The Comparison of Doxycycline Residue in the Meat of Broiler Chickens Administered in Feed and Water

The purpose of this research was to investigate the effect of doxycycline (a tetracycline derivative) administered at disease-prevention dose given daily in the feed and drinking water on the residue level in the broiler-chicken meat. Doxycycline at concentration of 100 ppm was mixed in the drinking water (1 g of doxycycline in 10 L of drinking water) and feed (1 g of doxycycline in 10 kg of feed). Samples of chicken meat were taken every week to measure their residue level. Analysis of doxycycline level was performed using high performance liquid chromatography with extraction method referring to the standard of Association of Official Analytical Chemistry. The result showed that the residue level in the group of chickens given doxycycline through drinking water was higher (P<0.05) than in the group given doxycycline through the feed, except for the result in the 5th week, that showed no significant difference. The highest residue level was found at the first week in the group given doxycycline in drinking water (0.96±0.15 µg/g), while the lowest was found at the sixth week in the group given doxycycline in the feed (0.10±0.01 µg/g). The level of doxycycline residue exceeding the level permitted by the Indonesian National Standard (0.1 µg/g). It can be concluded that doxycycline is not recommended to be given daily both through water or feed to commercial chickens as a disease-prevention agent.


INTRODUCTION
Commercial chickens diet always use feed additive, which includes antibiotics as one of its main ingredients. Some of farmer used to apply antibiotics for the disease prevention with unwise of application or recommended dosage. These antibiotics are expected to help increase the body-weight by inducing the production of vitamin B-complex in the chicken's digestive system (Chopra & Robert, 2001). The negative eff ect utilization of antibiotics for chicken will be deposit residue into eggs and meat. The residue of antibiotics in chicken meat consumed by people will cause residue in the human body and create resistance due to the continuous under-therapeutic-dose which was received, as well as misbalance of normal microbial in the gut that can reduce or eliminate the non pathogen bacteria (Rodvold, 2001). The high concentration of antibiotics can also create negative effects to consumers, such as allergic reactions, transfer of residue, lower meat quality, risk of carcinogenic eff ects, etc.
Research of residue on edible tissues in United States conducted by Donoghue (2003) revealed that many antibiotics including tetracyclines had found on many products. Some antimicrobials, such as tetracyclines, fl uoroquinolon, chloramphenicol, sulphonamid, and pesticides, such as organochlorine and organophoaphat, were found in chicken meat. The institute corporation deal to challenge the residues problem by conducting new techniques as confi rmation, such as pharmacokinetic study to measure the residue. Alhendi et al. (2000) conducted a research on oxytetracycline being used as curative and preventative medicine given in the feed to broiler chicken for 40 days, and found that the residue of oxytetracycline reached the level of 0.24-2.25 µg/g in the plasma, liver, kidney, and muscle. In the USA, 2.5 million of microbial is used every year to improve farm productivity, with 80% of it is used in poultry (Schneider & Lehotay, 2004).
This research was conducted to reveal the residual eff ect caused by the using of doxycycline as diseaseprevention-agent given through feed and drinking water during broiler rearing period and would have the product safe from farm to consume.

MATERIALS AND METHODS
Forty eight (48) day old chicks (DOC) were divided into two groups (24 each of group). The fi rst group was given doxycycline through the drinking water 1 g/100 (preventive dose of 100 ppm). The second group was given doxycycline into feed 1 g of doxycyline/10 kg (preventive dose of 100 ppm). All the groups were fed with diet contents of 22% crude protein and 3000 kcal of metabolizable energy (Table 1). Feed and drinking water was given ad libitum daily for 6 weeks. The chickens were also given vaccinations against Newcastle disease and Gumboro. Samples were taken each week (4 chickens were euthanized in each group), starting from week 1 to 6. Meat samples were taken from breast region as the part that commonly consumed and stored in the freezer before being analyzed.

