Three dimensional imaging of rumen tissue for morphometric analysis using micro-computed tomography
Rumen development in calves has been evaluated microscopically by measuring papillae height, width and density. Although common in the literature, there are disadvantages such as large variations in rumen papillae size and shape, small numbers of total papillae being measured and the time required. The objective of this study was to develop a more effective technique for assessing rumen papillae using micro-computed tomography (micro-CT) and to compare this technique with microscopy. Rumen tissue was collected from the ventral sac of 20 bull calves at 55 days of age, immediately fixed in 10% Neutral Buffered Formalin for 48 hours and stored in 70% ethanol at 4oC prior to the contrast enhancement. After evaluation of contrast enhancement protocols which included phosphotungstic acid, osmium tetroxide, and mercury chloride it was determined that mercury provided the most pronounced contrast for accurate micro-computed tomography imaging based on relatively density of the papillae. A 1 cm2 tissue section from the ventral sac of all bull calves was tensioned on rapid prototype curved plastic holders and imaged at 45µm resolution for 56 minutes using a GE Locus Explore micro-CT. MicroView V2.2 software created a three dimensional model of the entire sample. The height and width of 20 papillae per micro-CT section were measured three dimensionally and compared with measurements of 20 papillae under the light microscope taken from the same region using a mixed model equation with a random effect for calf. The length and width measurements using micro-CT (2.47±0.12mm and 0.55±0.01mm) compared to light microscope (2.96±0.03mm and 0.86±0.01mm) were significantly smaller (P<0.0001) . The difference may reflect a more accurate determination in the base of the rumen tissue with micro-CT or the specificity of mercury to bind only intact rumen tissue. The mean number of papillae per cm2 viewed using micro-CT was 128.5±33.9 with a total surface area of 681.8±112.4 mm2 and volume of 156.2±33.2 mm3 per sample. Micro-CT data showed that surface area and volume are positively associated (P=0.04) and that papillae length was negatively associated (P<0.001) with papillae per cm2 and positively associated (P=0.02) with total volume of tissue section as determined by Pearson Correlations. This study represents the first time that micro-CT has been being used to assess morphology of gastrointestinal tissue. Micro-CT has the potential to improve the accuracy and efficiency of rumen tissue measurements however more standardization of each factor involved in tissue preparation and imaging is required.
Keywords: Rumen, morphology, development