3.3.1. Dry matter (DM)
For the determination of DM, feed samples were samples collected directly from the feed supplied to the cows at feeding time. The DM content of the diet was determined according to the Association of Official Analytical Chemists (AOAC, 2012; 934.01). An accurate weight of the sample was oven dried (Bider, Coel-Palmers, UK) overnight at 105°C to constant weight. A desiccator was used to cool down the samples after being taken from the oven and reweighed. The DM was calculated according to the following equation:
DM (g kg) = Weight of dried sample (g)
Weight of fresh sample (g) × 1000
3.3.2. Crude protein (CP)
Crude protein concentration in the samples was determined according to (AOAC, 2012; 990.03). An element auto LECO FP 528 N analyser (Leco Corporation, Stockport, UK) was used to determine CP concentration in the samples by combustion method. Dried milled samples were accurately weighed (150 mg) onto aluminium foil. They were then placed into the auto analyser, and the CP content of the feed was calculated according to the following equation:
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3.3.3. Neutral detergent fibre (NDF)
The concentration of NDF of dried feed was measured using a Fibertec apparatus (Tecator Fibretec 1020 Hot Extractor, FOSS, Warrington, UK) in accordance to the methods described by Van Soest et al. (1991). The NDF reagent was previously prepared by mixing 93 g of disodium ethylene diamine tetraacetic acid dihydrate. Approximately 34 g sodium borate, 150 g sodium lauryl sulphate and 50 mL of tri-ethylene glycol with 22.8 g anhydrous di-sodium hydrogen phosphate were mixed and dissolved in distilled water then diluted to 5 litres with pH adjusted from 6.9 to 7.1.
Approximately 2.8 g of α-amylase enzyme from Bacillus subtilis spp (Sigma, Gillingham, UK) was dissolved in 90 mL of distilled water then 10 mL of tri-ethylene glycol was added for preparation of the α amylase solution.
Dried ground feed samples (0.5 to 0.6 g) was accurately weighed into previously dried and weighed crucible (sinter porosity 1, Suham Scientific, Ely, UK). The crucibles were placed into Fibretech® 1020 hot and 1021 cold extractor (Foss UK Ltd, Cheshire, UK) then about 25 mL of the cold neutral detergent reagent with a few drops of alcohol reagent grade (Sigma, Aldrich, Dorset, UK) were added to each of the crucibles. The samples were heated until starting of boiling; then the heat was reduced for about 30 min for digestion of samples. Following this the heat was switched off. Approximately 2 mL of α- amylase solution with an additional 25 mL of cold neutral detergent reagent were added, the samples were digested by boiling for an extra 30 min. Digested samples were then filtered and washed with 20 to 30 mL of hot distilled water (80°C). Additional α-amylase solution (2 mL) and distilled water (25 mL) were added to each sample and allowed to stand for 15 min. Then each sample was filtered and washed with hot distilled water 3 times. Crucibles were removed from the hot,and cold Fibretech® extractor then dried at 105°C overnight. After cooling using desiccator, crucibles were weighed and then placed in a muffle furnace for 4 h at 550°C. The crucible were cooled again to room temperature using the desiccators and NDF determined as follows:
NDF (g) = (crucible + dry fibre weight) – (crucible + ash weight)
NDF (g kg DM) = NDF weight (g)
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3.3.4. Ether extract (EE)
The ether extract content of feed samples was determined using the Soxtec apparatus (HT extraction apparatus, FOSS, Warrington, UK) according to the solvent procedure of Foss (1987). Approximately one gram (g) of dried sample was accurately weighed into a previously weighed cellulose extraction thimble (Warrington plc, Maidstone, UK). The cellulose extraction thimble was plugged with defatted cotton wool and fitted into the extraction unit. A cold petroleum ether (25mL) at 30–40°C (AnalaR, VWR International Ltd, Lutterworth, UK) was added to a pre-weighed extraction cup and placed under the extraction thimbles then boiled for one hour before additional rising 15 min. The final traces of the solvent were evaporated off, and the extraction cups were removed from the apparatus and moved into a fume cupboard. After the extraction cups were cooled, they were reweighed and the EE content determined:
EE (g kg DM) = Weight of fat (g)
Weight of dried sample (g) × 1000
3.3.5. Gross energy
The gross energy (GE) of feed was determined using anadiabatic bomb calorimeter apparatus (Parr 6200 Instrument Company, Moline, iL, 61265, USA). To make pelleted dried ground samples a pellet pressor (Parr 2811 pellet press, Parr Instrument Company, Moline, USA) was used. Pelleted samples were accurately weighed then placed into a crucible. Approximately 10 cm of fuse wire was inserted through the holes of the bomb, connected to both heads of the bomb without contact between the wire and the pelleted sample. The bomb was assembled and filled with O2 gas pressure, then placed into a bucket containing water (2L). The energy content of the sample was measured by burning the sample in the bomb calorimeter under enclosed conditions and a constant volume of O2 gas. The GE produced from the burning was measured as MJ/kg DM.
3.3.6. Ash and organic matter
Ash and organic matter were analysed according to AOAC (ash,AOAC, 2012; 942.05). Approximately 2g of dried and milled feed samples were weighed accurately into a labelled porcelain crucible and ashed by muffle furnace (Gallenkamp Muff Furnace, Size 3, GAFSE 620, Gallenkamp, Loughborough, UK) at 550°C for 4 h. A desiccator was used to cool down the samples and reweighed. The ash content of the feed was calculated according to the following equation:
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Ash (g kg DM) = Weight of ash (g)
Weight of dry sample before ashing (g) × 1000
Organic matter (g/kg DM) content of the feed was calculated as 1000 minus ash content.