Aquaculture research, tập 42, số 8, 2011

Aquaculture Research, 2011, 42, 1067^1078 doi:10.1111/j.1365-2109.2010.02690.x Feeding high inclusion of whole grain white lupin (Lupinus albus) to rainbow trout (Oncorhynchus mykiss): effects on growth, nutrient digestibility, liver and intestine histology and muscle fatty acid composition Aliro Borquez1, Edison Serrano1,2, Patricio Dantagnan1, Jaime Carrasco3 & Adrian Hernandez1 Escuela de Acuicultura, Universidad Cato¤lica de Temuco,Temuco, Chile Department of Animal and Aquacultural Sciences, Aquaculture Protein Centre, Centre of Excellence, Norwegian University of Life Sciences, Aas, Norway BioMar Chile S.A., Puerto Montt, Chile Correspondence: E Serrano, Escuela de Acuicultura, Universidad Cato¤lica de Temuco, Casilla 15-D,Temuco, Chile E-mail: eserrano@uct.cl Abstract The e¡ect of dietary inclusion of whole grain white lupin (Lupinus albus) on growth performance, histology, muscle fatty acid composition and nutrient digestibility was investigated in an 11-week growth and a 4-week digestibility trial with rainbow trout (initial body weight of 54.0 Æ 6.2 and 181.9 Æ 3.4 g respectively) Four experimental extruded diets were formulated to contain 0%, 30%, 40% and 50% of whole grain lupin and fed to triplicate groups of ¢sh twice a day until apparent satiation Faeces were collected daily from each digestibility tank by decantation No signi¢cant trends were observed with respect to growth, feed utilization, apparent digestibility coe⁄cients or whole-body composition (P40.05) Conversely, increasing levels of dietary lupin led to signi¢cant decreases in the Hepatosomatic index (R2 50.75, Po0.05) and slight lipid in¢ltration into hepatocytes and enterocytes Muscle fatty acid compositions were slightly a¡ected by the dietary treatment Polynomial regression of dietary inclusion of lupin and muscle fatty acid concentrations showed an increase in C18:1n-9, C18:2n-6 and C18:3n-3 and a decrease in C20:5n-3 with increasing dietary lupin level These results demonstrated that whole grain lupin can be included up to 50% in commercial rainbow trout diets without negative e¡ects r 2010 Blackwell Publishing Ltd Keywords: lupin, growth performance, digestibility, fatty acid, histology Introduction Historically, salmonid diets have been formulated to contain ¢sh meal as the most important source of dietary protein, comprising between 20% and 50% of the total ingredients (Watanabe 2002;Tacon & Metian 2008) The current production of ¢sh meal is not su⁄cient to cover increasing demand from the aquaculture sector (Tveteras & Tveteras 2010) This has led to the partial replacement of ¢sh meal with alternative sources of protein, primarily of plant origin such as legumes and oilseeds (Hardy1996; Glencross, Booth & Allan 2007) Lupin seeds have been used successfully as a replacement for ¢sh meal in aquaculture feeds of salmonids and other marine ¢sh (De la Higuera, Garcia-Gallego, Sanz, Cardenete, Suarez & Moyano 1988; Robaina, Izquierdo, Moyano, Socorro, Vergara, Montero & Fernandezpalacios 1995; Burel, Boujard, Corraze, Kaushik, Boeuf, Mol, Van der Geyten & Kuhn 1998; Burel, Boujard, Kaushik, Boeuf,Van der Geyten, Mol, Kuhn, Quinsac, Krouti & Ribaillier 2000; Carter & Hauler 2000; Farhangi & Carter 2001; Aslaksen, 1067 Whole grain white lupin in diets for rainbow trout A Borquez et al Kraugerud, Penn, Svihus, Denstadli, Jorgensen, Hillestad, Krogdahl & Storebakken 2007; Glencross, Hawkins, Evans, Rutherford, Dods, McCa¡erty & Sipsas 2008) Incorporation of between 40% and 50% of lupin seed meal into diets for rainbow trout has been considered as the maximum level of inclusion in terms of growth and nutrient digestibility Inclusion of lupin meal above this threshold can cause a drastic reduction in growth and increase lipid deposition (Burel et al 1998; Farhangi & Carter 2001) Similar to other plant proteins, inclusion of high amounts of lupin seed in salmonids feeds can a¡ect growth, feed intake and utilization of dietary nutrients due to the low content of lysine and methionine (Yanez, Ivanovic, Owen & Ballester 1983; Petterson, Sipsa & Mackintosh 1997), and also the presence of quinolizadine alkaloids and oligosaccharides (Francis, Makkar & Becker 2001; Krogdahl, Penn, Thorsen, Refstie & Bakke 2010) To date, most of the research using lupins as a replacement for ¢sh meal has been carried out with dehulled seed Removal of the lupin seed coat from the seed kernel has compositional and nutritional bene¢ts for experimental aquafeed (Glencross, Hawkins, Veitch, Dods, McCa¡erty & Hauler 2007) However, the practical application of this process under commercial conditions has yet to be demonstrated Whole grain lupin contains high levels of carbohydrates, mainly soluble and nonsoluble nonstarch polysaccharides and ¢bre (Daveby & Aman 1993) These components can cause negative changes in the chemical composition and nutritional value of the feed produced (Glencross, Booth et al 2007) Excessive quantities of carbohydrates and nonstarch polysaccharides in ¢sh diets have been reported to cause glycaemia, reduced digestibility, increased feed intake, decreased growth and faster gastric evacuation (Hilton, Atkinson & Slinger 1983; Francis et al 2001; Hemre, Mommsen & Krogdahl 2002; Glencross, Boujard & Kaushik 2003; Glencross 2009) On the other hand, as a consequence of the high inclusion of plant proteins, the chemical composition of the ¢sh tissues can be a¡ected considerably (De Francesco, Parisi, Medale, Lupi, Kaushik & Poli 2004) The fatty acid compositions of the ¢sh lipids are in£uenced by the fatty acid composition of dietary lipid and de novo synthesis of fatty acid in ¢sh tissues (Watanabe 1982; Sargent, Bell, McEvoy, Tocher & Estevez 1999) Fish oil and ¢sh meal are the main sources of fat in the diet for salmonids; these contain high levels of n-3 polyunsaturated fatty acids (PUFA) (Watanabe 2002) These fatty acids are required by salmonids in 1068 Aquaculture Research, 2011, 42, 1067^1078 order to achieve normal growth and development (Watanabe 1982; Sargent et al 1999) Generally, the utilization of a high inclusion of plant protein in diets for salmonids has resulted in higher levels of oleic acid and linoleic acid, and lower levels of n-3 PUFA, linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in tissues of rainbow trout (Gomes, Corraze & Kaushik 1993; De Francesco et al 2004; Morris, Gallimore, Handley, Hide, Haughton & Black 2005) This can cause changes in the nutritive value of the ¢sh and in£uence ¢sh physiology, as a result of the enzymatic competition among 18-C unsaturated fatty acid desaturation and elongation pathways in the ¢sh liver (Tocher, Bell, MacGlaughlin, McGhee & Dick 2001; Tocher 2003) Compared with other plant protein ingredients, commonly used in salmonid diets, white lupin seeds contain a larger amount of linolenic fatty acid (18:3n3), approximately 9% of the total fatty acids (Yanez et al 1983; Grela & Gunter 1995; Petterson et al 1997) However, around 50% of the total fatty acids of white lupin seed is 18:1n-9 and more than 17% is 18:2n-6 (Yanez et al 1983; Petterson et al 1997) These fatty acids could either cause some physiological metabolic and health problems in the ¢sh or a¡ect the nutritional value of the ¢sh £esh for human consumption, when they are present in commercial ¢sh feed at high concentrations The aim of this study was to evaluate the e¡ect of high inclusion levels of whole grain lupin in rainbow trout extruded diets on growth, nutrient utilization and fatty acids in the muscle Materials and methods Diets Four experimental extruded diets were evaluated: a control diet (L0) and three diets containing 30%, 40% and 50% of whole white lupin grain (Lupinus albus var Hamburg) (L30, L40 and L50 respectively) These diets were formulated to replace 0%, 25%, 33% and 42% of the total crude protein of the diet with lupin protein The diets were formulated using s the software SINGLE-MIX for Windows (FORMAT International, London, UK) and were manufactured by BioMar Chile S.A (Rancagua, Chile) All diets were formulated to be isonitrogenous and isoenergetic on a digestible basis, but consideration was also given to meet the minimum requirements of essential amino r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 Aquaculture Research, 2011, 42, 1067^1078 Whole grain white lupin in diets for rainbow trout A Borquez et al Table Composition of the ingredients used in this study Chemical composition (g kg À DM) LT fish meal Dry matter Crude protein Crude lipid Ash Carbohydratesà Gross energy (MJ kg À DM) Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine 908 680 106 140 74 215.1 39 25 28 49 52 19 27 28 33 Whole grain white lupin cv Hamburg 912 340 95 36 529 208.8 39 17 26 18 14 13 15 Wheat Sunflower defatted meal Feather meal 878 100 20 14 866 180.5 890 370 22 79 529 187.0 32 16 24 13 18 14 19 900 820 70 29 81 235.1 55 10 39 68 22 41 38 59 ÃCalculated as the remainder of crude protein1crude lipid1ash acids, extrudable starch and moisture content