• Journal of Embryo Transfer
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• v.30 no.1
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• pp.45-50
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• 2015

The purpose of this study is to produce wanted sex progeny of genetically confined White Hanwoo (albinism) with preselected sex sperm. One bull of White Hanwoo was chosen for semen donor and X sperm was sorted by MoFlo XDP cell sorter. To compare the pregnancy and birth rates, KPN straw was used as control, total number of unsorted sperm was $20{\times}10^6/straw$. Sexed X frozen semen with $20{\times}10^6$ cells or $4{\times}10^6$ cells per straw were in seminated twice on Hanwoo heifers. The abnormality of the sexed X semen was $24.9{\pm}7.31%$ and distal reflex abnormality of mid piece was significantly (p<0.05) higher (11.7%) compared with that of KPN 768 (5.6%). There were no differences on the pregnancy and birth rates between $2{\times}10^6$ cells or $4{\times}10^6$ cells of X-sperm but KPN semen showed significant differences (p<0.05). The pregnancy rates of KPN 768, $2{\times}10^6$ cells and $4{\times}10^6$ cells X-sperm of White Hanwoo cattle were 85.0%, 26.3% and 50%. The birth rates were 80.0%, 15.8% and 21.4%, respectively. The female offspring rates of KPN 768, $2{\times}10^6$ cells and $4{\times}10^6$ cells X-sperm of White Hanwoo cattle were 43.8%, 100% and 100% (p<0.05). These results indicated that sex sorted White Hanwoo could be used for the production of wanted progeny with $2{\times}10^6$ cells/straw for AI. To increase the efficiency of calf production, the sperm number of sex sorted semen will be optimized for sex selection of White Hanwoo progeny.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.701-710
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• 2002

Studies on the Viability of In Vitro-Matured Bovine Oocytes Vitrified by Microdrop and Straw Method To establish vitrification method for bovine oocytes, mature bovine oocytes were vitrified by microdrop (MD) or straw (Straw) method and the viability of vitrified oocytes with or without cumulus cells (CC) were examined by several methods; a) parthenogenetic activation; b) pronuclear formation after in vitro fertilization (IVF); and c) embryonic development after IVF. The survival rate of vitrified oocytes by MD was significantly higher than by Straw (92.50 vs. 74.19%, p<0.05). Most of the oocytes survived from vitrification using the MD methods. Cleavage and blastocyst development of parthenogenetically activated oocytes were higher in MD (45.05% and 10.81%, respectively; p<0.05)) than those in Straw method (27.17% and 6.52%, respectively; p<0.05). Male and female pronuclear formation of vitrified-thawed oocytes with or without cumulus cells (CC) after IVF were examined, respectively. The survival rate of vitrified oocytes by MD without CC was no difference between MD and Straw (80.368.14% vs. 67.31%). Normal fertilization (2PN) rates were not different among groups (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). While no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). The polyspermy (3PN) was appeared in the fresh (12.99%), but no appeared in the vitrified-thawed groups. In the without CC, normal fertilization (2PN) rates were significantly different between fresh and vitrified-thawed oocytes (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). Moreover, no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). The polyspermy (3PN, >4PN) was appeared not only fresh but vitrified-thawed groups. After IVF, two-cell developmental rates of vitrified oocytes with CC by MD and Straw were significantly low compared to fresh oocytes (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). Blastocyst developmental rates of vitrified oocytes also were significantly low compared to fresh oocytes (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). In the without CC, two-cell developmental rates were no difference between Fresh and MD (27.59% vs. 19.25%, p<0.05), while blastocyst rates were difference between Fresh and MD or Straw (4.31% vs. 0.62% and 0%, respectively; p<0.05). In conclusion, the results indicate that the vitrified bovine oocytes have the ability to develop to the blastocyst stage after IVF.

• Korean Journal of Ichthyology
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• v.32 no.2
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• pp.78-83
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• 2020

This study aimed to investigate the effects of different water temperature (5, 8 and 11℃) on egg hatch and larval growth of walleye pollock Gadus chalcogrammus to improve rearing techniques for this species. Survival of eggs maintained at 5℃ was higher than that of eggs maintained at 8℃ and 11℃. The greatest hatching rate was obtained for larvae maintained at 8℃. However, time to first, 50% and 100% hatch of eggs maintained at 11℃ was shorter than at 5℃ and 8℃. A higher survival was observed in larvae reared at 5℃ compared to 8℃ and 11℃ and the greatest growth was observed in larvae reared at 11℃ compared to 5℃ and 8℃ during the feeding trial. This study demonstrated the importance of water temperature, as it affects culture performance of eggs and larval pollock. The results from this study provides valuable information for further development of pollock aquaculture.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.459-465
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• 1997

