• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.805-812
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• 2006

The purpose of this study was to investigate the effect of Matrigel on the development of bovine embryos after in vitro fertilization. Bovine embryos were cultured in Ⅰ) SOF+ 0.8% BSA(SOF-B), Ⅱ) SOF+ 0.8% BSA plus 0.8% Matrigel(SOF-M) and III) SOF+0.8% BSA and 10% FBS(SOF-BF). The addition of Matrigel appeared not to increase the proportion of blastocysts (SOF-B, 26.6%; SOF-M, 28.2%; SOF-BF, 26.2%). However, the proportion of hatched blastocysts were significantly increased(P<0.05) by Matrigel(SOF-B, 23.7%; SOF-M, 48.7%; SOF-BF, 18.5%). The means of cell number blastocyst was not significantly different among the treatment groups(SOF-B, 172.7±35.5; SOF-M, 175.1±37.4; SOF-BF, 172.8±38.1). The proportion of apoptotic cells in blastocyst was also found to be not significant among the treatment groups(SOF-B, 3.6±3.2%; SOF-M, 4.3±2.6%; SOF-BF, 4.9±4.3%). In this experiment, Matrigel appeared to support embryonic hatching of bovine embryos. Results suggest that Matrigel, as extracellular matrix components, may be another avenue for formulating more physiological culture system in serum-free culture.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.239-249
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• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.229-234
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• 2014

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for in vitro culture (IVC) of bovine embryos. After in vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P<0.0001 and P<0.001, respectively). Although both serum-free conditions showed lower hatching rates than serum-added control, in serum-free groups, RD-mKSOM showed significantly higher hatching rate than mSOF (P<0.001). In addition, one-step using RD-mKSOM may facilitate IVC procedure than two-step culture system. In conclusion, the results indicate that one-step RD-mKSOM is more suitable defined culture system for IVC of bovine embryos than two-step mSOF.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.173-178
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• 1998

The objective of this study was to evaluate the embryonic development ability and the appearance of blastocysts of bovine in vitro fertilized oocytes cultured in different culture media, and also to evaluate survival rate after thawing of frozen embryos by using 1.5 or 1.8M ethylene glycol(EG) with sucrose or trehalose. Fertilized oocytes were divided into three groups; i ) monolayer of cumulus /granulosa cell prepared by TGM 199+5% calf serum(TGM199), ii)GRlaa+5% CS, iii)SOF+5% CS, and they were cultured after insemination for 9 days, at 39˚C, under 5% $CO_2$ in air, but SOF+5% CS was cultured at 39˚C, under 5% 02, 5% GO2, 99% N2. Blastocysts derived from GRlaa + 5% CS on day 7~8 after insemination were frozen by using 1.5M EG or 1.8M EG with/without 0.2M sucrose or O.1M trehalose. The development rate of blastocysts on day 7 after insemination in SOF+5% CS was significant higher than in TCM199 or CR1aa(P<0.05). The appearance rate of blastocysts on day 7-8 after insemination was higher than in TCM199, when fertilized oocytes were cultured in GRlas or SOF. The survival rate of frozen blastocysts after thawing tended to increase, when blastocysts were frozen by using 1.8M EG with 0.2M sucrose or O.1M trehalose. These results indicated that SOF or CRlaa media with amino acids was superior to TCM199 with monolayer in terms of blastocyst development in culturing of in vitro fertilized bovine nocytes, and sucrose or trehalose was supposed to prevent embryos from the freezing shock.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.235-243
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• 1998

In this study, we investigated the effects of three kinds of culture medium (Charles and Rosenkrans; CRlaa, Tyrode's; TALP, synthetic oviduct fluid: SOF), insulin transferrin + selenium complex (ITS), macromolecules(polyvinyl alcohol: PVA, fetalb-ovine serum: FBS) and NaCl on the development of early bovine embryos. In experiment 1, there were no differences in embryo development among three kinds of embryo culture medium (CR $l_{aa}$ , TALP, SOF). In experiment 2, BSA, FBS and PVA were added each in TALP as macromolecule sources. The developmental rates of embryos in BSA or FBS added TALP were significantly higher than in PVA added one (p〈0.01), but there was no difference between BSA and FBS added groups. In experiment 3, bovine embryos were cultured in TALP with the following supplements: BSA alone(1, 3 or 8 mg/ml, each) or BSA(1, 3 or 8 mg/ml, each)+ITS (10$\mu\textrm{g}$/m1 insulin, 5 $\mu\textrm{g}$/ml transferrin, 5 ng/ml selenium). In higher concentration of BSA and ITS supplemented groups, the developmental rates over compacted morula were higher than others, but there was a significant effect of ITS only in 1 mg/ml of BSA added group (p〈0.05). In experiment 4, the effect of reduced concentration of NaCl was evaluated. The developmental rate over compacted morula in the medium containing 90 mM of NaCl was higher than in 114 mM group (p〈0.05). In conclusion, BSA could be used as a macromolecule source in bovine embryo culture, and ITS, as a serum substitute, could be used for improving of embryonic development. Also, reduction of NaCl concentration from 114 mM to 90 mM may improve the development of bovine embryos.bryos.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.69-73
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• 2003

