Aquaculture Research, 2010, 41, 953^960 doi:10.1111/j.1365-2109.2009.02377.x Physiological responses of pink abalone Haliotis corrugata (Gray, 1828) exposed to different combinations of temperature and salinity Zarina Medina Romo1, Ana Denisse Re1, Fernando D|¤ az1 & Alfredo Mena2 Laboratorio de Eco¢siolog|¤ a de Organismos AcuaŁticos, Departamento de Biotecnolog|¤ a Marina, Centro de Investigacio¤n Cient|¤ ¢ca y de Educacio¤n Superior de Ensenada (CICESE), Ensenada Baja California, Me¤xico Departamento de Acuicultura-Biotecnolog|¤ a, Facultad de Ciencias Marinas (FACIMAR), Universidad de Colima, Manzanillo Colima, Me¤xico Correspondence: F D|¤ az, Departamento de Biotecnolog|¤ a Marina (CICESE), PO Box 430222, San Diego, CA 92143-0222, USA E-mail: fdiaz@cicese.mx Abstract Physiological responses of pink abalone Haliotis corrugata were determined under di¡erent temperature and salinity conditions Oxygen consumption rate was not a¡ected by temperature and salinity Ammonium excretion of pink abalone was inversely related to salinity The O:N ratio indicated that abalone maintained in lower salinities had an interval of 4.9^7.7, which is indicative of a protein-dominated metabolism, whereas the O:N in 35% was 28.8^35.5 for both temperatures, suggesting that carbohydrates were used as energy substrate Haemolymph osmolality of abalone exposed to 20 and 24 1C was slightly hyperiso-osmoconformic in salinity ranges of 20^35% The results of this study suggested that for optimized culture, pink abalone should be cultivated at 24 1C at a salinity of 35% Keywords: oxygen consumption rate, ammonium excretion, atomic ratio O:N, osmoregulation, Haliotis corrugata Introduction Haliotis corrugata (Gray 1828) is one of the species of economic importance and is actually cultivated in Baja California; it was observed that the growth of cultivated abalone is a¡ected by many factors such as temperature, salinity, dissolved oxygen, nitrogen subproducts, pH, density, food and water quality (Hahn 1989;Valde¤s-Urriolagoitia 2000) Temperature and salinity are two factors that control the life and distribution of aquatic organisms; both factors have direct e¡ects on the physiological responses of the marine and estuarine organisms, r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd such as oxygen consumption (Brown & da Silva 1979; Moore & Sander 1983; Saucedo, Ocampo, Monteforte & Bervera 2004; Soria, Merino & von Brand 2007), nitrogen excretion products (Livingstone,Widdows & Fieth 1979; Stickle & Bayne 1982; Regnault 1987; Saucedo et al 2004; Soria et al 2007), energy budget (Newell & Branch 1980; Bayne & Newell 1983; Bricelj & Shumway 1991; Beiras, Pe¤rez-Camacho & Albentosa 1993), endogenous substrate utilization (Barber & Blake 1985; Mayzaud & Conover 1988; Rosas, Cuzon, Gaxiola, Taboada, Arena & VanWormhoudt 2002) and osmoregulation pattern (Shumway 1977; Hildreth & Stickle 1980; Cheng, Yeh, Wang & Chen 2002) One of the physiological responses that can be correlated with a change in the environmental parameters is the oxygen consumption rate, because it is related to the metabolic work and energy £ow that organisms must channel to homeostatic control mechanisms (Salvato, Cuomo, Di Muro & Beltramini 2001) In aquatic organisms, the measurement of oxygen consumption is a valid method to assess the e¡ect of environmental factors such as temperature, salinity, exposure to pollutants, light intensity and dissolved oxygen It allows the estimation of the energy costs associated with the physiological stress that these factors impose on organisms (Brown & Terwilliger 1999; Lemos, Phan & Alvarez 2001; Altinok & Grizzle 2003; Brougher, Douglass & Soares 2005) Previous studies have investigated the e¡ects of salinity and temperature on the oxygen consumption in abalone such as Haliotis discus hannai (Ino) exposed to di¡erent environmental factors (Sano & Maniwa 1962) Uki and Kikuchi (1975) investigated the e¡ect 953 Physiological responses of pink abalone Z M Romo et al of temperature and weight on the oxygen consumption of H discus hannai In young disc abalone Nordotis discus discus (Reeve), Segawa (1995) determined in a preliminary study, the e¡ect of temperature on the oxygen consumption rate Paul and Paul (1998) determined the e¡ect of di¡erent temperatures on the respiration rate in Haliotis kamtschatkana (Jonas) Both osmotic and ionic regulation have been studied in a number of marine molluscs (Burton 1983) Changes in salinity may disturb the osmotic balance of marine molluscs However, nothing is known on the osmotic and ionic regulation of the Haliotis genus (Cheng et al 2002) Bivalve molluscs are osmoconformers in which the haemolymph is close to the osmotic pressure of seawater, and due to response, ammonium excretion increases with decreasing salinity (Shumway 1977; Bricelj & Shumway 1991) The role of ammonium in the osmoregulation processes has been studied in di¡erent organisms in two aspects: as a constituent of free amino acids (FAA) for intracellular osmotic regulation (Bishop, Gosselink & Stone 1980) and as an exchange ion for the regulation of Na1 in the haemolymph (Mangum, Silverthorn, Harris, Towle & Krall 1976; Pressley, Graves & Krall 1981; Re, D|¤ az & Go¤mez-Jime¤nez 2004) The atomic ratio (O:N) is an index that uses the integration of the values of the oxygen consumption and nitrogen excretion to determine which metabolic substrate is being used for the organisms (Mayzaud & Conover1988) This atomic ratio has also been used as a stress indicator due to the changes in the environment to which the organisms are exposed (Ikeda 1977; Cli¡ord & Brick 1979; Rosas, Cuzon, Gaxiola, LePriol, Pascual, Rossygnyol, Contreras, SaŁnchez & VanWormhoudt 2001) Finally, the O:N ratio is an important index that is used in both ecological and aquacultural settings, and so linked studies of oxygen consumption and nitrogen excretion in abalone would provide useful information on this index The main goal of this study was to determine the e¡ect of di¡erent combinations of salinity and temperature on di¡erent physiological responses in H corrugata Because of the commercial importance of this species, the information reported in this paper will be useful for managing these parameters under controlled conditions Material and methods About 720 juveniles with an average wet weight of 2.8 (Æ 0.9 g) were obtained from the commercial 954 Aquaculture Research, 2010, 41, 953^960 farm of B C Abalone Hatchery Ere¤ndira, Ensenada (Baja California, Me¤xico) The organisms were transported in a10 L-Styrofoam cage with seawater In the laboratory, the time to acclimate was weeks at 35%, 18 Æ 1C temperature, measured under farm conditions in three reservoirs of 2000 L with constant aeration and a 60 mm ¢ltered seawater £ow with an exchange rate of 100% daily Each reservoir was provided with abalone refuges and was heavily shaded; refuges and shaded were used to minimize disturbance from exterior movements The organisms were fed during the entire experimental period with macroalgae Macrocystis pyrifera and Egregia menziesii Acclimation of 489 abalones to the experimental temperature 20 and 24 1C was completed in 21days The temperature was increased at a rate of 1C everyday to reach the experimentalprogrammed temperature Both temperatures were achieved by titanium heaters of 1000 W controlled by Medusa devices (Sea Life Supply, Sand City, CA, USA) For experimental salinities 480 abalones were acclimated to 35%, 32%, 29%, 26%, 23% and 20%, where the lowest concentration was obtained by dilutions of seawater (35%) with tap water The rate of salinity decrease was 3% per days to reach the experimental salinity, and once the experimental salinity had been achieved, the abalones remained under those conditions for 15 days, which represent su⁄cient time to attain a steady internal medium of abalone according to Cheng et al (2002) To avoid interference with post-prandial metabolism of food and faeces production, acclimated abalone was kept unfed for 24 h Oxygen consumption and nitrogen excretion were measured with the organisms maintained at