Development of in vitro produced porcine embryos according to serum types as macromolecule

This study was conducted to establish an in vitro maturation (IVM) system by selection of efficient porcine serum during porcine in vitro production. To investigate the efficient porcine serum (PS), different types of PS [newborn pig serum, prepubertal gilt serum (PGS), estrus sow serum, and pregnancy sow serum] were used to supplement IVM media with or without gonadotrophin (GTH) and development rates of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos were then compared. The maturation rates of the PGS group was significantly higher when GTH was not added. Additionally, during development of PA embryos without GTH, the PGS group showed significantly higher cleavage and blastocyst formation rates. Moreover, the cleavage rates of IVF embryos were significantly higher in the PGS group, with no significant differences in the blastocyst formation. However, when GTH was supplemented into the IVM media, there were no significant differences among the four groups in the cleavage rates, development rates of the blastocyst, and cell number of the blastocyst after PA and IVF. In conclusion, PGS is an efficient macromolecule in porcine IVM, and GTH supplementation of the IVM media is beneficial when PS is used as macromolecule, regardless of its origin.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

Effect of IP3 and ryanodine treatments on the development of bovine parthenogenetic and reconstructed embryos

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.