Doxycycline is a commonly used tetracycline antibiotic used to treat a wide range of bacterial infections and tropical diseases such as malaria, Lyme disease, anthrax, respiratory and sexually transmitted infections, and severe acne (Styka & Savitz, 2020). Doxycycline’s main mechanism of action is preventing the association of aminoacyl-tRNA with the acceptor site of 30S ribosomal subunits within bacteria (Chopra & Roberts, 2001). Though widely available, inexpensive, and broad-spectrum, the FDA classifies doxycycline as a class D drug not recommended for use in pregnant individuals and children younger than 8 years old (Nahum et al., 2006). This classification is based on numerous studies done on tetracycline teratogenicity and embryotoxicity which show the effects of high concentrations of these drugs.  

Studies in rats show that administering doxycycline while pregnant causes the fetus to develop morphological anomalies, including a lack of ossification of multiple types of bones and decreased fetal weight (Abd-Allah, 2021). In addition to bone malformations, fetuses also showed cardiac arrhythmias, most likely due to the disruption of mitochondrial protein synthesis (Wüst, 2021). One study demonstrated that chicken eggs injected with doxycycline on the 7th day of incubation showed no macroscopic malformations; however, mortality increased in a dose-dependent manner (Arslan et al., 2025). Despite these reported risks at high doses and later developmental stages, there is limited research on how doxycycline affects fetal development in the earliest developmental stages at environmentally relevant doses.  

This study investigates possible teratogenic and embryotoxic effects of common doxycycline prescription doses at early stages of development using the chicken embryo as a model. This easily manipulated developmental model is relevant to human embryonic development due to the similar anatomical structure and formation of the four-chambered heart, as well as other tissues (Jiang et al., 2021). We exposed chicken embryos prior to incubation, incubated for seven days, then evaluated cardiovascular function and limb bud formation. We predicted that common human doses, when converted to relevant dosages, would cause decreased limb bud length and heart arrythmias.  

A total of 95 eggs were utilized to determine the effects of environmentally relevant doses of doxycycline on embryonic development. Of the 34 vehicle control eggs, 27 were fertilized. Of the 30 low dose eggs, 25 were fertilized. Of the 30 high dose eggs, 25 were fertilized. All unfertilized eggs were excluded from the data.  

The data for hindlimb and forelimb bud lengths presented similar negative trends (Figure 2, Figure 3). The high dose group exhibited the lowest hindlimb and forelimb bud lengths. The high dose group (n = 24) showed lower hindlimb bud length than the control group (n = 22) and the low dose group (n=25) (F(2, 68)=.4, p<0.01). The low dose group showed lower hindlimb bud length than the control group (F(2, 68)=.4, p=0.03). The high dose group (n = 23) showed lower average forelimb bud length than controls (n = 26) (F(2, 71)=10, p<0.01). The low dose group and control group showed no difference in forelimb bud length (F(2, 71)=10, p=0.20). The high dose group exhibited statistically significantly lower forelimb bud length than the low dose group (F(2, 71)=10, p=0.02). 

The control group (n = 27) displayed the highest heart rate (Figure 1). We found no statistically significant difference in the heart rates of the low dose (n = 25) and high dose groups (n = 25) (F(2, 60)=0.23, p =1.00). There was no difference between the control group  and the low dose group (F(2, 60)= 0.23, p =0.81). Additionally, there is no difference between the control group and the high dose group (F(2, 60)=0.23, p=0.85).  

Evaluating the effects of prescription-equivalent doxycycline doses at early embryotic developmental stages is imperative to performing appropriate risk analysis for administration. In assessing cardiovascular function via heart rate measurement, we found no statistically significant difference between control embryos and embryos treated with either low or high doxycycline prescription doses. However, forelimb and hindlimb bud lengths decreased in a statistically significant dose-dependent manner upon doxycycline administration. This finding suggests that prescription doxycycline dosages may exert adverse effects on the skeletal development of early-stage embryos. This implies that doxycycline limb effects are not limited to extreme doses or later embryonic development. The correlation offers further insight into the risk of doxycycline administration for providers to consider when treating severely ill pregnant individuals. 

There are not many studies done of doxycycline's effect on chicken embryos; however, there is some research on doxycycline’s effect on animal embryos. In previously done studies of doxycycline administration to pregnant rats, the results found that fetuses in the experimental group develop morphological abnormalities in the skeletal system. This included lack of ossification in multiple bone types, and a lower weight of the fetus (Abd-Allah, 2021). In addition, it was found that rat embryos had abnormal cardiac development that caused arrythmias (Wüst, 2021). Our research on doxycycline's effect on chicken embryos had both similar and contradictory findings. The embryos in both our low doxycycline dose and high doxycycline dose showed a statistically significant decreased length of limb bud formation compared to the control group. On the contrary, our experiment group, low doxycycline dose and high doxycycline dose, embryos showed no significant cardiac abnormalities compared to the control group. 

This study has several strengths, as it directly compares hindlimb and forelimb length in response to doxycycline injections in a dose-dependent manner. This helps us identify specific regions of development rather than head to tail measurements. Honing in on a specific region helps make the observed changes easier to interpret. The sample sizes were not large enough; some effects may have been harder to identify, so a larger sample size with a longer research period would be helpful for future research. We could not assess the molecular changes that could explain how and why the doxycycline is affecting the limb bud's length. For future research, having molecular analyses would be helpful to compare different doses to determine if the effects are threshold-based or dose-dependent. As the research progressed, the question of the mechanism of doxycycline arose, why and how does doxycycline affect limb bud length on a molecular basis. It would be interesting to understand how doxycycline interacts with specific signaling pathways involved in limb bud length.