Aquaculture research, tập 42, số 4, 2011

Aquaculture Research, 2011, 42, 469^479 doi:10.1111/j.1365-2109.2010.02640.x Growth and osmoregulation in Atlantic salmon (Salmo salar ) smolts in response to different feeding frequencies and salinities Albert Kjartansson Imsland1,2, Klement Arild VÔge1, Sigurd Olav Handeland1 & Sigurd Olav Stefansson1 Department of Biology, High Technology Center, University of Bergen, Bergen, Norway Akvaplan-niva, Iceland O⁄ce, Ko¤pavogur, Iceland Correspondence: A K Imsland, High Technology Center, Department of Biology, University of Bergen, N-5020 Bergen, Norway E-mail: imsland@vortex Abstract Introduction Groups of Atlantic salmon (Salmo salar) yearling smolts were reared in duplicate tanks supplied with freshwater or seawater, and subjected to di¡erent feeding frequencies, 100% (fed every day), 50% (fed every other day), 25% (fed every forth day) and 0% (starved), from 26 May to 26 July After weeks, all the groups were re-fed in excess for weeks Fish were maintained on their respective a priori salinity treatments during the 6-week follow-up period Starvation for a period of weeks in freshwater resulted in a loss of hypo-osmoregulatory ability when smolts were challenged with seawater and unfed smolts maintained in freshwater were unable to adapt to seawater in mid-July Ration levels in£uenced the growth rate and body size signi¢cantly The overall growth rate was higher in freshwater than at corresponding rations in seawater Partial compensatory growth was observed in the and 25% groups following re-feeding Branchial Na1,K1-ATPase (NKA) activity decreased rapidly in unfed smolts in freshwater and was the lowest in the starved group, whereas an initial increase was observed in those groups reared in seawater After re-feeding NKA activity di¡erences decreased between the former feeding groups Our results suggest that nutritional factors and/or energy levels are critical for the maintenance of hydro-mineral balance of salmon smolts In their natural habitat, smolti¢cation in Atlantic salmon is synchronized to occur during spring by seasonal changes in the temperature and photoperiod (Hoar 1988; Duston & Saunders 1989; Nilsen, Ebbeson, Kiilerich, Bj˛rnsson, Madsen, McCormick & Stefansson 2008; Stefansson, Bj˛rnsson, Ebbesson & McCormick 2008) Smolt development is normally accompanied by decreases in lipid reserves and liver glycogen, even when ¢sh are fed ad libitum (Sheridan 1989; Stefansson, Bj˛rnsson, Sundell, Nyhammer & McCormick 2003) This has led to the general concept that wild Atlantic salmon smolts are naturally ‘energy de¢cient’ during the downstream migration The ¢ndings of Stefansson et al (2003) indicated that smolts also have low energy reserves during the early marine phase If salmon smolts are prevented from reaching seawater, a partial readaptation to freshwater will occur through the abandonment of mechanisms, allowing survival in marine water and a re-establishment of adaptations to a hypo-saline environment This process is known as desmolting or parr-reversion (see Hoar 1988) Earlier research has focused on the e¡ect of di¡erent environmental factors on the desmolting process and the corresponding loss of seawater tolerance in Atlantic salmon (e.g Stefansson, Berge & Gunnarsson 1998; Handeland, Wilkinson, SveinsbÖ, McCormick & Stefansson 2004) However, the e¡ect of food deprivation on osmoregulatory mechanisms during desmolting is poorly understood Food deprivation is known to in£uence seawater adaptation in several ¢sh species, Keywords: Atlantic salmon, ration, food deprivation, osmoregulation, growth, salinity r 2010 Blackwell Publishing Ltd 469 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al Materials and methods Fish stock and rearing conditions The ¢sh were 1-year-old smolts from the AquaGen strain, hatched and reared at S×vareid Fish Farms (601N,Western Norway) The ¢sh were ¢rst fed in February 2002 at 13^14 1C, and later reared under natural light and temperature conditions until April 2003, when they were transferred to the Industrial Laboratory (ILAB) at the Bergen High Technology Centre A total of 1600 smolts were randomly distributed among sixteen 1m2 grey, covered ¢breglass tanks with a rearing volume of 450 L, and kept in running pH-adjusted (NaOH, 6.5opHo7.0) freshwater (7.5 L À 1) until 24 May under a simulated natural photoperiod (SNP) for Bergen (60125 N) At ILAB, water temperature increased from approximately 1C in early May to 8.5 1C in late May (Fig 1) From 20 May to 20 July, the temperature increased 470 14 Temperature (°C) including tilapia (Oreochromis mossambicus, Jˇrss, Bittorf, V˛lker & Wacke 1984; Kˇltz & Jˇrss 1991) and rainbow trout (Oncorhynchus mykiss, Jˇrss, Bittorf, V˛lker & Wacke 1987; Nance, Masoni, Sola & Bornancin1987) In contrast,Triebenbach, Smoker, Beckman and Focht (2009) found that spring increases in gill Na1,K1-ATPase (NKA) activity were una¡ected by a 16-week food deprivation in coho salmon (Oncorhynchus kisutch) and Chinook salmon (Oncorhynchus tshawytscha) Partially in accordance with the results of Triebenbach et al (2009) on coho and Chinook salmon, Dickho¡, Mahnken, Zaugg, Waknitz, Bernand and Sullivan (1989) showed that Atlantic salmon that were starved in November and December completed smolti¢cation the following spring and showed good post-smolt growth during the ¢rst months in seawater However, in starved Atlantic salmon smolts reared in freshwater, considerably reduced branchial NKA have been observed (Virtanen & Soivio 1985) Furthermore, Stefansson, Imsland & Handeland (2009) suggested that Atlantic salmon smolts in seawater lose their hypo-osmoregulatory capacity (measured as increased plasma ion levels and reduced NKA activity) during starvation Restricted feeding may, therefore, lead to a disruption of the smolti¢cation process, resulting in reduced hypo-osmoregulatory ability The present study was performed in order to describe the consequences of various degrees of food restriction in Atlantic salmon on growth and hypo-osmoregulatory ability during the critical early post-smolt phase Aquaculture Research, 2011, 42, 469–479 12 10 Treatment period May June July Aug Sept Month Figure Rearing temperatures for Atlantic salmon post smolts in seawater (whole line) and freshwater (dotted line) The temperature pro¢les are based on daily measurements in the experimental tanks from 8.5 to 12.0 1C, decreasing to 10 1C in late July, and remained around 10 1C until termination of the experiment on September Fish were fed a standard dry diet used throughout the experiment (47.0% protein, 18.5% fat, 19.0% carbohydrates, 9.0% ash, Ewos, Bergen, Norway) from automatic feeders at rates according to the temperature and ¢sh size (Austreng, Storebakken & —sgard 1987) during the photo phase Experimental design Between 24 and 26 May, water quality was changed in eight tanks from fresh water (FW) to full-strength sea water (34.5%, SW) of approximately 8.5 1C The eight remaining tanks were maintained on FW All groups were reared under an SNP during the entire experimental period Water £ow was maintained at approximately 7.5 L À in FW tanks and 8.5 L À in SW tanks, maintaining oxygen saturation above mg/L throughout the experiment All tanks were checked twice daily and dead ¢sh were removed immediately On 26 May, concurrent with the establishment of the two salinity regimes, four feeding regimes were initiated (0%, 25%,50% and100%) in a  factorial design, each group consisting of two replicate tanks The degree of food deprivation was obtained by withholding food at di¡erent days in a 4-day cycle, i.e 100%, fed continuously (control group);50%, fed every other day; 25%, fed every forth day and 0%, starved When