| The aim of this study was to determine the effects of trans-10, cis-12conjugated linoleic acid (t10c12CLA) supplementation on oocyte maturation, embryo development and cryopreservation in pigs.First, various concentration of tl0cl2CLA were supplemented in porcine in vitro maturation (IVM) medium. Compared with the control, supplementation of50μmol/L tl0cl2CLA to IVM medium significantly increased the proportion of oocytes at the metaphase-Ⅱ (MII) stage and subsequent parthenogenetic embryo development in terms of cleavage rate (89.5%±1.2vs.83.4%±1.6), blastocyst formation rate (51.9%±1.5vs.39.6%±1.5), and cell numbers in blastocysts (62.2±1.6vs.50.7±1.2). The state of nucleus was examined at22and36h maturation, the results showed that t10c12CLA treated oocytes resumed meiotic maturation and progressed to the MII stage significantly faster than those of control. The expression of phosphorylated mitogen-activated protein kinase3/1(p-MAPK3/1) and cyclooxygenase-2(COX2) in cumulus oocyte complexes (COCs) at5,10, and22h of IVM were significantly increased in the t10c12CLA treatment group. The level of p-MAPK3/1in t10c12CLA treated MII oocytes was also higher (p<0.05) than that of control. Moreover, t10c12CLA supplementation partially overcame the negative effects of U0126on cumulus expansion and nuclear maturation, and completely recovered COX-2protein levels in the presence of U0126. Treatment of COCs with NS398also significantly suppressed cumulus expansion and nuclear maturation, which was overcome by t10c12CLA. Yet, this stimulatory effect of t10c12CLA was blocked in the presence of both U0126and NS398. The maturation of cytoplasm was also evaluated; and founded that t10c12CLA treatment significantly reduced reactive oxygen species (ROS) level and increased glutathione (GSH) concentrations in MII oocyte.Second, the appropriate concentration (50μmol/L) of t10c12CLA was supplemented to IVM medium, and MII oocytes were subjected to Cryotop vitrification. The results showed that the formation rate of parthenogenetic blastocysts underwent vitrification was higher in t10c12CLA treated group (25.43%±2.14vs.11.76%±0.62, p<0.05), and t10c12CLA increased the percentage of normal oocytes (type N, lipid droplets with sharpen edge) significantly. Transmission electron microscope (TEM) was used to exam the ultrastructure of vitrified oocytes.t10c12CLA protected the lipid droplets from fusion and rupture. Moreover, the distribution and membrane potential of mitochondria was maintained in t10c12CLA treated group.Last, a serious of concentrations of t10c12CLA was added to in vitro culture (IVC) medium of porcine parthenogenetic embryos. Compared with the control, the blastocysts formation at5d IVC (34.54±0.95vs.26.48±2.19) and total cells in blastocysts (54.65±1.63vs.48.67±2.14) were increased in t10c12CLA treated group (50μmol/L). Based on the results mentioned above, the concentration of50μmol/L was used in the following experiments. Then, the expression of E-Cadherin in8-cell embryos was measured to analysis the effects of t10c12CLA on porcine early embryos development, and founded that t10c12CLA promoted the expression of E-Cadherin significantly as well as the percentage of blastocysts with more cell numbers (n>60)(40.79±1.61vs.28.43±1.14).In conclusion, an optimal concentration of t10c12CLA supplemented to IVM medium enhances the maturation and subsequent in vitro developmental competence of porcine oocytes. The beneficial effect of t10c12CLA on nuclear maturation in porcine oocytes appears to act indirectly through the cumulus cells by the regulation of both MAPK and COX2activity. Culture in the presence of t10c12CLA was also advantageous for cytoplasmic maturation of porcine oocytes, increasing intracellular MAPK activity and modulating the redox status of the cell. After treated with t10c12CLA, the distribution and membrane potential of mitochondria of vitrified MII oocytes was improved, and the lipid droplets were maintained regularly, which promoted the developmental competence of porcine oocytes underwent vitrification. Moreover, t10c12CLA supplemented during IVC affected the parthenogenetic embryos development posisitively through increasing expression of E-Cadherin, which leaded to the acceleration of morula compaction and blastocysts formation. |
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