• Journal of Embryo Transfer
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• v.17 no.3
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• pp.211-218
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• 2002

These studies were carried out to investigate the effects of the addition of amino acids and FBS as source of exogenous nitrogen fixation added to medium on in vitro production of blastocyst derived from bovine follicular oocytes. The base medium was TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and YS solution for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1) after IVF. Rmbryos were cultured in drop-culture that contained 25 embryos per 10 ${mu}ell$. The results obtained are as follows: 1 The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with NEAA derived from MEM alone were higher than those of YS solution without NEAA. 2. The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with EAA derived RPMI 1640 alone were significantly higher than those of YS solution without EAA (p<0.05). 3. When added to EAA on day 5 after NEAA supplementation on day 1, the developmental rates of hatched blastocyst and blastocyst to hatched blastocyst were improved. 4. When removed to EAA on day 3, day 4 and day 5 from medium after NEAA and EAA supplementation on day 1. the developmental rates of blastocyst to hatched blastocyst were reduced. 5. When added to FBS as source of exogenous nitrogen fixation, the developmental rates of blastocyst and hatched blastocyst that developed from the later culture higher(day 5) than those of the early culture.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.201-210
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• 1996

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.213-219
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• 1998

This study was carried out to investigate on the improvement of fertilizing ability of in vitro matured oocytes from sperm density, motility and polyvinylpyrrolidine (PVP) concentration, by intracytoplasmic sperm injection(ICSI) into the bovine oocytes. 1. The in vitro fertilization and cleavage rates of oocytes from 1.0, 2.0, 3.0, 5.0 ($\times$106/ml) sperm concentration by IVF and ICSI of bovine oocytes were 45.0%~65.0%, 65.0%~90.0% and 10.0%~30.0%, 35.0%~70.0%, respectively. 2. The in vitro fertilization and cleavage rates of oocytes from 20, 40, 60, 80% of sperm motility by IVF and ICSI of bovine oocytes were 47.8%~75.0%, 78.3%~90.0% and 8.7%~25.0%, 34.8%~70.0%, respectively. 3. The in vitro fertilization and cleavage rates of oocytes from 0.01, 0.02, 0.03, 0.05% of PVP concentration by microinjection of single into the bovine oocytes were 72.7%, 90.9%, 83.3%, 76.9% and 45.5%, 72.7%, 58.3%, 61.5%, respectively and these values of 0.02% addition of PVP were higher than other concentrations of PVP. 4. The in vitro fertilization and developmental rates of oocytes by IVF and ICSI methods were 63.3%~64.6%, 26.7%~29.2% and 88.2%, 47.1%, respectively. This ICSI method was improved high fertilization rates of bovined oocytes.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.71-81
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• 1994

These experiments were carried out to investigate the effects of human follicular fluid (hFF) as a protein supplement on development of mammalian embryo as well as to find out ways toward effective use of hFF. The developmental rates of mouse embryos to the blastocyst and implantation stages were significantly higher in T6 ＋hFF than T6＋hFCS. Classified hFF according to the maturity of contained oocytes (M-hFF and Im-hFF), and compared the rates of development of mouse embryo cultured in M-hFF or Im-hFF to culture medium T6. Total protein, albumin and estradiol concentrations were higher in M-hFF than Im-hFF (P<0.05). The developmental rates of mouse embryos to the blastocyst and hatching blastocyst stages cultured in Im-hFF were significantly lower than those in M-hFF and the basic medium. In accordance of the results of human IVF, hFF has been divided into 4 groups. The developmental rates of mouse embryos to the blastocyst stage in presense of hFF from pregnant patients, who have good grade embryos, were significantly higher than those in hFF from patients who have poor grade embryos or were not pregnant. In addition, the rates of development of human embryo were compared in presense of BSA, hFF or hFCS. The developmental rates of human embryos cultured in Ham's F10＋hFF were significantly higher than those in the Ham's F10＋BSA. These results suggests that the culture system using hFF could improve the development ability of mammalian embryos and the viability of blastocysts cultured in vitro.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.108-108
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• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.56-56
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• 2001

