Technical note: Three-dimensional imaging of rumen tissue for morphometric analysis using micro-computed tomography

Abstract

Rumen development in calves has been evaluated by measuring papillae length, width, and density using microscopy for over 50 yr. Although common in the literature, disadvantages to this method exist, 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 postweaned bull calves at 55 d of age, immediately fixed in 10% neutral buffered formalin for 48h, and stored in 70% ethanol at 4°C before the contrast enhancement. After evaluation of contrast-enhancement protocols, it was determined that mercury chloride provided the most pronounced contrast for accurate micro-CT imaging based on relative density of the papillae. A 1-cm2 tissue section from the ventral sac of all bull calves was tensioned on a rapid prototyped curved plastic holder and imaged at 45μm resolution for 56min using a GE Locus Explore micro-CT (General Electric, Milwaukee, WI). MicroView V2.2 software (General Electric) was used to create a 3-dimensional virtual model of the entire sample. The length and width of papillae were measured 3-dimensionally and compared with measurements of papillae under the light microscope taken from the same region. The length and width measurements using micro-CT (2.47±0.12 and 0.55±0.01mm) compared with light microscope (2.96±0.03 and 0.86±0.01mm) were significantly smaller. The difference may reflect a more accurate determination in the base of the rumen tissue with micro-CT or the specificity of mercury chloride to bind only to intact rumen tissue. The mean number of papillae per centimeter squared viewed using micro-CT was 128.5±33.9 with a total surface area of 681.8±112.4mm2 and volume of 156mm3 per sample. Micro-CT data demonstrated that surface area and volume are positively associated and that papillae length was negatively associated with papillae per centimeter squared and positively associated with total volume of tissue section. This study represents the first time that micro-CT has been being used to assess morphology of rumen 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, imaging, and location of papillae measurements is required.