• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.90-95
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• 2017

Objective: We investigated whether the insemination method (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]) affected morphokinetic events and abnormal cleavage events in embryonic development. Methods: A total of 1,830 normal fertilized embryos were obtained from 272 IVF and ICSI cycles that underwent ovum retrieval culture using a time-lapse system (Embryoscope) from June 2013 to March 2015. All embryos were investigated by a detailed time-lapse analysis that measured the developmental events in the hours after IVF or ICSI insemination. Results: No significant differences were observed between the two groups regarding clinical outcomes (p>0.05). ICSI-derived embryos showed significantly faster morphokinetics than those derived from conventional IVF, from the time to pronuclear fading to the time to 6 cells (p<0.05). However, no significant differences were found from the time to 7 cells to the time to expanded blastocyst (p>0.05). There were no differences in abnormal cleavage events between the two groups (p>0.05); they showed the same rates of direct cleavage from 1 to 3 cells, 2 multinucleated cells, 2 uneven cells, and reverse cleavage. Conclusion: The morphokinetics of embryo development was found to vary between IVF- and ICSI-fertilized oocytes, at least until the 6-cell stage. However, these differences did not affect the clinical outcomes of the embryo. Additionally, no significant differences in abnormal cleavage events were found according to the fertilization method.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.2
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• pp.79-84
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• 2017

Objective: Optimizing in vitro maturation (IVM) media to achieve better outcomes has been a matter of interest in recent years. The aim of this prospective clinical trial was to investigate the effects of different media on the IVM outcomes of immature oocytes at the germinal vesicle (GV) stage. Methods: A total of 400 immature oocytes at the GV stage with normal morphology were retrieved from 320 infertile women aged $31{\pm}4.63years$ during stimulated intracytoplasmic sperm injection (ICSI) cycles. They were divided into groups of homemade IVM medium (I, n = 100), cleavage medium (II, n = 100), blastocyst medium (III, n = 100), and Sage IVM medium (IV, n = 100) and cultured for 24 to 48 hours at $37^{\circ}C$. ICSI was performed, and the rates of fertilization and embryo formation were compared across the four groups. Results: In the 400 retrieved GV oocytes, the total maturation rates showed significant differences in groups I to IV (55%, 53%, 78%, and 68%, respectively, p<0.001). However, there were no significant differences in the fertilization, embryo formation, or arrest rates of metaphase II oocytes across these groups. In all groups, GV maturation was mostly completed after 24 hours, with fewer oocytes requiring 48 hours to mature (p<0.01). Moreover, the rate of high-quality embryos was higher in group IV than in the other groups (p=0.01). Conclusion: The quality of the IVM medium was found to affect clinical IVM outcomes. Additionally, blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.

• Journal of Korean Society of Forest Science
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• v.87 no.1
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• pp.57-61
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• 1998

Somatic embryo induction, plant regeneration, and field establishment were investigated from tissue cultured winter buds of a 10-year-old tree Aralia elata. Embryogenic calli were obtained from cultures of winter buds on MS medium supplemented with 2,4-D. A number of somatic embryos were regenerated from the calli on an embryo induction medium supplemented with 2,4-D and BA. Although abnormal somatic embryos were frequently observed, most of the embryos formed were morphologically normal. All somatic embryos at the later stage of maturity germinated successfully, but only 14% of them could be developed into plantlets on MS basal medium. The plants regenerated from the somatic embryos survived well in the field (survival rates : more than 95%) and have grown normally for three years after transplanting.

