• Reproductive and Developmental Biology
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• v.35 no.2
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• pp.185-190
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• 2011

To evaluate the effect of spermatozoa culture on glycosidase activity of frozen-thawed spermatozoa in human, the spermatozoa were treated experimentally and assayed for activities of ${\alpha}$-L-fucosidase, ${\alpha}$-D-mannosidase, ${\beta}$-D-galactosidase and N-acetyl-${\beta}$-D-glucosaminidase (${\beta}$-GlcNAc'ase). The ${\beta}$-GlcNAc'ase activity was at least two-folds higher than other glycosidases regardless of spermatozoa incubation (p<0.05). The spermatozoa motility was decreased with incubation periods, but no effects by different glycosidases on the changes of spermatozoa motility during the various periods of incubation. In all glycosidases, the spermatozoa-zona binding rates in spermatozoa without incubation were higher than in spermatozoa incubated for 2 h (p<0.05). ${\beta}$-GlcNAc'ase is present mainly in the plasma membrane of spermatozoa frozen-thawed in human. It was also shown that the glycosidase activity was increased in all glycosidases in spite of lower sperm-zona binding by spermatozoa incubation.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.269-271
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• 2005

The freemartinism is the most frequent form of intersexuality found in cattle, and females of heterosexual twins become sterile. With increase of twinning rates due to transfer of multiple embryos derived from in vitro fertilization, it is of great economic value to establish early diagnosis of freemartins to remove infertile individuals from breeding stock. In the present study polymerase chain reaction (PCR) of two different Y-chromosome specific segments (BRY.l and AMX/Y) was performed to identify freemartins from twins and less common single born freemartins in Korean Native Cattle (KNC). Two male-specific sequences were amplified in all heterosexual twins tested (n=5). In addition, Y-specific PCR products were detectable in one of the single born females (n=4) with visible genital abnormalities. These results suggest that the sensitivity of PCR-based assay may be sufficient to detect freemartinism in single born females as well as female partners of heterosexual twins in KNC.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.407-417
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• 2000

The present study was to examine effects of various electrical stimulus treatments used for electro-fusion on the preimplantation development of bovine nuclear transfer (NT) embryos with fetal fibroblast cells. Fetal fibroblast cells were isolated from one fetus at day 45 of gestation in Holstein cow, and passaged 3 to 4 times before being transferred into enucleated oocytes. Single fibroblast cells were individually placed into the perivitelline space of enucleated oocytes by using a micromanipulator. At first, the fusion and developmental rates of reconstructed oocytes were compared between different electric stimulation conditions. When fusion of the reconstructed oocyte was induced by different electric pulse periods (15, 30 and 45 $\mu$sec) at a DC pulse of 1.8 kV/cm, 15 (45.5%, 120/264) or 30 $\mu$ sec group (43.9%, 106/241) showed a higher fusion rate than 45 $\mu$sec group (23.2%, 58/250, P＜0.05). However, no difference was detected in the development rate of the fused oocytes to blastocysts between groups. Next experiment was to examine the effects of different electrical field strengths (1.5, 1.8 and 2.1 kV/cm) for 15 $\mu$sec at electrofusion on in vitro development of the NT embryos. As results, there was no difference in the fusion and developmental rates of the NT embryos between electrical strength (P＞0.05). Finally, developmental competence of bovine NT embryos with somatic cells was compared with IVF-derived embryos. Of enucleated oocytes fused with fibroblast cells, 27.4% (75/274) developed to the blastocyst stage, which is similar to that (24.5%, 58/237) of IVF-derived embryos. However, mean nuclei number of NT blastocysts was smaller than that of IVF-derived blastocysts. Thus, we have established an optimal condition (1.8 kV/cm, 15 $\mu$sec) for electric fusion of bovine NT oocytes with somatic cells. The present study indicates that bovine reconstructed embryos with somatic cells normally develop to blastocyst stage in vitro, although having smaller nuclei numbers of blastocysts as compared to IVF-derived embryos.

