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Development of Effective Cryopreservation Method for Mammalian Embryo (포유류 초기 배아의 효율적인 동결 보존 방법에 관한 연구)

  • Kim, Hyun;Cho, Young Moo;Ko, Yeoung-Gyu;Kim, Sung Woo;Seong, Hwan-Hoo;Yamanouchi, Keitaro
    • Journal of Embryo Transfer
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    • v.29 no.3
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    • pp.241-248
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    • 2014
  • This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by PMSG and hCG. Two-step EG, DMSO and 4-step EG, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in EG and DMSO was significantly higher than 4~8 cell (65.4% versus 61.2%, 81.1% versus 72.5%) (p<0.01, p<0.01), but the development rates of 4~8 cell embryos in EG and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4~8 cell embryos in EG were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in EG(77.0% versus 64.4%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was sinificantly higher in EG than in DMSO during 48 hr (p<0.01). The survival rate of 4~8 cell embryo was 62.5% in EG and 73.3% in DMSO. The development rates of embryo in EG were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. The survival rate of embryo in 2 cell stage was higher than in 4~8 cell stage, and EG appears more effective cryoprotectant than DMSO because EG showed better development rates of embryos in 2 and 4~8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

Effect of Dietary Plant Extracts (Coxynil®, Growell®, Respowell®) in Broilers (사료 내 식물추출물 복합제(Coxynil®, Growell®, Respowell®) 첨가가 육계의 성장에 미치는 영향)

  • Cho, Sang-Beum;Kwon, Seung-Hyun;Lee, Jun-Hyeong;Lee, Yun-Jeong;Kang, Chang-Won;Paik, Hyun-Dong;Chang, Byung-Joon;Kim, Soo-Ki
    • Journal of Life Science
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    • v.19 no.11
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    • pp.1547-1552
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    • 2009
  • This study was carried out to evaluate the supplementation effect of dietary natural plant extracts (NP: $Coxynil^{(R)}$, $Growell^{(R)}$ and $Respowell^{(R)}$) on broiler chickens. Forty thousand male broilers with 7 days adaptation after hatching were fed experiment diets for 34 days. The supplementation effects of NP on growth performance, blood parameters and biopsy were examined with twenty thousand broilers as the treatment group. Twenty thousand broilers for the control group (CON) were fed the diet with salinomycin-6, clopidol-25, enramycin-1, and BMD-2.5. In the diet of the treatment group, the antibiotics were replaced with 0.03%, 0.035% and 0.03% of $Coxynil^{(R)}$, $Growell^{(R)}$ and $Respowell^{(R)}$, respectively. The weight gain of the treatment group was increased but the feed intake was decreased, indicating that feed efficiency was increased compared to the CON. The mortality of the NP group was also lower compared to the CON group (1,008 birds to 1,693 birds), showing positive dietary effects from natural plant extracts. In the activity of infectious bursal disease virus (IBDV) and new cattle disease virus (NDV) antibodies, the NP showed lower antibody titer levels for both of IBDV and NDV compared to the CON. The levels of total cholesterol, HDL-cholesterol, globulin, and IgG in blood did not show significant differences between the groups. In the microscopic tissue analysis, no significant differences were detected. These results may suggest that a complex of three natural plant extracts can be used as alternative antibiotics in broilers.

A Study on the Evaluation and Maintenance for Alternative Habitats of the Narrow-mouth Frog (Kaloula borealis) - A Case Study on the Alternative Habitats of Kaloula borealis at the University of Seoul - (맹꽁이 대체서식지 조성 평가 및 유지관리 방안 연구 - 서울시립대학교 맹꽁이 대체서식지를 사례로 -)

