title: Refinements in primary rumen epithelial cell incubation techniques.
abstract: The objectives of this study were to 1) determine if the number of rumen epithelial cells in primary cell incubation affects the rate of metabolite production, and 2) determine the optimum mode of data expression to standardize reporting criteria. Sections of rumen epithelial tissue were excised from five Holstein heifers and subjected to serial tryptic digestion to isolate cells. Isolated cells had a mean viability of 86% (+/- 1.29) and were incubated at concentrations of 0.5, 1, 5, 10, 20, and 40 million cells per flask. Oxidation of [1-14C]butyrate to 14CO2 and production of acetoacetate (ACAC), beta-hydroxybutyrate (BHBA), lactate, and pyruvate were measured for cell dilution comparisons. Cell number, cell dry matter, cell crude protein, epithelial wet tissue weight, body weight, and metabolic body weight were measured to generate 12 different forms of data expression. Coefficients of variation were calculated for each type of expression. Expressing data per cell number resulted in the lowest variation. Oxidation of [1-14C]butyrate to 14CO2 and pyruvate production per million cells did not significantly differ between treatments for 90-min incubation. Acetoacetate and lactate concentrations were greatest at 0.5 and 1 million cells/flask, respectively, with no differences between 5 to 40 million cells/flask. Production of BHBA for 1 million cells/flask was greater than 0.5 and 40 million cells/flask, but did not change between cell concentrations 5 to 20 million. The BHBA:ACAC concentration ratios for 0.5 and 1 million cell dilutions were both 1.1 to 1 indicating low mitochondrial redox potentials. Concomitantly, lactate:pyruvate ratios for 0.5 and 1 million cells were greater than other cell dilutions, indicating a high cytosolic redox potential. The suggested range of rumen epithelial cells to include in incubations is 5 to 20 million cells/flask. This will minimize experimental error associated with using low cell numbers and the potential for reduced metabolite production caused by incubating large cell quantities. When rumen tissue taken from animals of the same species, size, and stage of development; data adjusted by cell number is preferred. However, it is recommended that cell protein, cell DM, and animal metabolic weight be included to facilitate future comparison between species and laboratories.
authors.affiliation: Department of Animal Science, The University of Tennessee, Knoxville 37901-1071, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Klotz
authors.forename: R L
authors.initials: RL
authors.lastname: Baldwin
authors.forename: R C
authors.initials: RC
authors.lastname: Gillis
authors.forename: R N
authors.initials: RN
authors.lastname: Heitmann
mesh_terms: D000818:Animals; D002087:Butyrates / Q000378:metabolism*; D002247:Carbon Isotopes; D002417:Cattle / Q000033:anatomy & histology / Q000378:metabolism*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D019344:Lactic Acid / Q000378:metabolism; D012417:Rumen / Q000166:cytology / Q000378:metabolism*
chemical_list: D002087:Butyrates; D002247:Carbon Isotopes; D019344:Lactic Acid