Kor J Fish Aquat Sci 42(6), 한수지, 42(6), , 2009 치어기조피볼락사료내 β-1,3 글루칸의첨가가성장, 혈액및전어체조성에미치는영향 김영철 김강웅 1 박건준 2 이준호 손맹현 1 배승철 부경대학교양식학과 / 사료영

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1 Kor J Fish Aquat Sci 42(6), 한수지, 42(6), , 2009 치어기조피볼락사료내 β-1,3 글루칸의첨가가성장, 혈액및전어체조성에미치는영향 김영철 김강웅 1 박건준 2 이준호 손맹현 1 배승철 부경대학교양식학과 / 사료영양연구소, 1 국립수산과학원양식관리과, 2 ( 주 ) 우성사료 Effects of the Dietary β-1,3 Glucan on Growth, Hematological and Body Composition in Juvenile Korean rockfish (Sebastes schlegeli) Young Chul Kim, Gun Jun Park 1, Jun Ho Lee, Kang Woong Kim 2, Maeng Hyun Son 2 and Sungchul C. Bai Dept. of Aquaculture / FFNRC, Pukyong National University, Busan , Korea 1 Aquaculture Management Division, NFRDI, Pohang , Korea 2 Woosung Co. Ltd, Daejun , Korea This study was conducted to investigate the effects of dietary supplementation of β-1,3 glucan on growth, hematological and body composition in juvenile korean rockfish, Sebastes schlegeli fed the white fish meal based diets for 6 weeks. Four experimental diets were formulated to contain control (without supplementation of glucan), 0.05%, 0.1% and 0.5% (this diet was fed to fish daily, bi-weekly as three different dietary treatments) β-1,3 glucan per kg diet (Control, D 0.05, D 0.1, D 0.5, B 0.05, B 0.1, B 0.5 ). Fish averaging 4.0±0.1g (mean±sd) were randomly distributed in each aquarium as triplicate groups of 23 fish. Weight gain (WG, %) and specific growth rate (SGR, %) of fish fed D 0.5 diet were significantly lower than those of fish fed the other diets (P<0.05). Feed efficiency (FE) of fish fed B 0.05 diet was significantly higher than those of fish fed D 0.1, D 0.5, B 0.1 and B 0.5 diets (P<0.05). Protein efficiency ratio (PER) of fish fed B 0.05 diet was significantly higher than those of fish fed D0.1 and D 0.5 diets (P<0.05). According to the results, the supplementation of 0.05% β-1,3 glucan daily and/or biweekly is optimum in juvenile Korean rockfish. Key words: β-1,3 Glucan, Korean Rockfish, Growth Performance, Blood Parameter, Body Composition 서론 양식어류의질병을예방하기위한면역증강물질 (Immunostimulant) 에대한연구가많은연구자들에의해진행되어지고있으며 (Sakai, 1999), 이와같은면역증강물질은어류의비특이적면역인자를증진시키는화학화합물과박테리아유도물등이있다. 면역증강물질중레바미솔 (Levamisole) 과 FK-565는합성화합물, MDP (Muramyl depetide), LPS (Lipopolysaccharide), 키토산 (Chitosan) 등은박테리아유도체로어류의비특이적면역인자를증강시켜질병에대한저항성을증강시켜준다는보고가있다 (Engstad et al., 1992; Kawakami et al., 1998; Kim et al., 1998; Kitao et al., 1987; Kodama et al., 1994; Sakai, 1999; Siwicki et al., 1990; Solem et al., 1995). 