4.2. Materials and Methods
5.2.4. Tissue collection and analytical procedures
By the end of week 6, all lambs were slaughtered in a commercial abattoir by electrical stunning and exsanguination similar to the lamb slaughtered on day 0. Whole livers were collected, weighed and samples (~35 g) were taken and stored at -20°C for subsequent analyses. The weekly concentrate samples were defrosted, bulked, and mixed then subsamples were taken and oven dried at 105°C overnight (Binder, Cole-Palmers, UK) (see Section 2.1.1). Dry feed samples were milled using a Delonghi KG79 grinder (Freemans PLC, Sheffield, UK) and analysed in duplicates for CP, EE, ash, and NDF in
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according to the procedure provided by Association of Official Analytical Chemist (AOAC, 2016) as described in Section 2.1.3 to 2.1.5 respectively.
Mineral concentration of diets was determined after digestion of dried samples with concentrated HNO3 (70%; Fisher Scientific., UK) and HCl (37%; Fisher Scientific., UK), using DigiPREP digestion system (QMX Laboratories Ltd., Essex, UK) and analysed by ICP-MS after diluted in 2% HNO3, 1% methanol, and 0.1% Triton X-100 (Sigma-Aldrich, Dorset, UK) as described by Cope et al. (2009) (see Section 2.6). Dry liver samples were analysed following digestion overnight at 60°C in concentrated HNO3. Plasma mineral concentration was determined after sample dilution (1:20) in 0.5% HNO3 (Cope et al., 2009) (see Section 2.8). Liver and hay reference samples were used to monitor consistency and reliability of specific minerals concentration (European Commission, Rerieseweg, Belgium).
Experiment 2:
In experiment 2, 56 store wethers (28 Texel cross and 28 Swaledale, with an initial BW of 28.2 ± 2.9 kg) were used in a 2 x 2 factorial design experiment. Prior to the commencement of the experiment, 8 lambs per breed were randomly selected and slaughtered, using the same procedure of experiment 1, to assess the initial liver mineral contents of lambs (first slaughter group) (Table 5.3). The 40 remaining lambs were blocked by LW and breed, and then randomly assigned to one of two dietary treatments, 10 lambs per treatment, for 10 weeks. All lambs were housed individually, in metal pens, and bedded on wood shaving in a good ventilated barn throughout the study.
Table 5.3. Liver mineral concentration of the first slaughter lamb group (mg/kg) (n=16 ±SD)
Mineral Swaledale Texel P-value
Cu 265 ± 90.7 159 ± 91.2 0.04 Fe 415 ± 91.6 369 ± 123.2 0.41 Mn 49.9 ± 24.3 34.7 ± 8.9 0.12 Mo 4.3 ± 0.5 3.4 ± 0.5 <0.01 Zn 215 ± 55.1 233 ± 63.8 0.55
5.2.5. Diet formulation
The raw feed ingredients used in the current experiment were similar to those used in experiment one except grass pallet nuts were used instead of NIS (Table 5.4). Supplemental feed grade urea (Trouw Nutrition, Northwich, Cheshire, UK) was used to balance N levels in all diets based on N proportion from (NH4)2SO4 inclusion to raise the
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rumen degradable nitrogen. The diets were mixed on farm using the similar procedure used in experiment 1. The basal diet was formulated to supply adequate level of Cu (NRC, 2007) required for a lamb weighing 30 kg to grow at a rate of 200 g/d (AFRC, 1993). The lambs were fed one of the following diets:
Control: Basal diet.
Fe +: Basal diet supplemented with 368.7 mg Fe/kg DM to obtain a total 800 mg Fe/kg DM.
Lamb groups have fed one of the following dietary treatments:
SW - Swaledale lambs given control diet
SW + Swaledale given Fe supplemented diet
T - Texel lambs given control diet diet
T + Texel lambs given Fe supplemented diet
Supplemental feed grade urea (Trouw Nutrition, Northwich, Cheshire, UK) was used to balance N levels in all diets based on N proportion from (NH4)2SO4 inclusion to raise rumen degradable nitrogen.
