Effects of Thyme Essential Oil and Disodium Fumarate on Ruminal Fermentation Characteristics, Microbial Population and Nutrient Flow in a Dual Flow Continuous Culture System

Document Type: Research Articles

Authors

Department of Animal Science, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

Abstract

The aim of the present study was to investigate the effects of di-sodium fumarate (DSF) and thyme essential oil (TEO) solely and simultaneously on ruminal fermentation properties and microbial abundance. A dual-flow continuous culture system (DFCC) with eight 1400-mL fermenters was used in a period of 12 d that divided to 9 d for adaptation and 3 d for sampling. Fermenters were fed 100 g d-1 [dry matter (DM) basis] of a 50:50 alfalfa hay:concentrate ration. Treatments were no additive (control), 10 mM of DSF, 125 µL/L of TEO and simultaneous use of 10 mM of DSF, and 125 µL/L of TEO (SIMTF). Treatments had no effect on organic matter (OM) and neutral detergent fiber (NDF) disappearance, total and cellulolytic bacteria and protozoa abundance, large peptides and N-NH3 concentration of the effluent, N-NH3 and dietary N flow and molar proportions of acetate (C2), butyrate and isovalerate. DSF significantly increased crude protein (CP) degradation, the molar proportion of propionate (C3) and reduced C2:C3ratio (P<0.05). TEO decreased (P<0.05) DM disappearance (-14.4%) and total volatile fatty acid (-19.4%) concentration, relative to the DSF. Relative to the control, small peptide plus amino acid N concentration was higher (P<0.05) in TEO treatment and DSF. SIMTF increased (P<0.05) the acid detergent fiber (ADF) disappearance and decreased the N-NH3 concentration from zero to 4 h after feeding. Total N, non-ammonia N and bacterial N flow and efficiency of microbial CP synthesis increased with SIMTF. Findings demonstrated that feed efficiency and ruminal fermentation were improved by the simultaneous use of DSF and TEO.

Keywords


INTRODUCTION

Intermediates in the succinate-propionate pathway such as organic acids can reduce the production of methane by acting as hydrogen acceptors (Martin, 1998; Lopez et al. 1999a; Newbold et al. 2005). Fumarate is a hydrogen sink and acting as C3 precursor in the rumen. Fumarate (salts and free acid form) and malate supplementation have been examined for manipulation and enhance the efficiency of the ruminal anaerobic fermentation process (Garcia-Martinez et al. 2005; Lin et al. 2013; Riede et al. 2013). Previous studies have reported that inclusion of fumarate in ruminant diets, decrease methane production, C2:C3ratio and ammonia nitrogen concentration, and increase the number of cellulolytic bacteria and organic matter (OM) disappearance (Lopez et al. 1999b). Newbold et al. (2004) demonstrated that sodium fumarate had efficient effects on fiber digestion, dry matter degradation and hydrogen acceptation in batch culture and rumen simulation technique (RUSITEC) compared with free acids and sodium acrylate. On the contrary, the effects of fumarate are unclear in vivo studies and presumably, it is due to the unknown of the optimum level of fumarate. Zhou et al. (2012) observed that the addition of 20 g/d di-sodium fumarate (DSF) in Hu sheep diet fed by a high-forage ration did not affect C3 proportion and C2:C3 ratio. Yang et al. (2012) indicated that the addition of 10 g/d DSF in goats fed according to maintenance requirements, increased C3 proportion, but not change C2:C3 ratio. Remling et al. (2011) reported that 100, 200 or 300 g/d of fumaric acid did not affect the performance of growing bulls fed with full forage diet. There is some evidence that essential oil (EO) and some secondary compounds can reduce C2:C3 ratio, methane and N-NH3 production through specific inhibition of ruminal gram-positive microbes that usually involvement in C2, methane and ammonia production (Szumacher and Cieslak, 2010). Thyme essential oil (TEO) as regards owning thymol, carvacrol, p-cymene and γ-terpinene have higher antibacterial properties (Ultee et al. 2002; Martinez et al. 2006). Researchers demonstrated that the use of a blend of essential oils includes thymol (Castillejos et al. 2006; Castillejos et al. 2007) and thyme EO (Jahani-Azizabadi et al. 2011) resulted in a decrease in C2:C3 ratio, methane and N-NH3 concentration. The addition of thymol and eugenol at 5, 50 and 500 mg/L in DFCC system implied that 5 mg/L of thymol tended to decrease the proportion of C2, and increase the large peptide (LPep N) concentration without reducing total volatile fatty acid (VFA) concentration (Castillejos et al. 2006). In our previous study, we examined the effect of different doses of TEO (from 100 to 400 µL/L) and DSF (from 8 to 12 mM) on in vitro batch culture rumen anaerobic fermentation characteristics. Baraz et al. (2018) demonstrated that 125 µL/L of TEO and 10 mM of DSF generally resulted improve in ruminal fermentation (decrease in methane, N-NH3, C2:C3 ratio and nonsignificant decrease in total VFA and significant increase organic matter disappearance). But, it is still unclear, what is the effect of TEO and DSF solely and simultaneously in the DFCC system that is much nearer to the active dynamic of ruminant’s rumen. In addition, when EO with high antimicrobial activity is applied for rumen manipulation and reduce methane production, the hydrogen produced from organic matter fermentation will be accumulated in rumen fluid (Lin et al. 2012; Lin et al. 2013), so, employ an alternative pathway for capturing of reducing equivalents can result to ultra-improve in the rumen anaerobic fermentation. Therefore, the objective of the present study was to evaluate the effect of TEO and DSF and their synergistic effects on rumen anaerobic microbial fermentation of a 50:50 alfalfa hay: concentrate diet in the DFCC system.