Chromium oxide (Cr2O3) was used as an inert marker The compositions of all of the ingredients used in the diet preparations are presented in Table 1, and the formulation and proximate composition of the diets are presented in Table The fatty acid and amino acid pro¢les are shown in Tables and respectively Growth experiment The growth trial was carried out at the experimental ¢sh farm of the Catholic University of Temuco (Los Laureles, Chile) Juvenile rainbow trout (Oncorhynchus mykiss) (initial mean weight of 54.0 Æ 6.2 g) were randomly distributed into twelve 500 L circular ¢breglass tanks (49 ¢sh tank À 1) supplied with freshwater (14.0 Æ 2.7 1C; £ow rate 12 L À 1) Before the start of the growth experiment, the ¢sh were acclimated for 10 days and fed a control diet Subsequently, triplicate groups of ¢sh were fed the experimental diets by hand, to apparent visual satiety twice a day, for 11 weeks At the beginning of the growth experiment,15 ¢sh were randomly sampled in order to determine the fatty acid pro¢le, body composition and histology At the end of the experiment, the ¢sh were fasted for a day, and then weighed individually Two ¢sh from each tank (six per treatment) were randomly taken for whole body composition and stored at À 20 1C for proximate analysis An additional three ¢sh were Table Ingredient and chemical composition of the experimental diets Diets L00 Ingredient composition (g kg À 1) 400.0 LT fish mealà Fish oilà 145.2 Whole grain white lupinw Wheat flourz 150.0 Sunflower defatted mealz 138.1 Feather meal‰ 122.6 Vitamin and mineral premixz 5.0 Monocalcium phosphatek 0.3 Ãà L-Lysine 0.2 Ãà 0.0 DL-methionine Chromium oxideww 10.0 Chemical composition (g kg À DM) Dry matter 918.9 Crude protein 491.3 Crude lipid 258.1 Ash 85.3 Carbohydrateszz 165.3 Gross energy (kJ kg À 1) 24.09 L30 L40 L50 350.0 136.2 300.0 63.5 49.0 85.4 5.0 5.7 0.5 1.4 10.0 300.0 134.0 400.0 63.5 0.00 107.5 5.0 10.5 1.9 2.4 10.0 250.0 131.6 500.0 63.4 0.00 102.5 5.0 14.3 3.1 3.1 10.0 927.7 527.7 254.1 77.6 140.7 24.49 919.0 527.0 243.7 72.4 156.9 24.30 911.2 484.4 223.0 71.0 221.6 23.05 ÃExapesca S.A (Talcahuano, Chile) wSel Chile S.A (Temuco, Chile) zGraneles Chile S.A (Santiago, Chile) ‰Terramar Chile S.A (Santiago, Chile) zBioMar Chile S.A (Puerto Montt, Chile) kAquafarma S A (Santiago, Chile) ÃÃEvonik Degussa wwSigma-Aldrich (St Loius, MO, USA) zzCalculated as the remainder of crude protein1crude lipid1ash r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 1069 Whole grain white lupin in diets for rainbow trout A Borquez et al Table Fatty acid compositions of the experimental diets containing increasing levels of whole grain lupin (as g100 g À of the total fatty acids) Aquaculture Research, 2011, 42, 1067^1078 Table Essential amino acid (EAA) requirement of rainbow trout (Oncorhynchus mykiss) compared with the amino acid pro¢le of the experimental diets (expressed as g100 g À diet) Diets L00 L30 Total lipid (g kg À 1) 237.2 235.7 Fatty acids (g 100 g À of the total fatty acids) C14:0 5.19 5.87 C15:0 0.76 0.81 C16:0 19.26 19.22 C17:0 1.26 1.15 C18:0 5.19 2.68 C16:1 4.70 5.10 C18:1n-9 22.71 25.02 C20:1 8.19 8.34 C22:1n-9 0.00 1.17 C18:2n-6 4.70 5.89 C18:3n-3 0.71 0.81 C20:4n-6 0.97 0.51 C20:5n-3 9.87 9.52 C22:6n-3 11.03 10.01 Sum SAFAà 34.43 31.29 Sum MUFAà 37.08 40.71 Sum n-3 PUFAà 21.75 20.46 Sum n-6 PUFAà 3.37 2.84 n-3/n-6 6.5 7.2 L40 L50 224.0 203.2 5.82 0.78 18.64 0.90 0.49 4.81 29.27 7.91 0.92 6.08 1.30 0.10 8.68 9.25 29.37 43.93 19.34 2.74 7.1 5.43 0.73 17.46 0.82 0.46 4.66 32.92 7.87 0.85 5.84 2.23 0.17 8.05 8.25 26.81 47.37 18.64 2.73 6.8 Amino acids ÃIncludes unlisted fatty acids: SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids removed from each tank (nine per treatment) and samples of dorsal muscle were dissected, skinned, deboned, homogenized and stored at À 20 1C for fatty acid analysis These same ¢sh were later sampled for liver weight determination, and both liver and intestine samples were collected for histological examination Fish were killed by a blow to the head after an overdose of benzocaine (BZ-20,Veterquimica Laboratories, Santiago, Chile) Digestibility experiment The digestibility trial was carried out at the school of aquaculture of the Catholic University of Temuco (Temuco, Chile) Apparent digestibility coe⁄cients (ADC) were determined using the modi¢ed Guelph method, using Cr2O3 as an inert indicator (Cho, Cowey & Watanabe 1985) Thirty Juvenile rainbow trout (O mykiss) (initial mean weight of 181.9 Æ 3.4 g) were randomly allocated into twelve 500 L cylindroconical ¢breglass tanks equipped with faecal settling columns connected to the outlet of each tank and supplied with well water (14.0 Æ 1C; £ow rate 1070 Methionine Lysine Threonine Arginine Isoleucine Leucine Valine Histidine Phenylalanine Cysteine Glycine Serine Alanine Aspartic acid Glutamic acid Trout EAA requirementà 1.40w 1.80 0.80 2.00 0.80 1.40 1.30 0.70 1.80z Diets L00 L30 L40 L50 0.97 2.77 1.88 2.94 1.97 3.38 2.51 1.28 2.04 0.58 3.06 2.62 2.56 3.85 6.30 1.04 2.49 1.92 2.77 2.01 4.47 2.46 1.26 2.29 0.65 2.76 2.72 2.98 3.86 7.34 0.99 2.37 1.93 2.92 2.08 4.72 2.49 1.24 2.35 0.67 2.69 2.79 3.01 3.94 7.66 0.72 2.20 1.71 3.18 1.85 3.63 2.15 1.09 1.96 0.58 2.43 2.55 2.30 3.75 6.83 ÃHardy (2002) wMethionine1cysteine zPhenylalanine1tyrosine 12 L À 1) Fish were fed the experimental diets in triplicate by hand, to apparent visual satiety twice a day, for weeks After an adaptation period to the dietary treatments of week, faeces were collected daily in each tank from settling columns, centrifuged at 1235 g for 15 and frozen at À 20 1C until analysis Calculations Growth was assessed using the thermal growth coef¢cient (TGC) and weight gain (G) Both variables were determined using the following equation: TGC [(W1/3 À W1/3 f i )]/S[T  D]  100, and G (Wf À Wi), whereWi and Wf are the initial and the ¢nal weights (tank means), respectively, D represents the number of feeding days and T corresponds to the average water temperature Feed conversion ratio (FCR) was calculated as: FCR F  G À 1, and protein e⁄ciency ratio (PER) was determined as: PER G  PIÀ 1, where F is consumption of dry matter from feed, G is the weight gain and PI is the protein intake The Hepatosomatic index (HSI) was calculated as: HSI 5100  (LW  FW À 1), where LWand FW represent wet liver weight and wet body weight respectively r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 Aquaculture Research, 2011, 42, 1067^1078 Whole grain white lupin in diets for rainbow trout A Borquez et al Apparent digestibility coe⁄cient was determined using the indirect method, as described by Cho et al (1985), dry matter ADC (%) were calculated as 100  [1 À (%Ifeed/%Ifaeces) and ADC of the nutrients as (%) 100 À [100 (%Ifeed/%Ifaeces)  (%Nfeed/ %Nfaeces)], where I is the inert marker and N the nutrient The digestible energy (DE) was calculated according to De la Higuera et al (1988) as DE (Fprotein  ADCprotein  Eprotein)1(Flipid  ADClipid  E lipid)1(Fnitrogen-free extract  ADCnitrogenfree extract  E nitrogen-free extract), where F is the nutrient content of the feed (g), ADC is the apparent digestibility of the nutrient and E is the theoretical energy content (kJ g À 1) Chemical analyses Proximate compositions (crude protein, crude lipid, ash and moisture) of diets and carcass were determined according to the methods of AOAC (1998) Carbohydrates were calculated by di¡erence Gross energy was estimated using the following coe⁄cients: 23.4 kJ g À for crude protein, 39.8 kJ g À for crude lipid and 17.2 kJ g À for carbohydrates (Cho, Slinger & Bayley 1982) The extraction of total lipids from diets and muscle tissues was carried out according to the method of Folch, Lees and Sloane-Stanley (1957) A sample of 1g was homogenized in a chloroform/methanol solution (2:1; v/v) and 0.01% butyl hydroxytoluene Fatty acids methyl esters (FAME) were prepared by acid catalysis transmethylation of total lipids (Morrison & Smith 1964) and analysed by gas chromatography (Hewlett Packard 6890 series II Plus, Wilmington, NC, USA) using a FID detector and a fused silica capillary column (SP-2398, 30 m, 0.25 mm i.d., 0.20 mm ¢lm thickness) Helium was used as a carrier gas Fatty acids were identi¢ed by comparison with fatty acid standards (Supelco 37 component FAME mix, Supelco, Bellefonte, PA, USA) and expressed as the percentage of total fatty acids identi¢ed The total amino acid contents of the experimental diets were determined using near-infrared re£ectance spectroscopy by Evonik Degussa (Hanau, Germany) Samples were ground to a 300 mm particle size before analyses Histological analysis Liver and intestine samples were ¢xed in Bouin solution for 24 h, and stored in 70% ethanol at 1C The tissues were subsequently dehydrated according to standard histological techniques in a graded ethanol series, and embedded in para⁄n Sections (4^6 mm) were cut and stained with haematoxylin and eosin and then blindly examined under a light microscope (Leica Microsystems model DM750, Leica, Bannockburn, IL, USA) Statistical