• Korean Journal of Animal Reproduction
• /
• v.23 no.3
• /
• pp.263-270
• /
• 1999

This study was conducted to investigate effects of L-ascorbic acid and selenium on maturation, fertilization, and development ablity of porcine follicular oocytes in vitro. When the follicular oocytes were cultured in the media containing 0, 62.5, 100 and 300 $\mu$M of L-ascorbic acid for 40~44h, the percentages of germinal vesicle breakdown were 86.8, 92.9, 91.7 and 92.6% respectively, and the nuclear maturation rates (M II) were 44.7, 57.1, 52.8 and 53.7%. The nuclear maturation rates of treated groups were significantly higher than those of non-treated group (p＜0.05). When the follicular oocytes were cultured at 0, 0.4, 0.8, and $1.5\mu$M of selenium for 40~44h, the nuclear maturation rates of treated groups were significantly higher than those of non-treated group (p＜0.05). The addition of L-ascorbic acid or selenium to the maturation medium, the incidence of male pronuclear formation was significantly increased (p＜0.05) and polyspermy rate was significantly decreased (p＜0.05). The addition of L-ascorbic acid or selenium to the maturation medium increased the clevage rate, morula and blastocyst rate (p＜0.05). These results suggested that the addition of L-ascorbic acid and selenium to maturation medium increase the nuclear maturation rates, male pronuclear formation and normal embryonic development: in porcine oocytes matured and fertilized in vitro.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.30 no.2
• /
• pp.203-210
• /
• 1997

The aim of this study was to determine the effect of steroids and human chorionic gonadotropin (HCG) on in vitro maturation and ovulation of oocyte in Pseudobagrus fulvidraco. Oocytes were incubated in the media Leibovitz L15 supplemented with the various concentration of $17\alpha,\;20\beta-dihydroxy-4-pregnen-3-one(17\alpha20{\beta}OHP),\;17\alpha-hydroxyprogesterone(17{\alpha}OHP),\;progesterone(P_4),\;estradiol-17\beta(E_2)and\;HCG$. After 60 hours incubation, the maturation ability of oocyte was assessed by the appearance of germinal vesicle breakdown (GVBD). GVBD was significantly enhanced by the addition of $17\alpha20{\beta}OHP,\;17{\alpha}OHP,\;P_4\;and\;HCD(P<0.05)$. The highest CVBD was observed when $17\alpha20{\beta}OHP$ and HCG were supplemented to media. When oocytes were cultured for 16 hours in media containing $10\~1,000\;ng/ml\;17\alpha20{\beta}OHP,\;17{\alpha}OHP\;and\;P_4$, the rate of GVBD in oocytes cultured in the medium supplemented with 100 ng/ml $17\alpha20{\beta}OHP(65\%)$ was significantly higher than that with $17{\alpha}OHP\;(40\%)\;and\;P_4(35\%)$. The efforts of $17\alpha20{\beta}OHP$ and HCG on GVBD were assessed by various concentration of these hormones. When oocytes were cultured for 60 hours in various media containing $1\~1,000\;ng/ml\;17{\alpha}20{\beta}OHP\;or\;5\~1,000\;IU/ml$ HCG, the GVBD of oocytes was significantly increased in the medium with $10\~100\;ng/ml\;17\alpha20{\beta}OHP$ and 500 IU/ml HCT. When oocytes were cultured in the various media supplemented with $1\~1,000\;ng/ml\;17\alpha20{\beta}OHP\;or\;5\~1,000\;IU/ml$ HCG for 60 hours, the media with $1\~100\;ng/ml\;17\alpha20{\beta}OHP\;or\;50\~1,000IU/ml$ HCG significantly increased in the rate of ovulation. However supplementation with $1,000\;ng/ml\;17\alpha20{\beta}OHP$or 5 IU/ml HCG did not improve the rate of ovulation compared to controls. This results indicate that supplementation of steroid and HCG except $E_2$ can improve the in vitro maturation and ovulation of oocyte in P. fulvidrac; HCG and $17\alpha20{\beta}OHP$ may be more effective than other steroids on oocyte maturation and ovulation in P. fulvidraco.

