The effects of fish hydrolysate (CPSP) level on Octopus maya (Voss and Solis) diet: Digestive enzyme activity, blood metabolites, and energy balance

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1 Plese note tht this is n uthor-produed PDF of n rtile epted for pulition following peer review. The definitive pulisher-uthentited version is ville on the pulisher We site Aquulture Deemer 27, Volume 273, Issue 4 : Pges Elsevier B.V. All rights reserved. Arhimer Arhive Institutionnelle de l Ifremer The effets of fish hydrolyste (CPSP) level on Otopus my (Voss nd Solis) diet: Digestive enzyme tivity, lood metolites, nd energy lne Josué Aguil, Gerrd Cuzon, Cristin Psul, Pedro M. Domingues d, Griel Gxiol, Aridn Sánhez, Teresit Mldondo e nd Crlos Ross, * Posgrdo en Cienis del Mr, Fultd de Cienis, UNAM. Puerto de rigo s/n, Sisl, Yután, Mexio IFREMER, Thiti, Frenh Polynesi Universidd Nionl Autónom de Méxio, Fultd de Cienis, Unidd Multidisiplinri de Doeni e Investigión, Administrión de orreos , C.P.9735, Hunumá, Yután, Mexio d CIFAP- Agus del Pino (IFAPA) Aprtdo 14, 2171 Huelv, Spin e Universidd Autónom de Cmpehe-Fultd de Cienis Químio Biológis. Av. Agustín Melgr s/n C.P Cmpehe, Mexio *: Corresponding uthor : Crlos Ross, emil ddress : rv@hp.fienis.unm.mx Astrt: As hs een demonstrted in previous studies, Otopus my n e fed on rtifiil diets. In the present study six different diets were ssyed. Five diets were designed to test the effet of perentge of inlusion of fish protein onentrte (CPSP:, 5, 1, 15, nd 2%) nd were offered to otopuses s speifilly designed rtifiil diet. The sixth diet onsisted of frozen r (Cllinetes spp) nd ws used s ontrol diet. Blood metolites nd energy udget of otopuses were evluted to determine how CPSP levels modulte the digestive pity nd llow retining energy for growth. Wild nimls (316.4 ± 9.8 g) were used in the study. Results showed tht CPSP produed positive speifi growth rte (SGR, % dy 1) with high vlue in otopuses fed 15% CPSP level. A mximum growth rte of.86% dy 1 ws reorded in these nimls, vlue tht is extremely low when ompred with the SGR otined when nimls were fed fresh r (3.7% dy 1). In generl, lood metolites were ffeted y diet omposition, inditing tht some metolites ould reflet the nutritionl nd/or physiologil sttus of otopus. Preliminry referene vlues for O. my fed r were found for gluose (.9 ±.2 mg/ml), ltte (.4 ±.2 mg/ml), holesterol (.16 ±.2 mg/ml), ylglyerol (.14 ±.1 mg/ml), protein (.37 ±.4 mg/ml), hemoynin (1.85 ±.4 mmol/l), nd digestive glnd glyogen (1.86 ±.3 mg/g). Totl energy ontent n e used s n inditor of tissue metoli reserves. In the present study, higher energy ontent in the digestive glnd nd musle ws oserved in otopuses fed r, followed y nimls fed 15% CPSP. Results from the digestive glnd indited tht the retined energy derived from glyogen, suggesting tht lipids nd protein were the min soures of vrition linked with energy ontent. In generl, digestive glnd proteses tivity nd trypsin were indued in otopuses fed 15% CPSP. The pity of O. my juveniles to djust their digestive enzymes to different types of food ws evidened. Essentil mino id ontent (EAA) of the diet ws not limiting ftor. When dietry EAA profiles were ompred with O. my EAA profiles, ll dietry EAA resulted in higher onentrtion thn whole ody otopus omposition. In the present study, ll experimentl groups ingested etween 33 nd 416 kj wk 1 kg 1 without sttistil differenes mong tretments, inditing tht experimentl diets were s ttrtive s r. Differenes were reorded in the proportion of sored energy (A, %) etween CPSP-sed nd r met diets, suggesting digestion limittions ssoited with rtifiil diets. The present results indite tht the 15% CPSP diet hd hrteristis tht stimulte digestive enzymes nd redue energeti osts ssoited with its digestion (HiE or SDA), hnneling more iomss prodution thn the other experimentl diets. Keywords: topus my; Nutrition; Blood metolites; Energy lne 1

2 Introdution Fish protein solule onentrtes (lso lled CPSP 8 ) re otined from the liqueftion of srps of mrine nimls nd re hrterized y higher ontent of peptides nd free mino ids s ompred with the omposition of ntive protein. Tking into ount their omposition, these produts hve een onsidered s omplement for ephlopod quulture diets (Le Bihn et l., 26). A reent study, nlyzing prodution osts of O. vulgris in Glii, Spin (Grí-Grí et l., 24), showed tht the tul tehnology for rering otopuses is low-profit usiness with high risk minly due to food osts. Nturl preys s food nd quisition of wild juveniles re expensive. For tht reson, n dequte nutritionl progrm is neessry to formulte ompounded feed for ommerilly profitle prodution of ephlopods. Reserh on rtifiil diets for ephlopods hs een inresing in importne during the pst dede. Cstro (1991), using rtifiil diets sed on shrimp pste, otined poor survivl nd growth. Cstro et l. (1993) used tfish fillet-sed surimi tht ws epted y uttlefish, lso with poor results. A similr diet ws then enrihed with protein nd other nutrients tht promoted moderte growth (Cstro & Lee, 1994). Finlly, Domingues (1999) nd Domingues et l. (25) used similr diets, supplemented with protein nd mino ids for uttlefish s well s diet with high protein ontent nd dequte mino id profile promoting moderte growth (<.5 %BW d- 1 ). Feeding rtes otined with ny of these diets (<3 %BW d -1 ) were low. Although there re studies relted with the effets of rtifiil diets on ephlopods (Cstro, 1991; Cstro et l., 1993; Cstro nd Lee, 1994; Domingues, 1999; Domingues et l., 25), urrently there is little informtion ville to formulte dequte diets for these orgnisms. Otopus my, s other ephlopods, is rnivorous speies nd protein is its min energy soure (Vn Heukelem, 1977; Vn Heukelem, 1983; Segw nd Hnlon, 1988; Ross et l., 27). In reent study using formulted diets we oserved tht dietry protein ontent modultes proteses tivity. In tht study, O. my juveniles hd the pity to djust their digestive enzymes to different types of food nd protein levels. Enzymes tivities ppered to e well orrelted with otopus growth. Generl proteses nd trypsin from the pnres where well relted with growth rtes. A low tivity ws oserved in otopuses fed 4%CP diet (negtive growth rte), while high tivity ws present in otopuses fed 6%CP diet nd rs. In ontrst, these sme enzymes were indued in the slivry glnds of otopuses fed 4%CP diet tht produed weight loss. Therefore, proesses involved in digestive enzyme prodution in different tissues re relted to the otopus pity to reognize nutritionl hrteristis of the ingested food. The sme trend ws oserved with O. vulgris (Best nd Wells, 1983; Best nd Wells, 1984). An energeti evlution ws lso mde to determine how dietry protein level modultes energy hnnelled to iomss prodution. Finlly, energy intke y otopuses fed 4 nd 6% CP or r met ws etween 1 to 13 kj kg -1.per week Otopuses fed rtifiil diets showed very low digestile energy (DE), inditing tht diets ould ontin ftor, suh s n nti nutritionl one, tht would limit their digestiility. Silges nd protein onentrtes hve een used s soure of short peptides nd mino ids for ephlopods. Le Bihn et l. (26) found tht uttlefish fed Crngon rngon surimi soked in silge (enrihed diet) hd etter growth nd onversion rte thn uttlefish fed non-enrihed diet. Differenes etween diets were the supply of low moleulr weight peptides nd mino ids from the ensilge tht were ingested, digested, nd sored rpidly y growing nimls. Nutritionl sttus is onsidered one of the importnt ftors tht determine the ility of nimls to use the ingested nutrients. In previous studies, we determined some lood metolites nd hemoynin, together with growth nd survivl in the shrimp L. vnnmei, L. stylirostris, nd L. setiferus to ssess the nutritionl role of dietry rohydrtes nd proteins (Ross et l., 2; 2