 

MATERIALS AND METHODS

Experimental design and treatments

A DFCC system with eight 1400-mL fermentors was used in a period of 12 d that divided into adaptation (9 d) and sampling (3 d) periods. On the first day to begin, 400 mL of strained rumen fluid and 1000 mL of pre-warmed McDougall’s buffer (McDougall, 1948) were added to each fermenter. Fermentors were fed 100 g/d DM (in three equal meals, every 8 h) of a diet included a 50:50 alfalfa hay:concentrate. Chemical composition and ingredients of offered ration presented in Table 1. Rumen fluid was taken from 10 slaughtered rams that fed with a 60:40 forage: concentrate diet. Fermentor's temperature was maintained at 38.5 ˚C by a circulating water bath. For maintaining the anaerobic conditions N2 gasat 40 mL min−1 infused into fermenters. Artificial saliva which contains urea solution (0.4 g urea/L) for simulate ruminal N recycling, was continuously infused into the fermenters. Liquid and solid dilution rates maintained at 10 and 5 percent h−1, respectively. Treatments were; control without additive (CON), DSF at 10 mM(DSF;Disodium salt; Sigma Chemical Co., Poole, and Dorset, UK), TEO at 125 µL/L (TEO;MONIN Company, France) and 10 mM of DSF plus 125 µL/Lof TEO (SIMTF). All treatments were incorporated directly into the contents of the fermentor at d 4 of the experiment before morning feeding. TEO was dissolved in ethanol (96%; 1:1 ethanol:TEO) before incorporating into fermenters. Fermenters selected for the CON group were also supplied 125 µL/Lof ethanol per feeding.

 

Sample collection and preparing

During sampling days (the final 3 d), 20 mL of filtrated (filter pore size was 48 µm) fermentors contents were taken at zero, 1, 2, 3 and 4 h after the feeding (on the morning meal) to determine N-NH3 concentration and pH. Moreover, at 4 h after the morning feeding, 1.5 mL of strained sample was removed and mixed with 375 µL of 25% orthophosphoric acid to determine VFA concentration and frozen at −20 ˚C. During sampling days, effluent collection vessels were maintained in cold water (4 ˚C) for the debarment of additional microbial fermentation. All content of collection vessels was mixed for 1 min, then immediately strained through cheesecloth to eliminate solid and liquid parts, and solid residues were dried in the oven (55 ˚C for 48 hours) and used to estimate DM, CP, OM, NDF and ADF digestibility. Subsamples of the filtrate were used for the determination of total N, ammonia N, tungstic acid (TA) soluble N and trichloroacetic acid (TCA) soluble N.

