• 한국동물생명공학회지
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• 제34권1호
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• pp.50-56
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• 2019

These studies were conducted to evaluate developmental competence of follicular oocyte collected from the ovaries of Hanwoo cows with the high offspring meat quality (1++ and 1+ grade). Cumulus oocyte complexes from individual cows were matured, fertilized and cultured using protocols of in-vitro maturation (IVM), invitro fertilization (IVF) and in-vitro culture (IVC). The rates of blastocyst development from Hanwoo cows with the offspring meat quality grades of 1++ and 1+ were 18.6 and 21.2%, respectively. The rates of blastocyst development were 26.3, 20.7, 20.7, 17.2 and 31.2% from Hanwoo cows with the meat quality grades of 1++, 1+, 1, 2 and 3, respectively. Fiftyseven transferable embryos were recovered from 11 Hanwoo donor cows (5.2/head) with the high offspring meat quality grades of 1++ and 1+ in vivo, and the pregnancy rate after embryo transfer was 61.1%. In conclusion, these results suggest that in vitro embryo production from the ovaries of cows with the high meat quality grades using individual culture system can be used an efficient method for livestock improvement. In addition, for the successful industrialization of embryo transfer, conception rate should be improved.

• Toxicological Research
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• 제22권2호
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• pp.127-133
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• 2006

Recently, we have reported that the environmental pollutant 2-bromopropane (2-BP) induces a significant embryo-fetal developmental toxicity in rats. However, the cause of developmental toxicity and the relationship between maternal and developmental toxicities could not be elucidated because the developmental toxicity of 2-BP was observed only in the presence of maternal toxicity The in vitro teratogenicity study using whole embryo culture was carried out to understand the teratogenic properties and the possible mechanism of teratogenicity induced by 2-BP in rats. Rat embryos aged 9.5 days were cultured in vitro for 48 hrs at medium concentrations of 0, 1, 3, or 10 mg/ml of 2-BP. Embryos were evaluated for growth, differentiation, and morphological alterations at the end of the culture period. At 10 mg/ml, 2-BP caused a delay in the growth and differentiation of embryos and an increase in the incidence of morphological alterations, including altered yolk sac circulation, abnormal axial rotation, craniofacial hypoplasia, open neuropore, absent optic vesicle and kinked somites. At 3 mg/ml, only a delay in the growth and differentiation of embryos was observed. There were no adverse effects on embryonic growth and development at the concentration of 1 mg/ml. The results showed that the exposure of 2-BP to rat embryos results in a developmental delay and morphological alterations at dose levels of 3 mg/ml culture media or higher and that 2-BP can induce a direct developmental toxicity in rat embryos.

• 한국가축번식학회지
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• 제10권1호
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• pp.76-82
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• 1986

This experiment was conducted to determine the effects of trophoblastic vesicles (TV) and estradiol-17$\beta$ on the development in vitro of rabbit embryos. Thirty matured female rabbits were treated with PMSG followed by HCG injection and mating. Embryos were recovered with D-PBS (Dulbecco's Phosphate Buffered Saline) after superovulation, and normally developed to two-to four-cell embryos were used in the subsequent in vitro culture. Basal medium was Medium-199 su, pp.emented with 1.5% bovine serum albumin. Embryo on Day 5 after mating (Day 0) was cut into two or three pieces to remove the embryonic disc. Each piece of tissue was cultured for 24 hours at 37$^{\circ}C$ in 0.5 mlMedium-199 in 5% CO2. During culture, peices of trophoblastic tissue changed into spherical vesicles which were used for co-culture. These spheres were called trophoblstic vesicles. Two-to four-cell embryos were cultured for 4 days in Medium-199 in the absence or presence of trophoblastic vesicle, and two-to four-cell embryos cultured with varing concentration (0, 0.1, 1, 10ng/ml) of estradiol-17$\beta$ for 4 dyas. Culture vessels used were watch glass for coculture with trophoblastic vesicles and micortube for estradiol-17$\beta$ infusion. Compared with the Medium-199 alone as basal culture medium, more blastocysts (46.7% vs 15.1%; P<0.01) and morulae (84.4% vs 56.6%; P<0.05) were developed in the co-culture with trophoblastic vesicles. Estradiol-17$\beta$ infused in culture medium was not effective for embryo development to blastocysts (78.3% in control, 50.0% in 0.1ng/ml, 61.5% in 1ng/ml and 64.4% in 10ng/ml) and also to morulae (91.3% in control, 84.2% in 0.1ng/ml, 92.3% in 1ng/ml and 91.1% in 10ng/ml). Compared with the watch glass culture mehotd, more (P<0.01) blastocysts were developed in microtube culture (78.3% vs 56.6%) and more (P<0.01) morulae in microtube culture (91.3% vs 56.6%).

