Materials and methods

All procedure involving animals were carried out according to the UK Animals (Scientific Procedures) Act 1986 and were approved by Harper Adams University Ethic Committee.

3.2.2. Animals and experimental design

A study was carried out at Harper Adams University (at 20th _{of June 2014) using 40 Texel-} cross breed lambs with an initial mean body weight of 28.3 kg (s.e.d; 0.65) over a period of 8 weeks. Eight representative lambs were slaughtered immediately prior to the start of the study in a commercial abattoir, and liver samples were collected and stored at -20 °C prior to serve as a baseline for liver Cu levels. The remaining 32 lambs (male 12 and female 20) were blocked according to liveweight (LW) and sex, and then randomly allocated to one of four treatments, with eight lambs per treatment. The lambs were housed in a well-ventilated shed in individual pens and bedded on wood shavings. They had free accessed to water.

3.2.3. Diets

Lambs were fed a diet with a forage to concentrate ratio of 60:40 (DM basis) to meet their requirements to grow at 200 g/day (AFRC, 1993). The forages were either dried grass pellets (DGP) (Graze-on grass Pellets, Northern Crop Driers, Melrose Farm, Melbourne, York, UK) or maize silage (MS), which was made at Harper Adams University. Appropriate concentrates were formulated to produce isonitrogenous, isoenergetic diets (Table 3.1). The predicted metabolisable energy (ME) for experimental diets DGP and MS (60:40) was 11.60 and 11.70 MJ/kg DM respectively (AFRC, 1993).

Table 3.1. Raw material composition of the experimental concentrates (g/kg DM) Ingredients, g/kg DM Concentrate Diets1 DGP MS Barley 643 256

Sugar beet pulp (Shreds) --- 125

Soya bean meal 192 487

Molasses 73 74

Megalac 35 ---

Mins/vits1 _{57 58}

Total 1000 1000

1 _{DGP= concentrate fed with dried grass pellets forage, MS= concentrate fed with maize silage.} 2 _{Mineral premix (25 kg/tonne)(Wynnstay Group P.L.C., Powys, Llansantffraid, UK). Major minerals} (g/kg DM): Calcium, 236.2; Phosphorous, 20; Magnesium, 80; Sodium, 49.2. Trace elements (mg/kg DM); Iron, 3226; Iodine, 630; Cobalt, 120; Manganese, 8065; Zinc (chelates of amino acids), 2000; Zinc (oxide), 8057; Selenium (sodium selenite), 75.6; Selenium (Selenised yeast inactivated), 500. Vitamins; Vit A {E 672}, 400000 IU/kg; Vit D3 {E 671}, 80000 IU/kg. Vit E (all-rac-alpha-tocopheryl acetate) {3a700} 1500 mg/kg. Vit B12 cyanocobalamin 500 mcg/kg.

Chemical composition and mineral concentration of the forages and concentrates were chosen based on predicted values of MAFF (1992). Basal diets (DGP and MS; 60:40; forage: concentrate ratio at DM basis) were predicted to supply 2.86 g/kg DM of S and 2.85 mg/kg DM of Mo. To evaluate the effect of antagonists on Cu metabolism, lambs received diets that were either unsupplemented (-) or supplemented (+) with Mo at 3.15 mg/kg DM as ammonium molybdate (NH4)6Mo7O24·4H2O (Fisher Scientific, Leicester, UK), and S (1g/kg DM) as ammonium sulphate (NH4)2SO4 (Alfa Aesar., Ward Hill, USA) to result in reducing Cu absorption by 50% (Suttle and MacLauchlan, 1976). The N content of the diets were balanced by the addition of 0.418 kg/tonne DM as feed grade urea (Trouw Nutrition, Cheshire, UK). Lambs were allocated by liveweight and sex to one of four dietary treatments (Table 3.2).

Table 3.2. Dietary treatments

Code Treatments

DGP- 0.60 dried grass pellets:0.40 concentrate (DM basis), no addition antagonists DGP+ 0.60 dried grass pellets:0.40 concentrate (DM basis), with additional Mo and S MS- 0.60 maize silage:0.40 concentrate (DM basis), no addition antagonists

Feed samples (forage and concentrates) were collected once weekly throughout the study. Then, at the end of the study, all feed samples were analysed for DM, Ash, CP, EE, NDF, and mineral contents as described in sections 2.1.1. to 2.1.5, and section 2.4.1 respectively. The chemical composition of the experimental diets are presented in Table 3.3.

Table 3.3. Analysed chemical and mineral composition of the experimental diets supplying 600 g/kg DM forage and 400 g/kg DM concentrates (60:40)1_.

