Comparative histomorphometric study of small intestine in broiler chicken using the closed-Clarke, opened-formalin and closed-formalin fixation methods

Tissue fixation is a vital step in preparation of samples for histological studies and mistakes at this stage could lead to irreversible damage. The aim of this study was to compare the effect of three sample preparation-fixation methods on histomorphometic indices of small intestine of broilers. Thirty 10 day old broilers of Ross 308 strain were used in the study. Segments of small intestine were washed and prepared as opened or closed sections. The closed sections were fixed in 10% saline-formalin. The opened samples were cut at the mesenteric line and were fixed by 10% saline-formalin. In Clarke’s fixation the ends of samples were sealed by string and Clarke’s solution was injected into the lumen. Fixed samples were subjected to paraffin embedding and 5 µm sections were cut using rotary microtome and sections were stained by Eosin-Hematoxylin and Alcian blue. In each section the villus height, villus thickness, crypt depth and goblets’ density were measured under light microscope and the villus surface index was also calculated. Data were subjected to ANOVA in a completely randomized design with three treatments and 30 observations. The results indicated that method of preparation-fixation had a significant effect on morphometric indices (p<0.01). The closed-formalin’s samples had the greatest villus height and crypt depth values in all three intestinal segments, while the lowest values were observed in closed-Clarke’s method (p<0.01). Closed-Clarke’s samples had the thickest villus in all three parts of small intestine followed by closed-formalin’s and opened-formalin’s samples (p<0.01). Villus surface area was highest in closed-formalin fixed samples (p<0.01). Considering simplicity of the method, low cost, better mucosal protection and lack of severe tissue changes observed in closed-formalin’s method, this method was considered as a method of choice in histomorphometric studies of poultry small intestinal mucosa.