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The in vitro embryo's competence is lower than in vivo counterparts and miRNAs found to affect the developmental competence, affecting pluripotency, stress level and apoptosis in embryos. We aimed to investigate the effect of miRNA-155 on parthenogenically activated early development porcine embryos at the preimplantation blastocyst stage. We designed miRNA-155 mimics and inhibitors and microinjected them into post-activated oocytes. The embryonic cleavage rate, blastocyst rate, and embryo development quality in terms of stress and apoptosis levels were investigated by confocal microscopy. Furthermore, we selected target genes, analyzed gene interaction and prediction networks, and compared the gene expression level in treatments and controls. miRNA-155 inhibition improved in vitro developmental competence by increasing cell numbers and reducing stress and apoptosis levels. The cleavage rate in the miRNA-155 inhibitor group was significantly higher (P 0.05) than that in the miRNA-155 mimic group, but not in the control, whereas the blastocyst rate of the miRNA-155 inhibitor group was statistically significantly higher (P 0.05) than in both control and miRNA-155 mimic groups. The relative gene expression level analysis showed downregulation of mRNAs related to stress and apoptosis, BAX, and the stress-induced autophagy gene ATF4, and TNF-ɑ in the miRNA-155 inhibitor group. Moreover, miRNA-155 inhibition showed upregulation of the relative expression of OCT4, ZEB2, BCL2, and IL-1 mRNA compared to control and mimic groups. However, the miRNA-155 mimic-injected group showed lower cleavage rates and blastocyst development rates than the other two groups. In conclusion, miRNA-155 inhibition in porcine in vitro embryos improved their preimplantation developmental competence and in vitro embryo production.

This study was performed to explore the effects of cobalamin treatment during in vitro maturation (IVM) of porcine oocytes. Specifically, the effects of cobalamin exposure on nuclear and cytoplasmic maturation of oocytes, diameter, glutathione (GSH) and reactive oxygen species (ROS) levels of matured oocytes, as well as development and gene expression of porcine parthenogenetic and cloned embryos were assessed. Cumulus-oocyte complexes were exposed to 200 pM cobalamin for 22 h or incubated for 22 h without cobalamin (controls). The mean diameter of cobalamin-treated oocytes was greater than that of control oocytes (160.0 vs. 154.5 μm; p 0.05). GSH level increased but ROS level decreased in the cobalamin-treated oocyte group. Parthenogenetic embryos derived from cobalamin-treated oocytes showed improved oocyte maturation (91.3% vs. 83.8%), cleavage (88.9% vs. 82.1%), and blastocyst formation (38.7% vs. 31.9%) rates compared with control embryos (p 0.05). Similarly, cloned embryos derived from cobalamin-treated oocytes showed higher oocyte maturation (89.2% vs. 82.2%), cleavage (87.5% vs. 80.3%), and blastocyst formation (30.0% vs. 23.8%) rates than control embryos (p 0.05). Furthermore, in parthenogenetic and cloned embryos, total cell number, inner cell mass (ICM), trophectoderm (TE) expression, and ICM: TE ratio were higher in the cobalamin-treated group compared to that in the control group (p 0.05). Cloned embryos in the cobalamin-treated group showed higher mRNA expression levels of POU5F1, DPPA2, and NDP52IL than control group embryos. Together, these results demonstrate that cobalamin treatment during IVM improves the developmental competence of porcine oocytes by neutralizing the free radicals produced in the IVM medium.

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts,16 being considered fresh and50 being long-term cultured; in adult ear fibroblasts,16 being considered fresh and30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.