 

Table 1 Ingredients and chemical composition of experimental diet

 

1 Each kg of premix containing: Ca: 190 g; P: 90 g; Na: 50 g; Mg: 19 g; Cu: 3 g; Fe: 3 g; Mn: 2 g; Zn: 3 g; Co: 100 mg; I: 100 mg; Se: 1 mg; vitamin A: 500000 IU; vitamin D3: 100000 IU; vitamin E: 100 mg and Antioxidant: 3 g.

2 NFC= 100 - (NDF+CP+EE+Ash).

 

Samples for determining bacterial N were procured from the 2 sampling d, followed the procedure of (Makkar et al.1982). The effluents (30 mL) were centrifuged in two-time at 3000 × g at 4 ˚C for 10 min to separate feed particles, in each time supernatants were separated and stored in another tube and pellets were washed with normal saline (0.9% NaCl) and centrifuged. Then the supernatants of previous steps were collected and centrifuged at 15000 × g for 20 min. The supernatants were discarded and the pellet (bacterial cells) dried in the oven (55 ˚C for 48 h) and used to determine bacterial N by the Kjeldahl procedure. The flows of total N, dietary N, bacterial N and non-ammonia N (NAN) were calculated as described by Stern and Hoover (1990). On the final day, 2 mL of fermenters' contents were taken at 4 h after the morning feeding to the enumeration of total viable and cellulolytic bacteria by most probable number (MPN) procedure from 3 replicate (Oblinger and Koburger, 1975). The medium samples were serially diluted (10 fold increments) in the liquid version of mediums in the Hungate tubes, 3 replicate per each dilution (Caldwell and Bryant, 1966). For cellulolytic bacteria, cellulose filters paper used as a single source of energy. The tubes were incubated at 38.6 ˚C for 14 d, and then growth was scored (positive or negative). For the enumeration of total viable and cellulolytic bacteria, was considered pH variation and disappearance of cellulose filter paper (Whatman® No. 7), respectively. For protozoa counting, 7 mL of fermenters' contents were fixed in an equal volume of a 50% formalin solution (18.5% formaldehyde concentration). Protozoa were enumerated microscopically in a Sedgwick-Rafter counting chamber according to Dehority (1984) method.

 

Chemical analyses

VFA concentration was determined using gas chromatography (GC, Philips, pu 4410). Solid residues or diet samples were dried using an oven at 55 ˚C for 48 h and analyzed for DM, CP, and OM based on the methods recommended by AOAC (1995). The crude protein of diet and solid residues, total N of effluent, bacterial N and N-NH3 concentration of the contents of the fermentor were determined using the Kjeldahl method (Kjeltec Vapodest 30s, Gerhardt). The TCA- and TA-soluble N concentration of effluents were determined as described by Winter et al. (1964). The feed and effluent samples were dried (at 55 ˚C for 48 h), ground to pass through a 1 mm screen. The ashless NDF and ADF content of the diet and solid phase of effluents were determined using the method recommended by Van Soest et al. (1991) and AOAC (1995), respectively.

 

Calculations and statistical analysis

Results of the TCA and TA soluble N concentration of liquid phase of effluents were used for calculation of large peptide (LPep (mg/dL)= LPep= TCA soluble N - TA soluble N) and small peptide plus amino acid N (SPep+AAN (mg/dL)= SPep + AAN =TA soluble N-ammonia N). All statistical analyses were conducted using generalized linear model (GLM) procedure of SAS (2001) with the following statistical model:

Yij= μ + Ti + eij 

Where:

Yij: dependent variable.

μ: overall mean.

Ti: effect of the treatments at each concentration.

eij: residual error.

The results of N-NH3 concentration and medium pH were analyzed as randomized block design. Tukey test was employed to compare the means (P<0.05).