• Development and Reproduction
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• v.24 no.1
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• pp.31-41
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• 2020

We investigated the effects of endoplasmic reticulum (ER) stress inhibitor and antioxidant treatments during the micromanipulation of somatic cell nuclear transfer (SCNT) on in vitro development of SCNT embryos. Tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor and vitamin C (Vit. C), an antioxidant, were treated by alone or in combination, then, the level of X-box binding protein 1 (Xbp1) splicing and the expressions of ER stress-associated genes, oxidative stress-related genes, and apoptotic genes were confirmed in the 1-cell and blastocyst stages. In the 1-cell stage, the levels of Xbp1 splicing were significantly decreased in TUDCA and Vit. C treatment groups compared to the control (p<0.05). In addition, the expression levels of most ER stress-associated genes and oxidative stress-related genes were significantly lower in all treatment groups than the control (p<0.05), and the transcript levels of apoptotic genes were also significantly lower in all treatment groups than the control (p<0.05). In the blastocyst stage, decreased expression of ER stress-, oxidative stress-, and apoptosis-related genes were observed only in some treatments. However, the blastocyst formation rates in TUDCA and Vit. C treatment groups (24.8% and 22.0%, respectively) and mean blastocyst cell number in all treatment groups (59.7±4.3 to 63.5±3.3) were significantly higher (p<0.05) than those of control. The results showed that the TUDCA or Vit. C treatment during micromanipulation inhibited both ER and oxidative stresses in the early stage of SCNT embryos, thereby reducing cell damage and promoting in vitro development.

• The Journal of Korean Obstetrics and Gynecology
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• v.23 no.4
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• pp.20-34
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• 2010

Purpose: These experiments were undertaken to evaluate the effect of administration of Palmulgunjatang gamibang on ovarian functions in female mice. Methods: We administered the Palmulgunjatang gamibang to 6-week-old female CF-1 mice for 4, 8, or 12 days. After administration of Palmulgunjatang gamibang with different concentration, the female mice were injected PMSG and hCG for ovarian hyperstimulation. The mice were divided into 3 different groups for each experiment. To compare the differences, we set a control group treated with plain water at the same volume by the same way. Results: In case of 4-day, 8-day, 12-day administration of Palmulgunjatang gamibang, the mean number of total ovulated oocytes and the number of morphologically normal oocytes were increased compared with control group. We were also examined the embryonic developmental competence in vitro. In case of 4-day administration of Palmulgunjatang gamibang, the rates of blastocyst formation from 2-cell stages were higher than control group. Conclusion: From our results suggested that the medication of Palmulgunjatang gamibang has beneficial effect on reproductive functions of female mice via promotion of cell proliferation.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.399-407
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• 2001

The microinjection of retroviral vectors into the perivitelline spaces of MII-stage oocytes increased production of transgenic bovine embryos. However, oocytes have various sizes of perivitelline space, and there is the tendency that the oocyte membranes are damageable by micropipettes during the injection of retroviral vectors into perivitelline spaces or oocytes. Thus, it was not always possible to stably inject retroviral vector into perivitelline spaces of oocytes. Here we used sucrose to minimize the damage of the oocyte membrane. When the oocytes were suspended in 0.5% sucrose, poor quality oocytes showed rough cytoplasmic membranes, while good quality oocytes maintained smooth membranes. However, when the tatters were subjected to in vitro fertilization, no significant differences were observed in cleavage rates (82% of control Vs. 84% of sucrose treated oocytes). The Same trends were obtained from the oocytes fertilized after microinjection of LN$\beta$-EGFP and LNC-hGH genes into the perivitelline spares. The rates of cleavage and blastocyst from microinjection of LN$\beta$-EGFP genes were 81 and 25%, and from microinjection of LNC-hGM genes were 53 and 30%, respectively. The result indicated that microinjected oocytes could develop to the blastocyst stages after in vitro fertilization with no significant difference from control group. Moreover, the integration of hGH-gene (by PCR analysis) was detected in 52% of infected cleaved embryos and the expression of EGFP-gene (under a fluoresrence microscope) was also observed in 34% of infected blastocyst. These results indicated that 0.5% sucrose treatment could be an efficient method not only to select good quality embryos but also to inject retroviral vectors into perivitelline spares without any harm and hence improving developmental rates.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.145-147
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• 2005

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of $2{\~}4$ embryos/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ drop ($68.5{\%}$). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.71-77
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• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.1
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• pp.22-29
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• 2015

Objective: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. Methods: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). Results: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. Conclusion: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.191-197
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• 2016

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.