We have previously determined phenylalanine (Phe) requirements in mature dogs; however, little information is available on differences of Phe minimum requirements on different breed sizes. The objective of this study was to determine Phe requirements in adult dogs of three different breed sizes using the direct AA oxidation (DAAO) technique. In total, 12 adult dogs were used, four Miniature Dachshunds (5.3 ± 0.6 Kg BW; 1.8 ± 0.1 years old; mean ± SD), four Beagles (8.3 ± 0.7 Kg BW; 6.7 ± 0.2 years old; mean ± SD), and four Labrador Retrievers (34.9 ± 2.2 Kg BW; 4.4 ± 1.4 years old; mean ± SD). A basal Phe-deficient diet with excess of tyrosine (Tyr) was formulated. Dogs were randomly fed the basal diet supplemented with increasing levels of Phe; the Phe content in the final experimental diets was 0.24, 0.29, 0.34, 0.44, 0.54, 0.64, and 0.74%. After 2 d of adaptation to the experimental diets, dogs underwent individual DAAO studies. During the DAAO studies, total daily feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1-13C]-Phe (9.40 mg/kg BW), and thereafter, L-[1-13C]-Phe (2.4 mg/kg BW) was supplied with every meal. Total production of 13CO2 (F13CO2) during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The mean requirement for Phe and the 95% confidence interval (CI) was determined using a two-phase linear regression model. To account for differences in feed intake, requirements were expressed in mg.kg BW-1.d-1. The mean requirement for Phe were 41.9, 41.3, and 42.6, and upper 95% CI of Phe requirements were 57.3, 58.4, and 64.8 mg.kg BW-1.d-1 for Miniature Dachshunds, Beagles, and Labrador Retrievers, respectively. The mean requirement and the upper 95% CI for the pooled data (all dogs) was 45.3 and 55.4 mg.kg BW-1.d-1, respectively. In conclusion, the Phe requirements for different breeds were similar among dog breeds studied. However, Phe recommendations proposed in this study are lower than those proposed by NRC and AAFCO (mg.kg BW-1.d-1).

Tryptophan (Trp) is an indispensable amino acid (AA) for dogs of all life stages; however, although Trp requirements for growing dogs are derived from 3 dose–response studies, there are no empirical data on Trp requirements for adult dogs at maintenance. The study objective was to determine Trp requirements of adult dogs of 3 different breeds using the indicator amino acid oxidation (IAAO) technique. Four spayed or neutered Miniature Dachshunds (5.28 ± 0.29 kg BW), 4 spayed Beagles (9.32 ± 0.41 kg BW), and 5 neutered Labrador Retrievers (30.51 ± 2.09 kg BW) were used. After a 14-d adaptation to a Trp-adequate basal diet (Trp = 0.482% dry matter), all dogs were fed a mildly Trp-deficient diet for 2 d (Trp = 0.092% dry matter) before being randomly allocated to receiving 1 of 7 concentrations of Trp supplementation (final Trp content in experimental diets was 0.092, 0.126, 0.148, 0.182, 0.216, 0.249, and 0.283% dry matter) and all dogs received all Trp treatments. After 2-d adaptation to the experimental diets, dogs underwent individual IAAO studies. Total feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1-13C]-Phenylalanine (Phe) (9.40 mg/kg BW), and thereafter, L-[1-13C]-Phe was supplied (2.4 mg/kg BW) with every meal. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The maintenance requirement for Trp and the 95% confidence interval (CI) were determined using a 2-phase linear regression model. Mean Trp requirements were estimated at 0.154, 0.218, and 0.157% (dry-matter) for Dachshunds, Beagles, and Labradors, respectively. The upper 95% CI were 0.187, 0.269, and 0.204% (dry-matter) for Dachshunds, Beagles, and Labradors. In conclusion, estimated Trp requirements are higher for Beagles compared with Labradors or Dachshunds, and all estimated requirements are higher than those currently recommended by the NRC and AAFCO.

