• Korean Journal of Animal Reproduction
• /
• v.13 no.2
• /
• pp.98-104
• /
• 1989

Successful techniques of in vitro fertilization(IVF) are valuable for studying the process of fertilization and for developing economical procedures for gene and nuclear transfer in farm animals. To date, bovine IVF system has been developed with oocytes in vitro or vitro, but the resulting zygotes exhibit limited embryonic development after in vitro culture. Even though in vitro matured oocytes achieved high fertilization and cleavage rates, these embryos appear extremly low rate of pregnancies when transferred to synchronized recipients. Development of early bovine embryos in vitro is generally arrested at the 8-to 16-cell stage. However, recent use of somatic cells such as trophoblastic vesicle, granulosa and oviduct epithelial cell for co-culture with early bovine embryos has proven effective for development of embryos, matured and fertilized in vitro, past the in vitro cell blocks. These factors clearly indicate the value of the co-culture system in promoting development of bovine oocytes matured and fertilized in vitro to morula or blastocyst stage in vitro. In addition, co-culture system may beome a tool for evaluation of viability of ova that have been manipulated by procedures such as splitting, microinjection and nuclear transfer.

• Asian-Australasian Journal of Animal Sciences
• /
• v.24 no.11
• /
• pp.1541-1546
• /
• 2011

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes.

• Korean Journal of Animal Reproduction
• /
• v.22 no.1
• /
• pp.101-104
• /
• 1998

This study examined the effects of growth factors in TCM199 on bovine 1-cell embryos development in vitro. After 6 day to 11 day in culture, 15.8%(19/120), 15.3%(20/130), 21.8%(35/160), 27.0%(56/207), 26.3%(53/201) and 30.7%(40/130) of the 1-cell embryos developed into expanding blastocysts in su, pp.ementing TCM199 with control, insulin, IGF-I, IGF-II, FGF and EGF, respectively. Hatching rate of 1-cell embryos in su, pp.ementing TCM199 with FGF, EGF and IGF-II were 21.4%(53/247), 20.3%(42/206) and 16.8%(41/243), respectively. The beneficial effect of growth factors on embryo development in vitro could be duplicated. These data indicate that the presence of FGF, EGF or IGF-II in the culture medium is beneficial for embryo development in vitro and accelerate cell differentiation.

• Journal of Embryo Transfer
• /
• v.11 no.3
• /
• pp.217-223
• /
• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula /blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine endometrial cell monolayers(PEM) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8~16-cell and morula /blastocyst stage were 49.6, 40.5, 28.2 and 15.3% in Ham's F-10 with PEM, and 55.3, 45.9, 32.7, and 17.6% in TCM-HEPES with PEM, respectively. The above development rates to morula /blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TGM-HEPES without PEM(P<0.05). The in vitro development rates to the morula /blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without PEM were 0~1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with PEM in the two different media enhanced the development of fertilized eggs to morula /blastocyst stages in vitro. However, we didn't find out any differences for the in vitro development to morula /blastocyst stages between Ham's F-10 and TcM-HEPES media.

• Korean Journal of Animal Reproduction
• /
• v.17 no.1
• /
• pp.69-74
• /
• 1993

This study was investigated to examine the effect of conditioned medium from bovine oviductal cell(BOEC) in the co-culture system with BOEC on in vitro development of in vitro produced bovine embryos. Oocyte-cumulus complexes were cultured for 24 hrs in TCM-199 supplemented with 10% fetal calf serum, 1$\mu\textrm{g}$/ml FSH and 21U hCG, 1$\mu\textrm{g}$/ml oestradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. In vitro fertilization was performed with epididymal sperm and heparin (10$\mu\textrm{g}$/ml, 15min.) or caffeine(2.5mM)-treated spermatozoa. Oocytes were incubated with 1$\times$106 spermatozoa/ml for 18 hrs and then cultured in various culture system for 7 days. The development rates of 16-cell or blastocyst stages were recorded on 4, 7 days, respectively, after incubating. The proportions ofembryonic development into molulae and blastocysts were higher in cumulus cell co-culture(23.4%) and BOEC co-culture(34.3%) than in M199-FCS(6.1%). Similarily, the development rates into molulae and blastocysts were significantly higher in BOEC-conditioned medium than those in M199-FCS. Therefore, it is suggested that BOEC co-culture and BOEC conditioned medium increase significantly the development of in vitro produced bovine embryos in in vitro system.