This study was carried out to investigate on the improvement of fertilizing and developing ability of in vitro matured oocytes from individuals of bulls, sperm type, pretreatment of sperm or oocytes obtained by intracytoplasmic sperm injection(ICSI). 1. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated individual of bulls were 73.9%-87.0% and 33.3%-60.9%, respectively. 2. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated fresh and frozen sperm, tail-cutting and tail-scoring sperm were 82.0%, 78.0%, 42.2%, 51.1% and 56.0%, 42.0%, 17.8%, 22.2% respectively. and these values of fresh sperm injection were higher than that of frozen sperm, tail-cutting and tail-scoring. 3. The male pronuclear formation and developmental rates of oocytes obtained by sperm pretreated heparin, BFF(bovine follicula fluid), His, Ca Ionophore(Ⅰ) and Ⅰ ＋ caffeine methods were 66.7%-82.2% and 33.3%-60.6%, respectively. and these values of treatment of Ⅰ＋ caffeine were higher than that of other methods. 4. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated with or without zona pellucida were 80.0%, 72.0% and 46.0%, 36.0%, respectively.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.133-139
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• 2005

Oxidative stress is one of the major causes of failure in in vitro storage of boar semen. Reactive oxygen species (ROS) are known to be important mediators of such stress. The present study examined the effects of pyruvate and taurine on sperm motility and expression of BAD, Cytochrome c, Caspase-3 and Cox-2 protein in in vitro storage of boar semen, and tested the effect of semen treated with antioxidant with or without hydrogen peroxide on the development of IVM/IVF porcine embryos. Semen samples were transported to the laboratory at $17^{\circ}C$ within 2 hr after collection and were treated with different concentration of pyruvate $(1\~10mM)$ and taurine $(25\~100mM)$ with or without 250uM $H_2O_2$ respectively. The supplementation of pyruvate and taurine increased sperm motility in boar semen during in vitro incubation at $37^{\circ}C$. Expression of apoptosis protein (BAD, cytochrome c, caspase-3 and cox-2) were reduced in the group of boar semen treated with pyruvate and taurine when compared to the other groups. The developmental rates of IVM/IVF porcine embryos fertilized by semen treated with pyruvate and taurine were significantly increased when compared to control (P<0.005). These results indicate that supplementation of pyruvate and taurine as antioxidants in boar semen extender can improve the semen quality and increase in vitro development of porcine IVM/IVF embryos when boar semen treated with antioxidants was used for in vitro fertilization.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.21-25
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• 2010

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% $CO_2$, 95% air, $38^{\circ}C$. The in vitro maturation rate to MII stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44~72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.143-150
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• 2023

Background: The successful production of superior or transgenic offspring from in vitro produced embryos in cattle relies heavily on the quality of blastocyst stage embryos. In order to enhance the developmental competency of these embryos, a novel culture method was devised. Methods: This study utilized stem cell culture medium (SCM) from hESCs as a supplement within the culture medium for bovine in vitro produced embryos. To gauge the efficacy of this approach, in vitro fertilized embryos were subjected to culture in CR1aa medium enriched with one of three supplements: 0.3% BSA, 10% FBS, or 10% SCM. Results: The blastocyst development and hatching rates of one-cell zygotes cultured in CR1aa medium supplemented with SCM (23.9% and 10.2%) surpassed those cultured in CR1aa medium supplemented with BSA (9.3% and 0.0%) or FBS (3.1% and 0.0%) (p < 0.05). Furthermore, post-zygotic gene activation, cleaved embryos cultured in CR1aa medium supplemented with SCM (57.8% and 34.5%) exhibited notably higher rates (p < 0.05) compared to those cultured with BSA (12.9% and 0.0%) or FBS (45.7% and 22.5%) supplementation. Furthermore, the microinjection of SCM into the cytoplasm or pronucleus of fertilized zygotes resulted in elevated blastocyst development and hatching rates, particularly when the microinjected embryos were subsequently cultured in CR1aa medium supplemented with SCM from the 8-cell embryo stage onwards (p < 0.05), in contrast to those cultured with FBS supplementation. Conclusions: In conclusion, this study conclusively demonstrated that the incorporation of SCM into the culture medium significantly enhances the developmental progress of preimplantation embryos.

• 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.