The semen was diluted at an ambient temperature (,278C) to a
final concentration of 25 106 spermatozoa per 0.25 mL per
straw and maintained at 48C for 5 h. The cooling curve was
,0.558C min1
. The straws were then frozen using a Digit Cool
machine (IMV, L’Aigle Cedex, France) and plunged into liquid
nitrogen and stored until evaluation. For post-freezing evaluation
or use, the semen straws were thawed in a water bath at 358C
for 20 s. For the cooled semen, the straws were kept in BotuTainer
boxes (Botupharma, Botucatu, Brazil) at 58C for 24 h
until use, without being placed in the water bath before FTAI.