0.05) increase in pH of culture. Results presented here are consistent with those reported in Fraser et al. (2007) in which cinnamon addition increased the fermenter pH in a RUSITEC. Similar results were obtained by Busquet et al. (2006) in which addition of cinnamon oil increased fermenter pH an in vitro fermentation system. Moreover Hodjatpanah‐Montazeri et al. (2015) showed the pH value of corn silage increased by cumin in in vitro rumen fermentation. Benchaar et al. (2007) reported an increase in ruminal pH when dairy cows received EO. In contrast to the effects observed in this study, other reports show that EO had no effect on pH (Yang et al. 2007). Whiles Jahani-Azizabadi et al. (2014) investigated EO of cumin at its 280 μL L-1 reduced pH. Increase in value pH may be related to the higher amount of ruminal NH3 concentration in our experiment. Results of different studies for the impact of EO on ruminal N-NH3 concentration are variable. In some studies, EO showed good potential to alter rumen microbial fermentation and ammonia producing bacteria in the rumen (McIntosh et al. 2003; Patra and Yu, 2014). The results of the current study on cinnamon and cumin impact showed a significant (P<0.05) decrease in N-NH3 concentration. Several authors have reported a reducing effect on N-NH3 concentration in the incubation medium by cinnamon (Jahani-Azizabadi et al. 2011; Jahani-Azizabadi et al. 2014). Similar results were obtained by Cardozo et al. (2005) when supplementing ocinnamon in an in vitro fermentation system fed a high-concentrate finishing diet. In addition, Busquet et al. (2006) demonstrated that cinnamon inhibited N-NH3 concentration at high concentration (3000 mg/L). Fraser et al. (2007) reported that cinnamon addition decreased N-NH3 in a RUSITEC. Macheboeuf et al. (2008) reported that cinnamon decreased ammonia in an in vitro fermentation system. Ferme et al. (2004) showed that cinnamon addition to an in vitro rumen simulation system resulted in a reduction in Prevotella spp. a group of bacteria known to be involved in deamination, providing evidence of a mechanism of activity. These results suggest that cinnamon extract inhibited peptidolysis. The results of this study indicated that black pepper significantly (P<0.05) decreased N-NH3 concentration of AH. These results are consistent with results reported in Cardozo et al. (2004) in which pepper addition decreased N-NH3 concentration in a continuous culture. Busquet et al. (2006) demonstrated that capsicum oil (active component of black pepper) inhibited N-NH3 concentration at high concentration (3000 mg/L). Cardozo et al. (2005) reported that capsaicin decreased N-NH3 concentration in an in vitro fermentation system fed a diet of straw/concentrate 10:90. Although several studies have demonstrated antimicrobial activity of capsicum oil, the lack of the effect of capsicum oil compared with other EO could be related to its low content of oxygenated hydrocarbons. Some studies have observed that hydrocarbons monoterpenes are in general significantly less active than oxygenated monoterpenes (Cox et al. 2001). Decreased N-NH3 concentration with EO suggests that these types of additives decrease deamination activity of bacteria that rely on peptides and AA as a source of N (Taghavi-Nezhad et al. 2014).
Figure 1Effect of treatments on quantity of the fungal population existing in the in vitro culture relative to total bacteria population (Ct fungi×10-8) of alfalfa hay (A), sugar beet pulp (B), barley grain (C)
Asterisk differ significantly from the control (P<0.05)
Wallace et al. (2002) suggested that anti-microbial properties of EO can be exploited to modulate activity of rumen microbial populations by reducing degradation of dietary protein and thereby enhancing rumen N escape. Also, recent studies indicated that application of EO resulted in a significant reduction in protozoal population in the rumen (Patra and Yu, 2014; Khorrami et al. 2015; Lin et al. 2013). Rumen protozoa are responsible for a considerable amount of rumen ammonia due to predation of rumen bacteria and consumption of feed proteins (Bonhomme, 1990; Firkins et al. 2007). Therefore, a portion of observed reduction in ammonia concentration may be due to anti-protozoa effects of the EO (Lorenz et al. 2011). Perhaps the different effects of EO on feed samples reflect that there are different species responsible for digestion of the various proteins. Beside these differences among studies, results may have been affected by method of experimentation. In the present study, cinnamon and cumin decreased disappearance of DM confirming the findings of some previous studies (Jahani-Azizabadi et al. 2011; Jahani-Azizabadi et al. 2014; Hodjatpanah‐Montazeri et al. 2015). These findings are similar with those of Fraser et al. (2007) who observed a decrease in DM digestibility when cinnamon leaf oil was supplied at 500 mg/Lin a RUSITEC. Because fiber disappearance was reduced by cinnamon, this can be lead to the conclusion that fungi were affected by cinnamon addition. However, the magnitude of the reduction in DM disappearance was greater for barley grain than for alfalfa. This implies that microbial populations involved in concentrate digestion were affected more by cinnamon than those involved in forage digestion. Reduction in DM disappearance might be nutritionally unfavorable for an animal but a decrease in concentration of N-NH3 because of an increase in ruminal escape dietary protein can improve efficiency of the nitrogen use by the ruminants (Van Nevel and Demeyer, 1988). Moreover, this study showed that EO resulted in a decrease in the rumen fungal population. These results are consistent with those reported in McIntosh et al. (2003) showing that addition of a blend of EO inhibited N. frontalis fungal activity in the rumen with H2 production at the concentration of 40 ppm. Similar results were obtained by Talebzadeh et al. (2013) in that fungal growth (Neocalimastix spp.) were inhibited and activity of fibrolytic fungal enzymes were reduced by adding ajowan EO. Kongmun et al. (2010) reported that ruminal anaerobic fungi population was not influenced by coconut oil and garlic powder. Although, garlic oil had fungicidal properties, as reported by Pai and Platt (2008). The antifungal effect of EO may also have contributed to decreased fiber digestibility. However very little information is available on the effect of EO on rumen fungal population. The results observed in the present trial suggest that careful selection of plant extracts may help to improve efficiency of rumen microbial fermentation. However, future research may help to establish the efficacy of EO as rumen microbial modulators.
The results obtained in the present study suggest that EO are able to modify rumen fermentation by changing gas production, protein metabolism or by causing disappearance of DM. In terms of protein metabolism, the mechanism of action may be related to inhibition of deamination, although the inhibition of peptidolysis has also been suggested for some EO. However, further research is necessary to determine optimal doses of EO, the potential for adaptation of rumen microflora to the actions of these additives, the fate of these products in an animals and the presence of residue in meat or milk, and effects on animal performance.
The authors gratefully acknowledge the technical and financial support of Ferdowsi University of Mashhad.