• 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.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.315-322
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• 1998

The objective of this study was to test the effect of catalase on penetration in vitro by spermatozoa preincubated with xanthine and/or xanthine oxidase. The penetration rates were, significantly (p<0.05) higher in spermatozoa preincubated without (66 and 38%) than with (40 and 15%) catalase for 0 and 30 min. When spermatozoa were preincubated and inseminated in medium with xanthine, the penetration rates were significantly higher (p<0.05) in medium with (68, 70 and 49% for 0, 30 and 60 min) than without (33, 41 and 19% for 0, 30 and 60 min) catalase. However, in oocytes were' inseminated with spermatozoa pre incubated with or without catalase in the presence of xanthine oxidase, no decrease in penetrations rates were observed for up to 60 min of preincubation. In another experiment, the penetration rates were significantly (p<0.00l) higher in medium with (75, 55 and 52%) than without (14, 4 and 8%) catalase when oocytes were inseminated with spermatozoa preincubated for 0, 30 and 60 min in the presence of xanthine plus xanthine oxidase. On the other hand, The rate of polyspermy in oocytes penetrated in medium without catalase in the presence of xanthine or xanthine plus xanthine oxidase decreased with time of spermatozoa preincubation. However, no differences were observed in polyspermy rates in the medium with xanthine oxidase alone despite presence of catalase. These results indicate the advantages of spermatozoa pre incubated with xanthine plus xanthine oxidase in the presence of catalase to increase penetration potential and with suppressed polyspermy in porcine.

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.179-187
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• 1991

These studies were carried out to investigate the effects of the size of follicles, semen types, capacitation methods, and additions of hormones, estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) to the medium on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48hrs. in an incubator with 5% $CO_2$ in air at $38.5^{\circ}C$ and then matured oocytes were again cultured for 18~20 hrs. with motile capacitated spermatozoas the TCF (Tyrode calcium free) solution containing $100{\mu}g/ml$ of heparin. The results obtained in these experiments were summarized as follows: 1. The oocytes classified as "A,B,C,D and Degenerative" depending morphological integrity and those 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1, 33.3%, respectively. 2. The average number of the follicular oocytes recovered from follicles size, 1~2mm, 3~5mm and above 5mm in diameter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TCM-199 medium were 56.7%, 82.5%, 46.0% and 44.8%, 71.4%, 28.6%, respectively. 3. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymal cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8% 38.3%, respectively. 4. The fertilization and cleavage rate of follicular oocytes, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0%, 53.8%, 34.2% and 38.3%, 23.1%. 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin. 5. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% and FSH, HCG, 17, $\beta$-estradiol were 76.0~82.3% and 26.2~70.0%, and those values were higher the supplementation than non-supplementation. 6. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5~20% ECS and FCS were 74.0~80.6%, 26.2~30.0% and 71.7~76.9%, 51.9~58.0%, and the values were higher the supplement of ECS than FCS. 7. The maturation rate (68.0~64.6%) and fertilization rate(59.6~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 8. The maturation rate(76.5%) and fertilization rate (61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and $1{\times}10^6/ml$ MCC were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and $1{\times}10^{{4}{\sim}{5}}/ml$ and $1{\times}10^8/ml$ cumulus cells.

• 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.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.19-28
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• 2001

The present study was undertaken to investigate the effects of cooling rate and equilibration time on the survival, in vitro maturation and development to embryos of frozen-thawed bovine immature oocytes(Germinal Vesicle Stage). The cryoprotectants are used 10% ethylene glycol(EG) as permeating cryoprotectant and 0.05M soc.ose(S) or trehalose(T) as low molecular weight nonpermeating cryoprotectants and 5% ficoll(F) or polyvinylpyrrolidone(PVP) as high molecular weight nonpermeating cryoprotectants. Four freezing solution were uysed in this experiment(EFT: 10% EG + 5% F + 0.05M T, EFS: 10% EG + 5% F + 0.05M S, EPT: 10% EG + 5% P + 0.05M T, EPS: 10% EG + 5% P + 0.05M S). The best equilibration time and freezing solution was 15 min in EPT(83% survival rate of frozen-thawed bovine immature oocytes). When frozen-thawed bovine oocytes were cultured following IVM and IVF, there was no significant difference in cleavage and development rates among the EFT, EFS, EPT and EPS solutions. When 9 blastocysts derived from frozen bovine oocytes were transferred to 6 recipients, two recipients were pregnant. And one was aborted at 45 days of pregnancy and the other had a stillbirth.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.65-70
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• 2011