fed, all groups were given excess food, with food distributed at hourly intervals during the photo phase After weeks on a di¡erent feed frequency (on 26 July), restricted frequencies were discontinued, 80 ¢sh from r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 Aquaculture Research, 2011, 42, 469^479 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al each group (i.e 40 ¢sh from each replicate tank) were ¢n-clipped (adipose ¢n or either pelvic ¢n) to identify the ration level, transferred to two 2000 L tanks supplied with running FWor SWand fed in excess until September, when the experiment was terminated When transferred to larger tanks, ¢sh were maintained on their respective a priori salinity treatments The experiment described has been approved by the local responsible laboratory animal science specialist under the surveillance of the Norwegian Animal Research Authority and registered by the Authority The experiment has thus been conducted in accordance with the laws and regulations controlling experiments in live animals on Norway, i.e the Animal Protection Act of 20 December 1974, No 73, chapter VI, sections 20^22 Mortality Acute mortality was observed among the FW groups during 7^11 June, in particular in the FW0% (50% mortality) Mortality was associated with reduced hyper-osmoregulatory ability in the ion-poor FW; hence on 11 June, a small amount of seawater was mixed with freshwater to increase the conductivity of the water from o70 mS cm À to approximately 200^ 250 mS cm À (YSI 556,YSI,Yellow Springs, OH, USA), which remained stable throughout the experiment Following this, water treatment mortality ceased immediately Mortality ranged between10% and 18% in the other FW groups (13%, FW25%; 10%, FW50%; 18%, FW100%), but was negligible in SW (1%, SW0%;1%, SW25%; 3%, SW50%;1%, SW100%) Seawater challenge test (SWCT) At approximately 14-day intervals between 24 May and 19 July, the seawater tolerance of 10 ¢sh from each group in FW was assessed in a 24-h SWCT (34.5%, Blackburn & Clark 1987) The SWCT was performed in similar types of tanks and with the same photoperiod (SNP) as in the main experiment The temperature during the SWCT was the same as in the FW tanks Sampling procedures Body weight (g) and fork length (cm) of 40 to 60 ¢sh in each tank were recorded at approximately 2-week intervals from 24 May until termination of the ex- periment on September The speci¢c growth rate for the experimental groups between two dates was calculated as: SGR (lnW2 À lnW1)  100/(T2 À T1), whereW1 andW2 are the mean weights (of all the ¢sh sampled in each tank) at daysT1 andT2 The condition factor (CF) was calculated as CF 5100  W/L3, where W is the individual weight and L is the corresponding fork length Concurrently, at each sampling, eight ¢sh from each group were killed by a blow to the head, blood was sampled from the caudal vein for the determination of plasma Cl À values and the second gill arch was removed for the determination of NKA activity Plasma was separated by centrifugation (10 at 1C and 2500 g) and stored at À 80 1C for subsequent analysis Plasma Cl À (mM) was determined in duplicate samples in a Radiometer CMT 10 chloride titrator (Radiometer, Copenhagen, Denmark) Gill tissue was quickly immersed in ice-cold SEI (Zaugg & McLain 1982) and frozen at À 80 1C and analysed according to the procedure of Zaugg and McLain (1982), modi¢ed by Berge, Berg, Fyhn, Barnung, Hansen and Stefansson (1995) Statistics All statistical analysis and graphics were developed using STATSOFT ^ STATISTICA 8.0 All data sets were tested for normality using a Kolmogorov^Smirnov test To ¢t normality, all data on branchial NKA activity and plasma Cl À levels were log transformed before the statistical tests A Hartley F-max test was used to test for homogeneity of variances, while a two-way ANOVA and a Tukey honest