  • Park, Seok-Cheol;Han, Bong-Ho;Park, Min-Jin
    • Journal of the Korean Institute of Landscape Architecture
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    • v.47 no.1
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    • pp.76-87
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    • 2019
  • The purpose of this study was to evaluate the performance of and to derive future maintenance-management measures of the constructed alternative habitat for the Kaloula borealis at the University of Seoul, examining the period between 2015-2017. The research was constructed in 2014 and in a $191m^2$ area. The performance evaluation was divided into maintaining the habitat of the target species, maintaining the population and reproduction rates of the target species, maintaining the habitat of the wild species, the resilience of natural ecosystems, and the harmony with the surrounding environment. In terms of maintaining the habitat of the target species, soil collected from the existing habitat of the Kaloula borealis and was the depth was increased to 30cm in the alternative habitat. An artificial water supply was required every year during the supporting the spawning and hatching of other amphibians along with the Kaloula borealis. The sources of water of the alternative habitat were both rain and tap water, as it cannot be maintained naturally. Additionally, the Kaloula borealis thrived because it inhabited the research site and the average temperature was $26.2^{\circ}C$ from April-June, which is when the Kaloula borealis spawns. In terms of maintaining the population and reproduction rates of the Kaloula borealis, they were evaluated to have stable rates of reproduction. In terms of maintaining the habitat of the wild species, studies on vegetation and the structure of the characteristics of prey or predators will be needed. Also, alien species, such as Humulus japonicus and Bidens frondosa needed to be removed to maintain the wetland ecosystem of the wild species. In the assessment of the resilience of the natural ecosystems, the mud was monitored, noting the changes in the depth of water, with steps taken to reduce the leakage of water. The mud collected from the Haneul Pond wetland, which is located around the research site was piled up. Also, partial mowing management and the inducement of a natural vegetation colony was required for vegetation management. It was also necessary to create porous spaces, such as old trees and tree branches to create a habitat with hiding places and feeding and spawning places for small organisms. In terms of the harmony with the surrounding environment, the following threat factors needed to be managed: amphibian roadkill by vehicles and pedestrians and artificial draining due to nearby user access. Based on the monitoring results, alternative habitat management measures presented the promoting various waterside structures, in which amphibians can spawn and hide in, managing the water environment consistently, managing the vegetation, focused on the habitat of the wild species, and managing the surrounding environment for the habitat. The creation of an alternative habitat should be managed through monitoring, reflecting the characteristics of the changes in the site. Also continuing efforts are also needed to improve the habitat of the target species.

Effects of Starvation and Delayed Feeding on Growth and Survival of Pacific Cod Gadus macrocephalus Larvae (대구, Gadus macrocephalus, 자어의 첫 섭식 시 기아와 늦은 먹이 공급이 성장과 생존에 미치는 영향)

  • Shin, Min-Gyu;Lee, So-Gwang;Jeon, Hae-Ryeon;Joo, Jae-Hyeong;Gwak, Woo-Seok
    • Korean Journal of Ichthyology
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    • v.31 no.1
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    • pp.7-15
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    • 2019
  • The present study aimed to investigate the influence of starvation on growth, survival and swimming ability of Pacific cod Gadus macrocephalus larvae. Notochord length, musculature height, body depth, gut height and volume of yolk of reared larvae were measured to determine the growth parameters. A significant difference was observed in all morphometric characteristics before 15 DAH (days after hatching). Body depth and volume of yolk of unfed larvae were significantly smaller than those of fed larvae from 9 DAH (P<0.05). Almost all yolk in fed group was consumed at 11 DAH. Survival and growth of larvae were observed to determine the effect of delayed initial feeding (2 DAH, 3 DAH, 4 DAH, unfed). All larvae in the unfed group died by 15 DAH and the larvae in other experimental groups survived until the end of the experiment to 21 DAH. Survival rate was not significantly different between the 2 DAH group ($17.5{\pm}4.27%$) and the 3 DAH group ($20.5{\pm}1.5%$) at 21 DAH (P>0.05). However, there was a significant difference in survival rate between the 3 DAH group and the 4 DAH group ($11.7{\pm}1.52%$) (P<0.05). There was no significant difference in notochord length among the groups fed from 2 DAH, 3 DAH and 4 DAH at 21 DAH (P>0.05). The swimming ability in fed group gradually increased in both cruising and burst swimming speeds, while those abilities in unfed group gradually decreased after reaching the peak at 6 DAH in both cruise ($18.7{\pm}6.56mm/s$) and burst swimming speed ($43.5{\pm}12.65mm/s$).

Study on the Characteristics of Feather Developing Pattern and Morphology in Early- and Late-Feathering Korean Native Chickens (한국재래닭에 있어 조우성과 만우성 깃털의 발생 양상 및 형태적 특성 고찰)

  • Bang, Min Hee;Cho, Eun Jung;Cho, Chang Yeon;Sohn, Sea Hwan
    • Korean Journal of Poultry Science
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    • v.45 no.3
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    • pp.155-165
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    • 2018
  • Chicken feathers could be classified into early-feathering (EF) and late-feathering (LF) depending on the development and patterns of the wing and tail feathers. Currently, feather-sexing is a widely used chick sexing method in the industry. This study was carried out to suggest the method of classifying of EF and LF chicks to establish auto-sexing Korean native chicken (KNC) strains. The development and morphology of wing feathers and tail feathers in 856 KNCs from hatching to 55-days old were analyzed to classify EF and LF chicks. We also performed PCR analysis using K-specific gene primers to confirm the agreement between the phenotypes and genotypes of EF and LF chickens. In the results, the EF chicks had long primaries and coverts, and there was a significant difference in length between primaries and coverts. The LF chicks had shorter primaries and coverts than the EF chicks, and showed little difference in the length between primaries and coverts. LF chicks could be classified into four groups: LF-Less, LF-Scant, LF-Equal and LF-Reverse according to their wing feather patterns. EF chicks had 1.5 times longer primaries than LF chicks until they were 15-days old, but the lengths were almost the same at 50-days old. The tail feathers of the EF chicks were apparent at 5-days old, but those of the LF chicks were short and indefinite at that time. When EF and LF chicks were classified by the length of primaries being more or less than 9 mm, the classification accuracies for EF and LF chicks were 96.2% and 85.4%, respectively, compared to the PCR results. In conclusion, juvenile EF and LF KNC showed distinct differences in feather development and morphology, and could be easily distinguished at one day-old.

Diapause and Voltinism in Ostrinia furnacalis (Lepidoptera: Crambidae) in Suwon, and Larval Instar Sensitivity to Diapause Induction (수원 지방에서 조명나방 휴면과 발생 세대수 및 휴면유도에 대한 유충의 민감성)

  • Kim, Eun Young;Kim, I Hyeon;Seo, Bo Yoon;Kim, Yonggyun;Park, Chang-Gyu;Jung, Jin Kyo
    • Korean journal of applied entomology
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    • v.59 no.3
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    • pp.185-202
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    • 2020
  • The diapause induction season in Ostrinia furnacalis (Lepidoptera: Crambidae) was estimated in Suwon. Three batches of adult generations were observed, the first one from early May to early July, the second from early or mid-July to early or mid-August, and the third from mid-August to October. In outdoor larval rearing, colony rearing occurring from mid-July to mid-August produced both non-overwintering and overwintering larvae, whereas late-reared colonies produced only overwintering larvae. Larvae collected during July and August in maize fields produced both non-overwintering and overwintering larvae, whereas late-collected larvae produced only overwintering larvae. The results indicated that O. furnacalis has a bi- or trivoltine complex life cycle in this area. In the laboratory, when larvae of all instars within 9 h after molting were first treated to a diapause induction condition (11:13 h = light:dark photoperiod and 20℃), almost all larvae were induced to diapause. However, when similar treatments were conducted age-specifically for the 5th instar larvae, diapause induction rates in 3- and 4-day-old larvae of the 5th instar decreased. In contrast, when larvae were subjected to the diapause induction treatment only during the periods from the hatching stage to the 2nd, 3rd, and 4th instar, almost all larvae were not induced to diapause. The results suggest that the early age of the 5th larval instar is the last stage for sensitivity to diapause induction stimuli. In the diapause-induced larvae, hemolymph trehalose content increased and body supercooling points dropped, compared with those in non-diapause larvae.

Meridic Diets for Rearing of Spodoptera frugiperda Larvae (열대거세미나방 유충 사육을 위한 반합성 인공사료)

  • Jung, Jin Kyo;Kim, Eun Young;Kim, I Hyeon;Ahn, Jeong Joon;Lee, Gwan-Seok;Seo, Bo Yoon
    • Korean journal of applied entomology
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    • v.59 no.3
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    • pp.243-250
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    • 2020
  • Two meridic diets, N4 and N6, containing pinto bean, wheat germ, soybean, whole milk and sucrose as main nutrients were developed for rearing Spodoptera frugiperda (Noctuidae) larvae. Six larval instars were observed when neonate larvae were individually raised on these diets in small petri-dishes (ø 50 × 10 mm, 19.6 ㎤) at 25℃ and 15:9 h (light:dark) photoperiod. The average pupation rate of 97.8% on the N4 diet was significantly higher than the rate of 85.6% on N6 diet. The emergence rates were 92.0% on N4 diet and 93.5% on N6 diet, with a non-significant difference. The larval periods were 17.9 and 17.7 days for females, and 18.7 and 18.5 days for males, for N4 and N6 diets, respectively, with non-significant differences between diets and sexes. The pupal periods on both diets were identical (11.1 days for females and 12.8 days for males), despite differences between sexes. The pupal weights of both sexes on N4 diet were identical with a value of 257 mg, whereas those on N6 diet were 256 and 263 mg for females and males, respectively, with a non-significant difference. The longevity of female adults that emerged on N6 diet was 13.8 days and longer than 8.6 days on N4 diet. The pre-oviposition periods were 5.0 and 4.2 days on the N4 and N6 diets, respectively, with a non-significant difference, however, the oviposition period of 6.5 days on N6 diet was longer than the period of 3.9 days on N4 diet. The effective fecundity on N6 diet was 1,392 eggs (maximum 1,776) and was higher than that of 942 eggs (maximum 1,694) on N4 diet, with a non-significant difference. The egg hatching rates on N4 and N6 diets were 79.2 and 79.8%, and egg periods were 3.0 and 2.9 days, respectively, with non-significant differences.

Artificial Mutation for Silkworm Molecular Breeding Using Gene Scissors (유전자 가위의 이용과 누에 분자 육종을 위한 인위적 돌연변이 유발)

  • Hong, Jeong Won;Jeong, Chan Young;Yu, Jeong Hee;Kim, Su-Bae;Kang, Sang Kuk;Kim, Seong-Wan;Kim, Nam-Suk;Kim, Kee Young;Park, Jong Woo
    • Journal of Life Science
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    • v.30 no.8
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    • pp.701-707
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    • 2020
  • Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and could be an effective way of shortening the time required.

PROPAGATION OF THE BLUE CRAB, PORTUNUS TRITUBERCULATUS (MIERS) (꽃게 Portunus trituberculatus (MIERS)의 종묘 생산에 관한 연구)

  • PYEN Choong-Kyu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.3 no.3
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    • pp.187-198
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    • 1970
  • The author succeeded in rearing the young blue crab from the first stage of zoe ato the true crab shape, and during this time he observed their growth and metamorphosis. The relationships between the number of eggs carried by female crabs (E) and the carapace width (C) and body weight (W) are shown as follows: E= 27.9049C-281.8155, E=0.5682 W-116.4606. There are five zoeal stages and a megalopa in the complete larval development of the blue crab. Water temperature in rearing aquaria ranged from 21.4 to $25.2^{\circ}C$. The duration of each zoeal stage was two days on the average. After the fifth moulting, the zoea becomes megalopa and 5 to 6 days later the megalopa moults and develops into the first stage of adult crab shape. The carapace width of megalopa measured about 1.70 mm and the carapace length, from the tip of the rostrum to the posterior dorsal margin of the carapace, was about 2.78 mm on the average. The carapace width and length of the first crab, 18 days after hatching, measured about 4.48 mm and 2.62 mm respectively. After two days, the first crab moulted and grew into the second crab with about 6.47 mm in carapace width and 4.66 mm in carapace length. The larval rearing in the outdoor tank shelved better results than in the indoor aquarium. The highest mortality occurred when the first stage of zoea moulted into the second stage. Percentage of crabs which survived, from the first crab to the ninth crab stages, was about $55\%$. The relationships between rearing days (D) and the carapace width (C), carapace length (L) and body weight (W) of the crab stages during 40 days of rearing are shown as follows. Carapace width, Indoor: C=1.1250D+1.7227 Outdoor C=1.3465D -0.2449 Carapace length, Indoor: L=0.6654D+1.6712 Outdoor: L=0.7893D+0.6919 Body Weight, Outdoor: $$W=1.15e^{0.12423D}$$ Indoor: $$W=6.759\times10^{-2}D^{1.2598}$$ (9-19 day old crabs) Outdoor: $$W=4.136\times10^{-2}D^{1.6024}$$ (21-40 day old crabs) During the crab stage, the following relationships between the number of moulting times and the carapace width (C), carapace length (L) and body weight (W) were found as follows: $$C=5.2e^{0.28119N}$$ $$L=3.65e^{0.26372N}$$ $$W= 0.14e^{0.7037N}$$ The relationships between the carapace length (L) and the carapace width (C) and body weight (W) of the crab stages are shown as follows: Carapace length, mm Formula 2.62-27.17 L=1.6864C-1.0387 7.47-18.53 $$W=9.367\times10^{-5}C^{3.5567}$$ 22.11-27.17 $$W=3.406\times10^{-5}C{3.8571}$$

