Alliance 2690

After homogeneous mixing, about 250 g of feed samples from each treatment diet in each period were collected in triplicate. All feed samples were dried in a 70°C oven for 72 h. Then, feed samples were ground and sieved with a 1‐mm sieve. Feed composition analyses were conducted on these feed samples. According to the procedure established by the AOAC (2000 ), the dry matter (method 930.15), crude protein (nitrogen × 6.25; method 968.06), crude fat (method 954.02), crude ash (method 942.05), calcium (method 984.01), phosphorus (method 965.17), and crude fibre (method 991.43) levels in the diet were analyzed. Then, the representative feed samples in each group were hydrolyzed with 6 N HCl for 24 h at 110°C. An amino acid analyzer (2690 Alliance, Waters, Inc., Milford, MA, USA) was used for the determination of amino acid contents in the diet. Energy in feed was measured by a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA). Phytate‐P in raw materials and diets was determined using the method described by Reichwald and Hatzack (2008 (link)). Absorbance was determined using a Media spectrophotometer (Marcel Lamidey S.A., Châtillon, France) at a 519 nm wavelength. Sodium was determined in accordance with AOAC (2005 ) using microwave plasma‐atomic emission spectrometry (4100 MP‐AES; Agilent Technologies, Santa Clara, USA).

The major flavonoids viz. pinocembrin, chrysin and galangin have been identified and quantified in the hydroalcoholic extract of propolis using Waters 2690 Alliance High performance liquid chromatography (HPLC) system equipped with a Waters 996 photodiode array detector (PDA). A Kromasil Eternity reversed phase column (C₁₈) was used with specifications (4.6 mm, 250 mm, 5μm particle size). Elution of major flavonoids in the propolis extract was achieved using 0.1% formic acid in water as solvent (A), and methanol 100% as solvent (B). The total run time was 65 min where gradient elution was adopted. The elution run started by 80% of solvent (A) 0–5 min, 40% (5–45 min), then 20% (45–60 min), then finally back to 80% (60–65 min). The flow rate was 1 mL/min with injection volume 10 μL. The UV detection was set at 290 nm for all flavonoids [18 (link)].

After homogeneous mixing, feed samples were collected from each dietary group. All feed samples were dried in a 70°C constant temperature oven for 72 h. Subsequently, feed samples were ground and sieved with a 1-mm sieve. According to the procedure established by the Association of Official Analytical Chemists (AOAC, 2000), the dry matter (method 930.15), crude protein (nitrogen × 6.25; method 968.06), calcium (method 984.01), and crude fiber composition (method 991.43) in the diet were analyzed. Then, the representative feed samples in each group were hydrolyzed with 6 N HCl for 24 h at 110°C. An amino acid analyzer (2690 Alliance, Waters, Inc., Milford, MA, United States) was used for determining amino acid content in the diet. In addition, the contents of neutral detergent fiber and acid detergent fiber in the diet were measured according to the method provided by Mertens (2002).

After homogeneous mixing, feed samples were collected from each dietary group. All feed samples were dried in a 70°C constant temperature oven for 72 h. Subsequently, feed samples were ground and sieved with a 1-mm sieve. The collected feed powder is with a diameter of <1 mm for feed composition analysis. According to the procedure established by the AOAC (14 ), the dry matter (method 930.15), crude protein (nitrogen × 6.25; method 968.06), and crude fiber (method 991.43) composition in the diet were analyzed. Then, the representative feed samples in each group were hydrolyzed with 6 N HCl for 24 h at 110°C. An amino acid analyzer (2690 Alliance, Waters, Inc., Milford, MA) was used for determining amino acid contents in the diet. In addition, the contents of neutral detergent fiber and acid detergent fiber in the diet were measured according to the method provided by Mertens (15 (link)).

After homogeneous mixing, about 250 g of feed samples from each treatment diet in each period were collected in triplicate. All feed samples were dried in a 70°C oven for 72 h. Then, feed samples were ground and sieved with a 1-mm sieve. Powder feed samples were collected for feed composition analysis.
According to the procedure established by the AOAC [21 ], the dry matter (method 930.15), crude protein (nitrogen ×6.25; method 968.06), crude fat (method 954.02), crude ash (method 942.05), calcium (method 984.01), phosphorus (method 965.17), and crude fiber (method 991.43) composition in the diet were analyzed. Then, the representative feed samples in each group were hydrolyzed with 6 N HCl for 24 h at 110°C. An amino acid analyzer (2690 Alliance; Waters, Inc., Milford, MA, USA) was used for determining amino acid contents in the diet. Energy in feed was measured by a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA). Phytate-P in raw materials and diets was determined using the method described by Reichwald and Hatzack [22 (link)]. Absorbance was determined using a Media spectrophotometer (Marcel Lamidey S.A., Châtillon, France) at a 519 nm wavelength. Sodium was determined in accordance with AOAC [23 ] using microwave plasma-atomic emission spectrometry (4100 MP-AES; Agilent Technologies, Santa Clara, USA).

In vitro release of CTS nanocrystals was performed using dialysis against 1% sodium dodecyl sulfate (SDS) in 20 ml pH progressive dissolution media (HCl buffer pH 1.2 for 2 h followed by phosphate buffered saline (PBS, pH 6.8). Lyophilized nanocrystals (1 mg CTS) were redispersed in buffers and placed in a dialysis bag (MWCO 14 kDa). Dialysate was collected and analyzed by HPLC (Alliance 2690; Waters, Milford, MA, United States). The detection conditions were referred to a published validated method with minor modifications as follows: Diamonsil C₁₈ column (250 × 4.6 mm, 5 µm); flow rate 1 ml/min, wavelength 269 nm. The mobile phase was methanol:water (75:25), column temperature 30°C. Linearity was good in the range of 0.75–50 μg/ml (y = 36,612x+13,691, R² = 0.9945) (Chu et al., 2014 (link)).