analyses Second-order polynomial regression models were used to describe the e¡ects of whole lupin grain dietary inclusion on di¡erent parameters studied The coe⁄cient of determination (R2) was used to evaluate the total variance explained by the model The significance level was set to Po0.05 All statistical analyses were conducted using SAS for Windows version 8.01 (SAS Institute, Cary, NC, USA), and data are presented as mean Æ standard error of mean Results Growth performance and feed utilization During the growth trial, all diets were well accepted by ¢sh and the survival rate was 98% The results of growth performance and feed utilization are presented in Table The mean treatment ¢nal body weights varied from 173.6 (L50) to 183.2 g (L40); ¢sh fed the diet containing 500 g kg À of whole lupin grain (L50) achieved the lowest weight gain (119.63 g) and the highest FCR (1.38), although there Table Growth performance, protein e⁄ciency ratio and Hepatosomatic index of rainbow trout (Oncorhynchus mykiss) fed increasing dietary whole grain lupin levelsà Growth performance Diets L00 L30 L40 L50 SEM Initial weight (g) 54.0 54.0 54.0 54.0 Final weight (g) 178.0 181.9 183.2 173.6 5.53 Gain (g) 124.0 127.9 129.3 119.6 5.52 Feed intake 169.3 172.8 180.0 163.2 11.58 (g fish À 1) TGC 0.13 0.13 0.13 0.13 0.00 FCR 1.28 1.26 1.32 1.38 0.09 PER 1.60 1.64 1.57 1.57 0.13 HIS 1.27 1.45 1.34 1.19 0.06 Pmodel R2 0.1689 0.33 0.1690 0.33 0.4604 0.16 0.1545 0.2512 0.8312 0.0018 0.34 0.26 0.04 0.75 ÃEach value is the mean of three replicates TGC, thermal growth coe⁄cient; FCR, food conversion ratio; PER, protein e⁄ciency ratio; HSI, Hepatosomatic index r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 1071 Whole grain white lupin in diets for rainbow trout A Borquez et al were no signi¢cant correlations with the dietary inclusion of whole lupin grain Correspondingly, no signi¢cant correlations were observed between the increase in the level of whole lupin grain in the feeds and feed intake, TGC and PER (R2 50.16, 0.34 and 0.04 respectively) Whole body composition and HSI The whole body composition and HSI are shown in Table In general, the inclusion level of lupin in the diet did not change the chemical composition of the ¢sh Hepatosomatic index, however, showed a significant decreasing trend (R2 50.75, Po0.05) The HSI values ranged from 1.19 (L50) to 1.45 (L30) Histology examination The cellular morphology of enterocytes showed a displacement of the nucleus in the direction of the distal cell pole Simultaneously, a reduction in the number of basophils and abundant number of lipid drops were also observed in the ¢sh fed the diet containing 40% whole grain lupin meal (L40) (Fig 1) Hepatocytes, on the other hand, showed a slight lipid in¢ltration in the ¢sh fed the control and L40 Surprisingly, the above e¡ect was observed to a lesser degree in the treatment L50 (Fig 2) Digestibility coe⁄cients The ADC are shown in Table The ADC values for dry matter, crude protein, lipid and carbohydrates showed slight variations among dietary treatments, but signi¢cant e¡ects of increasing dietary whole lupin grain were not observed Aquaculture Research, 2011, 42, 1067^1078 Protein digestibility ranged from 92.42% (L50) to 94.83% (L40) and lipid digestibility ranged from 98.62% (L50) to 99.06% (L30), whereas carbohydrate digestibility was high, considering the high incorporation of plant ingredient into the diets, and £uctuated from 66.6% (L50) to 75.9% (L40) Phosphorus digestibility was also not a¡ected by the dietary inclusion of whole lupin grain, varying from 62.3% (L30) to 75.2 (L40) Muscle fatty acid composition The fatty acid pro¢les in muscle of rainbow trout before starting the experiment and after feeding the experimental diets are shown in Table The incorporation of high levels of whole grain lupin into diets did not lead to signi¢cant changes in the total content of saturated fatty acids (SAFA) (R2 50.43, P 0.0788) Among the SAFA, the most abundant fatty acid was the palmitic acid (C16:0), which showed a slight decrease in the diets containing lupin; however, this trend was not signi¢cant (R2 50.38, P 0.1149) Total mono-unsaturated fatty acids (MUFA) showed an increase in the muscle as the dietary lupin inclusion was increased The change in the muscle content of MUFA was due to a signi¢cant increase in the oleic fatty acid observed in the ¢sh fed graded lupin diets (R2 50.69, P 0.0054) The total concentration of n-3 PUFA showed a slight decrease as the dietary lupin inclusion was increased; nevertheless, this tendency was not signi¢cant (R2 50.41, P 0.0906) There was a strong positive quadratic relationship between dietary lupin inclusion and the linolenic acid (C18:3n-3) in the muscle (R2 50.97, Po0.0001) On the contrary, a signi¢cant negative quadratic relationship was observed (R2 50.82, P 0.0004) between the dietary Table Whole body composition in rainbow trout (Oncorhynchus mykiss) fed diets (g kg À wet weight) with increasing dietary whole grain lupin levels, at the onset and after 120 days of feedingà Diets Initialw Dry matter Crude protein Crude lipid Ash 222.07 185.62 16.98 16.16 Æ Æ Æ Æ 0.15 1.14 0.28 0.44 L00 L30 L40 L50 SEM Pmodel R2 258.10 196.62 46.92 13.55 259.47 195.11 49.52 13.65 260.83 194.34 50.33 13.32 256.10 191.85 46.14 13.70 0.44 0.45 0.60 0.04 0.5576 0.4544 0.6812 0.9301 0.12 0.16 0.08 0.02 ÃEach value is the mean of three replicates (three ¢sh per replicate) wInitial values are expressed as means Æ SEM 1072 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 Aquaculture Research, 2011, 42, 1067^1078 Whole grain white lupin in diets for rainbow trout A Borquez et al Figure Cross section of middle intestine of rainbow trout fed control diet (a) 30% whole grain lupin meal (b) 40% whole grain lupin meal (c) and 50% whole grain lupin meal (d) (H&E x10) Arrows show an increasing lipid accumulation in the absorptive vacuoles of ¢sh fed the L50 diet Figure Hepatocytes of rainbow trout fed control diet (a) 30% whole grain lupin meal (b) 40% whole grain lupin meal (c) and 50% whole grain lupin meal (d) (H&E x40) Notable is the higher incidence of lipid vacuolization (indicated by black arrow) in the hepatocytes of ¢sh fed the L40 diet r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 1073 Whole grain white lupin in diets for rainbow trout A Borquez et al Aquaculture Research, 2011, 42, 1067^1078 Table Nutrient and energy digestibility (%) in rainbow trout (Oncorhynchus mykiss) fed increasing dietary whole grain lupin levelsà Diets Dry matter Crude protein Crude lipid Phosphorus Carbohydratesw DE (MJ kg À diet) L00 L30 L40 L50 SEM Pmodel R2 86.24 93.09 98.51 66.31 69.70 22.42 86.73 93.58 99.06 62.33 68.39 22.94 88.97 94.83 98.64 75.19 75.90 22.96 82.85 92.42 98.62 72.69 66.60 21.10 2.12 1.28 0.67 4.52 4.77 0.40 0.0757 0.3391 0.6729 0.6561 0.0746 0.0007 0.44 0.21 0.08 0.09 0.44 0.80 ÃEach value is the mean of three replicates wCalculated as the remainder of crude protein1crude lipid1ash DE, digestible energy Table Fatty acid composition in muscle of rainbow trout (Oncorhynchus mykiss) fed diets with increasing dietary whole grain lupin levels, at the onset and after 120 days of feeding (as g100 g À of the total fatty acids)à Diets Initialw Total lipid (g kg À 1) 16.8 Æ 1.3 Fatty acids (g 100 g À of the total fatty acids) C14:0 3.18 Æ 0.05 C15:0 0.57 Æ 0.01 C16:0 25.76 Æ 1.74 C17:0 0.83 Æ 0.03 C18:0 4.82 Æ 0.01 C16:1 4.39 Æ 0.28 C18:1n-9 21.39 Æ 0.91 C20:1 0.00 Æ 0.00 C22:1n-9 0.19 Æ 0.00 C18:2n-6 7.88 Æ 0.37 C18:3n-3 1.09 Æ 0.15 C20:4n-6 0.23 Æ 0.02 C20:5n-3 4.76 Æ 0.18 C22:6n-3 20.81 Æ 0.51 Sum SAFAz 37.40 Æ 1.04 Sum MUFAz 26.66 Æ 1.36 Sum n-3 PUFAz 26.83 Æ 0.58 Sum n-6 PUFAz 9.10 Æ 0.25 n-3/n-6 2.95 Æ 0.15 L00 L30 L40 L50 SEM Pmodel R2 42.7 35.2 37.9 33.9 0.76 0.4273 0.17 3.76 0.72 21.96 0.74 4.43 6.02 27.00 4.98 0.76 4.81 0.67 0.10 6.19 13.74 33.10 40.64 20.87 5.11 4.08 5.82 0.80 25.53 0.97 4.79 5.35 24.72 3.86 0.75 5.71 0.66 0.27 5.16 11.94 39.21 36.35 18.02 6.13 2.95 5.27 0.73 22.99 0.80 4.43 5.70 28.05 4.17 0.72 5.87 0.95 0.09 4.79 11.13 35.82 40.34 17.11 6.30 2.73 5.23 0.75 21.99 0.79 4.02 5.46 29.65 3.84 0.57 5.98 1.56 0.49 4.60 11.19 34.16 41.29 17.60 6.65 2.69 1.50 0.07 1.99 0.18 0.24 0.36 1.32 0.38 0.12 0.28 0.08 0.22 0.34 1.76 2.90 1.84 1.98 0.44 0.43 0.2984 0.7241 0.1149 0.3338 0.0145 0.1565 0.0054 0.0114 0.0798 0.0009 o0.0001 0.2917 0.0004 0.1744 0.0788 0.0329 0.0906 0.0055 0.0041 0.24 0.07 0.38 0.22 0.61 0.34 0.69 0.63 0.43 0.79 0.97 0.24 0.82 0.32 0.43 0.53 0.41 0.69 0.71 ÃEach value is the mean of three replicates (three ¢sh per replicate) wInitial values are expressed as means Æ SEM zIncludes unlisted fatty acids: SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids inclusion level of lupin and the muscle concentrations of EPA (C20:5n-3) Surprisingly, the concentration of DHA (C22:6n-3) remained steady in the muscle On the other hand, the total concentration of n-6 PUFA increased quadratically with an increase in the dietary inclusion of lupin (R2 50.69, P 0.0055) Correspondingly, the