Dimensión objetiva: “La dimensión objetiva de dichos derechos radica en que ellos son elementos constitutivos y

All samples were analysed in duplicate at Harper Adams University labs using methods in accordance with AOAC (2016).

2.1.1. Dry matter determination (DM)

Dry matter content of the diets and faecal samples were determined according to Association of Official Analytical Chemists (DM, AOAC; 930.15). Sub samples were accurately weighed and oven dried (Binder, Cole-Palmers, UK) at 105°C overnight or freeze dried (Edwards Modulyo freeze dryer, Sussex, UK) until stable weight. Samples were cooled in a desiccator after being taken from the oven and reweighed. The DM was then calculated:

DM (g

kg)

=

Weight_Weightof_of dried _freshsamplesample

_gg

×1000

2.1.2. Ash and Organic matter determination

Dried and milled feed and faecal samples were analysed for ash and organic matter (OM) according to AOAC (ash, AOAC; 942.05). Approximately 2 g of dried samples were accurately weighed into pre-weighed labelled porcelain crucibles. Samples were ashed in a muffle furnace (Gallenkamp Muffle Furnace, Size 3, GAFSE 620, Gallenkamp, Loughborough, UK) for 4 h at 550°_{C, cooled in a desiccator and reweighed. Ash content} was calculated:

Ash (g kg DM)= Weight of ash g

Weight of dry sample before ashing g ×1000

Equation 2.2.4

39

Organic matter (OM) of the samples were calculated as 1000 minus ash content

(g/kg DM).

2.1.3. Crude protein determination (CP)

Dried feed and faecal samples were analysed according to Association of Analytical Chemists (CP, AOAC, 2016, 968.06) by the use of an element auto analyser LECO FP528 (LECO Corp, Stockport, UK) operating the Dumas method. Approximately 150 mg of dried ground sample was accurately weighed into aluminium foil squares. This was then placed into the auto analyser, CP content of samples were calculated:

CP (g/kg DM) = Nitrogen content (g/kg DM) X 6.25 Equation 2.2.6

2.1.4. Ether extract (EE)

The ether extract content of feed samples was determined according to a solvent method of FOSS (1987) using Soxtec apparatus (HT 1043 extraction apparatus, FOSS, Warrington, UK). Approximately 1 g of dried ground sample was weighed accurately into a cellulose extraction thimble (Whatman Plc, Maidstone, UK). The thimbles were then plugged with de-fatted cotton wool and fitted in the extraction unit. Total fat was extracted in a pre-weighed extraction cup after boiling of the feed samples in 25 ml of cold petroleum ether (Fisher Scientific, UK) for 1 h. Samples were then removed from the solvent and rinsed for an additional 15 min. The final traces of the solvent were evaporated off, and the extraction cups removed from the apparatus and moved into a fume cupboard. After the extraction cups were cooled, reweighed and the EE determined: EE (g kg DM)= Weight of fat g

Weight of dried sample g ×1000

2.1.5. Neutral detergent fibre (NDF)

The neutral detergent fibre (NDF) of dried feed and faeces samples were determined according to the method of Van Soest et al. (1991) using Fibertec apparatus (1020, FOSS, Warrington, UK). The NDF reagent, was previously prepared from mixing 93 g of di-sodium ethylene diamine tetra acetic acid dehydrate (EDTA), 34 g sodium tetra borate (Na2B4O7.10H2O), 150 g sodium dodecyl sulphate (SDS), 50 ml tri-ethylene glycol, and 22.8 g anhydrous disodium hydrogen phosphate (Na 2HPO4) to make 5L solution with distilled water and the pH adjusted to approximately 6.9 to 7.1. Alpha amylase solution Equation 2.2.7

40

was prepared by dissolving 2.8 g of α-amylase (α-1, 4-glucan 4-gluconohydralase, Enzyme # 3.2.1.1 ~80EU/mg) from Bacillus subtilis spp (Sigma, Gillingham, UK) in 90 ml of distilled water followed by the addition of 10 ml of tri-ethylene glycol.

Approximately 0.4 to 0.6 g of dried ground samples was accurately weighed into glass crucibles (porosity 1, Soham Scientific, Ely, UK). Crucibles were tightly fitted onto the Fibretech® _{1020 hot and 1021 cold extractor (Foss UK Ltd, Cheshire,UK) making sure the} valves were in the closed position. Cold neutral detergent reagent (25 ml) followed by a few drops of octanol, reagent grade (Sigma, Aldrich, Dorset, UK) were added to each of the samples. The heat control knobs were turned to full and as samples started boiling, the heat was reduced. Samples were digested for 30 min then the heat was switched off. Another 25 ml of cold neutral detergent reagent and 2 ml of α-amylase solution were added and the samples brought to the boil and digested for an extra 30 min. Samples were then filtered and washed with 20-30 ml of hot distilled water (80°C). A further 2 ml of α-amylase solution and 25 ml of hot distilled water were added to the samples and allowed to stand for 15 min. Samples were then filtered and washed 3 times with hot distilled water, the crucibles removed from the Fibertech® _{hot and cold extractor and dried} overnight at 105°C. After cooling in a desiccator, crucibles were weighed and then placed in a muffle furnace at 550°C for 4 h. Crucibles were then allowed to cool in a desiccator to room temperature and reweighed:

NDF (g) = (crucible + dry fibre weight) – (crucible + ash weight) NDF (g kg DM)= NDF weight (g)

Weight of dried sample g ×1000

2.1.6. Gross energy determination (GE)

To determine the GE of the feed and faecal samples, an adiabatic bomb calorimeter (Parr 6200 Instrument Company, Moline, IL, 61265, USA) was used. Dried ground samples were pelleted using a 2811 Parr pellet press (Parr instrument Co., Moline. USA) and accurately weighed. Pelleted samples were then placed into a crucible. About 10 cm of fuse wire was inserted through the holes of the bomb; care was taken to ensure the wire did not contact with the sample. The apparatus was assembled, filled with O2 gas, and placed in a bucket containing 2 L of water and the wires connected. The bomb calorimeter measured the samples energy content by burning it with O2 under enclosed conditions at a constant volume. The energy produced was measured as MJ/kgDM.

41