Extracted Pearl Grass Effect on Broiler Carcass Kept on Escherichia Coli – Sprayed Litter

An experiment was conducted to determine the effect of hexane and acetic ethyl extracted pearlgrass as feed additive in the ration on carcass of broiler chickens where the litter was sprayed byEscherichia coli. The experiment was assigned to Completely Randomized Design with 5 treatmentsand 4 replications. The treatments were level of pearl grass extract added into the ration; 0 (R0, control),0.8 (R1), 1.6 (R2), 2.4 (R3) dan 3.2 (R4) gram per kilogram ration. After 3 weeks of age, litters weresprayed by Escherichia coli as much as 106 CFU/100 ml liquid agar. Then, chickens were kept until 6weeks of age. Result showed that there was no significant different among treatments groups on chickencarcass and there was no any adverse effect on liver and intestines. It is concluded that extracted pearlgrass could be utilized as natural feed additive source to produce carcass of broiler chicken where theirlitter was sprayed by E. coli bacteria.


INTRODUCTION
Utilization synthetical antibiotics, medicine or feed additive to prevent and treat animal diseases cause microbes resistance in digestive tract (Van den Bogaard et al., 2001), produce metabilte products of the antibiotics, medicine or feed additive in form of residue in animal products such as milk, meat, egg and animal tissues (Jetacar, 1999) those might be transferred to human who consumed it. Ladefoged (1996) reported that antibiotic residues in digestive tract could prevent microflora growth and digestion disorder. Mumtaz et al. (2000) reported that drug residue was detected in poultry meat in few days after drug ingestion and contaminated meat could disturb human health. Naeem et al. (2006) detected more Quinolones residue in liver and kidney than in chicken meat and egg. The residue might be accumulated into human body who consumed the animal products contained antibiotic residue and could cause allergy, resistance (Sundlof and Cooper, 1996), hypersensitive to stimulant, carcinogenic, mutagenic and toxicity (Voogd, 1981).
These circumstances and people realization to have healthy life and people need to back to nature persuade people to reduce and stop using synthetic antibiotics and drugs in their life and also in animal husbandry. They use alternative herbs such as pearl grass (Hedyotis corymbosa or Oldenlandia corymbosa L) that is classified into genus Hedyotis, family Rubiaceae. Pearl grass until the present time is usually used by community to treat digestion disorder, cancer, caecum sore, microbial infection (bacteria, protozoa and fungi) and (Dalimartha, 2002). Nurhayati et. al (2006) reported that pearl grass could inhibit bacterial growth including E.coli but it could not inhibit Candida albicans growth. The highest activity of extracted pearl grass was found in acid and base etyl acetate fraction and followed by methanol and n-hexane fraction. Nurhayati and Latief (2009) found that keton group (tersier butil isopropil keton) in extracted pearl grass could inhibit E.coli growth. Pearl grass contained 90.28% dry matter, 14.93% crude protein, and 556.81 kcal/kg gross energi. However, due to the lack of publication of pearl grass effect on animal especially on poultry causes this medicinal weed are not used either as antimicrobes or feed additive in livestock including poultry. Based on this reason the current study was conducted to determine the effect of hexane and acetic ethyl extracted pearl grass as feed additive in the ration on carcass of broiler chickens where the litter was sprayed by Escherichia coli.

Pearl Grass Extract, Birds, Diets and Housing
Pearl grass used in this study was collected from Jambi Regency. Then it was sorted, cleaned, chopped and extracted by maseration using organic solvent namely acetic etyl for 2 x 3 x 3 days until all active compounds were dissolved. Thereafter, all solvent were evaporated by vacuum rotary evaporator to get pearl grass extract. The extract was dried under room temperature before it was added into the basal diet based on the treatment level.
A hundred two days-old male broiler chicks with 47.69 ± 3.79 gram of average weight were purchased from a commercial hatchery in Palembang were used in this study. Chicks were randomly divided equally to 5 experimental treatments. The design of experiment was assigned into Completely Randomized Design. The treatments were : R0 : Basal diet + 0 g pearl grass extract R1 : Basal diet + 0.8 g pearl grass extract per kg diet R2 : Basal diet + 1.6 g pearl grass extract per kg diet R3 : Basal diet + 2.4 g pearl grass extract per kg diet R4 : Basal diet + 3.2 g pearl grass extract per kg diet Each treatment had 4 replicates of 5 chicks. Chicks of each replicate were housed in colony floor pens with deep litter and were fed the experimental diet for a period of 42 days.
Basal diet was differed into starter diet (until 3 weeks of age) and finisher diet (after 3 weeks of age) as shown in Table 1. Chemical composition of treatment diets are shown in Table 2 and Table  3. The diet was offered into the chicken ad libitum.
At 4 days of age, chicks were vaccinated by ND vaccine strain Lasota B1 through eye drops. Other vaccines were not injected. After 3 weeks of age, litter was sprayed with Escherichia coli as much as 10 6 CFU/100 ml of liquid agar. Then, chickens were kept until 6 weeks of age. At 42 days of age, 2 chickens with the nearest average live body weight was randomly selected from each treatment and were deprived of feed for 8 h prior to slaughtering. The chickens were weighed and were exsanguinated. Chickens were bled, scalded and feathered. Feet were removed manually by severing the intra tarsal joint. Carcass was manually eviscerated and abdominal fat, liver, gizzard, heart and giblets were removed and were weighed to calculate organ weight and carcass weight.