content of linoleic acid (C18:2n- 1074 6), the main n-6 PUFA, increased in response to the increasing contribution of lupin lipids to dietary crude fat (R2 50.79, P 0.0009) Given the decrease in n-3 PUFA and the increase in n-6 PUFA observed in the muscle of the ¢sh feed graded lupin diets, the n-3/n-6 ratio was reduced from 3.9 in the control diet to 2.42 in the diet containing 50% lupin (R2 50.71, P 0.0041) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 Aquaculture Research, 2011, 42, 1067^1078 Whole grain white lupin in diets for rainbow trout A Borquez et al Discussion The ¢ndings concerning growth performance and nutrient digestibility obtained in the present study have demonstrated the potential use of whole grain sweet white lupin (L albus) in commercial extruded diets for rainbow trout (O mykiss) The growth performances and feed intake of ¢sh fed diets containing up to 50% of whole lupin grain were comparable to those of ¢sh fed the control diet Similar results were obtained by Burel et al (1998) using dehulled white lupin kernel meal (L albus) and by Glencross, Evans, Hawkins and Jones (2004) using dehulled yellow lupin kernel meal (Lupinus luteus) at the inclusion levels of 50% The results in our experiment are also similar to those achieved by Farhangi and Carter (2001) using dehulled blue lupin kernel meal (Lupinus angustifolius), although at slightly lower inclusion levels of 40% High inclusions of lupin seed in diets for salmonids in general have been associated with a reduction in the growth rate and feed intake attributable to the de¢ciency in the essential amino acids (lysine and methionine) (De la Higuera et al 1988) and the presence of antinutritional factors (Francis et al 2001) In this trial, lysine and methionine amino acids were supplemented in the experimental diets in order to cover ¢sh needs (Hardy 2002) Moreover, white lupin cultivar Hamburg, used in the formulation, is characterized by a low alkaloid content (120 mg kg À 1), and therefore the e¡ects on palatability reported at higher alkaloid concentrations (Glencross, Evans, Rutherford, Hawkins, McCa¡erty, Dods, Jones, Harris, Morton, Sweetingharn & Sipsas 2006) were not observed The ADC of nutrients and dry matter were not affected by the dietary treatments The ADC values observed in our study are consistent with many other studies, which included dehulled lupin kernel meal in diets for salmonids (Burel, Boujard,Tulli & Kaushik 2000; Carter & Hauler 2000; Aslaksen et al 2007; Glencross, Hawkins et al 2007, 2008) The DE of pulses has been considered to be low when fed to ¢sh; this has been attributed to the high content of carbohydrates (Morales, Cardenete, De la Higuera & Sanz 1994) However, the DE values obtained in the present study were relatively high compared with other studies with pulses (Gomes et al 1993); the above is probably due to the high ADC value of carbohydrates achieved by the experimental diets Carter and Hauler (2000) reported that in diets for Atlantic salmon (Salmo salar), the incorporation of lupin showed the lowest PER value compared with soybean and pea at the same inclusion levels Conversely, the PER was not a¡ected by the increasing dietary inclusion of whole lupin grain, and the values were higher than those reported by these authors The high inclusion of vegetable ingredients in carnivorous ¢sh diets can increase glycogen deposition in the liver after long feeding periods (Russell, Davies, Gouveia & Tekinay 2001) This increase in gluconeogenesis has been related to the low carbohydrate digestibility (Brauge, Medale & Corraze 1994; Hemre et al 2002) Indeed, Farhangi and Carter (2001) suggested an increment in the glucogenic activity as the inclusion levels of lupin increased in the diet However, in our experiment, the HSI was decreased by dietary treatments, and according to histological examination, no signi¢cant decline occurred in glycogen and lipids These observations are in agreement with those of Robaina et al (1995), who found no alterations in lipid and glycogen storage in hepatocytes from Sparus aurata fed with an inclusion of up to 30% of dehulled lupin seed meal Histological analysis of the ¢sh fed diets containing 40% and 50% of whole grain lupin exhibited morphological changes in the mid intestine that included a decrease in the number of basophil granulocytes, distal displacement of enterocyte nucleus and an increment in lipid drops Several authors have reported histological alterations in the intestine such as a shortening of mucosal folds, a loss of the normal supranuclear vacuolization of the absorptive cells in the intestinal epithelium; a widening of the central stroma within the mucosal folding, with increased amounts of connective tissue; and a profound in¢ltration of in£ammatory cells in the lamina propria when plant ingredients are fed to carnivorous ¢sh (Krogdahl, Bakke-Mckellep, Roed & Baeverfjord 2000; Refstie, Korsoen, Storebakken, Baeverfjord, Lein & Roem 2000) In the case of dehulled lupin meal, Farhangi and Carter (2001) have observed that the increasing dietary inclusions of L angustifolius can slightly shorten the villous height in rainbow trout Glencross et al (2004) found no e¡ect of the dietary inclusion of yellow lupin (L luteus) on the histology of the intestine in rainbow trout, even though the lupin fed ¢sh had gastrointestinal weights that were higher than those fed other plant ingredient The fatty acid composition in rainbow trout muscle was directly related to the fatty acid pro¢le in the respective diets These results are in agreement with r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 1075 pH stress induced apoptosis in Chinese shrimp Y Wang et al Aquaculture Research, 2011, 42, 1214^1230 Figure Nucleotide and deduced amino acid sequence of FcCasp cDNA Nucleotides are numbered from the ¢rst base at the -end Amino acids are numbered from the initiating methionine An open reading frame of 972 nucleotides encoding 323 amino acids contains the single pattern hit of the caspase family cysteine active site (shadow) and a polyadenylation signal (boxed) The caspase family p20 and p10 domain are underlined with single lines and double lines respectively Primers used for caspase expression and for real-time PCR are underlined with single line and dashed line respectively The sequence had been deposited in GenBank with the accession no GU597089 cluded an His-tag (HHHHHH) and an S-tag (KET AAAKFERQHMDS), its calculated molecular weight was 41.4 kDa This was larger than that of the mature caspase (Fig 4) A band of approximately 50 kDa corresponding to the His-tag FcCasp fusion protein was observed after IPTG induction and was found to react with anti-FcCasp antibody No bands were found at the same position in either non-induced or induced transformed E coli with empty pET30a(1), or in the non-induced recombinant pET30a(1)-FcCasp plasmid 1220 (Fig 4a) The recombinant fusion protein FcCasp was successfully recognized by rabbit anti-FcCasp antibody (Fig 4b) To characterize the anti-FcCasp serum, 28^30 mg of total protein extract of haemolymph, haemocytes, gill, muscle, lymphoid organ, heart, stomach and hepatopancreas were subject to Western blot analysis The results showed that FcCasp was present in all tissues, whereas no target band was detected from the haemolymph fraction (Fig 5b) The reactive r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 Aquaculture Research, 2011, 42, 1214^1230 pH stress induced apoptosis in Chinese shrimp Y Wang et al Figure Multiple alignment of the deduced amino acid sequence of the FcCasp: sequences were from Fenneropenaeus chinensis caspase (GenBank accession no GU597089), Fenneropenaeus merguiensis (GenBank accession no AY839873), Penaeus monodon (GenBank accession no DQ846887), Litopenaeus vannamei (GenBank accession no EU421939), Penaeus monodon (GenBank accession no FE114674) and Marsupenaeus japonicus (GenBank accession no EF079670) Residues in the black background indicate higher levels of amino acid similarity Conserved residues sites, which are a histidine residue and a cysteine residue, are marked with an asterisk (Ã) r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 1221 pH stress induced apoptosis in Chinese shrimp Y Wang et al Aquaculture Research, 2011, 42, 1214^1230 Figure Phylogenetic relationships of caspase on the basis of the amino acid sequence using neighbour-joining distance analysis The reliability of each branch was tested by 1000 bootstrap replications Numbers at the nodes indicated bootstrap values Including caspase from Fenneropenaeus chinensis (Ã), caspase from Fenneropenaeus merguiensis (F merguiensis: AAX77407), caspase from Penaeus monodon (P monodon: ABI34434), caspase-3 from Litopenaeus vannamei (L vannamei: ABK88280), caspase from Penaeus monodon (P monodon: ABO38430), caspase from Marsupenaeus japonicus (M japonicus: ABK62771), caspase-1 from Musca domestica (M domestica: ACF71490), caspase-1 from Trichoplusia ni (Trichoplusia ni: ACI43910), caspase from Culex quinquefasciatus (C quinquefasciatus: XP_001842236), drICE protein from Drosophila melanogaster (D melanogaster: CAA72937), caspase-1 from Bombyx mori (B mori: NP_001037050), caspase-1 from Helicoverpa armigera (H armigera: ABO93468), caspase-3 from Culex quinquefasciatus (C quinquefasciatus: XP_001850594) and caspase-1 from Aedes aegypti (A aegypti: XP_001656809) bands from all tissues were slightly larger than the calculated molecular mass 36.0 kDa results suggest that the recombinant protein obtained was a Chinese shrimp caspase Identi¢cation of the recombinant protein Expression pro¢les of FcCasp in six di¡erent tissues after exposure to pH stress Peptide fragments of the recombinant caspase matched with the known amino acid sequence of caspase based on the comparison of peptide mass ¢ngerprint using the MALDI-TOF-MS analytical system The matched peptide fragments are as follows (Fig 6a): ^AEAQPNDGR^ located at 10^18; ^GRPTAY TEVDGLSER^ located at 52^66; ^YPMNHRPR^ located at 67^74; ^GSALIFAHSK^ located at 75^84, ^ ELQAVSKR^ located at 128^135; ^DHSGSDAFAIVFMSHGEVKTR^ located at 136^156; ^ELWINFTAER^ located at 173^182; ^LYFIQACR^ located at 192^199; ^GVNMSRAVR^ located at 206^214; and ^QVPYIHSTLLREIYF^ located at 309^323 In addition, the MS/MS spectrum of a single-charged ion with m/z at 1278.66 Da is shown in Fig 6b These The tissue-speci¢c expression pro¢les for FcCasp after pH stress are shown in Fig The number of FcCasp transcripts in the LpH group increased gradually in the haemocytes during the ¢rst 12 h, and then decreased to their lowest level by 48 h Following this, expression of the transcripts increased again to their highest level at 96 h before again decreasing The levels at the end of the experiment were signi¢cantly lower than those in the control group The magnitude of changes was lower in the HpH group than in the LpH group Levels of the caspase transcript also increased at h, and then decreased slightly by 24 h The highest levels were observed after 120 h Transcript levels had returned to baseline by 148 h (Fig.7a.) 1222 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 Aquaculture Research, 2011, 42, 1214^1230 Figure Expression and puri¢cation of the recombination caspase fusion protein (a) Protein samples were separated by SDS-PAGE and stained with Coomassie brilliant blue Lane M2, protein molecular-weight markers; lane 1, crude extract of BL21 (DE3) pLysS without plasmid; lane 2, crude extract of the transformed BL21 (DE3) pLysS with pET30a(1) not induced with IPTG; lane 3, crude extract of the transformed BL 21 (DE3) pLysS with pET30a(1) induced with IPTG; lane 4, crude extract of the transformed BL 21 (DE3) pLysS with recombined pET30a(1) plasmid not induced with IPTG; land e, crude extract of the transformed BL 21 (DE3) pLysS with recombined pET30a(1) plasmid induced with IPTG and lane 6, puri¢ed caspase The positions of the molecular-weight marker (kDa) are indicated on the right (b) Protein samples were analysed by immunoblotting using anti-FcCasp antibody FcCasp expression in the gill of the LpH group was almost always higher than in the controls, except at 48 and 120 h (Fig 7b) FcCasp transcript levels were signi¢cantly lower at h in the HpH group compared with the control group, but then gradually increased up to 24 h before decreasing until 48 h after initial exposure Following this, the levels in the treatment group were higher than in the control group between 72 and 120 h, and then decreased to their lowest levels at 148 h (Fig.7b) The transcript levels in the hepatopancreas of the HpH group increased after h, but then decreased and were slightly lower than the control group at 12 h The transcript levels peaked at 120 h and were higher than the control group from 24 to 148 h FcCasp expression was lower in the LpH group than pH stress induced apoptosis in Chinese shrimp Y Wang et al in the control group until the end of the experiment, except at 3, 48 and 96 h (Fig.7c) The pattern of FcCasp transcript expression was similar at the LpH and HpH groups in the muscle tissue (Fig.7d) Levels increased slightly h post stress, and then decreased signi¢cantly from 12 to 48 h Following this, levels increased again and peaked at 96 h At the end of experiment, the expression of caspase transcripts was signi¢cantly lower after 120 h and was lower than the control group by 148 h The number of FcCasp transcripts in lymphoid organ of shrimp in the LpH and HpH groups increased by h and then decreased to baseline by 12 h Levels increased (over six times and nine times than that of the control group) up to 24 h Thereafter, the expressions were almost always higher than in the control group (Fig.7e) The expression of FcCasp decreased signi¢cantly by h in the stomach tissue of shrimp in the HpH group Levels then increased gradually between 12 and 72 h, and decreased again up to 96 h before peaking at 120 h At the end of the experiment, the expression was lower than in the control group (P40.05) The expression of caspase was also lower after and 12 h in the LpH group, but had increased by 24 h Levels then decreased and were at their lowest by 72 h The highest expression levels were observed by 148 h (Fig.7f) TUNEL observation Brownish nuclei representing fragmented DNAwas observed in the groups exposed to pH stress (Fig 8a^f) These were similar to the TUNEL-positive nuclei found in the positive control (Fig.8k) Conversely,TUNEL-positive nuclei representing fragmented DNA were not observed in the control (Fig 8g^i) and negative control (Fig 8j) group Apoptotic cells were observed in hepatopancreas tissue after 12 h exposure to pH stress (Fig 8a and d) The cells with TUNEL-positive nuclei were also observed in hepatopancreas tissue at 48 and 148 h pH stress These results suggested that both neutral and alkaline pH stress induced apoptosis in hepatopancreas cells of F chinensis Discussion The caspase family of cysteine proteases plays a conserved role in the coordinate breakdown of cellular structures during programmed cell death in a range from species, from nematodes to humans (Colin, Emma, Sean & Seamus 2004) Given this role, the r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 1223 pH stress induced apoptosis in Chinese shrimp Y Wang et al Aquaculture Research, 2011, 42, 1214^1230 Figure Detection of FcCasp protein in 28^30 mg total protein derived from extracts of di¡erent tissues of Fenneropenaeus chinensis Western blot analysis was performed using anti-FcCasp antibody (a) Total extract protein of F chinensis tissues stained with Coomassie brilliant blue Lane M, protein molecular weight markers; lane 1, haemocytes; lane 2, haemolymph; lane 3, gill; lane 4, muscle; lane 5, lymphoid organ; lane 6, heart; lane stomach; and lane 8, hepatopancreas (b) Total extract proteins of F chinensis tissues were analysed by immunoblotting using anti-FcCasp antibody activation of cysteine proteases must be rigorously regulated to avoid cell degradation under physiological conditions when it is unwarranted or maladaptive (Menze et al 2010) In this study, a caspase with full-length cDNA sequence of 1329 bp was cloned from the hepatopancreas of F chinensis Consistent with other known shrimp caspase, it was hypothesized that the deduced F chinensis caspase was ¢rst synthesized as a zymogen The putative cleavage site was predicted to be located at Asp60^Gly61 and Asp221^Ser222 This is similar to the location in the caspase from P monodon (Asp55^Gly56 and Asp215^ Ser216) (Wongprasert et al 2007) and F merguiensis (Asp215^Asn216) (Phongdara et al 2006) Upon receipt of an apoptotic signal, the caspase zymogens undergo proteolytic processing to generate two subunits that comprise the active enzyme (Alexei, Michael & Junying 2003; Fuentes-Prior & Salvesen 2004) FcCasp contained the special amino acid residues (H150 and Cys198) to catalyse caspase enzyme as follows: the active site Cys198 acts as the nucleophile, which is a part of the conserved QACRG pentapeptide sequence; His150 acts as the general base to extract the proton from the catalytic Cys and promote the nucleophile The nucleophile or electrophilic character of one amino acid is noted as much for the char- 1224 acter of the amino acids near in the primary structure of the protein as for the character of the amino acids near in the tertiary structure Hence, other His amino acid in the primary may also act as a general base These observations are consistent with Deveraux, Stennicke, Salvesen and Reed (1999) and Stennicke and Salvesen (1999), who noted that all caspases contained a catalytic Cys^His pair The BLASTP search revealed a high degree of similarity between FcCasp and the caspases of other shrimp FcCasp had the highest homology (83^76%) with the penaeid shrimp caspase (P monodon, F merguiensis, L vannamei) In comparison of FcCasp with other penaeid shrimp caspases, the short N-terminal prodomain-containing caspases act as e¡ector caspases in much the same manner as mammalian caspase-3 and caspase-7 (Fraser & Evan, 1997; Song, McCall & Steller 1997) This