THE EFFECT OF RAMBUTAN PEEL (Nephelium lappaceum) AS REDUCING AGENT ON IN VITRO METHAN PRODUCTION WITHIN CREATING ENVIRONMENT FRIENDLY

FARMING
Siska Aditya

Dept. Animal Nutrition and Feed Science Faculty of Animal Science, Gadjah Mada University Indonesia, siskoaditya@yahoo.co.id

SUMMARY Syringes were filled with 300 mg mixture of forage and concentrate (60:40) and 30 ml medium consisted buffered rumen fluids derived from the gas production technique. Syringes were divided into four group of treatments with rambutan peel as saponin source at the level of 0% (P1), 0.2% (P2), 0.4% (P3), and 0.6% (P4) of the respective medium and incubated at 39C for 72 hours. Variables being measured were methan production, protozoal number, pH value, ammonia, and microbial protein. Additional rambutan peel of 0.2%, 0.4%, 0.6% decreased methan production as much as 6.63%, 14.90%, 25.65% (P<0,01) and decreased protozoal number as much as 17.50%, 63.28%, 72.02% (P<0,01). However, the treatments did not have any effect on together variables. It could be concluded that addition of saponin from rambutan peel as low as 0.2% of the medium decreased methan production and protozoal. Key words: Rambutan peels, Saponin, Rumen fermentation, Methan production

INTRODUCTION According to the Food Agriculture Organization (FAO) of the United Nations declared that the international meat industry is responsible for 18% of green house gas emissions and according to Guardian Unlimited in the UK, methane gas produced by cows partly responsible for 4 percent of greenhouse gas emissions.1.3 billion cows produce 100 million tonnes of methane. Cow manure also resulted in emissions of N2O and CH4 release dung decay. Methane gave a negative effect on the environment since estimated to contribute approximately 14 to 20% of total gas that cause the greenhouse effect. (Asanuma et al., 1999). Methane formation by Jouany (1991) that protozoa symbiotic with bacteria by producing H2 methanogenic which will be utilized by the bacteria, and then converted into CH4. Jouany (1991) states that defaunation is a process of elimination or reduction of protozoa in the rumen, although protozoan ciliates was removed digestive participation in the rumen, ruminant digestive function was still normal in its fermentatif. Further asserted that the defaunation process will increase the total bacteria in the rumen, because the protozoa are active against microbial pagosit especially rumen microbial amylolitic. Reducing the impact of protozoa would provide greater protection against protein degradation and increase the flow of feed protein into the duodenum. With descriptions of protozoa can be removed with defaunation agent, the agent of defaunation can be used as methane inhibitors or lower because if the protozoan is reduced, the symbiosis between protozoa and bacteria also reduced so that the H2 methanogenic utilized methanogenic bacteria are few and gas methane (CH4) produced also decreases. Saponin is a compound that can be used as agents defaunation. Saponin will react with the cholesterol contained in the membrane of eukaryotic cells including protozoan causing damage cell membranes and lyse cells of protozoa (Bangham and Home, 1962 cit. Klita et al., 1996). Defaunation action of saponins appear with the formation of an irreversible complex with cholesterol in the protozoal cell membrane causing cell lysis (Cheeke, 2005). Saponin found in several plants, one of which is the rambutan. Rambutan is a fruit of tropical fruit and inventory is abundant in Indonesia so that rambutan fruit peel waste was also abundant. Rambutan fruit peel contain saponin (Setiawan, 2003) so that the peel of rambutan fruit has potential for use as a feed additive that can reduce the formation of methane production in ruminants.

MATERIALS AND METHODS Materials were used in this research is the rambutan peel as a source of saponins, rumen fluid from cows fistula for rumen microbial inoculum source, reagents for testing of microbial protein and ammonia, as well as for the analysis of the number of protozoa and pH. Among other equipment used syringes as a fermenter, a microscope and haemocytometer to count the number of protozoa, waterbath, flasks, centrifuge, micropipet, pHmeter, spectrophotometer, CO2 gas cylinder, electric scales, and the oven.

Determination value saponin from rambutan peel 2 g samples and standards as much as 516.6 mg/50 ml hydrolyzed by adding 5 ml 1 N H2SO4, then direflux for 30 minutes. After the cold and then extracted with CHCl3 and added 5 ml of homogenized with a vortex of about 2 minutes. After that, separated using sentrifuge and will get the acid phase and organic phase. Obtained organic phase evaporated, dissolved with methanol, samples and standards were put on silica gel GF 254 plates, followed by elution. After that, the determined sample area and the area standard to determine levels of saponins in the sample (Wagner et al., 1984).

