Salmonellosis constitutes an important public health problem in both developing and developed countries, including Korea. The aims of present study were to investigate the serovar and antimicrobial resistance of Salmonella isolated from food animals and animal products in slaughterhouses and farms. A total of 323 Salmonella were isolated from food animals (n=277) and meats (n=46) during 2010. Of the isolates, 21 different serovars were identified. The predominant serovars were S. Rissen (35%) and S. Montevideo (24.3) in healthy pigs, while S. Enteritidis (25.5%) in healthy chicken. S. Typhimurium (88.8%) was predominant in disease pigs, while S. Gallinarum (29.2%) and S. Montevideo (26.9%) were in diseased chickens. Among meat samples, S. Typhimurium (57.1%) was the most common serovar in pork but S. Enteritidis (38.7%) and S. Montevideo(32.3%) were in chikcen meats. Analysis of antimicrobial resistance patterns revealed that 20.7% of the isolates were sensitive to all the 15 drugs tested. The isolates were frequently resistant to nalidixic acid (47.7%), tetracycline (38.4%), streptomycin (33.7%), and ampicillin (32.8%). The resistance to quinolone and 3rd generation cephalosporins was higher in chicken and chicken meat isolates. Of the 323 isolates, 174 (53.9%) were resistant to one or more CLSI subclass, and 117 (36.2%) showed multiple-resistance. Our findings showed that multiple resistant Salmonella organism are widespread in animals and animal products in Korea. To prevent the transmission or exposure for consumers of antimicrobial resistant Salmonella, policies and guidelines aiming at prudential use of critical antimicrobials for humans are needed.

Campylobacterosis is the most common food borne bacterial disease in many countries. Food animals and animal products are considered to be the reservoir of the Campylobacter species. The aim of this study was to investigate the antimicrobial resistance of Campylobacter spp. from food animals and raw meats in slaughterhouses. A total of 90 Campylobacter jejuni (C. jejuni) and 127 Campylobacter coli (C. coli) were isolated, for which antimicrobial susceptibility was examined using broth dilution method. Resistance to macrolide antimicrobials was higher among C. coli isolates than among C. jejuni. Among both C. jejuni and C. coli isolates, the most frequently observed resistance was to nalidixic acid, ciprofloxacin, and tetracycline. No erythromycin resistance was observed among C. jejuni isolates from cattle, pig and beef. However, 28.3% (n=13) and 25% (n=3) of C. coli isolates from pigs and pork showed resistance to erythromycin, respectively. The predominant profile of multiple resistance among C. jejuni and C. coli isolates was ciprofloxacin/tetracycline/nalidixic acid resistance (46.7%) and ciprofloxacin/nalidixic acid resistance (31.5%), respectively. This finding has important implication for food safety and public health.

Staphylococcus aureus is an important food-borne pathogen, which is present on the skin and mucosa of animals. Some of the S. aureus strains are causative agent of food poisoning syndrome. The aim of this study was to investigate the antimicrobial resistance of S. aureus isolates from raw meats in slaughterhouses during 2010. From 17,874 raw meat samples tested, a total of 190 S. aureus were isolated, for which antimicrobial susceptibility to 17 agents was examined using broth dilution method. Among isolates from beef, chicken and pork, 20 (51.3%), 20 (24.7%) and 9 (12.9%) were sensitive to all antimicrobials tested, respectively. Isolates from pork and chicken meats showed much higher resistance, compared to isolates from beef. Penicillin resistance was the most frequent among isolates from beef (35.3%) and pork (75.7%), while tetracycline resistance was among those from chicken meats (48.1%). A total of 3 methicillin resistant S. aureus (MRSA) were detected from beef (5.1%, 2/39) and pork (1.4%, 1/70). Although the prevalence of MRSA was low, the presence of antimicrobial resistant S. aureus such as MRSA suggests that further investigation and strict surveillance on MRSA and antimicrobial resistance are needed.

