• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.247-253
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• 1997

The objective of this study was to examine the fertilization and embryonic development rates of porcine oocytes matured in vitro according to the morphological normality of epididymal sperm in porcine IVF. The results obtained in this experiment were summarized as follows : 1. When the ratio of morphological normality of epididymal sperm was divided into the three groups with $\leq$ 10%, 10-30% and $\geq$ 50%, the fertilization and embryonic devel-opment rates of 능50% group (64%. 26%) w were significantly higher than those of other two groups ($\leq$10%: 27%, 6% and 10-30%: 36%. 5%) (p$\leq$50% group of morphologically normal epididymal sperm was adjusted to 100% (5 x 10$^5$ cells/ml), the fertilization a and development rates (63%, 27%) of epidymal sperm were similar to those of frozen-thawed ejaculated sperm (56%, 35%). 3. Also, when the fertilization and development rates of epididymal sperm were analyzed according to the oocyte : sperm ratio (1:6000, 1: 6650, 1: 7700, 1: 10000), the fertilization and development rates indicated high, in 1:6000 (68%, 32%) and 1:6650 (89%, 31%), the ratio of oocyte: sperm. Therefore, when the percentage of morphological normality of epididymal sperm is more than 50, the embryonic development a can be obtained similar to that of frozen thawed ejaculated sperm. Also, these result suggested that the morphological evaluation of normal sperm in porcine IVF using epididymal sperm sho비d be prerequisite for the more effective embryonic developm ment.

• Development and Reproduction
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• v.9 no.1
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• pp.49-52
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• 2005

The effects of cytochalasin B was studied for the cleavage and development of in vitro matured porcine follicular oocytes. The follicular oocytes were collected from slaughtered pig ovaries and matured for 65 hours. The matured oocytes were activated by 7% ethanol(v/v) in DPBS and the activated oocytes were subjected to cytochalasin B concentrations of 2.5, 5.0 and $7.5\;{\mu}g/mL$ for 3, 5 and 7 hours, and then the treated oocytes were cultured in NCSU23 with 0.4% BSA for 7 days. The cleavage rates were not different significantly in each treatment. However, the oocytes treated with $5.0\;{\mu}g/mL$ for 5 hours yielded a significantly higher morula rate(19.7%) than oocytes treated with $2.5\;{\mu}g/mL$ for 3 and 5 hours(9.4%). The sum rate of $2.5\;{\mu}g/mL$ concentration(10.5%) by hour was also significantly lower than those of 5.0(18.0%) and $7.5\;{\mu}g/mL$ concentration(14.6%). The blastocyst rate in oocytes treated with $5.0\;{\mu}g/mL$ for 3(9.4%) and 5 hours(9.0%) was significantly higher than the rate in oocytes treated with $2.5\;{\mu}g/mL$ for 3 hours(0%). The sum rate of $5.0\;{\mu}g/mL$ concentration also significantly higher than those of 2.5 and $7.5\;{\mu}g/mL$ concentration. The results demonstrated that the treatment of oocytes with cytochalasin B of $5.0\;{\mu}g/mL$ for $3{\sim}5$ hours was the optimal concentration and duration for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

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

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.19-26
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• 1996

In the present study, we investigated devel-opmental ability and transgene expression of IVM/IVF derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF derived embryos were used to examine developmental ability and transgene expression following DNA microinjection. After centrifugation, pronuclei were visible in 60.3% when examined between 18~21h after IVF. Development and transgene expression were assessed after 9 days in culture. The percentages of injected embryos reaching to the morula and blastocyst were significantly lower (P<0.05) than those of non-injected control embryos. However, the percentages of DNA microinjected embryos and non-injected embryos that developed to the blastocyst or hatched blastocyst stage in dual culture systems (NCSU23 and EMEM) were significantly higher (P<0.05) than those in NCSU23 medium alone. As the resuIt of X-gal staining, the proportion of positive embryos was 40~43% in morula and blastocyst stage embryos, however, mosaicism has been observed in the most putative transgenic morulae and blastocysts. In the PCR analysis, the percentages of embryos integrated gGH gene were 45.0 and 44.4% in morula and blastocyst stage, respectively. These results suggest that improved IVM /IVF system and culture condition increased the embryo viability and ex-pression of a microinjected transgene.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.117-122
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• 1997

