• Food Science of Animal Resources
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• v.28 no.2
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• pp.181-186
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• 2008

This study was conducted to investigate the effects of supplementation with transgenic Bacillus subtilis secreting chitinase on the growth performance, nutrient digestibility, blood characteristics and carcass traits in finishing pigs. A total of sixty-four pigs ($50.82{\pm}0.82kg$, average initial body weight) were assessed over a period of 84 days. Dietary treatments included: 1) CON (basal diet without antibiotics), 2) AD (basal diet + 0.1% Virginiamycin), 3) CD0.5 (basal diet + 0.5% transgenic B. subtilis), 4) CD1.0 (basal diet + 1.0% transgenic B. subtilis). Each dietary treatment had 4 replicates of 4 pigs per pen in a randomized complete block design. In terms of growth performance, the ADG (average daily gain) and gain/feed ratio were significantly increased with the CD1.0 diet compared to the AD diet during weeks 0-4 (p<0.05). During weeks 4-8, the ADG was significantly increased with the AD diet compared to the CON and CD0.5 diets (p<0.05). The ADFI was also significantly increased with the AD diet compared to the other diets (p<0.05). During weeks $8{\sim}12$, the ADFI was significantly increased with the CON diet compared to the other diets (p<0.05), and the gain/feed ratio was significantly increased with the CD0.5 and CD1.0 diets compared to the AD diet (p<0.05). Over the entire 84 day test period, the ADFI was significantly increased with the AD diet compared to the CD0.5 and CD1.0 diets (p<0.05). The gain/feed ratio was significantly increased with the CD0.5 and CD1.0 diets compared to the CON diet (p<0.05). In terms of meat color, the L value was significantly increased with the CD0.5 diet compared to the CON and AD diets (p<0.05), and the a value was significantly increased with the CON diet compared to the other diets (p<0.05). In terms of sensory evaluation, meat color was significantly improved with the CON, CD0.5 and CD1.0 diets compared to the AD diet (p<0.05). Marbling was significantly increased with the CON diet compared to the other diets (p<0.05). Firmness was significantly increased with the CD0.5 diet compared to the AD diet (p<0.05). In conclusion, supplementation with transformed B. subtilis secreting chitinase improved gain/feed ratios and influenced meat color. Thus, we suggest that transformed B. subtilis secreting chitinase can partially substitute for antibiotics.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.205-209
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• 2008

This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5' recombination arm and a 2.7-kb fragment as the 3' recombination arm. We used the neomycin resistance gene ($neo^{r}$) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.13-19
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• 2006

To search of new porcine pheromonal odorants for biostimulation control system technologies to improve reproductive efficiency in livestock species, the comparative molecular field analysis (CoMFA) for binding affinity constant $(p(Od)_{50})$ between porcine odorant binding protein (pOBP) and ligands of odorant 2-(cyclohexyloxy) tetrahydrofurane derivatives as substrate molecule was conducted and discussed. In the optimized CoMFA model AIV with chirality $(C_1'(R),\;C_2(S))$ in substrate molecule and atom based fit alignment (A) of odorants, the statistical results showed the best predictability of the binding affinities $(p(Od)_{50})$ based on the LOO cross-validated value $r^2_{cv}.\;(q^2=0.886)$ and non-cross-validated conventional coefficient $(r^2_{ncv}.=0.984)$. the binding affinity constants exhibited a good correlation with steric (40.8%), electrostatic (14.6%) and hydrophobic (44.6%) factors of the substrate molecules. from the analytical results of the contour maps, which may give us some valuable informations to the modification of odorants for effective binding affinity.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.169-174
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• 2006

To search of a new porcine pheromonal odorants, the comparative molecular similarity indices analysis(CoMSIA) between porcine odorant binding protein(pOBP) as receptor and ligands of green odorants 2-(Cyclohexyloxy)tetrahydrofurane derivatives as substrate molecule were conducted and disscused quantitatively. In the optimized CoMSIA model(I-AI) with chirality($I:\;C_{1'}(R),\;C_2(S)$) in substrate molecules and atom based fit alignment(AE) of the odorants the statistical PLS results showed the best predictability of the binding affinities based on the LOO cross-validated value ${r^2}_{cv.}\;(q^2=0.856)$ and non cross-validated conventional coefficient(${r^2}_{ncv.}=0.964)$). The structural distinctions of the highest active molecules were able to understand from the interaction between pOBP and green odorants in the contour maps with CoMSIA model.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.181-187
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• 2006

In recent years the number of patients waiting for organ transplantation has greatly outpaced the supply of human organs available, which leads to a renewed interest in pig-to-human xenotransplantation as an alternative. However, one of the biggest barriers in the xenotransplantation is presence of porcine endogenous retroviruses(PERV) that can infect human cells. In this study, to present a possible solution for this problem we tried to inhibit expression of PERVs using shRNAs(short hairpin RNA) at the level of RNA synthesis and virus release. The shRNA targeting the sequence of PERV A, B type was cloned into pSIREN-RetroQ vector under the control of polymerase-III U6-RNA gene promoter. Quantitative real-time PCR was performed to detect my alterations in mRNA production of PERV A, B targeted by the shRNA in each done. Depending on the target sequence of the shRNA, the transcription of PERV was decreased to as much as 4% and the number of progeny viruses was reduced to less than 1/200,000. Transgenic pigs producing such shRNAs may result in a highly reduced PERV expression in cells and organs, which is a prerequisite for safe xenotransplantations.

