Alzheimer's disease (AD) has caused by expression of amyloid precursor protein (APP), Tau and presenilin (PS) as known as plaques and tangle accumulation. AD transgenic porcine model is necessary for preclinical testing of therapeutic agent because of similar metabolic system between porcine and human. The objective of study was to generate AD transgenic pig by somatic cell nuclear transfer (SCNT) with multi-cistronic vector system. AD multi-cistronic vector was 6 well-known mutation on 3 AD related genes, hAPP (K670N/M671L, I716V, V717I), hTau (P301L) and hPS1 (M146V, L286P). Establishment of AD transgenic cell lines was used from Jeju black pig ear fibroblast cells (JB-PEFAD) with the AD multi-cistronic vector. The JB-PEFAD cell was confirmed on mRNA expression, protein synthesis of hAPP, hTau and hPS1 and identification of integration and karyotype. Although fusion rate was no difference in SCNT with JB-PEF AD (SCNTAD) embryos, cleavage and blastocyst formation rates were slightly lower than in SCNT with non-transgenic JB-PEF (SCNTnon-TG). Individual SCNTAD blastocysts were detected hAPP, hTau and hPS1 genomic integration which showed 93.2% (n=30) efficiency in genomic DNA (gDNA) level. It will give us a possibility to develop porcine animal model for AD study in the future.

Allicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study was to investigate the effect of allicin treatment during in vitro maturation (IVM) on porcine oocyte maturation and developmental competence. Porcine follicular oocytes were cultured in 0 (control), 0.01, 0.1, 1, 10, and 100 μM AL added IVM media. The rate of polar body emission was higher in the 0.1 μM AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 μM AL-treated group than in the control (p < 0.05). The reactive oxygen species level at metaphase II was not significantly different among all groups. In matured oocytes, the relative mRNA expression of both BAK and CASP3, and BIRC5 were significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Also, the mRNA expression of BMP15 and cyclin B, and the activity of phospho-p44/42 MAPK, was significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

Lysophosphatidic acid (LPA) is a member of the phospholipid autacoid family and has growth factor and hormone-like activities on various animal cells. In this study, we investigated the effect of LPA on porcine embryo development. Porcine parthenogenetic embryos were treated into various concentrations of 0 (control), 0.1, 1 and 10 μM LPA (0 LPA, 0.1 LPA, 1 LPA and 10 LPA) during in vitro culture for 7 days or cultured in basic culture medium until day 4 and treated LPA from day 4 to day 7. In the LPA treatment for culturing from day 0 to day 7, there was no significant difference on cleavage and blastocyst formation rate. In addition, the blastocyst development proportion which was classified as expanded, hatching, or hatched blastocystshas was no significant difference among all groups. In the LPA treatment for culturing from day 4 to day 7, 0.1 and 1 LPA groups were presented increased blastocyst formation compared to other groups, but cleavage rate and over-expanded blastocyst formation rate were not significantly different among all LPA treated groups. The total cell number was not different but apoptosis was reduced when 1 LPA treated from day 4 to day 7. The relative mRNA expression level of anti-apoptosis gene, BCL2L1 was higher and pro-apoptosis gene, BAK was lower in the 1 LPA treated group than the control. In comparison with the control and the 1 LPA treated group using time-lapse monitoring system, 1 LPA treated embryo was accelerated developmental speed via morula compaction and expanded blastocyst. The 1 LPA treated group significantly increased the relative expression levels of gap junction and tight junction related genes, GJD1, CDH1 and ZO-1 compared to the control. These results indicated that 1 μM LPA supplementation for culturing from day 4 to day 7 post activation is efficient in blastocyst formation and LPA may be helpful for embryo developmental capacity.