 

RESULTS AND DISCUSSION

Nutrients disappearance and microbial abundance 

The effects of treatments on the abundance of total viable and cellulolytic bacteria, protozoa and nutrients disappearance are presented in Table 2. Dry matter disappearance was significantly (P<0.05) decreased by TEO supplementation, relative to those of the DSF (596 vs. 696 g/kg, respectively). Relative to the control, DM, OM and NDF disappearance did not affect by treatments, although they were numerically lower in TEO. Supplementation of the TEO and DSF significantly increased (P<0.05) ADF disappearance, relative to the CON and TEO solely. Total viable bacteria, cellulolytic and protozoa abundance were not affected by treatments.

 

Crude protein degradation and N metabolism

Treatments had no effect on effluent N-NH3 concentration at 4h after morning feeding (Table 3). Relative to the control, DSF resulted in an increase in CP degradation. But, the addition of TEO relative to the SIMTF, decreased (P<0.05) total and non-ammonia N concentration. The SIMFT treatment resulted in an increase (P<0.05) in bacteria N flow and efficiency of microbial crude protein synthesis (EMPS) (Table 3). The results of our study showed that LPep N concentration was not affected by the addition of treatments (Table 3). However, the concentration of SPep + AA N was higher (P<0.05) in TEO compared to the CON and DSF. The concentration of medium N-NH3 was evaluated from zero to 4h post-feeding (Figure 1). Results showed that the simultaneous use of DSF and TEO (SIMTF) decreased N-NH3 concentration at zero, 2 and 4 h after feeding, compared to the CON (P<0.05).

 

Volatile fatty acid concentration and medium pH

In the present study, the pH of fermentors was evaluated from 0 to 4 h of post-morning feeding (Figure 2). The inclusion of DSF in the diet resulted in an increase (P<0.05) in the minimum and average of medium pH, relative to TEO and CON (Table 4). In addition, the medium pH at all recording time in DSF, TEO and SIMTF was higher than CON (Figure 2). The findings of the present study showed that the addition of DSF and TEO solely and simultaneously did not affect (P>0.05) proportion of C2, C4 and isovalerate (Table 4). Adversely, relative to the CON and use of TEO significantly decreased total VFA concentration (-19.4%, P<0.05). DSF and SIMTF treatments increased (P<0.05) the proportion of C3 (14.7 and 20.0%, respectively) and decreased C2:C3 ratio compared to those of the CON and TEO treatments (P<0.05). Fumarate affects rumen fermentation as H acceptor and as a precursor for C3 (Newbold et al. 2005), on other hand TEO effects on rumen fermentation because it's antimicrobial activity (Ultee et al. 2002). Therefore, it appears that simultaneously use of DSF and TEO may lead to synergistic effects on rumen microbial anaerobic fermentation. The decrease in DM disappearance with TEO supplementation was in agreement with previous studies (Castillejos et al. 2007; Jahani-Azizabadi et al. 2014; Pirondini et al. 2015). For instance, Jahani-Azizabadi et al. (2014) reported a 21.6 and 66% decrease in DM and NDF disappearance with supplemented 280 µL/L of TEO, which was associated with inhibitory and antibacterial effects on rumen bacteria (Ultee et al. 2002). Since the fumarate is an intermediate in rumen microbial metabolism, it appears that the addition of fumarate at 10 mM in SIMTF may be removed some negative effect of TEO on DM disappearance (Table 2). Relative to the control, we observed an increase (P>0.05) in cellulolytic bacteria with DSF (2.3%) and SIMTF (1.8%) supplementation. There has been some evidence that the use of fumarate leads to an increase in the abundance of cellulolytic and some fumarate-utilizing bacteria (Lopez et al. 1999b). It's assumed that the metabolism of fibrinolytic bacteria intermediate products (such as hydrogen) accelerate by fumarate-utilizing bacteria (Mao et al. 2007). On the other hand, an increase in ADF disappearance without significant change in abundance of cellulolytic bacteria, demonstrating that some obligate fiber fermenting bacteria that had a higher activity for fermentation of fiber fractions relatively increased. We observed that protozoa abundance did not affect with DSF and TEO. I agreeing to our findings, Lopez et al. (1999b) reported that fumarate was unable to changes in the protozoa population, likewise, Lin et al. (2012) observed that under in vitro condition, 500 mg/L of essential oil active compounds (EOAC) inhibited protozoa. Although, in the current study were observed a numerical decrease in the abundance of protozoa with the addition of TEO (P=0.12). It seems that a decrease in the protozoa population maybe one of the wide-range effects of essential oils on microbial activities. Higher NAN in SIMTF and consequently increased bacterial N (0.97 g/d) flow relative to the CON (0.69 g/d) demonstrated that SIMTF had more efficiency in increase organic matter fermentability and microbial protein synthesis. Results showed that SIMTF treatment resulted in a significant increase in the EMPS compared with those of the CON (15.76 vs. 11.25 g N/kg of digested OM, P<0.05).

