Objective

The purpose of this experiment is to determine how the development of bone and cartilage in a chick are affected by treating the embryo with nicotine at early stages of development.

Introduction

Maternal smoking during pregnancy has long been recognized as a hazard, increasing the incidence of premature delivery, spontaneous abortion, low birth weight, and neonatal mortality. There is also an increase in the occurrence of lower respiratory illnesses, which suggests that smoking can effect lung development of the fetus during pregnancy. Nicotine, which is able to cross the placental barrier, may be the primary cause of many of the effects of maternal smoking during pregnancy. In experimental studies, maternal nicotine exposure elicits the same effect on neonates as direct smoking by the mother (Pierce and Nugyen, 2001). Many studies have also found that, for example, women who smoke during pregnancy increased the risk that their children would develop asthma, and this is especially true in families with a history of asthma. Nicotine may adversely affect lung function by increasing airway resistance, diminishing airway conductance, decreasing peak tidal expiratory flow, and increasing respiratory time (Maternal Smoking During Pregnancy).

Nicotine, the chief alkaloid in tobacco, is known to have other adverse effects on body development also. Smoking during pregnancy is also known to affect the child’s intelligence in later years. One study offered evidence that maternal cigarette smoking during pregnancy poses a unique risk for neurodevelopmental impairment among children, which is manifested as early as the first four year where the child’s intellectual functioning is severely impaired (Olds et al. 1994). Nicotine exposure is also known to cause growth restriction, premature rupture of membranes, and an increase in heart rate. Nicotine is impairs the absorption of calcium, vitamin C, and other vitamins and minerals required by a developing fetus. Researchers have demonstrated similar effects similar to those in humans as a result of maternal nicotine exposure in rats, including reductions in ossification in the femur, forelimb, nasal bone, ribs, and the skull and face (Elliott and Unger, 2000; Paulson et al. 1994). The retardation of bone development may be due to and an inhibition of calcium absorption by nicotine. This suggests that nicotine causes abnormalities in embryonic bone development. This experiment is intended to examine the effects of embryonic exposure to nicotine on bone and cartilage formation in chicks.

Materials

24 - 3 day chick eggs nicotine stock solution (40% weight / volume) 4 % paraformaldehyde 100 % ethanol Alcian Blue Alizarin Red Howards Ringer's solution glacial acetic acid potassium hydroxide glycerol glass jars syringe incubator

Experimental Procedure

Preparation of Nicotine Solution and Treatment of Chick Embryos

Prepare nicotine solution by adding 25 µL of nicotine stock solution (40% weight / volume) to 10 mL of Howard’s Ringer’s Solution.

Make a small opening in the blunt end of the eggs, and administer 0.1 ml of nicotine solution to 8 of the eggs, 0.2 mL of the nicotine solution to 12 of the eggs, and 0.1 mL of Howard’s Ringer’s Solution to 2 of the remaining eggs and 0.2 mL of Howard’s Ringer’s Solution to the other two eggs as the controls.

Incubate eggs at 37 °C for 14 days.

After two weeks, remove the now 17-day-old chicks from the eggs and continue with the staining protocol.

Protocol for Staining Specimen for Bone and Cartilage

Fix specimen in 4% paraformaldehyde for at least 48 hours at low temperature.

Rinse embryos for at least 1 day in distilled water, with several changes.

Postfix in 70% ethanol. Embryos may be stored indefinitely.

Remove feathers and skin, and eviscerate specimens.

Place specimen in solution of Alcian Blue cartilage stain (20 mg Alcian Blue for every 100 mL of 70% ethanol in glacial acetic acid) for 2�3 days.

Destain specimen by transferring it to 70% ethanol in glacial acetic acid for 1 hour, then to 100% ethanol for 1 day. Change frequently.

Soak specimen in distilled water for 1 day.

Transfer embryo to 2% KOH overnight to clear embryo.

Transfer to 2% KOH to which enough saturated Alizarin Red has been added to turn the solution a very dark purple color.

Transfer to plain 2% KOH to rinse out Alizarin Red. The solution should be changed once or twice over a 24 hour period.

When cartilagenous skeletal structures, stained blue, and bone, stained red, become visible, transfer specimen to 1% KOH / 20% glycerol for one day to allow clearing of the tissue, occasionally gently swirling.

Store specimen in 50% ethanol / 50% glycerol in a screw top jar.

Photograph embryos and compare development of bone and cartilage.

Results

Embryos treated with nicotine exhibited stunted growth and some exhibited physical deformities of the head (Figure 1). Those treated with 0.2 mL of the nicotine were smaller than those treated with 0.1 mL of nicotine, which were, in turn, smaller than the control.

After being stained with Alcian Blue, embryos treated with 0.2 mL of nicotine solution exhibited a larger amount of cartilage in bones of the limbs and smaller gaps between the areas where the cartilage was stained blue than those treated with 0.1 mL of nicotine solution. Embryos treated with 0.1 mL of nicotine solution had a larger area of the limb bones stained blue with smaller gaps between the stained areas that the control. After being stained with Alizarin Red, however, the colors of stained skeletal structures could no longer be observed (Figure 2).

Discussion

Treatment of chick embryos with nicotine appears to have negative effects their development, the severity of which increases with the amount of nicotine to which the embryos are exposed. The treated embryos exhibited stunted growth and the embryos treated with the highest amount (0.2 mL) were the smallest. The treated embryos were also deformed, but there was no noticeable consistency in the deformities across the different treatment levels. For example, with the exception of their size, some of the embryos treated with 0.1 mL of the nicotine solution looked relatively similar to the control, but one of them was smaller and with a ventral midline that failed to fuse, so that the vicera were left completely exposed.

Comparative bone and cartilage development could not be fully examined because the staining procedure did not result as expected within the expected amount of time. For future experiments, it may be necessary to amend or allow more time to complete the staining protocol. Up until the point where the specimen is stained in Alizarin red, all steps proceed relatively quickly. After being stained with Alcian Blue and cleared in 2% KOH, the stained cartilaginous structures can be readily seen. The staining with Alizarin Red, however, makes the embryos a dark purple color, and the destaining process takes much longer. If this experiment is repeated, Alcian Blue staining alone should be considered as a means of examining bone and cartilage development.