3 Ross et l., 21; Ross et l., 21; Ross et l., 22). The results demonstrted tht lood gluose, triylglyerides, holesterol, nd ltte together with lood protein, hemoynin (OxyH), nd osmoti pressure were good inditors of nutritionl helth. In ephlopods there is not enough informtion to orrelte lood metolites with growth nd diet nd the wy in whih nutrients re moilized. At present, knowledge on lood hrteristis is sre nd onentrted on few temperte speies. Cephlopods hve losed irultory system in whih the lood is irulted through ontrtile lood vessels nd pillries y the ontrtion of three herts (Wells nd Smith, 1987). The lood onsists of hemoynin-rih plsm, hemolymph, nd lood ells, i.e., hemoytes. The hemolymph of ephlopods, like shrimp, ontins the respirtory pigment hemoynin dissolved in the lood. Hemoynin mounts to out 98% of the totl protein present in otopus lood. The totl lood volume of the Otopus dofleini ws demonstrted to e % of the ody weight (Mlhm et l., 1998). (Rögener et l., 1987) demonstrted tht totl protein nd rohydrtes onentrtion of the hemolymph ws 18 mg ml -1 nd.17 mg ml -1, respetively, in Otopus vulgris.. O. my n e fed on rtifiil diets nd our reserh group first used n rtifiil diet for O. my sed on pellet enrihed with squid pste (Domíngues et l., 27; Ross et l., 27). Suh diet hd 4% CP nd 21% lipids nd overed mintenne energy requirements; lipids were the limiting ftor for otopus growth. In seond study we tested two rtifiil diets with 4 nd 6% CP. Although otopuses fed 6% CP showed positive growth rte, energeti lne results showed tht there ws some dietry omponent tht limited digestion. Bsed on those results nd tking into onsidertion tht low moleulr weight of protein nd mino ids ould enhne digestiility of the diet, 6 different diets were ssyed in the present study. Five diets were designed to test the effet of perentge of inlusion of fish protein onentrte (CPSP 8 :, 5, 1, 15 nd 2%) nd offered to otopuses s n rtifiil diet speifilly designed. Frozen r (Cllinetes spp) ws used s ontrol diet in the experiment. The wy in whih CPSP 8 levels modulte the digestive pity, the umulted energy in tissue, lood metolites, nd energeti udget of otopuses ws evluted. 1. Mteril nd methods 1.1. Animls Speimens of O. my were ught using rtisn lines, with live rs s it, in front of Sisl hror (Yután, Méxio). Otopuses were trnsported from the port to the lortory situted 3 m inlnd, in 12 L irulr tnk with se wter. A totl of 6 otopuses were used for the experiments. In the lortory, otopuses were pled in individul flow-through 8-L tnks with erted se wter. A PVC tue (4 in dimeter) ws pled in eh tnk s refuge. Otopuses were fed different CPSP levels (1 nimls per diet):, 5, 1, 15 nd 2%. Ten other otopuses were fed frozen rs (Cllinetes spp). All the otopuses nd diet omintions were rndomly distriuted into the 6 tnks used. All groups hd similr weights (p>.5) t the strt of the experiment (men vlue of ± 9.8 g). Nturl filtered se wter (5 μm) flowed t 12 L h -1, llowing for omplete renewl in eh tnk every 4 minutes. During the experiment, se wter in the tnks ws mintined t C, 36 psu, dissolved oxygen higher thn 5 mg/l, ph ove 8. Eh tnk ws overed with green solid net, sewn round the top of the tnk to prevent otopuses from esping. The otopuses were kept in the tnks during the 4 experimentl dys Diets preprtion Experimentl diets were prepred y thoroughly mixing ingredients with oil nd then dding wter until semi-humid diet resulted (Tle 1). The rtifiil diet ws prepred on weekly sis nd stored t 6 C. Energy ontent ws mesured in lorimetri pump ) lirted with enzoi 3