is consistent with the observations of Wongprasert et al (2007) The FcCasp amino acid sequence shared 33% identity with H sapiens caspase-3,77% with L vannamei caspase-3 and 37^34% with insect caspase-1 (the e¡ector caspase in most insect species is typically named caspase-1) The results suggest that FcCasp is an e¡ector caspase and show characterization of a caspase-3-like protease The phylogenetic analysis revealed that r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 Aquaculture Research, 2011, 42, 1214^1230 pH stress induced apoptosis in Chinese shrimp Y Wang et al Figure Identi¢cation of the recombinant caspase (a) Peptide mass ¢ngerprint of the recombinant caspase using MALDI-TOF-MS and ten peptide fragments of the recombinant protein were matched with the amino acid sequence of FcCasp (b) MALDI-TOF/TOF MS/MS spectrum of a single-charged ion with m/z at 1278.66 Da matched with FcCasp shrimp caspases are distantly related to the insect caspases The results suggest that FcCasp formed a monophyletic subgroup with the caspases of P monodon, F merguiensis and L vannamei FcCasp was successfully expressed in E coli and the recombination protein was identi¢ed using MALDITOF-MS analysis The recombinant caspase protein was separated using SDS-PAGE and the theoretical molecular weight was 41.4 kDa However, the molecular weight of the recombination caspase protein in gel was larger than 45 kDa This was most likely caused by the fusing of the recombinant caspase protein with His-tag at the N terminus, resulting in a strong positive charge that reduces the mobility of the fusion protein bands in SDS-PAGE (Niu & Guiltinan 1994; Harwood 1996; Tang, Zhang,Wang & Hong 2000) In addition, Western blot analysis revealed that the target protein bound to the speci¢c rabbit anti-FcCasp polyclonal antibody The tissue distribution of FcCasp by Western blot illustrates the speci¢city of the antibody serum obtained from the rabbit Thus, this antibody o¡ers further potential for investigating the role of apoptosis in the response of Chinese shrimp to environmental changes The MALDI-TOF-MS analysis revealed ten peptide fragments of the recombinant protein that match with the amino acid sequence of FcCasp and several nontarget peptide masses It would appear to be that the target bands excised from the gels may be not a single band, and it also may include protein from E coli BL21 (DE) pLysS or bacterial proteins However, the MS/MS spectrum of a single-charged ion with m/z at r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 1225 pH stress induced apoptosis in Chinese shrimp Y Wang et al pH 7.0 pH 9.0 8.0 pH 8.2 (a) 10.0 Relative expression level of caspase in gill Relative expression level of caspase in haemocytes 12.0 Aquaculture Research, 2011, 42, 1214^1230 * 8.0 6.0 * * 4.0 * * 2.0 * 6.0 5.0 4.0 * * 3.0 2.0 12h 8.0 * 24h * 48h pH 7.0 72h 96h 120h 148h 0h pH 8.2 pH 9.0 * 3h Relative expression level of caspase in muscle Relative expression level of caspase in hepatopancreas 4.0 * * * * * * * * * 12h 24h 0.0 pH 7.0 * * 0h 3h 8.0 8.0 * * * * 4.0 * * * * Relative expression level of caspase in stomach Relative expression level of caspase in lymphoid organ * 12h 24h * 48h * 72h 96h 120h 148h pH 8.2 pH 9.0 * 6.0 5.0 4.0 * * 3.0 * * 2.0 * 48h 72h 96h 120h 148h Time interval after pH challenge (h) * * 0.0 0.0 3h * 24h (f) 1.0 0h 12h * * * * 7.0 10.0 2.0 * * pH 7.0 * * pH 8.2 Time interval after pH challenge (h) (e) 6.0 pH 9.0 2.0 120h 148h * * 120h 148h * pH 8.2 pH 9.0 96h 3.0 Time interval after pH challenge (h) 14.0 72h 4.0 * 96h 48h * 0.0 72h 24h 5.0 * 48h * (d) 1.0 1.0 * 12h pH 7.0 6.0 5.0 12.0 * Time interval after pH challenge (h) 6.0 2.0 * * * * 7.0 3.0 * 0.0 (c) 3h * * * pH 8.2 * Time interval after pH challenge (h) 0h pH 9.0 1.0 * 0.0 3h 7.0 * * * 0h pH 7.0 (b) 0h * * 3h 12h 24h * * 48h 72h 96h 120h 148h Time interval after pH challenge (h) Figure Analysis of caspase gene expression in six di¡erent tissues [(a) haemocytes, (b) hepatopancreas, (c) gill, (d) muscle, (e) lymphoid organ and (f) stomach] of Fenneropenaeus chinensis in di¡erent experimental pH treatments and the control group (pH 8.2) by SYBR green real-time RT-PCR after 0, 3, 12, 24, 48, 72, 96 and 148 h Each site represents the mean Æ standard deviation, n Signi¢cant di¡erences (Po0.05) of caspase gene expression between the experimental and the control groups at the same time intervals are indicated with asterisks (Ã) 1226 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 Aquaculture Research, 2011, 42, 1214^1230 pH stress induced apoptosis in Chinese shrimp Y Wang et al Figure TUNEL assay of Fenneropenaeus chinensis hepatopancreas tissues on control and pH stress group (a) pH 7.0 post-stress 12 h; (b) pH 7.0 post-stress 48 h; (c) pH 7.0 post-stress 148 h; (d) pH 9.0 post-stress 12 h; (e) pH 9.0 post-stress 48 h; (f) pH 9.0 post-stress148 h; (g) pH 8.2 control group12 h; (h) pH 8.2 control group 48 h; (i) pH 8.2 control group148 h; (j) negative control TUNEL; and (k) positive control TUNEL, indicating DNA cleavage Arrows indicate DNA 0-OH nicks Magni¢cation of the photographs was  40 1278.66 Da strongly supported the conclusion that the recombinant protein was Chinese shrimp caspase The roles of FcCasp in apoptosis were evaluated by examining the temporal pro¢le of FcCasp post pH stress The pattern of FcCasp expression di¡ered among the six tissues This likely relates to the di¡erent functions of these tissues in the response to the environmental changes In the present study, FcCasp mRNA expression was up-regulated in the haemocytes, gill, hepatopancreas, muscle, lymphoid organ and stomach tissue However, the timing of the in- crease di¡ered among the groups and tissues FcCasp expression was up-regulated in the haemocytes, gill, hepatopancreas, muscle and lymphoid organ during the ¢rst h in the LpH groups This suggests that FcCasp is involved in a transient response to the sudden environment pH stress, possibly in response to the associated respiratory burst and ROS generation (Wang et al 2009) The high level of FcCasp expression in shrimp gills and haemocytes 12 h post pH 7.0 stress and in gills 24 h post pH 9.0 stress suggests that apoptosis is initiated to remove unneeded or deleter- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 1227 pH stress induced apoptosis in Chinese shrimp Y Wang et al ious cells Moreover, this process is also essential for normal development and homoeostasis in a number of vertebrates and invertebrates (Ra¡ 1992; White 1996) We hypothesize that removal of unneeded or deleterious cells reduces energy consumption by these cells, thus allowing the organism to redirect energy to resisting pH stress The decrease in FcCasp mRNA expression in shrimp haemocytes and gills 48 h post pH 7.0 stress and in the gills 48 h post pH 9.0 stress may have been caused by feedback as a result of caspase-3 activity (Chiang, Grenier, Ettwiller, Jenkins, Ficenec, Martin, Jin, Distefano & Wood 2001) FcCasp expression was highest in the haemocytes, gills and hepatopancreas of the LpH and HpH group at the end of the experiment suggesting that apoptosis may be induced by the increase in FcCasp expression These observations also suggest that apoptosis plays a role in the poor tolerance of shrimp to long-time environmental stressors However, further research is needed to evaluate these hypotheses The lymphoid organ plays an important role in shrimp immunity (Hasson, Lightner, Mohney, Redman & White 1999) The expression of FcCasp in lymphoid organ was similar following exposure to 7.0 and 9.0 pH stressors At 24 h post pH stress, the increased expression of FcCasp observed in the treatment group may lead to an increase in caspase-3 activity in the cells undergoing apoptosis (Chiang et al 2001) At 148 h post pH stress, FcCasp expression remained higher than in the control group Together, these observations suggest that the pH stress induced the transcription of FcCasp mRNA, which in turn promoted the occurrence of lymphoid organ apoptosis The apoptosis of lymphoid organ cells following exposure to pH stress may increase the vulnerability of the shrimp to the bacteria normally present in culture ponds The TUNEL analysis suggested that 7.0 or 9.0 pH stress induced apoptosis in the hepatopancreas of F chinensis The hallmark of the programmed cell death is DNA degradation, which results in yielding doublestranded and single-stranded DNA nicks Both types of breaks can be detected using theTUNEL assay and can be visualized in situ (Wang, Lin, Zhang, Yan & Duan 2004) In the present study, the number of the TUNEL-positive nuclei seems positively to be correlated with the length of time the individuals were exposed to pH stress This is consistent with observations of a rapid time dependency in the level of DNA strand breakage in the hepatopancreas of L vannamei (Wang et al 2009) and in the grass shrimp embryos (Lee & Kim 2002) following exposure to acid or alkaline pH stress and phototoxicants Moreover, 1228 Aquaculture Research, 2011, 42, 1214^1230 