Optimization of human recombinant granulocyte-colony stimulating factor supplementation during in vitro production of porcine embryos to improve the efficiency of resource utilization of poor-quality cumulus-oocyte complexes

Granulocyte colony-stimulating factor (G-CSF), a pleiotropic cytokine, is secreted by the reproductive tract. Furthermore, our previous study indicated that human recombinant G-CSF (hrG-CSF) supplementation during porcine oocyte in vitro maturation (IVM) or during embryo in vitro culture (IVC) improved their quality and development potential when using cumulus-oocyte complexes (COCs) with more than three cumulus cell layers (CCL >3). Thus, in this study, we investigate the optimal conditions of hrG-CSF supplementation throughout the in vitro production (IVP: IVM + IVC) system to improve the embryo production efficiency of "poor-quality (CCL <= 3)" oocytes. COCs were classified into two groups according to the number of CCL (>3 and <= 3) and embryonic viability was analyzed after treatment with hrG-CSF during IVC. The mRNA transcription levels of G-CSF in COCs were compared based on their type and the period of IVM. Finally, developmental capacity and quality were evaluated after treatment with hrG-CSF for different periods of IVP. No marked effects on the developmental potential of embryos when using CCL <= 3 type COCs were observed after supplementing hrG-CSF only during IVC. Moreover, the mRNA transcription level of G-CSF increased gradually with IVM culture time and was higher in CCL <= 3 COCs than in >3. Supplementing hrG-CSF only during the IVM period resulted in the best embryo developmental potential, while supplementing hrG-CSF during the IVP period resulted in the best quality embryos, reflected in the increased total cell number and decreased apoptotic nuclei index of blastocysts. These findings indicate that "poor-quality" COCs may have a greater demand for G-CSF than "good-quality", meanwhile hrG-CSF supplementation throughout IVP improves resource utilization efficiency in poor-quality COCs.