The purpose of this study was to determine whether high voltage pulsed current stimulation (HVPCS) would enhance wound healing in neuropathic rabbits. Ten rabbits were assigned to either an experimental or a control group. The wounded part around the peripheral neuropathy of the experimental rabbits was stimulated for two hours twice a day for six days under the following conditions: pulse frequency 80 pps, pulse duration , and stimulation intensity 30~40 V. The results indicated that there was no difference in the wound closure between the experimental and control groups. The two groups showed similar aspects in collagen and reticulum, which were observed by colored Masson's trichome. While the rabbits in the control group had more or less thick fibers, the rabbits in the experimental group had thin and branched-shape fibers. The rabbits in the experimental group showed both strong responses in the shaping of elastic fibers and the increased aspects in fibroblast when compared with the control group.

The efficiency of transgenic livestock animal production may be improved by early selection of transgenci preimplantation embryos. To examine the possibility of GFP gene as a non-invasive marker for the early screening of transgenic embryo, the GFP gene was microinjected into rabbit zygotes and the later stages of preimplantation embryos were examined for the expression of GFP. The presence of injected DNA was detected by PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Out of 108 GFP gene-injected rabbit zygotes, seventy three(67.6%) were fluorescence-positive. When 11 fluroresecence-positive blastocysts were analyzed for the presence of GFP gene by PCR, 6(54.5%) were positive, and all of the 8 flrouescence-negative blastocysts were also negative by PCR. The results indicate that the screening of transgene in rabbit embryos by PCR analysis and GFP detection could be a promising method for the preselection of transgenic embryos.

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 sec) in 0.3 M mannitol solution supplemented with 100 M CaCl and MgCl. The nuclear donor embryos of 8-cell stage were synchronized to G phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

In order to improve the cryopreservation by vitrification or slow freezing of nuclear transplant rabbit embryos, the effects of factors affecting embryo cryopreservation such as cryoprotectants, equilibration, cooling rate and post-thaw dilution on post-thaw survial and development were determined using intact embryos of morular stage. And the post-thaw development of nuclear transplanted embryos cryopreserved under the optimal conditions examined was compared between vitrification and slow freezing. The cryoprotectant solution used was ethyleneglycol-ficoll-sucrose (EFS) or ethyleneglycol-poly-vinylpyrrolidone-galactose- I (EPG- I ) for vitrification, and EPG- II for slow freezing. To examine the viability of frozen-thawed embryos, the nuclear transplanted embryos were co-cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) for 24 hrs and the intact morulae were co-cultured with BOEC for 5 days and 3 days to hatching blastocyst stage in 39 ˚C 5% incubator. The results obtained were as follows: Following vitrification with EFS, the post-thaw development of rabbit morulae to hatching blastocyst was significantly(P<0.05) higher in compacted stage(82.4%) than in early morular stage(60.0%). The post-thaw development of compacted morulae to hatching blastocyst was similarly high in vitrification with EFS(82.4%), EPG- I (85.0%) and in slow freezing with EPG- II (83.3%). Following vitrification with EPG- I, the post-thaw development of intact rabbit morulae to hatching blastocyst was similar as 78.0% and 85.0% in 1-step and 2-step post-thaw dilution, respectively. The post-thaw development of nuclear transplanted rabbit embryos of compacted morulae stage to hatching blastocyst was similarly 43.6% and 40.0% in vitrification with EPG- Iand slow freezing with EPG- II, respectively. These results indicated that the rabbit nuclear transplant and intact embryos of morulae stage could be well cryopreserved with either vitrification or slow freezing procedure.

토끼에서는 수정란 이식과 같은 기본적인 번식공학적 방법의 효율성이 아직 생쥐와 같은 실험동물에 비해 떨어지고 있어 생물공학적인 기술을 응용하는데 큰 어려움이 있다. 특히 유전자 이식에 의한 형질전환 토끼의 생산과 같은 생물공학적인 기술을 실용화하는데 효율이 높은 수정란이식 기술의 개발이 필수적이라고 할 수 있다. 본 연구에서는 토끼에서 수정란 이식 기술의 첫 단계인 과배란 유도를 효율적으로 이용할 수 있는 방법을 정립하기 위해 반복적인 과배란 유도가 배

The growth inhibition effect of orally administrated yogurt ACE and Metchnikoff upon E. coli O157:H7 and S. typhimurium inoculated into gastric lumen of rabbits was investigated. The rabbits challenged with each 1 ml of suspension containing 10^8 CFU/ml of the pathogens were divided into 4 groups by the interval of yogurt administration: A group; preadministrated 7 days before inoculation of the pathogens and fed daily; B group; administrated daily after inoculation of the pathogens, C group; administrated every 3 days after inoculation of the pathogens; Control group, not fed after inoculation of the pathogens. Each 3 ml of yogurt containing 10^9 CFU/ml was orally administrated into rabbits. All yogurt administrated groups (A, B, C) showed growth inhibition effect on E. coli O157:H7 in one day after inoculation of the pathogen by the level of 0.8-1.0 log CFU/g, compared with the result differences between the control group and the yogurt administrated groups. In the control group after 5 days of inoculation, the number of colonized pathogens was 10^5-10^6 CFU/g, whereas 10³-10⁴ CFU/g was detected in the yogurt administrated groups. After 10 days of inoculation, the viable pathogen number per gram (g) of the rabbit feces was 10³ CFU/g in the control group, whereas the number below 10¹ CFU/g was detected in the group A, and 10² CFU/g in the group B and C. The growth inhibition effect of yogurt administration on E. coli O157:H7 was highly increased in the order of A, B, and C group. The same effect on S. typhimurium was observed at the level of 2 log CFU/g in the Metchnikoff yogurt administrated groups, compared with the control group result in one day after inoculation of the pathogen. In 7 days after inoculation of the pathogen, the viable number was increasingly decreased, and finally after 15 days no viable cell of S. typhimurium was discharged into the fecal samples in the group A, and the mean level of 10¹ CFU/g was detected in the group B, but there was no growth inhibition effect in the group C. The growth inhibition effect on S. typhimurium was observed at the same level of viable cell number between the yogurt ACE administrated groups and the control group in 5 days after inoculation. But, after 10 days of inoculation the viable cell number was started to decrease, and the viable cell of S. typhimurium was not discharged from rabbit intestinal contents after 15 days of inoculation in the yogurt ACE administrated groups. In such a case that yogurt was administrated in order to prevent the pathogens, pre-administration on a daily basis one week before inoculation of the pathogens exerted considerable effect in growth inhibition. In comparison with two kinds of yogurt tested in this study, the growth inhibition effect on two kinds of pathogens was observed more highly in the Metchnikoff administrated group than the ACE administrated group.

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.