• Journal of Embryo Transfer
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• v.28 no.3
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• pp.169-175
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• 2013

Recently, the transgenic animal production technique is very important for the production of bio-parmaceutical as animal bio-reactor system. However, the absence of survival evaluation in vitro produced transgenic embryos has been a problem of the low productivity of transgenic animal because of absent of pre-estimate of pregnancy after transgenic embryos transferred into recipient. Therefore, this study is conducted to improve efficiency of transgenic cattle production by improving the non-surgical embryo transfer (ET) method. Transgenic bovine embryos were produced by injection of feline immunodeficiency virus enhanced green fluorescent protein (FIV-EGFP) lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization (IVF) was occured. Normal IVF and EGFP expressing blastocysts were transferred into recipients. Results indicated that 2 expanded blastocysts (34.7%) transferred group showed significantly (P<0.05) higher pregnancy rate than 1 expanded blastocyst (26.8%) transferred group. In case of parity of recipient, ET to heifer (34.9%) showed significantly (P<0.05) higher pregnancy rate than ET to multiparous recipient (21.2%). However, there are no significant differences of pregnancy rate between natural induced estrus and artificial induced estrus groups. Significantly (P<0.05) higher pregnancy rate was obtained from recipient group which have normal corpus luteum with crown group (34.8%) than normal corpus luteum without crown (13.6%). Additionally, treatment of $100{\mu}g$ Gn-RH injection to recipient group (38.6%) 1 day before ET significantly (P<0.05) increase pregnancy rate than non- Gn-RH injection to recipient group (38.6%). We also transferred 2 EGFP expressing expanded blastocysts to each 19 recipients, 7 recipients were pregnant and finally 5 EGFP transgenic cattle were produced under described ET condition. Therefore, our result suggested that transfer of 2 good-quality expanded blastocysts to $100{\mu}g$ of Gn-RH injected recipient which have normal corpus luteum with crown is feasible to produce transgenic cattle.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.969-976
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• 2009

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.165-172
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• 2000

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.125-135
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• 1996

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$^{\circ}C$ to -35$^{\circ}C$ at -0.3$^{\circ}C$ or -O.6$^{\circ}C$ /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3$^{\circ}C$ /min and -0.6$^{\circ}C$ /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3$^{\circ}C$ /min(63.6%) than in -0.6$^{\circ}C$ /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$^{\circ}C$ /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.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.1-10
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• 2004

These studies were carried out to improve the reproductive efficiency through embryos transfer of Hanwoo IVM/IVF embryos. Following routine IVM/IVF procedure, oocytes and zygotes were cultured far 40 to 44 h in CRlaa medium with BSA. Then 2 to 8-cell embryos were removed the cumulus cell and were cultured in CRlaa medium containing 10% fatal bovine serum and 2.5 mM taurine in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. The fresh embryos of the morulae and blastocysts cultured for 6 to 9 days in vitro or the frozen-thawed embryos were transferred into recipients. The pregnancy rates of the blastocyst produced for 6, 7, 8, and 9 days in vitro culture were 59.4, 68.2, 66.0 and 100%, respectively. In the developmental stage, pregnacy rates of early blastocysts (61.1%), blastocysts(64.7%) and expanded blastocysts(69.5%) were higher than that of morulae stage(20.0%). The pregnancy rates according to the corpus luteum grades of A, B and C in recipients were 73.6, 62.9 and 50.0%, respectively. Effects of donor-recipients synchrony of after day 2, 1 and 0, before day 1 and 2 on the pregnancy rates were 35.7, 65.5, 72.6, 67.9 and 60.0%, respectively. Pregnancy rates of the body condition score of recipients $\leq$2(71.3%) were higher than those of $\geq$3.0 score(40.0%). The pregnancy rates according to the parity of recipients when embryo was transferred to cow(70.6%) was higher than in heifer(59.1%). The pregnancy rates according to hormone treatment before embryo transfer were 69.9% in hCG + GnRH administration group and 63.0% in control group. Fresh and frozen-thawed embryos on the pregnancy rates were 70.6 and 36.4%, respectively. Pregnancy rates in single and AI+single was 90.0% and 64.8%. Pregnancy rates in twin induction was better than in single. These results indicate that pregnancy rates after transfer were affected on the embryo ages, donor-recipient synchrony, body condition score of recipients, corpus luteum status, parity and hormone treatment to recipients.