Statistical Analysis
Parameters determined in this study were feed consumption, live weight, carcass percentage, liver percentage, gizzard percentage and intestinal percentage. The effect of diet on chicken carcass characteristics and organ weights were analyzed by one-way ANOVA using General Linear Model procedure of SAS (version 6.04) (SAS Institute, 1994). Significant differences among treatment means were separated using Duncan's Multiple Range Test. The statistical model used for analyzing data obtained was :

RESULTS AND DISCUSSION
Effect of dietary treatment diets on feed consumption, carcass characteristics and organ weight of broiler chicken is shown in Table 4.

Feed Consumption
Analysis of variance showed that there was no difference (P>0.05) effect of addition pearl grass extract into the ration of broiler chickens where their litter was sprayed with E. coli bacteria was found in feed consumption during the overall period. It means that pearl grass extract did not influence diet palatability and chicken appetite. It is due to that the experimental diet among treatment groups had similar quality (Table 2 and 3). Besides it might be due to the flavonoid, steroid and aromatic compound in pearl grass did not disturb chicken appetite. The similar effect also reported by other authors who offered herbs or medicinal plants as feed additive on broiler' feed consumption. Nurhayati et al. (2005) reported that chickens were offered 0, 2.5, 5.0, 7.5 and 10% of pace meal had similar (P>0.05) feed consumption and pace meal did not significantly (P>0.05) increase feed consumption. Lohakare et al. (2006) reported that lacquer is usually used as traditional medicine by Japanese, Chinese and Korean. There was no different effect among 0, 1, 2 and 4% of lacquer meal on increasing broiler feed consumption. The similar effect of pearl grass extract on feed consumption even though house litter was sprayed by 10 6 CFU E. coli bacteria per 100 ml liquid agar showed that the active compounds in pearl grass had synergetic effect on controlling and limiting bacteria growth. Nurhayati et al. (2006); Nurhayati and Latief (2009) found that pearl grass could inhibit E.coli growth in vitro. Inhibited microbes growth in the gut would cause the nutrient could be utilised by animal itself and he would not need more nutrient for maintenance, growth and production. Table 4 showed that feed consumption of broiler chicken during the experiment was 556.41 g/bird/week or 79 g/bird/day in average. This result was not significantly different by previous report by  who offered ration containing different medicinal weed namely bandotan (Ageratum conyzoides L), patikan kebo (Euphorbia hirta L) and sidaguri (Sida cordifolia) to the broiler chicken. Daily feed consumption of chicken fed 5% of bandotan, patikan kebo and sidaguri in the ration were 76.48, 78.40 and 74.29%, respectively.
Extracted Pearl Grass Effect on Broiler Carcass (Nurhayati et al.)

Liveweight and Carcass
Analysis of variance showed that hexane and acetic ethyl extracted pearl grass addition into the ration up to 3.2 g/kg did not significantly (P>0.05) influence live weight and carcass percentage. This result was parallel to feed consumption and daily weight gain those were also not significantly different among the treatment groups.
No difference on these parameters even though the litter was sprayed with E. coli bacteria might be due to that the nutrients and active compounds in the pearl grass have banded in nutrient digestive process and have synergetic effect to increase birds resistance on E-coli bacteria. As reported by Nurhayati et al. (2006); Nurhayati and Latief (2009) that pearl grass contained active ingredients those could inhibit bacterial growth including E.coli. Buchanan et al. (2009 ) reported that medicinal plants contained phytogenic compounds, essential oils and organic acid those could reduce pathogenic bacteria in the chicken. It was similar results were reported by previous authors (Dickens et al., 2000;Dorman et al., 2000;Abd El-Latif, 2002;Al-Harthi, 2002;Hassan, et al., 2004;Buchanan et al., 2008;Javed et al., 2009;Onibi et al., 2009) who fed briler chicken with the diet consists of herbs or medicinal plants. They reported that there was a positive effect of feeding herbal feed additive on broiler performance due to that herbal supplementation or medicinal plant supplementation could improve nutrient digestive process, thus, in could chicken body weight gain and at the end increase slaughter weight of chicken. Hernandez et al. (2004) stated that essential oils in the medicinal plants might increase broiler chicken digestion. Uunganbayar et al. (2006) concluded that green tea mixed into the broiler chicken had no adverse effect on broiler performance. Onibi et al (2009) found that quality and aroma of meat increased by supplemented garlic into the ration. The best level was 5 g/kg diet resulted 2.1 kg body weights at 56 days of age. Javed et al. (2009) reported that feeding some medicinal plants extract as much as 10 ml/L drinking water produced 1.4 kg body weight at 35 days old and carcass was 62% in average.

Organs Weight
Analysis of variance showed that hexane and acetic ethyl extracted pearl grass addition into the ration was not significantly (P>0.05) effect on organs (liver, gizzard, and intestine) weight. There was no adverse effect also on colour of liver. There was no blood spot on the intestine, no thickening on gizzard and intestine wall. These results mean that pearl grass extract had not toxic effect, thus, it would not cause gizzard, liver and intestine work harder than usual in digestion and absorbance of nutrients. The current result was similar to previous study on mice that ingested single dose pearl grass extract orally. There was no toxicity on mice those received. The current study also showed that E. coli could not grow and develop in the digestive tract. It is due to that E. coli might be inhibited by active compounds in the pearl grass extract those were fed by chickens. Nurhayati and Latief (2009) detected 3 active compounds in the pearl grass extract namely 4metil-2-en-heksanol, tersier butil isopropil keton, and stigmasta-5,22-dien-3-ol. The highest E. coli antibacterial activity in vitro was detected on tersier butil isopropil keton. Further, Buchanan et al. (2009) reported that essential oils, organic acid and phytogenic compounds in plants reduced number of bacteria in the animal body significantly.