EFFECT OF BOVINE SPERM SEPARATION BY EITHER SWIM-UP OR PERCOLL METHOD ON SUCCESS OF IN-VITRO FERTILIZATION AND EARLY EMBRYONIC-DEVELOPMENT

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface 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 the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P<0.05), with 40%+/-4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean+/-SEM of 74%+/-5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52%+/-8 (P<0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean+/-SEM of 1.25+/-.09 for swim-up separated spermatozoa and 1.14+/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by 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 procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.