4 id. Otopuses were fed twie dy (9 h nd 17 h) etween 7 nd 1% niml dw d -1 for ll regimes. This rtion is onsidered to e n dequte feeding rte for this speies nd ephlopods in generl, t these rering tempertures (Vn Heukelem, 1983; Domingues et l., 25) Amino id profiles Totl mino id omposition ws determined with n mino id uto nlyzer, nd id hydrolysis, using method (AOAC, 2). Tryptophn ws determined using lkline hydrolysis following method (AOAC, 2) Blood metolites Blood mesurements were otined in living fsted (12 h) otopuses t the end of experimenttion. Animls were pled in pre-hilled (15 ºC) nd erted sewter for 15 min to redue the effet of mnipultion efore hemolymph extrtion. Hemolymph (pproximtely 2-3 ml per niml) ws individully smpled through pre-hilled theter inserted t the dorsl rtery fter the otopus hd een dried with pper towel. The individul weight (+.5 g) ws reorded. For oxy hemoynin [OxyH] mesurements, 1 μl hemolymph were immeditely diluted with 99 μl distilled wter in 1-mm uvette, sorne ws mesured t 35 nm. Hemoynin onentrtion ws lulted using n extintion oeffiient of ε = lulted on the sis of the 74 D funtionl suunit (Chen nd Cheng, 1993). Commeril kits were used for ltte (Sigm-t. 735), ylglyerol (TAG; GPO-PAP, Merk, t 14354), nd holesterol (CHOD-PAP, Merk, t ). Determintions were dpted to miro plte using 2 µl of plsm otined t 8 g entrifugtion nd 2 µl of enzyme hromogen regent. Asorne ws reorded in miro-plte reder (BIO-RAD model 55) nd onentrtions were lulted from stndrd sustrte solution. Plsm ws further diluted 1:3 for protein (CP) determintion y the (Brdford, 1976) tehnique dpted to miro plte method using ommeril hromogen regent (Sigm, t. 61) nd ovine serum lumin s stndrd Enzymti tivity Enzymti tivity ws mesured individully in otopuses fed during 4 dys with eh experimentl diet. Slivry glnds (nterior nd posterior), pnres, digestive glnd, nd rop were disseted nd pled t -4 C until nlysis. All the nimls were fsted 12 h efore smpling. Frozen smples were homogenized t 4 C in 5 μl ie-old pyrogen-free wter. Homogentes were entrifuged t 132 rpm for 2 min t 4 C. The superntnt ws diluted in 1 volumes of ieold pyrogen-free wter. Homogentes were immeditely used for enzyme nlysis. The soluleprotein ontent ws mesured in diluted homogentes (Brdford, 1976) using the Bio-Rd protein determintion kit -5-6). Smples were red in Biord model 55 miropltes reder t 495 nm. Assys were mde in duplite for eh smple. Generl proteses tivity ws mesured in homogentes using zooll (Sigm A-4341) s sustrte in phosphte uffer, ph 7. Asorne ws red in spetrophotometer Spetroni model 21D t 52 nm. One unit is defined s the mount of enzyme tht tlyzes the relese of zo dye using ΔA/Δt =.1 min -1. Assys were mde in duplite for eh smple. Trypsin tivity ws mesured in diluted (1;1) homogentes using enzoil- rginine-prnitro-nilide (BAPNAl 1 mm) s sustrte in uffer (.1M TRIS, NCl.5 M, ph 7.5), t 4 C. Asorne ws red t 45 nm. Leuine minopeptidse ws mesured in diluted (1:1) homogentes using 1 mm L-leuine- p-nitronilide (LPNA) s sustrte in ufer (.1M TRIS, ph 8; 25 C). Asorne ws red t 41 nm. 4

5 1.6. Glyogen, totl lipids nd energy ontent of the digestive glnd nd musle After mesuring the totl weight, the musle nd digestive glnd were homogenized t 4 C for 5 min. Glyogen from the digestive glnd nd musle ws extrted in the presene of sulfuri id nd phenol (Duois et l., 1965). Tissues were first homogenized in ie-old trihloroeti id (TCA, 5%) t 3,34 g. After entrifugtion (7 g) the superntnt ws quntified; this proedure ws done twie. One milliliter of superntnt ws pipetted into tue nd mixed with 5 volumes of 95% ethnol. Tues were pled in n oven t 37-4ºC for 3 h. After preipittion, the tues were entrifuged t 7 g for 15 min. The glyogen pellet ws dissolved y dding.5 ml of oiling wter. Then, 5 ml of onentrted sulfuri id nd phenol (5%) were dded nd mixed. Tues ontent ws trnsferred to miro pltes nd red t 49 nm in miro plte reder (Bio-Rd 55). Extrtion of totl lipids followed the (Bligh nd Dyer, 1959) method. After phse equilirtion, the lower hloroform lyer (totl lipids) ws removed, onentrted nd weighed. Totl lipids were expressed s milligrms of lipids per grms of fresh tissue nlyzed. Energy ontent of digestive glnd nd musle ws determined using lorimetri pump ) lirted with enzoi id. Smples of oth tissues were dried t 6 C until onstnt weight Energy udget Food ingestion nd sorption effiieny Every dy, fter 4 hours in the tnks, uneten food ws removed, dried, nd weighed, to determine ingestion nd food onversion. The r given to the otopuses ws lulted on dry weight sis tking into ount tht 1 g fresh r orresponds to 18 g of dry iomss without shell. In this ontext rtifiil diets nd fresh r were offered to otopuses in similr rtions, onsidering the tul dry iomss given s food in ll experimentl diets. Fees were olleted one dy in the tnks, exept erly in the morning. Fees were immeditely dried in the oven to determine wter ontent nd lorimetri vlue. The ingestion rte (IR) ws lulted s the differene etween the delivered nd the remnnt food fter 4 h, nd orreted with the perentge of lehed nutrients. Remnnt food ws siphoned nd filtered through preweighed Wthmn filter numer 3 (5 μm).the lehing perentge of the pelletized diets ws mesured using the shking method (Oldo et l., 22). For this, 1g pelleted feed ws pled in 25 ml flsk pled in horizontl shker t 25 C during 2 h. After tht time, ll the wter ws filtered through Wthmn filter numer 3 (5 μm) to seprte the remining pellet from the lehed wter. The lehed nd originl feed smples were dried in onvetion oven t 15 C for 24 h, nd then ooled in dry environment. Dried feed smples were weighed nd nlyzed for dry mtter retention. Pellet stility ws lulted s the rtio of dry mtter retention fter lehing nd dry mtter of originl smples, expressed s perentge. The sorption effiieny of the diet (AE) ws determined y using fees reovered from otopuses fed eh diet. Fees were dried t 6 ºC until onstnt dry weight; prt of the dry fees ws used to determine lori vlue nd prt to determine sh ontent. The AE ws determined using the eqution: AE = [(DWAF/DWd DWAF / DWf))/(1 DWAF/DWf) DWAF/DWd)]x1, where DWAF/DWd is the rtio etween the sh-free dry weight nd the dry weight of the diet, DWAF/DWf is the rtio etween the sh-free dry weight nd dry weight of fees. The energy sored (kj week 1 kg -1 live) y otopuses ws lulted s: A = AE * I * FE where I is the ingestion rte (g dry weight week -1 kg live -1 ) nd FE is the food s energy ontent expressed s kj/g dry weight. Food nd fees energy ontent were determined with lorimetri pump ) lirted with enzoi id. All fees were pooled ording to diet nd dried t 6 C until nlysis. Fees energy ws otined from 3 smples per diet urned in the lorimetri pump. Totl fees energy ws expressed s kj/g of dry weight fees. 5

