This study was undertaken in an effort to develop a cryopreservation system of immature and mature porcine oocytes. For this aim, the experiments were designed to examine the effect of cryoprotectants and equdibration time on the viability of frozen-thawed oocytes by using trypan blue(TB) and fluorescene diacetate(FDA) test. The viability of frozen immature oocytes evaluated by TB test was slightly higher than that of frozen mature oocytes. The viability(25.O%) after IVM of frozen-thawed immature oocytes greatly decreased that(42.9%) of oocytes just after thawing, but it was higher than frozen-thawed mature oocytes(15.8%). When immature oocytes were equilibrated for 10, 20 and 30 minutes before freezing the oocyte viability was 20.0, 31.3 and 42.9%, respectively. There was a tendency for long equilibration before oocyte freezing to be more effective for the immature oocytes and a short equilibration time for mature oocytes. Although there was no difference in viability index of frozen oocytes hetween the viability test methods, the index of TB test was slightly higher than that of FDA test. The viability(FDA test) of frozen-immature oocytes with 3 different crtoprotectants was 22.2% for propylene glycol(PG), 9.3% for polyehtylene glycol(PEG) and 65.6% for PG+PEG, in which PG+PEG was more protective against freezing effect.

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

Escherichia coli O157:H7 and Salmonella ser. typhimurium are pathogens involved in food poisoning in numerous countries. This study aimed to obtain knowleges on the survival of E. coli O157:H7 KSC 109 and S. ser. typhimurium ATCC 14028 in fermented milk products which were on sale in Suwon Yakult supplier. To the final concentration of 10³-10⁴ cfu/ml of E. coli O157:H7 KSC 109 or S. ser. typhimurium ATCC 14028 in the fermented milks, Metchnikoff, Ace, Yakult, Mastoni and Super100 were inoculated with these pathogens and then were stored at 4 and viable cells of these pathogens were periodically counted. The results showed that the survival of two pathogens differed in the different types of fermented milks tested. Number of surviving E. coli O157:H7 KSC 109 and S. ser. typimurium ATCC 14028 cells (initial inoculum, 10³-10⁴ cfu/ml) were decreased to 10', 102 cfu/ml in Ace after 100 hours, and were decreased gradually to 10¹ cfu/ml in Yakult after 250 hours. In the other fermented milks, viable cells of E. coli O157:H7 KSC 109 was not drastically decreased but those of S. ser. typhimurium ATCC 14028 was decreased gradually to 10² (Mastoni), and to 10¹ cfir/ml (Super100) after 250 hours. It appeared that S. ser. typhimurium ATCC 14028 was more susceptible than E. coli O157: H7 KSC 109 at low pH. Vibale cells of E. coli O157:H7 KSC 109 was not drastically decreased in most of fermented milks tested except Ace and Yakult, but in general, S. ser. typhimurxum ATCC 14028 was drastically decreased in most of the fermented milks. The major inhibition factor against these pathogens in the fermented milks during storage at 4℃ appeared to be the acidity and the metabolites produced by the starters bacteria used in fermented milk products.

치밀 난구세포로 둘러싸인 소 난자를 . 5% 배양기에시 5% superovulated cow serum(SCS)이 첨가된 m-TCM 199 medium 으로 시간 배양하였으며, 수정능이 획득된 정자와 체외수정하였다. 7일8일경의 수정란을 1.3M methyl cellosolve(MC), 1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) 및 1.1M 1,3-butylene glycol(BG) 용액에서 10분간 평형시킨 후 0.25 ml 스트로내에 장전하였다. 스트로를 의 alcohol bath freezer에 넣고 까지 /분 속도로 냉각, 식빙 후 10분간 정체시켰으며, /분 또는 /분으로 까지 냉각 후 스트로를 액체질소에 침지하여 보관하였다. 수정란이 들어있는 스트로를 온수에서 융해하였으며, 수정란을 TCM 199 medium 으로 옮긴 후 5% SCS가 첨가된 TCM 199 medium 에서 48시간 배양하였다. 수정란이 양호한 형태를 유지하며 나중의 발육단계로 진행된 것을 생존한 것으로 간주하였다. 각 종류의 동해방지제에서 동결된 수정란의 일부는 융해 후 동해방지제를 제거하지 않고 직접 비외과적으로 이식하였다. 동결-융해 후 동해방지제의 종류에 따른 탈출배반포 발달율은 EG 50.0%, MC 53.6%, DEG 56.9%, PG 58.0% 그리고 BG 11.5%였다. /분 또는 /분 으로 냉각한 수정란의 생존율은 두 그룹간에 유의적인 차이가 없었으나 (P<0.05), 탈출배반포 발달율은 -0.5분 /분(22.6%, 12/53)보다 /분(64.6%, 31/48) 냉각시에 유의적으로 높았다(P<0.01). 동해방지제의 종류에 따른 수정란의 수태율은 MC 48%(10/21). DEG 30%(3/10), EG 74%(20/27) 및 PG 40%(4/10) 였다. 이러한 결과로 보아 MC, DEG, EG 그리고 PG는 소의 체외수정란의 동결을 위한 동해방지제로서 이용될 수 있음을 보여주었다.

