(T692) A SIMPLE MODIFIED ICSI TECHNIQUE RESULTING IN HIGH PREGNANCY RATES IN MERINO SHEEP(Ovis Aries).
Shin, Taeyoung 1, Long, Charles1, Foxworth, Gabriella1, Hanna, Carol1, Menges, Suzanne1, Bormann, Christy1, Almeida-Porada, Graca2, Porada, Christopher2, Glimp, Hudson2, Millsap, Laura2, Westhusin, Mark1, Kraemer, Duane1, 1 Texas A&M University, College Station, TX2 University of Nevada, Reno, NV
ABSTRACT- In order to establish a simple modified ICSI protocol to produce offspring in a sheep model for which sperm numbers are limited, we evaluated the developmental competence of ICSI embryos derived from in vitro or in vivo matured ova. Sperm injection procedures were modified using a 3-4 micrometer ID pipette instead of a conventional 8-10 micrometer ID pipette. In experiment I, we evaluated this modified ICSI technique with in vitro matured oocytes derived from the abattoir. Motile sperm were prepared by centrifugation of frozen-thawed semen on a percoll gradient. Out of 219 oocytes that were injected with motile sperm, 176 survived and 105 cleaved on d 3, and 30 developed to the blastocyst stage by d 7. The survival rate was 84 % and cleavage rate was 86.9 %, and blastocyst formation rate was 17.0 %. In experiment II, only MII oocytes with the 1st polar body, matured either in vivo or in vitro, were utilized. All ICSI embryos were transferred surgically into the oviduct of synchronized recipients on day 1 (Day 0=estrus) and pregnancy was diagnosed by ultrasonography. A total of 30 embryos derived from in vivo matured ova were transferred to 3 recipients, all three were pregnant at D56 after transfer. Seventy-five embryos were transferred to 7 recipients, 6 out of 7 were pregnant at d 56 after embryo transfer. Most of these pregnancies in both groups were observed to be multiples. In this study, in which up to 14 ICSI embryos were transferred to each recipient based upon Experiment I, a high pregnancy rate (12/13, 92.3 %) was achieved. This pregnancy rate was achieved using a simple modified pipette without sperm immobilization or additional activation, which some investigators believed to be necessary steps in sheep ICSI. Further study is needed to determine whether this simplified technique reduces physical damage, which may be associated with conventional ICSI. In conclusion, our results demonstrate that this protocol is useful for production of sheep embryos, which are capable of establishing pregnancies in this species.
KEY WORDS: ICSI, sheep