Vitamin A is a commonly used substance to regulate many physiological functions in a variety of organ systems. It is used in widespread practice for immunological function, cellular communication, reproduction in both sexes and general growth and development (Olsen 2001). It is found naturally in many foods such as fish, dairy, eggs, leafy greens, and orange and yellow vegetables and it can also be taken as a supplement to treat cases of acne (Michaëlsson et al., 1977). Many supplements and drugs that contain high vitamin A normally come with a warning label discouraging the use of the drug when pregnant because it can lead to embryonic birth defects. 

The parent molecule of vitamin A is known as retinol. Retinol is transferred through the bloodstream to eventually bind to a retinol-binding protien. An excess of retinoic acid and a lack of retinoic acid cause ectopic induction and the downregulation of genes dealing with gene transcription that ultimately end up changing the anatomy of the embryo (Zile 2001). Retinol also affects the central nervous system, specifically the hindbrain. A study showed that after a group of mice was injected with retinol the Hox genes of the hindbrain were altered which resulted in altered anatomy ( Marhsel et. al, 1992). These studies show that vitamin A does affect gene regulation and expression but the extent to how much vitamin A causes it is unknown. Acne medicine such as Accutane contains high levels of vitamin A that is not suitable in excess for pregnancy. Accutane is shown to cause birth defects when used during pregnancy. Some known effects include damage to the lungs and an increased risk of asthma (Parr et al., 2018) along with an overall inability for the infant to thrive which has proven vitamin A to be very harmful to pregnant women (Burnett et al., 1993). A study on vitamin A deficient (VAD) female rats showed that they failed to reproduce (Clagett-Dame & Knutson, 2011). This study suggests the need for vitamin A when it comes to proper reproductive function. That study also suggests another problem: the threshold for the amount of vitamin A that is good for the embryo, the point where it causes harm, and the point where it is lethal is not known. Vitamin A is predicted to cause other abnormalities such as heart formation (Osmond et. al 1991). When injected, the vitamin A inhibits the migration of the precardiac mesoderm that results in a stunted heart tube (Osmond et. al 1991).Out of the studies done on the specific concentrations that cause defects or lethality, the effect of vitamin A has on body length and mass is still poorly understood. 

Given the lack of research on the point at which the embryo causes death or birth defects and specifically what birth defects are, our group has chosen to examine the effects of this vitamin in excess on the embryonic body length and mass. The focus of our experiment was the embryo’s body length and mass. To test this literary gap, we used concentrations of 20,10,2, and 1 mg of vitamin A/mL and injected it into the air sack of a broiler chicken embryo.We picked those concentrations in order to see abnormalities. We would predict teratogenic effects, such as abnormal wing development and other vertebral and scapular defects (Larsen and Janners 1987). We predicted that if the concentration of the vitamin A mixture is increased, then the body mass and length will decrease significantly.

1. Administration of vitamin A

Two different concentrations of vitamin A (2.0 mg/mL and 1.0 mg/mL) were injected into fertile and unincubated chicken eggs from the University of Georgia’s Poultry and Diagnostic and Research Center, Athens, GA. The eggs were assessed at a one week checkpoint and two week checkpoint. These concentrations were chosen because the maximum solubility of vitamin A in DMSO is 40 mg/ mL. Only 0.1 mL of fluid can be injected into the egg. We diluted vitamin A in DMSO and corn oil to obtain the concentrations of 2.0 mg/mL vitamin A/ 0.05mL DMSO + 0.05 mL corn oil and 1.0 mg vitamin A/ 0.025 mL DMSO +0.075 mL corn oil. 0.1 mL of each concentration was injected into the eggs. The vitamin A was stored in a -20 degree celsius freezer and wrapped in tin foil because it is light sensitive. To disinfect the eggs, we used 70% ethanol and kimwipes. Once cleaned, the eggs were labeled with a sharpie and placed into a warm dish with cotton balls under a heat lamp for protection. The mass of each egg was recorded to 4 decimal places using an analytical balance. We then used a candler to identify the air sac, which we injected the vitamin A into to minimize variability among the eggs. An egg piercer was used to pierce the shells to make the injection with the syringe needle a little easier. For each trial, 4 eggs were injected with the 2.0 mg concentration and 4 eggs were injected with the 1.0 mg concentration. Once the vitamin A was injected, we carefully placed a small piece of tape over the hole to reseal the eggs to prevent any outside environmental factors affecting the egg through the egg and prevent water loss. They were then placed into an incubator (Brinsea Mini Advance) and were left alone for one week. After one week, 2 eggs from each concentration were opened and observations were recorded. The remaining two were left untouched in the incubator for one more week. The control eggs included 2 eggs that were not injected with anything and placed straight in the incubator after labeled. We had 2 more eggs that were injected with 0.05 mL DMSO + 0.05 mL corn oil to test if the solvent alone will impose any abnormalities with the chicken embryo. Those eggs were also first cleaned with 70% ethanol, labeled with a sharpie, injected into the air sac (found by using a candler), and resealed with a small piece of tape. One control from each group was opened at the one week and two week mark to use for comparison to the treated eggs. There were a total of 3 trials performed. 

