32 PREGNANCY OF EQUINE CLONED EMBRYOS MICROINJECTED WITH PLURIPOTENCY INDUCING GENES (Oct4, Sox2, c-Myc, K1f4)

Animal cloning is a high impact tool for scientific and economical production, but still with inefficient results. The efficiency of the cloning process depends on the state of differentiation of the donor cell. An adult equine somatic cell can be differentiated to a pluripotent stem cell (iPSC) inducing the expression of certain transcription factors (Oct4, Sox2, c-Myc, and K1f4; Breton et al. 2013). The objective of this work was to assess the effect of the intracytoplasmic injection of pluripotency inducing genes on embryo development and pregnancy rates of equine cloned embryos. Cumulus–oocyte complexes (COC) were obtained from slaughterhouse ovaries. Oocyte collection and maturation procedure were performed as described by Lagutina et al. (2007). After the removal of cumulus cells, oocytes showing first polar body were microinjected with a mixture 1/3 of plasmids/liposomes (Mi group). The plasmid used was the pEP4-E02s-EM2k, which encodes the human genes Oct4, Sox2, Myc, and K1f4. The DNA concentration was adjusted to 0.5 μg mL–1. Microinjected oocytes were enucleated using the zona free method. Adult male skin fibroblasts from the same animal were used as donor nucleus cells. These fibroblasts were attached to the ooplasts with phytohemagglutinin and then fused with an electric pulse. Activation was performed using 8.7 mM ionomycin for 4 min, followed by culture for 4 h in a combination of 1 mM 6-DMAP and 5 mg mL–1 cycloheximide. Zona free reconstructed embryos (ZFRE) were cultured for 7 to 8 days in DMEM-F12 in the well of the well (WOW) system, aggregating 3 embryos per well. A control group (CC group) of not microinjected embryos was included. Cleavage and blastocyst development was assessed at Days 2 and 7, respectively. Transcervical transfer of 49 Day 7 to 8 blastocysts was performed 6 days after ovulation. The mares received 2 blastocysts per transfer. Pregnancy was diagnosed by transrectal ultrasonography 15 days after ovulation. Cleavage and blastocyst rates were analysed by Chi-squared test and pregnancy rate by Fisher test (P < 0.05). Cleavage was 92.1% (n = 58/63) for the Mi group and 90.4% (n = 868/960) for the CC group. Blastocyst rate was statistically higher per well, 28.6% (n = 6/21) v. 13.4% (n = 43/320) but not per oocyte, 9.5% (n = 6/63) v. 4.5% (n = 43/960), for the Mi and CC groups, respectively. Pregnancy rate was 17% (n = 1/6) for the Mi group and 7% (n = 3/43) for the CC group. No twin pregnancies were found and all the pregnancies are still ongoing. The higher blastocyst rates obtained with the embryos microinjected with pluripotency inducing genes compared with the control group showed an improvement in embryo quality. In conclusion, the data presented indicate that the intracytoplasmic microinjection of pluripotency inducing genes in equine zona free cloned embryos improved blastocyst rates on a per well basis and showed a tendency to improve the pregnancy rates. The expression of the Oct4, Sox2, c-Myc, and K1f4 genes could be probably generating better reprogrammed donor nucleus compared with adult differentiated cells used in conventional cloning.