when the shrimp were exposed to the acid and alkaline pH stress, ROS were generated, which severely impaired normal cells function and may indirectly act as signalling molecules of DNA damage (Wang et al 2009) It is generally accepted that oxidative stress and ROS cause DNA damage, whereby insu⁄cient cellular repair mechanisms contribute to apoptosis (Franco et al 2009) Indeed, the addition of exogenous ROS is su⁄cient to trigger the apoptotic cascade Furthermore, the molecules involved in apoptosis (e.g caspases) are extremely sensitive to redox alterations (James, Maryanne & Thomas 2002) On the basis of the pattern of caspase gene expression, we hypothesize that there is a relationship between apoptosis and caspase gene expression following exposure to pH stress This is supported by the high levels of FcCasp expression in the 7.0 and 9.0 pH treatment group at the end of the experiment between 96 and 148 h in di¡erent tissues Increases in caspase-3 gene expression have been described in cells undergoing apoptosis (Chang et al 2009) Thus, it is reasonable to speculate that apoptosis of shrimp hepatopancreas may be activated by an elevation of FcCasp expression There are two pathways that have been described for the activation of apoptosis, extrinsic and intrinsic pathways (Hand & Menze 2008) It is thought that apoptosis is activated by intrinsic pathways in crustaceans (Menze, Hutchinson, Laborde & Hand 2005; Menze & Hand 2007) Over expression of the FcCasp gene was associated with the response to pH stress in shrimp Our results are consistent with other studies showing a link between caspase activity and the stress response For example, Brittany and Dietmer (2009) found that caspase 3/7 activity and apoptosis increased signi¢cantly in the gill epithelium of tilapia following exposure to a salinity stressor and remained elevated for the duration of time the tilapia exposed to the stressor Similarly, Rebecca and David (2004) concluded that apoptosis was induced in response to chemical and physical stress in sea urchin embryos Taken together, these observations provide support for a link between apoptosis and environmental adaptability In conclusion, a caspase gene was successfully cloned from the Chinese shrimp F chinensis The expression of FcCasp changed in response to pH stress in six tissues FcCasp expression remained up-regulated at the end of the experiment (96^148 h after initial exposure) suggesting that it was inducible and may play a role in adaptation to changes in the environment FcCasp is likely a member of the caspase-3 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1214^1230 Aquaculture Research, 2011, 42, 1214^1230 subfamily Apoptosis was induced by 7.0 and 9.0 pH stress in the Chinese shrimp.We hypothesize that this may explain the relatively poor ability for shrimp to adapt to environmental changes Further work will focus on the translational regulation of FcCasp in the response to aqueous environment changes Acknowledgments We thank other members of our laboratory for their assistance and discussion This work was ¢nancially supported by the National Key Technology R&D Program (No.2006BAD01A13), National Natural Science Foundation of China (No 40706052), National Special Research Fund for Non-pro¢t Sector (Agriculture) (No nyhyzx 07042) and the Earmarked Fund for Modern Agro-industry Technology Research System (No nycytx-46) References Alexei D., Michael B & Junying Y (2003) A decade of caspases Oncogene 22, 8543^8567 Assefa Z., Laethem A.V., Garmyn M & Agostinis P (2005) Ultraviolet radiation-induced apoptosis in keratinocytes: on the role of cytosolic factors Biochimica et Biophysica Acta 1775, 90^106 Bache're E (2000) Shrimp immunity and disease control Aquaculture 191, 3^11 Bache're E., Chagot D & Grizel H (1988) Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counter£ow centrifugal elutriation Developmental and Comparative Immunology 12, 549^559 Brittany D.K & Dietmer K (2009) Prolonged apoptosis in mitochondria-rich cells of tilapia (Oreochromis 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Renata Perpetuo Reis, Rafael Rodrigues Loureiro & Frederico Sampaio Mesquita Instituto de Pesquisas Jardim Bota“nico Rio de Janeiro, Rio de Janeiro, Brazil Correspondence: R P Reis, Instituto de Pesquisas Jardim Bota“nico Rio de Janeiro, Rua Pacheco Leaìo, 915, Rio de Janeiro, RJ, CEP 22460-030 Brazil E-mail: rreis@jbrj.gov.br Introduction Kappaphycus alvarezii (Doty) Doty ex P C Silva is one of the most important sources of raw material for the carrageenan industry (Ask & Azanza 2002) In Brazil, only Hypnea musciformis (Wulfen) J.V Lamouroux is used as raw material and its natural stocks are not su⁄cient to supply the Brazilian demand (Bulboa & Paula 2005; Reis, Yoneshigue-valentin & Santos 2008) This was one of the reasons why, in 1995, K alvarezii was introduced at the Brazilian Southeastern coast, in Saìo Paulo State (Bulboa & Paula 2005) The carrageenan yield (CY) can change in accordance to environmental parameters as a mechanism of prevention against stressful situations like salinity £uctuations (Hayashi, Oliveira, Bleicher-lhonneur, Boulenguer, Pereira,Von Seckendor¡, Shimoda, Le£amand,Valle¤e & Critchley 2007; Reis et al 2008) Few works discuss the salinity e¡ects on daily growth rate (DGR) and CYof eucheumatoids in spite of its importance (Ask & Azanza 2002) This information could help the identi¢cation of good sites for cultivation and would help mitigation activities (Ask, Batibasaga, Zertuche-gonzaŁlez & De San 2003) Thus, the aim of this study was to analyse the e¡ect of the salinity on DGR and CY values of K alvarezii in vitro Materials and methods Green, brown and red variants of K alvarezii were acquired at a farming located at Praia Grande, Itacuruc°aŁ Island, north of Sepetiba Bay, Rio de Janeiro State, Brazil (22157 00200 S and 43154 02200 W) Kappaphycus alvarezii voucher material was included in the Her- r 2010 Blackwell Publishing Ltd barium of the Botanical Garden of Rio de Janeiro (RB 425.507) The variants were cleaned of epiphytes, using clamps and paper towel and acclimated for 30 days in L glass tanks (water temperature ^ 22 Æ 1C, water surface irradiance ^ 130 Æ 20 mmoL m À s À photoperiod ^ 12-h day, salinity (PSS) ^ 35 Æ 2) After 35 days, the samples were weighted and the CY and quality were measured considering that a production cycle rates from 30 to 60 days (Neish 2006) The tested salinities (15, 25, 35 and 45) were obtained by freezing and defreezing seawater (Oliveira, Paula, Plastino & Petti 1995) and seawater (35) was used as control Six replicates of each salinity were tested Apical portions of each variant of K alvarezii (brown, red and green) with cm, making a ¢nal weight of g, were placed in Erlenmeyer £asks with 200 mL of seawater under aeration for weeks The seawater was ¢ltered in an ester cellulose membrane (0.45 mm, Millipore Corporate Headquarters, Billerica, MA, USA) and 10 mL L À of enriched seawater ^ ES/2 were added (Starr & Zeikus 1993) and weekly changed Growth was estimated as the mean DGR over a 35day period, calculated according to the formula DGR ln(Wf/Wo)/t  100, whereWo stands for initial dry weight, Wf for ¢nal and t for time of cultivation (Hurtado, Agbayani, Sanares & Castro-mallare 2001) The initial dry biomass was established with g of algal wet mass dried in an oven until constant weight (60 1C) The semi-re¢ned carrageenan extraction was obtained with hot alkali transformation in 0.2% KCl 1231 Does salinity a¡ect Kappaphycus alvarezii? R P Reis et al Aquaculture Research, 2011, 42, 1231^1234 and 6% KOH solution followed by successive water showering to remove any alkaline residues Carrageenan yield results were expressed as the percentage of carrageenan from a sample of the individual dry mass (Reis et al 2008) according to the formula: Yield (Wc/Ws)  100, where Wc is the extracted carrageenan dry weight and Ws is the dry seaweed weight used in the extraction The normality (Shapiro Wilk’s test) and homogeneity (Cochran test) assumptions of the variances were tested Carrageenan yield was transformed to its arcsine because it is recommended to transform percentages (Zar 1996) DGR were transformed using the equation x square root(x)1square root(x11) To verify the accuracy of the di¡erent tested salinities, obtained by the freezing and defreezing method, and the interaction of salinity and the variants of K alvarezii on DGR values and in the CY of the samples, the two-way analysis of variance (ANOVA) was used The post hoc Fisher’s LSD test was used to separate these di¡erences The salinity dependence on DGR and CY values was tested by polynomial regression tests STATISTICA 6.0 (Statsoft South America, Saìo Paulo, Brazil) was used on all analysis Tests were carried out at P 0.05 level of statistical signi¢cance and data are expressed as mean Æ standard deviation Results and discussion The accuracy of freeze^defreeze seawater method was attained because no di¡erence was obtained between the DGR (Two-way ANOVA, F 3.40, P 