Earticle

Electrical treatment has been widely used for porcine oocytes activation. However, developmental rates following electrical activation of porcine oocytes is relatively inefficient compared to other domestic animals. To investigate the effects of porcine oocytes on combined activation by both chemical and electrical treatment, in-vitro matured oocytes were activated by combined cycloheximide and electrical pulses treatment. Cumulus-free oocytes were exposed with NCSU-23 medium containing cycloheximide (10μgml) for 0, 5, 10, 20, 30 min and then activated by electrical pulse treatment and cultured in PZM-3 for 8 days. Also effects of exposure to 6.25μM calcium ionophore for 2 min for cumulus-free oocytes were tested. The percentage of blastocyst formation in 10 min exposure to 10μgml cycloheximide and electrical pulse treatment was significantly increased (P<0.05) than in the control group. And exposure to 6.25μM calcium ionophore for 2 min with 10μgml cycloheximide for 10min and electrical pulse treatment significantly increased (P<0.05) the percentage of blastocyst developmental rates than the control group. In conclusion, activation by combined cycloheximide and electrical stimulation treatment promoted the subsequent development of porcine oocytes and improved the subsequence blastocyst development

The aim of this study was to produce porcine tetraploid (4N) parthenogenetic embryos using various methods and evaluate their developmental potential. In method 1 (M1), porcine 4N parthenogenetic embryos were obtained by inhibiting extrusion of both first (PB1) and second (PB2) polar bodies; in methods 2 (M2) and 3 (M3), 4N parthenogenetic embryos were obtained by electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 or PB1 extrusion, respectively. We found no differences in the rates of cleavage or blastocyst formation or the proportion of 4N embryos among M1, M2, and M3 groups. The different methods also did not influence apoptosis rates (number of TUNEL-positive cells/number of total cells) or expression levels of apoptosis-related BAX and BCL2L1 genes. However, total cell and EdU (5-ethynyl-2’-deoxyuridine)-positive cell numbers in 4N parthenogenetic blastocysts derived from M1 were higher (p < 0.05) than those for M2 and M3 groups. Our results suggest that, although porcine 4N parthenogenetic embryos could be produced by a variety of methods, inhibition of PB1 and PB2 extrusion (M1) is superior to electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 (M2) or PB1 (M3) extrusion.

Allicin (AL) is one of the chemical substance from garlic and has strong antioxidant activity and considered to represent anti-aging effect in vitro. The objective of this study was to investigate the effects of allicin treatment during porcine oocyte aging and their in vitro developmental competency after somatic cell nuclear transfer (SCNT). Porcine oocytes were maturated in vitro for 44 h (control), 44+24 h and 44+24 h with 0, 0.1, 1, 10 and 100 μM allicin (0, 0.1, 1, 10 and 100 AL, respectively). We investigated the effects of appropriate concentration of allicin on nuclear, cytoplasmic maturation and the developmental capacity of aged porcine oocytes. The 1 AL significantly increased maturation rate and normal spindle formation rate compared to control and 0 AL. The mRNA expression of cytoplasm maturation marker genes (MOS, BMP15, GDF9 and CCNB1) in 1 AL was higher than control and 0 AL. Allicin enhanced the rearrangement of abnormal spindle in aged oocytes. Parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryos development in 1 AL were significantly increased compared to others. Allicin treatment on aged oocytes improved their poor developmental capacity by rearranging the cytoskeleton. Thus, the allicin is effective agent to prevent the poor developmental competence of porcine aged oocytes and enhanced PA and SCNT embryo developmental capacity. Therefore, AL may be helpful for improve the aged oocytes quality to use on in vitro experiments.

X‐box binding protein‐1 (XBP‐1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP‐1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP‐1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP‐1 was weak in mature oocytes and at the one‐cell, two‐cell, and eight‐cell stages of embryos, but abundant at the GV oocyte, four‐cell, morula, and blastocyst stages. In addition, RT‐PCR revealed that both spliced XBP‐1 (XBP‐1s ) and unspliced XBP‐1 (XBP‐1u) were expressed at the GV oocyte, four‐cell, morula, and blastocyst stages. Tunicamycin (TM), an ER stress inducer, blocked porcine embryonic development at the four‐cell stage, exhibiting the effect on embryonic genome activation. Next, porcine embryos cultured in the presence of tauroursodeoxycholate (TUDCA), an ER stress inhibitor, were studied. Total cell numbers and the extent of the ICM increased (p<0.05), whereas the rate of nuclear apoptosis decreased (p<0.05). Moreover, expression of the anti‐apoptotic gene Bcl‐2 increased whereas expression of the pro‐apoptotic genes Bcl‐xl and p53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress‐mediated apoptosis in vitro.

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, gene expressions in matured oocytes, cumulus cells, and IVF-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). In the nuclear maturation after 44 h IVM, the groups of 0.1, 0.5, and 2.0 μM (83.0%, 84.1%, and 88.3%, respectively) had no significant difference compared to the control (84.1%), but the group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (p<0.05). The groups of 0.5 and 2.0 μM showed a significant (p<0.05) increase in intracellular GSH levels compared to the control and 10.0 μM groups. Intracellular ROS level of oocytes matured with 2.0 μM resveratrol was significantly (p<0.05) decreased compared to the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rate, and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) compared to the control group. Cumulus-oocytes complex (COCs) treated with 2.0 μM resveratrol were showed lower (p<0.05) expressions of apoptosis-related genes in both matured oocytes (Bax, Bak, and Caspase-3) and cumulus cells (Bax). In IVF-derived blastocysts derived from 2.0 μM resveratrol treated oocytes had also decreased (p<0.05) expression of Bak compared to the control. In conclusion, the 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating apoptosis-related genes expression during oocyte maturation.