6 1.8. Oxygen onsumption nd nitrogen exretion The effet of ll diets on the physiologil ondition ws mesured in 9 nimls from eh diet. Otopuses were deprived of food for 12 h nd pled individully in 8-L respirometri hmers. to mesure oxygen onsumption nd nitrogen exretion. Tnks were seled nd onneted to flowthrough se wter reirulting system (Ross et l., 1993). To prevent hndling stress tht ould ffet redings, otopuses were onditioned in the hmers for 12 h efore ny reding. Oxygen onsumption ws lulted s the differene in onentrtions of the dissolved oxygen etween the input nd output of eh hmer, timing the wter flow. Dissolved oxygen ws mesured using the polrogrphi eletrode of digitl oxygen meter (YSI 5B ±.1 mg L-1). Redings were orreted with respet to the ontrol hmer, without otopuses. Oxygen onsumption ws otined every hour (etween 9:3 to 18:3 h) during 2-dy period. The first dy ws onsidered s fsting ondition nd on the seond dy nimls were fed fter the first (9:3 h) oxygen onsumption mesurement. Animls nd the ontrol hmer were fed with the frozen r or the rtifiil diet, using rtion etween 7 to 1% of wet weight (ww) d -1. At the end of the seond dy otopuses were weighed. Routine metolism (R rout ) ws estimted from the VO 2 (mg g -1 h -1 ) of unfed otopuses onsidering the time during the dy in whih otopuses do not feed. Apprent het inrese (R AHI ; j g -1 h -1 ) ws estimted from the differene etween VO 2 of unfed otopuses nd the mximum vlue ttined fter feeding nd onsidering the time needed for pek oxygen onsumption fter feeding nd the numer of rtions fed to otopuses per dy (n = 2) during growth rte experiments. A onversion ftor of oxygen onsumption of 14.3 J mg -1 ws used to trnsform unfed nd fed VO 2 to J g -1 wet weight (ww) (Lus 1993). The AHI oeffiient ws lulted s R AHI /I x 1. Respirtion (R) ws lulted s R rout + R AHI nd expressed s joules d -1 g -1 ww tking into onsidertion tht otopuses were fed twie dy during growth ssessments. Nitrogen exretion ws mesured oth efore nd fter feeding, t the sme time s oxygen onsumption. Ammoni onentrtion ws nlyzed using the olorimetri indophenol method (APHA, 1995). The U vlues were otined trnsforming mmoni vlues, using the vlue of 2.5 J mg -1 of mmoni exretion (Lus, 1993). The tomi rtio of O:N ws estimted for oth fsting nd feeding nimls, nd reorded vlues of oxygen onsumption nd mmoni exretion were trnsformed to μg At h -1 g dw -1. Feeding O:N ws otined using the mximum oxygen onsumption nd nitrogen exretion during the experimentl period Energy lne Energy udget ws estimted using the following equtions (Lus, 1993) : C = A = U + R + P, where C is the ingested gross energy, A is the energy sored or digestive energy (DE), U the energy lost in nitrogen produts, R indites respirtion (R = R rout + R AHI ), nd P is energy invested in iomss prodution. Assimilted energy (AS) ws lulted s R + P. All vlues were expressed s kj week 1 kg -1. Energy produed (P) ws lulted using the tul growth rte of the otopuses otined during the experimentl time (4 dys). The vlue of kj g -1 dw ws used to trnsform the growth dt into prodution units (P; kj week -1 kg -1 live). This vlue ws otined from nlyzing energy ontent pplied to 25 otopuses y mens of lorimeter ), previously lirted with enzoi id. Assimilted, respirtory nd prodution gross effiienies were lulted s As/ C x 1, R/C x 1, nd P/C x 1, respetively. Respirtory (R) nd prodution net effiienies (PE) were lulted s R/As x 1 nd P/As x 1, respetively Sttistil nlysis One wy ANOVA ws pplied to results of growth rte, survivl, nd energeti lne. Two wy ANOVA ws pplied to results of enzyme tivity, glyogen, totl lipids, nd energy ontent, to 6

7 determine the effets of ll diets on eh nlyzed tissue. Ar sine trnsformtion ws used efore proessing perentge dt (Zr, 1974). 2. Results 2.1. Growth All the diets were well epted y O. my. During the experimentl time 1% of survivl ws oserved with ll tretments. Otopus fed r showed higher growth rte in omprison to tht oserved in otopuses fed rtifiil pstes (P <.5; Fig. 1). Negtive growth rte ws oserved in otopuses fed the diet without CPSP 8. The rest of the tretments showed positive growth rte with mximum vlues in otopuses fed 15% CPSP (Fig. 1; P <.5). No differenes etween sexes were oserved mong tretments. The growth rte of r fed otopuses ws (3.71% per dy), wheres the mximum growth rte of otopuses fed lned food ws (.86 % per dy) (Fig. 1) Blood metolites Blood gluose vried etween.6 to.1 mg ml -1 with the highest onentrtion in otopuses fed 1% CPSP 8 nd the lowest in nimls fed 2% CPSP. Intermedite vlues were registered in otopuses fed, 5, 15% CPSP 8 nd r (Fig. 2). In ontrst, ltte onentrtion ws the highest in otopuses fed % CPSP (.3 mg ml -1 ), intermedite in nimls fed 5 to 2% CPSP 8 ( men vlue of.1 mg ml -1 ) nd the lowest ws reorded in otopuses fed r (.4 mg ml -1 ; Fig. 2). A similr pttern ws oserved for holesterol levels with high levels in otopuses fed % CPSP (.37 mg ml -1 ) nd low in the rest of the diets (men vlue of.14 mg ml -1 ) (Fig. 2). Aylglyerol vried etween.11 nd.21 mg ml -1 with low vlues in otopuses fed.5 nd 2% CPSP nd r nd high in nimls 5% CPSP. An intermedite vlue of.17 mg ml -1 ws reorded in otopuses fed 1% CPSP (Fig. 3d) (P <.5). The highest vlue of lood protein ws registered in otopuses fed 5% CPSP (9.5 mg ml -1 ) followed y nimls fed 1% CPSP (63 mg ml -1 ). Intermedite vlues were registered in otopuses fed 15, 2% CPSP nd r (44 mg ml -1 ). A onentrtion of 21 mg ml -1 ws oserved in otopuses fed ontrol diet. Tht vlue ws lower thn tht registered in the rest of the otopuses (Fig. 3; P <.5). A high vlue of oxy hemoynin ws otined in otopuses fed 1% CPSP (2 mmol l -1 ) nd 15% CPSP (1.95 mmol l -1 l). The rest of the otopuses showed vlues etween 1.65 nd 1.85 mmol l -1 with men vlue of 1.78 mmol l -1 (P>.5; Fig. 3)) Glyogen, totl lipids nd energy ontent Otopuses fed r showed the lowest vlue of digestive glnd glyogen (1.86 mg g -1 ; P <.5; Fig. 4). A men vlue of 3.8 mg g -1 of glyogen ws registered in otopuses fed to 2% CPSP diets (Fig. 4). Digestive glnd nd musle totl lipids were higher in otopuses fed r (79 nd 127 mg g -1 respetively) thn in the rest of the tretments (Fig. 5; P<.5). Among the CPSP 8 diets, the highest vlue of totl digestive glnd lipids were registered in otopuses fed the 15% CPSP 8 diet followed y otopuses fed 5, 1, nd 2% CPSP 8 (p<.5). The lowest vlue of totl digestive glnd lipids ws otined in otopuses fed the ontrol diet (Fig. 5). In ontrst, high vlues of totl lipids in musle were registered in otopuses fed ontrol, 5 nd 1% CPSP 8 -sed diets nd the lowest in nimls fed 15 nd 2% CPSP diets (P <.5; Fig. 5). Digestive glnd energy ontent ws higher in otopuses fed 15% CPSP 8 nd r (men vlue of 22 kj g -1 dw) followed y nimls fed 2% CPSP 8 (2 kj g -1 dw). Intermedite vlues were registered in otopuses fed 5 nd 1% CPSP 8 diets (men vlue 18.6 kj g -1 dw) nd low vlues were otined in nimls fed ontrol diet (14 kj g -1 dw) (P <.5; Fig. 6). Musle energy ontent 7

