Aquaculture Research, 2010, 41, 793^803 doi:10.1111/j.1365-2109.2009.02356.x Enrichment of the African catfish Clarias gariepinus (Burchell) with functional selenium originating from garlic: effect of enrichment period and depuration on total selenium level and sensory properties Edward Schram1, Rian A A M Schelvis-Smit1, Jan W van der Heul1 & Joop B Luten1,2 IMARES,Wageningen UR, IJmuiden,The Netherlands NoÂma Marine,Tromsệ, Norway Correspondence: E Schram, IMARES, Wageningen UR, PO box 68,1970 AB, IJmuiden,The Netherlands E-mail: edward.schram@wur.nl Abstract We wanted to optimize the procedure for the selenium enrichment of farmed African catÂsh, using garlic as dietary selenium source In the Ârst experiment we established the relation between the length of the selenium enrichment period and the resulting total selenium level in the Âllet of the Âsh It was found that at a dietary level of11.7 mg kg Se, a total selenium level in the Âllet of 0.7 mg kg was reached in a relatively short enrichment period of 10 days before harvest In the second experiment we studied the eĂect of depuration on the selenium level in the Âllet and the sensory properties of seleniumenriched African catÂsh It was found that total selenium levels in the Âllet were not aĂected during a 7-day depuration period, while garlic odours and Êavours in the raw and cooked Âllets were signiÂcantly reduced after days of depuration.We concluded that selenium enrichment of farmed African catÂsh can be obtained by selenium-enriched Ânishing diets, while garlic odours and Êavours resulting from dietary garlic can be eĂectively reduced in the Âllet during a short depuration period without negatively aĂecting Âllet levels of total selenium Keywords: African catÂsh, functional food, selenium, garlic, depuration, sensory properties Introduction Dietary selenium is essential for human health (Rayman 2000; Birringer, Pilawa & Flohe 2002) and at r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd concentrations above dietary requirement, selenium is reported in several studies as having anti-carcinogenic eĂects in humans (Ip 1998) However in some European countries the average daily intake is lower than the recommended daily intake (Rayman 2005) The need for selenium has resulted in an increase in selenium-rich functional foods (Dumont, Vanhaecke & Cornelis 2006), including the development of selenium-enriched farmed Âsh Functional food has been deÂned in a European consensus document as a food can be regarded as functional if it is satisfactorily demonstrated to aĂect beneÂcially one or more target functions in the body, beyond adequate nutritional eĂects, in a way that is relevant to either improved stage of health and wellbeing and/or reduction of risk of disease A functional food must remain food and it must demonstrate its eĂects in amounts that can normally be expected to be consumed in the diet: it is not a pill or a capsule, but part of the normal food pattern (Diplock, Aggett, Ashwell, Bornet, Fern & Roberfroid 1999) Aquaculture provides an excellent opportunity for the production of functional seafood products as many factors that determine the composition of the edible portion of Âsh can be controlled under farming conditions A number of studies have demonstrated the enhancement of the selenium concentration in the muscle tissue of Âsh as a result of elevated levels of dietary selenium (see Schram, Pedrero, Camara, Van der Heul & Luten 2008 for an overview) None of these trials used garlic as source of functional selenium species while Ip and Lisk (1996) showed that 793 Aquaculture Research, 2010, 41, 793803 Selenium enrichment of African catÂsh part II E Schram et al the anti-carcinogenic ecacy of the selenium species in garlic is superior to selenomethionine and selenite (in Ip 1998) In our previous work we took the Ârst step towards the development of farmed African catÂsh enriched with functional selenium using garlic as selenium source (Schram et al 2008) We established a dose^ response relation (feed to Âsh), the growth performance in relation to dietary selenium and garlic and a Ârst step was taken towards establishing the retention of functional selenium in the edible portion of the Âsh However, to eciently use the seleniumenriched garlic resources we also need to know the minimally required length of the selenium enrichment period to reach target concentrations in the Âsh Âllet In addition, the eĂect of depuration, a necessary procedure to eliminate oĂ-Êavours (Tucker 2000) during which Âsh are not fed, on the selenium level in the Âllet needs to be established to ensure targeted selenium levels are reached at harvest The use of garlic as a Âsh feed ingredient demands evaluation of the sensory properties of the Âsh at harvest and in relation to depuration, because the eĂects of dietary garlic on sensory properties of Âsh are unknown but likely to be present Feed component Control feed Seleniumenriched feed Wheat Wheat gluten Fish meal Fish oil Soybean meal Premix Binder Selenium-rich garlic Total selenium (mg kg 1) 223 100 450 60 125 17 25 2.1 223 100 450 60 111.5 17 25 13.5 11.7 All feed components are expressed as g kg wet weight, except for total selenium expressed as mg kg wet weight garlic with a high selenium content Garlic was supplied as a dry powder and included as such in the feed The control feed was the same except for the inclusion of garlic Experimental feeds were produced by Research Diets Services, the Netherlands, as mm steam pellets, with a proximate analysis of 46.8% crude protein,13.6% crude fat,1.1% crude Âbre and 10.1% crude ash The formulation of the experimental feeds is shown in Table Experiment 1: total selenium levels and selenium retention in relation to the length of enrichment period Materials and methods Introduction Two experiments were performed In the Ârst experiment Âsh were fed a selenium-enriched feed for six diĂerent periods before harvest to investigate the effect of the length of the enrichment period on the total selenium level in the Âllet at harvest Garlic was used as dietary selenium source Fillet samples were analysed for total selenium In the second experiment selenium enrichment was followed by a depuration period during which Âsh were sampled at intervals to assess the total selenium level and the sensory properties Experimental feeds For both experiments two feeds were used: seleniumenriched and a control feed Selenium enrichment was achieved by inclusion of selenium-enriched garlic This garlic was grown and processed by Plant Research International,Wageningen UR, the Netherlands, with selenium fortiÂcation of the soil as described in Larsen et al (2006), which resulted in 794 Table Formulation of the experimental feeds on the wet weight basis The six treatments consisted of six selenium enrichment periods of diĂerent length Groups of Âsh were fed the selenium-enriched feed for 35, 25, 17, 10 or days before harvest The experimental period lasted for 35 days for all treatments Fish were fed the control feed (Table 1) before receiving the seleniumenriched feed Table provides an overview of the treatments Treatments were randomly assigned to the tanks, with triplicate tanks for each of the six treatments The experimental set up consisted of 18, 30 L glass tanks placed in two rows of nine tanks Tanks were Êown through with tap water with a selenium level of o0.5 mg Se L 1at a rate of 10 L h Tank eƠuents were discharged as Âsh are known to take up waterborne selenium across the gills (Hodson et al 1980) and recirculation of water combined with leaching of selenium from the feeds and excretion by the Âsh could have resulted in transfer of selenium compounds between tanks.Water temperature was maintained at 25 1C throughout the experimental period Each tank was stocked with 14 African catÂsh r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 Aquaculture Research, 2010, 41, 793^803 Table Overview of the treatments in Experiment Control feed Selenium-enriched feed Treatment Feeding days Feeding days Proportion of total feed load (%) 110 118 125 130 135 135 1135 1935 2635 3135 100 76 53 34 13 Presented are for each treatment the experimental days during which the control (not selenium-enriched) feed and the selenium-enriched feed were fed and the relative proportion of the selenium-enriched feed load to the total feed load (Clarias gariepinus) at a sex ratio of one male to one female African catÂsh were obtained from FleurenNooijen BV, the Netherlands, a commercial African catÂsh hatchery After stocking, the Âsh were allowed to acclimatize to the experimental conditions for days, during which all Âsh were fed the control feed (Table 1) The day after the acclimatization period is referred to as day of the experimental period Fish were fed by hand to visually observe satiation twice daily (10:00 and 17:00 hours) The total feed load was documented for each tank Mortalities were recorded daily and dead Âsh were removed immediately from the tanks after detection On days and 35 (harvest), the total biomass and number of Âsh were determined for each tank The mean (SD) individual weight of 230 (6.7) g at day was not signiÂcantly diĂerent between tanks [analysis of variance (ANOVA), Po0.05] Before stocking of the experimental tanks on day 1, three males and three females were randomly sampled from the total stock On day 35, three males and three females were sampled randomly from each tank Sampled Âsh were Âlleted, Âllets were pooled per tank, homogenized and stored frozen at 70 1C Feed samples were taken at day of the experiment and stored frozen at 70 1C Selenium enrichment of African catÂsh part II E Schram et al during which Âsh were sampled at regular intervals The total experimental period lasted for 29 days Treatments were randomly assigned to the tanks, with triplicate tanks for each of the two treatments The experimental set up consisted of six, 400 L plastic tanks placed in two rows of three tanks Tank eƠuents were discharged as recirculation could have resulted in transfer of selenium compounds between tanks Each tank was stocked with 38 African catÂsh (C gariepinus) with a mean (SD) individual weight of 796 (11) g at a sex ratio of one male to one female The mean initial individual weight was not signiÂcantly diĂerent between tanks (ANOVA, Po0.05) After stocking, the Âsh were allowed to acclimatize to the experimental conditions for days, during which all Âsh were fed a commercial grower diet The day after the acclimatization period is referred to as day of the experimental period During the Ârst 21 days of experimental period the tanks were Êown through with 25 1C tap water with a selenium level of o0.5 mg Se L 1at a rate of 35 L h At day 22, the start of the depuration period, the Êow was increased to 60 L h On days and 22, the total biomass and number of Âsh was determined for each tank Before stocking of the experimental tanks on day 1, three males and three females were randomly sampled from the total stock On days 22, 23, 24, 25 and 29, concurring with days 0, 1, 2, and day of the depuration period, three males and three females were sampled randomly from each tank Sampled Âshed were Âlleted Per Âsh one Âllet was used for total selenium analysis and one Âllet for sensory analysis Fillets sampled for total selenium analysis were pooled per tank (six Âllets per tank per sampling point), homogenized and stored frozen at 70 1C Production parameters The speciÂc growth rate (SGR) for of the group of Âsh in each tank was calculated as follows: SGR ẳ lnWt ị lnW0 ịị Experiment 2: eĂect of depuration on total selenium level and sensory properties of the Âllet Treatments consisted of two diĂerent experimental feeds: control feed and selenium-enriched feed (Table 1) The experimental period consisted of two parts The Ârst part of the experimental period was a rearing period of 21 days The second part of the experimental period was an 8-day depuration period 100 T where SGR is the speciÂc growth rate (%BWday 1), BW is the body weight, Wt is the average