The aim of this study was to investigate the effect of the follicular culture from which the oocytes originate on their subsequent in vitro maturation ability. Ovarian follicles were isolated and cultured according to size(1~2mm, 2~6mm and 6~8mm) for 42~44 h. The rates of germinal vesicle breakdown(GVBD) in each groups were 87%(65/75), 82%(80/97) and 89%(47/53), but the oocytes maturation were su, pp.essed at anaphase-I stage. In spite of the adding porcine follicular fluid and/or hormones in maturation medium, maturation ability of oocytes from follicle cultured for 21~22 h were inhibited. When oocytes from follicle cultured for 4 h at various temperature were incubated for 38~40 h, the rates of oocytes maturation from follicle cultured at 2$0^{\circ}C$(51%, 26/51) and 39$^{\circ}C$(54%, 26/48) were significant higher(P<0.05) than group cultured at 4$^{\circ}C$(33%, 19/58). On the other hand, the GVBD were stared 2 h after culture of follicle of oocytes. To summairze, oocytes maturation by follicular culture were inhibited at anaphase-I stage in porcine. When the follicle cultured for 4 h, maturation were completed to metaphase-II stage. However, rates of GVBD in oocytes from follicular culture were higher than oocytes cultured in medium.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.187-194
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• 1998

In order to determine suitable conditions for rapid freezing of porcine embryos, the kind and concentration of cryoprotectants, sucrose concentrations, equilibration time and thawing temperature in freezing medium were examined in relation to the survival of frozen-thawed oocyte and embryos. The results obtained are as follows : 1. The suitable concentrations of cryoprotoctant in the freezing medium which consisted of TCM-199+20% FCS were 1.5M for glycerol, 2.0M for DMSO, 2.5M for ethylene glycol, and 2.0M for propanediol. The sucrose concentration of 0.25M in the medium was found to optimal because the survival rate was markedly higher at this concentration when compared to the others. The survival rate was relatively high when the frozen embryos were thawed at 30$^{\circ}C$ in the freezing medium containing 2.5M cryoprotectants. The equilibration periods of 2.0 and 5.0 minutes revealed the higher survival in the media containing 1.5 or 2.1M glycerol when compared to 10 and 15 minutes. 2. The fertilization rates of frozen-thawed follicular oocytes which matured in vitro for 1, 12, 24 and 48 hours were 6.7~26.7% depending on the maturation time, and the rates were relatively high for those matured for a short period of time. The survival rates of frozen-thawed oocytes which matured in vitro for certain periods and fertilized were 10.0~30.0% depending on the maturation time.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.139-146
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• 2017

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in vitro maturation of pig oocytes.

• Development and Reproduction
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• v.7 no.2
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• pp.119-126
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• 2003

In the previous study, we obtained list of differentially expressed genes between postnatal day 1 and day 5 mouse ovaries using suppression subtractive hybridization(SSH) and found that MT Oansposon-like element, clone MTi7(MTi7) was one of the highly expressed genes in the day 5 mouse ovary(Park et at., 2002). Results of in situ hybridization and RNA interference revealed that the expression of MTi7 is oocyte-specific in the ovary and may be involved in the regulation of oocyte maturation(Park et at., 2003). At present, MTi7 sequence has been known only in the mouse. Therefore, the present study was accomplished 1) to identify MTi7 sequence in the other mammalian species, such as bovine, porcine, rat, and human, and 2) to evaluate the flanking sequence of the mouse MTi7 since it has transposon characteristics. Using ovarian cDNAs derived from low different species, we cloned and identified new MTi7 sequence showing a high degree of sequence homology with the mouse MTi7(87∼98%). By using inverse PCR, we found that the mouse MTi7 may intercalated the beta-carotene 15, 15'-monooxygenase(Bcdo) gene and/or serine protease inhibitor, Kunitz Dpe I(Spint 1) gene. By finding the MTi7 sequences in the other mammalian species and the flanking gene of the MTi7 in mouse, it is expected to reveal the role(s) of MTi7 in the oogenesis as well as folliculogenesis in the near future.