• Journal of Embryo Transfer
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• v.9 no.1
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.43-49
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• 2008

This study was carried out to examine the efficiency of biopsy methods, and the pregnancy rate, calving and abortion rates, gestation length and birth weight of Hanwoo calves following transfer of fresh, frozen and sexed Hanwoo embryos produced in vitro. The survivability of biopsied embryos was 80.0 and 90.0% using aspiration and punching methods at 24 h after culturing, respectively. The ratios of male and female embryos were 42.1 and 52.6%, respectively, and the percentage of sex unidentified was 5.3%. Pregnancy rates was not significantly different between hCG and control group (46.4 vs. 38.5%), fresh and frozen embryos (41.3 vs. 35.0%), and sexed and IVP embryos (27.5 vs. 41.2%) (p>0.05). Calving and abortion rates of IVP and sexed embryos were not significantly different in calving (85.0 vs. 87.0%) and in abortion (15.2 vs. 13.3%) (p<0.05). Gestation length of IVP and sexed calves were 281.3 and 288.2 days in female and 283.0 and 282.3 days in male, and the birth weight of IVP and sexed calves were 23.6 and 25.0 kg in female and 24.6 and 23.8 kg in male, respectively. There were no difference in gestation length and birth weight between IVP embryos and sexed embryos (p>0.05). Administration of hCG to recipients did not improve the pregnancy rate following transfer of Hanwoo embryos produced in vitro and sexed embryos. Although the production of calves derived from sexed Hanweoo embryos cultured in vitro can be obtained, the efficiency of sexed calves production need to be improved in biopsy methods and pregnancy rate. Further study should be focused on the improvement of pregnancy rates for commercial application of embryo transfer.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.111-117
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• 2017

Objective: The aim of this study was to evaluate pregnancy outcomes and the live birth rate at 1-year age increments in women aged ${\geq}40years$ undergoing fresh non-donor in vitro fertilization (IVF) and embryo transfer (ET), and to identify predictors of success in these patients. Methods: This retrospective study was performed among women ${\geq}40years$ of age between 2004 and 2011. Of the 2,362 cycles that were conducted, ET was performed in 1,532 (73.1%). Results: The clinical pregnancy rate and live birth rate in women ${\geq}40years$ significantly decreased with each year of increased age (p<0.001). Maternal age (odds ratio [OR], 0.644; 95% confidence interval [CI], 0.540-0.769; p<0.001), basal follicle-stimulating hormone (FSH) levels (OR, 0.950; 95% CI, 0.903-0.999; p=0.047), the number of high-quality embryos (OR, 1.258; 95% CI, 1.005-1.575; p=0.045), and the number of transferred embryos (OR, 1.291; 95% CI, 1.064-1.566; p=0.009) were significant predictors of live birth. A statistically significant increase in live birth rates was seen when ${\geq}3$ embryos were transferred in patients 40 to 41 years of age, whereas poor pregnancy outcomes were seen in patients ${\geq}43years$ of age, regardless of the number of transferred embryos. Moreover, the cumulative live birth rate increased in patients 40 to 42 years of age with repeated IVF cycles, but the follicle-stimulating hormone in those ${\geq}43years$ of age rarely showed an increase. Conclusion: IVF-ET has acceptable outcomes in those < 43 years of age when a patient's own oocytes are used. Maternal age, basal FSH levels, and the number of high-quality embryos and transferred embryos are useful predictors of live birth.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.77-84
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• 1996

The purpose of this study was to evaluate effects of osmolarity and vitamins on the in vitro development of embryos. Bovine embryo that had been matured and fertilized in vitro were cultured in simple, chemically defined, protein free medium (mTLP-PVA). When the osmolarity of the medium supplemented 0.35 mM phos-phate and 19 amino acid was changed by NaCI concentration, significantly(p<0.05) higher pro-portion of 1-cell embryos in the medium of 265 or 290 mOsm developed to the morula (32 ~ 35%) and blastocyst(24 ~ 28%) stage. When embryos were transferred to fresh medium containing 5.56mM glucose at 120hrs post-insemination, the highest proportion of embryos developed to mor-ula(40%) and blastocyst(32%) stages at 290 mOsm(p<0.05), although the value in morulae was not significantly different with that(35%) at 315 mOsm. Vitamins in presence of glutamine and amino acids had no beneficial effects on the development of 1-cell embryos to the blastocyst.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.842-849
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• 2007