Determination of the dose levels of saponins rambutan fruit peel (on the basis of fermentation medium) Rambutan peel saponin content 7.86% or 7.86 mg/100 mg. To make a 0.1 mg/100 ml of saponin, the requires rambutan peel = (0.1 / 7.86) x 100 mg = 1.27 mg. In a 30 ml fermentation medium skin requires rambutan = (30/100) x 1.27 mg = 0.38 mg. Additional level of rambutan peel: 0% (0 mg/100 ml), 0.2% (0.2 mg/100 ml), 0.4% (0.4 mg/100 ml), 0.6% (0, 6 mg/100 ml). And the addition of rambutan peel: 0 mg/30 ml. 0.76 mg/30 ml. 1.52 mg/30 ml. 2.28 mg/30 ml.

In vitro fermentation The medium is the medium used for the measurement of gas production in vitro, by mixing the fluid with a solution of carbonate buffer. Rumen fluid taken from Ongole fistule cow. Intake of rumen fluid conducted in the morning before the cattle were given feed. Rumen fluid is inserted into the flask which was previously filled with water with a temperature of 39 C, which aims to adjust the temperature in the rumen, then removed just before it entered the rumen fluid. Flask filled to the brim and rumen fluid sealed to keep anaerobic conditions, then taken to the laboratory for use as a source of microbes. Fermentation medium prepared by mixing 474 ml H2O, 0.12 ml micro mineral solution, 237 ml of buffer solution, 237 ml mineral solution macro, resazurin 1.22 ml, and 49.5 ml of reducing solution. These materials were homogenized with a CO2 gas flowed, then rumen fluid was added in buffer solution after the solution is colorless which indicates conditions are anaerobic. Mixing ratio of rumen fluid: medium was 1:2 (v / v). The syringes were filled with 300 mg of a mixture of grass king with rice bran with counterweight 60; 40. The syringes that already contain the substrate and then put into an incubator at a temperature of 39 C for overnight. The syringes were then divided into four treatment levels with the rambutan fruit peel as a source of saponins with saponin levels 0%, 0.2%, 0.4% and 0.6%. Each treatment was done with three replications. Thirty milliliters of a mixture of rumen fluid fermentation medium and inserted into the syringe. Anaerobic process is carried out by the CO2 gas flowing into the syringe. Then the syringe sealed with clamps and incubated in an incubator at a temperature of 39 C for 72 hours. Results of fermentation and then filtered using a given cruss goch glass wool to separate the rest of the feed. Filtrate obtained partly used for the analysis to calculate the number of protozoa and pH test. Filtrate was sentrifuged with speed 3000 g for 15 minutes to separate feed particles, and then be repeated with speed 13 000 g for 15 minutes, the sediment was obtained for the test of microbial protein content, whereas the supernatant obtained was used as a sample for testing ammonia.

Observed variables Methane analysis. Total gas production measured after 72 h incubation with a view on syringes scale based on the increase in gas pressure caused the piston to the top (Getachew et al., 1998). To measure the levels of methane gas, samples gas were analyzed using gas chromatography. Total methane production is known to convert the methane gas levels in a sample of the total gas production. The number of protozoa. Preparation calculation of protozoa by Diaz et al., (1993). The degree of acidity or pH. pH is measured using a pH meter which was calibrated with buffers pH 4 and pH 7. pH measurements made at the end of fermentation. Microbial protein. Measurement of microbial protein by Lowry protein analysis method (Plummer, 1987). Ammonia levels. Determination of ammonia using the method of Weatherburn (1976). Data analysis. Data results were analyzed with one way analysis. The average difference was tested by Duncan's multiple range test test (DMRT) (Astuti, 1981).

Results

Table 1: Protozoa number, gas Production, methane production and characteristics of rumen fermentation at several level of rambutan peel addition as the saponin source.

Parameter Observation Protozoa number (103/ml) CH4 production (ml) pH valuens Microbial proteinns Ammonia concentrationns
abcd ns

Level saponin of peel rambutan (%) 0 0,2 0,4 0,6 6,89a 4,35b 2,53c 1,78d 17,64a 7,12 0,21 17,86 16,47b 7,10 0,17 17,05 15,01c 7,10 0,22 16,87 13,13d 7,11 0,21 16,99

The values in the same row with different superscripts show the different effects of treatment (P<0.01) not significant

