Fourteen chronically hypoxic chickens at 15 and 19 days of age each were taken for study; 14 control fetuses were of similar age. The hypoxic incubator was maintained at 14.5% O ₂ and normoxic incubator was maintained at 21% O ₂

Excised hearts were rinsed in PBS buffer and pinned to silicon plate. Through the two catheters inserted into pulmonary artery and aorta, PFA is flushed into the chambers and then stained with 1%menthylene blue in PFA. The heart is flushed with PBS and filled with embedding media (OCT gel). The heart is made frozen at -80°C and sections are made in cryostat. Sliced sections are captured with mediscope and pictures are analyzed with NIS-AR elements. Measurements are made manually and calibrated.

Cardiomyocytes are isolated through retrograde perfusion technique. Myocyte slurry is fixed with PFA and stained with DAPI stain. Processed slides are viewed under fluorescent microscope and cells pictures are captured with the help of QIMC camera. Cells are measured manually with NIS-AR elements software.  With the length and width measurements of cardiomyocyte, the cell volume is calculated.

The ventricular wall thickness was normalized based on the mean diameter of the heart. The mean axis was calculated using the formula

Mean axis= sqrt (long axis * short axis)

Two way ANOVA using embryonic age and incubation treatment (normoxia or hypoxia) was applied for the data obtained followed by the Newman-Keuls post hoc comparison in Statistica 9 (Statsoft Inc. Oklahoma, USA). Statistical significance was taken as p<0.05. All data presented as Mean (standard deviation).