어류의비특이적면역체계는세포성면역인자인 macrophage, granulocyte, nonspecific cytotoxic cell 등이있으며, 체액성면역인자로는 lysozyme, complement, interferon, transferrin, lectin 등이있다. 이러한비특이적면역인자의활성을증가시켜주기위하여인위적으로면역증강제 Corresponding author: scbai@pknu.ac.kr 를공급하게되는데면역증강제를사용하게됨으로써어류에 있어서식세포의활성 (Raa et al., 1992; Jørgensen et al., 1993a; Kim et al., 2006), natural killer cell 활성 (Kajita et al., 1992), 라이소자임 (Engstad et al., 1992; Jørgensen et al., 1993b) 과 보체의대사경로활성화 (Yano et al., 1989) 등을포함한 면역반응을증강시키는것으로보고되어져있다. 또한글루칸 을넙치사료내적정량을첨가하여공급할경우성장을향상 시킨다는보고가있었다 (Kim et al., 2006). 국내조피볼락양식은 1980 년말부터시작하여 2007 년 35,415 톤으로어류양식에중요한비중을차지하고있다 (NSO, 2009). 국내에서완전양식이가능한해산어주요종으 로사육관리가용이하여국내에서각광을받는종으로사료 첨가제로생균제 (Lee et al., 2008) 및구기자 (Lim et al., 2009) 를이용한보고가있다. 하지만아직까지조피볼락에서의글 루칸을이용한양어용사료첨가제로의활용보고가미흡하여 본연구를수행하고자한다. 따라서, 본연구는국내양식주종인조피볼락을대상으로면역증강물질인 β-1,3 글루칸에있어서농도별및투여주기별에따른성장, 혈액및전어체조성에대한미치는영향을평가하고자실시하였다. 609

2 610 김영철 김강웅 박건준 이준호 손맹현 배승철 112 재료및방법 실험어및사육관리실험어류는경남통영에서수송된조피볼락치어를부경대학교부설수산과학연구소로운반하여 2 톤원형수조에서실험환경에적응할수있도록 2 주간예비사육을실시하였으며, 예비사육기간동안상품사료를공급하였다. 2 주간예비사육후, 평균무게 4.0±0.10 g (mean±sd) 의조피볼락치어를 100 L FRP 원형수조에 23 마리씩수용하여 3 ( 투여량 ; 0.05, 0.1 및 0.5) 2 ( 투여시기 ; 매일및격주간 ) 다원변량분석 (factorial design) 으로총 7 가지실험사료를 3 반복으로무작위배치하였다. 사육수는고속모래여과기 ( 역여과방식 ) 에의해여과된해수를사용하였으며, 각실험수조는유수식으로유수량은실험시작시에 2 L/min 되도록하였고, 실험어류가성장함에따라 3 L/min 까지조절하였다. 수온은 20~22 로실험기간동안자연수온에의존하였으며, 실험기간은 6 주간실시하였다. Table 1. Composition and proximate analysis of the experimental diets (% of dry matter basis) Ingredients Con. D 0.05 D 0.1 D 0.5 B0.05 B 0.1 B 0.5 White fish meal Wheat flour Squid liver oil Vitamin premix Mineral premix CMC β-1,3 Glucan (65%) Cellulose Proximate analysis Moisture Crude protein Crude fat Ash Hanchang Co., Busan, Korea. 2 Young Nam Flourmills Co., Busan, Korea. 3 E-Wha Oil Co., Ltd., Busan, Korea. 4 Contains (as mg/kg in diets) : Ascobic acid, 300; dl-calcium pantothenate, 150; Choline bitartrate, 3000; Inositol, 150; Menadione, 6; Niacian, 150; Pyridoxine HCl, 15; Riboflavin, 30; Thiamine mononitrate, 15; dl-α-tocopherol acetate, 201; Retinyl acetate, 6; Biotin, 1.5; Folic acid, 5.4; Vit. B12, Contains (as mg/kg in diet) : Al, 1.2; Ca, 5000; Cl, 100; Cu, 5.1; Co, 9.9; Na, 1280; Mg, 520; P, 5000; K, 4300; Zn, 27; Fe, 40.2; I, 4.6; Se, 0.2; Mn, Carboxymethylcellulose. 실험사료및실험설계본실험에사용된사료의조성은 Table 1 에나타내었다. 실험사료는주단백질원으로북양어분을, 지질원으로오징어 간유를, 탄수화물원으로밀가루를사용하였다. 실험사료내 β-1,3 글루칸의첨가농도는글루칸이첨가하지않은사료구를대조구로하여 0.05%, 0.10% 및 0.50% 의 3 가지농도로첨가하였다. 실험사료는모든원료를혼합한후펠렛제조기로압출 성형하였으며, 입자크기는 Mesh 로고르게친후, 밀봉하여 -20 에냉동보관하면서사용하였다. 실험은글루칸의함량에따라 3 개의실험구로나누고, 각실험구는다시투여시기에따라각각두개의실험구로나누어총 7 가지실험구로실시하였다. 즉, 한실험구는 6 주간연속적으로실험사료를투여하고다른한실험구는글루칸이첨가되지않은대조구사료와실험사료를격주간격으로투여하였다. 