• Journal of Embryo Transfer
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• v.20 no.2
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• pp.97-104
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• 2005

This study was conducted to investigate the effects of various factors such as recipient parity, delivery season, offspring number, pregnancy period, delivery type, midwifery type and dystocia, on the viability of calves derived from embryos produced in vitro. There was no difference in the abnormality of calves among treatments ($0\~25\%$, respectively). The incidence of a disease was significantly higher in delivered by multiparous $(40\%)$ than nulliparous$(9.9\%)$, in eutocia than dystocia group, in delivered on spring $(20.4\%)$ and winter $(22.7\%)$ than summer$(4.3\%)$ and autumn $(0\%)$, in single offspring $(18.4\%)$ than twin offsprings $(6.7\%)$, and in eutocia group $(17\%)$ than dystocia $(2.7\%)$, respectively (p<0.05). The rate of mortality was significantly higher when transferred into mulliparous $(22.3\%)$ than multiparous$(0\%)$, when were delivered within 270 day $(53.3\%)$ than over 270 day $(14.3\~16.1\%)$, when were dystocia $(41.7\%)$ than eutocia$(14.1\%)$ group, when were induced delivery $(44.4\%)$ than self-delivery $(18.1\%)$, when were non-midwifery $(34\%)$ than midwifery$(10.8\%)$, and when delayed midwifery $(31.6\%)$ than earlier midwifery$(11.5\%)$, respectively (p<0.05). The present study suggested that the proper treatment of parturition may be increased the viability of calves derived from in vitro.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.141-145
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• 2004

Mucin coat is deposited on the embryos during passage through the oviduct in rabbit. When in vitro cultured blastocysts were transferred to the recipients, the lack of mucin coat might account in part for failure of pregnancy after transfer. The present study were carried out to investigate whether deposition of mucin coat were induced when in vitro cultured blastocysts were transferred to recipients. At 19 ～20 hours post-coitus one-cell embryos were collected by flushing oviducts. These embryos cultured for 72 hours were reached to blastocyst stage. And these blastocysts were transferred to the oviduct of asynchronized (one day later than the donors) and synchronized recipient. To confirm deposition of the mucin coat, blastocysts transferred to the oviduct were recovered at 24 and 48 hours after the transfer. Fifty eight percent of blastocysts recovered from uterus of asynchronous recipient at 24 hours after transfer and 92.9% of blastocysts recovered from uterus of synchronous recipient were 0～10 ㎛ of mucin coat thickness. And 11.8% of blastocysts of asynchronized recipients and 7.1% of blastocysts from asynchronized recipients were in 11～20 ㎛ of mucin coat thickness. When blastocysts were recovered from uterus at 48 hours after transfer, 87.0% of blastocysts from asynchronized recipients and 5.9% of blastocyst from synchronized recipients were in 0～10 ㎛ of mucin coat thickness. And 76.5% of blastocysts of synchronized recipients and 4.4% of blastocysts from asynchronized recipients were in 11～20 ㎛ of mucin coat thickness. From these results it is speculated that the low implantation rate of in vitro cultured rabbit blastocysts transferred to oviduct of recipient was caused by high degeneration of the embryo after transfer and inappropriate deposition of mucin coat.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.109-119
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• 2006

This study was conducted to examine relationship between vaginal cytology and reproductive hormones during the estrous cycle and to provide basic data to estimate for ovulation time and optimal mating time in 6 beagle dogs The duration of proestrus, estrus and diestrus were $8.5{\pm}1.4,\;10.0{\pm}1.4\;and\;54.0{\pm}2.8$ days at pregnant respectively, and $7.9{\pm}2.1,\;9.5{\pm}0.7\;and\;62.0{\pm}11.3$ days at non-pregnant respectively. The duration of interestrous intervals were $246.2{\pm}24.5$ days at pregnancy, and $175.3{\pm}34.5$ days at non-pregnancy. The duration of interestrous intervals at pregnancy was longer than that of non-pregnancy. A characteristic features of vaginal cytology during the estrous cycle were the high proportion of superficial cell, anuclear cell and erythrocyte in proestrus and estrus, parabasal cell, small intermediate cell and leukocyte in diestrus, and parabasal cell and small intermediate cell in anestrus, respectively. Cornification index (CI) in proestrus and estrus were significantly higher than that of CI in diestrus and anestrus. Plasma progesterone concentration was below 1.0 ng/ml at the first day of vulval bleeding at pregnancy and non-pregnancy, and then it was above 2.0 ng/ml at Day -2 in all bitches. When plasma progesterone concentration was first increased above 4.0 ng/ml, it was the second day after the first day of male acceptance. Plasma progesterone concentration showed above 40 ng/ml on Day $20{\sim}22$ in all bitches, and then it was gradually decreased until Day 35. Plasma progesterone concentration at pregnancy was higher than that of non-pregnancy from Day 35 to Day 63. Plasma estradiol-$17\;{\beta}$ concentration was above 9.0 pg/ml at the first day of vulval bleeding, and it showed 26.4 pg/ml on Day -2. When it was timed from the first day of male acceptance (Day 0), plasma estradiol-$17{\beta}$ concentration showed a peak on Day 0 and plasma progesterone concentration was first increased above 4.0 ng/ml on Day 2 which was the third day after plasma estradiol-$17{\beta}$ peak. CI was first increased above 80 and 90% on Day -1 and Day 1, respectively. CI was maintained above 80% from Day -1 to Day 8 (10 days) and above 90% from Day 1 to Day 6 (6 days), respectively. CI was maintained above 80% from Day 0 to Day 8 (9 days) and above 90% from Day 1 to Day 6 (6 days), respectively. Plasma progesterone concentration was first increased above 4.0 ng/ml on the second day after the day which CI was first increased above 90%. In conclusion, beagle bitches ovulated on the second day after the day which CI was first increased above 90% and on the day which plasma progesterone concentration was first increased 4.0 ng/ml, and it was estimated that the optimal mating time was the day which the second day after CI was first increased above 90% and plasma concentration was between $2{\sim}25ng/ml$. The measurement of plasma progesterone was used to determine of and accurate ovulation time and the optimal mating time, but vaginal cytology is low-priced and simple method to estimate estrous cycle, optimal mating time and ovulation time.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.61-61
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• 2001