• Journal of Embryo Transfer
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• v.21 no.4
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• pp.299-306
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• 2006

This study examined the effects of the in vitro produced (IVP) Hanwoo blastocyst stage (blastocyst, expanded blastocyst and hatched blastocyst), in vitro culture day (7, 8, and 9) and blastocyst grade (1, 2 and 3) on the pregnancy rate, gestation length, birth weight, the incidence of dystocia and twining rate after embryo transfer (ET). The pregnancy and abortion rates were significantly higher in the blastocyst (B) stage (64.4%) and in the hatched blastocyst (HB) stage (21.4%), respectively, than in those of the other developmental stages (p<0.05). The pregnancy rate of Day 7 embryos (49.0%) was significantly higher than those of Days 8 and 9 embryos (36.4 and 15.4%), but the abortion rates were similar (0 to 10.7%). There were no significant differences in the pregnancy (41.4 to 42.5%) and abortion (9.3 to 16.5%) rates among the three grades of embryos. There were no significant differences in gestation length, birth weight and the incidence of dystocia among the three development stages, but the twinning rate was significantly higher in the HB stage (p<0.05). The pregnancy rate, the incidence rate of dystocia and twinning rate were similar among the three different culture days, however birth weight was significantly heavier in calves from Day 9 embryos than in those from Days 7 and 8 embryos. The mean gestation length of grades 1 and 2 embryos (278.5 and 276.1 days) were significantly longer than that of grade 3 (p<0.05), but birth weight, the incidence of dystocia and twinning rate did not significantly differ. The mean gestation length in single calves was significantly longer than that in twin calves (278.5 vs. 272.5 days, p<0.05). In addition, the mean birth weight in single calves was significantly greater than that in twin calves (29.6 vs. 22.3 kg, p<0.05). Finally, the sex ratios and mean mortality rates between single and twin calves were similar.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.28-28
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• 2001

The objective of this study was to the effect on subsequent development of EGF present in defined medium during bovine 1)oocyte maturation or 2)embryo culture. The presence of EGF during IVM, irrespective of concentration(1, 10, 100ng/$m\ell$), stimulated cumulus expansion and significantly increased the proportion of oocytes attaining metaphaseII, the rate of cleavage, and develop to blastocyst. 1. In the group of EGF-added medium(1, 10, 100ng/$m\ell$), nuclear maturation rate for in vitro maturation was 91% to 92% but was not significantly higher than control group(87%). 2. For in vitro maturation, in the group of EGF-added medium(1, 10, 100ng/$m\ell$)the rate of cumulus cell expansion degree 2 ranged from 81% to 87%, which was significantly higher than the control group(medium with EGF not added). The rate of in vitro fertilization, developing to 2-to 4- cell stage, was 76% to 80%, which was also significantly higher(p<0.05)than control group(62%). 3. For in vitro maturation, in the group of EGF added in medium(1, 10, 100ng/$m\ell$)the development rate to blastocyst was 24.3% to 27%, which was significantly higher than control group(13.7%). The total cleavage rate in the group of EGF-added medium was 77% to 82%, which was higher than control group. 4. The development rate to blastocyst for 6 days of cultivation and the hatching blastocyst were 30.6% and 59.1%, respectively, in the group of 100ng/$m\ell$ of EGF, which were significantly higher(p<0.05)than control group(14.0% and 24%, respectively), The numbers of cells in blastocyst were 140.2 and 148, respectively, in 10ng/$m\ell$ and 100ng/$m\ell$ of EGF-added medium, which were higher than 108.5 in control group. 5. The development rate of in vitro fertilized embryos to blastocyst in 10ng/$m\ell$ of EGF-added medium co-cultured with somatic cell was 28%, which was significantly higher(p<0.05)than control group(11.8%). The numbers of cells in blastocyst were 141.6 for EGF-added medium and 145 for EGF+co-culture group, which were higher than control(101.6)and medium co-cultured with somatic cells(110.6). These results showed that in vitro maturation and fertilization, EGF was found a significant effect of increase of development rate to blastocyst and cell number.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.303-310
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• 1997

