Comparison of two methods for determination of intestinal villus to crypt ratios and documentation of early age-associated ratio changes in broiler chickens

The determination of intestinal villus to crypt ratios (VCR) is a common method utilized to evaluate effects of various diet regimens on gut microanatomy and for the histologic quantification of intestinal responses to disease processes. Two methods for the determination of small intestinal VCR were compared in early age chickens. A standard method for VCR determination based on 10 villus and crypt length measurements in the jejunal region of the small intestine was employed for the calculation of villus to crypt length ratio (VCLR). That method was compared to a new approach based on a single histomorphometric determination of the crypt and total mucosal areas using image analysis software. Subtraction of the crypt area from the total area provided the villus area and allowed for the subsequent calculation of villus to crypt area ratio (VCAR). At 4 and 18 h posthatch, VCLR was higher than that of VCAR, but there was no significant difference between VCLR and VCAR at 0 h (hatch) and at 168 h (d 7) posthatch. Nevertheless, the pattern of age-associated changes for VCLR and VCAR were comparable throughout the early posthatch period. Furthermore, the new method used in determining VCAR is subject to less human error, allows for an appreciable reduction in the number of measurements required, and facilitates a larger intestinal segment evaluation. Standard linear measurements require the selection of variable numbers of villi and crypts, whereas the area method only requires selection of a single region that incorporates numerous villi and crypts of variable sizes in providing a less subjective approach. This is particularly advantageous in studies on intestinal disease conditions resulting in marked multifocal variation in villus stature. This study further documented age-associated changes occurring in the VCR of the small intestine during the early posthatch period. Across the 2 methods used for VCR determination, a major and highly significant reduction in the VCR was observed to occur between 18 h and 168 h posthatch.