The purpose of the present study was to produce live offspring from in vitro fertilized goat embryos. Oocytes were collected from abattoir ovaries and kept in oocyte collection medium. Oocytes were washed 4-5 times with maturation medium containing medium-199 with 5 ${\mu}g/ml$ FSH, 100 ${\mu}g/ml$ LH, 1 ${\mu}g/ml$ estradiol-$17{\beta}$ 50 ${\mu}g/ml$ gentamycin, 10% inactivated estrus goat serum, and 3% BSA (fatty acid free). Oocytes were placed in 100 ${\mu}l$ drops of maturation medium containing granulosa cell monolayer and incubated in a 5% $CO_2$ incubator at $38.5^{\circ}C$ for 27 h. For capacitation of spermatozoa fresh semen was processed and mixed in 3 ml fertilization TALP medium containing 50 ${\mu}g/ml$ heparin and kept in the above incubator for 2 h. The capacitated spermatozoa were coincubated with matured oocytes for fertilization. Cleaved embryos were separated and cultured in embryo development medium with oviductal cells and 494 embryos were produced. Recipient goats were synchronized with two injections of 15 mg $PGF_{{2}{\alpha}}$/goat 10 days apart. Eighty early stage embryos were transferred into the uterotubal junction of 14 surrogate mothers using laparoscopy techniques. One recipient delivered twin kids, whereas another two recipients each.delivered a single kid The parentage of these kids was evaluated using highly polymorphic co-dominant microsatellites markers. From the present study, it was concluded that live goat kids can be produced from in vitro matured and fertilized goat embryos, to the best of our knowledge for the first time in India.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.225-228
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• 2015

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.147-156
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• 2006

The objective of this study was to supply excellent genetic resources to livestock farms by transferring embryos produced by genetically superior Korean cows (Hanwoo). Eighty Hanwoo donors were superovulated with gonadotropin ($Folltrpin^(R)\;or\;Antorin^(R)$) for 4 days combined with or without progesterone releasing intravaginal device (CIDR) insertion. The collected fresh or frozen-thawed embryos were transferred to 226 farm recipients. In this study, the effect of CIDR insertion in combination with gonadotropin ($Folltrpin^(R)$) treatments initiated at the random stage of estrous cycle on embryo production was evaluated and compared to conventional superovulation protocol. Moreover, the effect of gonadotropin ($Antorin^(R)$) dose in CIDR-treated Hanwoo donors on the embryo yield was determined. In addition, the effects of embryos (fresh vs. frozen-thawed), embryo transfer person, seasons and farms on the pregnancy rate were evaluated. In Hanwoo donors, CIDR insertion in combination with $Folltrpin^(R)$ treatments regardless of estrous detection resulted in increased numbers of total ova (6.5 vs. 5.8) and transferable embryos (3.9 vs. 3.2) compared to the conventional superovulation protocol (p<0.01). In CIDR-treated Hanwoo donors, the higher dose of $Antorin^(R)$ (36 vs. 28 mg) resulted in the increased number of transferable embryos (8.3 vs. 5.4, p<0.05). The embryos (fresh 43.9% vs. frozen-thawed 23.1%) and embryo transfer person (53.9 vs. $0{\sim}16.7%$) significantly affected the pregnancy rate after embryo transfer (p<0.01). These results suggest that CIDR-based superovulation protocol may be effectively used for production of superior Hanwoo embryos and, multiple ovulation and embryo transfer in Hanwoo might be effectively applied for livestock improvement if pregmancy rate with frozen-thawed embryos and embryo transfer skill would be improved.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.227-232
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• 2020

Objective: The aim of this study was to compare in vitro fertilization outcomes between fresh day 3 or day 4 embryo transfer cycles with dual progesterone (P) administration (intramuscular and vaginal) and cycles with single intramuscular P administration for luteal support. Methods: We selected 124 cycles from 100 women (under age 40 years) who underwent oocyte pick-up (number of trials ≤ 3, 4-14 oocytes obtained) and transfer of two or three day 3 or day 4 embryos at two infertility centers from January 2014 to June 2019. Dual P (intramuscular P [50 mg] daily+vaginal P) was used in 52 cycles and a single intramuscular administration of P (50 mg daily) was used in 72 cycles. Results: Women's age, infertility factors, number of oocytes retrieved, number of transferred embryos, and mean embryo score were similar between the dual P group and the single P group. Although the number of trial cycles was significantly higher (1.9 vs. 1.5), and the mean endometrial thickness on the trigger day (10.0 mm vs. 11.0 mm) was significantly lower in the dual P group, the implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and miscarriage rate for both day 3 and day 4 transfers were similar between the two groups. Conclusion: In fresh day 3 or day 4 embryo transfer cycles, dual P administration did not demonstrate any clinical advantages. Intramuscular P alone appears to be sufficient for luteal support.