일일사료공급량은어체중의 4~5% ( 건물량기준 ) 로 1 일 2 회 (10:00, 16:00) 공급하였다. 어체측정어체측정은 2 주간격으로실시하였으며, 성장률을측정하기위하여 24 시간절식시킨후 MS-222 (100 ppm) 로마취시켜전체무게를측정하였다. 실험종료후, 증중율 (weight gain, WG), 사료효율 (feed efficiency, FE), 단백질전환효율 (protein efficiency ratio, PER), 일간성장률 (specific growth rate, SGR), 비만도 (condition factor, CF) 및생존율 (survival rate) 을조사하였다. 간중량지수 (hepatosomatic index, HSI) 를조사하기위하여각수조별로 5 마리씩무작위로추출하여간의무게를측정하였다. 일반성분분석일반성분은실험사료와각수조별로 5 마리씩무작위로추출하여분쇄한전어체를 AOAC (2000) 방법에따라수분은상압가열건조법 (105, 4 시간 ), 조단백질은 Kjeldahl 질소정량법 (N 6.25), 조회분은직접회화법으로분석하였다. 조지방은샘플을 12 시간동결건조한후, Soxtec system 1046 (Tacator AB, Sweden) 을사용하여 soxhlet 추출법으로분석하였다. 어체의혈액및혈청성분분석혈액및혈청성분분석에있어서실험종료후, 증중율조사와함께혈액성분분석을위하여실험어를채혈하기전까지약 24 시간동안절식시켰다. 실험어를각수조당 3 마리씩무작위로추출하여일회용주사기를이용하여실험어의미부정맥에서혈액을채혈한후, micro-hematocrit 방법 (Brown, 1980) 에의해헤마토크리트 (hematocrit, PCV) 를측정하고, 동시에 Drabkin's 용액을사용하여 cyan-methemoglobin 방법 (Sigma Chemical, St. Louis MO; total hemoglobin procedure No. 525) 으로헤모글로빈 (hemoglobin, Hb) 을측정하였다. 혈청성분의분석을위하여채혈한혈액을항응고제가처리되지않은원심분리관에넣고실온에 30 분간방치한후 3,000rpm 에서 10 분간원심분리하여냉장보관하면서 16 시간이내에분석하였다. 혈청성분은임상용 kit ( 아산제약주식회사, 대한민국 ) 를사용하여총단백질 (total protein) 은 buret 법으로, 글루코스 (glucose) 는효소법으로그리고 glutamic oxaloacetic acid (GOT) 와 glutamic pyruvic acid (GPT) 는 Reitman- Frankel 법으로분석하였다.

3 127 치어기조피볼락사료내 β-1,3 글루칸의첨가가성장, 혈액및전어체조성에미치는영향 611 Table 2. Weight gain (WG), feed efficiency (FE), specific growth rate (SGR), protein efficiency ratio (PER), hepatosomatic index (HSI) and condition factor (CF) of juvenile Korean rockfish fed the experimental diets for 6 weeks Pooled SEM 8 WG(%) b ab b c a b b 6.14 FE(%) ab 91.0 ab 87.4 b 85.8 b 94.2 a 89.3b 88.4 b 1.02 SGR(%) b 3.24 ab 3.14 b 3.07 c 3.27 a 3.19 b 3.14 b 0.03 PER ab 2.27 a 2.21 b 2.19 b 2.34 a 2.24 ab 2.23 ab 0.02 HSI CF Values are means±sd from triplicate groups of fish where the means in each row with a different superscript are significantly different (P<0.05). 2 Percent weight gain : (final wt. - initial wt.) 100 / initial wt. 3 Feed efficiency : (wet wt. gain / dry feed intake) Specific growth rate : (log e final wt. - log e initial wt.) / days. 5 Protein efficiency ratio : wet wt. gain / protein intake. 6 Hepatosomatic index : (liver weight / body weight) Condition factor : {fish wt. (g) / fish length (cm) 3 } Pooled standard error of mean : SD/ n. 통계처리 모든자료는 Computer Program Statistix 3.1 (Analytical Software, St. Paul, MN. USA) 로 ANOVA(Analysis of variance) test 를실시하여최소유의차검정 (Least Significant Difference, LSD) 으로평균간의유의성 (P<0.05) 을검정하였다. 