Laboratory Methodology

The heparinised blood samples were kept on ice and within 10 hours of being taken the samples were centrifuged at 5000 rpm for five minutes. The plasma was then pipetted off into separate microcentrifuge tubes before being frozen and stored at -20°C until biochemical analysis could be performed.

The plasma was submitted to a commercial lab and batch-analysed at New Zealand Veterinary Pathology (Palmerston North, New Zealand). The biochemical parameters measured for all samples were CK, AST and glutamate dehydrogenase (GLDH). The plasma CORT concentration was analysed at the Institute of Veterinary, Animal & Biomedical Sciences, Massey University, Palmerston North, New Zealand. The concentration of CORT was measured by radioimmunoassay using a method that is well established for the measurement of CORT in birds (Cockrem et al., 2009; Adams et al., 2010; Cockrem et al., 2012).

2.2.2.1 Determining the concentration of CK in the plasma

The method used to determine the concentration of CK in the samples is derived from the formulation recommended by the International Federation of Clinical Chemistry (IFCC) (Schumann et al., 2002a). The CK assay used is an automated photometric assay using a Roche/Hitachi P800 analyser. The test principle is as follows: substrates (creatine phosphate

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and ADP) and buffer are added to the sample, and the CK in the sample transfers the phosphate group from creatine phosphate to ADP, giving creatine and ATP as products. A second enzyme (hexokinase) is added which converts glucose to glucose-6-phosphate (G6P) using the ATP produced in the first step. A third enzyme (glucose-6-phosphate dehydrogenase) is added which then oxidises the G6P produced in the second step to gluconate-6-phosphate while reducing NADP to NADPH. The rate of formation of NADPH is measured photometrically by the increase in absorbance and is directly proportional to the CK activity in the sample. Extra reagents are also added to activate the CK enzyme if it has been oxidised during sample collection, and to inhibit other enzymes which might otherwise interfere with the test.

2.2.2.2 Determining the concentration of AST in the plasma

The method used to determine the concentration of AST in the samples is an optimised method derived from the formulation recommended by the International Federation of Clinical Chemistry (IFCC) (Schumann et al., 2002b). The method used is an automated photometric assay using a Roche/Hitachi P800 analyser. The test principle is as follows:

substrates (aspartic acid, α-ketoglutarate) and buffer are added to the sample. The AST

present in the sample catalyses the reaction of the substrates into glutamic acid and oxaloacetate. The rate of oxaloacetate formation gives the AST activity in the sample so the increase in oxaloacetate is determined by adding a second enzyme (malate dehydrogenase) which converts the glutamic acid to malic acid while oxidising NADH to NAD. The rate of decrease of NADH is directly proportional to the rate of formation of oxaloacetate, thus photometrically measuring the rate of decrease in NADH gives the AST activity in the sample.

2.2.2.3 Determining the concentration of GLDH in the plasma

The method used to determine the concentration of GLDH in the samples is derived from the formulation recommended by the German Society for Clinical Chemistry (DGKC) (Anonymous, 1972). The GLDH assay used is an automated photometric assay using a Roche/Hitachi P800 analyser. The test principle is as follows: substrates (α-ketoglutarate, NADH, ammonia) and buffer are added to the sample and the GLDH in the sample acts a

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NAD as products. The amount of NADH consumed is measured photometrically by the decrease in absorbance and is directly proportional to the GLDH activity in the sample.

2.2.2.4 Determining the concentration of CORT in blood samples

Corticosterone concentrations in plasma diluted in phosphate buffered saline with gelatine (PBSG) were measured by radioimmunoassay by the method used by Cockrem et al. (2008). Plasma samples were initially spun for 10 minutes at 18, 000×g to separate lipid from the plasma. The clear plasma from below the lipid layer was transferred to another tube and diluted in PBSG for assay. Samples were assayed in duplicate. Ten microlitres of diluted plasma were incubated for 2 hours at room temperature (22–25°C) with iodinated corticosterone and antiserum from a Corticosterone Radioimmunoassay Kit (MP Biomedicals, USA). Precipitant solution (MP Biomedicals, USA) was added and each sample

vortexed thoroughly, then 50 μl egg white (10 g/l dried egg white [Sigma] in PBSG) was

added to increase adhesion of the pellet to the tube after centrifugation. The samples were incubated for 15 minutes at room temperature to separate bound and free corticosterone, and then centrifuged for 15 minutes, and the supernatant aspirated, and the pellets were counted on a LKB Wallac 1261 Multigamma gamma counter.

The sensitivity of the corticosterone assay was the minimum hormone level that could be consistently distinguished from zero. It was determined as the hormone concentration at the mean - 2 standard deviations from the zero hormone point on the standard curves. The assay sensitivity was 0.52 ng corticosterone/ml of plasma.

Solutions of corticosterone in PBSG at concentrations that gave approximately 80, 50 and 20% binding on the standard curve were used as low, medium and high quality controls in every assay. The mean concentrations of corticosterone in these solutions were 234.9+19.8, 574.2 + 47.2 and 1769.5 + 127.7 pg/ml respectively. The intra-assay coefficient of variation for each solution was determined by conducting an assay with twenty duplicates of each solution. The intra-assay coefficients of variation for corticosterone were 8.4%, 6.0% and 7.2% for low, medium and high solutions respectively. Inter-assay coefficients of variation were calculated from duplicates of the solutions included at the beginning and end of each assay. The inter-assay coefficients of variation for ten assays were 7.9%, 8.4% and 11.5% for low, medium and high solutions respectively.

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