In vitro Evaluation of Sheep Rumen Fermentation Pattern After Adding Different Levels of Eugenol – Fumaric acid Combinations

In vitro gas production technique was used to evaluate the effect of three different levels of eugenol + fumaric acid combinations on rumen fermentation. Rumen contents were collected from five rams immediately after slaughtering and used for preparation of inoculums of mixed rumen microbes that were used in generation of five –1 treatment systems, negative control with no additives (T1), fumaric acid 0.5 mg L (T2) and fumaric acid 0.5 mg –1 –1 L in combination with three different doses of eugenol, 100, 200 and 400 mg L (T3, T4 and T5 respectively). Incubations were conducted in triplicates with gas production, pH, ammonia nitrogen (NH -N), total and 3 fractional volatile fatty acids (VFAs) concentrations, cellulase activity, amount of substrate degraded, microbial yield (Y ), fermentation efficiency (FE) and VFAs utilization index (NGGR) were determined after 24 hours of ATP incubation. The results revealed that, different levels of eugenol + fumaric acid combinations were associated with decreased pH value, NH -N concentrations and methane production and increased valeric and isovaleric 3 acids molar proportions. T3 and T4 were associated with increased propionates at the expence of acetates (low A/P), decreased methane production and increased FE, microbial yield (Y ) and VFAs utilization. In contrast, ATP T5 showed decreased total VFAs concentrations, cellulase activity, the amount of substrate degraded, microbial –1 mass generated and VFAs utilization. In conclusion, the authors recommend using 200 mg L eugenol + fumaric acid combination as an alternative for antibiotic feed additives to optimize rumen fermentation pattern. Further investigations are required to apply this work in vivo experiments.


Introduction
Among these alternatives, essential oils and fumaric acid are most hopeful and have received Manipulation of rumen microbial fermentation much attention.Essential oils are secondary plant to decrease methane and ammonia nitrogen metabolites that generally recognized as safe for production using antibiotic feed additives has human consumption (FDA, 2004) and able to proved to be a useful strategy to improve modify rumen microbial fermentation through a production efficiency in ruminants (McGuffey et wide spectrum of antimicrobial activity al., 2001).However, the use of antibiotics as feed (Greathead, 2003) especially on Gram-positive additives in ruminants is banned in the European bacteria (Burt, 2004).Phenolic components such Union because of the risk of residues (Russell and as eugenol (one of the components of clove) are Houlihan, 2003) and emergence of multi-drug responsible for the antibacterial properties of resistant bacteria (Gustafson and Bowen, 1997) that may threat human health.This risk fueled the many essential oils (Dorman and Deans, 2000), and appear to act as membrane permeabilizers search for non-antibiotic alternatives, which might have similar effects on animal performance.(Helander et al., 1998).
The impact of a blend of feed additives that was not used as inoculum, immediately mixed with 0.3 mL H SO 10N and used for are currently registered for feeding to ruminants 2 4 on rumen fermentation pattern can be applied determination of total VFAs concentration before using In vitro gas production technique which is incubation.

Preparation of treatment systems and in vitro
proved to be a potentially appropriate and well fermentation: The method used for in vitro correlated with rumen fermentation pattern, fermentation was based on the in vitro gas microbial protein synthesis, in vivo digestibility production technique described by Menke and and animal performance (Kamalak et al., 2005).
Steingass (1988).Two-hundred milligrams of Several studies showed that eugenol could feed sample (Composition and chemical analysis manipulate rumen fermentation presumably is shown in Table 1), previously ground with a VFAs profile, ammonia N concentrations and gas pestle and mortar, were carefully dropped into production (Busquet et  with no additives (T1), fumaric acid 0.5 mg L alternative hydrogen sink within the rumen -1 (T2) and fumaric acid 0.5 mg L in combination (Itabashi, 2002).However, its maximum potential with three different doses of eugenol, 100, 200 to divert H away from CH is limited presumably  The objective of this in vitro study was to investigate effects of adding fumaric acid together with three different doses of eugenol using In vitro gas production technique to achieve auxiliary optimistic effects on rumen fermentation pattern.