individual weight at harvest (g), W0 is the average individual weight at stocking (g) and T is the number of days The feed conversion rate (FCR) per tank was calculated as follows: FCR ẳ r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 FL Wt Nt W0 N0 ị 795 Aquaculture Research, 2010, 41, 793803 Selenium enrichment of African catÂsh part II E Schram et al where FCR is the feed conversion rate (g g 1), FL is the total amount of feed administered to a tank during the experiment (g), Wt is the average individual weight at harvest (g), W0 is the average individual weight at stocking (g), Nt is the number of Âsh per tank at harvest and N0 is the number of Âsh per tank at stocking The retention of selenium in the Âsh Âllet was calculated as follows: ẵSet Wt Nt F ẵSe0 W0 N0 F Rẳ ẵSefeed FL 100% where R is the selenium retention rate in the Âsh Âllet (% of dietary intake), [Se]t is the total selenium concentration in the Âllet at harvest (mg kg 1),Wt is the average individual weight at harvest (kg), Nt is the number of Âsh per tank at harvest, F is the Âlleting yield (%), [Se]0 is the total selenium concentration in the Âllet at stocking (mg kg 1), W0 is the average individual weight at stocking (kg), N0 is the number of Âsh per tank at stocking, [Se]feed is the total selenium concentration in feed (mg kg 1) and FL is the total feed load (kg) Total selenium analysis Homogenized samples of the muscle tissue were microwave digested in microwave-lined digestion vessels using 70% (m/v) nitric acid (5 mL) and 30% (m/v) hydrogen peroxide (5 mL) as the oxidant mixture After digestion, the samples were reduced in the microwave by adding mL 37% (m/v) hydrochloric acid to convert Se61 into Se41 The resulting solution was diluted to a Ânal volume of 25 mL using 10% hydrochloric acid Blanks [5 mL 70% (m/v) nitric acid and mL 30% (m/v) hydrogen peroxide] were treated in the same way Total selenium was determined using hydride generation Flow Injection Analysis System^Atomic Absorption Spectrometry NaBH4 (0.2%) in NaOH (0.05%) was used to generate the H2Se The accuracy of the analyses was established by analysing the selenium content in the BCR-certiÂed Cod-CRM422 reference sample Sensory analysis The eĂect of dietary garlic and the eĂect of depuration on the sensory properties of the seleniumenriched African catÂsh were assessed in a sensory 796 intensity test in Experiment On days 19, 21, 22 and 26, concurring with days 0, 2, and of the depuration period, three males and three females were sampled randomly from each tank, yielding 18 Âsh per treatment From each Âsh, one Âllet was used for sensory analysis The Âsh were Âlleted on the day of slaughter and stored in vacuum at 25 1C On the day of testing six randomly selected Âllets per treatment were thawed for 60 in running cold tap water, pooled per treatment and homogenized by cooled mincing (type DRC compact 92, France) Samples (approximately 50 g) of the homogenized minced Âllets were put in small aluminium boxes with a three-digit random code and stored at 1C until preparation Samples were prepared by placing the aluminium boxes in 1cm boiling water in a hot air oven (Miele H 216, Miele, Germany) at 160 1C for Eight members of the Wageningen IMARES sensory panel participated in the sensory analysis of the African catÂsh samples All panelists were trained before sensory analysis in two1-h sessions using samples of all experimental treatments (selenium enriched, control) and sampling days (0, 1, and days of depuration) in accordance with international standards (ISO 1993) Panelists were trained to detect, recognize, describe and scale the intensity of odours and Êavours of raw and cooked catÂsh, using an existing sensory evaluation scheme for African catÂsh as a starting point The resulting vocabulary used to describe odours and Êavours (attributes) is listed inTable Intensities of each attribute were scaled using a nine-point intensity scale For each of the four sampling days, duplicates of the selenium-enriched Âllet Table Mean (SD, n 3) total selenium levels in African catÂsh Âllets and selenium retention (mean1SD, n 3) for diĂerent selenium enrichment periods before harvest No P-value LSD Treatment (no of selenium enrichment days) 35 25 17 10 Total selenium level (mg kg 1) Selenium retention (%) 0.99a (0.01) 0.87b (0.05) 0.80b (0.06) 0.71c (0.06) 0.41d (0.04) 0.21e (0.03) o0.05 0.075 6.46a (0.36) 6.83ab (0.50) 8.07bc (0.46) 9.05c (0.03) 7.92bc (1.34) 4.88d (0.99) o0.001 1.332 DiĂerent letters represent signiÂcantly diĂerent mean values (ANOVA, Po0.05) LSD, least square diĂerence of means at 5% signiÂcance level; SD, standard deviation; ANOVA, analysis of variance r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 Aquaculture Research, 2010, 41, 793^803 samples were evaluated in random order by each panelist in a single session A computerized system (FIZZ, version 2.10a 1994^2000, BiosysteÔmes, Couternon, France) was used for data recording Selenium enrichment of African catÂsh part II E Schram et al Table Mean (standard deviation, n 3) Ânal weight, speciÂc growth rate (SGR), feed conversion rate (FCR) for the treatments Statistics No Statistical analysis was conducted with GENSTAT 10.1 DiĂerences in mean values of SGR, feed conversion rate, weight gain, selenium retention and selenium levels between treatments were analysed using oneway ANOVA The relations between the length of the selenium enrichment period and the response variates selenium level in the Âllets and selenium retention were analysed with non-linear regression using a two straight-line model The individual mean body weights at the sampling days in the depuration period were tested for signiÂcant diĂerences between and within treatments using repeated measurements ANOVA Mean Âllet weights were tested for signiÂcant diĂerences among sampling days and between treatments using repeated measurements ANOVA In case of signiÂcance, one-way ANOVA and repeated measurements ANOVA were followed using the least significant diĂerence (LSD) post hoc analysis to detect the diĂerent mean values A signiÂcance level of 5% was used in all cases For the sensory analysis, the statistical analyses were performed in SPSS version 15.0 Mean values per sample and per attribute were calculated DiĂerences in the mean values were tested for signiÂcance using one-way ANOVA, followed by the least signiÂcant difference (LSD) post hoc analysis to detect the diĂerent mean values A signiÂcance level of 5% was used Pearsons correlation tests (two tailed) were performed to Ând correlations between attributes, for which a signiÂcance level of 1% was used P-value Results Experiment 1: total selenium levels and selenium retention in relation to the length of enrichment period The total selenium levels in the Âllets were diĂerent between treatments (ANOVA, Po0.001) and increased with increasing length of the selenium enrichment period (Table 4) The two straight-line regression model accounted for 98% of the variance and resulted in an inÊection point at a selenium enrichment period of 10.3 days (Fig 1) Selenium retention Treatment (Se feeding days before Final SGR FCR harvest) weight (g) (%BW day 1) (g g 1) 35 25 17 10 623 (25) 566 (6) 580 (27) 565 (46) 583 (18) 589 (3) 0.14 2.86 2.57 2.64 2.57 2.65 2.66 0.17 (0.14) (0.06) (0.14) (0.12) (0.21) (0.06) 0.79 0.84 0.83 0.89 0.86 0.90 0.56 (0.07) (0.01) (0.06) (0.10) (0.12) (0.02) 1.2 Total selenium level in the fillet (mg Se kg1) 1.0 Line 0.8 0.6 Line 0.4 0.2 0.0 10 15 20 25 30 35 Days of feeding selenium enriched feed prior to harvest 40 Figure Total selenium level (mg kg Se) in African catÂsh Âllets in relation to the length of the selenium enrichment period before harvest Line 1, selenium level Âllet 0.049 number of enrichment days10.214; line 2, selenium level Âllet 0.011 number of enrichment days10.612 InÊection point (10.3, 0.72) The two straightline regression model accounts for 98% of the variance in the Âllet was diĂerent between treatments (ANOVA, Po0.001) (Table 4) The two straight-line regression model accounted for 76% of the variance and resulted in a sharp inÊection point at a selenium enrichment period of 5.8 days (Fig 2) Selenium retention Ârst increased with an increasing length of the selenium enrichment period up to 5.8 days Beyond 5.8 days of feeding selenium-enriched feed before harvest, selenium retention decreased (Fig 2) Final weight (ANOVA, P 0.14), SGR (ANOVA, P 0.17) and FCR(ANOVA, P 0.56) were not diĂerent between treatments (Table 5) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 797 Aquaculture Research, 2010, 41, 793803 Selenium enrichment of African catÂsh part II E Schram et al 10 Table Sensory vocabulary for the selenium-enriched African catÂsh using dietary garlic as the selenium source Line2 Line1 0 10 15 20 25 30 35 Days of feeding selenium enriched feed prior to harvest 40 Figure Selenium retention (%) in African catÂsh Âllets expressed as the percentage of the total dietary selenium feed load in relation to the length of the selenium enrichment period before harvest Line 1, selenium retention 0.00762 number of enrichment days10.048; line 2, selenium level Âllet 0.001066 number of enrichment days10.099 InÊection point (5.81, 0.093) The two straightline regression model accounts for 76% of the variance Table Mean (SD, n 3) total selenium levels in the Âllets (mg kg 1) and mean (SD, n 3) Âllet weight (g) during the depuration period for the selenium-enriched Âsh and the Âsh that received the control feed Total selenium level in the fillet (mg kg ) Fillet weight (g) Depuration day Selenium enriched Control Selenium enriched Control 0.63 0.64 0.67 0.63 0.57 0.16 0.16 0.19 0.18 0.14 322 284 284 288 260 293 322 288 250 268 (0.02) (0.04) (0.01) (0.03) (0.06) (0.02) (0.04) (0.01) (0.00) (0.03) (7) (31) (32) (3) (51) (41) (12) (23) (37) (18) Depuration days 0, 1, 2, and concur with days 22, 23, 24, 25 and 29 of the experimental period Total selenium levels were equal within treatments for all sampling days during the depuration period for the selenium-enriched Âsh (ANOVA, P 0.10) and for the Âsh that received the control feed (ANOVA, P 0.14) Fillet weights were equal over time within and between treatments (repeated measurements ANOVA, Ptreatment time 0.31) SD, standard deviation; ANOVA, analysis of variance Experiment 2: eĂect of depuration on total selenium level in the Âllet After 21 days of Âsh rearing, the total selenium levels in the Âllets were 0.62 mg kg for the selenium- 798 Sensory attribute Short name Odour of the raw fillet R_O_metal Metallic R_O_garlic Garlic R_O_sour Sour Odour of the cooked fillet C_O_garlic Garlic C_O_cook potato Cooked potato Flavour of the cooked fillet C_T_musty Musty C_T_garlic Garlic C_T_cook potato Cooked potato Total selenium concentration in the fillet (mg Se kg1) Selenium retention in the fillet (% of Se load) Description of attribute Metallic odour Garlic odour, slight chemical Sour odour, spoilage Garlic odour, slight chemical Odour of cooked potatoes Like a wet cellar Garlic flavour, slight chemical Flavour of cooked potatoes 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 Depuration period (Days) Figure Total selenium concentration (mg kg Se) in the Âllet of African catÂsh in relation to the length of the depuration period for the selenium () and control treatment (^) For both the selenium treatment (simple linear regression, P 0.06, r2 50.25) and the control treatment (simple linear regression, P 0.31, r2 50.07), the linear relation is not signiÂcant enriched Âsh and 0.16 mg kg for the Âsh that received the control feed (Table 6) The Ânal total mean (SD, n 38) body weight after 21 days of Âsh rearing was 1083 (25) g for the selenium-enriched Âsh and 1071 (19) g for the Âsh that received the control feed Mean (SD, n 3) SGR was 1.46 (0.07)%BWday and 1.42 (0.10)%BWday Final weight (ANOVA, P 0.545) and SGR (ANOVA, P 0.614) were not diĂerent between treatments The length of the depuration period was found to have no eĂect on the total selenium levels in the Âllets