temperatures of 20 and 24 1C and experimental salinities of 35%, 32%, 29%, 26%, 23% and 20% by using a semi-closed respirometric system described by D|¤ az, Re, GonzaŁlez, SaŁnchez, Leyva and Valenzuela (2007), consisting of 21 chambers of 1000 mL each Twenty abalones of each temperature and salinity were individually introduced into the respiratory chamber 24 h before initiating measurements, which were made between 09:00 and 13:00 hours to avoid interference due to the rhythm circadian response Water £ow in the chambers remained open for h; before closing, one water sample was taken to measure the initial concentration of dissolved oxygen using aYSI 52 oxymeter (Yellow Spring Instruments, Yellow Springs, OH, USA), equipped with a polarographic sensor accuracy of Æ 0.03 mg L À 1, which was inside an acrylic hermetic chamber with a r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 Aquaculture Research, 2010, 41, 953^960 10 mL capacity with adequate stirring Subsequently, the chambers were kept closed for 1h, to avoid reduction in the dissolved oxygen of 25^30%, as this is a stress factor (Stern, Borut & Cohen 1984) Before reestablishing the water £ow, one water sample was taken to measure the ¢nal concentration of dissolved oxygen The 21st chamber was used as a control to measure oxygen consumption by the microorganisms present in the water, and the necessary corrections were made Two repetitions were carried out for each test The results of oxygen consumption are given in mg O2 kg À h À on a wet weight basis To determine the ammonium production (NH1 ), initial and ¢nal samples were obtained from the respirometric chamber in the same manner as that described for oxygen consumption; the di¡erence was that the water samples were of10 mL and the method for quanti¢cation was indophenol blue (Rodier 1998) The samples were analysed using the spectrophotometer El|¤ ptica (Ely-2000 Instruments, Ensenada, Me¤xico) at a wavelength of 640 nm Ammonium production was calculated as the di¡erence between the ¢nal and the initial measure and it was expressed as À1 À1 mg NH1 g wet weight (w.w.) h The O:N ratio was estimated using the oxygen consumption and the ammonium excretion values of the abalones, both were obtained using the respirometric system from all di¡erent experimental combinations The physiological rates were determined for both components and transformed to atom-gram in order to calculate the O:N index (Mayzaud & Conover1988) This index was used to estimate the ratio of proteins, lipids and carbohydrates that were used as energy substrates for the organisms under the di¡erent experimental conditions An individual sample of haemolymph was obtained from the membrane between muscle and mantle of the shell of abalones using a hypodermic needle, in a manner similar to that described by Cheng et al (2002) The samples of haemolymph,10 mL from 20 organisms from each experimental condition, were placed on a blotting paper disc in a Wescor 5520 vapour pressure osmometer (Wescor Logan, UT, USA) The osmolality of the internal as well as the external medium was expressed as mmol kg À The data for oxygen consumption and ammonium excretion of abalone exposed to di¡erent experimental conditions were plotted in parallel boxes (Tukey 1977).Within the boxes, 50% of the data were distributed around the median and the con¢dence intervals; the other 50% remained distributed in each bar The relationships between haemolymph osmol- Physiological responses of pink abalone Z M Romo et al ality and medium osmolality were determined using a linear regression after satisfying the test for suitability of ¢t to a linear model (SIGMA STAT version 3.1) A two-way analysis of variance (ANOVA) was used as previous determination of the normality and homoscedasticity of the data (SIGMA STAT), to determine the e¡ect of the temperature and salinity on oxygen consumption, ammonium excretion, atomic ratio O:N and the osmolality of the haemolymph of pink abalone (Zar 1999) Results The rate of oxygen consumption of pink abalone maintained at 20 1C and acclimated at di¡erent salinities was in the range of 0.58^0.79 mg O2 h À g À w.w In the organism acclimated to 24 1C, oxygen consumption was in the range of 0.64^0.81mg O2 h À g À w.w (Fig 1) An ANOVA indicated that temperature and salinity did not have a signi¢cant e¡ect on the oxygen Figure Oxygen consumption rate of Haliotis corrugata acclimated to two temperatures at di¡erent salinities The zone marked by circles represents the 95% con¢dence interval of the median; 50% of the data are distributed in vertical lines r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 955 Physiological responses of pink abalone Z M Romo et al Figure Ammonium excretion of Haliotis corrugata acclimated to two temperatures at di¡erent salinities The zone marked by circles represents the 95% con¢dence interval of the median; 50% of the data are distributed in vertical lines consumption rate of the pink abalone (d.f 55, 1, F 2.15,1.12, P 0.34) The ammonium excretion of the pink abalone at 20 1C increased at salinities of 20% and 23% and reduced when it was increased, yielding the lowest excretion value (0.25 mg NH4 h À g À w.w.) at the salinity of 35% In abalone maintained at 24 1C, the ammonium excretion rate increased when the organism was exposed to 20% and 23% salinities (Fig 2) Ammonium excretion decreased as salinity increased until the rate was in the range of 0.30^ 0.35 NH4 h À g À w.w An ANOVA indicated that there was a signi¢cant e¡ect of salinity on the ammonium excretion rate of H corrugata (d.f 55, 1, F 516.39, 12.72, Po0.05) The index of O:N values estimated for the juveniles of H corrugata acclimated to 20 1C was in the range of 23.9^28.8 in the organisms acclimated at salinities of 29^35% The lowest values of the O:N index found in the acclimated juveniles exposed to 20% and 23% salinities were 4.9 and 6.2 respectively (Fig.3) In abalones acclimated to 24 1C, the lowest values of the 956 Aquaculture Research, 2010, 41, 953^960 Figure Atomic ratio O:N (median Æ con¢dence interval) of Haliotis corrugata acclimated at two temperatures at di¡erent salinities O:N atomic ratio of 6.8 and 7.7 were obtained at salinities of 20% and 23%; the highest value of 35.5 was found in the juveniles acclimated to 35% salinity (Fig 3) An ANOVA indicated that salinity had a signi¢cant e¡ect (d.f 55,1, F 43.01,1.03, Po0.001) on the atomic ratio Osmolality of haemolymph in the abalone juveniles acclimated to 20 and 24 1C was related in a linear way with respect to the external medium, yielding the equations: IM in 20 C ¼ 69:25 þ 0:949X r2 ¼ 0:996 IM in 24 C ¼ À14:82 þ 1:045X r2 ¼ 0:986 where IM (internal medium) is the haemolymph osmolality and X is the external medium osmolality For the abalones acclimated to 20 1C and exposed to experimental salinities the haemolymph osmolality was in the range of 675^1002.7 mmol kg À 1, having a slightly hyperiso-osmoconformer pattern of osmoregulation (Fig 4) In the organism acclimated to 24 1C, haemolymph osmolality was in the range r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 Aquaculture Research, 2010, 41, 953^960 Figure Relation between haemolymph osmolality of Haliotis corrugata and medium osmolality when they were exposed to two temperatures at di¡erent salinities of 691^1054 mmol kg À 1, indicating that abalones had a slightly hyperiso-osmoconformer pattern (Fig 4) An ANOVA indicated that temperature and salinity had a signi¢cant e¡ect (d.f 55, 1, F 5168.3, 17.85, Po0.001) on the haemolymph concentration of pink abalone; the interaction between temperature and salinity did not have a signi¢cant e¡ect (P40.05) Discussion Oxygen consumption and ammonium excretion rates in marine invertebrates are a¡ected by body size, diurnal rhythm, feeding and environmental parameters such as temperature and salinity to which organisms are exposed (Crear & Forteath 2000; Salvato et al 2001; Ahmed, Segawa, Yokota & Watanabe 2008) Physiological responses of pink abalone Z M Romo et al In our experiment, the oxygen consumption rate was independent of salinity and temperatures, suggesting