Although in vitro production (IVP) techniques of porcine follicular oocytes have progressed and are well studied, the developmental potential of porcine oocytes matured in vitro remains low compared with those matured in vivo. It is well known that one of the reason occurred impair in vitro maturation (IVM) of porcine oocytes is the oxidative stress. Oxidative stress is mainly caused by reactive oxygen species (ROS) generation formed during cellular metabolism. β-cryptoxanthin (BCX) is one of the carotenoid pigment and possesses strong anti-oxidative and free radical scavenging activities and suppresses lipid peroxidation and nitrogen oxide production. The objective of this study was to examine the effects of BCX treatment on porcine oocyte during IVM and their in vitro developmental potential. The follicular oocytes were cultured in IVM medium supplemented with 0, 0.1, 1, 10 and 100 μM BCX (control, 0.1 B, 1 B, 10 B and 100 B). In analysis of intracellular ROS expression level after IVM, 1 B group was the lowest among all groups (p<0.05), while other BCX treated groups are similar to control group. Also, 1 B group was significantly decreased during the classified oocyte maturation stage (GVBD, MⅠ and MⅡ) than control (p<0.05). In addition, the relative mRNA expression level of antioxidant gene (superoxide dismutase-2 and peroxiredoxin-5) was significantly higher in 1 B group than control (p<0.05). After parthenogenetic activation, there was no different in the cleavage rate between two groups, however, the blastocyst formation rate was significantly higher in 1 B group than in control (p<0.05). In embryo quality, the total cell number and DNA fragmentation of blastocysts were no different between two groups. These results demonstrate that BCX is helpful for decreasing ROS level of porcine follicular oocytes and improves their developmental potential.

Allicin (AL) is one of the biologically active substance in garlic. Many researchers found that AL exhibits 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 development. The oocyte was maturated in vitro for 44 h (control) without AL or 44+24 h IVM (24 h aging) with 0, 0.1, 1, 10 and 100 μM allicin (0 AL, 0.1 AL, 1 AL, 10 AL and 100 AL). The 1 AL treated group was significantly increased on maturation rate compared to the 0 AL treated groups, but the other treated groups were not different compared to the 0 AL treated group (p < 0.05). The 1 AL treated group was significantly increased on normal spindle formation and chromosome alignment compared to 0 AL treated group. We checked the effect of AL on parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development using aged oocytes. PA embryos in 1 AL treated group significantly increased the cleavage and blastocyst formation rate compared to control (p < 0.05). However, SCNT embryos of 1 AL treated group were no significant differences in embryo development. In PA embryo quality, the total cell number was significantly higher in 0.1 AL, 1 AL and 10 AL treated groups than control and DNA fragmentation rate in 1 AL was the lowest among all groups. SCNT embryo in 1 AL significantly increased total cell number and decreased DNA fragmentation. The AL treatment on aged oocyte enhanced PA and SCNT embryo developmental capacity. Therefore, AL may be helpful for assisted reproductive technology applying aged oocytes.

Somatic cell nuclear transfer (SCNT) technique is a key point of producing transgenic animal disease models. During in vitro production of SCNT embryo, the quality of matured oocytes are one of the important factors that regulate embryo developmental capacity. In preliminary test, we confirmed the effect of fibroblast growth factor 10 (FGF10) on porcine oocyte maturation. In this study, we investigated the developmental potential of SCNT embryos treated with the 10 ng/ml FGF10 (10 F) during in vitro maturation of recipient oocytes. The polar body emission rate was significantly higher in the 10 F treated group than control group. After SCNT, although the rate of fusion was no significant difference, the rate of cleavage and blastocyst formation was significantly increased in the 10 F treated group (p<0.05). In 10 F treated group, the total cell number was increased and the percentage of apoptotic cell was decreased in the blastocyst stage at day 7 (p<0.1). The transcription level of apoptosis relative gene, Casp3 was significantly decreased, while anti-apoptosis gene BCL2l1 was increased in the 10 F treated group compared to control group. The 10 F treated group was highly expressed the reprogramming related genes, Sox2 and POU5f1. Also, the first cleaving time was more faster and the percentage of cell block was significantly lower in 10 F treated group than in control group. In this study, we confirmed that 10 ng/ml FGF10 has effect on enhance the oocyte maturation and developmental capacity. These results demonstrate that FGF10 treatment can be used for in vitro development of porcine SCNT embryos and subsequent production of transgenic animal model.