Journal of Coastal Life Medicine

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1 Journl of Costl Life Medicine 2017; 5(8): Journl of Costl Life Medicine journl homepge: Originl rticle y the Journl of Costl Life Medicine. All rights reserved. Influence of dietry mnnn oligoscchrides supplementtion on fingerling clownfish, Amphiprion ocellris (Cuvier, 1830) Hong Do-Huu *, Trung Kien Nguyen Vietnm Acdemy of Science & Technology (VAST) - Institute of Ocenogrphy, 01 Cu D, Nh Trng, Vietnm ARTICLE INFO Article history: Received 16 Jun 2017 Received in revised form 29 Jun 2017 Accepted 10 Jul 2017 Aville online 11 Aug 2017 Keywords: Amphiprion ocellris Clownfish Mnnn oligoscchrides Growth Body composition ABSTRACT Ojective: To test the effects of dietry mnnn oligoscchrides (MOS) on clownfish Amphiprion ocellris (A. ocellris). Methods: Six levels (0.5, 1.0, 1.5, 2.0, 2.5 nd 3.0 g/kg diet) of MOS were dded to the sl diet (used s control diet) nd fed clownfish A. ocellris [totl length (24.21 ± 0.16) mm] for 70 dys. Growth performnce nd tissue protein were mesured. Results: The result showed tht growth rte ws enhnced significntly in fish fed 0.10% nd 0.15% MOS in diets compred to fish fed other MOS levels or control (P < 0.05). Survivl rte significntly incresed in fish fed diets with 0.20% nd 0.25% MOS, compred to control. Frction rtes of protein growth were significntly higher in fish fed diets with 0.15%, 0.20% nd 0.25% MOS supplementtion thn those fish fed other MOS inclusion levels nd the control (P < 0.04). Dietry MOS concentrtions nd clownfish growth rtes were significntly correlted. Optiml MOS concentrtion ws predicted to e 0.185%, 0.173% nd 0.171% t Dys 14, 42 nd 70, respectively. Conclusions: In conclusion, MOS-supplemented diet enefits for oosting growth, survivl nd ody composition of the clownfish, A. ocellris. 1. Introduction The ornmentl clownfish Amphiprion ocellris (Pomcentride) (A. ocellris) is one of the most populr ornmentl mrine teleost species. They re rtificilly red worldwide to stisfy the demnd for the ornmentl fish s well s to reduce the ctching pressure on the nturl ornmentl fish stock. In Vietnm, this species ws successfully red nd grew in cptivity, however, growth nd survivl rtes re still low. Reserches hve reveled tht ntiiotics my oost growth nd improve feed consumption[1]. In contrst, the use of chemicls nd ntiiotics crries mny risks including toxicity, resistnce, residues nd possile hrm to humn helth[2]. This hs pved the wy in finding new replcements for ntiiotic[3]. Besides vccine, proiotics nd preiotics, such s mnnn oligoscchrides (MOS) hve received high ttention. Preiotics re non-digestile, ut it enefits the host y increse in growth nd metolism of helthy intestinl cteri of the host[3]. Previous reserch showed preiotics cn oost growth of mny quculture species[4-10], oost non-specific immune nd pthogen resistnce[11] nd improve intestinl function nd helth[4,10,12]. MOS is used commonly in fish nd crustcen diet. There re mny studies on effects of MOS on qutic species, however, to the uthors knowledge, MOS requirement for optiml growth of clownfish is still poorly understood. Inclusion dosge of the preiotics plys vitl role ecuse inpproprite dose my dversely ffect the nimls[4,13]. Therefore, the present study imed to define the optiml rnge of MOS inclusion in diets on growth performnce nd protein content of clownfish, A. ocellris. *Corresponding uthor: Hong Do Huu, Vietnm Acdemy of Science & Technology (VAST) - Institute of Ocenogrphy, 01 Cu D, Nh Trng, Vietnm. Tel: E-mil: dohuuhong2002@yhoo.com Foundtion Project: Supported y Institute of Ocenogrphy nd Vietnm Acdemy of Science nd Technology (Grnt No. DTCS-2013). The journl implements doule-lind peer review prcticed y specilly invited interntionl editoril ord memers. 2. Mterils nd methods 2.1. Experimentl fish nd culture systems Clownfish A. ocellris were red nd grew t the htchery t the Institute of Ocenogrphy, Nh Trng, Vietnm. A totl of 560 clownfish from the sme prents with men initil totl length