that abalone could adjust their metabolism after an acclimation period to di¡erent experimental conditions to which they were exposed In the abalone H discus hannai exposed to chlorinities higher than 14%, Sano and Maniwa (1962) obtained that the rate of oxygen consumption was kept constant In Argopecten purpuratus (Lamarck) exposed to a combination of two temperatures and three salinities for a period of 45 days; Soria et al (2007) reported that the rate of oxygen consumption was maintained independent In aquatic organisms that have been acclimated to di¡erent salinities, Kinne (1967) described four types of metabolic responses Pink abalone exposed to di¡erent salinities exhibited the type I response, because the oxygen consumption was not modi¢ed signi¢cantly For other marine organisms, it has been shown that salinity did not have a pronounced e¡ect on the oxygen consumption when the experimental organisms were acclimated to salinities and these are not extreme (Bishop et al 1980; Gaudy & Sloan 1981; Salvato et al 2001) Many osmoconforming marine invertebrates maintain FAA pools that vary directly with external salinity and this allows the preservation of cellular volumes by changing haemolymph osmolality (Tang, Liu, Yang & Xiang 2005) The ammonium excretion of pink abalone increased when salinity decreased from 35% to 23%, and this response may be related to an increase in catabolism of the amino acids to form intracellular osmolytes to regulate their osmotic equilibrium when exposed to lower salinities Under hyperosmotic stress, pink abalone shows an enhancement in the concentration of intracellular £uid by shrinking the cell volume from the environment absorbing ions and catabolizing self-tissue protein All these metabolic activities lead to increased ammonium excretion In Meretrix meretrix (Linnaeus) (Tang et al 2005) and A purpuratus exposed to decreasing and increasing salinities (Navarro & GonzaŁlez 1998); Soria et al (2007) reported an increase in the ammonium excretion rate, suggesting that it regulates the cellular volume by breakdown of amino acids as intercellular regulators with a reduction in salinity A high value of O:N is taken to represent a predominance of lipid and/or carbohydrate degradation over protein degradation (Mayzaud & Conover 1988) Ikeda (1977) reported an O:N ratio of 24 when protein and lipids were metabolized in equal quantities at the same time; hence, an O:N ratio o24 indicates a protein-dominant metabolism and a ratio 424 indicates r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 957 Physiological responses of pink abalone Z M Romo et al a lipid-dominant metabolism Factors such as season, temperature and salinity may in£uence the value of O:N (Ikeda 1977; Mayzaud & Conover 1988) In the present work, the O:N ratio found for abalone exposed to lower salinities had values of 4.9^7.7, implying that the organisms used protein catabolism as a primary catabolic substrate, due to the stress caused by exposure to lower salinities At intermediate salinities (23% and 26%), the organisms yielded an O:N of 16^24, indicative of protein and lipid catabolism in equal levels for organisms exposed to both temperatures In abalone acclimated to 24 1C and exposed to high salinities, the O:N ratios were 35.5, indicating that under these conditions, lipid and carbohydrate were the metabolic substrates used by abalones In Argopecten irradians concentricus (Say), Barber and Blake (1985) reported the same trend We observed a shift from protein to protein^lipid and lipid^carbohydrate as the source of energy metabolism associated with an increase in experimental salinities and a temperature In abalones exposed to high salinities and a temperature of 24 1C, the organisms found in environment optimum due to D|¤ az, Re, Medina, Re, Valdez and Valenzuela (2006) reported for H corrugata that the preferred and optimal of growth temperature were 25 and 24.5 1C for abalones maintained in 35%; under salinity condition, pink abalone used lipid^carbohydrates as the metabolic substrate This indicates that these combinations of salinity and temperature not produce stress in the organism, and therefore, we recommend these conditions for adequate maintenance of H corrugata under culture conditions Osmotic regulation in mollusks has been reported for some species of bivalves such as Argopecten ventriculosus-circularis (Sowerby II) (Signoret-Brailovsky, Maeda-Martinez, Reynoso-Granados, Soto-Galera, Monsalvo-Spencer & Valle-Meza 1996), Crassostrea gigas (Thunberg) (Hosoi, Kubota,Toyohara & Hayashi 2003) and the abalone H diversicolor supertexta (Lischke) (Cheng et al 2002) These papers indicated that the haemolymph osmolality varies directly with medium osmolality In the present study, pink abalone maintained an internal medium that was slightly hyperiso-osmoconformed, in contrast to the ¢nding of Cheng et al (2002) for Haliotis diversicolor supertexta, which had a slightly hypoiso-osmoconformed regulation pattern; the di¡erences in the osmoregulation pattern due to the process of transference to the experimental salinities in H diversicolor supertexta were drastic, and these conditions were maintained for days For pink abalone, the physio- 958 Aquaculture Research, 2010, 41, 953^960 logical changes were gradual and once the experimental conditions were reached, the abalones were maintained for 15 days In Argopecten ventriculosuscircularis, Signoret-Brailovsky et al (1996) observed an osmoconformer regulation pattern In C gigas exposed to gradual and sudden changes in salinities,an osmoregulation osmoconformator pattern was reported (Hosoi et al 2003) The osmoconformer marine organisms, including abalones, adapt to salinity changes using intracellular isosmotic regulation, in which intracellular FAA contribute predominantly to intracellular osmolality and to cell volume regulation (Shumway 1977; Signoret-Brailovsky et al 1996; Cheng et al 2002; Hosoi et al 2003) The slope regression line calculated from the relationship between haemolymph osmolality and medium osmolality was parallel to the isosmotic line, with values from 0.949 to 1.045 similar to those reported by Cheng et al (2002) for H diversicolor supertexta exposed to di¡erent salinity levels Acknowledgements We thank Jose M Dominguez and Francisco Javier Ponce from the Drawing Department of CICESE for preparing the ¢gures We are also grateful for the English language editing of the manuscript provided by Dr A Leyva References Ahmed F., Segawa S.,Yokota M & Watanabe S (2008) E¡ect of light on oxygen consumption and ammonia excretion in Haliotis discus discus, H gigantean, H madaka and their hybrids Aquaculture 279, 160^165 Altinok I & Grizzle J.M (2003) E¡ects of low salinities on oxygen consumption of selected euryhaline and stenohaline freshwater ¢sh Journal of the World Aquaculture Society 34, 113^117 Barber B.J & Blake N.J (1985) Substrate catabolism related to reproduction in the bay scallop Argopecten irradians concentricus, as determined by O/N and RQ physiological indexes Marine Biology 87,13^18 Bayne B.L & Newell R.C (1983) Physiological energetics of marine mollusks The Mollusca 4, 407^515 Beiras R., Pe¤rez-Camacho A & Albentosa M (1993) In£uence of food concentration on energy balance and growth performance of Venerupis pullastra seed reared in an open £ow system Aquaculture 116, 353^365 Bishop J.M., Gosselink J.G & Stone J.H (1980) Oxygen consumption and hemolymph osmolality of brown shrimp, Penaeus aztecus Fishery Bulletin 78, 741–757 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 Aquaculture Research, 2010, 41, 953^960 Bricelj V.M & Shumway S (1991) Physiology: energy acquisition and utilization In: Scallops: Biology, Ecology and Aquaculture (ed by S.E Shumway), pp 305^346 Elsevier, Amsterdam, the Netherlands Brougher D.S., Douglass L.W & Soares J.H (2005) Comparative oxygen consumption and metabolism of striped bass Morone saxatilis and its hybrid M chrysops , Â M saxatilis < Journal of theWorld Aquaculture Society 36, 521^529 Brown A.C & Da Silva F.M (1979) The e¡ects of temperature on oxygen consumption