This study was carried out to increase the viability of bovine frozen4hawed in vitro produced (IVP) embryos and pregnancy rate by direct transfer method. Cumulus-oocyte complexes were aspirated from excised Hanwoo ovaries and matured in TGM 199 for 20~22 hours at 38.5 in 2% in air. Matured oocytes were fertilized with capacitated sperm for 6 hours and then co-cultured with cumulus cells for 9 days. 63% of the oocytes cultured was deaved and 29% out of them developed into blastocysts. Good or excellent grade of blastocysts on D 7 or 8 were frozen with 1.8M ethylene glycol as a cryoprotectant for direct transfer. Frozen embryos were thawed at 2 water for 10 sec following 4~5 second in air. For the survival assay of frozen4hawed lVP blastocysts, they were cultured in TCM 199 supplemented with 100M -mercaptoethanol and 20% FCS for 72 hours. The percentage of embryos developed to re-expanded or hatched after 72 hours culture was 95. 5 and 77.3%, respectively. When frozen-thawed Ivp embryos were transferred to 43 synchronized recipients by direct transfer method, eighteen recipients (41.8%) was pregnant. The highest pregnant was in naturafly synchronized recipients (71.4%), but induced estrus by using PRID(29.2%) and PGF(20.0%) was showed lower pregnancy rate. The pregnancy rate was higher in day 7 blastocysts(56.0%) than day 8 blastocysts(22.2%). (Key words: in vitro produced, blastocyst, frozen-thawed, direct transfer)

The effect of superovulation (PMSG, FSH) on the ovarian response of matured cows were tested. The survival rates of bovine embryos and ovarian oocytes frozen by slow, rapid freezing and vitrification were investigated. A total of 15 heads of cow were devided into 3 groups by injection dose of GTH (PSMG, FSH). Each group was superrovulated with injections of 2, 500, 3, OOOJU PSMG and 40mg FSH followed by injection of 30mg PGF2a. Embryos were non-surgically recovered from superovulated cows 6~7days after estrus. The recovered embryos were frozen in 10% glycerol + 10% sucrose by slow and rapid freezing. Ovarian oncytes were frozen in 20% g]ycerol+l0% ethylene glycerol + 30% Ficol + 10% sucrose by vitrification and the survival of frozen embryos and ovarian oncytes were judged by FDA-test. The results are summarized as follows; 1. Estrus after the injection of 2500, 3000 I.U. PMSG and 4Omg FSH were 32.8, 35.0 and 43.4 and the duration of estrus were 18.6, 18.8 and 22.4 hours respectively. 2. The average sizes of the left ovaries were 5.4cm (2, 500 IU PMSG), 5.1cm (3, OOOIU PMSG) and 6.4cm (FSH), and the right were 6.2cm (2, 5001U PMSG), 5.7cm (3, OOOIU PMSG) and 7.&m (FSH) respectively. There were significant differences in the right overies among treatments (P<0.05). 3. The average number of ovarian follicles in the left ovaries were 4.8 (2, 500 IU PMSG), 5.2(3, 000 IU PMSG) and 7.8 (FSH) respectively. There were significant difference in the right ovaries among treatments (P<0.05). 4. In the average numbers of ovulation points in the left ovaries were 3.0 (2, 5001U PMSG), 3.2 (3, OOOIU PMSG) and 4.4 (FSH) respectively, and the right were 7.2 (2, 5001U PMSG), 7.8(3, 000IU PMSG) and 11.4 (FSH). There were significant differences in the right ovaries among treatments (P<0.05). 5. The numbers of the recovered embryos were 20 (2, 5001U PMSG), 19 (3, 000 IU PMSG) and 21 (FSH) respectively, and oncytes and degenerted oncytes were 6.5 and 11.0 Estrus periods of post parturation were 52.4days (2, 5001U PMSG), 69.8days (3, OOOIU PMSG) and 62.4days (FSH) respectively. 6. The FDA score of cow morulae frozen by slow freezing, sernirapid frezing and vitrified freezing was higher in slow (3.1) and vitrified freezing (3.0) than that in semirapid freezing (1.28). The FDA-scores of cow, pig and rabbit ovarian oocytes frozen in 20% glycerol + 10% ethylene glycol + 30% Ficoll + 10% sucrose by vitrification were higher in cows (3.3) than both in pigs (2.6) and rabbits (2.3).