결 6 주간의성장율, 사료효율, 일간성장율, 단백질전화효율, 간중량지수및비만도의실험결과는 Table 2 에나타내었다. 면역증강물질인 β-1,3 글루칸의첨가에따르는증체율과일간성장률에있어서 B 0.05 실험구가대조구와비교하여유의하게높은경향을나타내었으나 (P<0.05), D0.05 실험구와비교하여유의한차이를보이지않았다 (P>0.05). 사료효율과단백질전환효율에있어서 B 0.05 실험구가대조구및 D 0.05 실험구와비교하여유의한차이를보이지않았다 (P>0.05). 간중량지수, 비만도및생존율에있어서는모든실험구에서유의한차이를나타내지않았다 (P>0.05). β-1,3 글루칸의실험사료를섭취한조피볼락치어의혈액및혈청성분변화는 Table 3 에나타내었다. 헤모글로빈은모든실험구에서유의한차이가없었으며 (P>0.05), 헤마토크리트는 D 0.05, B 0.05, B 0.1 및 B 0.5 실험구는대조구와 D 0.5 사료구와 과 Table 3. Serological and hematological characteristics of juvenile Korean rockfish fed seven experimental diets for 6 weeks 1 Pooled SEM 2 Hb(g/dL) PCV(%) c 43.0 a 37.9 ab 36.6 b 39.8 a 42.2 a 41.8 a 3.49 T. P(g/dL) Glucose (mg/dl) 51.9 b 52.3 ab 56.2 a 56.8 a 56.3 a 55.9 a 57.2 a 3.42 GOT (IU/L) a 48.1b 47.6 b 51.5 ab 46.3 b 49.5 b 50.9 ab 2.56 GPT (IU/L) Means of triplicate groups, values in the same row with different superscripts are significantly different (P<0.05). 2 Pooled standard error of mean : SD/ n. 3 Glutamic oxaloacetic transaminase. One unit is defined as the amount of enzyme causing the transamination of 1.0μ mol of L-aspartate per minute at 25 and ph Glutamic pyruvic transaminase. One unit is defined as the amount of enzyme causing the transamination of 1.0 μmol of L-alanine per minute at 25 and ph 7.4. Table 4. Proximate analysis of whole-body of juvenile Korean rockfish fed four experimental diets for 6 weeks (% of dry matter basis) 1 Ingredients Pooled SEM 2 Moisture Crude protein Crude fat Ash Means of triplicate groups, values in the same row with different superscripts are significantly different (P<0.05). 2 Pooled standard error of mean : SD/ n. 비교하여유의하게높은값을보였으나, D 0.1 실험구는대조구와비교하여높은값을보였다 (P<0.05). 혈청내단백질과 GPT 는모든사료구에서유의한차이를보이지않았다 (P>0.05). 반면에혈청내글루코스에있어서 D 0.1, D 0.5, B 0.05, B 0.1 및 B 0.5 실험구는 D 0.05 실험구와비교하여유의한차이를보이지않은 (P>0.05) 반면대조구와비교하여유의하게높은값을보였다 (P<0.05). 혈청내 GOT 에있어서대조구는 D 0.5 및 B 0.5 실험구보다유의한차이를보이지않았으며 (P>0.05), D 0.05, D 0.1, B 0.05 및 B 0.1 실험구와비교하여유의하게높은값을보였다 (P<0.05). β-1,3 글루칸의실험사료를섭취한조피볼락치어의전어

4 612 김영철 김강웅 박건준 이준호 손맹현 배승철 112 체성분변화는 Table 4 에나타내었으며, 모든실험구에있어서유의한차이를보이지않았다. 고 본연구결과조피볼락사료내과다한글루칸의공급은조피볼락의성장을저해되는것으로사료되며, 넙치에서와유사한경향을확인할수있었다 (Kim et al., 2006). Won et al. (1998) 의보고에따르면, 성장기넙치 (60 g) 에있어서 7 주간 β-1,3/1,6- linked 글루칸을매일및격주간격으로투여한실험에있어서는성장면에서매일투여한 0.05% β-1,3 글루칸실험구와연속투여한 0.1% 글루칸실험구가높은성장률을보였으며, 면역반응에있어서도글루칸을투여한사료구에서높은값을나타내었다고보고하였다. 또한, Sung et al. (1994) 은새우 (tiger shrimp) 를 4 가지농도에서글루칸액을 3 일간침지하여 40 일간관찰한결과, 0.5, 1 및 5 mg/ml. 의사료구에서대조구와비교하여빠른성장을보였으나, 낮은농도인 0.25 mg/ml. 의사료구에서는대조구와유의한차이가없었다. 이와같은결과는상기실험과비교하여실험조건, 어종및개체간의크기에따른차이를보일수있을것으로사료된다. 