view - Department of Reproduction, Obstetrics and Herd Health

CHAPTER 5.2
The Flemish Veterinary Health Service (Dierengezondheidszorg-Vlaanderen, Torhout,
Belgium). All blood analyses were carried out on serum derived from the unheparinised,
silicone coated tubes except the one for glucose determination (NaF tubes). Milk protein
(MP) and MF content were analyzed by Fourier Transform Infrared spectrophotometry
(Lactoscoop FTIR Advanced, Delta Instruments, Drachten, The Netherlands). The fat and
protein corrected milk (FPCM) was calculated as [(0.337 + 0.116 x MF % + 0.06 x MP %)
x kg of milk] (CVB, 2007).
FATTY ACID ANALYSIS
Fatty acids in dietary components (200 mg freeze dried material), BP (EDTA
tubes) and FF (200 µL) were methylated by a base-catalyzed followed by an acidcatalyzed
step. Toluene (2 mL) containing the internal standard (13:0) and methanolic
NaOH (2 mL) was added and the mixture was incubated at 70°C (60 min) followed by 30
min at 50°C after addition of methanolic HCl (3 mL), prepared by dissolving 10 mL
acetyl chloride in 50 mL methanol. Fatty acid methyl esters (FAME) were extracted with
hexane. For feed samples, an aliquot of the hexane extract was directly used for injection.
Extracted FAME from BP and FF were dried under N2 and redissolved in 200 µL of
hexane prior to injection. Milk lipids were extracted as described by Chouinard et al.
(1997b) and FAME prepared as described by Stefanov et al. (2010).
To assess FA composition of lipid fractions in BP, plasma lipids (2 mL) of samples
taken on d -7, 14 and 46 were extracted with methyl-tert-butyl-ether as described by
Matyash et al. (2008). BP lipids fractions were separated using SPE-columns (Burdge et
al., 2000; Pinkart et al., 1998). Total plasma lipid extracts were dissolved in chloroform
(1.0 ml) and applied to an aminopropyl silica column (Pasteur pipette containing 100
mg aminopropyl silica gel) under gravity. Cholesteryl-esters (CE) and triacylglycerols
(TAG) were eluted with chloroform (1.0 mL and 0.5 mL), combined, dried under N2 and
dissolved in 1.0 mL hexane. NEFA were eluted with diethyl ether/acetic acid (100:2; 1.0
mL and 0.5 mL) and phospholipids (PL) with 1 mL methanol/chloroform (6:1) followed
by 0.5 mL 0.05M sodium acetate in methanol/chloroform (6:1). CE and TAG were futher
separated on a pre-packed 100 mg aminopropyl column (Bond Elut-NH2, Varian Medical
Systems Belgium, Diegem, Belgium). The CE and TAG fraction was loaded in 1 mL
hexane and CE were eluted with hexane (1.0 mL and 0.5 mL). TAG were eluted with
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