of both the selenium-enriched Âsh (simple linear regression, P 0.06, Fig 3) and the Âsh that received the control feed (simple linear regression, P 0.31, r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 Aquaculture Research, 2010, 41, 793^803 Selenium enrichment of African catÂsh part II E Schram et al Fig 3) Within treatments, the total selenium level in the Âllet was equal for all sampling days for the selenium-enriched Âsh (ANOVA, P 0.10) and for the Âsh that received the control feed (ANOVA, P 0.14) (Table 6) African catÂsh lost weight during the depuration period (Fig 4) Mean individual weights were not diĂerent between treatments for each of the sampling days during the depuration period (repeated measurements ANOVA, P treatment time 0.45) Overall mean weights were diĂerent for sampling days 0, and (repeated measurements ANOVA, P time o0.001, Fig 4) By day of the depuration period, the overall mean weight loss was 8.7% of the individual weight at the start of the depuration period Mean individual Âllet weights were equal for all sampling days during the depuration period between treatments and within treatments (repeated measurements ANOVA, P time treatment 0.31,Table 6) Experiment 2: sensory properties of seleniumenriched African catÂsh Âllets in relation to depuration Raw and cooked samples of African catÂsh Âllets fed with selenium-enriched feed (with garlic as selenium source) were found to have a rather intense garlic odour and Êavour at day of the depuration period, which had signiÂcantly decreased at day of depuration period (Table 7, Fig 5) No changes in the intensity of these attributes were observed between days and of the depuration period The intensity of the odour of both the raw and cooked samples was signiÂcantly reduced between days and of the depuration period, while the intensity of the Êavour of the cooked Âllet stabilized at a minimal detectable level in this period (Fig.5) The garlic odour correlates with the metallic odour and with the low values of sour Sensory score (1-9/ not at all-very much) Average weight (g) 1200 1150 1100 A B 1050 1000 C C C 950 0 Depuration time (days) Figure Overall mean (n 6) individual weight (g) of African catÂsh during the depuration period Data points marked with diĂerent letters diĂer signiÂcantly (repeated measurements analysisof variance, Ptimeo0.001) Depuration period (Days) Figure Sensory intensity (score 1^9) in relation to the length of the depuration period for the garlic odour of the raw African catÂsh (^), the odour of the cooked African catÂsh (&) and the Êavour of the cooked African catÂsh () Table Mean (standard deviation, n 2) intensity scores of the sensory attributes for the Âllets during the depuration period for the selenium-enriched African catÂsh Results sensory intensity score depuration day Attributes R_O_metal R_O_garlic R_O_sour C_O_garlic C_O_cook potato C_T_musty C_T_garlic C_T_cook potato 4.57 6.94 3.32 4.94 5.13 3.82 5.82 5.13 (0.09) (0.44)a (0.26)a (0.08)a (0.53) (0.45) (0.80)a (0.18) 3.13 3.75 2.82 3.07 5.57 3.19 3.76 5.75 (0.53) (1.41)b (0.92)a (0.45)b (0.92) (0.27) (0.53)b (0.71) 2.94 3.19 2.19 2.82 5.69 3.01 3.57 5.82 P-value (0.44) (0.44)bc (0.85)b (0.26)bc (0.27) (0.18) (0.26)b (0.45) 2.94 1.38 1.69 2.26 5.63 3.88 2.5 5.63 (0.79) (0.18)c (0.27)b (0.18)c (0.71) (0.35) (0.0)c (0.0) 0.091 0.008 0.004 0.002 0.642 0.122 0.011 0.454 Values with diĂerent letters within an attribute are signiÂcantly diĂerent (analysis of variance, Po0.05) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 799 Aquaculture Research, 2010, 41, 793803 Selenium enrichment of African catÂsh part II E Schram et al Table Correlation matrix for the intensity scores of the sensory attributes of the Âllets during the depuration period for the selenium-enriched African catÂsh Attribute R O metal R O garlic R O sour C O garlic C O cook potato C T musty C T garlic R_O_metal R_O_garlic R_O_sour C_O_garlic C_O_cook potato C_T_musty C_T_garlic C_T_cook potato 0.814 0.766 0.894 0.879 0.979 0.888 0.725 0.467 0.459 0.545 0.427 0.060 0.023 0.216 0.653 0.821 0.949 0.916 0.958 0.354 0.005 C T cook potato 0.329 0.396 0.384 0.516 0.392 0.537 0.416 For values marked with the correlation is signiÂcant (the two-tailed Pearson correlation test, Po0.01) odour (Table 8) The intensity scores of the other sensory attributes were the same for all sampling days during the depuration period (Table 7) No oĂ Êavours were detected in the Âllet samples Discussion Total selenium levels and selenium retention in relation to the length of enrichment period The daily selenium intake among Europeans is on average approximately 100 mg short of the recommended daily intake of 150 mg day 1.We aimed to Âll this gap with one 150 g portion of selenium-enriched African catÂsh Âllet, which demands a Âllet selenium concentration of approximately 0.70 mg Se kg This target concentration was reached after an enrichment period of 10 days before harvest, using a dietary selenium level of 11.7 mg kg This clearly demonstrates that selenium enrichment of African catÂsh can be achieved by feeding selenium-enriched feeds for a relatively short period before harvest We therefore consider the selenium-enriched feed a Ânishing diet Finishing diets for farmed Âsh have been studied for Atlantic salmon (e.g Jobling, Larsen, Andreassen, Olsen & Sigholt 2002; Bell, Henderson, Tocher & Sargent 2004) and red seabream (Glencross et al., 2003), all successfully aiming to boost n-3 polyunsaturated fatty acid (PUFA) levels before harvest to compensate for initially reduced n-3 PUFA levels due to growing Âsh on feeds containing vegetable oils Finishing diets aiming to boost levels in farmed Âsh of nutrients important in human nutrition other than n-3 PUFA, such as selenium, have not been studied to date Apart from the current study, there are no records of studies aiming to optimize the enrichment process in terms of the minimal required 800 feeding period for Ânishing diets to reach target levels at harvest The relation between length of the enrichment period and selenium level in the Âllet of African catÂsh as established in the present study, enables the design of a feeding programme that targets a certain selenium level in the Âllet We expect, however, that this relation is exclusive to the currently used dietary selenium level of 13.5 mg kg 1, but it can be used for other Âsh sizes by considering the proportion of selenium-enriched feed in the total feed load rather than the number of days the selenium-enriched feed was fed This was shown by the selenium level in the Âllet reached in the second experiment, where we aimed for a Ânal total selenium level in the Âllet of 0.70 mg kg This selenium level is comparable to the level reached in treatment of the Ârst experiment, where Âsh received 97 g of selenium-enriched feed per Âsh, concurring with 19% of the total estimated feed intake per Âsh (based on the Ânal mean weight of 565 g and an estimated overall FCR of 0.9 g g 1) In the second experiment we used this result to estimate the required amount of seleniumenriched feed at 180 g per Âsh (based on a Ânal individual weight of1kg, an overall FCR of 0.9 g g 1and a proportion of selenium-enriched feed of 20% of the total feed intake) Upon harvest, the African catÂsh in the second experiment had reached a mean individual weight over 1kg and a selenium level in the Âllet of 0.63 mg kg (Table 5), which is well in line with the targeted 0.70 mg kg Se The highest selenium retention from feed to Âllet was reached when feeding the Âsh the seleniumenriched feed during 10 days before harvest The higher retention of selenium in the treatment groups fed selenium-enriched feeds as compared with the control diet is most likely related to the presence of r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 Aquaculture Research, 2010, 41, 793^803 selenomethionine in the selenium-enriched feeds (Schram et al 2008), which can substitute for methionine in protein synthesis As a result, selenomethionine is more easily incorporated in the muscle tissue of growing Âsh than the inorganic selenium in the control feed (Waschulewski & Sunde 1988) The low retention of selenium obtained after days of selenium enrichment before harvest as compared with the other treatment groups that received seleniumenriched feed can be explained by the delay between feed intake and actual incorporation of selenium compounds in the Âllet Such a delay would have caused that the selenium intake from the last meals before harvest were not incorporated in the Âllet As this unknown number of last meals before harvest account for a larger proportion of the total selenium intake for short enrichment periods, the selenium retention is underestimated when based on the full enrichment period of days The decreasing selenium retention with increasing length of the enrichment period (Fig 2) can be a reÊection of a Êattening of selenium incorporation in the Âllet, which is likely to occur as the amount of selenium that can be incorporated in the Âllet is limited (Schram et al 2008) EĂect of depuration on total selenium level in the Âllet Depuration was found to have no eĂect on the total selenium level in the Âllet for both the seleniumenriched Âsh and the control treatments (Fig 3) As Âllet weight of the selenium-enriched African catÂsh did not decrease during the depuration period, despite the considerable total body weight loss (Table 5), the absolute amount of selenium in the Âllet was also not aĂected by depuration Sensory properties of selenium-enriched African catÂsh Âllets in relation to depuration The intensity of the garlic odour and Êavour of the African catÂsh samples were found to decrease signiÂcantly during the Ârst days of the depuration period (Fig 4), showing that depuration is probably an eĂective measure to eliminate the eĂects of dietary garlic on the sensory properties of the Ânal product, without aĂecting other sensory characteristics (Table 7) As we were mainly interested in the changes of the sensory properties of the garlic-fed African catÂsh Selenium enrichment of African catÂsh part II E Schram et al during the depuration period and due to the absence of information on the sensory properties of garlic-fed Âsh, we used a sensory intensity test in this experiment and not a sensory diĂerence test nor a sensory threshold test Using a sensory diĂerence test would have enabled us to detect diĂerences in the sensory properties of the garlic-fed and the non-garlic-fed African catÂsh for each of the individual sampling days during the depuration period However, we would not have been able to detect any changes in the sensory properties during the course of the depuration period Using a sensory threshold test we would have been able to determine exactly the required length of the depuration period to eliminate garlic odours and Êavours, but for such a test, preliminary information on the changes in the sensory intensity during the course of the depuration period is needed This means that the current results give a strong indication that it is possible to reduce the garlic Êavour of the garlic-fed African catÂsh to minimal sensory detection levels within days of depuration, but a sensory threshold test including samples of both garlic-fed and non-garlic-fed Âsh is needed to conÂrm this During the experiment, a strong garlic-like odour could be smelled in the experimental room, suggesting that African catÂsh excrete compounds causing a garlic odour after garlic consumption, but this remains to be conÂrmed by measurements of garlic odour causing compounds Odours resulting from garlic consumption have been studied in humans Garlic odour in human breath following garlic consumption is caused by allyl mercaptan, a water soluble and indigestible sulphide compound that is taken up in blood from the intestinal tract, followed by excretion via the skin and lungs (Takeshi, Boku, Inada, Morita & Okazaki 1989) A similar route in Âsh can explain our observations in this study Potential excretion routes of compounds