signi¢cance differences (HSD) post hoc test were applied to determine di¡erences between experimental groups In all cases, a signi¢cance level (a) of 0.05 was used Results Hypo-osmoregulatory ability Following a transient increase in the plasma Cl À levels in early June in the seawater-challenged FW0% group, a signi¢cant reduction in hypo-osmoregulatory ability was observed in SW-challenged ¢sh in early July, following weeks of starvation (Tukey HSD test, Po0.05, Fig 2) A further signi¢cant increase in the plasma Cl À levels was observed weeks later (Tukey HSD test, Po0.05), with the mean ( Æ SEM) levels reaching 184 Æ 11mM after a 24-h r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 471 Plasma Cl− (mM) E¡ect of feeding rations on growth and osmoregulation A K Imsland et al 195 190 185 180 175 170 165 160 155 150 145 140 135 a a b b b a b b b b b b May June Month July Figure Plasma chloride levels in Atlantic salmon post smolts fed di¡erent feeding regimes in freshwater and challenged in seawater (34.5%) for 24 h Symbols: 100% ( & ), 50% (D), 25% ( } ), and 0% ( ) Values are given as means Æ SE (n 6^10 in each experimental group) Signi¢cant di¡erences between the di¡erent experimental groups are given by di¡erent letters (Tukey post hoc test, Po0.05) Aquaculture Research, 2011, 42, 469–479 declined in the other three groups Accordingly, no signi¢cant di¡erences were seen in NKA activity among the FWgroups in September (Tukey HSD test, P40.25, Fig 3A) Following transfer to seawater, enzyme activity increased in all groups (Fig 3B) The activity levels did not di¡er signi¢cantly among feeding frequencies in seawater between May and July Following re-feeding, NKA activity increased transiently in the SW0% group to levels signi¢cantly higher than the controls and SW50% in August (Tukey HSD test, Po0.05), whereas the ¢nal NKA did not di¡er between the SWgroups in September (Fig 3B)  SW challenge No signi¢cant di¡erences were observed among the other FW groups after seawater challenge, although the Cl À levels were slightly elevated (albeit not signi¢cantly) after weeks in the FW25% group Plasma ion levels in unchallenged ¢sh grouped according to salinity, with Cl À levels of all the freshwater groups remaining around 135 mM, while all seawater groups showed levels around 145 mM No signi¢cant di¡erences (two-way ANOVA, P40.5) were found between unchallenged feeding groups in freshwater or seawater The exception was in freshwater in early June, when the levels were signi¢cantly reduced in all FW groups to 125 Æ mM, probably due to the inability of smolts to maintain hyper-osmoregulation in the ion-poor freshwater at this time No e¡ect of re-feeding on the plasma Cl À levels was observed as the values remained around135 mM in freshwater and around145 mM in seawater during the entire re-feeding period Branchial NKA Branchial NKA activity was in£uenced both by salinity and by ration (two-way ANOVA, Po0.05, Fig 3) In groups remaining in freshwater (Fig 3A), gill NKA activity di¡ered signi¢cantly among feeding groups, with the FW0% showing approximately 50% lower activity than controls from mid-June onwards (Tukey HSD test, Po0.05, Fig 3A) Following re-feeding in freshwater, NKA increased in the FW0% group and 472 Growth and condition Body length (Fig 4) did not change signi¢cantly in the 0% groups between May and July, while body weight (Fig 5) decreased during this period Body length (Fig 4) increased in the other groups at rates corresponding to the ration levels, while body weight (Fig 5) increased in the 50 and 100% fed groups only Accordingly, there were signi¢cant di¡erences among groups within each salinity regime at the end of the ration period (Tukey HSD test, Po0.05, Figs and 5) Except for the unfed groups, which did not di¡er in size after weeks of starvation, body length (Tukey HSD test, Po0.05, Fig 