• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.181-189
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• 2001

The objective of this study was to evaluate the development of porcine follicular oocytes fertilized by intracytoplasmic sperm injection (ICSI). Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2~7 mm in diameter from a local slaughterhouse. Oocytes were matured in vitro for 40~44 h, and spermatozoa were prepared by swim-up in the presence or absence of 5 mM dithiothreitol (DTT) and then M II stages of the oocyte were either centrifuged or not centrifuged for the following injection of ooplasm. Injected oocytes were cultured in NCSU 23 medium for 6 to 8 days. The results obtained were as follows: 1. The rates of cleavage and development rates into blastocyst by ICSI were not significantly different between the with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively (P＜0.05). 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5 mM DTT treated-sperm were not significantly different (60.4% vs 16.4% and 45.5% vs 22.2%), respectively (P＜0.05). 3. The cleavage and the developmental rates to btastocyst were not significantly different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%) (P＜0.05). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7$\pm$2.9 and 41.9$\pm$4.6).

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.265-271
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• 2014

Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production (OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmental competence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effect on follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated from Korean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two different seasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes were classified according to morphological categories and used for in vitro embryo production (IVEP). The mean number of total follicles was significantly higher (p<0.05) during the hot season ($18.32{\pm}2.26$) compared to cool season ($15.41{\pm}3.34$). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16% vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot ($1.75{\pm}1.86$) season compared to the cool season ($1.00{\pm}1.46$), but there was no significant difference of other grades oocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%) did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season (hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro cultured embryos.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.187-195
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• 2003

This study was carried out to improve of enucleation efficiency on porcine recipient oocytes preactivated. In ethanol or $Ca^{2+}$ ionophore, effect of repeating and combinational activation with 6-DMAP or cycloheximide compared with alone activated treatment. Recipient oocytes's activation by $Ca^{2+}$ ionophore combined with 6-DMAP or cycloheximide were significantly higher than alone treatment(P<0.05). Between repeating and alone treatments were not significantly different. In ethanol, repeating treatment was significantly lower than alone(P<0.05), and combination treatments were not significantly different. On the basis of these results, efficiency of enucleation, electrical fusion and in vitro development compared preactivated with non-preactivated recipient oocytes. Enucleation and fusion rates of preactivated oocytes were improved significantly compared with non-preactivated oocytes(90.7%, 71.8 vs 77.8%, 61.1%; P<0.05). Behind the back, cleavage and in vitro development rates were significantly lower than non-preactivated oocytes(38.7%, 19.3% vs 68.8%, 30.6%; P<0.05).

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.379-384
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• 1996

This study was undertaken to investigate the sister chromatid exchange (SCE) frequency and embryonic development after exposure to cryoprotectants and vitrification of mouse zygotes. Mouse IVF zygotes were cryopreserved by vitrification using vitrification solution, EFS40 (40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). After mouse zygotes were exposed to EFS40 at $25^{\circ}C$ for 30 sec., they were immediately plunged into $LN_2$ or cultured for cryoprotectant toxicity test without freezing. The results obtained in these experiments were summarized as follows: After thawing, survival rates to the 2-cell stage of zygotes exposed to or vitrified in EFS 40 (98.5%, 95.2%) were not significantly difference compared with that of control (100%). However, the developmental rates upto blastocyst and hatching blastocyst in vitrified groups (66.7, 50.0%) were lower than those of control (93.9, 81.8%) or exposed group (94.0, 78.8%) (p<0.05). When the influence of vitrification and exposure to cryoprotectant on the in vitro development of mouse zygotes was assessed by the SCE frequency, the SCE frequency in exposed ($20.2{\pm}2.1$) to or vitrified embryos ($21.4{\pm}3.2$) was higher than that in control embryos ($16.8{\pm}1.5$). These results suggest that the frequency of SCE was increased after cryoprotectant exposure or Vitrification although developmental rates of zygotes upto blastocysts and /or hatching blastocysts were not afected by cryoprotectant.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.1
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• pp.7-11
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• 2013

Objective: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. Methods: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. Results: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. Conclusion: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.119-126
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• 2004