causing garlic odour in Âsh include faeces and urine, but after uptake of the compounds from the intestinal tract in the blood, excretion (of metabolites) can also take place via the gills Next to uptake from the intestinal tract, the rearing water contaminated by faeces or urine with compounds causing garlic Êavours could be considered as a source of these compounds in the Âllets, as Âsh can take up contaminants from the surrounding water via the gills (Streit 1998) Either way, uptake in the blood of compounds causing garlic odour and Êavour is needed to explain their presence in the r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 801 Aquaculture Research, 2010, 41, 934^938 Discrimination of tilapia species J Toniato et al Table Summary of discrimination between tilapia species using PCR-RFLP patterns of 5S rDNA repeat units of Oreochromis niloticus (nil), Oreochromis aureus (aur), Oreochromis karongae (kar), Oreochromis mortimeri (mort), Oreochromis mossambicus (moss), Tilapia rendalli (rend), Tilapia zillii (zill) and Sarotherodon galilaeus (gal) nil aur kar mort moss zill rend gal nil aur kar mort moss zill rend gal ac ac X ac X X ac X X X abc abc abc abc abc abc abc abc abc abc b abc abc abc abc abc bc C The discrimination between species with the enzymes PvuII, PstI and HinfIII is indicated with the letters a, b and c respectively X represents the absence of discrimination between species pairs using any of the three enzymes PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism spacers in general are considered to show considerable nucleotide variability attributed to insertions/ deletions, minirepeats and pseudogenes (Nelson & Honda 1985; Leah, Frederiksen, Engberg & Sorensen 1990; Sajdak, Reed & Phillips 1998) The present data demonstrate the potential applicability of the 5S rDNA sequences as a genetic marker for the diĂerentiation of most tilapia species studied across three genera It is also worth mentioning that particularly in Âshery management and conservation, the 5S rDNA PCR-RFLP approach is relatively easy to apply Additionally, such technology could be applied on Âsh products that are commonly sold in markets, allowing an approach for the identiÂcation of species References Adepo-Gourene B., Gourene G & Agnese J.F (2006) Genetic identiÂcation of hybrids between two autochthonous tilapia species, Tilapia zillii and Tilapia guineensis, in the man-made lake Ayame Aquatic Living Resources 19, 239^245 Ahmed M.M.M., Ali B.A & EI-Zaeem S.Y (2004) Application of RAPD markers in Âsh: part I ^ some genera (Tilapia, Sarotherodon and Oreochromis) and species (Oreochromis aureus and Oreochromis niloticus) of Tilapia International Journal of Biotechnology 6, 86^93 Alves-Costa F.A.,Wasko A.P., Oliveira C., Foresti F & Martins C (2006) Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini Âshes Genetica 127, 243^252 Bardakci F & Skibinski D.O.F (1994) Application of the RAPD technique in tilapia Âsh: species and subspecies identiÂcation Heredity 73,117^123 Bardakci F & Skibinski D.O.F (1999) A polymorphic SCARRAPD marker between species of tilapia (Pisces: Cichlidae) Animal Genetics 30,78^79 Carrera E., Garcia T., CeÔspedes A., Gonzalez I., Fernandez A., Asensio L.M., Hernandez P.E & Martin R (2000) DiĂerentiation of smoked Salmo salar, Oncorhynchus mykiss and Brama raii using the nuclear marker 5S rDNA International Journal of Food ScienceTechnology 35, 401^406 DAmato M.E., Esterhuyse M.M., van der Waal B.C.W., Brink D & Volckaert F.A.M (2007) Hybridization and phylogeography of the Mozambique tilapia Oreochromis mossambicus in southern Africa evidenced by mitochondrial and microsatellite DNA genotyping Conservation Genetics 8, 475^488 Dover G (1982) Molecular drive: a cohesive mode of species evolution Nature 299,111^116 Drouin G & Moniz de Saỉ M (1995) The concerted evolution of 5S ribosomal genes linked to the repeat units of other multigene families Molecular Biology and Evolution 12, 481^493 Galls F & Metz J.A.J (1998) Why are there so many cichlid species? Trends in Ecology and Evolution 13,1^2 Greenwood P.H (1991) Speciation In: Cichlid Fishes: Behavior, Ecology and Evolution (ed by M.H.A Keenleyside), pp 86^102 Chapman & Hall, NewYork, NY, USA Gregg R.E., Howard J.H & Shonhiwa F (1998) Introgressive hybridization of tilapias in Zimbabwe Journal of Fish Biology 52,1^10 Klett V & Meyer A (2002) What, if anything, is a Tilapia? ^ Mitochondrial ND2 phylogeny of tilapiines and the evolution of parental care systems in the African cichlid Âshes Molecular Biology and Evolution 19, 865^883 Komiya H & Takemura S (1979) Nucleotide sequence of 5S ribosomal RNA from rainbow trout (Salmo gairdnerii) liver Journal of Biochemistry 86,1067^1080 Leah R., Frederiksen S., Engberg J & Sorensen P.D (1990) Nucleotide sequence of a mouse 5S rRNA variant gene Nucleic Acids Research 18,7441^7441 Lee B.Y., Lee W.J., Streelman J.T., Carleton K.L., Howe A.E., Hulata G., Slettan A., Stern J.E., Terai Y & Kocher T.D (2005) A second-generation genetic linkage map of tilapia (Oreochromis spp.) Genetics 170, 237^244 Long E.O & David I.B (1980) Repeated genes in eukaryotes Annual Reviews of Biochemistry 49,727^764 Martins C & Wasko A.P (2004) Organization and evolution of 5S ribosomal DNA in the Âsh genome In: Focus on Genome Research (ed by C.R Williams), pp 289^318 Nova Science Publishers, Hauppauge, NY, USA Martins C.,Wasko A.P., Oliveira C & Wright J (2000) Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiini cichlid Âsh, Oreochromis niloticus Hereditas 133, 39^46 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 934^938 937 Discrimination of tilapia species J Toniato et al Martins C., Wasko A.P., Oliveira C., Porto-Foresti F., PariseMaltempi P.P.,Wright J.M & Foresti F (2002) Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units, inverted sequences, pseudogenes and chromosome loci Cytogenetics and Genome Research 98,78^85 Mather P.B & Arthington A.H (1991) An assessment of genetic diĂerentiation among feral Australian tilapia populations Australian Journal of Marine and Freshwater Research 42,721^728 McAndrew B.J & Majumdar K.C (1983) Tilapia stock identiÂcation using electrophoretic markers Aquaculture 30, 249^261 Nagl S.,Tichy H., Mayer W.E., Samonte I.E., McAndrew B.J & Klein J (2001) ClassiÂcation and phylogenetic relationships of African Tilapiine Âshes inferred from mitochondrial DNA sequences Molecular Phylogenetics and Evolution 20, 361^374 Nelson D.W & Honda B.M (1985) Genes coding for 5S ribosomal RNA of the nematode Caenorhabditis elegans Gene 38, 245^251 Pendas A.M., MoÔran P., Mart|Ô nez J.L & Garcia-Vasquez E (1995) Applications of 5S rDNA in Atlantic salmon, brown trout and in Atlantic salmon x brown trout hybrid identiÂcation Molecular Ecology 4, 275^276 Pinhal D., Gadig O.B.F.,WaskoA.P., Oliveira C., Ron E., Foresti F & Martins C (2008) Discrimination of shark species by 938 Aquaculture Research, 2010, 41, 934^938 simple PCR of 5S rDNA repeats Genetics and Molecular Biology 31, 361^365 Pouyaud L & Agnese J.F (1995) Phylogenetic relationships between 21 species of three tilapiine genera Tilapia, Sarotherodon and Oreochromis using allozyme data Journal of Fish Biology 47, 26^38 Rognon X & Guyomard R (2003) Large extent of mitochondrial DNA transfer from Oreochromis aureus to O niloticus inWest Africa Molecular Ecology 12, 435^445 Sajdak S.L., Reed K.M & Phillips R.B (1998) Intraindividual and interspecies variation in the 5S rDNA of Coregonid Âsh Journal of Molecular Evolution 46, 680^688 Sambrook J & Russell D.W (2001) Molecular Cloning: a Laboratory Manual, 3rd edn Cold Spring Harbor Laboratory Press, 2344pp Sodsuk P & McAndrew B.J (1991) Molecular systematics of three tilapiine genera Tilapia, Sarotherodon and Oreochromis using allozyme data Journal of Fish Biology 39, 301^308 Trewavas E (1983) Tilapiine Fishes of the Genera Sarotherodon, Oreochromis and Danakilia British Museum (Natural History), London, UK, 583pp Keywords: species identiÂcation, Cichlidae,Tilipiinae, genetic markers r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 934^938 Aquaculture Research, 2010, 41, 939^944 doi:10.1111/j.1365-2109.2009.02369.x SHORT COMMUNICATION Occurrence of Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) in gilthead sea bream (Sparus aurata L.) from different mariculture systems in Spain Ariadna Sitjaỉ-Bobadilla, Ma JoseÔ Redondo & Pilar Alvarez-Pellitero Instituto de Acuicultura de Torre de la Sal, CSIC, CastelloÔn, Spain Correspondence: A Sitjaỉ-Bobadilla, Instituto de Acuicultura deTorre de la Sal, Consejo Superior de Investigaciones Cient|Ô Âcas,Torre de la Sal s/n, 12595 Ribera de Cabanes, CastelloÔn, Spain E-mail: ariadna@iats.csic.es The exponential increase in gilthead sea bream production in the Mediterranean basin in the last few years has been supported by the massive establishment of sea cages, although it has also contributed to the extension and dispersal of parasitic diseases (Sitjaỉ-Bobadilla 2004) The polyopisthocotylean Sparicotyle (sin Microcotyle) chrysophrii, is the most threatening ectoparasite for gilthead sea bream cultures Polyopisthocotyleans have been reported to be responsible for reduced catches in some wild populations, by altering their behaviour and making them more susceptible to predation (Shirakashi, Teruya & Ogawa 2008) They also induce mortalities in several cultured Âsh species (Ogawa 2002; Montero, Crespo, PadroÔs, de la Gandara, Garc|Ô a & Raga 2004; Hayward 2005) Some of them are responsible for 22% of the total production cost in Australian cultures (see Ernst, Whittington, Corneille & Talbot 2002), and are considered to be a serious risk for sea-cage aquaculture (Hutson, Ernst & Whittington 2007) Sparicotyle chrysophrii has been reported in wild and cultured gilthead sea bream in the Mediterranean basin (Oliver 1984; Mladineo 2005; Fioravanti, CaĂara, Florio, Gustinelli & Marcer 2006), sometimes associated with mortalities (Sanz 1992; Alvarez-Pellitero 2004; Vagianou, Athanassopoulou, Ragias, Di Cave, Leontides & Golomazou 2006) It is frequently found in mixed infections with other parasites and secondary bacterial infections (PadroÔs & Crespo 1995; Cruz e Silva, Freitas & Orge 1997; CaĂara, Quaglio, Fioravanti, Gustinelli, Marcer, Moscato & Caggiano 2005) However, there are few long-term r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd studies on this monogenosis, and the available data diĂer considerably depending on the surveyed area Risk assessment for parasites in aquaculture is of great interest and has to be determined for each particular area and environmental conditions (Hutson et al 2007) Furthermore, eĂective management strategies need to incorporate accurate knowledge of the mode of transmission and the inÊuence of environmental and culture conditions (Altizer, Dobson, Hosseini, Hudson, Pascual & Rohani 2006) Therefore, the aim of this work was to obtain data on the occurrence of this ectoparasite in Spanish Mediterranean and Atlantic facilities to design management strategies that could prevent monogenean epizootics For this purpose, a long-term parasitological survey was conducted in several aquaculture facilities from the Spanish coasts Two types of samplings were performed In study A (Table 1), a total of 360 Âsh from four diĂerent Mediterranean ongrowing systems were periodically sampled for years In all groups, the Ârst sampling was carried out before Âsh entered the corresponding facility (thus, it reÊects the previous preongrowing situation), and then every months until Âsh reached the market The surveyed systems included the intensive indoor experimental tanks of the Instituto de Acuicultura de Torre de la Sal (IATS), and three farms with two diĂerent growing systems: sea cages (F-1 and F-2) and intensive earth-ponds (F-3) In study B (Table 2), a total of 300 Âsh from sea-cage farms F1 and F2 and three additional ones (F-4, F-5 and F-6), and another earth-pond farm (F-7) were occasionally 939 Sparicotyle chrysophrii infections A Sitjaỉ-Bobadilla et al Aquaculture Research, 