The present study was undertaken to determine the effect of NT time on the rate of fusion a and suhseguent development In vitro and determine the optimal strength and duration of DC pulse for electrofusion of IVF donor embryo nuclei and IVM recipient oocytes. The recipient oocytes were enucleated 25 ~ 2Sh after IVM and further cultured for 18 ~ 20h prior to fusion for oocyte aging. IVF embryos as donor nuclei were C co cultured with BOEC for 16- to 32-cell stage development. The transfer time of donor bIas tomeres was 1~3h post-enucleation in early NT group and 1 ~ 18h post-enucleation in late NT group, respectively and fusion was performed 43~4Sh post-IVM. The fusion rate did not differ between the early NT and late NT group, but the rate of cleavage and 8- to 16-cell stage embryos in the late NT group was more higher than that in the early NT group. The fusion, cleavage and M+B development was high from O.7SkV /cm DC than from 1.0kV /cm DC voltage, resulting in 17.6% M+B from 0.75kV /cm DC voltage. No difference in fusion rate was among pulse durations, but 50 and 70 usec pulse duration showed slight high cleavage and M + B d development. The results indicate that the best NT time of IVF donor blastomeres into the enucleated oocytes was 42~44 post-IVM and the most suitable condition for electrofusion was a single 0.7SkV /cm DC voltage for SO~70$\mu$sec.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.117-117
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• 2003

체외에서 한우 난포란의 감수분열과 배발달 능력의 획득에는 단백질 합성이 수반되어야 한다. 그러나 이러한 변화와 관련된 연구 보고는 거의 전무한 실정이다. 본 실험은 난자의 핵성숙과 관련된 세포질내 단백질 변화를 파악하기 위하여, 체외성숙 시간에 다른 배발달율과 세포질내 단백질을 비교하여 배발달능력 획득과 관련 있는 단백질을 규명하고자 실시하였다. 한우 난소에서 2-8mm의 가시난포로부터 난포란을 회수하였다. 회수된 난포란은 10% FBS와 호르몬이 첨가된 TCM199 용액에서 18시간 또는 24시간 체외성숙을 실시하였다. 난자 세포질내 단백질 변화는 2D gel electrophoresis를 이용하였고, 유의적인 변화를 나타낸 spot은 peptide mass fingerprinting을 통하여 단백질 동정을 실시하였다. 체외수정은 fer-TALP 용액을, 체외배양은 CR1aa 용액을 배양 3일째까지는 0.3% BSA, 그 이후에는 10% FBS와 난관상피세포를 첨가하여 사용하였다. 통계분석은 t-test를 이용하였다. 난자의 세포질에 대한 이차원전기영동 결과 29개의 단백질 spot들을 확인하였다. 한편 체외성숙 18시간째에 PB가 출현된 난자는 PB가 출현되지 않은 난자에 비하여 15개의 spot에서 유의적인 변화를 나타냈다. 이들 중 4개의 단백질 spot은 낮았고, 11개 spot의 수준은 높은 경향이었다. 체외성숙 18시간째와 24시간째의 배발달율을 조사한 결과 18시간째에서 유의적으로 높은 배반포 발달율을 나타냈다. 그리고 체외성숙 18시간과 24시간째 난자의 세포질내 단백질 spot들의 변화를 비교한 결과 PB가 출현된 난자 세포질에서 단백질의 변화와 유사한 경향이었다. 그러나 2개의 단백질 spot은 상반된 경향을 나타냈다. 따라서 본 실험에서 난자의 핵성숙과 관련 있는 15개의 spot을 확인하였고, 이들 단백질 spot중에서 2개가 배발달 능력과 관련이 있을 것으로 사료된다.보다는 육질등급에 많은 영향을 받는 것으로 판단된다. 한편 육질 1등급에서 배발달율이 낮은 이유는 육질 향상을 목적으로 암소를 비육 하는 경우 발생하는 번식장애와 밀접한 관계가 있는 것으로 사료된다.각각 가장 높았다. 배양 8일째 배반포의 세포수에 있어서 총세포수와 TE 세포수는 차이가 없었으나, ICM 세포수가 l0mg 첨가군에서 가장 높았다. 본 실험 결과에서 체외성숙 배지에 NEAA와 EAA 첨가가 배발달율에는 효과가 없었지만, 첨가농도의 증가에 따라 ICM 세포수가 증가하였다. 한편 체외성숙 배지에 LAH 첨가는 첨가 농도가 높을수록 배발달율은 낮았지만 ICM 세포수는 증가하였다.에 Csk가 관여하고 있음을 알 수 있다. 결론적으로 성적 성숙에 따른 생쥐 정소 내 Src-Csk loop의 발현과 Src kinase 활성의 변동은 정소 내 간충조직, 세정관 상피의 증식 및 기능적 분화 과정을 매개하는 생리적 활성분자 수용체 하위의 신호전달 과정에 Src-Csk loop에 의한 조절가능성을 확인할 수 있었다.rugrene의 향기성분이 주요 성분군으로 확인되었다. 2. 생강나무에서 생강의 향기를 발산하는 성분으로는 $\beta$-myrcene, o-terpinolene, phellandrone, ι-limonene, $\beta$-eudesmol, $\delta$-cadinone, elemol, trans-caryophyllene으로 동정되었으며 그 중에서도 phellandrene, $\beta$-eudesmol이 주된 역할을 하는 성분으로 확인하였다. 유의적인 관련성이 나타났고, 복부 비만의 지표인 허리엉덩이둘레비는 GPT, alkaline phosphatase, 공복시 혈당 및 MCV 등 다양한 건강지표와 관련성을 나타내어 향후 비만에 있어 다양한 혈액 성상의 변화 및 역할규명에 대한 연구가 이루어져야 할 것으로 본다.hat

