Two fish per replication were sampled at random, and
astaxanthin, b-carotene, and total carotenoid were
measured using the following method: the semen of each
sample was collected and frozen using N2 gas, and the
samples were then placed inside an amber flask under an
N2 gas environment at 18 C. The procedure of carotenoid
extraction from the samples was conducted using pure
acetone specific to high performance liquid chromatography
(HPLC) and n-hexane (Hex, analytical grade) based
on a hexane/acetone ratio of 1:3. Afterward, 50 mL was
stored in a glass bottle and wrapped with aluminum foil,
and 5 mL of the hexane–acetone solution was added to the
mixture. The procedure was followed by a 10-minute
agitation of the mixture. The ambient temperature was
22 C under the dim lighting conditions during the
extraction of carotenoid from the sample [27].