2010, 41, 939^944 Table StudyA Sparicotyle chrysophrii Samplings Farm (province) IATS (Castellon) F-1 (Castellon) F-2 (Tarragona) F-3 (Tarragona) Time Fish weight (g) (mean ặ SD) Mar 99Jul 00 Mar 99z Jul 99 Oct 99 Feb 00 Jul 00 Apr 99z Jul 99 Oct 99 Jan 00 Apr 00 Jul 00 May 99Oct 00 20.5 21.3 92.3 222.9 313.5 339.8 8.5 31.8 110.3 142.4 175.7 281.3 2.9 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 3.7373.3 ặ 61.9 4.7 12.9 35.1 53.4 50.2 2.1 8.9 19.5 31.2 38.6 48.3 0.6414.1 ặ 76.5 Type of diagnosisw n P (%) MI Stages F F F F F F F F F F F F F 150 10 10 10 10 10 10 10 10 10 10 10 90 0 50 10 30 0 40 30 50 10 1.61 11 11 1.51 1.71 1.21 11 J, A A, E A J, A, E A, E A, E J Sampling conditions and infection levels in gilthead sea bream periodical samplings of indoor facilities (IATS), earth ponds (F-3) and sea cages (F-1, F-2) When the parasite was not detected in a facility, sampling, time period and Âsh weight at the initial and Ânal samplings are indicated, instead of the data at each sampling date wThe parasite was diagnosed with the fresh diagnosis (F) method, and the stages determined as J, juveniles; A, adults; E, eggs zFirst sampling before entering the cages P, prevalence; MI, mean intensity Table Study B Sparicotyle chrysophrii Samplings Farm (province) Time Months in cages Weight (g) (mean ặ SD) F-1 (Castellon) Apr 99 Mar 00 May 00 Jun 03 Oct 04 Mar 05 Jul 99 May 03 Jun 03 Nov 04 Jun 04 Oct 04 Nov 07 Jun 06 Jun 06 Oct 06 Oct 06 Sep 07 Sep 07 13 7.5 3.5 424 416 123.4 17.2 67.9 300.1 76.8 32.1 127.6 200.1 47.6 31.7 60.3 90.1 104.2 15.4 55.7 205.9 158.1 77.7 238.2 F-2 (Tarragona) F-4 (Castellon) F-5 (Castellon) F-6 (Valencia) F-7 (Cadiz) ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 36.7 5.9 11.7 48.3 15.4 5.2 49.6 20.3 6.9 4.3 8.9 13.7 15.3 3.2 14.8 44.9 51.9 20.9 85.8 Type of diagnosis n P (%) MI Stages F/H F/H F/H S S F/H F/H S S F/H S S F/H F/H F/H F/H F/H F/H F/H 10 10 10 15 10 20 8 13 20 20 20 18 20 20 20 20 17 21 20 100 93.3 100 60 25 100 100 100 100 11.1 0 0 0 1.51 3.81 8.6 13.6 2.21 11 6.9 70.6 12 52.4 11 J, A, J, A, J, A, A, E A, E A, E J, A, J, A, A, J J, A, A, J E E E E E E Sampling conditions and infection levels in gilthead sea bream in outbreak samplings of sea cages (F-1^6) and earth ponds (F-7) The monogenean was diagnosed by the fresh (F), histology (H) or stereomicroscope (S) diagnosis methods, and the stages determined as J, juveniles; A, adults; E, eggs P, prevalence; MI, mean intensity 940 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 939^944 Aquaculture Research, 2010, 41, 939^944 Sparicotyle chrysophrii infections A Sitjaỉ-Bobadilla et al 20 08 Ja n 20 07 Ja n Ja n 20 06 20 05 Ja n 20 04 Ja n 20 03 Ja n 20 02 Ja n 20 01 Ja n Ja n Ja n 20 00 28 26 24 22 20 18 16 14 12 10 19 99 Water temperature (C) Figure Approximate location of sampling sites (Instituto de Acuicultura de Torre de la Sal IATS and farms F) of Sparus aurata on the Spanish coasts that were checked for Sparicotyle chrysophrii infection Months Figure Monthly water temperatures at the indoor facilities of the Instituto de Acuicultura deTorre de la Sal (CastelloÔn province) sampled when mortalities or morbidity outbreaks occurred along a wide-ranging period (1999^2007) Most of the surveyed Âsh farms were located on the Western Mediterranean coast (from Tarragona to Valencia provinces), except F-7, located on the South Atlantic Spanish coast Figure shows the location of the sampling sites All Âsh were reared under natural temperature and photoperiodic conditions, following the standard procedures of each farm The mean annual water temperature ranges were 10.5^26.3 1C at IATS facilities, 10^28 1C at F-7, 7^30 1C at F-3, 12.5^25.9 1C at F-6 and 14.2^27.6 1C at the surface in the areas of F1, F2, F4 and F5 Figure shows the average monthly temperatures at IATS facilities, which are close to several sampling sites (Fig 1) IATS seawater supply (37.5% salinity) was from a pump on shore; F3 and F7 received water from marshland channels and have seasonal salinity oscillations from 34% to 40% The mean seawater salinity in the areas of F-1, F-2, F-4, F-5 and F-6 was 37%, with slight variations More details on the sampling conditions for both types of studies are shown in Tables and The monogenean was diagnosed by three methods Fresh (F) diagnosis: Âsh were anaesthetized with MS-222 (Sigma, St Louis, MO, USA) (100 mg L 1) and gill scrapings taken from the most external gill arch were examined using light microscopy (LM) The Ârst arch was selected because it is the most parasitized with respect to the remaining arches (Oliver 1984) Stereomicroscope (S) diagnosis: Âsh were killed by a blow on the head under anaesthesia (MS-222, 100 mg L 1), bled to reduce the blood content in gills, the whole gill arches were excised and r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 939^944 941 Sparicotyle chrysophrii infections A Sitjaỉ-Bobadilla et al examined under the stereomicroscope In both cases, the type of stages of the monogenean was recorded Histology (H) diagnosis: after necropsy, gills were Âxed in 10% neutral-buĂered formalin, embedded in Technovit resin (Kulzer, Heraeus, Germany), mm sectioned, stained with toluidine blue and examined at LM The anaesthetic procedure was checked previously not to aĂect monogenean attachment to gill arches.When more than one diagnostic method was applied at a particular sampling, the prevalence of infection was calculated considering any positive Âsh detected using any method.When using the F and H diagnosis, the intensity of infection was semiquantitatively evaluated following a conventional scale from to 61, according to the number of monogeneans per slide (40 observational Âelds) at 120 (11 51^2 parasites; 21 5, 3^5; 31 56^8; 41 9^11; 51 512^14; and 61 ! 15) Intensity was quantitatively registered (number of monogenean specimens per Âsh) using the S method Table shows the results of study A The monogenean was detected only in cage-cultured gilthead sea bream (F1 and F2) It was never found before Âsh entered the cages, but it appeared as early as the Ârst sampling after introduction (summer period) in both farms Table shows the infection levels of S chrysophrii during morbidity/mortality outbreaks (study B) Again, the parasite was not detected in earth-pond facilities (F-7) The prevalence of infection was high in most sea cages, with the presence of adults, juveniles and eggs, even at the end of the winter, regardless of the host weight Another monogenean, the diplectanid F echeneis, was usually found in the gills of S chrysophrii-parasitized Âsh, and in some outbreak samplings, epithelocystis organisms and the sanguinicolid Cardicola aurata were also present The concomitant presence of F echeneis and S chrysophrii has been reported previously, even in wild gilthead sea bream (Reversat, Silan & Maillard 1992) In the present survey, S chrysophrii was not found in indoor facilities, or in the two surveyed earth-pond facilities, even in Âsh that had spent more than years in the ponds In Italian extensive, earth/sand pond-based farms, the monogenean was not detected (Fioravanti et al 2006) However, in other natural environments with earth/sand bottoms and a lower water Êow than in open sea, like in coastal ponds (Oliver 1984) or lagoons (Vagianou et al 2006), the prevalence of infection in wild and cultured gilthead sea bream reached up to 85% and 100% respectively The apparent absence of this monogenean in the pond-culture facilities studied could be due to many 942 Aquaculture Research, 2010, 41, 939^944 diĂerent factors, such as higher water turbidity, lower water quality and a higher diurnal and seasonal oscillation of water temperatures due to the low depth of the ponds By contrast, S chrysophrii was very prevalent in sea cages, particularly during outbreak samplings This monogenean had a moderate prevalence (33.9%) of infection and low abundance (0.46) in Adriatic Sea cages (Mladineo 2005), but infection levels from Mediterranean waters diĂer depending on the country and the type of facility Thus, in Italian cages, the mean prevalence was 6.1% (Fioravanti et al 2006), whereas in Greek sea cages, the combined prevalence of S chrysophrii and F echeneis ranged from 61.5% to 13.3% (Vagianou et al 2006) Therefore, culture conditions and farm location have a clear eĂect on the infection levels Transmission of some monogeneans exhibits a clear seasonality, with invasion maximized during warmer months (Papoutsoglou, Costello, Stamou & Tziha 1996; Ogawa & Inouye 1997; Mladineo 2005; Rubio-Godoy & Tinsley 2008), and epizootics commonly occur with increasing water temperatures (Ernst et al 2002) Similarly, in the present survey, in both studies (A, B), the highest prevalence and intensity of infection seems to be coincident in most sampling sites with warm water temperatures, which, in the studied area, are registered in summer and early autumn The unexpectedly low values detected in some summer samplings, as in study A in July 2000 in both sea cages, could be due to formalin on-site treatments particularly performed by Âsh farmers in the previous spring However, from the data obtained, it seems that the parasite is capable of resisting winter conditions, as was detected in F-1in February and March in studies A and B respectively Moreover, the monogenean showed high infection levels and the presence of adults and eggs even in the winter of 2005, which was particularly cold (the minimum water temperature was lower than 1C) In the periodical survey (study A), it was established that Âsh entering the cages free of the monogenean can acquire the infection as soon as three months later, regardless of their initial age In fact, the infection can be experimentally transmitted to na|ô ve gilthead sea bream by contact with S chrysophrii eggs and by cohabitation with parasitized Âsh in a shorter time, reaching100% of prevalence in just weeks (Sitjaỉ-Bobadilla & Alvarez-Pellitero 2009) Because of the production cycle of gilthead sea bream, which takes about 18^24 months to reach the market size, newly introduced na|ô ve juveniles r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 939^944 Aquaculture Research, 2010, 41, 939^944 are held in cages in close vicinity to others holding infected adults Thus, cage-to-cage transmission could occur in gilthead sea bream stocks, as described for other Âsh^monogenean models (Chambers & Ernst 2005) However, no Âeld experimental data are available to conÂrm this From the information obtained in this and previous parasitological studies, we identify S chrysophrii as a real risk for gilthead sea bream sea cage cultures It is clear that sea cages provide the ideal conditions for the continuous exploitation of the host by the parasite, as it can survive winter conditions in the cages and spreads easily to new stocked animals Net biofouling not only provides a suitable place for the entanglement of monogenean eggs but also prevents a high water Êow, which is essential for animals with diminished gill function Thus, we strongly recommend synchronization of bath treatments, and net cleaning with the introduction of new animals in the facilities Knowledge of the minimum distance between neighbouring sea cages that would avoid parasite dispersal would help in the management of this monogenosis Acknowledgments This work was supported by research grants from the Spanish Ministerio de EducacioÔn, Cultura y Deporte (project MAR-98/1000), the Spanish Ministerio de Ciencia y Tecnolog|Ô a (project no AGL-2002-0475C02) and a research contract with the Âsh farm CUPIMAR S.A The authors are grateful to M Alonso and P Cabrera for excellent assistance with samplings, and to J Monfort and L Rodr|Ô guez for histological processing References Altizer S., Dobson A., Hosseini P., Hudson P., Pascual M & Rohani P (2006) Seasonality and the dynamics of infectious diseases Ecology Letters 9, 467^484 Alvarez-Pellitero P (2004) Report about Âsh parasitic diseases In: Mediterranean Aquaculture Diagnostic Laboratories, SeÔ rie B: Etudes et