• Journal of Embryo Transfer
• /
• v.16 no.3
• /
• pp.163-172
• /
• 2001

The present study was carried out to examine the effects of $O_2$concentrations and culture media on in vitro maturation and embryo development of porcine follicular oocytes. The results were summarized as fellows : 1. The rates of GVBD and nuclear maturation in NCSU-23 and TCM-199 media with 10% PFF under the conditions of 5% and/or 20% $O_2$concentrations were not different among the each treatment groups(P>0.05). 2. The rates of polyspermy and mean numbers of penetrated sperm were significantly lower in NCSU-23 medium than in TCM-199 medium (P>0.05). However, the rates of polyspermy and mean numbers of penetrated sperm were not different between 5% and 20% $O_2$concentrations. 3. The rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in NCSU-23 medium under the condition of 20% $O_2$concentration than in TCM-l99 medium under the condition of 5% or 20% $O_2$concentrations. However, the rates of blastocyst formation and total cell numbers in blastocysts were not different between 5% and 20% $O_2$concentrations. In conclusion, the use of NCSU-23 medium under the condition of 20% $O_2$concentration was beneficial for porcine oocyte maturation and in vitro development.

• Korean Journal of Agricultural Science
• /
• v.34 no.1
• /
• pp.13-18
• /
• 2007

This study was carried out to investigate the effects of ovarian estrus stage of oocytes and supplementation of medium with BSA, cysteine and myoinositol on in vitro maturation of canine oocytes. The in vitro maturation(IVM) rate to metaphase II (M II) stage of oocytes recovered from different stage of the reproductive cycle (inactive, follicular and luteal stage) cultured in TCM-199 media were $0.0{\pm}0.0%$, $10.0{\pm}4.1%$ and $5.7{\pm}1.6%$, respectively. The IVM rate of oocytes collected from follicular stage was significantly higher in inactive or luteal stage of oocytes. The IVM rates of oocytes recovered from different stage of the reproductive cycle cultured in TCM-199 media with 5% BSA and 0.1 mM cysteine were$0.0{\pm}0.0%$, $15.8{\pm}4.7%$, $5.6{\pm}1.5%$, respectively. The IVM rates of oocytes recovered from different stage of the reproductive cycle cultured in TCM-199 media with 5% BSA and 10 mM myoinositol were $0.0{\pm}0.0%$, $18.4{\pm}4.6%$ and $5.7{\pm}1.9%$, respectively. The IVM rate of follicular stage oocytes was significantly higher in oocytes collected from follicular stage and with cultured 5% BSA and 0.1 mM cysteine or 5% BSA and 10 mM myoinositol compared to other experimental group.

• Asian-Australasian Journal of Animal Sciences
• /
• v.14 no.10
• /
• pp.1360-1366
• /
• 2001

The aims of this study were first to evaluate the effects of different levels (20, 40 and 100%) and sources (follicular size: large, >7 mm; medium, >5-7 mm; small, 3-5 mm) of porcine follicular fluid (pFF) on the in vitro maturation (IVM) of porcine oocytes, and the effects of fertilization treatments and different culture conditions on development of fertilized oocytes were also investigated. No differences in the maturation (63.6-76.6%) and cleavage (24.8-34.3%) rates were observed among the 20,40 and 100% pFF groups (p>0.05). The cleavage rates of oocytes cultured and fertilized in 40% and 100% pFF maturation media were significantly higher than those fertilized in m199-NBCS (51.0-61.2% vs. 12.8-31.8%. p<0.05), regardless of sources of the pFF. When oocytes were fertilized in m199-NBCS followed by culture in rabbit oviducts for 4 days, the cleavage rate in 40% pFF group was better than that in 100% pFF group (46.9% vs. 32.5%, p<0.05). Two oocytes recovered from the oviducts in the 40% pFF group developed to blastocysts after IVC. However, none developed to blastocysts when fertilized in the IVM medium after being transferred to rabbit oviducts. In conclusion, addition of pFF accompanied with gonadotropins (FSH, LH) in IVM medium enhanced maturation and cleavage rates of porcine oocytes. Direct addition of sperm suspension to IVM medium may be an alternative to simplify the fertilization procedures and to reduce the mechanical lesion during manipulation. Furthermore, rabbit oviducts provide a better environment for the in vitro fertilized oocyte developing to the morula and blastocyst stages.