2 326 Hong Do-Huu nd Trung Kien Nguyen/Journl of Costl Life Medicine 2017; 5(8): [(24.21 ± 0.16) mm] were rndomly stocked in 28 glss tnks (30 cm 50 cm 45 cm). Ech tnk ws equipped with iofilter recircultion system with circulting rte of ~0.6 L/min. During the experiment, wter temperture, ph, mmoni (NH 4 /NH 3 ), nitrite (NO 3 ) nd slinity, were C, , mg/l, mg/ L nd ~35, respectively Experimentl design The experiment tested the growth response nd mortlity of fish fed sl diet dded with grded concentrtions of MOS for 70 dys. A design ws used in which the seven dietry tretments were hphzrdly llocted to 4 replicte tnks, ech with twenty nimls in ech tnk. A totl of 28 tnks nd 560 fingerling clownfish were stocked Experimentl diets nd feeding Six levels (0.5, 1.0, 1.5, 2.0, 2.5 nd 3.0 g/kg diet) of MOS were dded to sl diet (content 39.3% crude protein, 8.71% lipid nd gross energy MJ/kg). Experimentl fish were fed 5% of the verge iomss in ech tnk on the first dy, nd then every dy the mount fed ws djusted, sed on the mount of uneten or remining pellets. Rtions were offered to the fish twice dily (1/2 t 08:00 nd the remining 1/2 t 17:00). Sttus of fish ws oserved ech morning. Uneten feed nd feces were siphoned dily efore morning feed. These procedures were followed for 70 dys Smpling nd dt collection All the fish were weighed nd totl length ws mesured t Dys 0, 14, 42 nd 70. At the end of the experiment, one fish from ech cge (4 nimls in ech tretment) ws rndomly smpled nd scrificed for mesurement of muscle composition. Fish were strved for 24 h prior to mesuring or smpling Chemicl nlysis of culture fish A pooled smple of 10 fish efore the feeding tril ws rndomly collected nd then stored t 20 C for initil ody protein nlysis. Muscles of six fish in ech tretment were smpled for protein nlysis t the end (Dy 70). Crude protein ws mesured following the Kjeldhl stndrd method, with the procedure descried y Do Huu nd Jones[4] Dt clcultion Growth rte [ultiverge weekly length gin (LG, mm/week)], dily growth coefficient (DGC, %/dy) nd survivl were computed y following equtions[5]: LG (mm/week) = (L t L o )/week; DGC (%/dy) = 100 (L t 1/3 -L o 1/3 )/dys; Survivl rte = 100 (N t /N o ), where L t nd L o re totl lengths of clownfish t the end (Dy 70) nd initil length (mm) respectively, N o is the initil numer of fish nd N t is the numer of fish t the end of the experiment. Frction rte of protein growth ws clculted s K s (%/dy) = 100 {[ln(p t ) ln(p o )]}/dys[14], where P t nd P o re protein content of clownfish t time t (Dy 70) nd eginning, respectively Prediction of MOS concentrtion for highest growth response of clownfish The response of fish to dietry MOS concentrtions ws estimted from growth dt. Ech growth dt set t ech time point with respect to concentrtions of dietry MOS ws nlyzed y response models s descried y Do Huu et l.[15]. Dose-response nlysis ws performed using eight nutritionl models including 1 comprtmentl model, two roken lines (liner scending nd qudrtic scending), 1 sturtion kinetics, 2 logistic models (3 nd 4 prmeters) nd the two exponentil nd sigmoidl models. In ech cse, the levels of MOS were independent vrile nd growth ws the dependent vrile. The response prmeter tht gve the most significnt effect ws the men length of clownfish nd this ws selected s the output response for ech of the models. Asolute weight nd LG, DGC were lso considered ut rejected. The model selected for further nlysis of results ws sed on the low sum of squred residuls nd high vlues of R 2. In present study, the comprtmentl model y = e x (1 e c(x d) ) ws the most pproprite nd ws used for nlysis, where y is the growth response of clownfish, is theoreticl mximum, is intercept, c is nutrient rte constnt, nd d is kinetic order of the response when x = 0[16] Sttisticl nlysis Dt re presented s men ± SEM. To compre length nd growth rte of fish mong diet tretments, ANOVA tests nd lest significnt difference were used. Differences were significnt when P < Non-prmetric Kruskl-Wllis test ws used to compre survivl rte. The reltionships etween MOS levels supplemented in the diets nd growth dt were tested y Person correltion (r). All sttisticl nlysis were performed in SPSS 18 (IBM, Chicgo, IL). 3. Results 3.1. Growth performnce of clownfish fed different diets The length of fish t the strt of the experiment ws uniform mong the tretments [(24.21 ± 0.16) mm]. There ws no significnt difference in verge length of fish etween tretments t commencement, or t Dys 14 nd 42 (ANOVA, P 0.621). However, significnt differences of fish length mong diets were detected t the end of the tril (Dy 70). At the end of the experiment (Dy 70), fish fed with MOSsupplemented diets out-performed those fish fed on the sl diet. Men length rnged from mm to mm. The gretest increse in length ws in the group of fish fed 0.15% MOS [(30.53 ± 0.80) mm], followed y fish fed diet 0.10% MOS [(30.16 ± 0.09) mm] with no significnt difference (P = 0.320). The lengths of fish in those two diet tretments were significntly higher thn those fish fed the control diet nd other MOS-supplemented diets (P 0.001). The lowest length increment ws in the group of fish fed the control diet [(28.69 ± 0.50) mm], while the men lengths were mm, mm, mm nd mm in the groups of fish fed diets with 0.05%, 0.20%, 0.25 nd 0.30% MOS, respectively. There ws no significnt difference in the men lengths of those groups (P