• Journal of Veterinary Clinics
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• v.24 no.2
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• pp.94-98
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• 2007

This study was to investigate the effects of ascorbic acid and alpha-tocopherol on the attenuation of renal ischemia-reperfusion (IR) injury in pigs. Ten pigs were subjected to 60 minutes of warm unilateral renal ischemia followed by removal of contralateral kidney and then divided into two groups. Treatment group was performed ascorbic acid and alpha-tocopherol pretreatment 2 days before operation and ascorbic acid with heparin-saline solution irrigation-aspiration. Otherwise, control group used only irrigation-aspiration of heparin-saline solution. Blood samples were collected from these pigs for measurement of serum blood urea nitrogen (BUN) and creatinine values, antioxidant superoxide dismutase (SOD) at pre, day 1, day 3, day 7 and day 14. The kidneys were taken for histopathologic evaluation after euthanasia on postoperative day 14. The levels of BUN were significantly increased in the control group on day 1, day 3 and day 7 (P<0.05). And the level of creatinine was significantly increased in the control group on day 3 (p<0.05). Activity of antioxidant enzymes in plasma revealed significant difference (p<0.05) between control and treatment group at day 14. In histopathologic findings, treatment group was showed less damage than that of control group on the basis of renal tubular damage. It was concluded that ascorbic acid and alpha-tocopherol attenuated renal I/R injury in the pigs.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.153-158
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• 2008

Tissue-specific and temporal regulation of milk protein gene expression is advantageous when creating transgenic animal that produces foreign protein into milk. Gene expression, i.e. protein production, is regulated not only by promoter strength but also mRNA stability. Especially, poly A tail length by polyadenylation affects in vivo and in vitro mRNA stability and translation efficiency of the target gene. In the present study, nucleotide sequence of 3'-UTR was analyzed to evaluate the effects of mRNA stability on the target gene expression. Based on the poly A signal of 3' -untranslated region (UTR), nucleotide sequences of putative cytoplasmic polyadenylation elements (CPEs) and downstream elements (DSEs: U-rich, G-rich, GU-rich) were analyzed and used to construct 15 luciferase reporter vectors. Each vector was transfected to HC11 and porcine mammary gland cell (PMGC) and measured for dual luciferase expression levels after 48 hours of incubation. Luciferase expression was significantly higher in construct #6 (with CPE 2, 3 and DSE 1 of exon 9) and #11 (with CPE 2, 3 and DSE 1, 2 and 3 of exon 9) than construct #1 in the PMGC. These results suggest that expression of target genes in PMGC may be effectively expressed by using the construct #6 and #11 on production of transgenic pig.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Journal of Veterinary Clinics
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• v.24 no.3
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• pp.300-304
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• 2007

The purpose of this study was to determine the anesthetic effects of tiletamine-zolazepam (TZ) alone and azaperone plus tiletamine-zolazepam in growing pigs, and to compare the various physiological parameters in both treatments. Cross experiment was accomplished at 2-week interval. Group 1 (TZ group): six pigs ($31.4{\pm}4.83$ kg) received 4.4 mg/kg of TZ alone. Group 2 (ATZ group); the same six pigs ($43.6{\pm}4.31$ kg) received 4.4 mg/kg of TZ twenty minutes after receiving 2 mg/kg of azaperone. All of the anesthetic drugs were injected into the trapezius muscles. The pigs were fasted for 24 hours before the experiments. Induction and recovery values were determined. Heart rate, respiratory rate, rectal temperature, $pO_2,\;pCO_2$ and pH were determined before administration and 5, 25, 45, 65 and 85 minutes after administration. Induction time of ATZ group was more rapid than that of TZ group (p<0.01). During recovery, sternal recumbency time, standing time and walking time of ATZ group were longer than those of TZ group (p<0.01). Heart rate, respiratory rate, $pO_2,\;pCO_2$, and pH did not show especial differences between the two groups. However, rectal temperature was significantly different between the TZ and ATZ group (p<0.05). As a result, ATZ group had a faster induction and a longer duration of anesthesia than TZ group did. Thus, it was concluded that ATZ combination could be usefully used for chemical restraint in pigs.