• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.113-118
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• 1999

This study was carried out to investigate on the improvement of fertilizing ability of in vitro matured oocytes from sperm density and motility by intracytoplasmic sperm injection(ICSI) into the porcine oocytes. 1. The in vitro fertilization and cleavage rates of oocytes from 1.0, 2.0, 3.0, 5.0 ($\times$10$^{6}$ $m\ell$) sperm concentration by IVF and ICSI of porcine oocytes were 46.7%~75.0%, 60.0%~85.7% and 10.6%~25.0%, 20.0%~64.3%, respectively. 2. The in vitro fertilization and cleavage rates of oocytes from 20, 40, 60, 80% of sperm mortilty by IVF and ICSI of porcine oocytes were 46.4%~71.4%, 67.9%~85.7% and 7.1%~21.4%, 28.6%~60.7%, respectively. 3. The in vitro fertilization and developmental rates of oocytes by IVF and ICSI methods were 55.6%~60.0%, 77.8%~80.0% and 17.8%~24.0%, 42.2%~56.0%, respectively. This ICSI method was improved high fertilization rates of porcine oocytes.

• Development and Reproduction
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• v.16 no.4
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• pp.271-277
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• 2012

The effects of ovulation induction in ussurian bullhead, Leiocassis ussuriensis, were investigated by treating ussurian bullhead with hCG, LHRHa, GnRHa, ovaprim, and pimozide. hCG was injected to ussurian bullhead at 0.75% NaCl, 5,000, 10,000, 20,000, and 30,000 IU, respectively. The ovulation inducement rates were 100% in 20,000 and 30,000 IU. Fertilization rates were 82.7% and 79.8%. Hatching rates were 59.4% and 57.2%. Ovulation time was between 16-19 hr The concentrations of LHRHa injected were 0.75 NaCl, 50, 100, 200, 300, and $300{\mu}g/kg$. The ovulation inducement rates were 100% in 300 and $400{\mu}g/kg$. Fertilization and hatching rates were 84.9% and 68.4% at $200{\mu}g/kg$. The times to ovulation were between 23 hr and 34 hr. Ovaprim of 0.75% NaCl, 1.0, 1.5, 2.0, 2.5 and 3.0 ml/kg were injected to the abdominal cavity. The ovulation inducement rate was highest at 2.0 and 3.0 ml/kg to 92% and ovulation time was between 27-38 hr. LHRHa concentrations of 0.75% NaCl, 50, 100, 200, 300 and $400{\mu}g$ were injected with pimozide ($1,000{\mu}g$). Ovulation inducement rate was 100% from 200 to 400 IU with pimozide. Ovulation time was 22-36 h. Fertilization and hatching rates were 88.9% and 70.4% in $200{\mu}g/kg$ with pimozide.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.35-44
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• 2003

The present study was performed to investigate the first polar body(PB) extrusion during in-vitro maturation(IVM) and to examine the effect of different maturation time on the embryo development of Korean Native Cows(KNC) with regard to blastocyst(BL) cell numbers and pregnancy rates. PB extrusion did not take place for the first 12 hours(hr) of IVM, and most of KNC oocytes extruded PB from 14 to 20 hr after the onset of maturation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.05) in 18 hr maturation group(31.0$\pm$5.7 and 82.0$\pm$5.1%) than 22 and 24 hr maturation group. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in 18 hr maturation group(85%) than in 24 hr maturation group(55%). There was a significant difference(P<0.05) in inner cell mass, trophectoderm and total cell number between day 7 BL produced by in-vivo and IVM 18 hr and day 8 BL produced by IVM 18 hr and 24 hr. Pregnancy rates are also significantly higher(P<0.05) in in-vivo(56.3%) and IVM 18 hr day 7(50.0%) group than day 8 treatment groups(18 hr: 16.7%, 24 hr: 10.5%). These results suggest that KNC oocytes achieve developmental competency within 20 hr of IVM, and "short" IVM (18 hr) is more effective than "long" IVM(24 hr) in embryo development rates, BL cell numbers and pregnancy rates.

• Development and Reproduction
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• v.22 no.3
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• pp.245-252
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• 2018

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the postwarming pig oocytes were analyzed by fluorescein diacetate (FDA) assays with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.188-194
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• 1989

These experiments were carried out to investigate fertilizable and developmental ability after zona drilling the unfertilized eggs and the eggs not fertilized by the 1st insemination. The results of in vitro fertilization of the mouse eggs treated by using micromanipulation and acid tyrode's solution with capacitated epididymal spermatozoa were as follows. In the case of ovulated unfertilized eggs, according to sperm count(106, 105, 104 and 103/ml) the rates of in vitro fertlilization treated by zona drilling were 86.0%, 82.0%, 70.0% and 54.0%, respectively, and those of control were 58.0%, 52.0%, 12.0% and 8.0%, respectively. The rates of in vitro fertilization of zona drilled eggs were significantly high compared with those of control, and there were no significant difference between two groups. According to the sperm count the zona drilled eggs developed to the blastocysts were 51.4%, 40.5%, 23.3% and 17.4% and those of control were 35.7%, 26.3%, 0% and 0%, respectively. Also, in the eggs not fertilized by 1st insemination, the fertilization rates of oocytes reinseminated after zona drilling was significantly higher(83.5%) than that of control(34.7%), and the rates of polyspermy were similar. The rates of development to the blastocysts was 18.6% in the zona drilling treated eggs, and that of control was 27.3%, there was no significant difference between two groups. These results indicated that oocytes not fertilized by 1st insemination as well as ovulated unfertilized eggs could be fertilized, improve fertilizing rates by zona drilling treatment, and development potential were normal.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.155-159
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• 2004

This study was carried out to investigate the general characteristics and viability of sperm after freezing and thawing and the pregnancy rates after artificial insemination with thawed semen. The rates of viable sperm after slow and rapid freezing were 87.4±3.85% and 70.8±4.45%, respectively which were significantly lower than that of fresh semen control (91.7±3.45%). The mean concentration of epididymal sperm after dilution in 1.0 ml saline and. 3.0 ml extender in a various concentrations of cryoprotectants was 124.5±48.3 x 10/sup 6/ (range of 45 x 10/sup 6/ to 280 X 10/sup 6/ /ml). There was a significant difference not in the percentage of acrosome-reacted sperm, but in the percentage of capacitated sperm, between fresh and frozen-thawed epididymal semen. When frozen-thawed after diluting with tris-buffer extender containing glycerol, DMSO and ethylene glycol with concentration of 2 to 6%, the rates of epididymal sperm exposed to different cryoprotectants ranged from 14.4±4.7% to 20.7±5.8%, 17.8±5.2% to 36.5±4.9%, and 14.4±4.6% to 18.5±5.3%, respectively which were lower compare to fresh semen control. The pregnancy rate after artificial insemination with frozen semen was 70.6%, whereas that with fresh semen was 90.0% in dogs with naturally induced estrus.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.21-25
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• 2010

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% $CO_2$, 95% air, $38^{\circ}C$. The in vitro maturation rate to MII stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44~72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.111-115
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• 2008

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH ($67.4{\pm}1.8\;{\mu}m$) was significantly (p<0.05) smaller than that of the fresh preantral follicles ($69.1{\pm}2.3\;{\mu}m$). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.2
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.