2. Observing embryonic development

We observed two eggs from each concentration and one egg from each control after 7 days. The mass was first recorded using the same analytical balance so we could compare the embryo’s weight to the effects of vitamin A. This process was repeated for the 1.0 mg  eggs and then for the 2.0 mg eggs. We noted any unfertilized embryos and discarded them.  

The embryo length (cm) was measured using a ruler. The isolated embryo was weighed by tearing another small dish on the analytical balance and then transferring the embryo to the dish to be weighed using the embryo spoon. Other physically visible abnormalities were recorded as well. Pictures of each embryo were also taken using an iPhone 12 Pro. All eggs that were not yet opened were kept in the incubator to keep the embryos alive. 

3. Data analysis

We calculated the mean and standard deviation of the length, and mass were expressed statistically. Other visible abnormalities, because they were more variable between eggs, were recorded and presented using a simple bar graph for each different concentration group and the control group.

We originally predicted that the vitamin A would have negative effects on chicken embryo development. The results supported that prediction but to an extreme. All eggs treated with our low and high concentrations of vitamin A were undeveloped at both day 7 and 14. After reducing our concentrations further halfway through the experiment, all of our treatment groups' eggs were still undeveloped. Out of our 6 different types of control groups, almost all eggs developed “normally” with the exception of a few DMSO only embryos that were slightly smaller in length and weight. This shows that vitamin A does play a significant role in the overall body development of chicken embryos. We suspect that lack of development in the vitiamin A treated eggs was due to the mechanism of retnoic acid. It  binds to the binding portien which is then transported to the nucleus where it modifys gene transcription arresting development (Jelínek and Kistler 1981). In our case, there was too much Vitamin A which arrested development all together. Vitamin A is also considered to be embryolethal and cause high malformations (Jelínek and Kistler 1981). This implies that excess concentrations of vitamin A will result in abnormalities or death.This result was surprising because another study on chicken embryos treated with vitamin A at one of the concentrations we used did show development (Buskohl et. al 2012). We think that is due to the injection of the vitamin A in a different spot or the embryo was observed outside of the shell and didn’t have the permeability of gas exchange from the shell. Previous studies have done extensive research on the extent of vitamin A’s effect but it is still unclear where the threshold of concentrations that cause abnormalities or death for retinoic acid starts.

One of the biggest limitations to this study was having to scale down doses of both our treatment and control groups down to accomodate for the chicken embryo size. While chicken embryos show similar morphology and growth development as human embryos, they still have many differences. A human embryo takes approximately 9 months to develop, whereas a chicken embryo takes 21 days. On top of that, chicken embryos must remain in an incubator during development. Ultimately in our experiment, dosages used were too high to test for abnormalities due to the embryonic development being arrested. Further studies can be done with lower concentrations of vitamin A, injecting the vitamin A into a different part of the egg, or even letting the chicken embryo completely hatch to better see potential abnormalities.

                        Abby Ulrich                                                               Lauren Kelly                                                      Caroline Reeves

                amu52536@uga.edu                                  lmk86185@uga.edu                              cmr25954@uga.edu