Materials and Methods
This investigation was conducted in Department of Physiology and Rumenology laboratory in Department of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Egypt.Rumen contents used in preparation of the treatment Eugenol doses were previously prepared as systems were collected from five recently stock solutions by dissolving eugenol in 99.5% slaughtered rams and strained through four layers ethanol and a total of 0.2 ml of each dose was of cheesecloth into a separating flask previously added to the respective syringes.Equivalent gassed with oxygen-free CO and brought 2 amounts of ethanol (0.2 ml) were added to the immediately to the laboratory.Strained rumen syringes assigned for control and uncoupled liquor was mixed with the buffer solution of fumaric acid treatments.After gentle shaking, Goering and Van Soest (1970) in the proportion syringes were tapped and pushed upward by the 1:2 (v/v), flushed with oxygen-free CO and used 2 piston in order to completely eliminate air in the as inoculums of mixed rumen microorganisms.inoculums; the tips were then tightened by a Part of the buffered rumen fluid sample (blank) metal clip so as to prevent escape of gases.Incubation was carried out at 39°C and the equations of Wolin (1960), which has been volume of gas produced was recorded after 24 validated recently by Blummel et al. (1993).hours (The degradation time in the rumen needs 4-Percent of methane output per total VFAs 48 hours).After termination of incubation, the production.fluid samples were drawn into plastic bottles and 5-Fermentation efficiency (FE) was calculated on the basis of the equation worked out by pH values were immediately measured using a Orskov (1975) and modified by Baran and digital pH meter.Supernatant from each fluid Zitnan (2002).sample was separated by centrifugation at 7,000 x 6-VFAs utilization index was expressed by g for 10 minutes.For determination of total VFAs non-glucogenic VFAs/glucogenic VFAs concentrations and individual VFAs proportions, ratio (NGGR) and estimated according to one mL of 25% meta-phosphoric acid was added Orskov (1975).to 5 ml of supernatant and stored at -20°C until analyzed.For NH -N determination, two milliliters 3 Measurement of cellulase activity: From each of supernatant was acidified with 2 ml of 0.2 N crude enzyme solution of the supernatant 0.5 ml HCl and were analyzed by spectrophotometry for was mixed with 0.5 ml of 1% carboxymethyl NH -N according to Chaney and Marbach (1962)

Results
Methane production was greatly reduced by Data presented in tables 2 and 3 showed that, the different levels of fumaric acid -eugenol the effects of eugenol + fumaric acid combinations combinations (table-3) and this was most obvious on rumen fermentation pattern were different in T4.Means of the calculated fermentation depending on the dose.Table 2 revealed that, the efficiencies reveal the following order: T4 > T3 > pH value of the fermentation fluid was decreased T2 > T1, while T5 did not alter fermentation by all levels of eugenol + fumaric acid combinations efficiency with reference to control.In contrast, relative to control.Adding fumaric acid singly or NGGR was decreased in T3 and T4, increased in in combinations with eugenol at 100 and 200 mg T5 and did not differ by addition of fumaric acid -1 L levels did not alter total VFAs concentrations singly in T2.Furthermore, microbial mass -1 generated during fermentation was increased in but when was added with eugenol at 400 mg L T3 and T4, decreased in T5 while, it also was not level resulted in reduced VFAs concentrations.
affected by addition of fumaric acid singly (T2).

The means of VFAs molar proportions identify
Relative to control, microbial yield / mmole ATP that, T2, T3 and T4 were associated with generated during fermentation (Y ) was increased propionate at the expense of acetate ATP without detectable alteration in butyrate molar increased in T3 and T4 while, it was not affected proportions.In contrast, T4 was associated with by addition of fumaric acid singly or in -1 increased butyrates at the expense of both acetates combination with eugenol at 400 mg L level.and propionates.Furthermore, the molar