Item DGP- DGP+ MS- MS+ Chemical composition, g/kg DM DM, g/kg 899.1 899.5 559.2 559.6 CP, 166.7 166.3 146.4 142.1 EE, 22.1 23.0 20.1 20.9 NDF, 302.7 298.8 283.4 283.9 Ash, 85.2 87.9 68.0 66.2 Mineral composition, mg/kg DM Cu, 9.3 9.5 7.9 7.8 Mo, 1.9 4.6 2.7 4.8 S, g/kg DM 3.7 4.3 3.5 3.9 Fe, 458.5 462.1 263.3 256.5 Zn, 214.1 210.2 196.1 190.3 Mn, 198.9 193.7 155.7 158.2

1 _{Diets consists of either dried grass pelleted (DGP) + concentrate or maize silage (MS) + concentrate} at a ratio of 60:40 forage: concentrate. Diets DGP+ or MS+ received additional Mo and S, resulting in Mo ent 5 mg/kg DM and S content 4 g/kg DM.

3.2.4. Experimental routine

All lambs were offered fed twice a day at (08:30 and 16:30h). Forages (dried grass pellets and maize silage) were put into wooden troughs, and concentrates placed into plastic buckets. Feed refusals were collected twice a week (every Monday and Friday until the end of experiment) to estimate individual feed intake and feed conversion efficiency. The quantity of both diets offered was adjusted weekly according to the liveweight of the animal taken on the day of liveweight determination (section 3.2.4.2) to meet AFRC (1993) requirement. At the end of the study, lambs were sent to a commercial abattoir for slaughter. All lambs, including the eight representative lambs on day 0, were slaughtered following electrical stunning. Livers were collected immediately after slaughtering, weighed, and stored at -20°C for subsequent mineral content determination.

3.2.4.1. Blood sample collection

Blood samples were collected by jugular vein puncture (section 2.2.) once a week on a Tuesday at 11:00h for plasma and serum (sections 3.2.5). On weeks 0, 4, and 8 an additional EDTA tube was collected for haematology analysis and an aliquot stored at -20°C for SOD analysis (section 3.2.5). Blood samples (week 0) were collected at 20th _{of June} 2014.

3.2.4.2. Liveweight determination

Lambs were weighed once a week on Friday at 11:00 using Standard Operating Procedure as described in section 2.3. Daily liveweight gain (DLWG) was calculated using regression analysis.

3.2.5. Blood analysis

Fresh blood samples after being collected were analysed immediately for haematocrit (Hct), haemoglobin concentration (Hb), red blood cell counts (RBC), and while blood cell counts (WBC) using using a Vet Animal Blood Counter (section 2.2.1). Frozen samples of whole blood, plasma, and serum were defrosted thoroughly at room temperature. Whole blood samples were analysed for SOD activity using a Cobas mira plus as described in section 2.2.3.1. Plasma samples were used to determine mineral concentrations (section 2.2.2), and trichloroacetic acid soluble concentration (section 2.2.2.1). Blood serum samples were also analysed for ceruloplasmin activity (Cp) using a Cobas mira plus (section 2.2.3.2).

3.2.6. Liver mineral analysis

Liver samples were analysed for mineral concentrations using an ICP-MS as described in section 2.4.2. Whole liver minerals content was determined by multiplying liver mineral concentrations by liver weight and liver DM. Liver minerals retention was determined by substracting whole liver minerals content of the initial slaughter group from final whole liver mineral content and divided by days of the whole study period, which was 8 weeks.

3.2.7. Rumen pH determination

Rumen fluid samples were collected immediately after slaughter of the lambs, put into 100 ml plastic pots and stored on ice prior to measuring pH within an hour after slaughtering (section 2.1.6).

3.2.8. Statistical analysis

Performance, plasma minerals, haematology, and enzyme activities were analysed by repeated-measures ANOVA as a 2x2 factorial randomise block design with the main effects of forage type (F), antagonists (Ant.), and interaction between forage type and antagonists (Int.). Daily Liveweight gain (DLWG) was calculated by regression analysis and analysed by ANOVA. For plasma Cu concentration, Cp activity, Cp:Pl-Cu ratio, SOD activity, haemoglobin concentration, WBC counts, week zero was used as a covariate. All statistical analysis were conducted using Genstat version 17.1 (Lawes Agricultural Trust, VSN International Ltd, Oxford, UK). Significance was set at P < 0.05 and trends at P < 0.10. Significant differences between means were tested using the protected least significant difference (LSD) (Snedecor and Cochran, 1989).