Recherches, No 49, Options MeÔ diterraneÔ ennes (ed P Alvarez-Pellitero, J.L Barja, B Basurco, F Berthe & A.E Toranzo), pp 103^129 CIHEAM/ FAO, Zaragoza CaĂara M., Quaglio F., Fioravanti M.L., Gustinelli A., Marcer F., Moscato M & Caggiano M (2005) Coinfezione da Polysporoplasma sparis (Myxozoa) e Sparicotyle chrysophrii (Monogenea) in orata (Sparus aurata In: Atti del XII Convegno Nazionale S.I.P.I., 47 pp Cesenatico, Italy Sparicotyle chrysophrii infections A Sitjaỉ-Bobadilla et al Chambers C.B & Ernst I (2005) Dispersal of the skin Êuke Benedenia seriolae (Monogenea: capsalidae) by tidal currents and implications for sea-cage farming of Seriola spp Aquaculture 250, 60^69 Cruz e Silva M.P., Freitas M.S & Orge M.L (1997) Co-infection by monogenetic Trematodes of the genus Microcotyle, Beneden & Hese 1863, Lamellodiscus ignoratus Palombi, 1943, the protozoan Trichodina sp Ehrenber, 1838 and the presence of Epitheliocystis,Vibrio algynoliticus and V vulniÂcus in cultured seabream (Sparus aurata L.) in Portugal Bulletin of the European Association of Fish Pathologists 17, 40^42 Ernst I., Whittington I., Corneille S & Talbot C (2002) Monogenean parasites in sea-cage aquaculture Austasia Aquaculture 2, 46^48 Fioravanti M.L., CaĂara M., Florio D., Gustinelli A & Marcer F (2006) A parasitological survey of European sea bass (Dicentrarchus labrax) and Gilthead sea bream (Sparus aurata) cultured in Italy.Veterinary Research Communications 30, 249^252 Hayward C.J (2005) Monogenea Polyopisthocotylea (Ectoparasitic Êukes) In: Marine Parasitology (ed by K Rohde), pp 55^63 CSIRO Publishing, Canberra, Australia Hutson K.S., Ernst I & Whittington I.D (2007) Risk assessment for metazoan parasites of yellowtail kingÂsh Seriola lalandi (Perciformes: Carangidae) in South Australian sea-cage aquaculture Aquaculture 271, 85^99 Mladineo I (2005) Parasite communities of Adriatic cagereared Âsh Diseases of Aquatic Organisms 64,77^83 Montero F.E., Crespo S., PadroÔs F., de la Gandara F., Garc|Ô a A & Raga J.A (2004) EĂects of the gill parasite Zeuxapta seriolae (Monogenea: Heteraxinidae) on the amberjack Seriola dumerili Risso (Teleostei: Carangidae) Aquaculture 232, 153^163 Ogawa K (2002) Impacts of diclidophorid monogenean infections on Âsheries in Japan International Journal for Parasitology 32, 373^380 Ogawa K & Inouye K (1997) Heterobothrium infection in cultured tiger puĂer, Takifugu rubripes ^ a Âeld observation Fish Pathology 32, 15^20 Oliver G (1984) Microcotyle chrysophrii Van Beneden and Hesse, 1863 (Monogenea, Polyopisthocotylea, Microcotylidae) parasite de Sparus aurata Linnaeus, 1758 (Teleostei, Sparidae) dans les eÔtangs littoraux du Languedoc-Roussillon (France) Bulletin de la Societe Zoologique de FranceEvolution et Zoologie 109,113^118 PadroÔs F & Crespo S (1995) Proliferative epitheliocystis associated with monogenean infection in juvenile sea bream Sparus aurata in the North East of Spain Bulletin of the European Association of Fish Pathologists 15, 42^44 Papoutsoglou S.E., Costello M.J., Stamou E & Tziha G (1996) Environmental conditions at sea-cages, and ectoparasites on farmed European sea-bass, Dicentrarchus labrax (L.), and gilt-head sea-bream, Sparus aurata L., at two farms in Greece Aquaculture Research 27, 25^34 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 939^944 943 Sparicotyle chrysophrii infections A Sitjaỉ-Bobadilla et al Reversat J., Silan P & Maillard C (1992) Structure of monogenean populations, ectoparasies of the gilthead sea bream Sparus aurata Marine Biology 112, 43^47 Rubio-Godoy M & Tinsley R.C (2008) Transmission dynamics of Discocotyle sagittata (Monogenea) in farmed rainbow trout interpreted from parasite population age structure Aquaculture 275, 34^41 Sanz F (1992) Mortality of cultured sea bream (Sparus aurata) caused by an infection with a trematode of the genus Microcotyle Bulletin of the European Association of Fish Pathologists 12,186^188 Shirakashi S., Teruya K & Ogawa K (2008) Altered behaviour and reduced survival of juvenile olive Êounder, Paralichthys olivaceus, infected by an invasive monogenean, Neoheterobothrium hirame International Journal for Parasitology 38, 1513^1522 Sitjaỉ-Bobadilla A (2004) Parasites in Mediterranean aquacultured Âsh: current impact and future research direc- 944 Aquaculture Research, 2010, 41, 939^944 tions In: Multidisciplinarity for Parasites, Vectors and Parasitic Diseases (ed by S Mas-Coma), pp 301^311 Medimond, Bologna, Italy Sitjaỉ-Bobadilla A & Alvarez-Pellitero P (2009) Experimental transmission of Sparicotyle chrysophrii (Monogenea: Polyopisthocotylea) to gilthead sea bream (Sparus aurata L : an haematological and histopathological approach Folia Parasitologica 56,143^151 Vagianou S., Athanassopoulou F., Ragias V., Di Cave D., Leontides L & Golomazou E (2006) Prevalence and pathology of ectoparasites of Mediterranean sea bream and sea bass reared under diĂerent environmental and aquaculture conditions Israeli Journal of Aquaculture-Bamidgeh 58,78^88 Keywords: ectoparasites, gills, aquaculture, environmental conditions, Sparidae r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 939^944 Aquaculture Research, 2010, 41, 945^948 doi:10.1111/j.1365-2109.2009.02371.x SHORT COMMUNICATION Accumulation of tannin in different tissues of Indian major carps and exotic carps Sudipta Mandal & Koushik Ghosh Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Burdwan, West Bengal, India Correspondence: K Ghosh, Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal, India E-mail: kghosh_bu@yahoo.co.in Tannin is a phenolic derivative of Êavone and occurs as glycosides in the natural state Tannins are widespread in nature and are probably present in all plant materials starting from phytoplankton to macrophytes Indian major carps and exotic carps cultured in India are mostly herbivorous or omnivorous (Jhingran 1997) Therefore, they depend on phyto- and zooplankton or other plant materials for their feeding in natural habitats In addition, supplementary feeds may also contain tannin-like compounds as there is a thrust to substitute Âsh meal in aquafeed with the less expensive and protein-rich plant ingredients for economic Âsh production in most developing countries (Mukhopadhay & Ray 1996; Becker & Makkar 1999) Conversely, high levels of tannin in feed have been shown to have adverse eĂects on herbivorous and omnivorous Âsh (Olvera, Martinez, Galvan & Chavez 1988; Al-Owafeir 1999; Becker & Makkar 1999) Interference of tannin with protein and dry matter digestibility has also been reported (Krogdahl 1989) Tannins can be grouped into condensed and hydrolysable tannins Condensed tannins are complex and large molecules; therefore, they are not properly absorbed into the blood It seems that the adverse eĂects are probably due to tannic acid, which is hydrolysable (Prusty, Sahu, Pal, Reddy & Kumar 2007) However, there is a dearth of information on the accumulation of tannin in Âsh tissues when ingested by Âsh with food containing tannin The present study, therefore, was undertaken to determine the accumulation of hydrolysable tannin in diĂerent tissues of Indian major carps and exotic carps that feed on plant materials and/or plant detritus containing tannins Subsequently, the concentration of r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd tannin has been determined in phytoplankton, zooplankton and some aquatic macrophytes that usually form natural food for the carps to determine any apparent link with the accumulation of tannin in Âsh tissues Some other plant ingredients with potential for use as aquafeed ingredients were also evaluated in this respect In the present study, accumulation of tannin was determined in the muscle, liver and kidney tissues from three Indian major carps: rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) and three exotic carps: silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella) and common carp (Cyprinus carpio) The average weight of the examined Âsh and their feeding habits are presented in Table Specimens were collected from a local composite carp culture pond located at Burdwan (23114 N, 87139 E) The pond was free from any Table Food habits and the average weight of Âsh examined Fish species Food habits Average weight (g) (ặ SD) Catla catla Hypophthalmichthys molitrix Labeo rohita Zooplanktophagous Phytoplanktophagous 235.5 ặ 10.3 115.7 ặ 6.2 Omnivorous, mostly plant matter Herbivorous, mostly macrophytes Detritivorous Detritivorous 212.3 ặ 8.1 Ctenopharyngodon idella Cirrhinus mrigala Cyprinus carpio 242.6 ặ 9.2 147.4 ặ 4.5 205.7 ặ 6.9 Values are a mean ặ SD of Âve specimens SD, standard deviation 945 Bioaccumulation of tannin in carps S Mandal & K Ghosh Aquaculture Research, 2010, 41, 945^948 Table Accumulation of tannin (mean ặ SE of three observations) in diĂerent tissues of the major carps (mg g^1 wet weight) Tissue Fish species Muscle Catla catla Hypophthalmichthys molitrix Labeo rohita Ctenopharyngodon idella Cirrhinus mrigala Cyprinus carpio 0.436 0.912 0.384 0.615 0.861 0.814 ặ ặ ặ ặ ặ ặ Liver 0.004b 0.006f 0.004a 0.005c 0.006e 0.007d 1.41 1.87 1.42 2.4 2.95 2.79 ặ ặ ặ ặ ặ ặ Kidney 0.03a 0.01b 0.06a 0.06c 0.05d 0.08d 1.61 1.85 2.26 2.93 2.99 3.15 ặ ặ ặ ặ ặ ặ 0.05a 0.02b 0.02c 0.01d 0.08de 0.01e Values with the same superscript in the same column are not signiÂcantly diĂerent (Po0.05) from each other sewage discharge or other anthropogenic activities Along with natural feeding, the Âsh were fed a mixture of Âsh meal, rice bran and diĂerent oil cakes as supplementary feed The temperature, pH and dissolved oxygen content of the collection pond were 23.8^28.2 1C, 7.2^7.8 and 7.2^8.4 mg L 1, respectively, during the period of sampling Five specimens of each Âsh species were analysed for each replicate and there were three replicates for each tissue type The tissues were dissected, washed in normal saline and pooled together, separately Tannin from Âsh tissues, plankton and plant materials was extracted following the method described by Schanderi (1970) The concentration of tannin in the extracts was measured using the Folin^Denis method (Schanderi 1970) with minor modiÂcations The crude extract (0.2 mL) was diluted with 8.3 mL of distilled water and then mixed with 0.5 mL of Folin^Denis reagent The reaction mixture was alkalinized by addition of 1mL of 15% (w/v) sodium carbonate solution and kept in the dark for 30 at room temperature The absorbance of the solution was read at 700 nm using a spectrophotometer (Shimadzu UV/VIS-1700, Kyoto, Japan), and the concentration of tannin in the extract was determined using pure tannic acid (Merck, Mumbai, India) as a standard There were three replicates for each determinations Statistical analysis of the data was performed using ANOVA, followed by Tukeys test using SPSS ver 10 (Kinnear & Gray 2000) software Accumulation of tannin was observed in tissues of all the six Âsh species studied (Table 2) Among the tissues studied, tannin content was elevated in the kidney and liver compared with the muscle in all six species This is evident from the post hoc Tukeys test, which revealed a signiÂcant diĂerence in the level of tannin between the tissue types (between muscle and liver jqj 51.47, between muscle and kidney 946 Table Concentration of tannin (mean ặ SE of three observations) in natural and potential food items/ingredients for Indian and exotic major carps Potential fish food Amount of tannin (mg g^1 dry weight) Phytoplankton Zooplankton Lemna minor Lemna major Azolla sp Pistia sp Ipomoea sp Sunflower oil cake Coconut oil cake Mustard oil cake Soybean seed 34.3 6.58 5.83 7.5 19.58 26.67 5.81 26.25 38 21.25 10.42 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.67i 0.19c 0.21b 0.27d 0.38f 0.41h 32b 38h 0.18a 0.35g 0.26e Values with the same superscript in the same column are not signiÂcantly diĂerent (Po0.05) from each other jqj 1.79 and between liver and kidney jqj 0.32; SE 0.022; d.f 536,2 Po0.05; jqj student range) (Zar 1999) Deposition of tannin in the kidney ranged between 1.61 ặ 0.05 (C catla) and 3.15 ặ 