3 Hong Do-Huu nd Trung Kien Nguyen/Journl of Costl Life Medicine 2017; 5(8): ) (Figure 1). Men length (mm) dys 42 dys 70 dys Figure 1. Averge DGC (%/dy) (± SEM) of clownfish fed different concentrtions of MOS. 0.20% MOS; D6: 0.25% MOS; D7: 0.30% MOS. Different letters indicte significnt differences etween tretments. At Dy 70, DGC ws highest in the fish fed 0.15% MOS (DGC = 0.34%/dy), followed y the group of fish fed 0.10% MOS in the diet (DGC = 0.32%/dy), ut there ws insignificnt difference (P = 0.24). The DGCs of those two groups were significntly higher thn fish fed the control diet nd those groups of fish fed other concentrtions of MOS (P 0.03). There ws no significnt difference (P 1.27) in DGCs of fish fed 0.05%, 0.20% 0.30% of MOS inclusions or the control diet Survivl rte At the end of the experiment (Dy 70), survivl rtes of fish rnged from 90.00% to 98.33% (Figure 2). The highest survivl rtes were for fish fed diets 0.20% nd 0.25% MOS which were oth 98.33%. The survivl rtes of those two groups of fish were significntly higher thn those fish fed the control nd 0.3% MOS inclusion diets (P 0.04). The lowest survivl rtes were for fish fed control nd 0.3% MOS, which were 90.00% nd 90.95%, respectively, nd with no significnt difference (P = 0.81). Survivl rte of fish fed 0.05% nd 0.25% MOS oth ws 96.67%. 100 Survivl rte (%) 50 0 Figure 2. Survivl rte of clownfish fed different levels of MOS (0% 0.3%). 0.20% MOS; D6: 0.25% MOS; D7: 0.30% MOS. Different letters indicte significnt differences etween tretments Protein content nd K s (%/dy) At Dy 0, protein content in muscle of clownfish ws uniform mong the tretments (12.33% ± 0.01%) without significnt difference (ANOVA, P 0.371). At Dy 70, our results reveled tht protein content in the muscle of clownfish rnged from 13.15% in the group fed the control diet to 16.25% in the group fed diet 0.2% MOS. There ws no significnt difference in protein content of fish fed the different diets (P 0.05). K s of fish fed diets 0.15%, 0.20% nd 0.25% MOS were highest, nd there ws no significnt difference etween them (P = 0.645). The K s of those three groups of fish ws significntly higher thn those fish fed the control diet, diets 0.05% nd 0.10% MOS (P 0.038). The lowest K s were in groups of fish fed control diet, diets supplemented with 0.05% nd 0.10% MOS, with no significnt difference etween them (P = 0.231) (Figure 3). Frction rte of protein growth (%/dy) c Figure 3. Frction rte of protein growth. 0.20% MOS; D6: 0.25% MOS; D7: 0.30% MOS. Different letters indicte significnt differences etween tretments Reltionship etween MOS levels nd growth of fish Person correltion nlysis showed tht dietry MOS concentrtions were significnt positive correltions to growth of clownfish (P 0.017). Among growth performnce dt, MOS levels showed higher correltions with the men totl length of fish (P = 0.001) (Tle 1). Tle 1 Person correltion (r) mtrix mongst dietry MOS inclusion levels (%) nd growth rte of clownfish t Dys 14, 42 nd 70. Time L (mm) W (g) LG (mm/week) DGC (%/dy) Dy 14 r ** * ** ** Significnce Dy 42 r ** * * * Significnce Dy 70 r ** ** ** * Significnce * : Correltion is significnt t the 0.05 level (2-tiled); ** : Correltion is significnt t the 0.01 level (2-tiled). L: Length of fish; W: Weight of fish. c c

4 328 Hong Do-Huu nd Trung Kien Nguyen/Journl of Costl Life Medicine 2017; 5(8): Predicting optiml level of MOS requirement for mximl growth of clownfish When dose response ws nlyzed y comprtmentl model[17], growth t Dy 70 nd MOS-suplemnted levels positively correlted (R 2 = 61.19%). The predicted optiml levels of MOS supplementtion in the diet reduced s the weight of fish incresed. The predicted optiml MOS requirement for mximl growth of clownfish ws 0.185%, 0.173% nd 0.171% t Dys 14, 42 nd 70, respectively (Figure 4, Tle 2). Men length (mm) MOS inclusion in diet (%) 14 dys 42 dys 70 dys Figure 4. Estimtion of optiml MOS concentrtion in the clownfish diet for mximl growth (men totl length) of clownfish, A. ocellris using comprtmentl model. Tle 2 Nutritionl requirement of MOS sed on men length over time for clownfish, A. ocellris fed diet supplemented with MOS. Time Mximl MOS Goodness c d SSE requirement of fit (R 2 ) 14 dys dys dys It is determined y comprtmentl model y = e -x (1 e -c(x-d) ). y is men length (mm), x is concentrtions of MOS in diets (%), is the intercept, is the theoreticl mximum, c is the nutrient rte constnt, nd d is the kinetic order of the response when x = 0. SSE: Sum of squred errors. 