We compared the effects of the three aforementioned euthanasia methods on metabolic biomarkers in serum (n = 8 per protocol). All rats were individually handled during the week prior to testing and killed by one experienced person. The rats in the first group were killed by decapitation, using a guillotine. The rats in the second and third groups were killed by CO2 inhalation – 2 min. 30 sec., fixed time and gradually increased concentration – and an overdose of pentobarbital – 120 mg/kg intraperitoneal injection, in a volume of 1 ml/100 g of body-weight – respectively. Blood samples from all groups of animals were collected using a standardized protocol. After decapitation, 1 ml trunk blood was collected at the decapitation site and allowed to coagulate before centrifugation at 1000 × g for 10 min. and the serum was stored at −80°C until analysis.
The analysis of corticosterone and insulin was conducted using the Coat-A-Count Rat Corticosterone 125I RIA kit (Siemens Medical Solutions, Los Angeles, CA, USA) and the Mercodia Rat Insulin ELISA (Mercodia, Uppsala, Sweden) following the instructions of the manufacturer. Triglycerides, cholesterol and glucose were analysed with enzymatic colorimetric methods using an automated chemistry analyser Architect c4000 (Abbott Diagnostics, Lake Forest, IL, USA). FFAs were extracted from serum by protein precipitation and quantified by mass spectrometry. In short, 10 μl of serum was added to an equal volume of an internal standard mix (2H2-16:0, 13C16-16:1n-7, 2H2-18:0, 2H2-18:1n-9, 2H4-18:2n-6, 2H6-20:3n-6, 2H8-20:4n-6, prepared in methanol), and 80 μl of methanol. Samples were vortexed, and precipitated proteins were removed by centrifugation. Supernatants were diluted in 10 volumes of methanol in glass autosampler vials, and immediately quantified by liquid chromatography–tandem mass spectrometry as previously described [9 (link)]. Twelve FFA – myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid – were analysed. The analytes were separated on a Kinetex 2.6-μm core shell pentafluorophenyl column (100 × 2.1 mm, 100 Å; Phenomenex, Macclesfield, UK) using a Prominence UFLCXR system (Shimadzu, Milton Keynes, UK), and detected by ‘pseudo-molecular’ scheduled multiple reaction monitoring transition on a QTRAP 5500 hybrid triple quadrupole mass spectrometer (AB Sciex, Warrington, UK). Analyst software version 1.5.1 (AB Sciex) was used for data acquisition and analysis.
The analysis of corticosterone and insulin was conducted using the Coat-A-Count Rat Corticosterone 125I RIA kit (Siemens Medical Solutions, Los Angeles, CA, USA) and the Mercodia Rat Insulin ELISA (Mercodia, Uppsala, Sweden) following the instructions of the manufacturer. Triglycerides, cholesterol and glucose were analysed with enzymatic colorimetric methods using an automated chemistry analyser Architect c4000 (Abbott Diagnostics, Lake Forest, IL, USA). FFAs were extracted from serum by protein precipitation and quantified by mass spectrometry. In short, 10 μl of serum was added to an equal volume of an internal standard mix (2H2-16:0, 13C16-16:1n-7, 2H2-18:0, 2H2-18:1n-9, 2H4-18:2n-6, 2H6-20:3n-6, 2H8-20:4n-6, prepared in methanol), and 80 μl of methanol. Samples were vortexed, and precipitated proteins were removed by centrifugation. Supernatants were diluted in 10 volumes of methanol in glass autosampler vials, and immediately quantified by liquid chromatography–tandem mass spectrometry as previously described [9 (link)]. Twelve FFA – myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid – were analysed. The analytes were separated on a Kinetex 2.6-μm core shell pentafluorophenyl column (100 × 2.1 mm, 100 Å; Phenomenex, Macclesfield, UK) using a Prominence UFLCXR system (Shimadzu, Milton Keynes, UK), and detected by ‘pseudo-molecular’ scheduled multiple reaction monitoring transition on a QTRAP 5500 hybrid triple quadrupole mass spectrometer (AB Sciex, Warrington, UK). Analyst software version 1.5.1 (AB Sciex) was used for data acquisition and analysis.
