Embryotoxicity of Doxycycline HCl in Gallus domesticus: Morphometric Changes and External Anomalies

Poultry is among the world’s fastest rising sources of meat (Apata, 2009). The commercial and health benefits of using antibiotics have revolutionized rigorous poultry and livestock production (Mobarak, 2010). Antibiotics use in food-producing animals has remarkably increased animal health by lessening mortality and diseases (Cooper et al., 2009). However, use of antibiotics in poultry production cause an upsurge in resistance to antibiotics in hazardous bacterial strains and also in commensal bacteria (Lukasova and Sustackova, 2003).

Doximycine is a newly presented product comprising of the active material, an antibiotic, Doxycycline HCL 10g. Doxycycline is very active, low-cost with a comprehensive beneficial spectrum and remarkable bioavailability. How-ever its importance has been masked by its classification together with tetracyclines - class D drugs (Ruby et al., 2016, Zakeri and Wright 2008).

Infectious agents are significant and momentous hazard for animal and human health. In veterinary medicine, antibiotics are used to eradicate pathogens and transmission of disease, but the effects of drugs are the main concern (Javakkoli, 2014). However, to the best of our knowledge, teratogenic effect of Doxycycline HCL on Gallus domesticus embryos has not reported. Therefore, the present experiment was planned to evaluate the embryotoxicity of Doxycycline HCL on growing chick embryos.

Seventy fertilized eggs of Fayoumi breed were procured from a local layer farm. Eggs were assorted into seven groups (A, B, C, D, E, F and G) with 10 eggs each without considering the size and color of eggs. Eggs were marked according to their groups after cleaning with a sterile cotton plug using 70% Ethyl alcohol. The test substance, Doxycycline HCL was purchased from a local veterinary shop. Considering group A, as a control group (without treatment) whereas groups B, C, D, E, F and G were treated with different doses of antibiotic (0.1, 0.5, 1.0, 1.5, 2.0 and 3.0µg/100µl distilled water /egg respectively. The required sequential doses of antibiotic were formulated by dissolving the antibiotic in distilled water and equal volume of distilled water was injected into controls by using “window technique” (Jelink, 1982), using syringe. Shell membrane was not removed. Shell particles were removed by aspirator. The needle (1 inch long) was introduced into the egg yolk (Blankenship et al., 2003). The shell hole was closed with cellophane tape immediately after the injection. To avoid contamination, this was performed in laboratory in pretreated germ free atmosphere in an incubator. An incubator model DH3600BII (Huanghua Faithful Instrument CO. China) were used for this experiment Experimental eggs were kept in incubator and were normally rotated every 6 hours and were kept at an optimum temperature of 38 ± 0.5°C and 60% humidity. Dead embryos were removed by candling after every four days. After 12 days post infection, eggs were removed from incubator and examined. At the end of the trail, mortality rate of the embryos were recorded and morphological observations were noted. Weight of embryos was measured by digital balance (SF-400). Crown rump length (C-R), anterior posterior (A-P) head diameter, neck length, beak length and eyes diameter measurements, were taken using digital vernier calipers while forelimbs and hind limbs measurements were made by using compass and scale. External malformations were noted. Recorded data was tabulated for studying differences with control group. The 12-days-old chick embryos were photographed by using Digital Samsung Camera (WB25OF). Recorded data of different measurements were presented as Mean ± S.E (Standard Error). Statistical significance was tested by applying One-way ANOVA by using Statistical Package for Social Sciences (Version21).

Mortality rate
 
Mortality was noted in chick embryos after recovery at 12th day treated with dose (0.1, 0.5, 1.0, 1.5, 2.0 and 3.0µg/100µl) of Doxycycline HCL. The embryos that were given 0.1, 0.5, 1.0, 1.5, 2.0 and 3.0µg/100µl doses of Doxycycline showed 20, 20, 50, 60, 60, 80% mortality respectively (Table 1).


 
Morphometric measurements
 
The standard error and mean of measurements of wet body weight, C-R length, A-P head and eye diameter, beak length, neck length, humerus, radius and ulna, metacarpus, femur, fibula and metatarsus length were noted in treated and negative control groups (Table 2). Wet body weight, neck length and crown rump length among experimental groups revealed significant (p<0.05) decrease compare to untreated group and this trend was also noted in treated groups from lower to higher dose. Anterior-posterior head diameter of all groups treated with Doxycycline HCL showed significant (p<0.05) difference compared to negative control. Like-wise eye diameter and beak length of all treated groups exhibited significant (p<0.05) difference compared to control group. In average length of forelimb, humerus and radius of Gallus domesticus embryos revealed significant (p<0.05), while metacarpus revealed non-significant (p>0.05) difference as compared to control group. Femur and fibula of chick embryos declined significantly (p<0.05) at high dose levels, while metatarsus length shows non-significant (p>0.05) decrease compared to control group.