4) and weight (Tukey HSD test, Po0.05, Fig 5) were signi¢cantly higher in freshwater groups than in the corresponding seawater groups Growth rate was signi¢cantly in£uenced by both ration and salinity, with an overall negative SGR between May and July in the starved groups (Table 1) After re-feeding, the growth rate increased in the groups previously held on reduced ration levels, with SGR in the previously restricted groups exceeding SGR of the fully fed controls (Tukey HSD test, Po0.05,Table 1) However, despite the higher growth rates in the previously reduced ration groups, body weight di¡erences persisted during the weeks of feeding, with the ¢nal body size being larger in the fully fed groups (Fig 5) The CF decreased in all groups between mid-May and mid-June (Fig 6) Condition factor remained low (0.8^0.85) in the 0% groups during July, with no signi¢cant di¡erences between salinities (two-way ANOVA, P40.3, Fig 6) Condition factor in the fully fed groups remained relatively constant between 1.0 and 1.05 and was not signi¢cantly di¡erent between sali- r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 Aquaculture Research, 2011, 42, 469^479 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al 22 (A) Freshwater Na+K+ ATPase activity (µmol Pi mg protein–1 t–1) 20 18 a 16 14 bc c cd ab b b b b bc c cd cd 12 10 b b b b b b b c Treatment b d period May June d July Aug Sept Month 22 a (B) Seawater a Na+K+ ATPase activity (µmol Pi mg protein–1 t–1) 20 * * a 18 a a a a 16 a a a 14 ab ab ab 12 a a ab ab ab ab ab 10 a ab b b Treatment period May June July Aug Sept Month Figure Gill Na1,K1-ATPase activity in Atlantic salmon post smolts fed di¡erent feeding regimes in freshwater (A) and seawater (B) Symbols: 100% ( & ), 50% (D), 25% ( } ) and 0% ( ) Values are given as means Æ SE (n 6^10 in each experimental group) Signi¢cant di¡erences between the di¡erent experimental groups (all groups in FW and SW tested simultaneously) are given by di¡erent letters (Tukey post hoc test, Po0.05) ÃA signi¢cant interaction between feeding regimes and salinity  nities Following re-feeding, the CF increased in all previously reduced ration groups Discussion The restricted feeding frequency signi¢cantly in£uenced the growth rate and CF of Atlantic salmon in both seawater and freshwater During the feed deprivation period, the growth rate decreased systematically with the ration level, causing signi¢cant reductions in body weight compared with the fully fed controls Changes in length were less a¡ected by feed restriction Positive growth in length (i.e skele- tal growth) during periods of starvation and weight loss (i.e little or no muscle growth) seems to be a general phenomenon in ¢sh, and may be adaptive in ¢sh experiencing £uctuating (seasonal) changes in food abundance (Nicieza & Metcalfe 1997) In ¢sh, muscle growth occurs by both hyperplasia (cell proliferation resulting in the generation of new myotubes) and hypertrophy (increase in myotube size) (Rowlerson & Veggetti 2001; Johansen & Overturf 2006) Kiessling, Storebakken, Asgard and Kiessling (1991) suggested that in rainbow trout, periods of rapid growth favoured ¢bre hypertrophy and periods of slow growth favoured ¢bre recruitment In Atlantic salmon (Higgins & Thorpe 1990), ¢bre recruitment (hyperplasia) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 473 Aquaculture Research, 2011, 42, 469–479 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al 30 (A) Freshwater Body length (cm) a ab ab a a 28 26 a ab a ab 22 a 20 ab ab ab cd b a ab ab 24 c c d e b e Treatment period 18 May June July Aug Sept Month 30 * (B) Seawater * * 28 Body length (cm) b b c 26 24 b b ab ab 22 ab ab 20 ab b c d ab b bc c d e c cd d c d Treatment period 18 May June July Aug Sept Month Figure Mean length (cm) in Atlantic salmon post smolts fed di¡erent feeding regimes in