in Bulla digitalis (Gastropoda, Prosobranchiata) Comparative Biochemistry and Physiology 62A, 573^576 Brown A.C & Terwilliger N.B (1999) Developmental changes in oxygen uptake in Cancer magister (Dana) in response to changes in salinity and temperature Journal of Experimental Marine Biology and Ecology 241,179^192 Burton R.F (1983) Ionic regulation and water balance In: The Mollusca Physiology,Vol (ed by A.S.M Saleuddin & K.M.Wilbur), pp 291^352 Academic Press, NewYork, NY, USA Cheng W.,Yeh S.P.,Wang C.S & Chen J.C (2002) Osmotic and ionic changes in Taiwan abalone Haliotis diversicolor supertexta at di¡erent salinity levels Aquaculture 203, 349^357 Cli¡ord H.C & Brick R.W (1979) A physiological approach to the study of growth and bioenergetics in the fresh water shrimp Macrobrachium rosenbergii Proceedings of World Mariculture Society 10, 701–719 Crear B.J & Forteath G.N.R (2000) The e¡ect of extrinsic and intrinsic factors on oxygen consumption by the southern rock lobster Jasus edwardsii Journal of Experimental Marine Biology and Ecology 252, 129^147 D|¤ az F., Re A.D., Medina Z., Re G., Valdez G & Valenzuela F (2006) Thermal preference and tolerance of green abalone Haliotis fulgens (Philippi, 1845) and pink abalone Haliotis corrugata (Gray,1828) Aquaculture Research 37, 877^884 D|¤ az F., Re A.D., GonzaŁlez R.A., SaŁnchez L.N., Leyva G & Valenzuela F (2007) Temperature preference and oxygen consumption of the largemouth bass Micropterus salmoides (Lace¤pe'de) acclimated to di¡erent temperatures Aquaculture Research 38,1387^1394 Gaudy R & Sloane L (1981) E¡ect of salinity on oxygen consumption in postlarvae of the penaeid shrimp Penaeus monodon and P stylirostris without and with acclimation Marine Biology 65, 297^301 Hahn K.O (1989) Survey of the commercially important abalone species in the world In: Handbook of Culture Abalone and Other Marine Gastropods (ed by K.O Hahn), pp 3^11 CRC Press, Boca Raton, FL, USA Hildreth J.E & Stickle W.B (1980) The e¡ects of temperature and salinity on the osmotic composition of the southern oyster drill Thais haemastoma Biological Bulletin 159, 148^161 Hosoi M., Kubota S., Toyohara M., Toyohara H & Hayashi I (2003) E¡ect of salinity change on free amino acid content in Paci¢c oyster Fisheries Science 69, 395^400 Physiological responses of pink abalone Z M Romo et al Ikeda T (1977) The e¡ect of laboratory conditions on the extrapolation of experimental measurements to the ecology of marine zooplankton IV Changes in respiration and excretion rates of boreal zooplankton species maintained under fed and starved conditions Marine Biology 41, 241^252 Kinne O (1967) Physiology of estuarine organisms with special reference to salinity and temperature In: Estuaries (ed by G.H Lau¡), pp 525^540 American Association for the Advance of Science,Washington, DC, USA Lemos D., Phan V.N & Alvarez G (2001) Growth, oxygen consumption, amomonia-N excretion, biochemical composition and energy content of Farfantepenaeus paulensis Perez-Farfante (Crustacea, Decapoda, Penaeidae) early postlarvae in di¡erent salinities Journal of Experimental Marine Biology and Ecology 261, 55^74 Livingstone D.R.,Widdows J & Fieth P (1979) Aspects of nitrogen metabolism in the common mussel Mytilus edulis: adaptation to abrupt and £uctuating changes in salinity Marine Biology 53, 41^55 Mangum C.P., Silverthorn S.U., Harris J.L.,Towle D.W & Krall A.R (1976) The relationship between blood pH, ammonia excretion and adaptation to low salinity in the blue crab, Callinectes sapidus Journal of Experimental Zoology 195, 129^136 Mayzaud J.C & Conover R.J (1988) O:N atomic ratio as a tool to describe zooplankton metabolism Marine Ecology Progress Series 45, 289^302 Moore E.A & Sander F (1983) The e¡ect of temperature^salinity combinations on oxygen consumption of the tropical gastropod Murex pomun: a response surface approach Comparative Biochemistry and Physiology 77A, 679^683 Navarro J.M & GonzaŁlez C.M (1998) Physiological responses of the Chilean scallop, Argopecten purpuratus to decreasing salinities Aquaculture 167, 315^327 Newell R.C & Branch G.M (1980) The in£uence of temperature on the maintenance of metabolic energy balance in marine invertebrates Advances in Marine Biology 17, 329^396 Paul A.J & Paul J.M (1998) Respiration rate and thermal tolerances of pinto abalone Haliotis kamtschatkana Journal of Shell¢sh Research 17,743^745 Pressley T.A., Graves J.S & Krall A.R (1981) Amiloride-sensitive ammonium and sodium ion transport in the blue crab AmericanJournal of Physiology 241, 370^378 Re A.D., D|¤ az F & Go¤mez-Jime¤nez S (2004) Oxygen consumption, ammonium excretion and osmoregulatory capacity of Litopenaeus stylirostris (Stimpson) exposed to di¡erent combinations of temperature and salinity Ciencias Marinas 30, 443^453 Regnault M (1987) Nitrogen excretion in marine and freshwater crustacea Biological Reviews 62, 1^24 Rodier J (1998) AnaŁlisis de las aguas: aguas naturales, aguas residuales y agua de mar Omega, Barcelona Rosas C., Cuzon G., Gaxiola G., LePriol Y., Pascual C., Rossygnyol J., Contreras F., SaŁnchez A & VanWormhoudt A r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 959 Physiological responses of pink abalone Z M Romo et al (2001) Metabolism and growth of juveniles of Litopenaeus vannamei: e¡ect of salinity and dietary carbohydrate levels Journal of Experimental Marine Biology and Ecology 259, 1^22 Rosas C., Cuzon G., Gaxiola G., Taboada G., Arena L & VanWormhoudt A (2002) An energetic and conceptual model of the physiological role of dietary carbohydrates and salinity on Litopenaeus vannamei juveniles Journal of Experimental Marine Biology and Ecology 268, 47^67 Salvato B., Cuomo V., Di Muro R & Beltramini M (2001) Effects of environmental parameters on the oxygen consumption of four marine invertebrates: a comparative factorial study Marine Biology 138, 659^668 Sano T & Maniwa R (1962) Studies of the environmental factor having an in£uence on the growth of Haliotis discus hanai Bulletin Tohoku Regional Fisheries Research Laboratories 21,79^86 Saucedo P.E., Ocampo L., Monteforte M & Bervera H (2004) E¡ect of temperature on oxygen consumption and ammonia excretion in the Cala¢a mother-of-pearl oyster, Pinctada mazatlanica (Hanley,1856) Aquaculture 229, 377^387 Segawa S (1995) Preliminary experiment on the e¡ect of temperature on rates of oxygen consumption and ammonia excretion of young disk abalone Nordotid discus discus Suisanzoshoku 43, 219^224 Shumway S.E (1977) E¡ect of salinity £uctuation on the osmotic pressure and Na1, Ca21, and Mg21 ion concentrations in the hemolymph of bivalve molluscs Marine Biology 41,153^157 Signoret-Brailovsky G., Maeda-Martinez A.N., ReynosoGranados T., Soto-Galera E., Monsalvo-Spencer P & 960 Aquaculture Research, 2010, 41, 953^960 Valle-Meza G (1996) Salinity tolerance of the catarina scallop Argopecten ventricosos-circularis (Sowerby II, 1842) Journal of Shell¢sh Research 15, 623^626 Soria G., Merino G & von Brand E (2007) E¡ects of increasing salinity on physiological response in juvenile scallops Argopecten purpuratus at two rearing temperatures Aquaculture 270, 451^463 Stern S., Borut A & Cohen D (1984) The e¡ect of salinity and ion composition on oxygen consumption and nitrogen excretion of Macrobrachium rosenbergii Comparative Biochemistry and Physiology 79A, 271^274 Stickle W.B & Bayne B.L (1982) E¡ects of temperature an salinity on oxygen consumption and nitrogen excretion in Thais (Nucella) lapillus (L) Journal of Experimental Marine Biology and Ecology 58, 1^7 Tang B., Liu B.,Yang H & Xiang J (2005) Oxygen consumption and ammonia-N excretion of Meretrix meretrix in different temperature and salinity Chinese Journal of Oceanology and Limnology 23, 469^474 Tukey J.W (1977) Exploratory Data Analysis Adisson-Wesley, Reading, MA, USA Uki N & Kikuchi S (1975) Oxygen consumption of the abalone Haliotis discus hanai in relation to body size and temperature Bulletin Tohoku Regional Fisheries Research Laboratories 35, 73–84 Valde¤s-Urriolagoitia A.A (2000) Efecto de tres densidades de cultivo en la sobrevivencia y crecimiento de