0.01 (C carpio) mg g 1wet weight In the liver, the concentration of tannin varied between1.41 ặ 0.03 (Catla catla) and 2.95 ặ 0.05 (C mrigala) mg g wet weight, whereas accumulation of tannin in the muscle was between 0.384 ặ 0.004 (L rohita) and 0.912 ặ 0.006 (H molitrix) mg g wet weight A signiÂcant concentration of tannin was also found in the phyto- and zooplankton as well as in the plant materials studied (Table 3) The concentration of tannin was signiÂcantly higher in phytoplankton (34.3 ặ 0.67 mg g dry weight) compared with zooplankton (6.58 ặ 0.19 mg g dry weight) Tannin in plant matters varied between 5.38 ặ 0.18 (coconut oil cake) and 26.67 ặ 0.41 (Pistia sp.) mg g dry weight r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 945^948 Aquaculture Research, 2010, 41, 945^948 The results of the study revealed that accumulation of tannin varied signiÂcantly in the diĂerent tissues studied as well as among diĂerent Âsh species The results might signify a correlation between the feeding habit of the Âsh species studied (Table 1) and the degree of tannin accumulation in their tissues For instance, signiÂcantly higher concentration of tannin in the liver and kidney of C mrigala and C carpio may be associated with their feeding habits towards the plant detritus, which originate from decomposing macrophytes containing a high level of tannin (Table 3), whereas, the signiÂcantly lower accumulation of tannin in the liver and kidney of C catla and L rohita may be related to their planktivorous- or omnivorous-feeding habit Elevated levels of tannin in the tissues of C idella strongly suggest their feeding of the aquatic macrophytes rich in tannin However, in addition to the comparatively lower levels of tannin in the kidney and liver, the highest level in the muscle tissue is paradoxical in H molitrix, although the highest accumulation of tannin in the muscle of H molitrix may be correlated to their feeding on the phytoplankton and minute aquatic vegetation Tannins are considered to be a very complex group of plant secondary metabolites, which are distinguished from other polyphenolic compounds by their ability to precipitate proteins (Silanikove, Perevolotsky & Provenza 2001) It has been postulated that tannins interfere with protein and dry matter digestibility by inhibiting protease and also by forming indigestible complexes with dietary protein (Krogdahl 1989) However, little is known about the eĂect of tannin on protein digestibility in Âsh (Becker & Makkar 1999) Hossain and Jauncy (1989) observed a poor growth response in common carp (C carpio) fed diets containing 0.57% and 1.14% tannins The extents of these growth-inhibitory eĂects varied in different Âsh species studied and also plant material used in the Âsh-feed formulation The reason behind this growth-inhibitory eĂect of tannin has been attributed to the inhibition of digestive enzymes by tannin Bean tannins were shown to strongly inhibit pancreatic trypsin, chymotrypsin and a-amylase (Sing 1984; Carmona, Seild & JaĂe 1991) Studies of Maitra and Ray (2003) revealed that tannin extracted from Acacia inhibits the intestinal enzymes of L rohita Ângerlings to a signiÂcant level However, there is hardly any report on the eĂect of tannin on metabolic enzymes of Âsh and other organisms The present study revealed signiÂcant accumulation of tannin in Âsh muscle and metabolically active tissues like the Bioaccumulation of tannin in carps S Mandal & K Ghosh liver and kidney Therefore, the interference by tannin with the activity of metabolic enzymes at the tissue level may be assumed to serve as a basis to explain the poor growth response of Âsh fed tannin-containing feed (Olvera et al 1988; Al-Owafeir 1999; Becker & Makker 1999) The growing demand to substitute Âsh meal in aquafeed has led to a search for less expensive and protein-rich nonconventional feed resources of vegetative origin (Edwards, Kamal & Wee 1985; Wee & Wang1987; Ray & Das1992,1994,1995; Mondal & Ray 1999) However, the results of the present study emphasize that the presence of tannin should be well thought out while incorporating plant matter into formulated diets Acknowledgments The authors are grateful to the Head, Department of Zoology, The University of Burdwan, West Bengal, India, and The Department of Science and Technology (FIST programme), New Delhi, India, for providing research facilities References Al-Owafeir M (1999) The eĂects of dietary saponin and tannin on growth performance and digestion in Oreochromis niloticus and Clarias gariepinus PhD Thesis, Institute of Aquaculture, University of Stirling, UK, 220pp Becker K & Makkar H.P.S (1999) EĂects of dietary tannic acid and quebracho tannin on growth performance and metabolic rates of common carp (Cyprinus carpio L.) Aquaculture 175, 327^335 Carmona A., Seild D.S & JaĂe W.G (1991) Comparison of extraction methods and assay procedures for the determination of the apparent tannin content of common beans Journal of the Science of Food and Agriculture 56, 291^301 Edwards P., Kamal M & Wee K.L (1985) Incorporation of composted and dried water hyacinth in pelleted feed for the tilapia Oreochromis niloticus (Peters) Aquaculture and Fisheries Management 16, 233^248 Hossain M.A & Jauncey K (1989) Nutritional evaluation of some Bangladeshi oilseed meals as partial substitutes for Âsh meal in the diet of common carp, Cyprinus carpio L Aquaculture and Fisheries Management 20, 255^268 JhingranV.G (1997) Fish and Fisheries of India, 3rd edn Hindustan Publishing Corporation, Delhi, India pp 335^337 Kinnear P.R & Gray C.D (2000) SPSS forWindows Made Simple Release 10 Psychology Press, Sussex, UK Krogdahl A (1989) Alternative protein sources from plants contain anti-nutrients aĂecting digestion in salmonids In:The Current Status of Fish Nutrition in Aqua-culture Pro- r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 945^948 947 Bioaccumulation of tannin in carps S Mandal & K Ghosh ceedings of the Third International, Symposium on Feeding and Nutrition in Fish, 28 August^1 September 1989, Toba, Japan, (ed by M Takeda & T Watanabe), pp 253^261 Tokyo University of Fisheries,Tokyo, Japan Maitra S & Ray A.K (2003) Inhibition of digestive enzymes in rohu, Labeo rohita (Hamilton) Ângerlings by tannin, an in vitro study Aquaculture Research 34, 93^95 Mondal T.K & Ray A.K (1999) The NutritiveValue of Acacia auriculiformis leaf meal in compounded diets for Labeo rohita Ângerlings In: The Fourth Indian Fisheries Forum Proceedings, (ed by Mohan Joseph Modayil), pp 295^298 Asian Fisheries Society, Indian Branch, Mangalore, India Mukhopadhyay N & RayA.K (1996) The potential of deoiled sal (Shorea robusta) seed meal as a feedstuĂ in pelleted feed for Indian major carp, rohu, Labeo rohita (Hamilton) Ângerlings Aquaculture Nutrition 2, 221^227 Olvera N.M.A., Martinez P., Galvan C.R & Chavez S.C (1988) The use of seed of the leguminous plant Sesbania grandijlora as a partial replacement for Âsh meal in the diets for tilapia (Oreochromis mossambicus) Aquaculture 71,51^60 Prusty A.K., Sahu N.P., Pal A.K., Reddy A.K & Kumar S (2007) EĂect of dietary tannin on growth and haematoimmunological parameters of Labeo rohita (Hamilton) Ângerlings Animal Feed Science andTechnology 136, 96^108 Ray A.K & Das I (1992) Utilization of diets containing composted aquatic weed (Salvinia cuculata) by the Indian major carp, rohu, (Labeo rohita Ham.) Ângerlings Bioresource Technology 40, 67^72 948 Aquaculture Research, 2010, 41, 945^948 RayA.K & Das I (1994) Apparent digestibility of some aquatic macrophytes in rohu, Labeo rohita (Ham.) Ângerlings Journal of Aquaculture in theTropics 9, 335^342 Ray A.K & Das I (1995) Evaluation of dried aquatic weed, Pistia stratiotes meal as a feedstuĂ in pelleted feed for rohu, Labeo rohita Ângerlings Journal of Applied Aquaculture 5, 35^44 Schanderi S.H (1970) Methods in Food Analysis Academic Press, NewYork, NY, USA,709pp Silanikove N., Perevolotsky A & Provenza F.D (2001) Use of tannin-binding chemicals to assay for tannins and their negative post ingestive eĂects in ruminants Animal Feed Science and Technology 91, 69^81 Singh U (1984) The inhibition of digestive enzymes by polyphenols of chick pea (Cieer arrierinum L.) and pigeon pea (Cajamus cajan (L.) Millsp.) Nutrition Reports International 29,745^753 Wee K.L & Wang S.S (1987) Nutritive value of Leucaena leaf meal in pelleted feed for Nile Tilapia Aquaculture 62, 97^108 Zar J.H (1999) Biostatistical Analysis, 4th edn Pearson Education Singapore Pte Ltd (Indian Branch), New Delhi, India 663pp Keywords: tannin, Âsh tissue, bioaccumulation, carps r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 945^948 Aquaculture Research, 2010, 41, 949^951 doi:10.1111/j.1365-2109.2009.02382.x SHORT COMMUNICATION Effect of feeding level of Ugachick feed on the reproductive performance and growth of Nile tilapia (Oreochromis niloticus) in ponds in Uganda David Semu Matsiko1, Eugene Rurangwa2, Harrison Charo-Karisa3 & Musiimbi Fred1 National Agricultural Research Organisation (NARO), Aquaculture Research and Development Centre (ARDC) Kajjansi, Kampala, Uganda Laboratory of Aquatic Ecology and Evolutionary Biology, Katholieke Universiteit Leuven, Leuven, Belgium Kenya Marine and Fisheries Research Institute (KMFRI), Sangoro Aquaculture Centre, Pap-Onditi, Kenya Correspondence: D S Matsiko, National Agricultural Research Organisation (NARO), Aquaculture Research and Development Centre (ARDC), Kajjansi, P.O Box 530, Kampala, Uganda E-mails: smatsiko2002@yahoo.co.uk, matsikodavidsemu@aqua.go.ug Introduction For successful and sustained aquaculture, a continuous supply of high-quality tilapia seed is essential Increased production of Nile tilapia has been attributed mainly to the intensiÂcation of hatchery seed production (Little, Coward, Bhujel, Pham & Bromage 2000) and appropriate feeding and management of broodstock (Gunasekera, Shim & Lam 1995; Bhujel, Little & Hossain 2007; El-Sayed & Kawanna 2008) Both the quality and the quantity of food aĂect the frequency of spawning and the number of seed per clutch (Bhujel,Yakupitiyage,Turner & Little 2001; ElSayed & Kawanna 2008) In Uganda, eĂorts to intensify tilapia production are hampered by insucient supply of quality fry and appropriate broodstock feeds Ugachick has produced Ugachick Âsh feed but its eĂect on seed production among tilapia broodÂsh has not been evaluated In attempts to meet the high demand for tilapia fry, farmers in Uganda tend to use high feeding levels of Ugachick feed This experiment aimed at determining the optimal feeding level for maximum fry production in Nile tilapia broodstock using Ugachick feed Nine-month-old Nile tilapia broodÂsh (n 240) from Lake Victoria were used in the trial for 94 days BroodÂsh were randomly assigned to100 m2 earthen ponds and acclimated to experimental conditions until swim-up fry were observed The water exchange r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd rate per treatment was 10 L During this period, they were fed Ugachick feed at 1% of their body weights twice daily Four treatments corresponding to feeding levels used by farmers were used for the trial: 0.5%, 2%, 3% and 5%, each in randomly arranged triplicate ponds Higher feeding levels, as high as 5%, are preferred by farmers because they are thought to increase the quantity and quality of fry production Ten males and ten females were weighed individually and stocked in each pond Fish were fed twice a day (10:00 and 17:00 hours) on Ugachick feed containing 32% crude protein, 7% crude fat, 25% carbohydrates, 3% minerals, 2% vitamins, 6% Âbre and 9% ash From day 14 of stocking, ponds were observed daily for swim-up fry The time from stocking to sighting of swim-up fry was then recorded, the pond was harvested and fry was counted The rest of the ponds in the same treatment were seined days later regardless of fry sighting Ponds were completely drained and pond water was passed through a Âne mesh hapa at the outlet to prevent the escape of fry The fry were placed on a dry towel and bulk weighed