one from Select Sires, Plain City,
OH, USA), and two of the bulls used for Dairy 1 were used
for Dairy 2 (one each from XY Inc. and Select Sires). Semen
was thawed, no more than two straws at a time, in a 37 C
water bath for 30 seconds. The X-sorted spermwas thawed,
layered on top of a mini-Percoll gradient (500 mL 45% Percoll
over 450 mL 90% Percoll in a 1.5-mL conical tube), and
the suspension was centrifuged at 600 g for 10 minutes.
The supernatant was removed with a pipette, and 1 mL
HEPES-CDM-1 (H-CDM-1) was added to resuspend the
sperm pellet. The tubewas then centrifuged at 300 g for 5
minutes and the supernatant removed to leave a pellet and
small volume of 50 to 100 mL. The sperm concentrationwas
then determined using a hemacytometer, and sperm was
diluted to 10 106 sperm per mL so that the final
concentration in the fertilization drop would be 2 106
sperm per mL. The fertilization drop was constituted with
30 mL of X-sorted sperm, 25 to 35 COC in 10 mL, and 110 mL
F-CDM in a 60-mm dish under mineral oil.