juveniles de abulo¤n rojo Haliotis rufescens en un laboratorio comercial Tesis Facultad de Ciencias Marinas UABC Zar J.H (1999) Biostatistical Analysis Prentice-Hall, Englewood Cli¡s, NJ, USA r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 953^960 Aquaculture Research, 2010, 41, 961^967 doi:10.1111/j.1365-2109.2009.02378.x Use of commercial fermentation products as a highly unsaturated fatty acid source in practical diets for the Pacific white shrimp Litopenaeus vannamei Tzachi M Samocha1, Susmita Patnaik1, Donald A Davis2, Robert A Bullis3 & Craig L Browdy4 AgriLife Research Mariculture Laboratory, AgriLife Research & Extension Center, Corpus Christi,TX, USA Department of Fisheries and Allied Aquacultures, Auburn University, Auburn, AL, USA Advanced BioNutrition, Columbia, MD, USA South Carolina Department of Natural Resources, Charleston, SC, USA Correspondence: Tzachi M Samocha, AgriLife Research Mariculture Laboratory, AgriLife Research & Extension Center, 4301 Waldron Rd., Corpus Christi,TX 78418, USA E-mail: t-samocha@tamu.edu Abstract Introduction Removal or reduction of marine ingredients (MI) from feed formulations is critical to the sustainability of the aquaculture industry By removing MI, diets may become limiting in several nutrients including highly unsaturated fatty acids (HUFA) such as docosahexaenoic acid (DHA) and arachidonic acid (ArA) To reduce reliance on MI in shrimp diets, two trials were conducted with Litopenaeus vannamei juveniles to determine the feasibility of using fermentation meals rich in DHA and ArA as the primary source for HUFA A practical diet with no MI was formulated with/without DHA and ArA supplements and fed in the ¢rst trial A diet with menhaden ¢sh oil or a combination of plant oil with/without DHA and ArA supplements was used in the second trial To determine whether HUFA is only needed in the early growth stages, we also fed one group a HUFA-supplemented diet to g and then switched them to a HUFA-supplement-free diet In both trials, the weights were reduced when HUFA supplements were not provided either throughout the trial or from g to harvest (o16 g) These results suggest that supplementation of plant oils with DHA- and ArA-rich oils from fermented products is a viable option to replace marine ¢sh oil for L vannamei Marine ¢sh meals and ¢sh oils are excellent sources of high-quality essential amino acids, lipids, vitamins, minerals and attractants in aquaculture diets (Tacon & Akiyama 1997) However, the unstable prices associated with £uctuations in the supply of these marine ingredients and the sustainability of these practices are of prime concern (Chamberlain 1993; Tacon & Akiyama 1997; Naylor, Goldberg, Primavera, Kautsky, Beveridge, Clay, Folk, Lubchenco, Mooney & Troell 2000) Hence, replacement of these marine ingredients with cost-e¡ective alternative sources of proteins and lipids in aquaculture feeds is a high-priority task for feed mills and aquaculturists (Tacon & Akiyama 1997) Previous studies (Lim 1996; Davis & Arnold 2000; Samocha, Davis, Saoud & DeBault 2004; Menoyo, Lopez-Bote, Obach & Bautista 2005) showed that either animal or plant sources can be used as suitable substitutes for ¢sh meal and ¢sh oil in a small-scale tank system In their e¡orts to replace ¢sh meal, researchers used plant protein sources such as soybean meal (Sudaryono, Hoxey, Kailis & Evans 1995; Hertrampf & Piedad-Pascual 2000; Olvera-Novoa & Olivera-Castillo 2000), solvent-extracted cotton seed meal (Lim 1996), lupin meals (Sudaryono et al 1995), legumes, leaf meals (Eusebio & Coloso 1998; Li, Robinson & Hardy 2000) and papaya or camote leaf meal (Pena£orida 1995) in feed formulations for aquatic animals, with varying Keywords: DHA, ArA, practical diets, Paci¢c white shrimp, Litopenaeus vannamei r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd 961 Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al Table Results of MIC test of enro£oxacin against six strains of bacteria Aquaculture Research, 2010, 41, 1095^1100 Table Inhibitory test of berberine hydrochloride combined with enro£oxacin to Streptococcus dysgalactiae (0722XY) Enrofloxacin concentrations (lg mL À 1) Stains 1.6 0.8 0.4 0.2 0.1 0.05 0.1/4 0.1/8 XS9141 56-12-10 0722XY HSN-1 Ecgy0204 ATCC25922 À À À À À À À À À À À À 1 À À 1 À À 1 À À 1 À À 1 À À 1 1 1 Enrofloxacin (lg mL À 1) Agents and concentration 1, represents visible bacterial growth; À, represents no visible bacterial growth ATCC, American Type Cultures Collection; MIC, minimum inhibitory concentration Table Inhibitory test of berberine hydrochloride combined with enro£oxacin to Escherichia coli (ATCC25922) Enrofloxacin (lg mL À 1) Agents and Concentration À À À À À À À À À À 1 À À 1 1 À À 1 1 £oxacin against E coli decreased further to 0.00625 mg mL À In the same way, sub-MIC of enro£oxacin also enhanced the antibacterial e¡ect of berberine hydrochloride, lowered its MIC against E coli from 300 to 50 mg mL À when 1/2MIC or 1/4MIC of enro£oxacin was used The calculated FIC was 0.67, indicating a synergism e¡ect A similar synergistic action against E ictaluri between berberine hydrochloride and enro£oxacin was observed (Table 6) Sub-MIC (450 mg mL À 1) of berberine hydrochloride can reduce the MIC of enro£oxacin on E ictaluri two to eight times, i.e., from 0.025 to 0.003125 mg mL À In the same way, subMIC of enro£oxacin can enhance the antibacterial activity of berberine hydrochloride, and allows the MIC against E ictaluri to decrease from 300 mg mL À go down to 50 mg mL À The FIC calculated was 0.625 0.8 0.4 0.2 0.1 0.05 À À À À À À À À À À 1 1 À À À 1 1 À À À 1 1 À À À 1 1 À À À 1 1 1, represents visible bacterial growth; À, represents no visible bacterial growth Table Inhibitory test of berberine hydrochloride combined with enro£oxacin to Edwardsiella ictaluri (HSN-1) Enrofloxacin (lg mL À 1) Agents and concentration À À À À À À À 1, represents visible bacterial growth; À, represents no visible bacterial growth ATCC, American Type Cultures Collection 1098 Berberine hydrochloride (mg mL À 1) 300 À 200 À 150 À 100 À 70 À 40 À À 0.1/2 0.1/4 0.1/8 0.1/16 0.1/32 Berberine hydrochloride (mg mL À 1) 400 À 300 À 200 À 150 À 100 À 50 À À 1.6 0.1/2 0.1/4 0.1/8 0.1/16 0.1/32 Berberine hydrochloride (mg mL À 1) 400 À 300 À 200 À 150 À 100 À 50 À À À À À À À À À À À À À À À À À À 1 1 À À 1 1 À 1 1 1 1, represents visible bacterial growth; À, represents no visible bacterial growth Berberine hydrochloride did not increase the inhibitory e¡ect of enro£oxacin against S dysgalactiae (Table 5) The FIC calculated was The MBCs of berberine hydrochloride for E coli, S dysgalactiae, E ictaluri were 400, 300 and 500 mg mL À respectively The MBCs of enro£oxacin for E coli, S dysgalactiae and E ictaluri were 0.2, 3.2 and 0.1 mg mL À respectively The MBCs of berberine hydrochloride in combination with enro£oxacin for E coli, S dysgalactiae and E ictaluri were 0.00625 mg mL À of enro£oxacin plus 300 mg mL À of berberine hydrochloride, 0.4 mg mL À of enro£oxacin plus 200 mg mL À of berberine hydrochloride and 0.00625 mg mL À of enro£oxacin plus 300 mg mL À of berberine hydrochloride respectively These results demonstrated that the enro£oxacin enhanced the bactericidal e¡ect of berberine hydrochloride and vice versa r 2009 Aihua Li Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100 Aquaculture Research, 2010, 41, 1095^1100 Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al Discussion The MICs of berberine hydrochloride for theA hydrophila, P £uorescens, V harveyi, E coli, E ictaluri and S dysgalactiae stains were ! 500, ! 500, ! 