The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and 100 μM) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with 50 μM ALA were fertilized and cultured in IVC medium with ALA (25, 50 and 100 μM) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with 25 μM ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by 50 μM ALA treatment groups compared with control groups (p<0.05). Treatment of 25 μM ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by 25 μM ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (ICR ESCs). Similar to those from 129/Ola mouse blastocysts (E14 ESCs), the established ICR ESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, ICR ESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts (ICR MEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred ICR MEFs, hybrid B6CBAF1 MEFs as feeder cells could sufficiently support in vitro maintenance of ICR ESC self-renewal. Additionally, ICR ESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ICR ESCs cultured for 40th subpassages (164 days) on B6CBAF1 MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ICR ESCs could be maintained on B6CBAF1 MEFs in culture.

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In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and β subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α , α , α , α , α , and β subunits, but not the α , α , α , α , and α subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C, and vitronectin were observed and functional blocking of integrin heterodimer α β , α β , or α β showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α β heterodimer-mediated adhesion to laminin was detected. These results demonstrate that active α β , α β , and α β integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α (presumed) and α subunits.

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