Abstract
Determining protein requirements (PR) for dog maintenance using welfare-conscious methods is challenging. This study aimed to establish a minimally invasive and efficient protocol for applying the indicator amino acid oxidation (IAAO) technique using L-[1-13C] phenylalanine (13Phe), while minimizing animal handling and cost (through lower isotope doses and fewer meals) and eliminating the need for respiration chambers. Two diets were extruded: a complete and balanced for maintenance based on poultry by-product meal (PBM) with 28% crude protein (CP); a low-protein formulation where PBM was replaced by maize starch (SBD) with 6% CP (DM basis). Ingredients were analyzed, and the SBD was supplemented so both formulations presented 22 g/kg of phenylalanine + tyrosine (DM basis). The dilution technique was applied to create five levels of CP: 6%, 8%, 10%, 12%, and 14% (DM basis). A sixth diet with 11% CP was used in the adaptation period. To test three feeding, isotope-enrichment, and sampling protocols (P1, P2, and P3), a block design was used with 10 dogs assigned to each protocol (14.8 ± 1.1 kg; 5.8 ± 2.0 years). After two days of adaptation (11% CP), the daily dog food was divided into 10 (P1), 7 (P2), or 5 (P3) meals. A priming dose (PD) of 0.18 mg/kg of 13C-Bicarbonate (13Bic) and 0.66 mg/kg of 13Phe were offered on the fourth, sixth, or fifth meal, followed by six, four, and four subsequent doses of 1.33 mg/kg of 13Phe in capsules every 30 min (P1, P2, and P3, respectively). Two basal samples of expired air were collected using an adapted mask, and after PD, eight samples were collected, starting 30 (P1), 90 (P2), and 90 (P3) min after enrichment. The CO2 volume was measured by the 13Bic method using masks. The time required to reach isotopic steady state (ISS) and the accuracy of measurements varied across protocols. P1 required the longest time to reach ISS (183.7 ± 8.4 min) and showed lower accuracy (r2 = 0.39; mean absolute percentage error [MAPE] = 17.1%; P < 0.05). P3 reached ISS more quickly (105.4 ± 6.1 min) but demonstrated moderate accuracy (r2 = 0.67; MAPE = 7.1%; P < 0.05). P2 represented an intermediate condition, reaching ISS at 170.6 ± 6.7 min while providing higher accuracy (r2 = 0.81; MAPE = 1.26%; P < 0.05). It was concluded that P2 is a minimally invasive and efficient protocol for IAAO application in dogs, potentially providing valuable insights for future research involving dogs living outside of a laboratory setting.