This study was undertaken to investigate the effect of three different porcine IVM media, TCM-199, mSOF and NCSU-23 on early development of porcine spontaneous parthenogenotes. Spontaneous parthenogenotes were induced by a prolonged culture of porcine oocytes in each medium. In Experiment 1, oocytes derived from gilts were matured in three IVM media and maturation of oocytes was evaluated by the status of chromatin configuration. Oocytes matured in mSOF, NCSU-23 and TCM-199 showed no significant difference (P>0.05) in maturation. Maturation rates at 48h after IVM were 83.1$\pm$2% (mSOF), 78.0$\pm$3% (NCSU-23) and 83.5$\pm$2% (mSOF), 78.0$\pm$3% (NCSU-23) and 83.5$\pm$2% (TCM-199). In Experiment 2, pronucleus formation and development to 6~8 cell stage of pig oocytes activated spontaneously. Pronucleus formation, cleavage rate and development to 6~8 cell embryos of porcine spontaneous parthenogenotes were assessed at 55~58 h, 96 h and 168h after IVM, respectively. Pronucleus formation (5.4$\pm$2% and 3.7 $\pm$ 1% vs 1.7 $\pm$ 3%) and development to the 6$\pm$8 cell (3.2$\pm$3% and 4.0$\pm$1% vs 1.4$\pm$3%) was significantly (P<0.05) higher in mSOF or NCSU-23 than TCM-199. In conclusion, the present study showed that oocytes matured in mSOF and NCSU-23 were spontaneously activated with higher frequency.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.21-26
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• 2007

본 연구는 mSOF(modified synthetic oviduct fluid medium) 배양액을 이용하여 $100{\mu}1$와 $10{\mu}1$ 배양 소적에서 일본 흑우의 수정란 생산 효율을 개선하기 위하여 수행하였다. 난구세포가 부착된 미성숙 난자는 각각 단독 배양조건($S;\;10{\mu}1$ 소적) 및 그룹 배양 조건 ($G;\;100{\mu}1$ 소적)에서 실시하였고 배양액은 TCM-199의 기본 배지에 10% FCS, 0.02IU/ml FSH와 $1{\mu}g/ml$ $estradiol-17{\beta}$를 첨가하여 사용하였다. 배반포 단계로 발육한 수정란은 1.5M ethylene glycol로 직접 이식법에 의한 동결 방법으로 동결을 실시하였고, 세포수는 융해 후 생존 수정란에 대해 조사하였다. 체외 배양 시간이 $16{\sim}17$시간 배양 조건에서 난자의 성숙율은 그룹 배양 조건$(27.1{\pm}16.8%)$보다는 단독 배양조건$(57.1{\pm}15.0%)$에서 성숙율이 높았다(p<0.05). 그러나 체외 배양 시간이 $18{\sim}19$ 시간과 $20{\sim}21$시간 배양시는 유사한 성숙율을 보였다. 난자의 체외 배양율은 체외 배양 시간의 증가에 의해 성숙도가 $86.3{\pm}9.9%$로 증가하였다. 접합체(zygote)의 분할율은 단독이나 그룹 배양 조건에서도 유사한 결과를 얻었다. 배반포 발달율은 배양 $7{\sim}8$일째에 조사한 결과 단독 배양 방법보다는 그룹 배양 방법에서 발달율이 높았으나, 분할된 접합체를 기준으로 한 경우 배반포 발달율$(S;\; 21.4{\pm}10.6%,\;G;\;39.0{\pm}13.1%)$에서는 유의적인 차이가 없었다. 단독 배양과 그룹 배양에서 $6.5{\pm}8$일 사이에 배반포로 발달된 수정란의 세포수 조사에서는 유사한 결과를 얻었다. 동결 융해 후 24시간 배양 후 배반포 생존율(5; 24.2%, G; 30.2%), 부화율(S: 20.9%, G: 12.7%) 및 생존 수정란수(S; 45.2%, G: 42.8%)에서도 배양 조건에 따른 유의적인 차는 없었다. 결론적으로 mSOF배양액을 이용하는 경우 미성숙 난자의 체외 성숙 유도 배양 시 단독이나 그룹 배양 시 배반포 발달율에서 그룹간에 유의적인 차가 인정되었다(p<0.01).

