• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.185-192
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• 1999

The present study was performed to investigate the efficacy and safety of laser assisted hatching (AH) on mouse embryos. Non-contact $1.48{\mu}m$ diode laser system used to create a precise hole on zona pellucida. 2-cell embryos were collected from the mice (ICR) that had the coitus vaginal plug confirmed at 48 hours after hCG injection. Collected 2-cell embryos were cultured in the HTF medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after 18-22 hours in culture. After assisted hatching, the embryos were further cultured in HTF medium containing 0.1% PVP (anti-hatching system) for 3 days. For evaluate efficiency of laser on mouse embryo hatching, the effect of AH methods (acidic tyrode, pronase and laser), the number of artificial holes (1, 2 and 3 hole) and the irradiation time of laser (2, 4, 6, 8 and 10 ms) were examined. Hatching rates of laser AH group (95.2%) was significantly higher than that of control group (50.8%), but there was no differences among the laser (95.2%), acidic tyrode (100%) and pronase (98.5%) groups. Hatching rates of the number of zona pellucida opening by laser, there were no differences among the 1 hole (87.5%),2 hole (92.1%) and 3 hole (85.9%) groups. Developmental and hatching rates of embryos according to laser irradiation time were similar in the treatment groups. Therefore, these results suggest that laser AH using non-contact $1.48{\mu}m$ diode laser is a simple and accurate and effective procedure for AH. Based on these results, laser AH could be use safely for human ART program.

• 생명과학회지
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• 제9권3호
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• pp.333-340
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• 1999

The toxic effect of TBTO on Chlorella and Rotifer was observed. The value of 48hr-LC50 for Chlorella (3.3$\mu\textrm{g}$/L) estimated to be almost 500 times as high as that for Rotifer (6.7ng/L). A fertilized egg of olive flounder exposed in an embryo-formation stage was mostly influenced by TBTO toxicity when the fertilized egg at each stage until hatching was exposed to TBTO at the concentrations of 5 to 200ng/L. The values of LT50 were estimated to be 68.0, 41.0, 21.0, 13.0, 7.7 and 4.7 hours at 5, 10, 25, 50, 100 and 200ng/L of TBTO, respectively when the fertilized egg in a morula stage was exposed to TBTO, and the 48hr-LC50 was 8 ng/L. In case of TBTO treatment in an embryo-formation stage, the values of LT50 were 33.0, 12.5, 3.5, 1.3, 0.5 and 0.2 hours at 5, 10, 25, 50, 100 and 200ng/L of TBTO, respectively, and the value of 48hr-LC50 was 4ng/L. The values of LT50 were estimated to be 17.0, 11.0, 6.2, 4.0, 2.6 and 1.7 hours at 5, 10, 25, 50, 100 and 200ng/L of TBTO, respectively when the fertilized egg in a stage just before hatching was exposed to TBTO, and the 48hr-LC50 was below 1ng/L. The percentages of hatching were 46.2, 20.6, 21.9, 20.6 and 13.2% at 25, 50, 100, 250 and 500ng/L of TBTO, respectively when the fertilized egg in the stage just before hatching was exposed to TBTO and the measurement was done at second day after the completion of hatching. However no survival after the completion of hatching was found in all cases. With the treatment of 1, 5 and 10ng/L of TBTO, the percentages of hatching were 80.5, 70.0 and 44.1%, respectively. The percentages of survival until second day after the completion of hatching were 80.0, 63.3 and 9.1%, respectively. The percentages of hatching and survivability after the completion of hatching for the control were 84.5 and 82.5%, respectively.

• International Journal of Industrial Entomology and Biomaterials
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• 제46권1호
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• pp.16-23
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• 2023

