Cryopreservation of canine spermatozoa affords potential exchange of genetic material, and thus may lead to improvement in the breeding management. However, canine spermatozoa undergo many damages such as, cold shock, ice crystal formation, oxidative stress during cryopreservation. In this study used the CASA for investigating the effect of various trehalose concentrations and thawing temperatures on the sperm viability. In addition, the efficacy of the most optimal of the tested cryopreservation protocols in this study was verified by AI as the in vivo test. Also, this study evaluates the variation of frozen- thawed canine spermatozoa during different incubation condition. The addition of trehalose 25 mM was optimal concentration and frozen-thawed semen quality was significantly higher better than control (Glucose) and other concentration groups. In effect of thawing temperature on frozen-thawed sperm movement and intact acrosome evaluations, which result enhance the sperm motility and movement value depending on increase temperature condition at 36, 54 and 72℃. Also, in the effect of different incubation condition on frozen-thawed sperm after thawing at 36℃ for 60 sec, that the results trehalose 25 mM was significantly better (p<0.05) than glucose in general as well as, the post-thawed sperm motility and intact acrosome was reduced depending on increase the incubation time. Especially, incubation at 4 to 8 hour was rapidly depreciation of movement value and the rate of intact acrosome was dropped similar tendency. Thus, incubation 17℃ was better than other incubation groups on sperm motility and acrosome integrity. For the in vivo evaluate of spermatozoa survival and is the most definitive test of sperm function, we performed artificial insemination in estrous bitch. The semen was prepared for intrauterine insemination using the 25 mM trehalose freezing extender and thawing at 36℃, and 2 bitches were inseminated with 1×106 motile spermatozoa by surgical method. The results of AI, the pregnancy rates, mean litter size and oocyte fertilization rate were 16.6% (1/6), and 50% (2/4), respectively. In conclusion, based on the results of these experiments, the effect of addition of trehalose on extender improves the movement and intact acrosome of frozen-thawed semen. In particular, trehalose 25 mM groups was higher than other different concentration group on movement value and acrosome integrity of frozen-thawed sperm. Also, through incubation condition, this study identify the optimal incubation temperature after thawing was 17℃. Furthermore, the information will be contributed to develop the canine ART including AI, IVF and canine ICSI. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Cell transplantation therapy using adult stem cells has recently been identified as a potential treatment for spinal cord injury (SCI). But, recovery after traumatic SCI is very limited. As dogs are physiologically much more similar to human compared with other traditional mammalian models in disease presentation and clinical responses, a number of researches demonstrated canis familiaris is a suitable model for human diseases. This study investigated the effect of transplantation of canine Mesenchymal Stem Cells (cMSC) and neural-induced cMSC (nMSC) to understand how these cells improve neurological function in canine SCI model. The differentiation of cMSC into neural precursor cells was induced in dulbecco’s modified eagle’s medium supplemented with N2-supplement, dibutyryl cyclic adenosine monophosphate, and butylated hydroxyanisole. SCI was induced between T1 and T2 by surgical hemi-section in adult dogs, and then assigned to two groups according to the applied cell types (cMSC vs nMSC). Pelleted cMSC or nMSC were transplanted directly into the injured site after SCI, respectively. Analysis of motor function after transplantation was evaluated by modified Olby score. Magnetic resonance imaging (MRI), histological and immunohistichemical analysis were also performed. Functional recovery in group of cMSC was increasing gradually after transplantation and was higher than nMSC. In MRI, we could not confirm any difference between the cMSC and nMSC experimental groups. Immunohistochemically, beta3-tubuline and nestin were observed in injury site of two experimental groups with the expression level close to non-injured groups. Transplantation of mesenchymal stem cells could promote neuronal reconstruction and repair motor function in SCI. These showed mesenchymal stem cells could be a great candidate as a therapeutic tools in degeneration disease, and dogs could be used to explore human regenerative medicine as a promising animal model. This research was supported by iPET (Grants 110056032CG000), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside (verbascoside) is a typical phenylethanoid glycoside, extracted from various plants. It has various biological functions such as anti-oxidant, anti-inflammation, and anti-hypertension. Specially, it was powerful anti-oxidants either by direct scavenging of reactive oxygen and nitrogen species, or by acting as chain-breaking peroxyl radical scavengers. We examined the role of acteoside in IVM medium on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. The selected COCs were cultured in TCM-199 with various concentration of acteoside: 0 (control), 10, 30, and 50 μM. After 22 h of maturation with hormones, the oocytes were washed twice in a fresh maturation medium before being cultured in hormone-free medium for additional 22 h. The oocytes maturation rates of supplemented with acteoside were no significantly different compared with control group (71.13, 75.96, 72.95 and 73.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (40.03 vs. 22.95%). During IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with non-treated control oocytes. And reverse transcription polymerase chain reaction (RT-PCR) witarthenogenetic blstocysts revealed that acteoside increased the anti-apoptoticgenes, otherwise reibued pro-apoptotic genes. In conclusion, our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes such as viability and activation, providing a improved method for porcine oocytes in vitro.