Table 5.4. Diet formulation and chemical composition of the basal diet (study 2)
Ingredient Basal diet (g/kg) Fe supplemented (g/kg)
Dried grass nuts 500 500
Barley grain 200 200
Sugar beet pulp 85 85
Soybean meal 110 110 Molasses 50 50 Megalac 30 30 Mins/vits premix 1 25 25 Chemical compotation (g/kg DM) DM (g/kg fresh) 934.2 935.8 CP 162.4 174.2 EE 24.0 26.6 NDF 406.8 403.3 Ash 89.1 98.5 ME (MJ/kg DM)2 11.14 11.1 MP (g/kg DM)2 93.82 93.8 Mineral concentration (mg/kg DM) Cu 13.6 12.2 Fe 532.4 968.2 S (g/kg) 2.9 2.8 Mn 78.1 58.89 Mo 2.1 2.2 Zn 83.8 78.8
1Mineral premix (25 kg/ ton) (RUMENCO LTD., Burton upon Trent, UK) containing 320,000 IU/kg Vit A, 100,000 IU/kg Vit
D3, 2,000 IU/kg Vit E, 18.5% calcium, 2.0% phosphorous, 1.0% magnesium, 12.0% sodium, 25% chloride, 20 mg/kg selenium, 90 mg/kg cobalt, 150 mg/kg iodine, 3000 mg/kg manganese, and 3000 mg/kg zinc.
2
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5.2.6. Experimental routine
Lambs were fed at a restricted level, twice a day, at 08:00 and 16:00 h, using a metric scale to support a predicted LWG of 200 g/day (AFRC, 1993). Daily feed was offered in individual clean plastic buckets. Feed samples were collected weekly and stored at -20°C for subsequent chemical analyses. Water was available ad libitum. Feed refusals, if present, were recorded twice a week. The LW of lambs was recorded weekly using a weight crate (Shearwell Data Ltd., Somerset, UK). DLWG was calculated by regression analysis based on the weekly LW. The weekly live weights of lambs were used to calculate the daily feed offered in the following week to achieve the targeted DLWG. of 200 g/d (AFRC, 1993).
5.2.7. Blood sampling and analysis
Jugular blood was taken weekly, on a Thursday, at 11:30 h for plasma, serum, and fortnightly for the whole blood. Blood was collected in vacutainer coated with K2EDTA designed for trace mineral analysis (Becton Dickinson Vacutainer systems, Plymouth, UK) (see Section 2.2). Whole blood samples were collected in 4.0 ml vacutainer, coated with K2EDTA (Becton Dickinson Vacutainer systems, Plymouth, UK) and run on Vet Haematology Analyser device (Woodley Equipment Co Ltd., Bolton, UK) as described in Section 2.3. Subsamples of whole blood were also taken to determining SOD activity using a Cobas Mira Plus auto-analyser (ABX Diagnostics, Bedfordshire, UK) as described in Section 2.4. Vacutainers designed for serum collection (silica coated) were stored overnight at 4°C before being centrifuged as described in Section 5.2.3. Serum was used for determining Cp activity based on the method of Henry et al. (1974), using Cobas Mira Plus auto-analyser (ABX Diagnostics, Bedfordshire, UK), as described in Section 2.5.
5.2.8. Tissue collection and analytical procedures
By the end of week 10, all lambs were slaughtered in a commercial abattoir using a similar procedure used for the group of lambs slaughtered at day 0. Liver weights were recorded and samples (~ 35 g) were taken and stored at -20°C for further analysis. Weekly concentrate samples were bulked and oven dried as described in Section 2.1.1. Dry feed samples were milled using a Delonghi KG79 grinder (Freemans PLC, Sheffield, UK) and analysed in duplicate for CP, EE, ash, and NDF according to AOAC (2007) as described in Sections 2.1.3, 2.1.4, 2.1.2, and 2.1.5, respectively. Concentrations of mineral in feed was determined as described in Section 5.2.4.
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5.2.9. Data analysis
Weekly live weights, blood components, plasma minerals, Cp, Cp:Pl-Cu ratio and SOD activity were analysed by repeated measures analysis of variance with week 0 as a covariate for plasma mineral using GenStat 17th edition (VSN Int. Ltd., Hempstead. UK). All data were analysed by a two way ANOVA as a 2 x 2 factorial design experiment with breed of sheep and dietary treatments being the main factors. Week 0 served as a covariate for plasma mineral concentrations analysis. The significance difference between means was determined using the protected least significant difference (LSD) (Snedecor and Cochran, 1989).
5.3. Results
Experiment 1:
Collection of livers was problematic in the abattoir setting so this has been excluded from the statistical analysis due to unreliable sample identification post slaughter. The liver Cu, Mn, Mo, and Zn content of lambs slaughtered on day 0 were not significantly different between lambs of both breeds (Table 5.1). Texel lambs of the first slaughter group had higher liver Fe concentration compared with Scottish Blackface lambs; however, there was no significant difference between breeds (659.0 vs. 301.0 mg/kg DM).