4. Discussion This study emphsized the enefit of dietry MOS to growth nd survivl of clownfish, A. ocellris. Growth nd survivl of fingerling fish were improved when fed diets with 0.10% 0.15% MOS inclusion. This ws in greement with other studies tht reported the importnt role of MOS on oosting growth nd survivl of qutic species[4,10,18-20]. In contrst, dietry MOS supplementtion did not improve growth in sturgeon, Acipenser oxyrinchus desotoi[21] or in common crp, Cyprinus crpio[22]. The enefit of MOS inclusion my e species-specific. There is no significnt difference in the protein content of clownfish fed different concentrtion of MOS. Our results re similr to Do Huu nd Jones[4] findings tht protein content in loster fed control nd MOS-supplemented diets did not differ significntly, nd similr ody composition did not chnge in Asin sess (Ltes clcrifer) fed MOS[23]. However, this contrdicts to the study tht showed the protein content in se ss (Dicentrrchus lrx) incresed with the incresed levels of dietry MOS[18]. Although our study showed protein content of clownfish did not differ etween the tretments, K s of fish fed diets contining 0.15%, 0.20% nd 0.25% MOS were highest. K s vlues of those fish were significntly different in comprison to fish fed control nd fish fed 0.05% nd 0.10% MOS-supplemented diet. Therefore, K s might e etter indictor to evlute protein utiliztion in clownfish with respect to dietry MOS supplementtion. Preiotic inclusion in the diet significntly improved protein utiliztion in striped ctfish, Pngsinodon hypophthlmus[19]. Similr inclusion of MOS in the diet enhnces utiliztion of essentil ftty cid in gilthed se rem, Sprus urt[10]. Similrly, the feed conversion rtio nd protein efficiency rtio were significntly higher in Mcrorchium rosenergii fed Agrimos[24]. Determintion of optiml nutrient requirement plys vitl role in quculture. Providing nutrient t levels which re higher or lower thn required my result in dverse effects[4,13]. Most studies on enefits of MOS hve focused on one or two recommended inclusion concentrtion, rther thn exmining the inclusion rtes directly. There re some studies which investigte rnge level of MOS in the diet such s 0% 0.3% in crp (Cyprinus crpio)[25] nd 0% 0.8% MOS in the diet of shrimp Litopeneus vnnmei[26], however, they did not determine n optiml level of MOS in the diet. The only prediction of MOS requirement for mximl growth ws done on loster, Pnulirus homrus[4]. There were eight models of nutrient requirement used in this study in which the model tht hs the highest goodness of fit (R 2 ) ws selected to estimte the optiml level of MOS required for growth. According to Ryn[27], the goodness of fit (R 2 ) is the min element to evlute the coreltions etween the model nd the effect. In the present study, the use of numerous nutritionl models is very similr to previous reserch on crustcens[4,13]. Similr to the finding in this study, oth the reserch on crustcens lso found negtive effect on growth when nutrient supplements were out of the optiml dose. The optiml MOS inclusion diminishes s size increses for Pnulirus homrus loster[4]. As size influences the dietry MOS requirement on other species, further testing on different sizes of clownfish should e conducted experimentlly. The feed tril reported here hs confirmed tht the inclusion of MOS in diets promotes high growth, survivl rtes of clownfish, A. ocellris. The optiml level of dietry MOS for mximl growth of this species ws lso determined, indicting tht MOS t 1.0 to 1.5 g/kg is recommended. Further reserch is needed to determine MOS concentrtion to optimize growth of clownfish exposed to vrious environmentl conditions nd stressors. It is lso recommended tht the effects of dietry MOS on different life stges should e exmined. Conflict of interest sttement We declre tht we hve no conflict of interest. Acknowledgments This project is supported y Institute of Ocenogrphy nd

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