• Applied Microscopy
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• v.26 no.3
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• pp.253-266
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• 1996

To investigate the effects of mercuric chloride ($HgCl_2$) on the differentiation of the cerebral neuron of chick embryo 9 days, the ultrastructural changes in nerve cells injected with a various doses of mercuric chloride were observed with transmission electron microscope. The enzyme activity of the some dehydrogenases, cerebral proteins and adenosine triphosphate (ATP) were also analyzed. The results obtained are as follows: The ultrastructural changes in 0.5 and 1.0mg-injected groups were undetectable, but in 2.0mg-injected group, the nuclear envelops were very irregular and mitochondria, were swelled and destroyed partly. The number of polypeptide bands separated by SDS-PAGE in the normal group were 37 bands. According to the in creased dose of mercuric chloride, contends of the bands were increased in 7 bands. The activities of dehydrogenases were declined by increasing the dose of mercuric chloride. Lactate dehydrogenase (LDH) activity failed to 78% in 1.0mg-injected group and greatly to 68% in 2.0 mg-injected group. Malate dehydrogenase (MDH) activity failed to 81% in 2.0 mg-injected group. On the other hand, succinate dehydrogenase (SDH) activity decreased to 80% in 1.0 mg-injected group and greatly to 63% in 2.0 mg-injected group. ATP content in 1.0 mg-injected group was increased slightly and in 2.0 mg-injected group was increased greatly.

• Journal of Plant Biotechnology
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• v.7 no.1
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• pp.75-80
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• 2005

Eleutherococcus senticosus (also called Acanthopanax senticosus), belonging to Araliaceae family, has been used as an important medicinal woody plant. Mature seeds of Eleutherococcus senticosus have rudimentary (extremely immature) zygotic embryos and require a long-term stratification for about 18 months to induce germination. Here, through the methods of endosperm removal and other exogenous treatments, we investigated the factors for inducing rudimentary embryos by in vitro culture, Rudimentary zygotic embryos in seeds were at globular to heart-shaped stage at about $250{\mu}m$ in length just after harvest of fruits. When the seeds without testa were cultured on 1/2 MS (Murashige and Skoog 1962) medium, they did not germinate regardless of medium and sucrose concentrations but the removal of endosperm tissue markedly stimulated the growth of rudimentary zygotic embryos. The embryo reached ear-lier maturation, once when the endosperm surrounding the rudimentary embryos was removed. Rudimentary zygotic embryos developed cotyledons within 3 weeks of culture after endosperm emoval. However, post-mature zygotic embryos failed to germinate though they were morphologically normal, indicating another dormancy of embryos. $GA_3\;(2.0\;\cal{mg/L})$ and/or charcoal ($0.2\%$) treatment rapidly enhanced the germination of zygotic embryos. These results suggest that E. senticosus seeds have double dormancy; i. e. morphological rudimentary dormancy influenced by surrounding endosperm and physiological dormancy after post-maturation of zygotic embryos. Based on the above findings, we established the rapid germination of rudimentary zygotic embryos by in vitro culture of excised seeds with endosperm removal and $GA_3$ treatment.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.77-86
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• 1998

A combined technology of transvaginal ovum pick-up(OPU) system with in vitro-oocyte manipulation technique can be used for improving reproductive efficiency in the cattle. The objective of this study was to establish a newly-conceived breeding program using OPU in the pregnant cows. The OPU trial was performed in pregnant cows every 10 days from 40 through 90 days of artificial insemination (Al), and number of follicles in ovary, number of retrieved oocytes and embryo development following in vitro-fertilization, were evaluated. Reduced number of follicles in the ovaries of pregnant cows was firstly detected from 70 days after A' and a significant (P<0.05) decrease in the follicle number (5.4 follicles /donor) was found at 90 days than at 40, 50, 60 and 80 days after Al (8.0~9.2). A similar pattern was also observed in the number of oocytes retrieved by OPU apparatus during experimental period. When retrieved oocytes were matured and inseminated in vitro with frozen bull semen, development of the oocytes to the blastocyst stage was not significantly affected by the retrieval time. Four embryos (morula or blastocyst stage) derived from oocytes retrieved from pregnant cows were nonsurgically transferred to four recipient cows on day 7 of estrus cycle. For the first time in Korea, three of four transferred embryos developed to live calves with normal physiological parameters. In conclusion, an effective breeding program employing pregnant cow can be developed by use of OPU trial and in vitro culture techniques of oocytes ; OPU system could be repeated in pregnant cows with no risk of abortion and viable offsprings were borne after transfer to the recipients.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.281-287
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• 2013

