Soxtec extraction unit

The experimental food and fecal samples were dried in a freeze dryer for 72 h before further analysis. For DM determination, the samples were dried at 103 °C for 16 h, followed by cooling in a desiccator before weighing [33 ]. Ash was determined by incinerating the dried samples in an oven at 550 °C for three hours and then cooling in a desiccator before weighing. Total nitrogen was determined according to the Kjeldahl method [34 ], using a 2020 digester and a 2400 Kjelltec analyzer (FOSS Analytical A/S, Hilleröd, Denmark), and crude protein was calculated as N × 6.25. Crude fat was analyzed according to Commission Directive EC/152/2009 [35 ] using a Soxtec extraction unit (FOSS Analytical A/S, Hilleröd, Denmark). Crude fiber was analyzed by boiling a sample first in H₂SO₄ solution and then in KOH solution [36 ]. Neutral detergent fiber was analyzed as previously described [37 (link)]. Gross energy (GE) was measured on a Parr isoperobol Bomb Calorimeter 6300 (Parr Instrument Company, Moline, Illinois, USA). Titanium dioxide was analyzed according to Short et al. [38 (link)] and ATTD was calculated as: ATTD (%) = 100-[(% TiO₂ in food/% TiO₂ in feces) × (% Nutrient in feces/% Nutrient in food) × 100] [26 (link)].

The dog food samples were analyzed without pre-drying. To determine dry matter (DM) content, the samples were dried at 103 °C for 16 h, and then placed in a desiccator to cool before weighing³⁶ . For ash determination, samples were incinerated at 550 °C for three hours and then cooled in a desiccator before weighing. The Kjeldahl method³⁷ was applied to determine nitrogen content, using a 2020 digester and a 2400 Kjeltec analyzer (FOSS Analytical A/S, Hilleröd, Denmark). Crude protein was then calculated as N × 6.25. Crude fat was analyzed in accordance with Commission Directive EC/152/2009³⁸ , on a Soxtec extraction unit (FOSS Analytical A/S, Hilleröd, Denmark). Crude fiber was analyzed as previously described³⁹ . Gross energy (GE) was measured on a Parr isoperobol Bomb Calorimeter 6300 (Parr Instrument Company, Moline, Illinois, USA). Total dietary fiber (TDF) as well as soluble and insoluble dietary fiber were analyzed in accordance with AOAC 991.43 using a Total Dietary Fiber Assay Kit (Megazyme, Bray, Ireland), while resistant and non-resistant starch were analyzed according to AOAC 2002.02 using a Resistant Starch Assay Kit (Megazyme, Bray, Ireland).