The silkworm's dormancy and embryonic development are accomplished through the interaction of various genes. Analysis of the expression of several interacting genes can predict the embryonic stage of silkworms. In this study, we analyzed the changes in the expression level of genes at each stage during the embryonic development of dormant silkworm eggs and selected genes that can predict the hatching time. Jam123 and Jam124 silkworms were collected after egg laying, and the silkworm eggs were preserved using a double refrigeration method and expression analysis was performed for 23 genes during embryogenesis. There were 5 genes showing significant changes during embryogenesis: UDP-glucuronosyltransferases (BmUGTs), heat shock protein hsp20.8 (BmHsp20.8), Cytochromes b5-like proteins (BmCytb5), Krüppel homolog 1 (BmKr-h1), and cuticular protein RR-1 motif 41 (BmCpr41). As a result of quantitative comparison of the expression levels of these 5 genes through real-time PCR, the BmUGTs gene showed a difference between Jam123 and Jam124, making it difficult to see it as an indicator for predicting hatching time. However, the BmHsp20.8 gene had a common expression decreased at the imminent hatching stage. In addition, it was confirmed that the expression level of the BmCytb5 gene decreased to the lowest level at the time of imminent hatching, and the expression of the BmKr-h gene was made only at the time of imminent hatching. The expression of the last BmCpr41 gene can be confirmed only at the time of imminent hatching, and it was confirmed that it shows a rapid increase right before hatching. Taken together, these results suggest that expression analysis of BmHsp20.8, BmCytb5, BmKr-h1, and BmCpr41 genes can determine the stage of embryogenesis, predict hatching time, which facilitate better management of silkworm eggs.

• Asian-Australasian Journal of Animal Sciences
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• 제15권12호
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• pp.1702-1707
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• 2002

The effects of dark-control (D) and continuous green light (GL) exposure of incubated meat-type breeder eggs (Hybro) on embryonic growth from 5 to 15 days of age, hatching time, hatchability per cent and chick hatching weight were investigated in three consecutive experiments at 33, 38, and 41 weeks of age. A total of 798 eggs were used in this study. Eggs were set in an incubator on trays either in the D or under two tubes of 20-watt green fluorescent light during the first 18 days of incubation. Eggs from both treatments were transferred to the dark hatching compartment at 19 days of incubation. The light intensity was in the range of 1,340 to 1,730 lux at the surface of the eggs. GL incubation of eggs significantly (p<0.01) increased weight (expressed as an absolute value) and daily weight gain of embryos at 11 and continued to 15 days of age, hatchability per cent by 4.8%, reduced dead embryos per cent and chick weight at hatch by 37 and 2%, respectively and accelerated hatching time by about 24 h when compared with the D-control incubation. Chicks hatched at 504 h of incubation had significantly (p<0.01) higher body weight, expressed as an absolute value or as a percentage of egg weight, than those hatched earlier at 456 h of incubation. It was concluded that the GL incubation of meat breeder eggs reduced incubation period and chick weight at hatch and increased embryonic growth and hatchability per cent.

• Fisheries and Aquatic Sciences
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• 제21권9호
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• pp.23.1-23.8
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• 2018

Background: Russian sturgeon (Acipenser gueldenstaedtii) is an emerging candidate species in the Korean aquaculture domain owing to its highly valued caviar. Although the embryonic development of this species was previously described, the complete image data on the morphological differentiation of developing embryos have not been yet fully available. Further, with the viewpoint of larval production in hatchery, the effects of temperature on embryonic viability and the temporal window of hatching event have not been extensively studied. Hence, the objective of this study was to provide a complete set of photographic image data on the embryogenesis and also to examine the effects of incubation temperatures on embryonic viability and hatching event in farm-bred Russian sturgeon. Results: Typical characteristics of embryonic development including uneven, holoblastic cleavages with unequal blastomeres, followed by the formation of germ layer, neurulation, and organogenesis until hatching, were documented. Under different temperature conditions (12, 16, or $20^{\circ}C$), viability of embryos incubated at $12^{\circ}C$ was significantly lower as relative to those of 16 and $20^{\circ}C$ incubated embryos. Hatchability of embryos was higher, and the timing of hatching event was more synchronized at $20^{\circ}C$ than at 12 and $16^{\circ}C$. Conclusion: Data from this study suggest that the incubation of Russian sturgeon embryos at $20^{\circ}C$ would be desirable in the hatchery practice with respect to the good hatchability of embryos and the synchronization of hatching events. Additionally, the updated image data for complete embryonic development could be a useful reference guide for not only developmental researches but also artificial propagation of Russian sturgeon in farms.

