Developmental competence and pluripotency gene expression of cattle cloned embryos derived from donor cells treated with 5-aza-2'-deoxycytidine

Reconstructed embryos from terminally differentiated somatic cells have revealed high levels of genomic methylation which results in inappropriate expression patterns of imprinted and non-imprinted genes. These aberrant expressions are probably responsible for different abnormalities during the development of clones. Improvement in cloning competency may be achieved through modification of epigenetic markers in donor cells. Our objective was to determine if treatment of donor cells for 72 hours with 5-aza-2'-deoxycytidine [5-aza-dc; 0-0.3 microM], a DNA methyl transferase inhibitor, improved development and expression of Oct-4. In comparison with untreated cells, 0.01 and 0.08 microM 5-aza-dc treated cells insignificantly decreased the blastocyst rate [32.1% vs. 28.6% and 27.2%, respectively] while it was significant for 0.3 microM treated cells [6.5%]. Embryo quality as measured by the total cell number [TCN] decreased in a dose-related fashion, which was significant at 0.08 and 0.3 microM 5-aza-dc treated cells when compared with 0 and 0.01 microM 5-aza-dc treated cells. Although reconstructed embryos from 0.08 and 0.3 microM 5-aza-dc treated cells showed lower levels of DNA methylation and histone H3 acetylation, development to blastocyst stage was decreased. The epigenetic markers of embryos cloned from 0.01 microM 5-aza-dc remained unchanged. These results show that 5-aza-dc is not a suitable choice for modifying nuclear reprogramming. Finally, it was concluded that the wide genomic hypomethylation induced by 5-aza-dc deleteriously impacts the developmental competency of cloned embryo

Effect of ovarian cyclic status on in vitro embryo production in cattle

The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle [LF], 2. the presence of a corpus luteum [CL] and 3. ovaries without LF or CL [WLCF]. Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group [36.6%], but non-significantly higher than that of the ST group [45.7%]. The highest blastocyst rate belonged to the CL group [54.6%] which was significantly greater than the WLCF group [32.9%] and non-significantly higher than the LF group [52.4%]. There was no significant difference in blastocyst rates in the CL and LF groups. Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and noninvasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage