Development of an Enzyme-linked Immunosorbent Assay Using Vitellin for Vitellogenin Measurement in the Pale Chub, Zacco platypus

Eun-Suk Lim,Eun Hee Lee,Myung Hee Kim,Chang-Hee Han,Sung-Kyu Lee,Jiwon Kim 환경독성보건학회 2013 환경독성보건학회지 Vol.28 No.-

Objectives Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. Methods Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. Results The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was 99.5±5.5%. Conclusions A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.

Development of an Enzyme-linked Immunosorbent Assay Using Vitellin for Vitellogenin Measurement in the Pale Chub, Zacco platypus

Lim, Eun-Suk,Lee, Eun Hee,Kim, Myung Hee,Han, Chang-Hee,Lee, Sung-Kyu,Kim, Jiwon The Korean Society of Environmental Toxicology 2013 환경독성보건학회지 Vol.28 No.-

Objectives Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. Methods Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. Results The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was $99.5{\pm}5.5%$. Conclusions A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.

옵셋배플형 암거식 어도의 수리특성 및 어류이동효과에 관한 연구

박성용,최지웅 윤병만,김서준 한국수자원학회 2008 한국수자원학회논문집 Vol.41 No.1

피라미는 국내 하천에서 서식하며 국지적인 회유를 하는 대표적인 어종이다. 피라미와 같이 작은 몸집의 국지회유성 어종은 서식처간의 이동이 생존을 위해 필수적이다. 그러나 어류 서식처간의 연결부가 배수암거(culvert)로 설치되어 있는 경우에는 평상시 빠른 유속이 발생되어 어류 이동에 많은 장애를 주고 있다. 외국에서는 배수암거의 설계유량과 유속기준이 홍수기간과 어류가 상류로 올라가는 기간별로 필요한 기능에 따라 제시되어 있어 홍수시에는 홍수소통기능, 평 The pale chub (Zacco platypus) is a dominant species that migrates locally and inhabits in rivers in Korea. The fish movement at confluences or hydraulic connections is very important especially for the life of small fish as pale chub. If main stream and

횡성호에 분포하는 피라미(Pale chub: Zacco platypus) 개체군의 Length-weight Relationship 및 Condition Factor

장영수 ( Young Su Jang ),최재석 ( Jae Seok Choi ),이광열 ( Kwang Yeol Lee ),서진원 ( Jin Won Seo ),김범철 ( Bom Chul Kim ) 한국하천호수학회 2007 생태와 환경 Vol.40 No.3

피라미(Zacco platypus)의 난자형성에 관한 연구

장성재,김동희,류동석,등영건,Jang, Seung-Jae,Kim, Dong-Heui,Reu, Dong-Suck,Deung, Young-Kun 한국현미경학회 1995 Applied microscopy Vol.25 No.3

The development of pale chub oocyte from the immature oogonium to mature oocyte was investigated by light and electron microscope. The cytoplasm of pale chub oogonia was acidic and many vesicles were located at inner side of nuclear membrane. In primary oocytes, yolk vesicles were distributed in cytoplasm. Also, fibrous materials and protuberances were distributed on the surface of zona radiata. The nucleus of secondary oocyte was enlarged and yolk vesicles in cytoplasm migrated to zona radiata. In early egg, yolk mass are formed and yolk vesicles were located at inner side of zona radiata. Three-layered zona radiata was about $3{\mu}m$ in thickness. The three layers were an outer fibrous material layer, a middle nurse cell layer in which microvilli of early egg cytoplasm contact with processes of nurse cells, and an inner layer with high electron density. In mature egg, euchromatin and a germinal vesicle were developed, mitochondria, free ribosomes, and yolk mass were distributed in cytoplasm. But, yolk vesicles were disappeared. Specially, zona radiata of matured eggs were better thin than the one of immature eggs In conclusion, it is summerized that the oogenesis of pale chub were the increase of cell size, the formation and accumulation of yolk, the decrease in nucleat electron density, changes of zona radiata, and the development of microvilli.

Effects of Dietary Paprika and Lipid Levels on Growth and Skin Pigmentation of Pale Chub (Zacco platypus)

Lee, Choong-Ryul,Pham, Minh Anh,Lee, Sang-Min Asian Australasian Association of Animal Productio 2010 Animal Bioscience Vol.23 No.6

Two feeding experiments were conducted to investigate the effects of dietary paprika (DP) and lipid (DL) levels on growth performance and skin pigmentation of pale chub, Zacco platypus. Six diets (designated as $P_0L_8,\;P_0L_{17},\;P_8L_8,\;P_8L_{17},\;P_{16}L_8\;and\;P_{16}L_{17}$) were formulated to contain 0%, 8% and 16% paprika with 8% and 17% lipid, respectively. For the growth experiment (Exp I), three replicate groups of fish (average weight 2.6${\pm}$0.2 g) were fed one of the six experimental diets for 8 weeks. At the end of the feeding period, survival was above 94% and not significantly different among dietary treatments. Weight gain, feed efficiency and protein efficiency ratio of fish fed the $P_{16}L_8$ diet were lower than for fish fed the $P_0L_8$ diet. The highest total carotenoid (TC) content was observed in fish fed the $P_{16}L_8$ diet. For the pigmentation experiment (Exp II), each experimental diet was fed to two replicate groups of fish (average weight 9.0${\pm}$0.5 g) for 8 weeks. At the end of the feeding period, TC content of the skin was significantly affected by DP (p<0.05). The highest TC content of the skin was observed after 6 weeks of feeding at all dietary treatments. Astaxanthin content of the skin was not affected by DP and DL (p>0.05). The capxanthin and zeaxanthin contents of skin increased significantly with increasing DP, whereas the opposite trend was observed for lutein and ${\beta}$-cryptoxanthin contents. The skin lightness ($L^*$ values) significantly decreased whereas the values of $a^*$ and $b^*$ were significantly increased in fish fed the diets containing paprika (p<0.05). The present results suggest that feeding a diet containing 8% paprika and 8% lipid for 6 weeks could improve skin pigmentation of pale chub without any adverse effects on growth performance.

피라미(Zacco platypus)의 정자형성과정에 관한 미세구조적 연구

조장현,류동석,Cho, Jang-Hyeon,Reu, Dong-Suck 한국현미경학회 1998 Applied microscopy Vol.28 No.2

The spermatogenesis of pale chub (Zacco platypus) was investigated morphologically. The testis of pale chub contained numerous testicular sacs. These testicular sacs were bounded on neighboring sacs by single layer of squamous cells. Also, differentiated sperms were filled in the sacs. In the stage of spermatogonium, the germ cells had a large nucleus and a distinct nucleolus, and mitochondrial development was prominent. In the primary and secondary spermatogonia, these cells had a round electron-dense nucleus, reduced cytoplasm, and mitochondria were congregated in the side of cytoplasm. The highly condensed chromatin of sperms was electron-dense, the acrosome was not found in the head of the sperm and a motile flagellum consisted of an axoneme with a typical 9+2 pattern of microtubules.

경골어류 잉어과 피라미의 수정란 난막 미세구조

등영건,김동희,류동석,Deung, Young-Kun,Kim, Dong-Heui,Reu, Dong-Suck 한국현미경학회 2000 Applied microscopy Vol.30 No.4

경골어류, 잉어과에 속하는 피라미 수정란을 실험재료로 사용하여 난문의 미세구조, 난막의 표면 및 단면구조를 밝힘으로써 계통분류학적 기초자료를 제시하고자 주사전자현미경과 투과전자현미경을 이용하여 관찰하였다. 피라미의 수정란은 부착성란으로 동물극 쪽에 수정을 위한 정자의 통로로 생각되는 한 개의 난문이 관찰되었고 이 난문은 5개의 돌출물에 의해 둘러싸여 있었다. 난막의 표면은 부속구조물들은 관찰되지 않았고, 수많은 pore canal들이 분포하고 있었으며 난막은 모두 3층으로 부착성인 전자밀도가 낮은 외층, 전자밀도가 높은 중층 및 9층의 층상구조를 이룬 내층으로 구성되어 있었다. 이상과 같은 피라미 수정란 난막의 미세구조적 특징은 피라미의 수정란이 가지는 독특한 형태학적 형질로서 종을 분류하는데 사용될 수 있을 것으로 사료된다. The ultrastructure of the fertilized egg envelope from pale chub , Zacco platypus belong to Cyprinidae was studied using scanning and transmission electron microscopes. The fertilized egg was an adhesive type, have a single micropyle resembling the pathway of sperm in the area of the animal pole. The micropyle was surrounded by 5 peaks of hill. An outer surface of the fertilized egg envelope was arranged by pore canals irregularly. In section of fertilized egg, the egg envelope consistes of three layers, an outer adhesive layer, a middle electron dense layer and an inner lamellae layer consisting of 9 layers. These ultrastructural characters of fertilized egg envelope from pale chub can be utilized in taxonomy of teleost.

하천차수에 따른 금강수계 피라미(Zacco platypus)의 물리적 서식지 평가

허준욱 ( Jun Wook Hur ),박상영 ( Sang Young Park ),강신욱 ( Shin Uk Kang ),김정곤 ( Jeong Kon Kim ) 한국환경생물학회 2009 환경생물 : 환경생물학회지 Vol.27 No.4

2007년 10월부터 2008년 10월까지 하천차수에 따른 금강수계에서 피라미, Zacco platypus를 대상으로 서식지평가를 실시하였다. 하천차수에 따라 28개 지점을 설정하여 모니터링을 실시하여 총 2,362마리를 채집하였다. 3차하천 이상에서 수심 및 유속은 0.8m와 0.8 m s-1에서 대부분 서식하는 것으로 나타났다. 전장 8~10 cm, 유속 0.3 m s-1, 수심 0.4 m, 모래 (0.1~1.0 mm)~굵은자갈(50.0~100.0 mm), 유수역에서 가장 많이 서식하였다. 전장 6.9 cm 이상에서 암수구별이 가능하였으며, 이에 따라 수컷과 암컷에서 전장 (total length, TL)과 체중(body weight, BW)의 상대성장식은 각각 BW=0.0068×TL3.0274(r2=0.9102) 및 BW=0.0075×TL2.9995 (r2=0.8517) (n=1059)로 나타났다. 부화일수(days after parturition, DAP) TL=0.0108DAP+1.5795 (r2=0.9721)로 나타났다. 식에 의해 6.9 cm일 때 무게는 2.7 g (수컷)과 2.4 g (암컷)로 나타나 부화일수로부터 약 490일 성장한 것으로 추정되었다. We assessed the physical habitat conditions of pale chub (Zacco platypus) considering various stream order in the Geum river basin. Field monitoring was conducted for ecological and habitat conditions for 28 sites from October 2007 to October 2008. The number of Z. platypus sampled during this period was 2,362. In more than 3rd stream, water depth and velocity showing the highest number of samples were 0.8 m and 0.8 m s-1, respectively. In addition, the highest number of samples was observed under the condition of total length (TL) (8~10 cm), velocity (0.3m s-1), water depth (0.4 m), substrate size (send, 0.1~1.0 mm~coarse gravel, 50.0~100.0 mm) and habitat type (run). Based on the monitoring data (n=1059) the relationship between body weight (BW) and TL in male and female were estimated as BW=0.0068×TL3.0274 (r2=0.9102) and BW =0.0075×TL2.9995 (r2=0.8517), respectively. Growth equations on days after parturition (DAP) to the TL were estimated as TL=0.0108DAP+1.5795 (r2=0.9721). By equations (TL 6.9 cm), BW were 2.7 g (male) and 2.4 g (female), and DAP was about 490 days.

위천에 서식하는 피라미(Zacco platypus)의 난 발생 및 초기생활사

박재민,한경호,Park, Jae-Min,Han, Kyeong Ho 한국어류학회 2017 韓國魚類學會誌 Vol.29 No.3

The egg and larval development of pale chub, Zacco platypus were determined by observation of laboratory reared larvae from hatching. The experimental adult fish were collected in Wicheon of Nakdong river in June 2016, The eggs and larvae were reared in the experimental tanks, and observed their morphological change. The egg diameter ranged from 1.68 to 1.78 mm ($mean{\pm}SD$, $1.73{\pm}0.07mm$, n =30). The eggs were hatch 70 hours after fertilization. The total length (TL) of the newly hatched larvae ranged from 5.39 to 5.42 mm ($5.40{\pm}0.02mm$, n=10). On the 8 days after hatching, 10 fin rays were formed in caudal fin at 7.89~7.93 mm TL ($7.91{\pm}0.02mm$, n=10). On the 44 days after hatching, the juveniles ranged from 16.1~16.5 mm TL ($16.3{\pm}0.28mm$, n=10). 피라미의 수정란을 부화 사육하여 난 발생 및 자치어 형태발달을 관찰하였다. 실험어는 2016년 6월 낙동강 수계인 위천에서 어미를 포획하여 인공수정 하였다. 난의 크기는 1.68~1.78 mm (평균${\pm}$SD, $1.73{\pm}0.07mm$, n=30)였고, 부화 시간은 70시간이 소요되었다. 부화 직후 자어는 전장 5.39~5.42 mm ($5.40{\pm}0.02mm$, n=10)였고, 부화 후 8일째 후기 자어는 전장 7.89~7.93 mm ($7.91{\pm}0.02mm$, n=10)로 꼬리지느러미에 10개의 줄기가 형성되었으며, 부화 후 44일째 치어는 전장 16.1~16.5 mm ($16.3{\pm}0.28mm$, n=10)로 각 부위별 지느러미 줄기수가 정수에 달하였다.