This study was conducted to investigate the survival rate of AndroMed and Tris-egg yolk extender for cryopreservation of Korean Native Bull Semen (Chick Cow). Semen was collected from a Korean Native Bull Semen over 3 year's old. The semen was diluted 1:1 by AndroMed and Tris-egg yolk extender. The pellet was diluted to final sperm concentration of $5{\times}10^7$ cell/ml by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hrs at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes. And then the frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. The survival rates was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender ($89.7{\pm}19.8$ vs. $73.4{\pm}11.2$). However, motility was no significant differences ($78.4{\pm}18.7$ vs. $67.9{\pm}14.6$). Survival rate in time of equilibration between visual and CASA program had higher in 2 h ($86.33{\pm}9.4$ vs. $92.32{\pm}12.4$) than in 5 h ($78.20{\pm}7.8$ vs. $88.28{\pm}13.1$) 15 h ($65.24{\pm}6.6$ vs. $76.48{\pm}17.3$) 20 h ($56.26{\pm}4.6$ vs. $67.73{\pm}18.4$). The development rates to cleavage was higher in Tris-egg yolk extender than AndroMed extender (82.2% vs. 81.7%). Similarly, the development rates to blastocyst was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender (42.3% vs. 29.6%). In conclusion, the obvious impact of this study will be its practical application to improve viability and fertilizing ability of cryopreserved spermatozoa used for in vitro fertilization (IVF) and AI, Which in turn will be beneficial to animal genetic resources conservation.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.165-176
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• 1994

This study was carried out to investigate the effect of the timing and dose of human chorionic gonadotropin(hCG) on oocyte recovery, in vitro fertilization, and preimplantation development in superovulation of mouse. F1 hybrid($C57BL{\times}CBA$) mice were obtained and superovulation was induced in female mice by sequential intraperitoneal injection of PMSG and hCG. In the first series of experiments, mice received 5 IU of PMSG given intraperitoneally, and 48 hours later were injected 1 IU, 5 IU, or 10 IU of hCG respectively. In the second series of experiments, mice received 5 IU of PMSG given intraperitoneally and were injected 5IU of hCG 36, 48, or 60 hours later respectively. 1. When the mice received 5 IU of PMSG given intraperitoneally and 48 hours later were injected 1 ItT, 5 IU, or 10 IU of hCG respectively, there were no differences in the total number of the oocytes obtained from the three experimental groups. When the cultures were examined 48 hrs after the termination of insemination the proportion of unfragmented oocytes which had developed over two-cell stage was observer to be lowest in 10 IU hCG group. When the cultrues were examined 120 hour after termination of insemination the proportion of embryos which had developed to the blastocyst stage was observed to be significantly higher in 10IU hCG group than 5IU hCG group(p<0.05), but there was no difference between 10 IU hCG group and 1IU hCG group. 2. When the mice received 5 IU of PMSG and were injected 5 IU of hCG 36, 48, or 60 hours later respectively, there were no differences in the total number of oocytes obtained from the three experimental groups. When cultures were examined 48 hour after the termination of insemination the proportion of unfragmented oocytes which had developed over two-cell stage was observed to be significantly lower in 36 hour interval group than 48 hour interval and 60 hour interval group(p<0.05). When the cultures were examined 120 hour after termination of insemination the proportion of embryos which had developed to the blastocyst stage was found to be higher in 60 hour interval group than 36 interval or 48 hour interval group (P<0.05), and the proportion of hatching blastocyst was found to be higher in 60 hour interval group as well. In this study, it was concluded that the administration of adequate dose of hCG, and long (60 hour) PMSG-hCG interval were necessary in superovulation of mice($C57BL{\times}CBA$) in order to get a large number of oocytes which had an early oocytes which had an early embryonic developmental capability when fertilized in vitro, and especially it had better have been avoided to administer a large dose of hCG.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.97-108
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• 2003