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External anomalies
 
Normal body parts were observed in embryos of control group (Fig 1 A-H). Omphalocele were poorly developed in 12 days old chick embryo treated with 0.1µg Doxycycline HCL. Hematoma, Damaged auditory meatus and Swelling were noted around eyes in chick embryos treated with 0.5µg Doxycycline HCL. Embryos treated with 1.0µg Doxycycline HCL showed many anomalies, Anophthalmia, Hematoma, swelling around eyes, Edematous swelling, Short beak, Microphthalmia, Amelia, Agnathia, Omphalocele and Damaged auditory meatus (Table 3). Malformations as the result of treatment of 1.5µg of Doxycycline HCL on chick embryos are shown in. Anomalies including Agnathia, Short beak, Micromelia (under sized limbs), Microcephaly and Microphthalmia (undersized eye) are shown by embryos treated with 1.5µg of Doxycycline HCL. Swelling around eyes, Agnathia, Microphthalmia, Short beak, Omphalocele and Ectopia cordis were found in embryos treated with 2.0µg of Doxycycline HCL. By treatment 3.0µg of Doxycycline HCL, chick embryos showed anomalies including swelling around eyes, Edematous swelling, Agnathia, Microphthalmia, Micromelia and Omphalocele.

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The present design was planned to evaluate the embryotoxic impacts of Doxycycline HCL on death rate, morphology, crown rump length, eye, beak, neck, fore and hind limbs, length and weight of growing chick embryos. Moreover malformations like microcephaly, hydrocephaly, edema, hematoma, abnormal body coloration, anophthalmia, microphthalmia, deformed beak, agnathia, wry neck, micromelia, amelia, meromelia, damaged auditory meatus, omphalocele and ectopia cordis were noted in experimental groups as compared with untreated groups.
 
Mortality
 
The lowest mortality rate of chick embryos was found with 0.1 and 0.5µg/100µl and the highest mortality rate were found with 3.0µg/100µl dose. This shows that high embryotoxic effects of Doxycycline HCl antibiotic on chick embryos depend on concentration. Embryo toxicity increased with high doses. Likewise (Mobarak, 2010) observed that the death rate increased in antibiotic (enrofloxacin) treated Gallus gallus domesticus embryos as compared to negative control group.
 
Morphometric Measurements of Doxycycline HCl Treated Twelve Days Old Chick Embryos
 
Due to the fact that this antibiotic, Doxycycline HCl is toxic enough to disturb the morphology of developing embryo. Similarly, Mobarak (2010) revealed a significant decrease in body weight, anterior posterior head diameter and crown-rump length in 6 and 13-day-old enrofloxacin treated chick embryos.
 
External Morphology of Doxycycline HCl Treated Twelve Days Old Chick Embryos with comparison to control
 
This study revealed that embryos from control group showed all normal body parts. The same normal body characters were observed by (Mobarak, 2010) in 12-days-old Gallus gallus domesticus embryos of control group.

Similarly, (Mobarak, 2010) revealed that the chick embryos treated with Enrofloxacin showed oedema, omphalocele and other malformations in limb and head.

As Doxycycline HCl treatment showed external malformations in treated specimens. It is sensible to understand that these developmental defects were due to chicken treatment with Doxycycline HCl as such results were not observed in control embryos and this may be due to the fact that this tetracycline antibiotic tend to accumulate in eggs and edible chicken tissues. It’s essential to understand that, in chicks’ mortality rate and developments defects could increase due to continuous doxycycline HCl exposure.

According to our results it is summarized that Doxycycline HCl treated 12-days-old embryos shows significantly high percentages (compared to controls) of external anomalies. There-fore it is obvious from the results obtained that, Doxycycline HCl, an antibiotic is potentially hazardous to avian development and may be dangerous to different animals including human beings. However, further study is important to assess teratological effects of Doxycycline HCl on humans and animals.

The authors extend their appreciation to the RSP. No-RSP2019-110, King Saud University Riyadh, Saudi Arabia for financial support.