• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.240-240
• /
• 2004

The modification of in vitro maturation (IVM) conditions should be required to efficient production of matured porcine oocyte in vitro. Estradiol-17β (E₂) as steroid hormone exists in ovarian follicular fluid and plays a major role in ovulation. It has been reported that estradiol as well as other steroids are involved in keeping the oocytes in meiotic arrest. (omitted)

• Reproductive and Developmental Biology
• /
• v.29 no.2
• /
• pp.121-125
• /
• 2005

The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as $<100{\mu}m,\;<100{\mu}m\;to\;<110{\mu}m,\;<110{\mu}m,\;to\;<120{\mu}m,\;>120{\mu}m,$. Oocytes were cultured in culture medium supplemented with $10\%\;FBS,\;0.4\%\;BSA,\;10\%$ porcine follicular fluid (pFF), $10\%$ canine serum (CS), or $10\%$ canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes $(>120{\mu}m)$ was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of $>110{\mu}m$ groups was significantly higher than that in $<100{\mu}m$ group (p<0.05). The oocytes cultured in $10\%$ FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in $10\%$ FBS-, $0.4\%$ BSA-, and $10\%$ pFF-supplemented groups were significantly higher than those in $10\%$ CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.70-70
• /
• 2002

Sphingosine-1-phosphate(S1P) is one of the sphingolipid metabolites which affect a variety of cellular processes including the proliferation, differentiation, growth, survival, migration and gene expression. The present study was undertaken to investigate the effect of SIP on nuclear maturation of porcine oocytes. In vitro maturation frequency of porcine oocytes were compared in three different media; group Ⅰ: NCSU23＋0.1% PVA, group Ⅱ: NCSU23＋10% PFF(porcine follicular fluid), and group Ⅲ: NCSU23＋10% PFF＋10 ng/㎖ EGF＋2.5 mM β-mercaptoethanol. Each group containing 0.1 ㎎/㎖ cysteine was divided into 4 sub-groups of SIP concentration(0, 50, 500 and 5000nM). Porcine oocytes were incubated in each maturation medium supplemented with hormones(10 IU/㎖ PMSG and 10 IU/㎖ hCG) for 22h and then further cultured in the same medium without the hormones for 22h. After completion of in vitro maturation, the oocytes were fixed and stained to examine nuclear maturation by using a rapid stain method. In the group Ⅰ, the proportions of metaphase Ⅱ stage among oocytes cultured in 0nM(control), 50 nM, 500nM and 5000nM S1P were 45.5%, 66.7%, 56.6% and 48.7%, respectively. (omitted)

• Korean Journal of Agricultural Science
• /
• v.43 no.1
• /
• pp.52-60
• /
• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• Korean Journal of Animal Reproduction
• /
• v.20 no.4
• /
• pp.413-421
• /
• 1997

In vitro culture has provided new information on the mechanisms involved in fertilization how sperm and oocyte fuse together. At the same time, results obtained in vitro have led to new questions. Techniques for In vitro maturation of porcine oocytes have progressed such that the problem of the low rate of pronucleus formation with in vitro matured oocytes after in vitro fertilization has been nearly improved. On the other hand, porcine spermatozoa have been shown to be capacitated if the fertilization medium contains caffeine and Ca$^2+$, but the incidence of polyspermy in IVM-IVF oocytes is still high. To prevent polyspermy, co-culture with oviductal cells, sperm preincubation with porcine follicular fluid or control of sperm concentration, have been examined with significant effects but still remarkably high rates of polyspermy. The under standing of these influences is a prerequisite to enhancing in vitro production of porcine em bryos.

• Asian-Australasian Journal of Animal Sciences
• /
• v.26 no.4
• /
• pp.501-508
• /
• 2013

The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, modified Tyrode's albumin lactate pyruvate (mTALP) medium, and modified North Carolina State University (mNCSU)-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln)+L-glycyl-L-glutamine (GlyGln) significantly (p<0.05) improved oocyte maturation, fertilization and the incorporation and oxidation of 14C(U)-glucose when compared to the control group and other treatment groups. Additionally, 2-4 cell, 8-16 cell, morula and blastocyst development increased significantly (p<0.05) following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media.