using an electronic balance (two decimal digits) and their weight was recorded Water quality was monitored weekly at 08:00 hours throughout the experiment Dissolved oxygen (DO) and temperature were measured using YSI meter model 550A (Environmental Incorporated, Yellow Springs, OH, USA), pH with YSI 60 while unionized 949 EĂect of feeding level of Ugachick feed D S Matsiko et al ammonia (NH3) was determined using the Kjeldahl method Percentage feed conversion to seed was calculated as: Percentage feed conversion to seed Weight (g) of seed/Weight (g) of feed 100 (Bhujel et al 2007) The proÂt index was calculated as: ProÂt index (Total value of fry-total value of feed used)/ Total value of feed used SPSS version 11.5 software was used for data analysis Homogeneity of variances was tested using Levenes statistic, and pairwise comparisons between treatments were performed using the Duncan multiple range test The eĂect of increasing feeding rates on the inter-spawning interval and number of fry produced per batch/female was evaluated using one-way ANOVA The relationship between feeding rates and time to spawn was determined using regression analysis Data on feed conversion to seed, proÂt index, total feed up to spawning and cost of feed were log transformed for normal distribution before analysis by one-way ANOVA as well There was a signiÂcant diĂerence in feed conversion to seed between the 0.5% feeding rate and the other three treatments Feed conversion to seed at 0.5% was 4, and 11 times better than at 2%, 3% and 5% feeding rates respectively In this study, percentage feed conversion to seed decreased with increasing feeding rates This is similar to earlier observations that Nile tilapia invests more in reproduction at the cost of somatic growth in times of food scarcity (Siddiqui, Al-harbi & Al-hafedh 1997; Bhujel et al 2007; Tsadik & Bart 2007) Bhujel et al (2001) advise against high feeding rates and high crude protein in tilapia broodÂsh feeds because they support somatic growth at the cost of reproductive output and associated high costs for feeding large broodstock biomass Aquaculture Research, 2010, 41, 949^951 Fry production per square metre per day ranged from 1.09 to 1.27 (Table 1) and was not signiÂcant between treatments This is very low and can be attributed to the large size of spawners and the low stocking density adopted to simulate farmer practices in the country The mean time to sighting of swim-up fry per treatment was the shortest (28.7 days) at 0.5% and the longest (32.7 days) at the 2% feeding rate but the differences were not signiÂcant (P40.05) There was a signiÂcant positive correlation between time taken to spawning and fecundity [r 0.424 (Po0.05)] but the relationship was weakly negative [r 0.037 (P40.05)] with increasing feeding rate A positive correlation implies that more females spawn with time while a negative relationship can be attributed to deteriorating water quality with an increase in the feeding level At the end of the experiment, all broodÂsh showed an increase in the mean weights (Table 1), with the highest growth increment at the highest feeding rate Throughout the experiment, pH was 6.5 DiĂerences in DO, temperature, NH3 or pH were not signiÂcant across treatments However, NH3 increased with increasing feeding rate Although NH3 was higher than the limits recommended for best Nile tilapia growth (Pillay 1992; Tran-duy, Schrama, Van Dam & Verreth 2008), its eĂect was not signiÂcant, suggesting that the waterexchange rate adopted was eĂective Tsadik and Bart (2007) recommend continuous water exchange as a way of improving Âsh reproductive performance High feeding rates should be avoided if there is no water exchange as this can lead to deterioration in water quality, causing low fertilization and poor via- Table Means and SD of broodstock weight, feed input and its usage, fry output, proÂt index and water quality parameters Feeding level (percentage biomass) Parameter 0.5% Initial broodstock weight (g) Final broodstock weight (g) Feed given up to spawning (kg) Cost of feed to spawning ($) Number of fry per female per spawning Seed per square metre per day Time taken to fry sighting (days) % feed conversion to seed Profit index Dissolved oxygen (mg L 1) Nitrogen ammonia (mg L 1) Temperature ( 1C) 399.4 430.0 1.21 0.55 369.8 1.27 28.7 9.79 165.54 3.6 0.2 24.8 2% ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 32.3a 33.7a 0.1a 0.05a 72.1a 0.56a 3.0a 4.79b 81.54b 0.28a 0.17a 1.0a 393.6 426.1 5.58 2.51 358.1 1.14 32.7 2.2 36.37 3.4 0.30 25.1 3% ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 11.4a 14.6a 0.9b 0.41b 87.0a 0.73a 4.4a 1.41a 23.93a 0.20a 0.21a 0.89a 377.2 443.8 8.39 3.72 371.0 1.09 30.5 1.31 21.3 3.5 0.30 24.8 5% ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 18.0a 37.0a 3.4b 1.46b 320.5a 0.9a 10.7a 1.1a 17.84a 0.12a 0.21a 0.89a 375.5 447.7 14.27 6.31 363.3 1.17 31.3 0.87 13.88 3.3 0.8 24.9 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 30.1a 44.6a 5.6c 2.51c 54.6a 0.29a 10.3a 0.24a 4.0a 0.31a 0.04a 0.90a Similar superscripts imply no signiÂcant diĂerence 950 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 949^951 Aquaculture Research, 2010, 41, 949^951 bility of eggs or embryos, resulting in low seed output (Bhujel et al 2001) The cost of feed was signiÂcantly lower (Po0.05) at the 0.5% feeding rate compared with the other feeding rates There was a signiÂcant diĂerence (Po0.05) in the proÂt index between 0.5% and the other feeding rates However, there was no diĂerence between 2%, 3% and 5% feeding rates The proÂt index at the 0.5% feeding rate was 5, and 12 times higher than that at 2%, 3% and 5% body weight respectively The present study was aimed to determine the most costeĂective feeding rate The proÂt margin from the sale of fry is a function of female fecundity, the amount of feed utilized and the cost of each kilogram of the same feed In this study, the 0.5% feeding rate yielded the best results as feeding at higher rates did not signiÂcantly increase the number of fry Therefore, 0.5% is the recommended feeding rate for farmers using Ugachick feed EĂect of feeding level of Ugachick feed D S Matsiko et al References Nile tilapia (Oreochromis niloticus) broodÂsh at varying feeding rates Aquaculture 273,71^79 El-Sayed M.A.-F & Kawanna M (2008) EĂects of dietary protein and energy levels on spawning performance of Nile tilapia (Oreochromis niloticus) broodstock in a recycling system Aquaculture 280, 179^184 Gunasekera R.M., Shim K.F & Lam T.J (1995) EĂect of dietary protein level on puberty, oocyte growth and egg chemical composition in tilapia, Oreochromis niloticus (L.) Aquaculture 134, 169^183 Little D.C., Coward K., Bhujel R.C., Pham T.A & Bromage N.R (2000) EĂect of broodÂsh exchange strategy on spawning performance and sex steroid hormone levels of Nile tilapia (Oreochromis niloticus) broodÂsh in hapas Aquaculture 186,77^88 Pillay T.V.R (1992) Aquaculture and the Environment, 1st edn Cambridge University Press, Cambridge, p.189 Siddiqui A.Q., Al-Harbi A.H & Al-HafedhY.S (1997) EĂect of food supply on size at Ârst maturity, fecundity and growth of hybrid tilapia Oreochromis nilotocus (L.) Oreochromis aureus (Steindachner), in outdoor concrete tanks in Saudi Arabia Aquaculture Research 28, 341^349 Tran-Duy A., Schrama J.W., van Dam A.A & Verreth J.A.J (2008) EĂects of oxygen concentration and body weight on maximum feed intake, growth and haematological parameters of Nile tilapia Oreochromis niloticus Aquaculture 275, 152^162 Tsadik G.G & Bart A.N (2007) EĂects of feeding, stocking density and water-Êow rate on fecundity, spawning frequency and egg quality of Nile tilapia, Oreochromis niloticus (L) Aquaculture 272, 380^388 Bhujel R.C.,Yakupitiyage A.,Turner W.A & Little D.C (2001) Selection of a commercial feed for Nile tilapia (Oreochromis niloticus) broodÂsh breeding in a hapa-in-pond system Aquaculture 194, 303^314 Bhujel R.C., Little D.C & Hossain A (2007) Reproductive performance and the growth of pre-stunted and normal Keywords: feeding rate, Ugachick feed, broodstock, Oreochromis niloticus, reproductive performance Acknowledgments Mr Kawoya Godfrey and Maurice Ssebisubi are acknowledged for their help with data analysis Mr Owori W.A and Kasibante M are thanked for their support during the experimental period r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 949^951 951 [...]... problems in aquaculture Reviews in Fisheries Science 8, 45^88 Waschulewski I.H & Sunde R.A (1988) EĂect of dietary methionine on utilization of tissue selenium from dietary selenomethionine for glutathione peroxidase in the rat Journal of Nutrition 118, 367^374 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 803 Aquaculture Research, 2010, 41, 804^813... genus Leuciscus Archives of Polish Fisheries 16, 319^340 Kupren K (2005) Thermal conditions for embryonic development of Âsh from the genus of Leuciscus Doctoral thesis, University of Warmia and Mazury, Olsztyn, 95pp (in Polish) r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 804^813 Aquaculture Research, 2010, 41, 804^813 Lahnsteiner F., Berger B., Horvath... Kucharczyk D & Ciereszko A (2003) Characterization of protease inhibitors of seminal plasma of cyprinids Aquatic Living Resource 16, 461^465 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 804^813 813 Aquaculture Research, 2010, 41, 814^827 doi:10.1111/j.1365-2109.2009.02358.x Growth, survival and immune activity of scallops, Chlamys farreri Jones et Preston,... strategies in Norway: a review Aquaculture International 9, 305^318 Broom M.J & Mason J (1978) Growth and spawning in the pectinid Chlamys opercularis in relation to temperature r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 814^827 825 Growth, survival and immune activity of scallops Z Yu et al Aquaculture Research, 2010, 41, 814^827 Grant J., Emerson... when milt makes contact with water or other activating solutions (Poupard, Paxion, Cosson, Jeulin, r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 804^813 Aquaculture Research, 2010, 41, 804^813 Fierville & Billard 1998) During milt collection, uncontrolled (and unwanted) spermatozoa activation can occur through their contact with urine released during... 3 56, 233^241 Rayman M.P (2005) Selenium in cancer prevention: a review of the evidence and mechanism of action Proceedings of the Nutrition Society 64, 527^542 Schram E., Pedrero Z., Camara C.,Van der Heul J.W & Luten J.B (2008) Enrichment of African catÂsh with functional r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 793^803 Aquaculture Research, 2010, ... batch (10 000 g) for 10 min and next the supernatant obtained was transferred into test tubes 806 r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 804^813 Aquaculture Research, 2010, 41, 804^813 Hormonal stimulation of ide males B I Cejko et al and stored at 80 1C until analyses The total protein content in seminal plasma (mg mL 1) was determined using... longline at about 5 m below the sea surface One bottom culture system r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 814^827 815 Growth, survival and immune activity of scallops Z Yu et al Aquaculture Research, 2010, 41, 814^827 Figure 1 Study site on cultured scallops, Chlamys farreri, during 2007^2008 in Haizhou Bay, theYellow Sea, China (sleeves) used... nets (about 100 ind) and three diĂerent compartments of sleeves (about 1000 ind) were counted to r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 814^827 Aquaculture Research, 2010, 41, 814^827 Growth, survival and immune activity of scallops Z Yu et al evaluate survival at the end of this study; the time of death of the scallops was estimated retrospectively... 7.2 8.6 9.4 11.7 9.0 8.7 7.2 8.3 9.2 11.4 8.8 DO, dissolved oxygen r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 814^827 817 Growth, survival and immune activity of scallops Z Yu et al Aquaculture Research, 2010, 41, 814^827 30 Surface Bottom Water temperature (C) 25 20 15 10 5 0 Jun-07 Jul-07 Sep-07 Oct-07 Dec-07 Feb-08 Mar-08 May-08 Jul-08 Figure