Semen Processing:Semen samples were diluted with
TRIS extender and used as control and other aliquots of
pooled semen samples were diluted with TRIS extenders
containing the different concentrations of honey bee
solution in order to provide concentration of 60 million
sperm/ml. Extended semen was slowly cooled
(approximately for 2 hrs) to 5°C and equilibrated for 2 hrs.
Semen was packed into 0.25 ml polyvinyl French straws.
After equilibrium periods, the straws were horizontally
placed on a rack and frozen in a vapour 4 cm above liquid
nitrogen for 10 minutes and were then dipped in liquid
nitrogen. A fraction of extended semen from control and
each concentration of the additives was kept at 5 °C for 7
days (chilling) and sperm motility was evaluated daily.