In vitro fertilization of ovulated and ovarian ovine oocytes

In vitro fertilization of ovine ovulated and ovarian oocytes was attempted after capacitation of ejaculated spermatozoa by a variety of methods. All sperm incubations and oocyte cultures were performed in a modified Krebs Ringer bicarbonate (KRB) medium. High ionic strength (HIS) medium was prepared by adding NaCl to provide an osmolarity of approximately 370 mOsmol/kg. All oocytes were obtained from progestogen-synchronized and follicle-stimulating hormone-treated ewes. Ovulated oocytes were recovered 64 h after progestogen withdrawal. Ovarian oocytes were recovered 45 h after progestogen withdrawal. Oocytes were incubated with spermatozoa in microdrops for 6 h, transferred to fresh medium and cultured for 48 h. In the first series of experiments, capacitation was attempted by either preincubation in KRB, washing spermatozoa with KRB medium or treatment of spermatozoa with HIS medium. Spermatozoa washed with KRB penetrated three of 61 (5%) oocytes and spermatozoa incubated in HIS medium penetrated eight of 209 (4%) oocytes. In the second series of experiments, spermatozoa were capacitated by incubation with ovine cumulus cells or in the uterus of a rabbit. A 33% fertilization rate was observed with spermatozoa recovered from the rabbit uterus and a 28% rate with spermatozoa incubated with cumulus cells. Treatment with HIS medium previously shown to capacitate bovine and rabbit spermatozoa failed to capacitate ram spermatozoa; however, fertilization was observed after capacitation by cumulus cells or in the rabbit uterus.