The present study evaluated the effect of protein supplementation in potassium simplex optimization medium (KSOM) on bovine preimplantation embryo development. The in vitro fertilized (IVF) (Experiment 1), non-transgenic (Experiment 2) and transgenic cloned embryos (Experiment 3) were cultured for 192 h in KSOM supplemented with 0.8% BSA (KSOM-BSA), 10% FBS (KSOM-FBS) or 0.01% PVA (KSOM-PVA). Transfected cumulus cells with an expression plasmid for human alpha1-antitrypsin gene and a green fluorescent protein (GFP) marker were used to produce transgenic cloned embryos. Modified synthetic oviductal fluid (mSOF) supplemented with 0.8% BSA (mSOF-BSA) was used as a control medium. In Experiment 1, cleavage rate was significantly (P < 0.05) lower (69.1%) in IVF embryos cultured in KSOM-FBS than in KSOM-BSA (80.3%). The rate of hatching/hatched blastocyst formation was significantly (P < 0.05) lower in embryos cultured in KSOM-PVA than in KSOM-FBS (2.2% versus 10.8%). Blastocysts cultured in KSOM-FBS contained significantly (P < 0.06) higher numbers of inner cell mass cells (50.4 +/- 20.2) than those cultured in mSOF-BSA (36.9 +/- 19.2). In Experiment 2, the rate of blastocyst formation was significantly (P < 0.05) lower (20.5%) in embryos cultured in KSOM-PVA than in other culture media (33.3-38.5%). The rate of hatching/hatched blastocysts was significantly (P < 0.05) lower in KSOM-PVA (13.9%) and KSOM-FBS (17.1%) than in KSOM-BSA (30.8%) and mSOF-BSA (33.9%). The numbers of total and trophectoderm cells (104.6 +/- 32.2 and 71.7 +/- 25.5, respectively) were significantly (P < 0.05) lower in blastocysts cultured in KSOM-PVA than in KSOM-BSA (125.7 +/- 39.7 and 91.7 +/- 36.2, respectively). In Experiment 3, no significant differences in embryo development, GFP expression and blastocyst cell numbers were observed among the culture groups. In conclusion, the present study demonstrated that KSOM and mSOF supplemented with BSA were equally effective in supporting development of bovine non-transgenic and transgenic cloned embryos. Moreover, different developmental competence in response to protein supplementation of KSOM was observed between bovine non-transgenic and transgenic cloned embryos.

We conducted this study to examine whether serum starvation in culture contributes to better development of bovine reconstructed oocytes and to evaluate which serum-starved somatic cell is the most effective for cloned calf production. In Experiment 1, donor cells of four different types (cumulus cells, ear fibroblasts, oviduct cells and uterine cells) were either serum-starved or not before fusion with enucleated oocytes, and reconstructed oocytes were further cultured for 168 h. Regardless of serum starvation, cumulus cells or ear fibroblasts yielded higher (P < 0.05) rates of fusion than other cells (62.6-69.3 versus 33.3-38.7%). In the serum-starved group, the first cleavage after reconstruction was significantly increased in cumulus cells and ear fibroblasts, compared with oviduct cells (93.4-94.3 versus 78.8-86.0%), and oocytes reconstructed with either of these yielded more blastocysts than oocytes reconstructed with oviduct or uterine cells (40.6-43.8 versus 20.3-19.0%). We observed a similar pattern in the non-starved group, but we found a significant increase in blastocyst formation was found only in cumulus cells compared with other donor cells (42.6 versus 15.4-27.7%). Overall comparison showed that serum starvation increased the rates of cleavage and development to the blastocyst stage, but we found a statistical significance only in the cleavage rate (80.0 versus 89.5%). In Experiment 2, we transferred randomly selected 59 blastocysts that were developed from oocytes reconstructed with serum-starved cells to 44 synchronised recipients. Of those recipients, 23 became pregnant on Day 60 after transfer (52.3%) and 12 (27.3%) delivered cloned calves. The mean gestation length and birth weight was 275 +/- 8 days and 39.6 +/- 15.6 kg, respectively. Although there was no significant difference among donor cells, blastocysts that were derived from oocytes reconstructed with ear fibroblasts yielded the highest rates of pregnancy (50.0%) and delivery (27.3%). In conclusion, serum starvation is effective for improving preimplantation development of oocytes reconstructed with cumulus or ear fibroblast cells and it may positively influence on obtaining better pregnancy outcome.