Gene microinjection in bovine embryos facilitated by centrifugation

Abstract

Twenty-seven Merino and Merino × Border Leicester ewes were superovulated in the following manner 10 days after sponge insertion: Day 10 — 500 i.u. PMSG + 5 mg FSH-P; Day 11 — 300 i.u. PMSG + 4 mg FSH-P; Day 12 — 3 mg FSH-P + sponge removed. Twenty-four hours after sponge removal, 100 μg GnRH was administered. Tubal oocyte collections were performed at 18–21 (Group A), 21–25 (Group B) or 25–29 (Group C) hours after GnRH administration. Oocytes were recovered by a retrograde flush of the oviducts. Ovulation rates were 3.9 ± 1.7, 9.6 ± 1.5 and 11.9 ± 1.5 for Groups A, B and C respectively.The level of cellular vestment surrounding collected oocytes was noted. Oocytes were grouped into one of four categories: I — surrounded by an expanded cumulus/corona complex; II — surrounded by a corona only; III — devoid of all cellular vestments; IV — abnormal or degenerating oocytes. In Group A, 100% (n = 5) oocytes were Type I. In Group B (n = 44), 18.2% were Type I, 61.4% Type II, 18.2% Type III and 2.2% Type IV. In Group C (n=70), 10% were Type II and 90% were Type III.Results indicate that ovulation had begun 18–21 h after GnRH, and was mostly complete by 25 h after GnRH. There was a marked rapid cumulus loss from superovulated sheep oocytes in the oviduct — within 4–6 h, oocytes were devoid of cellular vestments. The implications of these results are discussed.