8 followed similr tendeny s tht oserved in the digestive glnd (Fig. 6). However, in musle, otopuses fed 15% CPSP 8 showed lower energy ontent (18.9 kj g -1 dw) thn tht oserved in nimls fed r (19.7 kj g -1 dw). In musle, low vlues were oserved in nimls fed ontrol nd 1% CPSP diets with men vlue of 18.2 kj g -1 dw Digestive glnd enzymes. Ativity of generl proteses followed urve with mximum vlues in otopuses fed 1 nd 15% CPSP nd lower vlues in nimls fed, 5, 2% CPSP nd r (Fig. 7). Trypsin tivity ws high in nimls fed 15% CPSP wheres leuine mino peptidse ws high in nimls fed, 1, 15 nd 2% CPSP nd low in otopuses fed 5% CPSP nd r (Figs.7 nd ) Amino id omposition. In generl, essentil mino ids (EAA) ontent of experimentl diets nd r ws similr. Only Arg nd Lys were higher in r feed thn in experimentl diets (Tle 2). Essentil mino ids from diets,were higher onentrted thn in O. my. Only Lys ontent ws similr in the ontrol diet nd in O. my (Fig. 8). Non essentil AA (NEAA), Cys, Trp, nd Tyr, were higher in r thn in experimentl diets. The rest of the NEAA were similr etween diets, r nd otopus. Almost ll EAA nd NEAA were less onentrted in O. my thn in the experimentl diets nd r. Totl EAA inresed ording to the proportion of CPSP 8 in the diet with low vlues with % CPSP (42.5 mg 1 g -1 protein) nd high with 2% CPSP 8 (47 mg 1 g -1 protein) (P <.5; Tle 2). This vlue ws similr to those otined in r (48 mg 1 g-1 protein) (P >.5). NEAA inresed ording to the CPSP level in the diet until 1%, fter tht redution in NEAA ws registered with 15 nd 2% CPSP 8 levels (Tle 2). There were no differenes etween NEAA in CPSP 8 diets nd r (P >.5). NEAA of the diets were onentrted higher thn in O. my su dults Oxygen onsumption nd mmoni exretion Oxygen onsumption ws lower in fsting thn in feeding nimls, with vlues etween 49 to 89 J dy -1 g -1 ww, 15 nd nd1% CPSP 8 (Fig. 9). Intermedite vlues were registered in otopuses fed 5 nd 2% CPSP 8 nd r (men vlue of 63 J dy -1 g -1 ww (Fig. 9). Feeding oxygen onsumption ws higher in otopuses fed, 5, nd 1% CPSP (men vlue of 12 J dy -1 g -1 ww) thn in nimls fed 15, 2% CPSP nd r (Fig. 9). Ammoni exretion ws lso lower in fsting otopuses thn in feeding nimls, with vlues tht osillted etween 8.5 joules/dy/g ww (5% CPSP 8 ) nd 12.2 J dy -1 g -1 ww (r diet) (P <.5; Fig. 9). Feeding mmoni exretion ws high in otopuses fed 2% CPSP nd r (24.5 J dy -1 g -1 ww) nd low in nimls fed, 5, nd 1% CPSP diets (14.1 J dy -1 g -1 ww). Fsting nd feeding O:N rtio showed tht otopuses fed ny of the diets hd protein metolism (vlues etween 6 to 14) with higher vlues in nimls fed thn in fsting nimls, s well s eing high in otopuses fed, 5 nd 1% CPSP nd low in nimls fed 15, 2% CPSP 8 nd r (Fig. 1). Apprent het inrement (AHI) ws higher in nimls fed 5% CPSP nd lower in otopuses fed r with intermedite vlues in nimls fed, 1, 15, nd 2% CPSP 8 (Fig. 1) Energy lne Ingestion rte vried etween 3368 to 441 kj week -1 kg -1. There were no sttistil differenes mong tretments, for tht reson men vlue of 362 kj week -1 kg -1 ) ws lulted (P >.5; Tle 3). Type of diet ffeted the sorption effiieny (A E, %) with low vlues in nimls fed nd 1% CPSP 8 (men vlue of 34%), intermedite vlue in otopuses fed 5, 15 nd 2% CPSP 8 (44.7%) nd high vlues in nimls fed r (82%) (Tle 3). In onsequene, the digestile 8