• Korean Journal of Veterinary Research
• /
• v.59 no.2
• /
• pp.81-88
• /
• 2019

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF ($113.0-114.8{\mu}m$) were significantly larger than that of no IVG-SAF ($111.8{\mu}m$). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

• Asian-Australasian Journal of Animal Sciences
• /
• v.19 no.4
• /
• pp.495-499
• /
• 2006

In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p<0.05) and FBS+VEGF (42.1%, p<0.05) groups than in the PVA group (17.1%). Two- factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

• Journal of Acupuncture Research
• /
• v.23 no.2
• /
• pp.103-111
• /
• 2006

Objectives : Deer antler Water Extract(DAE), prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong), a traditional immuno-suppressive and immuno-activating Korean herbal-acupuncture, is thought to play an important role in human bone remodeling. Methods : To determine whether DAE can induce the differentiation of resting zone chondrocytes(RC) or not, confluent cell cultures were pretreated for 24, 36, 48, 72, and 120hrs with DAE. At the end of pretreatment, the media were replaced with new media containing $10^{-10}{\sim}10^{-8}M\;1,25-(OH)_2D_3$ and the cells incubated for an additional 24hrs. Results : This second treatment was chosen because prior studies had shown that only the more mature growth zone chondrocytes(GC) respond to this vitamin $D_3$ metabolite. The effect of DAE pretreatment on cell maturation was confirmed by measuring alkaline phosphatase (ALPase)-specific activity. Changes in matrix protein synthesis were examined by measuring collagen synthesis, as well as $^{35}SO_4$ incorporation into proteoglycans. When RC cells were pretreated for 120h with DAE, treatment with $1,25-(OH)_2D_3$ caused a dose-dependent increase in ALPase-specific activity and collagen synthesis, however, the proteoglycan production was not affected. RC cells pretreated with $1,25-(OH)_2D_3$ responded like RC cells that had not received any pretreatment. Conclusion : These results indicate that DAE directly regulates the maturation of RC chondrocytes into GC chondrocytes. Therefore it was indicated that DAE may play a significant role in regulating chondrocyte maturation during endochondral ossification.

• The Korean Journal of Pain
• /
• v.35 no.2
• /
• pp.160-172
• /
• 2022

Background: The authors established an in vitro model of diabetic neuropathy based on the culture system of primary neurons and Schwann cells (SCs) to mimic similar symptoms observed in in vivo models of this complication, such as impaired neurite extension and impaired myelination. The model was then utilized to investigate the effects of insulin on enhancing neurite extension and myelination of diabetic neurons. Methods: SCs and primary neurons were cultured under conditions mimicking hyperglycemia prepared by adding glucose to the basal culture medium. In a single culture, the proliferation and maturation of SCs and the neurite extension of neurons were evaluated. In a co-culture, the percentage of myelination of diabetic neurons was investigated. Insulin at different concentrations was supplemented to culture media to examine its effects on neurite extension and myelination. Results: The cells showed similar symptoms observed in in vivo models of this complication. In a single culture, hyperglycemia attenuated the proliferation and maturation of SCs, induced apoptosis, and impaired neurite extension of both sensory and motor neurons. In a co-culture of SCs and neurons, the percentage of myelinated neurites in the hyperglycemia-treated group was significantly lower than that in the control group. This impaired neurite extension and myelination was reversed by the introduction of insulin to the hyperglycemic culture media. Conclusions: Insulin may be a potential candidate for improving diabetic neuropathy. Insulin can function as a neurotrophic factor to support both neurons and SCs. Further research is needed to discover the potential of insulin in improving diabetic neuropathy.

• Journal of Life Science
• /
• v.13 no.5
• /
• pp.732-739
• /
• 2003

The purpose of this study was to determine if the addition of spermatozoa into the culture medium could influence the nuclear maturation of denuded porcine germinal vesicle (GV) oocytes in vitro. Cumulus-oocyte complexes were collected from follicles of 3 to 5 mm in diameter, The cumulus and corona cells were removed from oocytes. Porcine denuded oocytes were cultured in tissue culture medium containing spermatozoa. After 48 h culture, oocytes were examined for the evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II (M II). The proportion of oocytes reaching M II stage was significantly (P<0.01) increased in the oocytes cultured in media containing spermatozoa compared to those in media without spermatozoa $(31.9\pm1.8%\; vs\; 14.9\pm1.0%)$.No differences in the rates of M II were observed among the different period of spermatozoa exposure nor among the spermatozoa from different species. The proportion of oocytes reaching M II stage was significantly different between high and low concentrations of spermatozoa. The present study suggests that mammalian spermatozoa contain a substance(s) that improves nuclear maturation in vitro of GV oocytes. Enhancing effect of spermatozoa for oocytes maturation in vitro is a highly dose-dependent.