In vitro embryo production in sheep: Pregnancy after long periods of oocyte and embryo transport

Abstract

To evaluate the feasibility of long distance transport of ovine oocytes and in vitro produced embryos. Santa Ines pluriparous sheep (n=5) were synchronized with intravaginal devices containing 0.3mg progesterone (CIDR®, Pfizer, Netherlands) for 10 days. Thirty-six hours prior to progesterone device removal and follicular aspiration, 0.04mg d-cloprostenol sodium (Ciosin®, Coopers, Brasil), 200 IU FSH (Folltropin®, Bioniche, Canada) and 300IU eCG (Novormon®, Syntex, Argentina) were administered intramuscularly. Oocyte retrieval was performed via mid-ventral laparotomy, under general anesthesia. The oocytes were evaluated and classified under a stereomicroscope and placed in cryovials with TCM 199, aerated with 5% CO2, covered with mineral oil, sealed, wrapped with parafilm and stored in a transport incubator at 38.5°C. Oocytes were then transported via the road for 14h to the laboratory, where the oocytes then completed more than 10h of IVM. In vitro fertilization (IVF) was performed with frozen sperm from a single ram. On Day 3 the embryos were placed in cryovials containing SOFovine (In Vitro Brasil, Sao Paulo, Brazil), aerated with 5% CO2, covered with mineral oil, sealed, wrapped with parafilm and placed into the same portable incubator. The embryos were returned to the farm under the same transport conditions (km and h). Upon arrival at the farm, all embryos were evaluated and transferred into recipient ewes 4 days after the initial oocyte retrieval. Embryos were transferred in sets of 3 to 4, into the apex of the uterine horn, ipsilateral to the corpus luteum, by means of mid-ventral laparotomy, under general anesthesia. Pregnancy was diagnosed using transrectal ultrasonography 30 days after embryo transfer. On average 9.8±5.7 oocytes (49 oocytes/5 donors) were obtained per donor, and 25 grade I morulae were transferred into 7 recipients. One pregnancy (14.3%) was confirmed resulting in a healthy lamb. These results indicate the feasibility of in vitro embryo production in sheep after maturation and embryonic development following transport over long distances.