• Development and Reproduction
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• v.11 no.1
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• pp.43-47
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• 2007

Smad proteins mediate transforming growth $factor(TGF)-{\beta}$ signaling and play a pivotal role in embryonic development. The estrogen receptor-related receptors(ERRs), which are structurally similar to estrogen receptors, are members of orphan nuclear receptor in the nuclear receptor superfamily and their functions are known to be involved in the formation of extra-embryonic ectoderm. To investigate the involvement of Smad3 and $ERR{\beta}$ like 1 in reproductive activities and embryogenesis in marine invertebrate, we examined gene expression of Smad3 and $ERR{\beta}$ like 1 in Strongylocentrotus nudus during their seasonal changes and embryonic development using real-time polymerase chain reaction. The Smad3 mRNA levels in gonad showed an increasing pattern from February to June 2004 but decreased at August(spawning season) followed by an elevation of the levels at October and December 2004. The mRNA levels of the $ERR{\beta}$ like 1 significantly elevated during the spawning season. During embryonic development, Smad3 mRNA levels at $8{\sim}16$ cell stages were significantly higher than those of other stages, whereas the mRNA of the $ERR{\beta}$ like 1 was significantly high levels at late development stages, i.e., blastular, gastrula and plutei stages. These results suggest that the Smad3 could be involved at least in part in the early cleavage stages and the $ERR{\beta}$ like 1 may play an important role in the spawning season and late developmental stage in the sea urchin.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.307-311
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of several cells. In our previous study, inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the pig embryonic and primary cells was reported. However, its role during early bovine embryonic development is not sufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on early bovine embryonic development. We also investigated several indicators of developmental potential, including structural integrity, gene expression (apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Bovine embryos were cultured in the CR1-aa medium with or without 17-AAG for 7 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG ($33.1{\pm}9.6$ vs $21.7{\pm}8.3%$). The structural integrity of the blastocysts was examined by differential staining. Blastocysts from the dbcAMP-treated group had higher numbers of ICM, TE, and total cells than those from the untreated group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (11.2 vs 3.9, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation bovine blastocysts. The mRNA expression of the pro-apoptotic gene (Bax) increased in 17-AAG treated group, whereas expression of the antiapoptotic gene (Bcl-XL) decreased. In conclusion, Hsp90 also appears to play a direct role in bovine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with apoptosis-related genes expression in developing bovine embryos.

• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.115-121
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• 2014

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, $0.1{\mu}g/mL$, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by $0.01{\mu}g/mL$ compared with other groups (p< 0.05). Moreover, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p<0.05). Otherwise, treatment with $0.01{\mu}g/mL$ of Humulus japonicus extract not increased the total cell number but reduced apoptotic-positive nuclei number. In conclusion, our results indicate that supplementation of Humulus japonicus extract in IVF medium may have important implications for improving early embryonic development in bovine embryos.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.