조피볼락에있어서사료첨가제로의생균제 (Lee et al., 2008) 및구기자 (Lim et al., 2009) 를이용할경우성장, 사료효율, 비특이적면역반응및질병저항성이향상된보고하였으나, 본실험의경우비특이적면역반응을확인하지못하여좀미흡한부분이있으나, Kim et al. (2006) 의보고에따르면치어기넙치에서비특이적면역력향상을확인함으로써치어기조피볼락에서면역력향상이된것으로사료된다. 또한본실험에서사료효율및단백질전환효율에있어서는글루칸을첨가하지않은실험구가가장높은값을나타내었는데, 글루칸제재는다이어트건강보조식품으로판매되어조피볼락에서도다이어트효과가나타난것으로사료되어지며, 이부분에대해서는좀더구체적인연구가필요하다. 조피볼락의치어에있어서 β-1,3 글루칸의생리상태에미치는영향을조사에하기위해, 헤모글루빈, 헤마토크리트, 혈청내총단백질량, 글루코스량, GOT, GPT 의변화를생리적지표로하여조사하였다. 헤마토크리트는글루칸을공급한사료구에서높은수치를나타내었으며, 특히성장이가장좋았던글루칸 0.05% 사료구에서가장높은값을보였다. Sim et al. (1995) 은넙치의질병에대한저항력이높아질때, 헤마토크리트가정상어류보다높아질수있다고보고하였으므로, 본실험의결과도글루칸의투여에의해저항력이변화한것과관련이있을것으로생각된다. 글루코스농도는글루칸의농도가높을수록다소높은경향을나타내었으며, 대조구가다른사료구와비교하여가장낮은값을보였다. 혈청내 GOT 의경우대조구가다른실험구들에비하여높은값을나타냄으로써사료내 β-1,3 글루칸을첨가하여공급할경우간기능향상이되는것으로사료되어졌다. 따라서, 상기실험의결과를토대로면역증강물질인 β-1,3 글루칸을치어기조피볼락에첨가할경우 β-1,3 글루칸을 0.05% 매일또는격주간공급하는것이가장적합한것으로 찰 사료된다. 사 본연구는국립수산과학원 ( 고효율배합사료개발및실용화연구, RP-2008-AQ-163) 및부경대학교사료영양연구소의지원에의해운영되었습니다. 사 참고문헌 AOAC, Official Methods of Analysis, 16th Ed., Association of Official Analytical Chemists. Arlington, Virginia, USA. Brown BA Routine hematology procedures. In: Hematology Principles and Procedures. Lea and Febiger, Philadelphia, Engstad RE, Robertson B and Frivold E Yeast glucan induce increase in activity of lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish Shellfish Immunol 2, Jørgensen JB, Lunde H and Robertsen B. 1993a. Peritoneal and head kidney cell response to intraperitoneally injected yeast glucan in Atlantic salmon, Salmo salar L. J Fish Dis 16, Jørgensen JB, Sharp GJE, Secombes CJ and Robertsen B. 1993b. Effect of a yeast-cell wall glucan on the bactericidal activity of rainbow trout macrophages. Fish Shellfish Immunol 3, Kajita Y, Sakai M, Kobayashi M and Kawaushi H Enhancement of non-specific cytotoxic activity of leucocytes in rainbow trout Oncorhynchus mykiss injected with growth hormone. Fish Shellfish Immunol 2, Kawakami H, Hirastsuka M and Dosako S Effects of ironsaturated lactoferrin on iron absorption. Agri Biol Chem 52, Kim KH, Hwang YJ and Bai SC Enhancement of chemiluminescent response of phagocytic cells from junvenile Rockfish, Sebastes schlegeli, by oral administration of levamisole. J Fish Sci Tech 1, Kim YC, Kim KW, Lee S, Park GJ, Okorie OE, Kang YJ and Bai SC Effects of dietary β-1,3 glucan on growth and immune responses in juvenile olive flounder, Paralichthys olivaceus. J Aquaculture 19, Kitao T, Yoshida T, Anderson DP, Dixon OW and Blanch A Immunostimulation of antibodyproducing cells and humoral antibody to fish bacterins by a biological response modifier. J