• 한국환경생태학회지
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• 제37권3호
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• pp.192-197
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• 2023

본 연구는 서울대공원 인공사육장에서 남생이(Mauremys reevesii) 알의 부화온도 차이에 따른 부화기간과 성장에 영향을 주는지 알아보고자 연구했다. 전체 201개의 남생이 알을 26(n=89)℃, 28(n=75)℃ 그리고 32(n=37)℃에서 각각 부화시켰다. 남생이 알의 부화온도와 부화기간은 온도에 따라 유의미한 차이를 나타내었다. 본 연구에서 부화 온도가 높을수록 부화율이 증가하였다. 26℃, 28℃, 그리고 32℃에서 부화한 알의 부화 기간은 각각 66.1(±4.0, n=52)일, 65.3(±3.3, n=44)일 그리고 58.8(±7.7, n=31)일이었다. 부화율은 32℃(83.8%)가 26℃(58.4%)와 28℃(58.7%)보다 상대적으로 높았다. 32℃에서 부화한 14일 된 새끼의 몸무게가 26℃와 28℃에서 부화한 14일 된 새끼의 몸무게보다 높았다. 그렇지만 부화 후 180일과 270일에 새끼의 몸무게는 부화 온도에 따라 큰 차이가 없었다. 본 연구에서 한국산 남생이(M. reevesii) 알의 부화 온도가 부화 기간과 성장 초기 남생이의 체중에 유의한 영향을 미치는 것으로 나타났다.

• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.345-351
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• 2000

Objective: Hatching of the blastocyst from the zona pellucida (ZP) is a key event in mammalian implantation. In vivo, two factors have been identified as possible mediators of hatching: lysis of the ZP by substances elaborated either from the embryo or female reproductive tract and pressure exerted on the zona by expansion of the blastocyst. Two methods of zona manipulation were already in use to enhance the ability of embryos to hatch: mechanical PZD and chemical ZD by acidic Tyrode's solution. But several controversies of each method have been reported. The purpose of this study was to investigate the effect of pronase for mouse embryo hatching. Methods: Mouse embryos were obtained following ovulation induction of $F_1$ animals. Fresh and cryo-thawed morula embryos were exposed to 0.5, 1.0, 2.0, 5.0 ${\mu}g/ml$ pronase in Ham's F10 for 72 hrs. Main outcome measures were the rates of partial hatching and completely hatched blastocysts, and cell number of it. Results: In fresh and cryo-thawed group, the rates of completely hatched blastocyst were significantly higher in 5 ${\mu}g/ml$ pronase treatment group than control group. There was no difference in completely hatched blastocyst total cell number between pronase treatment group and control group. This suggest that pronase treatment did not harmful in mouse embryo development. In pronase treatment group, zona pellucida were thinner than control group. Conclusion: The addition of pronase to culture media may accelerate the hatching of embryo. So, enzymatic treatment of the zona may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.

• 한국패류학회지
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• 제32권2호
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• pp.133-139
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• 2016

The incubation time required for hatching of O. ocellatus were investigated through the processes of egg and embryonic developments by the dissecting microscopic and visual observations. And differences in ecological characteristics of the plankton mode of life or the benthic mode of life according to total numbers of the suckers on each short arm of the hatched juvenile larvae of O. ocellatus were studied by comparisons with other octopodidae species. Compared with the recent a few results reported by other researchers associated with the incubation time required for hatching by female adult mother of O. minor (73-90 days after spawning at $20.9-21.5^{\circ}C$ ranges), in this study, the incubation time required for hatching by female adult mother of O. ocellatus was 56-57 days after spawning at $11.0-20.4^{\circ}C$. Therefore, the incubation time required for hatching by female adult mother varied with Octopodidae species. In this studies, each ovarian egg laid by a female was connected to an egg string attaching to the surface of the wall or bottom of vacunt shell of Rapana venosa. Egg and embryonic developments of this species were studied in the indoor aquaria, in the specific gravity ranging 1.024-1.025. the hatched juvenile of O. ocellatus is 10.3 mm in the mean total length and 4.5 mm in mantle length, and each of its short arms has 18-20 suckers. The just hatched juvenile larvae of O. ocellatus enter the benthic mode of life (benthic larval stage) after hatching. In particular, regarding differences in ecological characteristics of the mode of life according to total numbers of the suckers, O. vulgaris may not need to have many suckers because they enter the planktonic mode of life after hatching, however O. ocellatus may need to have many suckers, because they should adapt to the benthic mode of life. And also the just hatched juvenile larvae of O. minor (bearing many suckers more than O. ocellatus) enter the benthic mode of life (benthic larval stage) after hatching. Therefore, the total number of the suckers on each short arm of the hatched juvenile larvae can be used for determining whether an octopus species has planktonic larval stages or benthic larval stage (benthic mode of life). In particular, The intracohort cannibalism phenomena appeared at the hatched juvenile larval stage because the larval stage of O. ocellatus and O. minor enter into the benthic larval stage in the early stage, unlike entering into the plaktonic larval stage in other Octopus species such as O. vulgaris: at this time, the early hatched larvae fed the late hatched larvae (they are the same species and almost same ages). Therefore, the intracohort cannibalism pheneomena occur in the just hatched juvenile stage of only O. ocellatus and O. minor.

