• Korean Journal of Animal Reproduction
• /
• v.16 no.3
• /
• pp.277-284
• /
• 1992

The cattle follicular oocytes matured for 26~28h in culture condition were examined at 4, 5, 6, 7, 8, 10, 12, 14, 16 and 18h after insemination with bull spermatozoa preincubated for 4.5h in the uter isolated from estrous hamsters. After further culture with spermatozoa for 4~18 h, 73~89% of the total oocytes had matured to the second metaphase. None of the follicular oocytes matured in culture, were fertilized 5h after insemination. But when the oocytes were examined at 6, 8, 10, 14 and 18h after insemination, 60, 73, 82, 80 and 87% of oocytes were fertilized, respectively. The majority of the fertilized oocytes had enlarged sperm head at 6h after insemination and a part of the fertilized oocytes begun to develop from enlarged sperm head to male pronuclear stage at 8h after insemination, and most of them developed to male and female pronuclear stage at 10h after insemination. The results suggest that the penetration of spermatozoa into the oocytes may occur earlier than 6h after insemination and development of their pronuclear stage may occur at 8h after insemination.

• Asian-Australasian Journal of Animal Sciences
• /
• v.18 no.11
• /
• pp.1557-1563
• /
• 2005

Ovarian follicular fluid contains both stimulatory and inhibitory agents that influence the growth and maturation of oocyte. In the present study, an attempt was made to isolate and study the biological properties of ovarian follicular fluid peptide(s) in buffaloes. Bubaline ovarian follicular was made steroid- and cell-free. A protein fraction was obtained by saturation (30-35% level) of the follicular fluid with ammonium sulfate. The protein fraction was purified with Sephadex-G 50 gel filtration chromatography and a single peak was obtained in the eluant volume, which was lyophilized. SDS-PAGE of the lyophilized fraction revealed a single band and the molecular weight of the peptide was 26.6 kDa. The peptide stimulated the cumulus cell expansion and in vitro maturation rate of oocytes in buffaloes in a dose dependent manner when it was incorporated at different dose levels (0, 10, 25, 50, 100 and 1,000 ng $ml^{-1}$ of maturation medium). The basic culture medium consisted of TCM 199 with Bovine serum albumin (0.3%). The in vitro maturation rates were comparable to those obtained with a positive control medium (TCM 199+20 ng EGF $ml^{-1}$+steer serum (20%)). Further purification and biological assays may throw more light on the nature and functions of this peptide.

• Journal of Embryo Transfer
• /
• v.19 no.1
• /
• pp.11-18
• /
• 2004

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37$^{\circ}C$/with hFF, Group II: 32$^{\circ}C$/withGroup III: 37$^{\circ}C$/without, Group IV:32$^{\circ}C$ /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.9$\pm$1.9%. After 24 hours culture, sperm motility was 23.5$\pm$2.1% (Group I), 8.1$\pm$1.1% (Group II), 10.4$\pm$ 1.4% (Group III) and 4.0$\pm$0.8% (Group IV), respectively. After 48 hours, the motility had been changed as 32$\pm$2.3% (Group I), 14.3$\pm$1.7% (Group II), 5.3 $\pm$1.4% (Group III) and 4.3$\pm$0.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37$^{\circ}C$ was higher than those of group II at 32$^{\circ}C$. But, sperm viability of group I cultured at 37$^{\circ}C$ was lower than those of group II at 32$^{\circ}C$ (54.4$\pm$4.1% vs. 59.4$\pm$3.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37$^{\circ}C$. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.