proportions of the major branched chain VFAs
Results reveal that the effects of eugenol + (valeric-isovaleric -isobutyric) were not affected fumaric acid combinations on rumen microbial by addition of fumaric acid singly but combinations activity varied greatly with regard to dosage of with different levels of eugenol achieved an  eugenol added.Presence of a hydroxyl group in the recorded reduction in total VFA concentration the phenolic structure of eugenol is responsible at this level warns that high doses of eugenol are for its high antimicrobial activity (Dorman and likely to be detrimental for rumen microbial Dean, 2000; Burt, 2004) and hence its dose-fermentation if the same effects were expressed dependent properties.T3 and T4 achieved additional in vivo.Increased concentrations of both valeric improvements in VFAs profile towards increased and isovaleric acids at all levels of eugenol + propionates at the expense of acetates, while at T5 fumaric acid combinations indicates enhanced combination, the results were frustrating as both microbial deaminative activity as deamination of acetic and propionic acids were reduced relative branched chain amino acids represents the major to control.It is predictable that, each mole of source of BCVFAs (Hino and Russell, 1985).fumaric acid when converts into propionic acid This result is analogous to that obtained by would stoichiometrically decrease CH Busquet et al. (2006) when added eugenol at the 3 associated with all levels of fumaric acid + eugenol 1999).The additional increase in propionic acid combinations despite the suggested increase in proportions achieved in T3 and T4 corresponded deaminative activity.However, in T3 and T4 well to the additional decrease in CH production 4 combinations, these low NH3-N concentrations by these treatments relative to uncoupled could be attributed to greater ammonia utilization addition of fumaric acid.This result confirms the by rumen microbes as both microbial mass and issue that hydrogen diverted away from methane microbial yield (Y ) were increased in accordance is used in reduction of fumarates into propionates ATP to the decremental effect of each of the previous with little production of acetates and butyrates et al treatments.Reduction of methane production by (Bayaru ., 2001).all levels of fumaric acid -eugenol combinations The pattern of VFAs observed in T5 mix was efficient especially in T4 combination and could stem from inhibition of gram-negative, propionate-producing bacteria and activation of this probably saved more energy to meet the gram-positive, butyrate-producing bacteria.Burt synthetic needs of rumen microbes.Huber and Herrara (1994) suggested that a synchronous (2004) suggested that gram-positive bacteria supply of energy and NH -N is required for appear to be more susceptible to the antibacterial concentration of 800 mg L , while at 500 mg L achieved in T3 and T4 combinations could also be eugenol, Castillejos et al. (2006) observed no related to the additional increase in propionate at the expense of acetates associated with these effect on the molar proportions of acetate and combinations.Leng (1993)  electron transport phosphorylation coupled with that when used at the concentration of 300 mg L , propionate producing bacteria but absent in eugenol did not change molar proportions of acetate acetate producing bacteria, which implies that and propionate, but increased the proportion of these bacteria acquire energy from hydrogen ions butyrate.
which otherwise are used to produce methane as Therefore, this VFA pattern suggests that, in suggested by Asanuma et al (1999).The recorded T5 combination, gram-negative bacteria were pH values here were within the normal range more sensitive to the antibacterial activity of required for optimum microbial activities eugenol than gram-positive bacteria.Furthermore, (Russell and Welson, 1996).VFAs and ammonia microbial yield (Y ).200 mg L eugenol + fumaric acid combination negative drawbacks on cellulolytic bacterial activity.Recorded reduction associated with T5 combination as an alternative for antibiotic feed additives to points to harmful effects on cellulolytic bacterial optimize rumen fermentation pattern.Further activity.The fermentation efficiency calculated for investigations are required to apply this work in T4 combination amounted to 79.6% and vivo experiment.outperformed the T3 combination by 1.27% and L (T3, T4 and T5 respectively).because methanogen utilize H more rapidly than 2 fumarate-utilizing bacteria (Asanuma et al., 1999).
mg L but incosistent to that production by 5.6 liters (Newbold et al., 2005); observed by Castillejos et al. (2006) when used the inability of fumarate-reducers to compete for -1 eugenol at 500 mg L concentration.More puzzling H with methanogens limits the efficacy of 2 is the tendency to lower NH -N concentrations fumaric acid to play this role (Asanuma et al.,

3
properties of plant extracts than gram-negative ammonia utilization in microbial protein bacteria.Similar VFA patterns were observed by synthesis.et al The substantial microbial cell yield (Y ) Benchaar .(2007) when used eugenol at the ATP -1 -1 ATP concentrations of the fermentation fluid are the Conclusion principal determinants of pH values, as they are + -Form the three different levels of eugenolthe main sources of H and OH and hence, fumaric acid combinations tested in this study, reduction of pH associated with all levels of -1 fumaric acid -eugenol combinations could be only 100 and 200 mg L eugenol + fumaric acid attributed to the recorded reduction in NH -N combinations improved VFAs profile, VFAs 3 utilization, microbial cell yield and rumen concentrations.fermentation efficiency.High doses of eugenol Cellulase activity and the amount of substrate -1 (400 mg L ) could be detrimental on rumen degraded did not alter in T3 and T4 combinations which informs on efficient H disposal without microbial fermentation.It is advisable to use the 2 -1

Table - 2. Effect of treatment systems on fermentation pattern by mixed rumen micro-organisms after 24 hours in vitro incubation (measured parameters)
. estimated according to the then estimated according to Grings et al.(2005).Data presented as means ± SE, N =5 (prepared in triplicates), Values having different letters in the same raw are significantly different at P> 0.05, *Cellulase activity (mmol glucose eq/ min) ** Amount of substrate truly degraded (mg)In vitro Evaluation of Sheep Rumen Fermentation Pattern After Adding Different Levels of Eugenol -Fumaric acid CombinationsMicrobial yields (Y ) were calculated as the mg increase in valeric and isovaleric acids molar

Table - 3. Effect of treatment systems on fermentation pattern by mixed rumen micro-organisms after 24 hours in vitro incubation (calculated parameters)
Data presented as means ± SE, N =5 (prepared in triplicates) Values having different letters in the same raw are significantly different at P> 0.05 In vitro Evaluation of Sheep Rumen Fermentation Pattern After Adding Different Levels of Eugenol -Fumaric acid Combinations