This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

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The aim of this study was to clone native Anatolian Grey cattle by using different donor cell types, such as fibroblast, cartilage and granulosa cells cryopreserved in a gene bank and oocytes aspirated from ovaries of Holstein cows as the recipient cytoplasm source. One male calf from fibroblast, three female calves from granulosa cells and one female calf from cartilage cells were born healthy and at normal birthweights. No calves were lost after birth. The results demonstrated that the cloned calves had the same microsatellite alleles at 11 loci as their nuclear donors. However, the mtDNAs of the five Anatolian Grey cloned calves had different haplotypes from their donor cells and mtDNA heteroplasmy could not be detected in any of the clones. The birth of healthy clones suggests that the haplotype difference between the cell and oocyte donor did not affect the pre- or post-implantation development of the bovine nuclear transfer derived embryos in our study. The results showed that well established nuclear transfer protocols could be useful in conserving endangered species. In conclusion, somatic cell banking can be suggested as a tool in conservation programmes of animal genetic resources.

Embryonic stem (ES) cells have a great interest for tissue engineering because of their pluripotent nature and proliferative capacity. The objective of this study is to constitute a synthetic microenvironment to support the in vitro propagation of murine ES cells in an undifferentiated state. That is why we used a three-dimensional matrix, nonwoven polyester fabric (NWPF), which was formed from poly(ethylene terephthalate) (PET) fibers. NWPF discs were partially hydrolyzed, and then the carboxyl groups were coupled with leukemia inhibitory factor (LIF) in the presence of water-soluble carbodiimide. The effectiveness of immobilization process was checked with ATR-FTIR spectroscopy, fluorimetry, and cell culture studies. ES cell colony morphology, alkaline phosphatase (AP) activity, stage-specific embryonic antigen-1 (SSEA-1) immunoreactivity, and SEM analysis following a 72 - 96-h culture period upon hydrolyzed and LIF-immobilized surfaces were assessed to determine the pluripotent status of ES cells. Results revealed that LIF was active in immobilized form; undifferentiated colonies had not only a significant AP and SSEA-1 immunoreactivity, but also a higher undifferentiated colony ratio on LIF-immobilized surfaces than that of hydrolyzed surfaces. The immobilized LIF protein might be a good model to provide a feeder-free system, but the physical properties of the scaffold is more convenient for differentiation studies.