9 energy (DE) ws higher in nimls fed r thn in the rest of tretments. A high mmoni exretion U vlue ws registered in nimls fed 2% CPSP 8 nd r (men vlue of 257 kj week -1 kg -1 ). The rest of the tretments showed men vlue of174.5 kj week -1 kg -1 (P <.5; Tle 3). An inverse reltion etween CPSP 8 % nd R ws oserved with high vlues in nimls fed ontrol diet nd low in otopuses fed r (Tle 3). Three groups of R vlues ould e oserved. The first group ws of nimls fed ontrol diet, 5 nd 1% CPSP (men vlue of 85 kj week -1 kg -1 ). A seond group ws of otopuses fed 15 nd 2% CPSP (men vlue of kj week -1 kg -1 ), nd dt from nimls fed r (65 kj week -1 kg -1 ) (Tle 3; P <.5). The highest prodution vlue (P) ws registered in nimls fed r (1411 kj week -1 kg -1 ). This vlue ws 8- times higher thn the highest vlue registered in otopuses fed 15% CPSP 8 (P <.5; Tle 3). Intermedite vlues were registered in nimls fed 5 nd 1% CPSP 8 with men vlue of 78.5 kj week -1 kg -1 ). A negtive P vlue of kj/week/kg ws lulted for nimls fed ontrol diet. The proportion of energy ssimilted tht ws hnnelled to iomss prodution ws 7% in nimls fed r, whih used the rest of the ssimilted energy in metolism (Tle 3). In ontrst, nimls fed CPSP 8 used etween 8 to 93% of the ssimilted energy in respirtory metolism nd the rest of the ssimilted energy, etween 6 to 2%, ws hnnelled into growth. 3. Disussion In the present study we used different CPSP 8 levels in n ttempt to enhne the digestiility of the rtifiil diet nd to otin growth rte similr to tht oserved when nimls re fed r. Results showed tht CPSP produed positive SGR (% dy -1 ) with high vlue in otopuses fed 15% CPSP level. A mximum growth rte of.74 % dy -1 ws reorded on these nimls; this vlue ws quite low ompred to the SGR otined when nimls were fed fresh r (3.71 % dy -1 ). Ross et l. (27) using rtifiil diets enrihed with squid pste otined negtive growth nd only 6% of ssimiltion effiieny. In tht study, we onluded tht the lipid level in ommeril pellets, nerly 2%, ould e the min reson limiting the digestive feed oeffiient. In more reent study, different pellet ws designed in n ttempt to redue dietry lipid onentrtion; n ssimiltion effiieny ner 5% ws otined with SGR round zero (.8% dy -1 ) when otopuses were fed 6% CP (Aguil et l., 27). In the present study etter growth rte ws oserved (.7 % dy -1 ) higher thn with other rtifiil diets. Although the 15% CPSP diet showed lower SGR thn tht otined in otopuses fed r, the present results suggest tht the use of fish hydrolyste ould e nutritionl soure to mke rtifiil diets fesile. Differenes etween CPSP 8 diets nd r ould e relted with differenes in protein levels (36.8 nd 58% CP, respetively) tht ould limit growth rte of otopus. Diets with 15% CPSP 8 nd high protein level (round 5% CP) should e mde to test this hypothesis. In the present study we re reporting for the first time some lood metolites tht ould e used s nutritionl ondition indies. In this sense, preliminry vlues otined in O. my fed r for gluose ( mg ml -1 ), ltte ( mg ml -1 ), holesterol ( mg ml -1 ), ylglyerol ( mg ml -1 ), protein ( mg ml -1 ), hemoynin ( mmol l - 1 ) nd digestive glnd glyogen ( mg g -1 ) ould e proposed s referenes tking into ount tht r hs een reported s the est diet for growth rte for O. my nd other otopus speies (Grí-Grí nd Agudo-Giménez, 22; Giménez nd Gri, 22; Agudo-Giménez nd Grí-Grí, 23; Miliou et l., 25; Rodríguez et l., 26; Ross et l., 27). In generl lood metolites were ffeted y diet omposition nd/or presenttion, inditing tht some metolites ould reflet the nutritionl nd/or physiologil sttus of otopus. Severl uthors hve oserved tht otopuses hve limittion for rohydrtes metolism (Bouhe- Rodoni, 1973; Bouud-Cmou et l., 1976). Consequently, lood gluose ws lmost onstnt mong tretments inditing tht, independently of the CPSP 8 level, rohydrtes metolism did not hnge. It is interesting to note tht the gluose levels oserved in O. my were lower thn 9

10 those of ultured shrimp Litopeneus vnnmei (.42 mg ml -1 ; (Psul et l., 23) ) nd wild shrimp L. setiferus (.2 mg ml -1 ; (Ross et l., 24). Agin, it is n indition of lower rohydrtes metolism in otopus thn in shrimp. In the present study ltte lood levels were higher in nimls fed ontrol diet thn in otopuses fed CPSP 8 levels or r, inditing tht otopuses fed the ontrol diet ould e under stress onditions. In rustens, ltte is known s stress inditor nd hs een used to evlute the effets of ir exposure in depods (Pterson, 1993; Sntos nd Keller, 1993; Rivonker et l., 1997). In ft ltte n redue CO 2 trnsport induing nrolepsy in r when nimls re stored out of wter (Morris nd Tylor, 1988). In the otopus, the metoli pthwy for ltte synthesis is relted with protein metolism. Ltte n e synthesized from threonine (thr), n essentil mino id minly loted in musle tentles (Akgi nd Ohmori, 24). In this sense, high ltte level in lood ould reflet high thr metolism in musle, inditing tht otopuses re using their essentil mino ids to produe physiologil fuel. Animls fed the ontrol diet used musle protein (presumly thr) s n energy soure, produed higher levels of ltte, nd lost more weight thn nimls on the other tretments. In ephlopods, lipids re stored in the digestive glnd nd then trnsported to the musle to e used s n energy soure (Ros et l., 24). Voogt (1973) reported tht ephlopods re le to synthesize sterols, lthough mollusks, in generl, pprently n only rry out slowly this iosynthesis. For this reson the min lipids soure re from food, even during reprodution (Ros et l., 25). One ingested, lipids re stored in the digestive glnd, used lolly or trnsported through different tissues for energy soure (Ros et l., 25). Ftty ids re stored s ylglyerol in the digestive glnd. But, there is n inverse reltion etween totl lipids from digestive glnd (TLDG) nd lood AG levels in otopuses fed CPSP 8. Animls fed diets low in CPSP 8 levels ould moilize more lipids from the digestive glnd to ompenste for low peptides nd mino id onentrtion. This type of response ould e explined s strtegy of otopus to ompenste for qulity protein nd quntity tht were insuffiient to stisfy nutritionl requirements. Redution in totl musle lipids oserved in reltion with inrements in CPSP 8 levels ould prove tht nimls utilized energy ontent from lipids to ompenste for n pprent low protein level in these diets. In ft, high totl lipids oserved in the digestive glnd nd musle of otopuses fed r evidened tht when protein qulity nd quntity re met, otopuses umulte lipid s metoli reserve. Totl energy ontent n serve s n inditor of tissue metoli reserves. In the present study, higher energy ontent in the digestive glnd nd musle were oserved in otopuses fed r, followed y nimls fed 15% CPSP 8 level. Results on digestive glnd glyogen indite tht this umulted energy ws not originted y glyogen suggesting tht lipids nd protein were the min soure of vrition ssoited with energy ontent. In the digestive glnd, energy ontent followed the sme ehvior s totl lipids, evidening tht the energy ontent of the digestive glnd is more relted with these moleules thn with protein. In ontrst, in musle there is no reltion etween energy ontent nd totl lipids suggesting tht energy ontent in this tissue is minly relted with musle protein onentrtion. These results reinfore the ide tht CPSP 8 -sed diets worked metolilly well, ut did not provide enough protein to stisfy musle tissue uild-up. Digestive glnd totl proteses nd trypsin were indued in otopuses fed 15% CPSP. Thus, O. my juveniles hve the pity to djust their digestive enzymes to different types of food. The digestive dpttion ould involve different retions, whether the food meets nutritionl requirements or not. Digestive enzymes n e indued s response to growth, or in n ttempt to drive more nutrients from defiient mel (Vn Wormhoudt et l., 198). Our results showed tht totl proteses nd trypsin from the digestive glnd ould e indued y CPSP 8. This ompound ould t vi seretgoge role of peptides nd mino ids s shown in fish (Kiy et l., 23). Similr results were oserved in O. my fed different protein levels (Aguil et l., 27). Current results suggest tht enzyme pity of otopuses nd, in onsequene, food digestiility ould e improved using nutrients tht stimulte enzyme seretion. In this sense, it ws found (Best nd Wells, 1983; Best nd Wells, 1984) tht enzyme seretion n e regulted y visul, hemil 1

11 nd endorinologil stimulus. For exmple, 15% CPSP 8 n stimulte trypsin nd totl proteses tivity presumly y enhning food digestion nd, onsequently, improve growth rte. Dietry essentil mino ids ontent (EAA) ws not limiting. When dietry EAA profiles were ompred with O. my s EAA profile, ll dietry EAA hd higher onentrtion thn whole ody omposition. However, in reltion with r, EAA profiles (Arg, Ile nd Lys) were t higher levels thn in CPSP 8 diets. These EAA ould mke differene etween rtifiil diets nd r. Although the role of these EAA in O. my is undefined yet, results otined in O. vulgris prlrve suggest tht y their levels, Lys, Arg, nd Ile ould ply protgonist role in protein metolism of ephlopods (Villnuev et l., 24). Arg is vigorously metolized in ephlopods (Hohhk et l., 1983). During neroi work, Arg-phosphte is hydrolyzed, leding to inresed vilility of Arg for ondenstion with gluose-derived pyruvte to form otopine. Otopine is the min neroi end-produt tht umultes in dult ephlopods during periods of exerise nd stress (Hohhk et l., 1976; Storey nd Storey, 1978). In dult uttlefish S. offiinlis, Argderived ron ppers in CO 2, ure, ornithine, glutmte, itrulline, lnine, otopine, nd proline (Hohhk et l., 1983). On the other hnd, Lys hs een identified s growth promoter in S. offiinlis su-dults (Domingues, 1999) suggesting not only high onentrtion ut relevnt metoli role. In onsequene Lys nd Arg should e tested for their nutritionl requirement. All experimentl groups reeived etween 3.3 nd 4.1 MJ wk -1 kg -1.Sttistil differenes mong tretments did not exist, inditing tht experimentl diets where s ttrtive s r. Differenes where registered in the proportion of energy sored (A, %) etween CPSP 8 diets nd r met, suggesting n inferior digestion oeffiient linked to rtifiil diets. While CPSP 8 -sed diets reveled n A etween 33 to 45%, otopuses fed r sored 82%, inditing tht ll rtifiil diets were limiting in terms of digestion. Previous reserh on O. my fed ommeril diet enrihed with squid (Ross et l., in press) nd high protein rtifiil diet (6% CP) (Aguil et l., in press) showed tht rtifiil diets were prtilly digested. Ross et l. (27) showed tht in ommeril pellets enrihed with 2% squid lipid ontent would e limiting ftor. Thus, low ft nd high protein diet ws designed nd gin low digestiility ws oserved. For this reson, diet with more digestile protein ws designed in n ttempt to improve its digestiility nd enhne otopuses weight gin. In spite of the improved growth rte in omprison to other diets, A % ws still low. There re other dietry spets tht would limit nutrient sorption. Differenes in pprent het inrement (AHI) were oserved mong CPSP 8 tretments with low vlues in nimls fed 15% CPSP 8 nd high in otopuses fed nd 1% CPSP 8, suggesting tht 15% CPSP 8 diet hd higher ost-enefit rtio thn the other diets. AHI mirrors energy invested into mehnil nd iohemil food trnsformtion. Aording to the present results, the 15% CPSP 8 -sed diet improved mehnisms of O. my to e more effiient to hnnel energy to iomss. Although tht diet ws poorly digested, O:N vlues reorded in otopuses fed 15, 2% CPSP 8 nd r indite tht, with suh diets, otopuses utilized more protein s fuel thn nimls fed other diets. Effiieny to derive energy from diet egins when nimls ingest food nd end up when tht energy is deposited s iomss or used s metoli energy. In ephlopods, this proess is minly driven y protein metolism. A 15% CPSP 8 -sed diet hs hrteristis tht stimulte digestive enzymes nd redue energeti osts linked with digestion. It helped iomss prodution more thn the other experimentl diets. An mino ids requirement definition is needed. The use of other protein soures will e tested further in n ttempt to otin lned ompound feed le to sustin otopus iomss prodution. Animls fed r will still serve s referene. Aknowledgments The present study ws prtilly finned y DGAPA-UNAM projet No: IN2166-3, nd SAGARPA-CONACYT Thnks re given to CONACYT for the fellowship to JA nd we ppreite the tehnil ssistne of Drwin Chy nd Juli Tut during the ourse of the experiments. 11

12 Referene List Agudo-Giménez,F., Grí-Grí,B., 23. Growth nd food intke models in Otopus vulgris Cuvier (1797): influene of ody weight, temperture, sex nd diet. Aquulture Interntionl 1, Akgi,S., Ohmori,S., 24. Threonine is the est sustrte for D-ltte formtion in otopus tentle. Amino Aids 26, APHA, Stndrd methods for the exmintion of wter nd wstewter., 19 ed. Amerin Puli Helth Assoition. Best,E.M.H., Wells,M.J., The ontrol of digestion in Otopus. I. The ntiiptory response nd the effets of severing the nerves to the gut. Vie et Millieu 33, Best,E.M.H., Wells,M.J., The ontrl of digestion in Otopus. II. The role of internl stimulus. Vie et Millieu 34, 1-7. Bligh,E.G., Dyer,W.J., A rpid method of totl lipid extrtion nd purifition. Cn. J. Biohem. Physiol. 37, Bouud-Cmou,E., Bouher-Rodoni,R., Mngold,K., Digestive sorption in Otopus vulgris (Cephlopod: Otopod). J. Zool. 179, Bouhe-Rodoni,R., Vitesse de digestion d'otopus yne (Cephlopod: Otopod). Mr. Biol. 18, Brdford,M.M., A refined nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. Anl. Biohem. 72, 248. Cstro,B., Cn Sepi offiinlis L. Be rered on rtifiil food? Mr. Behv. Physiol. 19, Cstro,B., DiMro,F.P., DeRush,R., Lee,P.G., The effets of surimi nd pelleted diets on the lortory survivl, growth nd feeding rte of the uttlefish Sepi offiinlis. J. J. Exp. Mr. Biol. Eol. 17, Cstro,B., Lee,P.G., The effets of semi-purified diets on growth nd ondition of Sepi offiinlis L. (Mollus: Cephlopod). Comp. Biohem. Physiol. 19A, Chen,J.C., Cheng,S.-Y., Hemolymph PCO 2, hemoynin, protein level nd ure exretions of Peneus monodon exposed to mient mmoni. Aquti Toxiology 27, Domingues,P., Development of lterntive diets for the mss ulture of the Europen uttlefish Sepi offiinlis. University of the Algrve, Portugl, pp Domingues,P., DiMro,F.P., Andrde,J.P., Lee,P.G., 25. Effet of rtifiil diets on growth, survivl nd ondition of dult uttlefish, Sepi offiinlis Linneus, Aquulture Interntionl 13, Duois,M.K., Lilles,L.A., Hmilton,J.C., Reers,P.A., Smith,F., Cholorimetri method for determintion of sugrs nd relted sustnes. Anlyt. Chem. 28,