This study was conducted to improve the in vitro maturation(JVM), in vitro fertilization (IVF) and in vitro developmental capacity of oocytes derived from slaughtered Korean native cattle. The recoverd oocytes, obtained from a local slaughter house, were used completely surrounded by at least 3 layers of cumulus cells in combination with a homogeneous cytoplasmic pigmentation. In vitro maturation was induced in TCM-199 or Ham's F-10 supplemented with LH(1O g/rnl), FSH(35 g/ml), estradiol-17(1 g/ml) at 39 under 5% in air for 24 hours. Sperm from caudal epididyrnis and previously matured cumulus-oocytes complexes were cultured for 24 hours in 100 l droplets of fertilization media under paraffin oil. The zygotes were cultured with media(TCM-199 with bovine oviductal epithelial cells or CRlaa) for 7 to 10 days. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following maturation using Ham's F-10 (59.9%) than TCM-199 (51.6%). Development to the blastocysts among cleaved embryos was not signficantly different between maturation media: Ham's F-10 (16.0%) and TCM-199(11.9%). However, the hatching rate was affected significantly (P<0.05) on rnaturation media as 62.9% in Ham's F-10, compared with 41.2% in TCM-199. The cleavage rate of IVM-IVF oocytes was significantly (P<0.05) higher following IVF using m-TALP medium (80.1%) than BO medium (51.6%). The percentage of in vitro developed blastocysts among cleaved embryos was not signficantly different between fertimization media: BO (11.7%) and m-TALP (17.6%). The cleavage and the developmental rate to the blastocysts after IVF in m-TALP or condition medium(CM) with or without oviduct epithelial cell monolayer(OECM) was similar(80.1% and 17.6% in m-TALP, 83.8% and 19.4% in M-TALP with OECM. 82.9% and 18.9% in CM, 87.6% and 16.0% in CM with OECM, respectively). The percentage of in vitro developed blastocysts among cleaved embryos was significantly (P<0.05) higher in TCM-199 medium co-cul tured with bovine oviduatal epithelial cell monolayers(35.2%) than CRlaa medium(1.9%). These results stggest that the most transferable IVF embryos could be produced from Ham's F-10, m-TALP and TCM-199 medium with bovine oviductal epithelial cell monolayers for IVM, IVF and IVC, respectively.

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39 in a 5% incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

The present study was undertaken to determine the optimal condition for parthenogenetic activation of rabbit oocytes by electric stimulation in vitro in an attempt to develop nuclear transplantation techniques for cloning mammalian embryos and animals. Freshly ovulated oocytes were collected from superovulated rabbits from 13 to 26 hrs. after hCG injection. The cumulus-free oocytes were activated parthenogetically by repeated stimuli of square direct electric pulses in O.3M mannitol solution. After applying electric stimulations of different voltages, pulse durations and pulse times, all of the oocytes were cultured in TCM-199 with 10% FCS for 96 hours in a 5% incubator, and their developmental potential in vitro was examined. The higher activation rate (68.9%) was achieved at the voltage of 2.0kv/cm, the pulse duration of 60 sec and three pulse times and the activation rate of 100% was achieved at the pulse duration of 100 and 200 sec, the voltage of 1.5kv/cm and three pulse times. Although the higher rates of activation of oocytes were achieved at 100 and 200 sec, none of them developed to blastocyst in vitro. The oocytes collected 18~20 hours post hCG injection showed the highest rate of activation and development to blastocyst in vitro than the oocytes collected 13~15 or 25~26 hours post hCG injection. Therefore, it can be suggested that the application of electric stimulation of 2.0kv/cm, 60 sec and three pulse times to the oocytes collected at 18~20 hours post hCG injection would be more beneficial for the parthenogenetic activation of oocytes in rabbits.

The nudear changes of bovine oocytes during 24 hrs. of culture for mejotic maturation were examined. Bovine oocytes were collected from small(<2 mm), medium(2~6 mm) and large(>6mm) follicles and classified into three grades by their morphological characteristics. A total of 242 oocytes collected were obtained:from 184 small, 157 medium and 1 large follicles, respectively and were classified into 95 grade I, 155 grade H and 92 grade III oocytes. All the bovine oocytes collected and graded were washed with a basal medium and incubated in groups of 10 for 24 hrs in 5% and 39. The basal medium used was composed of TCM-199 supplemented with sodium bicarbonate, sodium pyruvate, streptomycin, penicillin G and 10% FCS. The oocytes were cultured in drops of 50,l basal medium supplemented with 35g /ml FSH, 10g /ml LH and 1g /ml estradiol-17. The oocytes were fixed and examined on their chromosomal status by 1% acetorcein staining in the interval of 3 hrs. Most of the grade I oocytes developed to germinal vesicule stage at 0 to 3 hrs., germinal vesicle breakdown at 6 hrs., metaphase I at 9 to 15 hrs., anaphase I and telophase I at 18 hrs., and metaphase II and the first polar body at 24 hrs. after culture for meiotic maturation. However, it was found that compared to grade I oocytes, grade H and W oocytes reached earlier to germinal vesicle breakdown and most of them developed earlier to M II stage at 21 hrs. after culture.

These experiments were carried out to determine the effect of cell stage in embryo bisection on the sub-Sequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgicaily bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using a microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stage. The successful rate of mouse embryo bisection at 4-cell stage (59.0%) was significantly (p <0.05) lower than those at 8-cell (75.6%), 2ce11 (80.7%) or morula stage (84.8%), and highest at blastocyst stage (95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst,the in vitro de'velopment of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-em-bryos bisected at 2- to 8-cell stage was signiflcantiy (p <0.05) lower.The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage,respectively and not significantly (P <0.05)different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2- or 8-cell stage were significantly (P <0.05) lower than the result from the intact embryos of the corresponding stage.