5 127 치어기조피볼락사료내 β-1,3 글루칸의첨가가성장, 혈액및전어체조성에미치는영향 613 Fish Biol 31, Kodama H, Mukamoto M, Baba T and Mule DM Macrophage-colony stimulating activity in rainbow trout Oncorhynchus mykiss serum. Modators Fish Immune Responses 1, Lee SH, Yoo GY, Choi SM, Kim KW, Kang YJ, Bai SC Effects of dietary probiotics supplementation on juvenile korean rockfish Sebastes schlegeli. J Aquaculture 21, Lim DK, Yoo KY, Shin DG, Kim JE, Bae JY, Bai SC Effects of dietary kugija lycium chinense supplementation on juvenile Korean rockfish Sebastes schlegeli. Kor J Fish Aquat Sci 42, NSO Servey on the status of fish cultureal. Retrieved from on October 15. Raa J, Tørstad G, Engstad R and Robertsen B The use of immunostimulants to increase resistance of aquatic organisms to microbial infections. In: Dis Asian A 1. Sharif M, Subasighe RP and Arthur JR eds. Fish Health Section. Asian Fish Soc Manila Philippines, pp Saki M Current research status of fish immunostimulants. Aquaculture 172, Sim DS, Jung SH and Lee SD Changes of blood parameters of the culture flounder (Paralichthys olivaceus) naturally infected with Staphylococcus epidermidis. Bull Nat Fish Res Dev Age 49, Siwicki AK, Anderson DP and Dixon OW In vitro immunostimulation of rainbow trout Oncohynchus mykiss spleen cells with lavamisole. Dev Comp Immunol 14, Solem ST, Jørgensen JB and Robertsen B Stimulation of respiratory brust and phagocytic activity in Atlantic salmon Salmo salar L. macrophages by lipopolysaccharide. Fish Shellfish Immunol 5, Sung HH, Kou GH and Song YL Vibriosis resistance induced by glucan treatment in tiger shrimp (Penaeus mondon). Fish Pathol 29, Won KM, Kim SM and Park SI The effects of β-1,3/1,6-linked glucan in the diet on immune response of olive flounder, Paralichthys olivaceus by oral administration. J Fish Pathol 17, Yano T, Mangindaan REP and Matsuyama H Enhancement of the resistance of carp Cyprinus carpio to experimental Edwardsiella tarda infection, by some β-1,3 glucans. Nippon Suisan Gakkaishi 55, 년 10 월 15 일접수 2009 년 12 월 1 일수정 2009 년 12 월 12 일수리

한수지 47(6), , 2014 Original Article Kor J Fish Aquat Sci 47(6), ,2014 배합사료및습사료공급에따른넙치 (Paralichthys olivaceus) 의성장및어체성분비교 김강웅 이진혁 배기민 김경덕

한수지 47(6), , 2014 Original Article Kor J Fish Aquat Sci 47(6), ,2014 배합사료및습사료공급에따른넙치 (Paralichthys olivaceus) 의성장및어체성분비교 김강웅 이진혁 배기민 김경덕 한수지 47(6), 810-817, 2014 Original Article Kor J Fish Aquat Sci 47(6),810-817,2014 배합사료및습사료공급에따른넙치 (Paralichthys olivaceus) 의성장및어체성분비교 김강웅 이진혁 배기민 김경덕 이봉주 한현섭 김성삼 * 국립수산과학원사료연구센터 Comparison of Extruded