• Korean Journal of Animal Reproduction
• /
• v.14 no.1
• /
• pp.84-91
• /
• 1990

Experiments were disigned to define and optimize efficiency of a system whereby pig follicular oocytes could be matured and fertil ized in vitro. The pig oocytes removed from 1- 2 mm and 3-7 mm follicles were cultured in vitro in the mKRB(-BSA) solution containing estrous sow serum (ESS), FCS or dialyzed pig follicular fluid for 24 to 48 hr at 37$^{\circ}C$. The oocytes matured in vitro were evaluated after epididymal spermatozoa-oocyte incubation for 24 hr for pronucleus formation. 50-60% of the oocytes reached metaphase II during 36 to 48 hr of culture. There was no differernce in oocyte matura¬tion between two groups of follicular size but meiosis was slightly faster in the 3-7 mm follicular oocytes. The oocytes matured in mKRB (-BSA) plus 5% ESS, 15% FCS or dialyzed follicular fraction showed slightly higher maturation rates than the control mKRB. in vitro fertilization, pronucleus formation, tended to be increased when mKRBi-BSA) plus 5% ESS or 15% FCS was used for oocyte maturation and in vivo -capacitated spermatozoa were inseminated, respectively. It is concluded that ESS, FCS and dialyzed pig follicular fluid may be effective factors for in vitro maturation and fertilization of pig follicular oocytes.

• Clinical and Experimental Reproductive Medicine
• /
• v.19 no.2
• /
• pp.125-131
• /
• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Korean Journal of Animal Reproduction
• /
• v.13 no.1
• /
• pp.32-39
• /
• 1989

The present studies were conducted to investigate the possibility of capacitation of bull spermatozoa in the uteri isolated from estrous hamsters and to investigate the possibility of in vitro fertilization of follicular oocytes matured in culture, using ejaculat spermatozoa preincubated in the uteri isolated from estrous hamsters. The follicuar oocytes matured in culture were not fertilized after insemination spermatozoa preincubated for 3 and 5.5-7 h in the uteri isolated from estrou hamsters, but 5, 77, 85 and 50-67% of those oocytes were fertilized by spermatozoa preincubated for 3.5, 4, 4.5 and 5h in those uteri, respectively.

• Reproductive and Developmental Biology
• /
• v.30 no.3
• /
• pp.163-167
• /
• 2006

The purpose of the present study was to establish culture conditions for the in vitro study of the neonatal piglet Sertoli cell. Isolation for the culture of Sertoli cell was established using collagenase and pancreatin digestion of testicular tissues. The effects of various culture media, fetal bovine serum(FBS), follicular stimulating hormone(FSH), epidermal growth factor(EGF) and insulin-transferrin-sodium selenite(ITS) on growth of neonatal piglet Sertoli cells were investigated. The mitogenic effects of Dulbecco's modified Eagle's medium+Ham's F-12 medium was higher than other media used in this experiment. The addition of 1% FBS in cultures was necessary for attachment of Sertoli cell clusters. However, except FBS and EGF, FSH and ITS did not stimulate Sertoli cell proliferation. When Sertoli cells isolated from neonatal piglets were cultured in Dulbecco's modified Eagle's medium+Ham's F-12 medium supplemented with 1% FBS, FSH EGF and ITS, the yield and plating efficiency of Sertoli cells were largely increased. Confluency of Sertoli cells was reached as early as 4 days of culture. The method described here reduces or eliminates many of the drawbacks of the conventional procedures used to isolate and culture of Sertoli cells, thus providing a useful tool in studies of growth kinetics and regulation of cell proliferation in vitro.

• Korean Journal of Animal Reproduction
• /
• v.22 no.1
• /
• pp.95-99
• /
• 1998

Three experiments were conducted with follicular oocytes, to compare some somatic cells, growth factors and media for in vitro maturation of bovine oocytes. In the first experiment, the type of somatic cells had no effects on in vitro maturation of bovine follicular ooctyes. In the second experiment, oocytes were matured in TCM199 su, pp.emented with growth factors on IVM of bovine follicular oocytes, then all were co-cultured with cumulus cells. The proportion of used oocytes that developed to expanding blastocysts was 22.2%, 20.2%, 17.7%, 22.2%, 24.4% and 20.2% after maturation in TCM199 su, pp.emented with control, insulin, IGF-I, IGF-Ⅱ, FGF and EGF, respectively. In the third experiment, oocytes were matured in BO, Ham's F10 and TCM199, then all were fertilized in BO, and embryos cultured in BO, Ham's F10 and TCM199, respectively. Cleavage rates in BO were 90%, had higher than in Ham's F10(80%) or in TCM199(64%). But production of expanding blastocysts in TCM199(21%) or Ham's F10(20.6%), had higher than in BO(4.6%).