Threonine (Thr) requirements for immature (growing) Beagles have been determined, but little knowledge is available on Thr requirements for maintenance in mature dogs. Moreover, differences of Thr requirements among different breeds or sizes of adult dogs have not been investigated. The objective of the present study was to determine Thr requirements in adult dogs of three different breeds using the indicator amino acid oxidation (IAAO) technique. In total, 13 adult dogs were used, 4 Miniature Dachshunds (5.8 ± 0.4 kg body weight [BW]; 3 spayed and 1 neutered), 4 spayed Beagles (9.3 ± 0.6 kg BW), and 5 neutered Labrador Retrievers (30.5 ± 1.7 kg BW). Dogs were fed a Thr-deficient diet (Thr = 0.23%) and randomly allocated to receiving one of seven concentrations of Thr supplementation (final Thr concentration in experimental diets was 0.23%, 0.33%, 0.43%, 0.53%, 0.63%, 0.73%, and 0.83%; as fed basis) for 2 d. After 2 d of adaptation to the experimental diets, dogs underwent individual IAAO studies. During the IAAO studies, total daily feed was divided into 13 equal meals; at the sixth meal, dogs were fed a bolus of l-[1-13C]-Phenylalanine (Phe) (9.40 mg/kg BW), and thereafter, l-[1-13C]-Phe (2.4 mg/kg BW) was supplied with every meal. Before feeding the next experimental diet, dogs were fed a Thr-adequate basal diet for 4 d (Thr = 0.80% as fed basis) in known amounts that maintained individual dog BW. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total production of CO2 measured using indirect calorimetry. The mean requirements for Thr, defined as the breakpoint, and the 95% confidence interval (CI) were determined using a two-phase linear regression model. For Miniature Dachshunds, the two-phase model was not significant, and Thr requirements could not be determined. Mean Thr requirements for Beagles and Labradors were 72.2 and 64.1 mg/kg BW on an as-fed basis, respectively. The requirement for Thr between these two dog breeds was not different (P > 0.10). Thus, the data for Beagles and Labradors were pooled and a mean requirement for Thr was determined at 66.9 mg/kg BW, and the 95% CI was estimated at 84.3 mg/kg BW. In conclusion, estimated Thr requirements for Beagles and Labradors did not differ, and these recommendations are higher than those suggested by NRC (2006) and AAFCO (2014) for adult dogs at maintenance.

There is a lack of knowledge regarding the lysine (Lys) requirements of mature dogs and whether there are breed differences. The present study aimed to determine the Lys requirement in 3 breeds of mature dogs using the indicator amino acid oxidation (IAAO) technique. Thirteen adult dogs were used, 4 Miniature Dachshunds (5.39 ± 0.71 kg; 1.05 ± 0.02 yr old, mean ± SD), 4 Beagles (8.09 ± 0.40 kg; 5.03 ± 0.09 yr old, mean ± SD), and 5 Labrador Retrievers (29.42 ± 2.04 kg; 3.30 ± 0.69 yr old, mean ± SD). After 14 d of adaptation to a basal extruded kibble diet, dogs were fed a test diet mildly deficient in Lys (Lys concentration = 0.36%) at 17 (Miniature Dachshunds) or 13 g/kg BW (Beagles and Labradors) for 2 d. The test diet was supplemented with 1 of 7 isonitrogenous Lys-Ala solutions, resulting in a final dietary Lys concentration of 0.36, 0.40, 0.44, 0.50, 0.54, 0.58, and 0.62% (as fed basis). Dogs received dietary concentrations of Lys in random order and no dog received the same order. Following 2 d of adaptation to the experimental diets, the dogs underwent IAAO studies. During the IAAO studies, total daily feed was divided in 13 equal meals. At the sixth meal, dogs were fed a bolus of L-[1-13C]-Phe (9.40 mg/kg BW); thereafter, L-[1-13C]-Phe was supplied with every meal (2.4 mg/kg BW). Total production of 13CO2 (F13CO2) during isotopic steady state was determined by enrichment of 13CO2 of breath samples and total production of CO2, measured using indirect calorimetry. A 2-phase linear regression model was used to derive the mean Lys requirement, defined as the breakpoint, and the upper 95% confidence limit was calculated as the recommended allowance (RA) for Lys intake. For Miniature Dachshunds, the study was repeated with a feed intake of 14 g/kg BW, but Lys requirements could not be determined at either feed intake, suggesting a requirement below the lowest concentration and intake. Mean Lys requirements for Beagles and Labradors were 0.455% (59.16 mg/kg BW) and 0.440% (57.19 mg/kg BW), respectively, on a dry matter basis. Pooling the data for these breeds provides a mean estimate of the Lys requirement at 0.448% (58.21 mg/kg BW) with an upper 95% CL of 0.526% (68.41 mg/kg BW) on a dry matter basis. In conclusion, the Lys requirements of Beagles and Labradors are similar while the requirement for Miniature Dachshunds is undetermined and likely lower. The estimated Lys requirement for Beagles and Labradors is higher than the NRC (2006) recommendation.

The objective of this study was to determine the minimum requirement (MR) for methionine (Met), when cyst(e)ine (Cys) is provided in excess, in adult dogs of 3 different breed sizes using the indicator AA oxidation (IAAO) technique. In total, 12 adult dogs were used: 1 neutered and 3 spayed Miniature Dachshunds (4.8 ± 0.4 kg body weight (BW), mean ± SD), 4 spayed Beagles (9.5 ± 0.7 kg BW, mean ± SD), and 4 neutered Labrador Retrievers (31.8 ± 1.7 kg BW, mean ± SD). A deficient Met basal diet with excess Cys was formulated. Dogs were fed the basal diet randomly supplemented with different Met-Alanine (Ala) solutions to achieve final Met concentrations in experimental diets of 0.21, 0.26, 0.31, 0.36, 0.41, 0.46, and 0.66% (as fed basis). After 2 d of adaptation to the experimental diets, dogs underwent individual IAAO studies. During the IAAO study day, total feed was divided in 13 equal meals; at the sixth meal, dogs were fed a bolus of L-[1- 13C]-Phenylalanine (Phe), and thereafter, L-[1- 13C]-Phe was supplied with every meal. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples, and total production of CO2 measured using indirect calorimetry. The mean maintenance requirement (MR) for Met and the upper 95% confidence limit (CL) were determined using a 2-phase linear mixed-effects regression model. For Miniature Dachshunds, the MR for Met was between the first 2 dietary Met concentrations and is therefore between 35.7 and 44.1 mg.kg BW -1.d -1 (0.21-0.26%, as fed basis; no requirement could be determined on a metabolic BW basis). For Beagles and Labrador Retrievers, the MR for Met was 57.5 and 50.4 mg.kg BW -1.d -1, 107.7 and 121.8 mg/kg BW ^0.75 as fed basis, and 0.338 and 0.360%, as fed basis, respectively. The upper 95% CL of Met requirements were 77.9, and 72.4 mg.kg BW -1.d -1 or 147.8 and 159.6 mg/kg BW ^0.75 or 0.458, and 0.517%, (as fed basis) for Beagles, and Labradors, respectively. When pooling data from Beagles and Labrador Retrievers, the MR and upper 95% CL were 56.0 and 75.8 mg.kg BW -1.d -1 or 118.4 and 150.5 mg/kg BW ^0.75 or 0.360 and 0.482% (as fed basis). In conclusion, the MR and the upper 95% CL for Met is different for Dachshunds when compared to Beagles and Labrador Retrievers. Using this low protein diet, the estimated upper 95% CL Met requirement for Beagles and Labrador are higher than those recommended in the NRC (2006), but NRC is similar to the estimated upper 95% CL for Dachshunds.