• Journal of Embryo Transfer
• /
• v.22 no.4
• /
• pp.245-249
• /
• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Korean Journal of Animal Reproduction
• /
• v.23 no.4
• /
• pp.323-331
• /
• 1999

The effects of insulin-like growth factor-I (IGF-I) and cumulus cells during in vitro maturation in porcine oocytes were examined. When follicular oocytes were cultured in medium with different concentrations of IGF-I, maturation rates were 60, 61 and 62 and 72% for 0, 15 and l0ng/$m\ell$ IGF-I. In medium with 10ng/$m\ell$ IGF-I, maturation rates were not significantly difference between oocytes with (68%) and without (52%) cumulus cells during the culture. In medium with-out IGF-I, however, the maturation rates in oocytes with cumulus cells (63%) was significantly (P＜0.05) higher than oocytes without cumulus cells (32%). On the other hand, when IGF-I was added for first 24 h period or later 24 h period of culture, maturation rates were higher in oocytes with (61 and 49%) that than without (49 and 45%) cumulus cells. In experiment used medium without fetal calf serum (FCS) and porcine follicular fluid (PFF), the maturation rates in oocytes with cumulus cells for 48 h (48 and 67%) or first 24 h (46 and 63%) period after culture were significantly (P＜0.01) higher than in oocytes without cumulus cells (16 and 18%) in the presence or absence of IGF-I. These results indicated that cumulus cells is essential on maturation in vitro in porcine oocytes, but IGF-I can promote oocytes maturation of oocytes without cumulus cells in medium with FCS and PFF.

• Development and Reproduction
• /
• v.11 no.3
• /
• pp.263-272
• /
• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.10
• /
• pp.1403-1411
• /
• 2002

The aims of this study were, firstly, to analyze the biochemical compositions of serum and follicular fluid (FF) from prepubertal gilts after PMSG (1,000 IU) treatment. The concentrations of total proteins, lipids, cholesterol, glucose and sex hormones (progesterone, $P_4$; estradiol-$17{\beta}$, $E_2$; testosterone, T) were measured. Secondary, the effects of porcine FF (pFF) addition (40% and 100%) in IVM media and different culture conditions [Exp. 1: mBMOC-2+20% porcine serum (PS), fresh IVM medium, filtered IVMconditioned medium, or rabbit oviducts; Exp. 2: mBMOC-2+20%PS or stepwise medium replacement procedures (SMRP) cocultured with or without cumulus cells] on the in vitro development (IVD) of porcine oocytes were also examined. Results showed that no significant differences were found in total protein levels between serum and pFF from different sizes (large, >7 mm; medium, ~5-7 mm; small, <3-5 mm) of follicles (75-85 and 49-90 mg/dl; p>0.05). Total lipid concentrations remained constant in serum (395-472 mg/dl), and reduced significantly in the pFF from large follicles (287 mg/dl) at 132 h after PMSG treatment when compared to those at other time points (441-480 mg/dl). Basal cholesterol levels in serum and pFF at 12 h were similar (153-161 mg/dl), but increased at 36 h (186-197 mg/dl). Basal P4 and E2 levels in serum (0.1 ng/ml and 5.5 pg/ml) were low, but increased from 0.34 ng/ml and 12.13 pg/ml at 24 h to 0.81 ng/ml and 61.70 pg/ml at 98 h, respectively, after PMSG treatment (p<0.05). P4 levels increased linearly in pFF from large follicles during 12 through 132 h (138-1,288 ng/ml). A similar increase was also observed in $E_2$ levels (22-730 pg/ml) before 60 h post PMSG treatment, and then dropped afterwards (730-121 pg/ml). The development of the oocytes fertilized in 40% pFF-medium was greater than that in 100% pFF-medium group without gonaodtropin addition (31% vs 10%, p<0.05). However, both were lower than those in mBMOC-2+20%PS and in rabbit oviducts (p<0.05). When cocultured with cumulus cell monolayers, a greater cleavage rate was observed in the group cultured in filtered IVM-conditioned medium than the SMRP group (36% vs 18%, p<0.05). A similar phenomenon was also observed in the culture without cumulus cell monolayers (33% vs 19%, p<0.05). It is concluded that neither the fresh IVM nor filtered IVM-conditioned medium has positive effect on the IVD of oocytes. Coculture with cumulus cell monolayers and the SMRP were not beneficial to the development of IVF pig oocytes.