소 난포란의 체외성숙시 성숙배지에 FSH 및 LH의 첨가가 체외성숙난자의 calcium 반응과 체외수정란의 발달에 미치는 영향을 조사하였다. 난포란의 체외성숙은 TCM199을 기초로 한 4가지의 배양조건 하에서 : 1) 0.5$\mu\textrm{g}$/ml FSH＋5$\mu\textrm{g}$/ml LH, 2) 0.5$\mu\textrm{g}$/ml FSH, 3) 5$\mu\textrm{g}$/ml LH 및 4) 무 호르몬 첨가구로서 5% CO2에 24시간 동안 체외성숙을 유도하였다. 체외성숙 24기간째에 난포란의 과립막세포는 1ml PB1＋에서 4분 동안 vortexing을 하여 완전히 제거하였다. 세포 내 calcium 반응을 측정하기 위하여 2mM Fura-2 AM ester 및 0.02% Pluronic F-127가 첨가된 PB1-용액에 39$^{\circ}C$ in cubator에서 40분 동안 배양하였다. 30${\mu}\ell$ M2 medium drop을 30mm plastic dish에 만들어 20$\times$ 형광대물렌즈가 장착된 Nikon Diaphot 현미경의 장착된 Nikon Diaphot 현미경의 warm stage에 설치하였다. 세포 내 calcium 방출을 자극하기 위하여 난자에 25mM inositol 1, 4, 5-trasphophate(IP3)로 1.21kV/cm의 전기자극 또는 20mM ryanodine으로 미세주입을 실시하였다. 이러한 처리를 하지 않은 난자는 체외수정 후 CR1aa와 BRL monolayers의 공배양조건 하에서 체외발달을 유도하였다. 분할율(Day 2)과 배반포기발달율(Day 9)을 조사하였다. FSH와 LH의 처리구에서 IP3 또는 ryanodine으로 자극된 난자(1.79$\pm$0.05, 1.66$\pm$0.06)는 FSH, LH 및 무 호르몬처리구에 비하여 유의적으로 높은 calcium 반응을 보였다(1.00$\pm$0.03, 1.28$\pm$0.04, and 0.53$\pm$0.02 in IP3 elctroporation; 0.68$\pm$0.05, 1.03$\pm$0.05, and 0.47$\pm$0.04 in ryanodine microinjection). FSH와 LH, FSH, LH처리구에서 분할율(87.9, 71.5 및 75.6%)은 무 호르몬처리구(60.7%)(P<0.05)에 비하여 유의적으로 높았으며, FSH와 LH처리구(29.3%)에서의 배반포기 발달율은 FSH, LH 처리구뿐만 아니라 무 호르몬처리구보다 유의적으로 높았다(16.5, 19.0 and 9.8%)(P<0.05). Bovine FSH 및 Ovine FSH의 처리구에서의 calcium 반응은 유의적인 차이가 없었다(1.72$\pm$0.05, 1.61$\pm$0.06). 또한 분할율(82.2 and 84.0%) 및 배반포기(27.8 and 27.1%) 발달율도 bovine 및 ovine FSH처리구간에는 유의적인 차이가 없었다. 이상의 결과에서 전기자극에 의한 세포 내 calcium 반응은 체외성숙배지에 첨가하는 호르몬의 처리에 따라서 유의적인 변화를 보였다. 비록 분할율은 처리구간에 유의적인 차이가 없었지만 배반포기 발달율은 FSH와 LH 공동처리구에서 FSH, LH 단독처리구 및 무 호르몬처리구에 비하여 유의적으로 높은 발달율을 보였다. 체외성숙기간에 FSH와 LH의 공동첨가는 체외성숙 및 체외발달의 생리적인 교정을 위하여 요구되는 것으로 사려된다.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.215-220
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• 2007

This study was conducted to examine the establishment of bovine ES-like cells having pluripotency. The hatched blastocysts derived from culture of in vitro fertilized embryos for 10 to 12 days dissociated mechanically into ICM-and trophectoderm-rich clumps using needle, and cultured onto mitotically-inactivated MEF feeder layer. The primary colonies originated from ICM cells were detached mechanically 7 days after seeding and subsequent subculture was conducted at intervals of every 5 to 7 days. Two ES -like cell lines were established and maintained over 40 passages. Self-renewal of the established lines was confirmed by examining the alkaline phosphatase activity, stem cell-specific marker profiles including SSEA isotopes, Oct-4 and STAT3. Moreover, the established cell lines could produce anchorage-independent embryoid bodies (EBs) with gradual decrease of Oct-4 transcript level in time-dependent manner.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.193-201
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• 1999

In this study we determined fertilization processes and developmental ability of porcine oocytes following injection of round spermatid in the presence of artificial activation. Electrical stimulation at 3 h before spermatid injection significantly increased the incidence of normal fertilization as compared to those following injection without stimulation or with stimulation immediately after injection. The incidences of two pronuclear formation and apposition were not different in oocytes between following intracytoplasmic spermatid and spermatid nucleus injection. Indirect immunocytochemistry and laser scanning confocal microscopy study revealed that micro tubules were organized from the oocyte cortex following round spermatid injection, and this seemed to move both male and female pronuclei into the oocyte center. Paternal mitochondria which are introduced with spermatid have been observed up to 4-cell. Our study indicated that either round spermatid or it's nucleus can be used to produce viable bovine embryos by injection into unfertilized oocytes.