Development of In Vitro Derived Sheep Embryos to the Blastocyst Stage

Authors: SEMA BİRLER, SERHAT PABUCCUOĞLU, SERHAT ALKAN, ÖZEN BANU ÖZDAŞ, HATEM ATALLA, İRFAN KAMURAN İLERİ

Abstract: The objective of the present study was to mature and fertilize primary sheep oocytes in vitro and to develop them to the blastocyst stage. Ovaries from slaughtered Kıvırcık ewes were used. The ovaries were transported to the laboratory in a vacuum flask in which 30-35 °C PBS (phosphate buffered saline) was included. The cumulus-oocyte complexes were collected by rupturing and washing the follicle wall with oocyte washing medium. They were then selected under a stereo microscope and maturated for 23-24 hours in medium 199 supplemented with sodium pyruvate, follicle stimulating hormone (FSH), luteinizing hormone (LH) and 10% fetal calf serum (FCS), under 5% CO2 atmosphere and at 38.5 °C temperature. Semen collected and pooled from three Kivircik rams by electro-ejaculation and prepared for in vitro fertilization by Percoll-gradient method was incubated for 20-21 hours with oocytes in BSOF medium supplemented with 2% sheep estrous serum (SES) (0.8x106 spermatozoon/ml). Presumptive zygotes were cultured in SOF medium for 8 days in synthetic oviduct medium (SOF) under an atmosphere of 5% CO_2, 5% O_2 and 90% N_2. Glucose (1.5mM) was added to the culture medium on Day 4. Out of 171 oocytes, 151 cleaved (88.3% ). Fifty-six morulae (37.1% ) and 14 blastocysts (9.3% ) were obtained from these in vitro derived embryos.

Keywords: Sheep, in vitro fertilization, in vitro culture, development to blastocyst stage

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