Total fatty acid composition of spermatozoa and seminal plasma
was investigated in fresh and in stored semen. Five semen samples
were used. Washed spermatozoa from the 5 samples were re-diluted
in seminal plasma from the same males at sperm densities of 5–
7 × 107 cells ml−1 to obtain a final volume of 5 ml. Low sperm
densities were used to avoid sperm death due to anoxic conditions
(Lahnsteiner et al., 2004). Sperm suspensions were stored in 10 ml
centrifuge tubes at 4 °C for 48 h. A 2.5 ml subsample was taken
immediately after dilution and served as a control while the other
subsample was taken after 48 h. Immediately after collection, each
subsample was centrifuged at 300 × gravity for 10 min. Lipids were
extracted from seminal plasma and spermatozoa into a chloroform:
methanol mixture (2:1/v:v) using the procedure of Bligh and Dyer
(1959). After extraction, the extraction solution was evaporated
under a stream of nitrogen and the lipid was stored at −20 °C until
analysis