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Effect of Cryoprotectant Kinds and Cell Stages on the Viability of Mouse Embryos Cryopreserved by OPP Vitrification (동결보호제의 종류 및 배발달단계가 OPP Vitrification 동결보존시 생쥐수정란의 생존성에 미치는 영향)

  • 공일근;조성균;조성근
    • Korean Journal of Animal Reproduction
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    • v.23 no.1
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    • pp.85-92
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    • 1999
  • This study was designed to determine effect of cryoprotectant kinds and cell stages on OPP vitrification method in mouse embryos. The freezing speed, cryoprotectants and cell stage could affect of embryo viability following various vitrification methods. The vitrification solution used were consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose solution in holding medium (D-PBS supplemented with 5% FCS: HM) (EFS) or 16.5% ethylene glycol , 16.5% dimethyl sulfoxide, 0.5 M sucrose in HM (EDS). The embryos were collected from oviduct at 18 h after hCG injection and then washed and cultured in mHTF medium until use. In experiment 1, the blastocysts were vitrified by OPP straw to determine the optimal vitrification solution of EFS or EDS. The post-thaw survival rates at re-expanded stage rates were significantly different between EFS and EDS (95.0 vs 100%), but at hatching stage was not different between EFS and EDS (90.0 vs 95.0%). respectively. In experiment 2, zygotes, 2-, 4-cell, morula and blastocysts were vitrified by OPP method to determine the acceptable of early stage embryos. The development rates to expanded blastocyst in zygote (70.0%) were significantly lower rather than those in 2-, 4- 8-cell, compacted morula or blastocyst (89.7, 90.0, 92.8, 97.6 or 97.5%), respectively. However, the cell number of post-thaw developed to expanded blastocyst in blastocyst and control blastocyst stage (39.6$\pm$2.81, 35.7$\pm$2.98) were significanty higher than those in zygote, 2-, 4-, 8-cell, compacted morula (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 or 30.8$\pm$2.93). In experiment 3, the zygotes were exposed in VSl for 1, 2, and 3 min to the optimal exposed time. The cleavage rates (91.6, 88.5, 88.9%) and develop mental rates to blastocyst (83.3, 74.3 and 69.4%) depends on the exposed time in VSl were not significantly different among 1, 2, or 3 min, respectively. The cell number also were not significantly different among exposed time in VS1. respectively. These results indicate that OPP method could be useful for vitrification either EFS or EDS vitrification solution. The post-thaw survival rates at zygote were significantly lower than those at 2-, 4-, 8-cell, morula or blastocyst, respectively. The zygote stage were more sensitive rather than late stage embryos. The exposing time in VS1 for 1 min was better than that for 2 or 3 min, even it was not significantly different. The OPP vitrification method could be useful of mouse embryos either with EFS or EDS vitrification solution.

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