500, 400, 300 and 150 mg mL À 1, respectively, which are much higher than those of commonly used antibiotics S dysgalactiae was most sensitive to berberine hydrochloride among the organisms tested in the present study This ¢nding is in coincidence with a previous study, which showed that the antibacterial e¡ect of berberine on Staphylococcus aureus, Bacillus subtilis were stronger than on E coli (Wang, Yan & Gao 2006) This results show that berberine hydrochloride can inhibit the growth of E ictaluri, E coli and S dysgalactiae at a concentration o400 mg mL À 1, but berberine hydrochloride alone did not show e¡ective inhibition against A hydrophila, P £uorescens andV harveyi in vitro even at a concentration of 500 mg mL À Combination of berberine hydrochloride and enro£oxacin showed a signi¢cant synergistic action against E ictaluri and E coli (FIC 0.625 and 0.67 respectively), but did not show such a e¡ect on S dysgalactiae (FIC 51) Considering S dysgalactiae is a Gram-positive bacterium, and E ictaluri and E coli are Gram-negative bacteria, does this mean that the synergistic action between berberine hydrochloride and enro£oxacin can only occur on Gram-negative bacteria? Of course, it needs a more detailed study This result clearly indicated that berberine hydrochloride has signi¢cant antibacterial activity against E ictaluri and E coli when used together with enro£oxacin The synergistic action, however, has not been demonstrated in vivo studies The ¢nding that the antibacterial e¡ect of berberine hydrochloride on Gram-positive bacteria (S aureus, B subtilis and S dysgalactiae) was stronger than on the Gram-negative bacteria tested (A hydrophila, P £uorescens, V harveyi, E ictaluri and E coli) probably owes to the di¡erence of structure and function of bacterial cell wall partly Whether the combination of berberine hydrochloride and enro£oxacin can reduce the risk of development of resistant bacteria to both antimicrobials also need further investigations Our results show that E ictaluri and E coli had higher susceptibility to enro£oxacin than A hydrophila, P £uorescens, S dysgalactiae and V harveyi had The MIC value of enro£oxacin against E coli (ACTT25922) was in accordance with that reported previously (Lykkeberg, Halling-SÖrensen & Jensen 2007) This result can be taken as a quality control for MIC assay There were several researchers exploring the mechanism behind the antimicrobial activity of berberine There was a report that berberine sulphate was bacteriostatic for streptococci and that sub-MICs of berberine blocked the adherence of streptococci to host cells (Sun, Courtney & Beachey 1988) Berberine speci¢cally blocks the synthesis and assembly of Pap ¢mbriae on the surface of E coli CI6 cell in the presence of 200 mg mL À berberine for 18 h (Sun, Abraham & Beachey 1988) In the presence of 1^50 mg mL À berberine, adhesion and intracellular invasion ability of methicillin-resistant S aureus were notably decreased (Yu, Kim, Cha, Kim, Lee, Choi & You 2005) Berberine was also found to have e¡ects on toxins and plasmids of bacteria Berberine might not allow the formation of active and intact cholera toxins (Modak, Modak & Venkatraman 1970) Berberine also markedly inhibited the secretory response of E coli heat-stable enterotoxin in the infant mouse model (Sack & Froehlich 1982) Berberine sulphate has some e¡ects to eliminate the plasmids of E coli (Li, Wang & Hu 1994) In conclusion, berberine is a multifunctional antimicrobial agent, which has a potential to be used in aquaculture Further studies are de¢nitely needed to determine the exact underlying mechanism of the antibacterial e¡ect of berberine and the combination with antibiotics Coptis chinensis Franch, which is an important Chinese herb and is an important source of berberine, has been used widely in Chinese aquaculture to treat ¢sh diseases and to promote ¢sh immune system with di¡erent levels of success (Commission of Chinese Veterinary Pharmacopoeia 2005) Disease problem is a major threaten to aquaculture in China Unfortunately, antimicrobial treatment is currently the major control method However, as in many of other countries, antibiotics are facing tougher control on the use on food-producing animals, including on aquaculture ¢sh in China Products from plants are receiving greater attention for the substitution of chemical antimicrobials for this purpose The ¢ndings of the present study proved that a Chinese herb extract, berberine, was e¡ective in controlling some ¢sh pathogenic bacteria, suggesting its potential as an antimicrobial agent for aquaculture use, especially when used in combination with antibiotics By this way, it will reduce the usage of antibiotics to a certain extent To our knowledge, this is the ¢rst study on the inhibitory e¡ect of berberine hydrochloride against r 2009 Aihua Li Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100 1099 Antibacterial e¡ect of berberine hydrochloride and enro£oxacin D Zhang et al ¢sh pathogenic bacteria when in combination with antibiotics Acknowledgments We thank W M.Yang, C Ji, J.Y Liu and X N Gong for their technical assistance This study was supported by the National Science and Technology Pillar Programs (Grants No 2006BAD03B04 and No 2006BAK02A22) The authors are indebted to the National Science Foundation of China for their support of the project (No.30670112) References Amin A.H., Subbaiah T.V & Abbasi K.M (1969) Barbering sulfate: antimicrobial activity, bioassay and mode of action Canadian Journal of Microbiology 15,1067^1076 Casolari C., Rossi T., Baggio G., Coppi A., Zandomeneghi G., Ruberto A.I, Farina C., Fabio G., Zanca A & Castelli M (2004) Interaction between saquinavir and antimycotic drugs on C albicans and C neoformans strains Pharmacological Research 50, 605^610 Commission of Chinese Veterinary Pharmacopoeia (2005) Veterinary Pharmacopoeia of The People’s Republic of China,Vol.2 (pp.311^317 China Agriculture Press, Beijing, China Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards (2006) Methods forAntimicrobial Disk SusceptibilityTesting of Bacterial Isolated from Aquatic Animals, Approved Guideline (CLSI/ NCCLS Document M42-A) CLSI/NCCLS,Wayne, PA, USA Donovan T.J & van Netten P (1995) Culture media for the isolation and enumeration of pathogenicVibrio species in foods and environmental samples International Journal of Food Microbiology 26,77^91 Enriz R.D & Freile M.L (2006) Structure-activity relationship of berberine and derivatives acting as antifungal compounds The Journal of the Argentine Chemical Society 94, 113^119 1100 Aquaculture Research, 2010, 41, 1095^1100 Li L.J., Wang Z & Hu W.Z (1994) Elimination of drug resistance plasmids from Shigella by nor£oxacin and berberine ChineseJournal of Infectious Diseases 12, 4^8 Lu A.P., Ding X.R & Chen K.J (2008) Current situation and progress in integrative medicine in China ChineseJournal of Integrative Medicine 14, 234^240 Lykkeberg A.K., Halling-SÖrensen B & Jensen L.B (2007) Susceptibility of bacteria isolated from pigs to tiamulin and enro£oxacin metabolites Veterinary Microbiology 121,116^124 Modak M.J., Modak S & Venkatraman A (1970) E¡ect of berberine on the fatty acid composition of Vibrio cholerae and Vibrio cholerae El tor The IndianJournal of Medical Research 58,1523^1525 Nakamoto K., Sadamori S & Hamada T (1990) E¡ects of crude drugs and berberine hydrochloride on the activities of fungi The Journal of Prosthetic Dentistry 64, 691^694 Quan H., CaoY.Y., Xu Z., Zhao J.X., Gao P.H., Qin X.F & Jiang Y.Y (2006) Potent in vitro synergism of £uconazole and berberine chloride against clinical isolates of Candida albicans resistant to £uconazole Antimicrobial Agents and Chemotherapy 50,1096^1099 Sack R.B & Froehlich J.L (1982) Berberine inhibits intestinal secretory response of Vibrio cholerae and E coli enterotoxins Infection and Immunity 35, 471^475 Subbaiah T.V & Amin A.H (1967) E¡ect of berberine sulfate on Entamoeba histolytica Nature 215, 527^528 Sun D.X., Abraham S.N & Beachey E.H (1988) In£uence of berberine sulfate on synthesis and expression of Pap ¢mbrial adhesin in uropathogenic E coli Antimicrobial Agents and Chemotherapy 32, 1274^1277 Sun D.X., Courtney H.S & Beachey E.H (1988) Berberine sulfate blocks adherence of Streptococcus pyogenes to epithelial cell, ¢bronectin, and hexadecane Antimicrobial Agents and Chemotherapy 32, 1370^1374 Wang J.T.,YanY.M & Gao Q.Z (2006) Study on the bacteriostasic activity of berberine by micocalorimetry Journal of Qufu Normal University (NATURAL SCIENCE) 32, 99^103 Yu H.H., Kim K.J., Cha J.D., Kim H.K., LeeY.E., Choi N.Y & You Y.O (2005) Antimicrobial activity of