Technical note: Evaluation of 3 methods to determine mucin protein concentration in ileal digesta of young preweaning calves

Abstract

The use of alternative sources of protein to substitute for milk proteins in milk replacers (MR) can increase the synthesis of endogenous proteins and therefore alter ileal or total-tract digestibility calculations. Mucin is the main component of gastrointestinal mucus and represents the greatest contribution to total endogenous protein. Mucin is difficult to isolate and has not been extensively studied in dairy calves. We explored 3 different procedures to analyze and estimate mucin protein (MUP) in ileal digesta of young dairy calves. Ileal digesta samples were collected from nine 30-d-old ileal-cannulated calves that were enrolled in a 3 × 3 replicated Latin square with 5-d periods. The 3 diets were a control whey protein-based MR (WPC), an isonitrogenous MR in which 50% of the protein was from enzyme-treated soybean meal (ESBM), and an N-free MR (NFREE). Mucin protein concentration and flow were analyzed by fractionation of the digesta and ethanol precipitation; this process served as the reference method. Alternative methods to estimate MUP consisted of using commercial enzymatic kits to analyze glucosamine (N-acetylglucosamine, GlcNAc) and galactosamine (N-acetylgalactosamine, GalNAc), 2 amino-sugars that are highly enriched in mucin. Before GlcNAc determination, samples were processed using 3 different procedures: sample clarification (GLCL), clarification plus hydrolysis (GLCH), and hydrolysis alone (GLHL). The MUP was estimated by regression of the GlcNAc and GalNAc values using previously validated equations. According with the bias and agreement analysis, none of the methods yielded MUP values similar to the reference method. However, GLHL showed a strong association with the reference method (ρ = 0.73). It allowed identifying the smaller MUP flows with NFREE compared with the other 2 diets and detecting the greater flow of ESBM than WPC, as observed with the reference method. Using the GlcNAc values from GLHL and the MUP measured with the reference method, we were able to establish a linear relationship between both methods (adjusted R2 = 0.75). We found that the GLHL method enabled detecting differences in MUP ileal flows between diets differing in protein level and source. Inferences about MUP secretions must be done cautiously because many dietary and physiological factors are involved. The adoption of practical techniques to determine MUP can help to increase our knowledge about gastrointestinal tract function and to improve the accuracy of MR digestibility calculations.