Quantification of total endogenous nitrogen losses in broilers using a minimally invasive 15N-isotope dilution method

Total endogenous nitrogen (N) losses (ENLs) measurement in broilers is important for quantifying dietary true N digestibility. Basal ENLs are commonly quantified using enzymatically hydrolysed casein (EHC) or EHC-ultrafiltration (EHC-UF), whereas diet-specific ENLs require regression methods. Total (tENL; basal + diet-specific) ENL can be measured using the ¹⁵N-isotope dilution (ID) method. This study aimed to quantify tENL using a minimally invasive ID method based on urinary ¹⁵N-enrichment as a proxy for ¹⁵N-enrichment in the precursor pool for endogenous protein synthesis, and to compare ID-derived tENL with estimates from EHC and EHC-UF methods. A total of 432 day-old female broilers were randomly assigned to 48 pens (nine birds/pen), and fed one of five maize-based diets varying in fibrous ingredients (intact or ground soybean hulls with or without purified arabinoxylans, or ground sugar beet pulp), or a semisynthetic diet containing EHC (molecular weight <5 000 Da) as the only protein source. Titanium dioxide (4 g/kg) was added as an inert digestibility marker. At day 26, three birds per pen received a daily oral dose of ¹⁵N-isotope solution containing 2.4 g ¹⁵N-milk protein concentrate (atom- = 2.27) and 0.12 g ¹⁵NH₄Cl (atom- ¥ 98); between days 27 and 31, the milk protein dose was reduced to 1.2 g. From days 31 to 37, excreta were collected, and at day 37, birds were euthanised for ileal digesta and tissues sampling. Nitrogen and tracer concentrations in digesta and excreta, ¹⁵N-enrichment in samples, and high-molecular weight N in digesta (EHC-diet) were quantified. Urinary ¹⁵N-enrichment dropped within 2 days postdosing, then reached a steady state. Total ENLs were calculated from ¹⁵N-enrichment in digesta or faeces, relative to ¹⁵N-enrichment in the precursor pool, assuming that it can be reflected by (1) the weighted average of ¹⁵N-enrichment in liver-, pancreas-, and ileum tissues, or (2) urinary ¹⁵N-enrichment. Estimates of tENL based on these assumptions positively correlated (r = 0.84-0.90, P < 0.001). Ileal tENL estimates were smaller with the ID compared with EHC (75.6, P < 0.001) and EHC-UF (70.3, P < 0.001) methods, resulting in lower true ileal N digestibility estimates (1 to 4-units, P < 0.001). In conclusion, the minimally invasive ID method can be used for quantifying tENL in broilers under close-to-practical conditions. Using urinary ¹⁵N-enrichment to represent ¹⁵N-enrichment in the precursor pool enabled tENL quantification across diets varying in fibre and protein sources. The ID method provides a useful tool to compare total ENL and dietary true N digestibility. © 2026 The Author(s)