123 AGGREGATION OF CLONED EQUINE EMBRYOS: IMPROVEMENT OF IN VITRO AND IN VIVO DEVELOPMENT

Abstract

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measured. In advanced pregnant mares, combined thickness of the uterus and the placenta (CTUP) and serum progesterone levels were also determined. The remaining embryos obtained from each group were maintained in culture from day 7 until day 15. Blastocysts growth was determined every 24 h. In vitro development, on a per-well and RE basis, was compared using the chi-square test. Statistical differences were observed in cleavage among groups I and II (P = 0.0088) and groups I and III (P = 0.0004): (I: 91/111, 82%; II: 74/78, 95%; III: 62/62, 100%). Blastocyst rates differed between groups I and III (I: 10/111, 9%; III: 23/62, 37%); no difference was observed with group II (11/78, 14%). There was no difference on blastocyst rates based on the number of aggregated RE (I: 10/111, 9%; II: 11/156, 7%; III: 23/184, 12.5%). The highest pregnancy rate was obtained in group III (I: 1/3, 33%; II: 2/5, 40%; III: 3/4, 75%). Sizes of vesicles and embryos did not differ statistically in such groups. The CTUP and serum progesterone levels were considered normal (<1.2 cm; >8 ng mL^–1, respectively) in ongoing pregnancies. We did not observe any differences in Oct-4 expression patterns among groups. Even though statistical differences were found, surprisingly all embryos grew in vitro until day 15 with good rates and the biggest embryo reached 4.25 mm. Embryo aggregation improved in vitro development of equine cloned embryos until day 7, and pregnancies rates were higher. The in vivo sizes of vesicles and embryos were normal for all groups, and in vitro development beyond day 7 showed the high viability of embryos. To conclude, aggregation of cloned equine embryo does not imply extra oocytes because there is no statistical difference in the number of blastocysts obtained per oocytes used to achieve RE. It is also a good strategy to improve in vitro embryo development without alterations on in vivo progress. This is the first report of pregnancies from aggregated equine cloned embryos, and the first healthy cloned foal from South America, confirmed by STR analysis, was born recently derived from group II.