 

Table 2 Effects of di-sodium fumarate and thyme essential oil solely and simultaneously (SIMTF) on nutrients digestibilities and abundance of total and cellulolytic bacteria in a dual-flow continuous culture system

 

CON: control; DSF: 10 mMdi-sodium fumarate; TEO: 125 µL/L thyme essential oil and SIMTF: 10 mMDSF and 125 µL/L TEO.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 3 Effects of di-sodium fumarate and thyme essential oil solely and simultaneously (SIMTF) on the flow of N in a dual-flow continuous culture system

 

CON: control; DSF: 10 mMdi-sodium fumarate; TEO: 125 µL/L thyme essential oil and SIMTF: 10 mMDSF and 125 µL/L TEO.

EMPS: efficiency of microbial crude protein synthesis; LPep N: N from large peptides and SPep + AA N: N from small peptides and AA.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Figure 1 The effect of di-sodium fumarate and thyme essential oil solely and simultaneously (SIMTF) on medium NH3-N fluctuation in a dual-flow continuous culture system


 

 

Figure 2 The effect of di-sodium fumarate and thyme essential oil solely and simultaneously (SIMTF) on medium pH fluctuation in a dual-flow continuous culture system

 

 

Table 4 Effects of di-sodium fumarate and thyme essential oil solely and simultaneously (SIMTF) on total and individual volatile fatty acid (VFA) concentration at 4 h post-feeding and pH in the dual-flow continuous culture system

 

CON: control; DSF: 10 mMdi-sodium fumarate; TEO: 125 µL/L thyme essential oil and SIMTF: 10 mMDSF and 125 µL/L TEO.