• Korean Journal of Animal Reproduction
• /
• v.14 no.4
• /
• pp.253-261
• /
• 1990

This study was carried out to investigate the factors affecting development in vitro of follicular oocytes fertilized in vitro in Korean Native Cattle. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follciles of 3~6mm. The bovine oocytes were matured in vitro for 20~24 hours in TCM0-199 containing 10% FCS and hormones (0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml estradiol-17$\beta$). The matured oocytes were fertilized in vitro using Percoll-separated frozen-thawed spermatozoa in BO solution containing caffeine(5mM) and heparin(10$\mu\textrm{g}$/ml). Twenty-four hours after insemination, the oocytes were cultured in vitro and then the effects of cumulus cell layer, co-culture with cumulus cells, bovine oviduct epithelial cells from ampulla or isthmus on development of ova, were studied. The results obtained are summarized as follows : 1. The in vitro development degree of oocytes attached with compact and dense layered cumulus cells was higher than that with 3~4 layered cumulus cells to be 9~16cells(P<0.01). 2. When the in vitro fertilized oocytes were co-cultured with bovine oviduct epithelial cells or cumulus cells, the development rate to be morula was 20.2% and 12.7%, respectively and the rates were higher than that of control, 2.1%(P<0.05). 3. The development rate to be morula was 15.8% and 23.8%, respectively when the in vitro fertilized oocytes were co-cultured with bovine oviduct epithelial cells from ampulla or isthmus, and the rates were higher than that of control, 0%(P<0.05%).

• Korean Journal of Animal Reproduction
• /
• v.20 no.3
• /
• pp.299-305
• /
• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula/blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine oviductal epithelial cell monolayers(POEC) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8∼16-cell and morula/blastocyst stage were 57.2, 48.2, 37.2 and 19.3% in Ham's F-10 with POEC, and 51.4, 41.2, 31.1, and 15.5% in TCM-HEPES with POEC, respectively. The above development rates to morula/blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TCM-HEPES with out POEC(P<0.05). The in vitro development rates to the morula/blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without POEC were 1.1∼1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with POEC in the two different media enhanced the development of fertilized eggs to morula/blastocyst stages in vitro. However, we didn't find out any difference for the in vitro development to morula/blastocyst stages between Ham's F-10 and TCM-HEPES media.

• Korean Journal of Animal Reproduction
• /
• v.8 no.2
• /
• pp.110-115
• /
• 1984

These experiments were carried out to obtain the information about the optimal pH osmolality affecting in vitro fertilization of the mouse eggs, to elucidate the 2-cell block to development in vitro and to find out the method of controlling the subsequent embryo development in vitro. pH and osomlality was adjusted by adding NaCl or NaHCO3 to the basic salt solution. In vitro fertilization were carried out by inroducting the cumulus masses to the suspension of epididymal spermatozoa at each pH, osmolality, and 10${\mu}$M-EDTA medium. The results obtained in these experiments were summarized as follows: 1. The fertilization rates in vitro at each medium of 235, 252, 269, 286, 306, 323, 345, 368, 393 mosmol were 15.6, 38.2, 65.7, 75.6, 80.9, 74.3, 58.1, 35.1, 24.3, 11.1%, respectively. 2. The fertilization rates in vitro at each medium of pH 6.1, 6.4, 6.7, 7.0, 7.3, 7.6, 7.9, 8.1 were 11.8, 17.9, 32.4, 61.9, 79.5, 76.7, 53.5, 13.6%, respectively. 3. In case of ICR female x ICR male embryos, the development rate of 2-cell embryos to 4-8 cell embryos was 16.2% at normal medium, but the rate was increased to 49.3% in medium containging 10 ${\mu}$M-DETA; In case of C3H female x ICR male embryos, the development rate was 41.0% at normal medium, but the rate was increased to 71.7% at 10 ${\mu}$M-EDTA-medium.

• Journal of Animal Science and Technology
• /
• v.66 no.2
• /
• pp.374-386
• /
• 2024

To predict the apparent total tract digestibility (ATTD) of crude protein (CP) in dogs we developed an in vitro system using an in vitro digestion method and a statistical analysis. The experimental diets used chicken meat powder as the protein source, with CP levels of 20% (22.01%, analyzed CP value as dry-based), 30% (31.35%, analyzed CP value as dry-based), and 40% (41.34%, analyzed CP value as dry-based). To simulate in vivo digestive processes a static in vitro digestion was performed in two steps; stomach and small intestine. To analyze ATTD the total fecal samples were collected in eight neutered beagle dogs during the experimental period. CP digestibility was calculated by measuring CP levels in dog food, in vitro undigested fraction, and dog feces. In result, CP digestibility at both in vivo and in vitro was increased with increasing dietary CP levels. To estimate in vivo digestibility the co-relation of in vivo ATTD and in vitro digestibility was investigated statistically and a regression equation was developed to predict the CP ATTD (% = 2.5405 × in vitro CP digestibility (%) + + 151.8). The regression equation was evaluated its feasibility by using a commercial diet. The predicted CP digestibility which was calculated by the regression equation showed high index of similarity (100.16%) with that of in vivo in dogs. With that, it would be a feasible non-animal method to predict in vivo CP digestibility by using in vitro digestion method and the proposed linear regression equation in adult dogs.

• Journal of Embryo Transfer
• /
• v.12 no.3
• /
• pp.283-291
• /
• 1997

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.