The objective of this study was to investigate the effect of milrinone supplementation as a phosphodiesterase 3A inhibitor during in vitro maturation (IVM) to coordinate the cytoplasmic and nuclear maturation of porcine oocytes and subsequent development of porcine cloned embryos. Brilliant cresyl blue (BCB)-stained (BCB +) oocytes, classified as well-developed, and BCB− oocytes were used in parthenogenesis (PA) and cloning, and their preimplantation development was compared. In PA embryos, BCB + oocytes had significantly higher rates of development than BCB− oocytes in terms of maturation (87.5 vs. 71.3%), cleavage (88.6 vs. 76.3%), and blastocyst development (34.3 vs. 25.3%) and also had higher cell numbers (46.9 vs. 38.9%), respectively (p < 0.05). In cloned embryos, the BCB + group also had a significantly higher blastocyst formation rate than the BCB− group (30.6 vs. 20.1%; p < 0.05). Supplementation with 75 μM milrinone during IVM of BCB− oocytes showed improvement in maturation and blastocyst development rates, which may be due to the coordinated maturation of the cytoplasm with the nucleus as an effect of milrinone. Moreover, the analysis of nuclear reprogramming via the examination of the expression levels of the reprogramming-related genes POU5F1, DPPA2, and NDP52IL in milrinone-supplemented BCB− oocytes showed higher expression levels than that in non-treated BCB− oocytes. These findings demonstrate that milrinone is useful in improving developmental competence in less competent oocytes during IVM and for proper nuclear reprogramming in the production of porcine cloned embryos by coordinating cytoplasmic and nucleus maturation.

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus–oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25 and 50 µg/ml) during in vitro maturation (IVM). Oocytes treated with 25 µg/ml CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs10 and CNPs50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25‐treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25‐treated group was significantly higher than that in the CNPs50‐treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25‐treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25 µg/ml CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.

Molecular approaches have been used to determine metabolic substrates involved in the early embryonic processes to provide adequate culture conditions. To investigate the effect of modified Spirulina maxima pectin nanoparticles (MSmPNPs) on oocyte developmental competence, cumulus–oocyte complexes (COCs) retrieved from pig slaughterhouse ovaries were subjected to various concentrations of MSmPNPs (0, 2.5, 5.0, and 10 µg/mL) during in vitro maturation (IVM). In comparison to the control, MSmPNPs-5.0, and MSmPNPs-10 groups, oocytes treated with 2.5 µg/mL MSmPNPs had significantly increased glutathione (GSH) levels and lower levels of reactive oxygen species (ROS). Following parthenogenetic activation, the MSmPNPs-2.5 group had a considerably higher maturation and cleavage rates, blastocyst development, total cell number, and ratio of inner cell mass/trophectoderm (ICM:TE) cells, when compared with those in the control and all other treated groups. Furthermore, similar findings were reported for the developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos. Additionally, the relative quantification of POU5F1, DPPA2, and NDP52 mRNA transcript levels were significantly higher in the MSmPNPs-2.5 group than in the control and other treated groups. Taken together, the current findings suggest that MSmPNP treatment alleviates oxidative stress and enhances the developmental competence of porcine in vitro matured oocytes after parthenogenetic activation and SCNT.

This study examined the effects of a caffeine treatment to improve nuclear reprogramming in porcine cloned embryos. Embryonic development and the expression of genes related to pluripotency ( POU5F1 , SOX2 , NANOG , and CDX2 ) were compared after caffeine supplementation during manipulation at different concentrations (0, 1.25, 2.5, and 5.0 mM) and after varying the delayed activation time (control, 1, 2, and 4 h) after fusion. Caffeine added to media during manipulation produced a higher rate of development to blastocysts in the 1.25 mM group than in the other concentration groups (22.8% vs. 16.1%, 16.2%, and 19.2%; p < 0.05). When caffeine was added during the 4 h delayed activation, the 1.25 mM caffeine concentration produced a significantly higher rate of development than those in the other 4 h-activation-delayed caffeine concentration groups (22.4% vs. 9.4%, 14.0%, and 11.1%; p < 0.05). On the other hand, no significant improvement over that in the control group was observed when caffeine was supplemented during both the manipulation period and delayed activation period (16.0% vs. 15.2%), respectively. The levels of POU5F1 , SOX2 , and NANOG expression in blastocysts were significantly higher in the delayed activation caffeine group (4 h, 1.25 mM) than in the control group (1 h, 0 mM; p < 0.05). In conclusion, a caffeine treatment at 1.25 mM during delayed activation for 4 h can improve the preimplantation development of porcine somatic cell nuclear transfer embryos by activating nuclear reprogramming.