Jj. Parrish et al., EFFECT OF BOVINE SPERM SEPARATION BY EITHER SWIM-UP OR PERCOLL METHODON SUCCESS OF IN-VITRO FERTILIZATION AND EARLY EMBRYONIC-DEVELOPMENT, Theriogenology, 44(6), 1995, pp. 859-869
The objectives of these experiments were to characterize separation of
frozen-thawed bovine spermatozoa on a Percoll gradient and then to co
mpare sperm separation by either a swim-up or Percoll gradient procedu
re for the ability of spermatozoa to fertilize oocytes in vitro. The P
ercoll gradient was a 45 and 90% discontinuous gradient. Initial exper
iments found that centrifugation of semen on the Percoll gradient for
15 min at 700 g was sufficient to obtain optimal recovery of motile sp
ermatozoa. Most of the nonmotile spermatozoa were recovered at the int
erface of the 45 and 90% Percoll layers, while the motile spermatozoa
were primarily in the sperm pellet at the bottom of the gradient. When
frozen-thawed semen from each of 7 bulls was separated by swim-up, a
mean+/-SEM of 9%+/-1 of the motile spermatozoa were recovered after th
e procedure. In contrast, more spermatozoa were recovered after Percol
l gradient separation (P<0.05), with 40%+/-4 of the motile spermatozoa
recovered. The effect of separation procedure on in vitro fertilizati
on found swim-up separated spermatozoa penetrated a mean+/-SEM of 74%/-5 of the oocytes, while fewer oocytes were penetrated by Percoll sep
arated spermatozoa at 52%+/-8 (P<0.05). There was no effect of the sep
aration procedure on the rates of polyspermy as measured by sperm/pene
trated ova, with a mean+/-SEM of 1.25+/-.09 for swim-up separated sper
matozoa and 1.14+/-.07 for Percoll separated spermatozoa (P>0.05). A c
arry over of Percoll into the fertilization medium with the Percoll se
parated spermatozoa was found not the cause for the decreased penetrat
ion of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decre
ased penetration of oocytes by Percoll separated spermatozoa could be
overcome by increasing the sperm concentration during fertilization fr
om 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized b
y either swim-up or Percoll separated spermatozoa was compared for the
semen from 2 bulls, a difference in cleavage rate was found in favor
of swim-up separated spermatozoa (P<0.05), but there was no effect of
separation procedure on development (Day 7) to the morula + blastocyst
or blastocyst stage (P>0.05). The disadvantages of the Percoll proced
ure could easily be overcome and the procedure was faster and yielded
a six-fold greater recovery of motile spermatozoa than the swim-up met
hod.