0.07) and CY (Two-way ANOVA, F 0.01, P 0.90) values on the variants of K alvarezii ^ brown, red and green (Fig a^f) An interactive e¡ect of the variants and di¡erent the salinities on DGR values (Two-way ANOVA, F 6.41, Po0.001, Fig 1) was observed After 35 days in vitro, the variants presented higher DGR values in salinities of 25, 35 and 45 The lowest DGR of all variants occurred in salinity 15; however, the brown variant (Fig 1a) showed higher DGR results in this salinity level when compared with the other variants (red ^ Fig.1b, green ^ Fig1c) In other studies, K alvarezii presented lower DGR values in periods of rainfall at Brazilian southeastern coast (Paula, Per- Figure Daily growth rate (% day À 1) of the brown (a), red (b) and green (c) variants of Kappaphycus alvarezii and carrageenan yield (%) extracted from the brown (d), red (e) and green (f) variants of this species Square: median; rectangle: standard error; vertical bar: standard deviation and circle: outliers 1232 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1231^1234 Aquaculture Research, 2011, 42, 1231^1234 eira & Ohno 2002; Bulboa & Paula 2005) The DGRs of the brown and the green variants in salinity 45 were higher than the red variant (Po0.001, LSD test, Fig 1a) There is no clear reason why the brown variant obtained higher DGR values at this given salinities (15 and 45 PSS) when compared with the green and red variants Santelices 1999 relates seaweed growth to a complex interaction between irradiation, water motion, temperature and nutrients Munìoz, Freilepelegr|¤ n and Robledo 2004 correlate these factors explaining that when a major decline of one particular factor occurs it can be compensated by another, thus regulating the growth of the a¡ected individual The brown variant could show higher DGR values in lower salinity levels due to its more e¡ective ion sorbing capabilities when compared with other variants (Kumar, Ganesan & Rao 2007) as a direct response to this particular stress No di¡erence was observed between the variants in salinity 35 Higher DGR values were obtained by the brown variant in farmings at Kenya (Wakibia, Bolton, Keats & Raitt 2006) and by the green variant at the Philippines (Hurtado et al 2001) There is not an interactive e¡ect of the variants and the di¡erent salinities on the CY (Two-way ANOVA, F 51.2, P 0.33) All the variants produced higher CY values at salinity 15 (F 536.6, Po0.001, Fig 1d^f); on the other hand, the DGR values of K alvarezii (r2 50.68, Po0.001, Fig 2a) and its CY (r2 50.94, Po0.001, Fig 2b) had a high dependence on salinity This environmental factor is one of the most important in£uences on the CYof K alvarezii (Hurtado et al 2001) and Hypnea musciformis (Reis et al 2008) It is a natural defensive response to stressful conditions for most red algae in particular Gracilaria sp and carragenophytes like Chondrus sp and Solieria sp., to increase the cell-wall contents of such components in order to rapidly mitigate any harmful consequences caused by either salinity, light or water motion (Goulard, Diouris, Quere, Deslandes & Flocapos 2001; Freile-pelegr|¤ n, Robledo, Pederse¤n, Bruno & R˛nnqvist 2002; Villanueva, Hilliou & Sousa-pinto 2009) They directly contribute to the osmotic equilibrium of the cell (Percival 1979) and it is important to the survival of the algae in sites with £uctuating salinity conditions (Percival & McDowell 1967) It is di⁄cult to compare the CY results obtained in this work with others studies due to the di¡erent extraction techniques applied (Munìoz et al 2004; Hayashi, Oliveira et al 2007) The CY of K alvarezii from Sepetiba Bay in vitro in salinity 35 was higher than Does salinity a¡ect Kappaphycus alvarezii? R P Reis et al Figure Linear regression of daily growth rate (a) and carrageenan yield (b) of Kappaphycus alvarezii under di¡erent salinities the semi-re¢ned carrageenan (Hayashi, Paula & Chow 2007) and re¢ned carrageenan extracted from seedlings cultivated in the Brazilian Southeastern (Hayashi, Oliveira et al 2007) from Mexico (Munìoz et al 2004) and lower than the ones cultivated at Philippines (Trono & Lluisma 1992) Because Estuaries and coastal lagoons are common in southern Brazil and these environments represent 15% of coastal areas (Barnes 1989), the information found in this study could be important to consider the expansion of Kappaphycus cultivation Acknowledgments We are grateful to Henrique Gerome¤l de Go¤es (Sete Ondas Biomar) for ¢eld assistance, Fernando Azeredo for technical support, the National Research Grant Institution (CNPq) and Sete Ondas Biomar for ¢nancial support and the National Meteorology Institute (INMET) for all meteorological data provided for this work References Ask E.I & Azanza R.V (2002) Advances in cultivation technology of commercial eucheumatoid species: a review r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1231^1234 1233 Does salinity a¡ect Kappaphycus alvarezii? 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(2000) Digestibility of extruded peas, extruded lupin, and rapeseed meal in rainbow trout (Oncorhynchus mykiss) and turbot (Psetta maxima) Aquaculture 1 88, 285^298 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 Aquaculture Research, 2011, 42, 1067^1078 Whole grain white lupin in diets for rainbow trout A Borquez et al Caballero M.J., Obach A., Rosenlund G., Montero D., Gisvold... lacustris L., Coregonus sp Aquaculture Research 26, 801^807 Lahnsteiner F.,Weismann T & Patzner R (1996) Cryopreservation of semen of the grayling (Thymallus thymallus) and r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1096^1100 1099 Cryopreservation of Arctic char semen G F Richardson et al Aquaculture Research, 2011, 42, 1096^1100 the Danube salmon (Hucho hucho) Aquaculture 144, 265^ 274... cultivation in Northeast America World Aquaculture 29, 26^29 Zhang X.C., Brammer E., PederseÔn M & Fei X.G (2006) Effects of light photon Êux density and spectral quality on photosynthesis and respiration in Porphyra yezoensis (Bangiales, Rhodophyta) Phycological Research 45, 29^37 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1079^1086 Aquaculture Research, 2011, 42, 1087^1095 doi:10.1111/j.1365-2109.2010.02693.x... amino acids in shrimp Journal of Liquid Chromatography 18, 2059^2068 Wongso S & Yamanaka H (1998) Extractive components of the adductor muscle of Japanese baking scallop and changes during refrigerated storage Journal of Food Science 63,772^776 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1087^1095 1095 Aquaculture Research, 2011, 42, 1096^1100 doi:10.1111/j.1365-2109.2010.02695.x Effect... be helpful for the new breeding technology applied to Porphyra aquaculture production Conclusions The objective of this study was to produce critical information as a base for breeding with free-living conchosporangia in P yezoensis The optimum r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1079^1086 Aquaculture Research, 2011, 42, 1079^1086 Formation and growth of free-living conchosporangia... Ivanovic D., Owen D.F & Ballester D (1983) Chemical and nutritional-evaluation of sweet lupines Annals of Nutrition and Metabolism 27, 513^520 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1067^1078 Aquaculture Research, 2011, 42, 1079^1086 doi:10.1111/j.1365-2109.2010.02691.x Formation and growth of free-living conchosporangia of Porphyra yezoensis: effects of photoperiod, temperature and light... were grown in pyxises with 5 mL of VSE medium shaken twice every day The VSE culture medium was changed once a week (He & Yarish 2006) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1079^1086 Aquaculture Research, 2011, 42, 1079^1086 Formation and growth of free-living conchosporangia of P yezoensis X Li et al According to preliminary experiments, the eĂects of photoperiods (16 L:8 D, 12... (Xactics International, Cornwall, Ontario, Canada) connected to a pure compressed oxygen tank Sensors of a YSI oxygen monitor and a digital r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1087^1095 Aquaculture Research, 2011, 42, 1087^1095 The shipment eĂect on the physiological condition V M Ocanỡo-Higuera et al Oximeter Digital Thermometer Cooler Ice bag Wood Oxygen Rubber sponge Rubber sponge... 90 1C for 5 min The reaction r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1087^1095 1089 The shipment eĂect on the physiological condition V M Ocanỡo-Higuera et al was cooled down in ice and the absorbance was read in a spectrophotometer under the same conditions to quantify the content of glycogen Aquaculture Research, 2011, 42, 1087^1095 The concentrations of ATP, ADP and AMP in the adductor... monophosphate 1090 Statistical analysis Analyses were performed using the NCSS 2000 statistical software (NCSS, Kaysville, UT, USA) Descriptive r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 1087^1095 Aquaculture Research, 2011, 42, 1087^1095 The shipment eĂect on the physiological condition V M Ocanỡo-Higuera et al statistics (mean and standard deviation) and one-way SigniÂcance level was set at