The present study was carried out to examine the effect of four different media (NCSU (North Carolina State University)-23, PZM (Porcine Zygotes Medium)-3, PZM-4 and TCM (Tissue Culture Medium)-l99) and two oxygen concentrations (39 , 5% $O_2$, 5% $CO_2$ and 90% $N_2$, 5% $CO_2$ in air) on in vitro production of porcine IVM/IVF embryos. The results were summarized as follows: The rates of GVBD and nuclear maturations were not significantly different (p>0.05) for 44 hours of culture with four media in two oxygen concentrations. The rates of polyspermy, penetrated sperm(s) and male and female prouclei formation were not significantly different (p>0.05). among four media in two oxygen concentrations. The cleavage rates were not significantly different (p>0.05) among four media in two oxygen concentrations. At day 7 under gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$, the blastocyst formation was significantly higher (p<0.05) in PZM-3 (19.9$\pm$2.4) than other media. Also, NCSU-23 medium gave high rate of blastocyst formation at day 7 under gas atmosphere of 5% $CO_2$ in air (p<0.05). Based on the result of differential staining of porcine blastocyst at dat 7, inner cell mass cell and total cell numbers were not significantly different (p>0.05) among four media in two oxygen concentrations. However, the observed total cell number was higher in PZM-3 medium (36.8$\pm$6.5) than other madia. In conclusion, these results suggested that in vitro production of porcine embryos in PZM-3 medium under a gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$ was effective on the blastocyst formation rate and total blastocyst cell number.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.117-123
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• 1993

The condition of the endometrium is an important factor which may influence the success or failure in IVF-ET. This study was undertaken for evaluation of the value of endometrial growth as an early predictor for the success of IVF. Ultrasonographic endometrial measurement were performed in 43 IVF cycles that conceived, 101 cycles that did not with an IVF-ET There was no significant difference in the endometrial thickness and the serum concentration of estradiol in the pregnant versus nonpregnant group(10.4 vs. 9.9 mm: 2348 vs. 2017 pg/ml no hCG administration day). No correlation was found between the ultrasound image and serum estradiol levels around the time of hCG administration(r=0.54, p=0.13 no Day 2; r=0.45, p=0.14 no Day 1). The duration of gonadotropin treatment, number of follicles, number of oocytes retrieved, and fertilization rate were not statistically different in the two groups, however, there was a significant difference in the number of embryos in the pregnant versus nonpregnant group)p< 0.05). A higher pregnancy rate and ongoing pregnancy rate occured with an endometrial thickness over 11 mm compared with below 7mm(p< 0.05, p< 0.005). however, no significant differences were noted in the implantation rate and abortion rate among the groups that classified according to their endmetrial thickness. The endometrial growth(${\Delta}$) from hCG administration day(DO) to D6 was greater in the women who achieved pregnancy than in the nonpregnant group(p< 0.01). There were no significant differences in serum estradiol levels, implantation rate, pregnancy rate, and abortion rate among the groups that classified according to the pattern of echogenesity of endometrium, however, significantly higher ongoing pregnancy rate was noted in group A, B compared with group C.(p< 0.0001, p< 0.001) These results suggest that there were no ultrasonographically detectable differences in the patterns of endometrial growth and development around the time of hCG administration in patients who conceive versus those that do not in IVF-ET.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.81-87
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• 2008

Production of transgenic animals for studying specific gene has been limited due to a low efficiency, lack of skilled researchers and the need for expensive equipment. Currently, the boar spermatozoa as a vector to deliver exogenous DNA into the oocyte were used to improve the efficiency of transfection rate. In this study, we revealed that the optimal conditions for DNA uptake in spermatozoa by liposome were to 90 min of incubation, $17^{\circ}C$, $10^5$ spermatozoa, 4 ng/ml of exogenous DNA and 0.5% (v/v) liposome, without damage to fertility. In addition, the developmental rate to the blastocyst stage of embryo in control group was significantly higher than those embryos with exogenous DNA and liposome, whereas there were no significant differences in embryo development between the liposome and type of DNA. The transfection rates of embryo using treated spermatozoa with both liposome and circular DNA were higher than those using linear DNA. These findings raise the possibility thattreated spermatozoa with liposome/DNA complexes could be used in in vitro fertilization, and the exogenous DNA transferred into the oocytes. Taken together, we demonstrated that liposome a vector for the uptake of exogenous DNA in boar spermatozoa could improve the efficiency of sperm-mediated gene transfer in creating transgenic pig and the other domestic transgenic animals.