Analysis of Residue Level
Analysis of doxycycline level was performed by referring to the standardized method of tetracycline residue analysis authorized by AOAC (Association of Offi cial Analytical Chemistry) (AOAC, 2005). Five gram of sample was homogenized with 20 ml of Mc Ilvine buff er-EDTA, shaken at high speed, and centrifuged at 2,500 g for 10 minutes. The next step was rinsed with 2 ml of Mc Ilvine buff er-EDTA and centrifugation with the same speed. After three (3) repetition of centrifugation, the supernatant was collected and centrifuged at 2,500 g for 20 min. A 20 µl of supernatant was then injected to HPLC system.

Data Analysis
Data of doxycycline level in the meat (muscle tissue) from group 1 and 2 was analyzed with student T-test to evaluate the comparison of the level from week 1 to 6.

RESULTS AND DISCUSSION
Measurement of residue in group 1 showed high accumulation in the fi rst week (0.96±0.15), followed then by decline of residue level, and became constant starting from week 2 until week 6. In the second group, residue level kept increasing started from week 1 to reach maximum level in week 3 (0.15±0.04), and then declined to reach the lowest level in week 6 (0.1±0.015) ( Table 2). The residue level of group 1 was higher at week 1 than constant at the next weeks. The level of doxycycline was quite constant in group 2. This proved that daily administration of the medicine is able to maintain the level of medicine that has been gained. Table 3 showed the feed and water consumption during weeks 1 until 6 for both treatments.
Based on that result, it could be compared that in the group given doxycycline through the drinking water showed higher level of residue compared to the feed (P<0.05), with the exception for the comparison in week 5. Shargel et al. (2005) stated that the distribution volume of medicine would increase when the medicine was diluted in a solution, thus increase absorption rate.
distribution-volume when mixed into drinking water, so the residue of the medicine in the muscle was higher in group 1, as reported by Alhendi et al. (2000) and Basha et al. (2006) that examined the residue of oxytetracycline (another derivative of tetracycline) that was given as medication or disease-prevention given for 40 days. Residue of oxytetracycline in the broiler muscle in this research reached 0.24-2.25 µg/g. administered at dose of 20 mg/kg bodyweight could still be detected in the broiler muscle after 5 days of administration. Laczay et al. (2001) reported that doxycycline level in the tissues of liver, kidney, muscle, and lungs reached a range of 0.028-0.20 µg/g was able to be measured until the fi fth day after single-dose of intravenous administration. This research in line with Wijayanti et al. (2009) who revealed that the elimination half life (T1/2) of doxycycline in the muscle was 55.5 hours and the period of elimination time was long. Alsarra et al. (2005) found that the T1/2 of doxycycline in plasma was 16-17 hours given by oral formulation. The long elimination time and continuous administration will cause the persistence of drug level in high concentration for a long period.
Medication using doxycycline has been widely used penetration and higher absorption, thus reduce frequency of administration (Nicolau, 2003). Prats et al. (2005) older tetracycline group because of its good penetration and lower bound to calcium. This drug has proven the effi cacy base from its pharmacokinetic profi le to concur the poultry diseases caused by infections of Micoplasma gallisepticum, Staphylococcus aureus, Streptococcus pneumoniae, Pasteurella multocida, Bordetella bronchiseptica, and Escherichia coli (Wijayanti et al., 2009). At European countries farms, doxycycline has been used as curative and preventative medicine, and based on the research conducted by Craigmill (2003), the presence of residue in the muscle was quite high. Administration of higher level doxycycline into drinking water made it eff ective for medication, but this gave higher residue level.
Minimum level of doxycycline residue in the meat according to Indonesia National Standard (SNI-01-6366-2000) is 0.1 mg/kg or 0.1 µg/g, so administration of doxycycline at preventative dose given daily during rearing period is not recommended, even though from  the point of medication, level above 0.1 µg/g was proved eff ective.

CONCLUSION
The administration of doxycycline that of drinking water gave the higher level of residue than that of feed administration. Doxycycline administration at preventive dose is not recommended to be given daily to broiler chickens.