In-vitro fertilized Hanwoo embryos were biopsied for sex determination by PCR. Biopsied embryos were incubated for 1∼2 hours for the recovery. Those sexed Hanwoo embryos were transferred to 49 Hanwoo and 16 Holstein recipients from February 2000 to February 2001. Of 65 recipients, 14 cows(12 Hanwoo and 2 Holstein) delivered the same offspring as sex-determined by PCR, therefore the conception rate was 21.5%. 1. Total 65 embryos(male 35, female 30) were transferred to recipients, and 14 calves (male 6, female 8) were delivered. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex determination was 100.0%. 2. The conception rate after transfer with biopsied embryo between Hanwoo and Holstein was 24.5% and 12.5% 3. The conception rate after transfer with biopsied embryo between fresh and frozen-thawed embryos was 23.5% and 14.3%. 4. The conception rate according to the season when embryo was transferred was 11.8, 29.4, 23.5 and 20.0% for spring, summer, autumn and winter, respectively. 5. The conception rate according to embryo quality after biopsy was 41.7, 30.0 and 0.0% for excellent, good and fair quality. 6. The conception rate according to thickness of uterine horn was 71.4, 18.9, 11.8 and 0.0% for 0, +, ++ and +++ thickness. 7. The conception rate according to the site in the uterine hem where embryo was put was 30.0, 20.0 and 10.0% for cranial, mid, and caudal part of uterine horn. 8. The conception rate according to the quality of corpus luteum ipsilateral to the uterine horn where embryos was transferred was 41.2, 14.3 and 15.4% for excellent, good and fair quality. 9. The conception rate according to the time required for embryo transfer was 18.2, 30.0, 30.0, 0.0 and 25.0% for 10, 15, 20, 25 and 30 minutes. 10. The conception rate according to parity of recipients was 26.5, 19.1, 14.3 and 0.0% for the primiparous, the 2nd parous, the 3rd parous and the 4th parous recipients. These results indicated that fresh embryos are more demanded than frozen-thawed embryos for good conception rate in embryo transfer with biopsied-sexed embryo. Also, it was indicated that we should consider embryo-recovering condition, recipient's uterine thickness, transfer site in uterine horn, quality of corpus luteum, time required for transfer and parity of recipient to achieve good conception rate in ET with biopsied-sexed embryos.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.409-419
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• 2001

This study was to examine whether the in vitro friability, motility and intact acrosome of frozen-thawed bovine and human sperm can be improved by adding Pentoxifylline (PF) or Fertilization Promoting Peptide (FPP). Human semen was frozen ultra-rapidly using Test yolk-buffer (TYB) freezing medium. Additive (PF, FPP) effects in frozen-thawed bovine and human sperm were analyzed by microscopic count for sperm motility and coomassie brilliant blue staining method f3r sperm acrosome intact. The in vitro motility of frozen-thawed bovine sperm with 5 mM PF treatment group (50.0%) was significantly higher than that of control (34.0%) (P＜0.05). In the frozen-thawed bovine sperm was examined, the intact acrosome rate of 50 nM FPP treatment (49.0%) was significantly higher than those of control (30.0%) and 25 nM FPP (38.0%) treatment groups (P＜0.01). In human semen, when in vitro motility of sperm with PF addition prior to freezing was examined, the result of 5 mM treatment group (51.0%) was significantly higher than those of control and 2.5 mM treatment group (39.0, 40.0%) (P＜0.01). In addition, 50 nM (75.5%) FPP adding in all treatment procedures for human semen freezing (before freezing, freezing and after thawing) was significant effect on maintenance of the sperm intact acrosome percentage (control: 45.0; 25 nM: 53.0; 100 nM: 68.0%) (P＜0.01). Also, the intact acrosome rate of human sperm with FPP (65.0%) was significantly higher than that with PF (43.0%) (P＜0.05), although sperm motility was slightly higher in PF treatment group. These results suggest that improved sperm motility and intact acrosome of frozen thawed bovine and human sperm can be obtained by addition of PF or FPP, and that the enhanced in vitro viability, motility and intact acrosome can be obtained by addition of FPP in all semen freezing procedures.