The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.371-378
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• 1997

Human lactoferrin (hLF) was expressed in the mammary gland of transgenic mice. Expresion of hLF was achieved by palcing its cDNA under the control of bovine $\beta$-casein gene. To improve the hLF expression level, two artificial introns were introduced into the expression vector. One intron is a hybrid-splice consisting of bovine $\beta$ casein intron 1 and rabbit $\beta$-casem intron II. The other intron is a DNA fragment spanning intron 8 of bovine $\beta$ casein gene. Trans sgenic mice were developed which expressed hLF in their milk. Twenty lines of transgenic mice were produced. hLF was present in the milk at concentrations of 1 ~ 200 ${\mu}\textrm{g}$ / ml. hLF RNA was only detected in the mammary gland of transgenic mice. The expressed RNA was cor r rectly spliced at the exon /intron junctions. To generate transgenic cows secreting active hLF in their milk, we transferred the DNA-injected bovine embryos to recipient heifers by surgical a and non-surgical methods out of 68 embryos transferred to 51 recipients by surgical or non-surgical method, 7 calves were normally born. Effect of embryo quality of DNA-injected blastocysts on pregnancy rate after transfer was investig a ated. Higher pregnancy rate of (38.9%) DNA-injected embryos was shown in excellent embryos. Pregnancy rates in the groups of good a and fair embryos were 15.4 and 14.3%, respectively. Effect of culture period of DNA-injected b bovine embryos on pregnancy rate after transfer was investigated. When Day-6 blastocysts of cuI ture were transferred, there was no pregnancy. Pregnancy rates of Day-7 and -8 blastocysts were 28.6 and 33.3%, respectively. There was no difference on pregnancy rate between Day-7 a and -8 bovine blastocysts after DNA injection. Thus, we established the techniques for transfer a and culture of DNA-injected bovine embryos. In a addition, factors affecting the pregnancy rate of DNA-injected embryos after transfer were investigated .

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.177-181
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• 2008

This study was performed to investigate the effects of hCG treatment on pregnancy and delivery rates in the Hanwoo recipients. There were significantly higher pregnancy and delivery rates in the recipients treated with hCG at 7 days after artificial insemination (p<0.05), respectively. The SCNT embryos from bovine fetal fibroblast cells were transferred into the synchronized recipients. The recipients were administered saline (n=89) or hCG (1,500 IU) (n=48) at 7 days after heat, respectively. The pregnancy rate was significantly higher in the recipients treated with hCG compared to that of saline treated group (p<0.01), however, the delivery rate was not different in both treated groups. The concentration of plasma progesterone (P4) was not different in both groups before hCG treatment, but the P4 level was increased significantly in hCG treated group after hCG injection (p<0.05). Although the pregnancy rate was very high in early stage of pregnancy, it was decreased dramatically after 50 days of pregnancy and maintained basal level. Taken together, the treatment of hCG in the SCNT recipients after day 7 of heat was effective method to increase the P4 concentration and to increase the pregnancy rate. But it did not affect directly to delivery.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.195-202
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• 1997