Discussion Methane gas production with the addition of saponin levels rambutan fruit peel at 0%, 0.2%, 0.4% and 0.6% respectively was 17.64 ml, 16.47 ml, 15.01 ml, and 13.13 ml. The results showed that methane production decreased (P <0.01) at 6.63%, 14.90%, 25.65%. These results indicate that the higher levels of saponins in the diet of cattle given the lower production of methane in the rumen. The decrease in methane gas production is due defaunation causing protozoan symbiosis between methanogens and protozoa in the rumen decreased so that the methane gas production also declined. Cheeke (2005) states that the saponins has the ability to reduce the number of protozoa. In addition, Miller (1995) stated that bacteria and protozoa that no one can directly ferment carbohydrates into CH4 (methane). Species of methanogens and then transform the H2 and CH4. Format and H2 is the primary substrate to produce methane. By the action of the protozoa defaunation, methane production is reduced because the supply of hydrogen is lower, thus reducing bacterial activity in the form of methane methanogenik (Finlay et al., 1994). Number of protozoa with the addition of rambutan fruit peel with saponin levels of 0%, 0.2%, 0.4% and 0.6% respectively was 6.89 x103/ml, x103/ml 4.35 x103/ ml, 2.53 x103 / ml, and 1.78 x103/ml. The results showed that the number of protozoa decreased (P <0.01) by 17.5%, 63.28% and 74.61% compared to controls. These results indicate that the higher the lower the saponin content of protozoa in the rumen. These results are in accordance with the revelation Cheeke (2005), that the saponin had the effect of fermentation in the rumen and inhibit the growth of cilliata protozoa in the rumen . Decrease the number of protozoa occurs because cholesterol in the membrane of eukaryotic cells including protozoan react with saponin so will damage the cell membranes and lyse cells. The population of protozoa in the rumen is influenced by pH, type of substrate, and the competition between both the protozoa eat bacteria and protozoan predation among themselves (Church, 1988). In addition, the presence of protozoa is influenced by the additive compound (Hart et al., 2007). One of the additive compounds that affect the number of protozoa is a saponin. Saponin causes damage cell membranes and lyse cells of protozoa (Bangham and Home, 1962 cit. Klita et al., 1996). pH value with the addition of rambutan peel with saponin levels of 0%, 0.2%, 0.4% and 0.6% respectively were 7.12, 7.10, 7.10, and 7.11 . The results showed that the pH for four different levels of saponins are not real. According to Deacon (2004) in rumen pH optimum ranging from 6.3 to 7.5. This is in accordance with the statement Jouany (1991), that although protozoa digestive removed from participation in the rumen, ruminant digestive function was still normal in fermentation. Microbial protein value with the addition of rambutan peel with saponin levels of 0%, 0.2%, 0.4% and 0.6% respectively was 0.21 mg / ml, 0.17 mg / ml, 0,22 mg / ml, and 0.21 mg / ml. The results showed that the addition of rambutan peel did not affect microbial protein. Addition of rambutan fruit peel as a source of saponins caused a significant decrease in the number of protozoa, however, microbial protein synthesis results showed diffrrence no significant. According Jouany (1991) reduction will increase the total number of protozoa rumen bacteria. Increased protein synthesis by rumen bacteria with the increase in total rumen bacteria. Thus, microbial protein fixed or not there is a decrease or increase. The main factors affecting microbial protein synthesis is the availability of precursors in sufficient concentration in rumen fluid. Widyobroto (1992) states that the precursor for microbial protein synthesis is the availability of sufficient carbon skeleton, NH3, energy and minerals. Besides, that takes place under ideal conditions microbial protein synthesis would be achieved if the source of carbohydrate fermentation, are available simultaneously with the source of protein. Based on table 5. ammonia content with the addition of rambutan skin with saponin levels of 0%, 0.2%, 0.4% and 0.6% respectively was 17.86 mg/100 ml, 17.05 mg/100

ml, 16.87 mg/100 ml, and 16.99 mg/100 ml. The results showed that the ammonia content for the fourth level was not significantly. Although not significantly but ammonia levels were within normal range. It was in accordance with that stated by Leng (1985), maximum growth and microbial activity necessary concentration of ammonia in the rumen fluid for 5 to 23.5 mg/100 ml. According to

Arora (1989) level of 5.8 mg/100 ml of rumen fluid was able for the formation of microbial protein from rumen. Ammonia content was influenced by the level of rumen wall absorption speed. Ammonia released quickly if it will increase the absorption of ammonia by the rumen wall (Noor, 1990) and the less ammonia is available that can be used by bacteria (Orskov, 1992).

Conclusion It could be concluded that addition of saponin from rambutan peel as low as 0.2% of the medium decreased methan production, protozoal and did not have any effect on rumen fermentation.

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