Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

Abstract

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 swimup, 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 × 10 6 to 5 × 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.