This study was carried out to investigate the effect of equilibration time, sucrose concentration and age of embryo on survival and developmental rates of bovine IVF expanding blastocysts frozen-thawed by direct transfer method. The bovine oocytes were collected from 2~5mm follicles, matured for 20~24hrs in 5% incubator and then fertilized with frozen-thawed semen. Expanding blastocysts at day 7, 8, 9, 10 and 11 after IVF were frozen in 1.8M ethylene glycol(EG). Survival and hatching rates of frozen-thawed IVF embryos were examined. The results were as follow ; Survival and hatching rate of TVF expanding blastocysts after 10, 20, 3Omin exposure in 1.8M EG were 100,0,90.9, 47.1, 85.0, 75.0 and 62.5% respectively. Survival rates of IVF expanding blastocysts frozen with 1.8M EG and various concentration(0, 0.25, 0.5, 1M) of sucrose were 73.3, 25. 0, 16.7, 9.1% respectively. Survival and hatching rates of IVF expanding blastocysts frozen-thawed according to age of embryo(Day 7, 8, 9,10, 11) were 86.1, 84.8, 79.3, 61.4, 51.3, 74.2, 76.9, 71.7, 63.0 and 65.0% respectively. In conclusion, the age of the embryo(Day 7, 8) is very important for the successful freezing of IVF bovine embryos and 1.8M ethylene glycol not containing sucrose may be effective cryoprotectant for direct transfer method.

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6 to -35 at -0.3 or -O.6 /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3 /min and -0.6 /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3 /min(63.6%) than in -0.6 /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3 /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

This experiment was carried out to investigate the ovarian responses of the ovulation point, ovarian weight and size, the number of ovarian follicles and collected embryos, and to study the effects of the developmental stages (oocytes, 2-4 cell. 8-16 cell and morulae), additional levels of Ficoll (0, 15, 30%) on the survival rate (FDA-test) of rat embryos frozen in vitrification solution (20% glycerol + 10% ethylene glycol + 10% sucrose). Sunanarized results was as follows; 1. The mean ovulation point per head was 7, and the weight of ovaries was 0.03g. The size of ovary was 5.9 mm(L) and 4.6 mm(W), and the number of ovarian follicles over and below 2 mm was 4.7 and 8.7, respectively. The number of the collected embryos per head was 5.5 (79%). 2. 2. The FDA score of embryos frozen in 20 G 10 E 10 S without Ficoll was 2.8 (oocyte), 2.6 (2-4 cell), 3.9 (8-16 cell) and 3.6 (morula), respectively. However, there were no significant differences among treatments. 3. The FDA score of embryos frozen in 20 G 10 E 10 S with 15% Ficoll was 3.4 (oocyte), 4.0 (2-4 cell), 4.7 (8-16 cell) and 4.8 (morulae), respectively (P>0.05). 4. The FDA score of embryos frozen in 20 G 10 E 10 S with 30 % Ficoll was 3.7 (oocyte), 3.2 (2-4 cell), 4.4 (8-16 cell) and 4.4 (morulae), respectively (P>0.05). 5. As shown in the above results, the higher survival rate was obtained in the treatment of 15% Ficoll than that of 30%. And the survival rate (FDA-test)of the oocytes and 2-4 cell stages of the rat embryos was lower than that of 8~16 cell and morulae stages. It was considered that 8-16 cell and morulae could be available for the successful freezing by vitrification of rat embryos with 15% Ficoll except for oocytes.

This study was carried out to select the best cryoprotectant and to establish optimal concentration of the cryoprotectant in ultrarapid freezing of mouse 4-cell embryos and morulae, respectively. We investigated survival of ultrarapid frozen embryos according to various cryoprotectants such as glycerol, ethylene glycol, propylene glycol and dimethyl sulfoxide (DMSO). Suvival of the embryos frozen at different concentrations (3.0, 4.0 and 5.0 M) of indivisual cryoprotectant was also tested. Preimplantation developmental rate (96.3%, 83/86) of 4-cell mouse embryos treated with 4.0 M ethylene glycol after ultrarapid freezing and thawing was higher than those of other cryoprotectants (glycerol, propylene glycol and DMSO). In the ultrarapid freezing of mouse morulae, the highest developmental rate (98.8%, 89 /90) of the embryos to blastocysts was shown in the group of 5.0 M glycerol. Thus, these results demonstrate that 4.0 M ethylene glycol and 5.0 M glycerol are optimal for ultrarapid freezing of 4-cell mouse embryos and morulae, respectively.