After milk removal, milk was homogenized, and
samples of 250 mL were collected from individual milk
buckets and reserved at 4°C. Samples from 4 consecutive
milkings were composited and centrifuged at 1,000
× g for 10 min at 4°C to separate milk fat. The supernatant
fat was kept in a freezer (−20°C) overnight, and
the transesterification of triacylglycerol was performed
according to the method of the International Organization
for Standardization (ISO, 1978). The methyl esters
were prepared according to method 2301 (IUPAC,
1987) and analyzed by gas chromatograph model 14-B
(Shimadzu, Kyoto, Japan), along with fused silica
capillary column, type Omewax250 (30 m × 0.25 mm
× 0.25 μm) Cat. No. 24136-Supelco, with the following
analytical conditions and programing: 100°C for 2
min; heating 4°C/min up to 220°C and maintaining this
temperature for 25 min; injector temperature of 250°C;
detector temperature of 280°C; carrier gas velocity (H2)