• Journal of Embryo Transfer
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• v.18 no.1
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• pp.61-68
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• 2003

Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to improve reproductive efficiency of artificial insemination with fresh- and frozen-semen following estrus induction in dog. Fifty infertilie dogs (age 2~3 years) were selected fur the study and divided into three different estrus induction treatment groups. Group 1 : dogs (n=15) were given clomifene (0.1 mg/kg) orally f3r five days at 12 hr intervals. Croup 2: dogs (n=15) were given bromocriptine (50 $\mu$g/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Croup 3, n=20) when pro-estrus occurred. After being treated, the dogs were evaluated fur the rates of estrus induction and time interval lapses from treatment to beginning of the pro-estrus. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The ejaculated semen was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM; TES, 209 mM; citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 co above the surface of liquid nitrogen (L$N_2$) for 23 min. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, there was no significant differences in the pregnancy rate of dogs between group treated with a combination of GnRH and bromocriptine and group treated clomifene or bromocriptine only. However, frozen-thawed semen can be used successfully fur artificial insemination in dog.

• Korean Journal of Plant Tissue Culture
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• v.27 no.6
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.207-214
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• 1996

To examine the developmental capacity of manipulated embryos after ultrarapid refreezing and thawing, mouse embryos were biopsied at 4-cell stage, frozen twice at 4-cell and morula stages, respectively, and then transferred to rec-ipients. Single blastomeres were biopsied from 4-cell embryos by a modified aspiration method. Biopsied 4-cell embryos were equilibrated into freezing medium at room temperature for 2.5 min, loaded into 40 $\mu$I of freezing medium in 0.25 ml plastic straw and then directly immersed into liqiud nitrogen. Freezing medium for 4-cell embryos consisted of 4.0 M ethylene glycol and O.25 M sucrose in dPBS supplemented with 6 mg/lm BSA. Morulae were frozen into freezing medium containing 5.0 M glycerol instead of ethylene glycol. Thawing was conducted by agitating each straw in 3TC water for 20 sec. The c content of each straw was expelled into 0.5 ml of dilution medium, which consisted of 0.25 M sucrose and 3 mg/ml BSA in dPBS. The thawed embryos were rehydrated in dilution medium for 10 min, washed 3 times with dPBS and then cultured in M16 medium at 37$^{\circ}C$, 5% CO$_2$ in air. Blastocysts that developed from frozen or refrozne biopsied embryos were transferred to recipients on Day 3 of pseudopregnancy, respectively. In vitro and in vivo developmental rates of the biopsied and intact 4~cell embryos after freezing and thawing were 78 (10l/130) and 25% (10/40), and 91 (114/125) and 30% (12/40), respectively. Although the rates of in vitro development of biopsied and intact embryos to blastocyst stage were significantly different after freezing and thawing (P

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.269-278
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• 2002

Objective s: Chromosome aneuploidy is associated with recurrent abortion and congenital anomaly and genetic diseases occur repeatedly in the specific families. Preimplantation genetic diagnosis (PGD) can prevent aneuploidy or genetic disease by selecting normal embryos before implantation and is an alternative to prenatal diagnosis. The aim of this study is to assess the outcome of PGD cycles by using FISH or PCR, and to determine the clinical usefulness and values in patients with risk of chromosomal aneuploidy or genetic disease. Materials and Methods: From 1995 to Apr. 2001, a total of 108 PGD cycles in 65 patients with poor reproductive outcome were analyzed. The indications of PGD were translocation (n=49), inversion (n=2), aneuploidy screening (n=7), Duchenne muscular dystrophy (n=5) and spinal muscular atrophy (n=2). PGD was applied due to the history of recurrent abortion, previous birth of affected child or risk of aneuploidy related to sex chromosome aneuploidy or old age. Blastomere biopsy was performed in 6$\sim$10 cell stage embryo after IVF with ICSI. In the single blastomere, chromosome aneuploidy was diagnosed by using FISH and PCR was performed for the diagnosis of exon deletion in DMD or SMA. Results: The FISH or PCR amplification was successful in 94.3% of biopsied blastomeres. The rate of transferable balanced emb ryos was 24.0% in the chromosome translocation and inversion, 57.1% for the DMD and SMA, and 28.8% for the aneuploidy screening. Overall hCG positive rate per transfer was 17.8% (18/101) and clinical pregnancy rate was 13.9% (14/101) (11 term pregnancy, 3 abortion, and 4 biochemical pregnancy). The clinical pregnancy rate of translocation and inversion was 12.9% (11/85) and abortion rate was 27.3% (3/11). In the DMD and SMA, the clinical pregnancy rate was 33.3% (3/9) and all delivered at term. The PGD results were confirmed by amniocentesis and were correct. When the embryos developed to compaction or morula, the pregnancy rate was higher (32%) than that of the cases without compaction (7.2%, p<0.01). Conclusions: PGD by using FISH or PCR is useful to get n ormal pregnancy by reducing spontaneous abortion associated with chromosome aneuploidy in the patients with structural chromosome aberration or risk of aneuploidy and can prevent genetic disease prior to implantation.