• Korean Journal of Animal Reproduction
• /
• v.17 no.2
• /
• pp.113-120
• /
• 1993

To provide the optimal culture conditions for the developm,ent of in vit개 produced embryos, we have been investigated various culture media as well as co-cultrue systems using porcine cumulus cells or mouse fetal fibroblast cells. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles(3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated(39$^{\circ}C$, 5% CO2 in air) in various maturation media for 42 hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were rpepared for fertilizing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${\mu}\ell$ fo capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three times with culture media and transferred to the various culture media, to the culture media with a monolayer of somatic cells. The in vitro development rates of 1-cell embryos cultured with three different media, m-KRB, BECM and TCM-HEPES were 0~1.0%, showing extremely lower rates. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. The rates of embryos developed to 2-, 4-, 8-, 16-, 32-cell and morula or blastocyst stage in co-culture with porcine cumulus cells and mouse fetal fibroblast cells were 61.1~67.0%, 59.0~58.0%, 42.5~43.1%, 28.4~30.2% and 20.4~21.0%, respectively. These development rates upto morula or blastocyst stages were significantly higher than those of the embryos cultured in the basic culture medium(P<0.01). These findings suggest that co-culture of in vitro fertilized eggs with porcine cumulus cells or mouse fetal fibroblast cells enhance the development of fertilized eggs to morula or blastocyst stage in vitro.

• Korean Journal of Animal Reproduction
• /
• v.14 no.4
• /
• pp.263-271
• /
• 1990

This study was carried out to investigate the factors affecting maturation in vitro of follicular oocytes 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 follicles of 3~6mm. The bovine oocytes were matured in vitro in TCM-199 containing FCS and hormones. The effects of TCM-199 salt type, number of oocytes per drop, incubation time and co-culture with granulosa cells on maturation of oocytes, were investigated. The results obtained are summarized as follows. 1. The maturation rates of follicular oocytes cultured for 22, 25 and 28 hours in Hank's TCM-199 or Earle's TCM-199 were 59.3, 59.6, 80% and 80.0, 90.0, 93.7%, respectively. The maturation rate of follicular oocytes in Earle's TCM-199 was faster and higher than in Hank's TCM-199(P<0.05). 2. The maturation rates of oocytes were 54.5, 62.5 and 62.0% when cultured the oocytes number 10, 20 and 40 per 200${mu}ell$ drop for 18 hours. 3. The maturation of follicular oocytes in the Korean Native Cattle was induced at 14 hours culture, by giving the maturation rate of 90.0% after 20 hours. 4. The maturation rates were 63.3% and 66.7%, respectigely when the oocytes were co-cultured with granulosa cells from medium-size follicles used immediately after recovery in the presence or absence of hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$). When the oocytes were co-cultured with granulosa cells from medium-size follicles cultured for 3 days, the maturation rates were 20.8% and 76.2%, respectively(P<0.05). 5. The maturation rate were 88.0% and 70.0%, respectively when the oocytes were co-cultured with granulosa cells from large-size follicles used immediately after recovery in the presence or absence of hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$)(P<0.05). When the hormones added(0.02AU/ml FSH, 10$\mu\textrm{g}$/ml LH and 1$\mu\textrm{g}$/ml estradiol 17-$\beta$)(P<0.05). When the oocytes were co-cultred with granulosa cells from large-size follicles cultured for 3 days, the maturation rates were 41.2% and 73.3%, respectively(P<0.05).