C2:C3: acetate:propionate ratio.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Relative to the CON, solely the use of TEO did not affect the N flow. Similarly, in the previous studies that evaluated the effect of supplementation of thymol (a main active component of thyme essential oil) in the DFCC system (Castillejos et al. 2006; Castillejos et al. 2007), observed that N-NH3 concentration, N flows, EMPS and CP degradation were not affected by treatments. Supplying of DSF significantly increased CP degradation around 13% compared to those of the CON (P<0.05). Numerically this effect appeared with an accumulation of LPep N (P>0.05), which was higher in DSF relative to the CON treatment. The increase in CP degradation without an increase in N-NH3 concentration suggested that DSF supplementation may result in an increase in ruminal efficiency use of dietary protein and a decrease in microbial deamination of amino acids. The accumulation of SPep + AA N with TEO supplementation, suggests that peptidolysis stimulated or deamination was inhibited by TEO. In agreement with our findings, Castillejos et al. (2006) observed an increase in the concentration of LPep and SPep + AA N by thymol supplementation at 500 mg/L. Our findings showed that the simultaneous use of DSF and TEO (SIMTF) decreased the rate of N-NH3 production from 0 to 4 h after morning feeding, relative to the CON (Figure 1). McIntosh et al. (2003) reported that a commercial blend of EO compounds containing thymol, through inhibiting the growth of ammonia hyper-producing bacteria, resulted in a significant decrease in the rate of amino acid deamination. Castillejos et al. (2007) reported that the addition of a commercial blend of EO (Crina®)resulted in a higher accumulation of SPep + AA N and a decline in the N-NH3 concentration. Similarly, several in vitro and in situ studies demonstrated that the concentration of N-NH3 (Jahani-Azizabadi et al. 2011) was lower (-16 to -44%) and deaminase inhibited (Newbold et al. 2004)by the addition of TEO and Crina® in the diet, respectively. Moreover, there are a few data on the impacts of DSF and TEO on ruminal fermentation in DFCC, but evaluated the effects of some EOAC (contained thymol) along with monosodium fumarate in vitro studies (Lin et al. 2012; Lin et al. 2013). Consistent with our findings, Lin et al. (2012) and Lin et al. (2013) observed that the addition of 200 or 500 mg/L of a blend of EO with 0, 5, 10 or 15 mM of monosodium fumarate, after 24 h of incubation decreased the N-NH3 concentration relative to the control and EO solely. Higher medium pH at 0 to 4 h of morning feeding with supplementation of DSF, TEO, and SIMTF observed in the present study, confirms findings of previous in vitro batch culture and RUSITEC studies (Callaway and Martin, 1996; Asanuma et al. 1999; Carro and Ranilla, 2003; Newbold et al. 2005). In the ruminal fermentation process, conversion of hexose to VFAs results in an overall net release of reducing equivalents. In the rumen anaerobic condition, H2 is used to reduce fumarate, therefore fumarate as H2 acceptor resulted in a decrease in the availability of H2 in the internal environment of the rumen, which was associated with the inhibitory effect of fumarate on methane production (Callaway and Martin, 1996). In addition, at least a part of higher medium pH in DSF, TEO and SIMTF treatments compared to those of the control, related to their buffering capacity, decrease in overall fermentation and VFA production and their synergistic effects, respectively. Previous evaluation of the short and long-term effects of some EO showed that high dose (5000 or 500 mg/L) of thymol strongly decreased total VFA concentration in the DFCC system and in vitro batch culture (Castillejos et al. 2006). Similarly, Pirondini et al. (2015) reported that the supplementation of the medium with thymol or TEO (500 mg/L) after 24 h of incubation significantly decreased total VFA concentration, relative to the control. It seems that reducing in total VFA concentration is consistent with the known antimicrobial properties of TEO on a wide range of gram-positive and -negative bacteria in the rumen (Ultee et al. 2002). Increase in C3 and a decrease in C2:C3 ratio, with DSF and SIMTF supplementation, confirm previous findings in the batch culture system (Lopez et al. 1999b; Carro and Ranilla, 2003; Garcia-Martinez et al. 2005). Propionate is considering a valuable energy fuel for the ruminants, and this is a highly desirable effect on animal performance and efficient use of feed energy. In contrast to our observation, Lopez et al. (1999b) reported an 11.6 and 24.6% increase in the molar proportion of C2 at 5 and 10 mMof sodium fumarate in a 75: 25 forage: concentrate diet. However, Garcia-Martinez et al. (2005) also observed a 7.7% increase in C3 proportion at 8 mMof DSF in a medium-forage diet. At least, a part of inconsistencies in the studies results may be related to the basal diets (medium forage vs. higher forage diets). Similarly, Lin et al. (2013) observed that the simultaneous use of monosodium fumarate and a blend of EO resulted in a significant increase in the molar proportion of C3 and a decrease in C2:C3 ratio. Fumarate can be converted to C3 (48% of supplemented fumarate) and C2 (20% of fumarate) via different pathways (Ungerfeld et al. 2007).

 

CONCLUSION

In the present study, use of DSF along with TEO resulted in higher modulatory effects on total VFA, the proportion of C3 and C2:C3 ratio compared to TEO treatment (P<0.05). Relative to the CON, our findings demonstrated a numerical decline in lipogenic (C2 and butyrate) precursors and significant enhance (20%) in glucogenic (C3) precursor with the simultaneous addition of DSF and TEO. Probably, this glucogenic impact can improve effectiveness production of liver glucose, glucose provides to the mammary gland and lactose and milk production in the high-producing dairy cow.

 

ACKNOWLEDGEMENT

This work was funded by the University of Kurdistan (Iran). The authors are indebted to Mr Hossein Souri and Ms Somayeh Khodamoradi from the University of Kurdistan for their assistance throughout the experiments.

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