The present study was carried out to evaluate the effect of superovulation treatments on ovarian responses, oocyte recovery rates and grades of collected oocytes using an ultrasound-guided transvaginal approach in Korean native cows. Superovulation in cows was induced with two different regimenes: 1) FSH-decreasing dose(n=8): the cows were received twice per day for three days of the total dose of 400 mg of FSH-p, 2) FSH-single dose(n=9): the cows were administrated a single dose of 400 mg of FSH-p in 25% PVP. The Observation of visible follicles and collection of oocytes were performed 12 hours following the last FSH in FSH-decreasing dose group and 48 hours after the FSH-single dose injection. All visible follicles larger than 6 mm were punctured and aspirated with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use. The mean number of visible follicles(> 6 mm) was significantly(P<0.05) higher in the FSH-decreasing dose treatment (22.811.9) and FSH-single dose treatment (20.612.0) groups than the non-treatment group(7.0$\pm$8). The mean recovery rate of oocytes was not significantly(P<0.05) different between the treatment and control groups, but the mean number of collected oocytes was significantly(P<0.05) higher in the FSH-decreasing dose treatment( 12.611.5) and FSH-single dose treatment (11.813.6) groups than the non-treatment group(3.7$\pm$0.5). In conclusion, the FSH-single dose treatment at superovulation in cows for ultrasound-guided aspiration might increase the number of aspiratable follicles and the recovery rate of follicular oocytes as the FSH-decreasing dose treatment.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.63-69
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• 2005

This study were carried out to evaluate the possibility of nuclear progression of canine immature oocytes, of which was cultured in a reproductive tract, such as oviduct, ovarian bursa and uterus of estrus bitch for 4, 5 and 6 days following immediately collection. Cumulus intact oocytes(COC) fore collected from domestic dog following ovariohysterectomy at local veterinary clinics. In Exp. 1, COCs $of>110\;{\mu}m$ diameter were selected and cultured in vitro at $39^{\circ}C$, $5\%\;CO\_{2} $ in air atmosphere. The nuclear progression of canine oocytes checked at 24, 48 or 72 h after in vitro maturation. There was not increased the nuclear progression to the M II stage depending on culture periods at 24, 48 and 72h $(1.3\%,\;3.7\%\;and\;4.7\%)$. In Exp. 2, to evaluate of nuclear progression of immature oocytes, collected or in vitro cultured oocytes were transfer into a canine reproductive tract (oviduct, ovarian bursa and uterus). The recovery rates of canine oocytes from a reproductive tract after 4 days $(33.7\%)$ in vivo culture were significantly higher than those 5 $(17.7\%)$ 6 day $(3.4\%)$ (P<0.05). The survival rates of collected oocytes after 4 days $(60.0\%)$ were also significantly higher than those of 5 days $(30.2\%)$ and 6 days $(38.9\%)$ (P<0.05). The meiotic resumption rates of canine oocytes were not significantly difference among the culture periods at 4 days $(5.9\%)$, 5 days $(0.0\%)$ and 6 days $(0.0\%)$. These results show that the nuclear progression of canine immature oocytes from in in vivo culture was not affect the nuclear resumption of oocytes.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.55-62
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• 2005

This study was undertaken to access the effect of collection methods on the collection efficiency, blastocyst rate and pregnancy rate after IVP embryo transfer. The ovaries of Hanwoo were obtained from an abattoir and kept on 25 to $28^{\circ}C$ and transported to laboratory within 4 hrs. The oocytes were collected by aspiration of follicles $(2\~6\;mm)$ with or without slicing of ovaries after aspiration. The oocytes were matured in vitro (IVM) for 20 to 24 hrs in TCM-199 supplemented with $10\%$ fetal bovine serum at $39^{\circ}C$ under $5\%\;CO_{2}$ in air. Following routine IVM/IVF procedure, the oocytes and presumed zygotes were cultured for three day in CRlaa medium with BSA. The cumulus cells at 2 to 8-cell stage of embryos removed then the embryos and were cultured in CRlaa medium containing $10\%$ fetal bovine serum in $5\%\;CO_{2}$ at $39^{\circ}C$. The fresh blastocysts cultured for 7 to 9 days were transferred into recipients. The numbers of oocytes recovered form two different methods, the aspiration and slicing after aspiration, were compared to know what. The number of oocytes per ovary was 8.2 and 6.5 in aspiration combining slicing, and aspiration groups, respectively (p<0.05). The cleavage rate in aspiration method are significantly (p<0.05) high than those in slicing post aspiration $(27.9\%)$, and aspiration $(25.5\%)$. The pregnancy .ate in aspiration method $(62.5\%)$ was high than that in slicing method after aspiration $(54.4\%)$. The pregnancy rates of aspiration method and slicing method after aspiration in nullipara $(58.1\%\;vs\;68.2\%)$ was high than that in pluripara $(49.5\%\;vs\;53.2\%)$. The results obtained that the increased number of oocytes per ovary in slicing method after aspiration could be better than that in aspiration method. Pregnancy rate in aspiration method was slightly higher in than that in slicing method after aspiration.