This study was conducted to find out the effect that κ-Carrageenan has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% κ-carrageenan (64.26 ± 0.49) was significantly higher than control (40.24 ± 8.27) (p < 0.05). RPMs of extender with 0.1%, 0.2% κ-carrageenan (57.64 ± 6.34, 56.47 ± 1.35) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% κ-carrageenan (61 ± 8.03) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of κ-carrageenan of 0.1% (0.81 ± 0.05), 0.2% (0.85 ± 0.05) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.

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.

In this study, we examined the effects of porcine granulocyte-macrophage colonystimulating factor (pGM-CSF) on in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) at first time. The objective of present study was to verify effects of pGM-CSF on SCNT-derived blastocyst formation and evaluate gene expressions and qualities of the blastocyst formed after pGM-CSF treatment. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. A total 522 cloned embryos in 6 replicates were treated with 10 ng/ml concentration of pGM-CSF during in vitro culture (IVC). It was demonstrated that treatment of 10 ng/ml pGM-CSF could increase blastocyst formation and total cell number in blastocyst significantly (p<0.05) compared to the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no any effect on cleavage rate. It was found that the number of cells in the inner cell mass (ICM) and trophectoderm (TE) were significantly increased compared to the control (4.4 and 31.9, respectively) when cloned embryos were cultured with 10 ng/ml pGM-CSF (6.0 and 43.0, respectively). It was also found that treatment of 10 ng/ml pGM-CSF significantly (p<0.05) increased POU5F1 and Cdx2 mRNA expressions in blastocysts. In addition, Bcl-2 mRNA expression was found to be significantly (p< 0.05) up-regulated in blastocysts in the pGM-CSF supplemented group compared to the control. In conclusion, these results suggest that pGM-CSF may improve the quality and developmental viability of porcine cloned embryos by enhancing nuclear reprogramming via regulating transcription factors expression.