Marker-based approach to estimate apparent and true total tract nutrient digestibility in ruminants: comparative analysis and practical implications for nitrogen balance

This study evaluates the effectiveness of internal markers, specifically uNDF, uNDFom, and AIA, in estimating total-tract apparent digestibility (ttaD) and total-tract digestibility (ttD) across various ruminant species and conditions. Five trials were conducted focusing on small ruminants, dairy buffaloes, dairy cows, and beef cattle, with a final trial providing a comprehensive discussion on the technical application of uNDF as marker to assess Nitrogen (N) balance (NB), and marker utilization in dairy cows fed hay-based diets within the Parmigiano Reggiano region. In Trial 1, total collection, uNDF, uNDFom, and AIA were compared in small ruminants. The results demonstrated significant differences among markers, with uNDF providing more consistent and reliable estimates, while AIA tended to predict lower values of digestibility, compared to total collection. In Trial 2, digestibility in Mediterranean buffaloes was analyzed. The uNDF and AIA were used as markers, with AIA producing higher estimates. However, due to variability in ash content across diets and in accordance with literature and the following study, uNDF was considered a potential option for providing more accurate fiber digestibility predictions. In Trial 3, the comparison of uNDF and uNDFom markers was extended to dairy cows. Both markers performed similarly across most nutrients, but uNDFom offered a slight advantage in accuracy when estimating fiber digestibility, making it the preferred marker for high-fiber diet. Trial 4 focused on beef cattle, comparing tta of dry matter (ttaDMD) when estimated by uNDF, ADL, and ADIA. The findings suggest that uNDF could again provide more accurate estimates in fiber-rich diets between the three markers, being the experimentally less variable and again aligning with literature. Trial 5 provided a conclusive assessment of NB in dairy cows fed hay-based diets, specifically within the Parmigiano Reggiano production system, starting from markers-based calculation of 3 total fecal and urine output. The study highlighted the challenges of using hay-based diets, which resulted in a negative nitrogen balance (-60.77 g/day), reflecting inefficiencies in N utilization, but also focusing on the potential technical bias that can be due to inappropriate choice, or use, of markers to estimate total fecal and urine output, namely uNDF and creatinine. In the technical annexes I and II the potential impact of urine acidification on creatinine measurement, used as marker for total urinary N output estimation, and the evaluation of an in vitro method to assess the digestibility of rumen-protected amino acids are briefly described and discussed. In the first annex, it is suggested that, while acidification may alter creatinine concentration, the method could remain valid for estimating urine output. In the second annex, an in vitro method for ruminal and intestinal digestibility was used to evaluate a novel chitosan-based biopolymer as an alternative encapsulating agent for methionine. In conclusion, uNDF and uNDFom are reliable markers for estimating nutrient digestibility, particularly in fiber-rich diets, while AIA may introduce variability due to ash content; the choice of marker needs to reflect the species of interest, the dietary preferences, the diet composition, and the sampling protocol. These findings are critical for optimizing feeding strategies and improving N utilization efficiency in ruminants, especially in systems heavily reliant on hay- based diets.