In mammal, unfertilized oocytes remain in the oviduct or under in vitro culture, which is called "oocyte aging". This asynchrony negatively affects fertilization in pre- and post-implantation embryo development. Caffeine a phosphodiesterase inhibitor is known to rescue oocyte aging in several species. The objective of this study is to determine the cytoskeleton distribution in aged oocytes and the embryo developmental ability of aged oocytes in the present or absence of caffeine during maturation. Caffeine treatment increased the incidence of normal spindle assembly of aged oocytes (treatment, $67.57{\pm}4.11%$ aging, $44.61{\pm}6.4%$) and no significant differences compared to control group. Fluorescence values were compared using ROS (Reactive oxidation species) stain. Fluorescence values appear of control group intensity rate ($51.53.{\pm}3.80$), aging group ($68.10{\pm}5.54$) and treatment of caffeine ($45.04{\pm}2.98$). Aged oocytes that were derived from addition of caffeine to the IVM (in vitro maturation) medium had significantly increased 2-cell that developed to the blastocyst stage compared to the aging group. Blastocysts, derived from caffeine treatment group, significantly increased the total cell number compare aging ($90.44{\pm}10.18$ VS $67.88{\pm}7.72$). Apoptotic fragments of genomic DNA were measured in individual embryo using TUNEL assay. Blastocyst derived from caffeine treatment group decreased significantly the apoptotic index compared to blastocyst derived from aging group. In conclusion, we inferred that the caffeine treatment during oocyte aging can improve the developmental rate and quality in bovine embryos developing in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.3
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• pp.141-145
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• 2017

Objective: Delaying embryo transfer (ET) enables us to select among the embryos available for transfer and is associated with positive effects on implantation and pregnancy outcomes. However, the optimal day for ET of human cleavage-stage embryos remains controversial. Methods: A retrospective study of 3,124 in vitro fertilization/intracytoplasmic sperm injection cycles (2,440 patients) was conducted. We compared the effects of day 2 and 3 ET on rates of implantation and pregnancy outcomes between young maternal age (YMA; < 38 years old, n = 2,295) and old maternal age (OMA; ${\geq}38years\;old$, n = 829) patient groups. Results: The YMA and OMA groups did not differ in terms of patient characteristics except for the proportion of unexplained factor infertility, which was significantly greater in the OMA group, and the proportion of arrested embryos, which was significantly greater in the YMA group. However, the biochemical pregnancy, clinical pregnancy, ongoing pregnancy, abortion, and implantation rates per cycle were not significantly different between day 2 and 3 ET in the YMA group or the OMA group. Conclusion: We suggest that offering patients the opportunity to decide which day would be suitable for ET could be part of a patient-friendly protocol that takes into consideration an infertile woman's circumstances and work schedule by allowing ET to be performed on day 2 instead of the traditional transfer on day 3.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.341-344
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• 2015

A 4-day-old, male Poodle dog was presented with dull, depressed and exhausted activity after the birth. On physical examination, the puppy showed arthrogryposis, muscular atrophy and no movement of hindlimbs. Palpation on dorsum revealed an absence of lumbar and sacral vertebrae. On prenatal and postnatal radiography, lumbar vertebrae, sacrum and coccygeal vertebrae were not visualized. On ultrasonography, bilateral kidney and urinary bladder were observed. On computed tomography, there were no apparent abnormalities in the forelimbs, cervical vertebrae or head, while lumbar vertebrae, sacrum and coccygeal vertebrae were not observed. At necropsy examination, the liver, stomach, intestine, kidney and urinary bladder were normal. This congenital anomaly was consistent with Perosomus elumbis. Perosomus elumbis in dogs is a rare condition of unknown etiology. In this report, Perosomus elumbis was evaluated with radiography, ultrasound and computed tomography.