Effect of nicotine and camellia sinensis (green tea extract) on histomorphology of developing thigh bone of chick embryo

Material and Method
This study was carried out at the poultry research institute (PRI) Rawalpindi and at Anatomy department of Army Medical College Rawalpindi. All the procedures were approved by Ethical Review Committee of Army Medical College Rawalpindi.
3.1 Sample Collection
40 fertile eggs of Fyoumi species of chick , were selected at zero hour of incubation.
3.1.1 Species selection
Fyoumi species of chick were selected for obtaining the specimen.
The eggs were collected from Poultry Research institute Punjab, Rawalpindi.
3.1.2 Specimen Collection
Sampling technique was random , eggs of abnormal shape, colour, size, texture and refrigerated eggs were excluded from the study.
3.1.3 Incubation
Eggs were placed in hatchery after properly fumigating and clearing the hatchery. Temperature was maintained at 37.5 degree centigrade ,the relative humidity was kept between 75% and proper ventilation was maintained. Rotation of eggs were done 4 hourly . Placement of eggs in hatchery was taken as day zero.
3.1.4 Study Groups
This study design comprises of 4 groups each comprising of 20 eggs of fouymi species.
Control group 1:(G1) Injected 0.1m of Normal saline.
Experimental group2:(G2)Injected 0.1m of Camellia sinensis (green tea extract).
Experimental group 3:(G3)Injected 0.1m of Nicotine solution.
Experimental group 4:(G4)Injected 0.1m of Nicotine solution and 0.1ml of Camellia sinensis.
Each group were injected with 0.1ml concentrations of respective solution.
3.2. Administeration of Different Working Solution
3.2.1 Control Group 1
Eggs were injected with insulin gauge needle, 0.1ml normal saline under laminar flow. First holes were made at the blunt end of eggs with the help of egg driller and then the 0.1ml quantity of solution were given to the eggs at 48 hr of incubation. Holes were sealed with paraffin wax after the injection.
3.2.2 Experimental Group 2
For this group 8g of dried leaves of Camellia sinensis in 100ml of hot distilled water for 15 minutes then filtering the solution. This solution of 0.1ml in quantity was administered to the experimental group 2 at 48 hr of incubation. Holes were sealed with paraffin wax after the injection.
3.2.3 Experimental Group 3
For this group 0.1ml of nicotine solution of 0.0001% concentration after preparing Nicotine stock solution from 1mg/ml,Nicotine Vial solution (purchased from Sigma Aldrich) were injected to group 3 at 48 hour of incubation . Holes were sealed with paraffin wax after the injection.
3.2.4 Experimental Group 4
In this group 0.1ml of Nicotine solution injected through the hole made at the blunt end of the eggs after the hole got sealed with paraffin wax, second hole made for the injection of 0.1% of camellia sinensis extract to the eggs at 48 hr of incubation. Holes were sealed with paraffin wax after the injection.
3.3 Collection of Specimen at 17 Days of Incubations
After incubating the eggs for 17 days, 10 eggs from each group were sacrificed and specimens were collected. Samples were collected by breaking the blunt end of eggs with the help of forceps. Separating the embryo from the yolk sac placing them in the formalin filled jars for 48 hour.
Results And Observation:
Aim of this study was to evaluate the effect of nicotine on the histomorphology of developing thigh bone of chick and role of camellia sinensis (green tea extract ).
SURVIVAL OF EMBRYO
The survival of control group (G1 )and experimental group (G2) at 17th day of incubation was significantly higher than the experimental group(G3) and (G4).Whereas in comparison of (G3) and (G4) the survival rate of G4 is significantly higher than that of (G3).
GROSS EXAMINATION
For 17th day old embryo the crown rump length was taken from vertex that is highest point between the eyeballs and tip of coccyx .A thread was taken whose one end was placed on the vertex and other end followed the curve of the back of embryo to the tip of coccyx . the distance covered by the thread was measured .Mean length of embryo in control group and experimental group(G2) was significantly more than that of(G3)and(G4).
Length of femur at 17th day of incubation was measured from the greater trochanter to the lateral condyle ( K.A. Alfonso-Torres etal., 2009).length of femur at 17th day of incubation were done it was significantly more in control group (G1 )and experimental group (G2) than the experimental group(G3) and (G4).
Histological Observation
By observing the height of proliferating zone and the hypertrophy zone by doing micrometery it was found that it was more in the experimental group (G3)and(G4) than control group (G1)and(G2) .
DISCUSSION
Nicotine is responsible of sever oxidative stress and camellia sinensis decrease it as the green tea resembles with control group but when it is administered along with nicotine it tries to overcome the oxidative stress of nicotine.Nicotine decrease the bone matrix formation by looking at the histomorphological changes.In a recent study of the eating habits of 2,018 women, consumption of mushrooms and green tea was linked to a 90% lower occurrence of breast cancer ( Zhang, M 2009).It has been suggested that antioxidant can modify some of histological changes produced by exposure to smoke (Klesges LM etal., 1998).Antioxidant provides protection against ischemia it can be useful in treating clinical states involving excess free radical production, such as fetal growth restriction and fetal hypoxia( OkataniYetal.,2001) Nicotine have a direct effect on bone metabolism by influencing the bone remodelling process. Nicotine suppressed cellular proliferation in rat osteosarcoma cells (Fang etal.,1991) and suppressed osteogenesis through a decrease in alkaline phosphatase (ALPase) and type1 collagen production by osteoblast(Tanka etal.,2005).In contrast, nicotine appears to stimulate osteoclast resorption by inducing bone resorbing cytokines, interleukin-1 (Norazlina etal.,2004).From different studies it has been suggested that nicotine was not only a direct inhibitor of osteoblast differentiation but also a regulator of osteoclast activity.

3.13 Photography
For photomicrography Olympus digitial camera (10mega pixel ) Stylus 1010 was used through the ocular of the Olympus CX21FSI having light microscope with the tube length of 160mm.The images were corrected and adjusted with respect to contrast, brightness, sharpness and color balance. The labelling of the pictures was done using computer softwares. The magnification was calculated according to the following formula (Anonymous,N.D): Power of eyepiece x Power of objective x Camera zoom.
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