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.221-226
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• 2016

The aim of the present study was to assess the embryo development and survivability of post-thawed bovine embryos produced in vitro by addition of cysteine. The rates of metaphase II formation were not differed significantly among three groups(TCM199 73.8%, TCM199 with 0.3% cysteine 76.9%, TCM199 with 0.5% cysteine 83.8%, respectively). No difference of cleavage rate(70.6~74.6%) was seen among three culture medium(TCM199 70.6%, CR1aa 71.3%, SOF 74.6%) with 0.5M cysteine. however, Significantly(P<0.05) higher development rate into blastocyst stage by 0.5M cysteine addition was obtained in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%), however no significant differences in the cleavage rates were among three culture medium. After frozen the blastocysts cultured with 0.5M cysteine, The re-expansion rates were 61.3%~86.4% among groups, and hatching rates were 26.3%~46.9% among groups, the rates of re-expansion and hatching were significantly(P<0.05) higher in SOF medium(86.4% and 46.9%) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate was significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC medium with 0.5M cysteine improved the quality of in vitro production embryo and post- thawed embryo. Future studies comparing these media systems in well-designed trials should be performed.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.249-253
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for studying the interactions between an oocyte and somatic nucleus. The object of this study was to investigate the developmental competence of in vitro-matured porcine oocytes after transfer of the somatic cell nuclei of 2 different species (goat and rabbit). Porcine cumulus oocytes were obtained from the follicles of ovaries and matured in TCM-199. The reconstructed embryos were electrically fused with 2 DC pulses of 1.1kV/cm for $30{\mu}s$ 0.3M mannitol medium. The activated cloned embryos were cultured in porcine zygote medium-3 (PZM-3), mSOF or RDH medium for 7 days. The blastocyst formation rate of the embryos reconstructed from goat or rabbit fetal fibroblasts was significantly lower than that of the embryos reconstructed from porcine fetal fibroblast cells. However, a significantly higher number of embryos reconstructed from goat or rabbit fetal fibroblasts cultured in mSOF or RDH, respectively, developed to the morular stage than those cultured in PZM-3. These results suggest that goat and bovine fetal fibroblasts were less efficacious than porcine-porcine cloned embryos and that culture condition could be an important factor in iSCNT. The lower developmental potential of goat-porcine and porcine-bovine cloned embryos may be due to incompatibility between the porcine oocyte cytoplasm and goat and bovine somatic nuclei.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.181-185
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• 2004

This study was conducted to examine whether activation treatments, source of oocytes and culture conditions affect in vitro developmental ability of caprine oocytes. Mature Korean native goats were pretreated with intravaginal CIDR for 10 days. The goats were then treated with a single intramuscular injection of 1,000 IU PMSG on Day 8 or twice daily injection of a total of 70 mg FSH for 3 days from Day 8 of CIDR insertion for superovulation. All the goats were injected with 10 mg PGF/sub 2a/ on Day 8 and 400 IU hCG on Day 10 of CIDR. Oocytes were surgically collected by oviduct flushing(in vivo maturation) or direct follicle aspiration(in vitro maturation) through mid-ventral incision at 35 h after hCG injection. Fifteen to twenty oocytes were placed in TCM-199 medium containing 25 mM Hepes and hormones under mineral oil at 39℃ in a humudified atmosphere of 5% CO₂ in air for 22 to 24 h. After maturation, the oocytes were activated by electric stimulation or ionomycin + 6-DMAP. The activated oocytes were then cultured in M16, TCM-199 and mSOF media supplemented with proteins at 39℃ for 6 to 7 days. Activation treatments did not affect cleavage of the oocytes. The cleavage rates were 64.1% (41/64) in oocytes activated by electric stimulation and 76.5% (218/285) in oocytes activated by ionomycin + 6-DMAP. The proportion of development to blastocyst was 15.6% (34/218) in oocytes activated by ionomycin + 6-DMAP, but activation by electric stimulation did not support embryos developed beyond morula stage. There were no differences in the cleavage rates of activated oocytes experiencing in vivo (86.8%, 66/76) and in vitro maturation (69.0%, 127/184). However, the development rate to blastocyst stage was significantly (P<0.05) higher for oocytes matured in vivo (50.0%, 33/66) compared to in vitro (0.8%, 1/127). Culture conditions did not affect the cleavage of -activated oocytes. The cleavage rates were 51.6% (49/95) in M16, 64.3% (18/28) in TCM-199 and 81.0% (145/179) in mSOF, respectively. By contrast, the development rate of activated oocytes to stage was greater (P<0.05) for oocytes cultured in mSOF medium (23.4%, 34/145) than in M16 or TCM-199 (0.0%). Our results suggest that source of oocytes and culture conditions are major factors affecting in vitro development of caprine parthenogenetic oocytes.