berberine alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus Journal of Medicinal Food 8, 454^461 r 2009 Aihua Li Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1095^1100 Aquaculture Research, 2010, 41, 1101^1106 doi:10.1111/j.1365-2109.2009.02391.x SHORT COMMUNICATION Gastrointestinal evacuation time in gilthead seabream (Sparus aurata) according to the temperature Ana AŁlvarez, Benjam|¤ n Garc|¤ a Garc|¤ a, Jesu¤s Cerezo Valverde, Felipe Aguado Gime¤nez & Mar|¤ a Dolores HernaŁndez IMIDA Acuicultura Consejer|¤ a de Agricultura yAgua de la Regio¤n de Murcia, Murcia, Spain Correspondence: Ma D HernaŁndez, IMIDA Acuicultura Consejer|¤ a de Agricultura y Agua de la Regio¤n de Murcia, PO 65, 30740 San Pedro del Pinatar, Murcia, Spain E-mail: mdolores.hernandez6@carm.es From a bioclimatic point of view, Mediterranean aquaculture is characterized by temperate-warm waters, with the typical winter^summer range along its coasts being 13^29 1C (Person-Le Ruyet, Mahe¤, Le Bayon & Le Delliou 2004) As a result, temperature is an important factor to consider for aquatic production in this area Gilthead seabream is one of the most widely cultivated species of ¢sh in the Mediterranean The optimal temperature for its culture ranges from 24 to 26 1C (Ravagnan1984; Barnabe¤ 1991; Garc|¤ a Garc|¤ a1994), and the ¢sh are sent to market when they attain a body weight between 300 g and 1kg, requiring 15^18 months in order to reach this minimum commercial weight During this period of time, gilthead seabream are subjected to temperature variations that occur in the Mediterranean throughout the year In ¢sh farming, in order to obtain ¢sh with completely empty digestive tracts (free of any food remains), the normal procedure is to fast them during the days before slaughter The minimum required fasting time depends mainly on the gastrointestinal evacuation rate (ER) Temperature is one of the variables that have been studied the most regarding its e¡ect on gastric evacuation (Bromley1994) However, to date, there is no information available on the e¡ect of temperature on gilthead seabream ER According to recent data (AŁlvarez, Garc|¤ a Garc|¤ a, Garrido & HernaŁndez 2008), starvation periods longer than that required to empty the digestive tract have detrimental e¡ects on the quality of the ¢nal product, which must be considered r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd The objective of our study was to estimate, using laboratory data, the time required for complete evacuation of the gastrointestinal tract in commercialsized gilthead seabream (400^500 g) at the average temperatures typically found in the Mediterranean during the di¡erent seasons of the year (15, 20 and 25 1C) This information would allow us to determine the minimum fasting times the ¢sh must be subjected to before being slaughtered, depending on the season of the year in which they are harvested Materials and methods Facilities and experimental design Gilthead seabream with an average weight of 200 g were obtained from a ¢sh farm located on the eastern Mediterranean coast of Spain and kept at the IMIDA aquaculture facilities (San Pedro del Pinatar, Murcia, Spain) until they reached the required experimental size A total of 120 commercial-size ¢sh (479.8 Æ 23.3 g) were used, divided among six 850-L circular tanks supplied with running seawater (salinity: 37 g L À 1; NO2À o0.1mg L À 1; NO3À o0.1mg L À 1; NH3o0.5 mg L À 1; pH:7.7) These tanks formed a part of a recirculating system and were equipped with biological ¢ltration, an ultraviolet lamp and a thermostat used to control the experimental temperature The tanks were placed under natural photoperiod conditions (37150 N, 0146 W) The dissolved oxygen level, which was measured twice a day, was maintained above saturation level (80%) 1101 Gastrointestinal evacuation time in gilthead seabream A AŁ lvarez et al The ¢sh were fed by hand to satiety twice a day, using a dry commercial feed from Skretting (Cojobar , Burgos, Spain) (EXCEL,6-mm-diameter pellets) (Table 1) The ¢sh were divided among three experimental groups of 40 animals each, with each experimental group being distributed into two tanks (20 ¢sh tank À 1) The circuit temperature was initially maintained at 24.9 Æ 0.6 1C during the weeks before the slaughter of the ¢rst experimental group Temperature was lowered (0.7 1C day À 1) until it reached a value of approximately 20 1C, and then maintained at 19.8 Æ 0.7 1C for weeks before the slaughter of the second group Finally, it was lowered (0.5 1C day À 1) until it reached approximately 15 1C, resulting in an average temperature of 15.0 Æ 0.4 1C over the weeks before the slaughter of the last group Each experimental group was fasted for days before slaughter in order to ensure evacuation of the digestive system, and then fed by hand to satiety (for approximately 20 at 25 and 20 1C, and 10 at 15 1C) Five animals were slaughtered at 0.5, 3, 6, 12, 24,36, 48 and 72 h after feeding In order to minimize the disturbance of the animals, these ¢sh were alternately taken from each of the two tanks into which each experimental group had been divided The ¢sh were anaesthetized with 2-phenoxyethanol (0.2 mL L À 1) and then weighed and slaughtered by a cut to the spinal cord They were then dissected in order to remove their digestive tracts The digestive tracts were weighed whole and then frozen until the time of the analysis After partially thawing the digestive tract, the stomach and the intestine were separated in order to remove the contents of each part separately Partial thawing facilitated the extraction of the intestinal contents and minimized contamination from material from the intestine itself (mucus or À1 Table Ingredients (g kg ) and proximate composition (g kg À of dry matter) of the diet Fish meal Soybean meal Corn gluten meal Fish oil Broad bean Peas Soybean oil Corn DDGS Wheat gluten Protein Fat Ash DDGS, distiller’s dried grains with soluble 1102 270 210 150 130 90 50 50 30 20 440 220 85 Aquaculture Research, 2010, 41, 1101^1106 sloughed epithelium) The contents from both sections were maintained for 24 h in an oven at 105 Æ 1C in order to obtain the dry weight The total digestive system contents were calculated by adding the gastric contents to the intestinal contents Statistical analysis Geometric mean and standard deviation values were calculated for each set of replicates, and a regression procedure was conducted in each of the datasets, using STATISTICA software The ER for each of the temperatures (T) studied was calculated based on the ratio between the time elapsed after feeding (t) and the gastrointestinal contents (GC), ¢tting the data to the following equation by means of regression analysis: GC ¼ n þ mt ð1Þ À1 where m, the straight-line slope, is the ER (g h ) The e¡ect of T on the ER, as well as the interaction between t and T, was analysed using multiple regression analysis; the data were ¢t to the following equation: GC ¼ a þ bT þ ct þ dtT ð2Þ This may be expressed as GC a1bT1(c1dT)t, where t coe⁄cient has a linear relationship with T in the following manner: b c1dT, when b, c and d are signi¢cantly di¡erent from zero The evacuation time was then calculated based on eqn 2, with GC 0, according to the following expression: t ¼ Àða þ bTÞ=ðc þ dTÞ ð3Þ Results and discussion Table shows the average weights and the average daily intake rate (DIR) for each experimental group Feed intake increased as temperature increased, with a greater daily intake at 25 1C (1.12%) as compared with 20 1C (0.65%) or 15 1C (0.18%) It is also well known that when food is unlimited, ingestion in- Table Average weight (g) and average daily intake rate (DIR) for each experimental group Temperature 24.9 Æ 0.6 1C 19.8 Æ 0.7 1C 15.0 Æ 0.4 1C Body weight DIR 491.6 Æ 16.6 1.12 Æ 0.07 484.3 Æ 22.2 0.65 Æ 0.05 463.1 Æ 21.1 0.18 Æ 0.01 DIR [(feed intake/mean body weight)/no days] Â 100 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1101^1106 Aquaculture Research, 2010, 41, 1101^1106 Gastrointestinal evacuation time in gilthead seabream A AŁlvarez et al Fitting the GC versus time (Fig.1) relationship at 25 and 20 1C yielded high R2 values, 0.9946 and 0.9463 respectively, that were highly signi¢cant (Po0.005) For 15 1C, the data demonstrated a poorer, albeit still statistically signi¢cant ¢t (0.806, Po0.006) This poorer goodness-of-¢t may be due to a greater data dispersion, particularly during the ¢rst 10 h, resulting from a reduced ER due to the temperature; in our work, the R2 value decreases with temperature It may also be that at low temperatures, the GC^t relationship is better ¢t by an exponential equation In fact, in this case the data were better ¢tted by the equation LnGC 0.1808 À 0.0042 t, with an R2 value of 0.9578 (Po0.006) However, this better ¢t may be more because of the result of the data distribution observed than the physiological process itself, which is something that must be experimentally veri¢ed In any case, the ER increased as the temperature rose, with values of À 0.0042 g h À at 15 1C, À 0.0093 g h À at 20 1C and À 0.0273 g h À at 25 1C Similar results have been obtained for salmonids (Elliot1972; Sweka, Cox & Hartman 2004; Kawaguchi, Miyasaka & Genkai-Kato 2007; Handeland et al 2008) and other species (Parrish & Margraf 1990; Santulli et al 1993; PÌÌkk˛nen & MarjomÌki 1997; Temming et al 2002; Hurst 2004; Miyasaka et al 2005; Vinagre et al 2007) The point of intersection with the y-axis (n) re£ects the level of intake before slaughter (Fig 1) This level increased with temperature, and was far greater in the 25 1C group (0.652) A higher ER reduces the time required to empty the digestive tract The estimated evacuation times, indicated by the intercept of the regression line with the x-axis (Santulli et al 1993) are 23.6 h at 25 1C and 26.5 h at 20 1C (Fig 1) These values are similar; how- creases with increasing temperature, reaching a peak at the optimum temperature before declining steeply as the temperature approaches the species’ thermal limit (Jobling 1993; Yamashita, Tanaka & Miller 2001) For gilthead seabream, this occurs at around 27 1C (Garc|¤ a Garc|¤ a 1994) with the temperature limit being 30 1C Similar results have been obtained for other species (Santulli, Modica, Cusenza, Curatolo & D’Amelio 1993; Temming, Bohle, Skagen & Knudsen 2002; Handeland, Imsland & Stefansson 2008) A number of models have been proposed to describe the gastric ER in ¢sh (Bromley 1994) Linear models (Santulli et al 1993; Miyasaka, Kawaguchi, Genkai-Kato, Yoshino, Ohnishi, Kuhara, Shibata, Tamate, Taniguchi, Urabe & Nakano 2005; Adamidou, Nengas, Alexis, Foundoulaki, Nikolopoulou, Campbell, Karacostas, Rigos, Bell & Jauncey 2009) and exponential models (Vinagre, Maia & Cabral 2007) have probably been the most used Few studies exist that consider intestinal evacuation The gastric and intestinal contents extracted at each of the post-ingestion sampling points are shown in Table As the food bolus leaves the stomach, the intestinal contents increase, reaching a maximum value and then decreasing until they are evacuated The initial gastric contents at 25 1C were greater than at 20 or 15 1C Evacuation, both gastric and intestinal, was delayed in the experimental group subjected to 15 1C, as compared with the other two temperature groups After 24 h, ¢sh subjected to 25 and 20 1C conditions had completely emptied their stomachs, while the 15 1C group still had 0.009 g of contents At 36 h post ingestion, when the 25 and 20 1C groups had emptied their intestines, at 15 1C, the ¢sh had an average content of 0.016 g Table Gastric and intestinal content calculated as grams of dry matter as the percentage of body weight for each experimental group 25 1C 20 1C Time (h) Stomach 0.5 12 24 36 48 72 0.647 0.485 0.403 0.197 0.000 0.000 0.000 0.000 Æ Æ Æ Æ 0.16 0.29 0.11 0.08 Gut 0.001 0.054 0.086 0.079 0.001 0.000 0.000 0.000 15 1C Stomach Æ Æ Æ Æ Æ 0.01 0.02 0.01 0.03 0.01 0.206 0.179 0.126 0.080 0.000 0.000 0.000 0.000 Æ Æ Æ Æ 0.14 0.06 0.05 0.07 Gut 0.006 0.052 0.061 0.069 0.009 0.000 0.000 0.000 Stomach Æ Æ Æ Æ Æ 0.01 0.01 0.01 0.02 0.03 0.228 0.159 0.071 0.035 0.009 0.000 0.000 0.000 Æ Æ Æ Æ Æ 0.19 0.19 0.22 0.02 0.02 Gut 0.004 0.013 0.028 0.067 0.052 0.016 0.008 0.000 Æ Æ Æ Æ Æ Æ Æ 0.01 0.03 0.05 0.05 0.02 0.02 0.01 Geometric mean values of ¢ve samples Æ standard deviation r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1101^1106 1103 Gastrointestinal evacuation time in gilthead seabream A AŁ lvarez et al 25°C 0.8 GC=0.643 –0.0273t R2=0.9946; P[...]... (1993) Aquaculture trends and feed projections.World Aquaculture 24, 19^29 Davis D.A & Arnold C.R (2000) Replacement of ¢sh meal in practical diets for the Paci¢c white shrimp Litopenaeus vannamei Aquaculture 185, 291^298 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 961^967 Aquaculture Research, 2010, 41, 961^967 Alternative HUFA source for Litopenaeus... Publishing Ltd, Aquaculture Research, 41, 961^967 967 Aquaculture Research, 2010, 41, 968^972 doi:10.1111/j.1365-2109.2009.02379.x Initial influence of fertilizer nitrogen types on water quality Charles C Mischke1 & Paul V Zimba2 1 National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS, USA US Department of Agriculture, Agriculture Research Service, National Warmwater Aquaculture. .. P.V., Tucker C.S., Mischke C.C & Grimm C.C (2002) Short-term e¡ect of diuron on cat¢sh pond ecology North American Journal of Aquaculture 64, 16^23 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 968^972 Aquaculture Research, 2010, 41, 973^981 doi:10.1111/j.1365-2109.2009.02380.x The antioxidant capacity response to hypoxia stress during transportation... the guppy, Poecilia reticulate, for air transport in a closed system Aquaculture 78, 321^332 Terao J (1989) Antioxidant activity of beta-carotene-related carotenoids in solution Lipids 24, 659^661 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 973^981 Aquaculture Research, 2010, 41, 973^981 Antioxidant capacity response to hypoxia of characins C-H... ponds had higher concentrations of both nitrate and nitrite rela- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 968^972 969 Fertilizer nitrogen types and water quality C C Mischke & P V Zimba Aquaculture Research, 2010, 41, 968^972 3.00 Ammonium chloride Ammonium nitrate 2.50 Nitrate (mg N L-1) Calcium nitrate 2.00 Sodium nitrite Urea 1.50 1.00 0.50 0.00... concentrations in the pond, minimal deleterious effects on water quality (e.g., changes in ammonia and r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 968^972 Aquaculture Research, 2010, 41, 968^972 Fertilizer nitrogen types and water quality C C Mischke & P V Zimba Ammonium chloride 400 Desirable Zooplankton (Number L-1) Ammonium nitrate 350 Calcium nitrate... test diets were prepared in the feed laboratory of Auburn University, Auburn, AL, USA, using stan- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 961^967 Aquaculture Research, 2010, 41, 961^967 Alternative HUFA source for Litopenaeus vannamei T M Samocha et al Table 1 Diet formulation (% as is basis) for practical diets designed to contain 35% protein... laboratory in a 0.5 tonne tank, ¢sh were fed the control diet for 2 weeks to equalize their body CD r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 973^981 Aquaculture Research, 2010, 41, 973^981 Antioxidant capacity response to hypoxia of characins C-H Pan et al Table 1 Proximate analysis of the control and carotenoid diets Control diet Carotenoid type... superoxide dismutase; GPx, glutathione peroxidase; ALT, alanine transaminase; AST, aspartate transaminase r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 973^981 Aquaculture Research, 2010, 41, 973^981 Antioxidant capacity response to hypoxia of characins C-H Pan et al Table 3 After hypoxia stress,Ã the average and standard deviation (in parentheses) activities... This clearly shows that AX can protect liver damage caused by hypoxia stress better than BC and MX r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 973^981 Aquaculture Research, 2010, 41, 973^981 Antioxidant capacity response to hypoxia of characins C-H Pan et al In this study, ¢sh fed the CD supplement had lower SOD, GPx and ALT activities than control