• 한국가축번식학회지
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• 제21권1호
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• pp.61-69
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• 1997

본 연구는 EGF와 anti-EGF가 초기 생쥐배아의 발생 및 부화에 미치는 영향을 알아보고자 실행되었다. 초기 2세포기부터 상실배까지의 배아를 EGF와 anti-EGF를 각각 처리한 Ham's F10 배양액에서 배양하여 그 발생률과 부화율을 대조군과 비교하였다. EGF 처리시 배양시간에 따른 발생률은 증진되었으나 통계학적 유의성은 없었다. EGF 처리군에서의 부화율(57.5, 62.5, 65.0, 62.5%)은 대조군(35%)에 비하여 유의하게 (p<0.01) 높았다. Anti-EGF 처리시 각 발생시기별 1:1000 실험군의 발생률은 대조군과 차이가 없었다. 그러나, 1:100 실험군의 경우, 2∼4세포기의 배아는 모두 4∼8세포기에서 정지되었고, 8세포기와 상실배의 포배형성은 48시간 이상 지연되었으며 부화 역시 대조군에 비해 억제되었다(8세포기; 2%, 44%, 상실배; 6.2%, 58.3%). 이 실험에서, EGF는 생쥐 배아의 포배형성과 부화를 증진시키는 반면, anti-EGF는 이를 억제하였다. Anti-EGF 처리시 나타나는 발생정지 현상은 anti-EGF가 배아에서 만들어지는 EGF와 반응하여 EGF의 작용을 억제시키기 때문으로 사료된다. 그러므로 EGF는 paracrine mode로서 뿐만 아니라 antocrine mode로서 생쥐 초기배아의 발생에 중요한 인자로 작용함을 알 수 있었다.

• 한국양식학회지
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• 제21권3호
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• pp.176-180
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• 2008

본 연구는 인위적으로 배란시킨 뱀장어 Anguilla japonica 배란란의 과숙특징을 생화학적으로 밝히고, 과숙을 방지하기 위한 하나의 방법을 제시하였다. 사용된 뱀장어 성숙란의 수정률과 부화율, 부화 후 3, 10일 후 생존율과의 관계는 Y=0.7954X+32.448($R^2=0.8176$, P=6.35E-7), Y=1.0241X-26.401($R^2=0.8121$, P=5.95E-27) 및 Y=0.9314X-24.814($R^2=0.7619$, P=7.33E-14)였다. DNA 함량은 부화율의 감소와 함께 20% 이하의 부화율을 보이는 배란란에서 유의적으로 가장 낮은 0.653 pg/ug protein을 보였다(P<0.05). 그리고 이들의 비는 부화율이 20% 이하의 난에서 1.058로 유의적으로 가장 높은 비를 보였다(P<0.05). 또한 부화율에 따른 total alkaline protease와 ACPase 활성은 모든 부화율 범위에서 유의적인 차이를 보이지 않았다(P>0.05). 단백질 함량은 부화율 20% 이하 배란란에서 유의적으로 가장 높은 186.16 ug/mg eggs의 함량을 보였다(P<0.05). 따라서 뱀장어의 과숙란은 지속적인 단백질의 공급으로 정상란의 과숙화를 촉진하여 부화율이 저하된다. 결국 세포는 과숙으로 인해서 죽었기 때문에, 간에서 생성된 난황단백질의 공급을 차단하거나 공급속도를 늦추어서 복강 내 배란 후 과숙을 방지해야 할 것으로 판단된다.