34 EFFECT OF DONOR CELLS SERUM STARVATION ON THE DEVELOPMENT OF AGGREGATED ZONA FREE CLONED EQUINE EMBRYOS

Abstract

Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning procedure, and it has been shown that different methods affect embryo development. The goal of the present study was to determine the effect of serum starvation in combination with growth to confluence of the somatic donor cells, on in vitro embryo development and quality of aggregated cloned equine embryos. Oocyte collection, maturation, cloning, and activation procedures were performed as described previously by (Gambini et al. 2012 Biol. Reprod. 87, 15). Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of 2 horses. They were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. After cell multiplication, they were cryopreserved and stored in liquid nitrogen. After thawing, 2 groups of cell synchronization were established. Group I: growth to confluence for 3 to 5 days before NT followed by serum starvation in DMEM supplemented with 0.5% FBS for 24 h prior to NT; group II: growth to confluence for 3 to 5 days before NT. After activation, reconstructed embryos (RE) were cultured in SOF in the well of well system, placing 3 RE per well. Cleavage and blastocyst formation at Day 7 to 8 were assessed. At Day 7 to 8, embryos were measured and some of each group were fixed with paraformaldehyde to measure DNA fragmentation through the DeadEnd fluorometric TUNEL system (Promega, Madison, WI, USA). In vitro embryo development, on a per embryo and cleaved RE basis, and blastocyst size was compared using the chi-squared test; the proportion test was used for statistical analysis of DNA fragmentation levels (fragmented DNA cells/total cells). There were no statistic differences on cleavage per RE (I: 136/177, 80.2%; II: 142/171, 79.6%), blastocyst rates at Day 7 (I: 29/58, 49.1%, II: 22/56, 39.2%), or on blastocyst size at Day 8. Statistical differences were observed in blastocyst rate at Day 8 (I: 42/58, 72.4%, II: 30/56, 53.5%) and in blastocyst DNA fragmentation levels (I: 202/2464, 8%; II: 173/1440 12%). In conclusion, the restriction of FBS to the cell culture medium 24 h before cloning seams to improve embryo development at Day 8 and also reduces the level of apoptosis in cloned blastocyst, suggesting a better embryo quality. For these reasons, we consider that the addition of the restriction of FBS to the growth to confluence is beneficial for cloned equine embryo development.