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14 Rögener,W., Renwrntz,L., Uhlenruk,G., Anlysis of Otopus vulgris hemolymph ontining glyoprotein with lood group A-like properties. Comp. Biohem. Physiol. 86, Ros,R., Cost,P.R., Bndrr,N., Nunes,A.J.P., 25. Chnges in Tissue Biohemil Composition nd Energy Reserves Assoited With Sexul Mturtion in the Ommstrephid Squids Illex oindetii nd Todropsis elne. Biol. Bull. 28, Ros,R., Cost,P.R., Nunes,L., 24. Effet of sexul mturtion on the tissue iohemil omposition of Otopus vulgris nd O. defilippi(mollus: Cephlopod). Mr. Biol. 145, Ross,C., Cooper,E.L., Psul,C., Brito,R., Gelert,R., Moreno,T., Mirnd,G., Sánhez,A., 24. Inditors of physiologil nd immunologil sttus of Litopeneus setiferus wild popultions (Cruste, Peneide). Mrine Biology 145, Ross,C., Cuzon,G., Gxiol,G., LePriol,Y., Psul,C., Rossignyol,J., Contrers,F., Sánhez,A., Vn Wormhoudt,A., 21. Metolism nd growth of juveniles of Litopeneus vnnmei: effet of slinity nd dietry rohydrte levels. Journl Experimentl Mrine Biology nd Eology 259, Ross,C., Cuzon,G., Gxiol,G., Aren,L., Lemire,P., Soyez,C., Vn Wormhoudt,A., 2. Influene of dietry rohydrte on the metolism of juvenile Litopeneus stylirostris. Journl Experimentl Mrine Biology nd Eology 249, Ross,C., Cuzon,G., Gxiol,G., Psul,C., Tod,G., Aren,L., VnWormhoudt,A., 22. An energeti nd oneptul model of the physiologil role of dietry rohydrtes nd slinity on Litopeneus vnnmei juveniles. Journl of Experimentl Mrine Biology nd Eology 268, Ross,C., Cuzon,G., Psul,C., Gxiol,G., Chy,D., López,N., Mldondo,T., Domingues,P.M., 27. Energy lne of Otopus my fed r nd rtifiil diet. Mrine Biology In press. Ross,C., Cuzon,G., Tod,G., Psul,C., Gxiol G., Vn Wormhoudt,A., 21. Effet of dietry protein nd energy levels (P/E) on growth, oxygen onsumption, hemolymph nd digestive glnd rohydrtes, nitrogen exretion nd osmoti pressure of Litopeneus vnnmei nd L. setiferus juveniles (Cruste, Depod ; Peneide). Aqu Reserh 32, 1-2. Ross,C., Vnegs,C., Tres,I., Rmirez J., Energy lne of Cllinetes rthune Contrers 193 in floting ges in tropil ostl lgoon. Journl of the World Aquulture Soiety 24, Sntos,E., Keller,R., Effet of exposure to tmospheri ir on lood gluose nd ltte onentrtions in two rusten speies: role of the rusten hyperglyemi hormone (CHH). Comp. Biohem. Physiol. 16A(2), Segw,S., Hnlon,R.T., Oxygen Consumption nd Ammoni Exretion Rtes in Otopus my, Loligo foresi nd Lolligunul revis (Molluss : Cephlopod). Mr. Behv. Physiol. 13, Storey,K.B., Storey,J.M., Energy metolism in the mntle musle of the squid Loligo peleii. J. Comp. Physiol. 123, Vn Heukelem,W.F., Otopus my. Cephlopod Life Cyles. Ademi Press, London., pp

15 Vn Heukelem,W.F., Lortory mintenne, reeding, rering nd iomedil reserh potentil of the Yutn otopus (Otopus my). L. Anim. Si. 27, Vn Wormhoudt,A., Celdi,J.H., Mrtin,B.J., 198. Adpttion de l teneur en enzymes digestives de l heptopnres de Plemon serrtus (Cruste Depod) l omposition dáliments experimentux. Aquulture 21, Villnuev,R., Ri,J., Ruíz-Cpills,C., González,A.V., Bet,M., 24. Amino id omposition of erly stges of ephlopods nd effets of mo id dietry tretmets on Otopus vulgris prlrve. Aquulture 242, Wells,J., Smith,P.J.S., The performne of the otopus irultory system: triumph of engineering over design. Experienti 43, Zr,J.H., Bioesttistil Anlysis. Prentie Hll, Englewood Cliff. 15

16 Figures Speifi growth rte,,% dy -1 4,5 3,5 2,5 1,5,5 -,5 d Cr CPSP level,% Fig.1. Speifi growth rte (% dy -1 ) of O. my fed different CPSP 8 levels or fresh r. Men + S.E. Different letters men differenes (p<.5) mong tretments. 16

17 Gluose, mg/ml A Cr Ltte, mg/ml B Cr CPSP level,% Cholesterol, mg/ml C Cr AG, mg/ml D Cr Fig. 2. Blood gluose, ltte, holesterol nd ylglyerol (AG) of O. my fed different CPSP levels or fresh r. Men + S.E. Different letters men differenes mong tretments t p <.5 level. 17

18 Protein, mg/ml d A Hemoynin, mmol/l B Cr Cr Fig. 3. Blood protein (A) nd hemoynin (B) of O. my fed different CPSP levels or fresh r. Men + S.E. Different letters men differenes etween tretments t p <.5 level. Glyogen DG mg/g Cr Fig. 4. Digestive glnd glyogen of O. my fed different CPSP levels or fresh r. Men + S.E. Different letters men differenes etween tretments t p <.5 level. 18