• Plant Biotechnology Reports
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• 제2권2호
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• pp.153-161
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• 2008

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation ($57.22{\pm}2.02$), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation ($58.80{\pm}1.47$) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyl-adenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• 한국수정란이식학회지
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• 제15권1호
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• pp.15-21
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• 2000

These studies were undertaken to evaluate morphological normality and development competence in vitro of hyman oocytes following vitrificatioin using ethylene glycol and electron microscopic grid. Human immature oocytes retrieved from natural and stimulated cycles was vitrified at 0 or 48 h and 0, 8 to 15 or 24 to 28 h after maturation culture, respectively. In oocytes retrieved from unstimulated cycle, no signifciant differences were found in morphological normality (56 to 63%) and fertilization (31 to 37%) rates between the times of vitrification. In stimulated patients, however, more oocytes were morphologically normal when vitrified at 24 to 28 h than when vitrified at 0 or 8 to 15 h after maturation culture. Regardlesss of the hormonal stimulation, high cleavage rates(83 to 100%) were obtained in all treatment groups but did not differ significantly. Twenty to 43% of cleaved oocytes developed to the blastocyst stage at 6 days after IVF. These results suggest that vitrified oocytes from unstimulated and stimulated cycles could develop to the blastocyst stage, regardless of the stages of vitrification.

• 한국수정란이식학회지
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• 제27권3호
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• pp.189-192
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• 2012

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, ${\alpha}$-MEM or KnockOut$^{TM}$-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, ${\alpha}$-MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and ${\alpha}$-MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, ${\alpha}$-MEM tended to be more suitable after passage 6. Although ${\alpha}$-MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

• 한국수정란이식학회지
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• 제19권1호
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• pp.11-18
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• 2004

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37$^{\circ}C$/with hFF, Group II: 32$^{\circ}C$/withGroup III: 37$^{\circ}C$/without, Group IV:32$^{\circ}C$ /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.9$\pm$1.9%. After 24 hours culture, sperm motility was 23.5$\pm$2.1% (Group I), 8.1$\pm$1.1% (Group II), 10.4$\pm$ 1.4% (Group III) and 4.0$\pm$0.8% (Group IV), respectively. After 48 hours, the motility had been changed as 32$\pm$2.3% (Group I), 14.3$\pm$1.7% (Group II), 5.3 $\pm$1.4% (Group III) and 4.3$\pm$0.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37$^{\circ}C$ was higher than those of group II at 32$^{\circ}C$. But, sperm viability of group I cultured at 37$^{\circ}C$ was lower than those of group II at 32$^{\circ}C$ (54.4$\pm$4.1% vs. 59.4$\pm$3.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37$^{\circ}C$. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.

• 한국수정란이식학회지
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• 제31권3호
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• pp.221-226
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• 2016

The aim of the present study was to assess the embryo development and survivability of post-thawed bovine embryos produced in vitro by addition of cysteine. The rates of metaphase II formation were not differed significantly among three groups(TCM199 73.8%, TCM199 with 0.3% cysteine 76.9%, TCM199 with 0.5% cysteine 83.8%, respectively). No difference of cleavage rate(70.6~74.6%) was seen among three culture medium(TCM199 70.6%, CR1aa 71.3%, SOF 74.6%) with 0.5M cysteine. however, Significantly(P<0.05) higher development rate into blastocyst stage by 0.5M cysteine addition was obtained in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%), however no significant differences in the cleavage rates were among three culture medium. After frozen the blastocysts cultured with 0.5M cysteine, The re-expansion rates were 61.3%~86.4% among groups, and hatching rates were 26.3%~46.9% among groups, the rates of re-expansion and hatching were significantly(P<0.05) higher in SOF medium(86.4% and 46.9%) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate was significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC medium with 0.5M cysteine improved the quality of in vitro production embryo and post- thawed embryo. Future studies comparing these media systems in well-designed trials should be performed.

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.