• Journal of Embryo Transfer
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• v.15 no.3
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• pp.237-245
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• 2000

This study was carried out to elucidate whether sperm contain a factor inducing second polar body extrusion and to search for an effective collection method of the sperm factor Thus, sperm extract, dialyzed sperm-extract or liquid chromatographic fractions of sperm extract was microinjected into ovulated oocytes. And the microinjected oocytes were incubated for 24 hours to investigate about the extrusion of second polar body. The results obtained were as follows; 1. Sperm extract significantly increased the second polar body extrusion. 2. Sperm extract showed five major fractions at retention volumes (RVs) 1.25, 1.37, 1.84, 2.10 and 2.67ml after separation with Superose 12 column. These sperm extract fractions did not significantly increase the second polar body extrusion. 3. Dialyzed sperm-extract significantly increased the second polar body extrusion 4. Dialyzed sperm-extract showed three maior fractions at RVs 1.88, 2.14 and 2.77ml after separation with Superose 12 column. Of these fractions, the fraction RV2.14 significantly increased the second polar body extrusion. In conclusion, sperm extract contained a factor inducing the second polar body extrusion and the factor was contained largely in fraction RV2.14 after dialysis and liquid chromatographic fractionation of sperm extract.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.97-101
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• 2010

Intracytoplasmic sperm injection (ICSI) is one of the artificial fertilization methods when only a few sperm are available for insemination, and an important tool for the preservation of genetic materials of endangered animal species, especially the male is infertile. Different from other species such as mice and pigs, the conventional ICSI method which uses spiked pipette for injection (Spike-ICSI) is exhibited low success rates in cattle because the bovinesperm head membrane is hard to break during injection procedure. We chose piezo-assisted ICSI (Piezo-ICSI) for the improvement of the injection procedure including sperm head membrane rupture and efficient puncture of the plasma membrane of the oocytes. In this experiment, we compared the efficacy of the bovine ICSI embryo production between the Piezo-ICSI and Spike-ICSI. The second polar body extrusion, pronuclear formation, cleavage and blastocyst formation were evaluated after implementation of two different ICSI techniques. The Piezo-ICSI tended to show comparably higher rates of the second polar body extrusion (41.7%), the pronuclei formation (42.9%) and the two-cell cleavage (41.4%) than Spike-ICSI does (33.3%, 28.6% and 23.5%, respectively) although there is no statistic significance between two groups. In addition, the blastocysts were only obtained from the Piezo-ICSI group (10.3%). Our finding shows that the Piezo-ICSI may be used as an artificial fertilization method in cattle when in vitro fertilization is not applicable.

• Journal of Animal Science and Technology
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• v.61 no.4
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• pp.225-233
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• 2019

The aim of this study was to produce porcine tetraploid (4N) parthenogenetic embryos using various methods and evaluate their developmental potential. In method 1 (M1), porcine 4N parthenogenetic embryos were obtained by inhibiting extrusion of both first (PB1) and second (PB2) polar bodies; in methods 2 (M2) and 3 (M3), 4N parthenogenetic embryos were obtained by electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 or PB1 extrusion, respectively. We found no differences in the rates of cleavage or blastocyst formation or the proportion of 4N embryos among M1, M2, and M3 groups. The different methods also did not influence apoptosis rates (number of TUNEL-positive cells/number of total cells) or expression levels of apoptosis-related BAX and BCL2L1 genes. However, total cell and EdU (5-ethynyl-2'-deoxyuridine)-positive cell numbers in 4N parthenogenetic blastocysts derived from M1 were higher (p < 0.05) than those for M2 and M3 groups. Our results suggest that, although porcine 4N parthenogenetic embryos could be produced by a variety of methods, inhibition of PB1 and PB2 extrusion (M1) is superior to electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 (M2) or PB1 (M3) extrusion.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.223-231
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• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

• Journal of Aquaculture
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• v.10 no.2
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• pp.123-131
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• 1997

Growth of triploid abalone, Haliotis discus hannai induced by cody (3$^{\circ}C$) shock and its feed efficiency were investigated from larva to adult for 51 months. After 51 months from triploidy induction, the triploid abalones have outgrown to diploid abalones in shell length and total weight. Triploid abalones with inhibition of extrusion of first polar body (3n-1pb) were outgrown to diploid abalones, however, triploid abalones with inhibition of extrusion of second polar body (3n-2pb) were not significantly different from diploid controls in shell length and total weight through the whole rearing period (P<0.05), because of their heterozygosity differences. Daily feeding rates and feed conversion rates decreased with the growth of abalones and both rates had no differnce between two experimental groups. After 51 months from inducing triploid, conditin index of triploid abalone (64.1%) was higher than that of diploid control (59.4%) (P<0.05). Survival rate was 63.0% in triploid group (3n-1pb 62.0%, 3n-2pb 64.0%) and 62.0% in diploid group during the experimental period.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.3
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• pp.119-124
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• 2019

Objective: It is widely accepted that aging decreases women's fertility capacity. The aim of this study was to assess correlations between maternal age and the morphokinetic parameters and cleavage pattern of embryos. Methods: The morphokinetics of embryos derived from women < 30, 30-35, 36-40, and > 40 years of age were compared retrospectively in terms of time of second polar body extrusion, time of pronuclei appearance, time of pronuclei fading, and time of two to eight discrete cells (t2-t8). Furthermore, abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), and trichotomous mitoses (TM) were assessed. Results: Only t5 occurred later in women aged 36-40 and > 40 years when compared with those aged < 30 and 30-35 years (p< 0.001). Other morphokinetic timing parameters, as well the presence of uneven blastomeres, were comparable between the groups (p> 0.05). However, Fu and TM were more common in women aged > 40 years than in younger women (p< 0.001). Conclusion: Maternal age was correlated with the cleavage pattern of embryos. Therefore, evaluating embryo morphokinetics may contribute to optimal embryo selection, thereby increasing fertility in patients with advanced maternal age.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.1
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• pp.48-54
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• 2022

Parthenogenesis is maternally uniparental reproduction through the embryonic development of oocytes without fertilization. Artificial activation of mature oocytes could generate homozygous haploid embryos with the extrusion of the second polar body. However, the haploid embryos showed low embryo development in preimplantation embryos. In this study, we investigated whether the electronic fusion of the haploid embryos could enhance embryo development and ESC establishment in mice. Haploid embryos showed the developmental delay from 4-cell to the blastocyst stage. The haploid blastomeres of the 2-cell stage were fused electronically, resulting in that the fused embryos showed a significantly higher rate of blastocysts compared to non-fused haploid embryos (55% vs. 37%). Further, the embryonic stem cells (ESCs) derived from the fused embryos were confirmed to be diploid. The rate of ESC establishment in fused embryos was significantly higher compared to non-fused ones. Based on the results, we concluded that the electronic fusion of haploid embryos could be efficient to generate homozygous ESCs.

• Journal of Aquaculture
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• v.20 no.3
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• pp.184-189
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• 2007

Treatment conditions for the induced diploid gynogenesis, a maternal-exclusive form of artificial parthenogenetic reproduction, were optimized in bagrid catfish (Leiocassis ussuriensis, Siluriformes). Optimal amounts of ultraviolet (UV) irradiation for the genetic inactivation of spermatozoa in bagrid catfish and Pseudobagrus fulvidraco were proven to be ranged from 3,600 to 4,800 $ergs/mm^2$ based on the examination of viability of embryos and haploid incidence. Haploid embryos were restored to diploidy by preventing the extrusion of the second polar body using cold shock treatment. Thermal treatments (4 or $6^{\circ}C$ for 30, 40 or 50 min) were carried out 3, 5 or 7 min post insemination. Best scores for embryo viability (38.6% of total eggs taken) and incidence of normal diploidy (87.9% of hatched larvae) were observed at the embryo group treated at $4^{\circ}C$ for 40 min, 5 min after insemination. Restoration of gynogenetic diploidy was confirmed based on the absence of haploid syndrome, cell size and/or nucleolar organizing region (NOR) counts.

• Journal of Life Science
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• v.20 no.11
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• pp.1634-1639
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• 2010

This study was conducted to increase the efficiency of farming practice in rainbow trout, Oncorhynchus mykiss, by sex reversal and chromosome-set manipulation techniques. To obtain phenotypic males, hormonal sex reversal was carried out using an exogenous hormone treatment method. 5 mg of 17 alpha-methyltestosterone per kg diet was supplied for 82 days after first feeding at $10^{\circ}C$ and $13^{\circ}C$. More than 93% of the male population was produced by this method and growth of hormone-treated fish at $13^{\circ}C$ was faster than that of untreated bi-sexual groups. Induced diploid gynogenesis was carried out using artificial insemination of UV-irradiated sperm into haploid eggs. Based on the appearance of the rate of haploid syndrome and survival of embryo, a UV ray dose of at least $3,600\;erg/cm^2$ was required to inactivate rainbow trout sperm genetically. Haploid embryos were restored to diploid by blocking the extrusion of the second polar body using heat shock treatment at $28^{\circ}C$ for 20 min, 10 min post insemination. Gynogenetic diploid sex ratios were confirmed after maturation of the fish erythrocyte measurements and chromosome counts.

• Parasites, Hosts and Diseases
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• v.31 no.1
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• pp.57-66
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• 1993

The potential of fumagillin dicyclohexylamine salt to treat and prevent intestinal giant-cystic disease in Israel carp, Cyprinus carpio nudus, was monitored in field experimental studies. In experiment 1 (therapeutic), most fish were already naturally infected with more advanced stage of Relohqnellu: kitnuet. Fumagillin was administered to ash (mean body weiht of 830 g for a Penod of one month at a dose of 10.62 mg in the first group and 5.3 mg in the second group per fi sh per day. In experiment 2 (fprophylactlcl), most flesh also were already naturally infected with an early developmental stage of the protozoa and fish (average body weight of 484 g) were administered fumagillln for 45 days at a dose of 3.95 mg per fish per day. In both experiments, the cumulative mortalities of fish and the extrusion rates of the polar filaments of the spores were significantly decreased in a dose-independent fashion. In experiment 2 no dead fish were observed. No adverse side effects of the drug were observed among fish from any dosage group. In experiment 2, an oval or dot-like concave lesion of most cysts developed at the 7th day and the vegetative form was never observed at the 17th day postmedication and the cysts were grossly reduced in size as compared with the control group, beginning at the 24th day until the end of the study. In contrast, it was scarcely effective to the cysts in experiment 1. Taking the seasonal development of the protozoa into consideration, the above results revealed that oral administration of fumagillin at 3.95 mg/500 g body weight/day for a month Is the optimal dose for the treatment and prevention of thelohanellosls caused by T kitnuei among C. carpio nudus.