freshwater (A) and seawater (B) Symbols:100% ( & ),50% (D), 25% ( } ) and 0% ( ).Values are given as means Æ SE (n 516^93 in each experimental group) Signi¢cant di¡erences between the di¡erent experimental groups (all groups in FWand SW tested simultaneously) are given by di¡erent letters (Tukey post hoc test, Po0.05) ÃA Signi¢cant interaction between feeding regimes and salinity  seemed to be most apparent in periods with unfavourable growing conditions, e.g feed restriction, which could explain the positive skeletal growth seen in periods of no muscle growth Decreased density of ¢bres with increasing size has also been found for Atlantic salmon, while the estimated total ¢bre number increased simultaneously (Johnston, Manthri, Smart, Campell, Nickell & Alderson 2003) The intuitive explanation for this is that as the ¢sh grow in size, more ¢bres are recruited, resulting in a higher total number of ¢bres, while at the same time, the ¢bres increase in diameter at a higher rate than does the recruitment of cells, resulting in a lower density (Johnston et al 2003) After re-feeding, all previously 474 feed-restricted groups showed an increase in the growth rates, i.e compensatory growth (CG) However, only partial weight compensation was achieved within the weeks of re-feeding in the previously feed-deprived groups in seawater, whereas full weight compensation was reached in the FW25% and FW50% groups The results of the seawater groups are comparable with previous experiments, where partial CG as a result of starvation has been found, i.e Arctic charr, Salvelinus alpinus (Jobling, Jorgensen & Siikavuopio 1993), and Atlantic halibut, Hippoglossus hippoglossus (Heide, Foss, Stefansson, Mayer, Roth, Norberg, Jenssen, Nortvedt & Imsland 2006) In contrast, experiments performed on Atlan- r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 Aquaculture Research, 2011, 42, 469^479 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al 250 (A) Freshwater 230 210 Body weight (g) a a ab a ab cd 190 a b c 170 a a a 150 130 ab ab ab ab a ab ab 110 a b c c e 90 b 70 b Treatment period d e 50 May June July Aug Sept Month 250 * (B) Seawater * * 230 Body weight (g) 210 b d d 190 170 150 130 110 a ab ab ab 90 b b b 70 ab cd cd a ab ab b d d ab b b c e b b Treatment period e e 50 May June July Aug Sept Month Figure Mean weight (g) in Atlantic salmon post smolts fed di¡erent feeding regimes in freshwater (A) and seawater (B) Symbols: 100% ( & ), 50% (D), 25% ( } ) and 0% ( ) Values are given as means Æ SE (n 516^93 in each experimental group) Signi¢cant di¡erences between the di¡erent experimental groups (all groups in FWand SW tested simultaneously) are given by di¡erent letters (Tukey post hoc test, Po0.05) ÃA signi¢cant interaction between feeding regimes and salinity  Table Mean speci¢c growth rate (SGR) in Atlantic salmon post smolts fed di¡erent feeding regimes in fresh- and seawater Periodà FW100% FW50% FW25% FW0% SW100% SW50% SW25% SW0% 26 May to June 0.31 (0.03)a À 0.11 (0.05)c 0.02 (0.06)b À 0.31 (0.04)d 0.01 (0.02)b 0.07 (0.04)b À 0.25 (0.04)d À 0.27 (0.04)d June to 21 June 0.20 (0.01)b 0.52 (0.04)a À 0.12 (0.04)c À 0.10 (0.06)c 0.40 (0.04)a 0.16 (0.06)b 0.23 (0.03)b À 0.11 (0.03)c 22 June to July 1.36 (0.06)a 0.34 (0.02)d 1.14 (0.04)b À 0.65 (0.06)f 0.85(0.05)c 0.93 (0.05)c À 0.28 (0.03)e À 0.77 (0.06)f July to 26 July 1.28 (0.04)a 1.01 (0.05)b 0.18 (0.06)e À 0.29 (0.06)f 0.85 (0.05)c 0.33 (0.06)d 0.48 (0.05)d 0.04 (0.06)e -27 July to 16 0.94 (0.08)c 1.38 (0.05)b 1.60(0.06)a 1.51 (0.05)a 1.08(0.08)c 1.05 (0.05)c 1.57 (0.03)a 0.91 (0.09)c August 17 August to 0.81 (0.07)e 1.02 (0.05)d 1.27 (0.06)c 1.58 (0.05)b 0.82 (0.06)e 1.04 (0.05)d 1.05 (0.06)d 1.82 (0.06)a September ÃThe experimental groups were fed di¡erent feeding rations from 26 May to 26 July, but from 27 July to September, all groups were re-fed in excess (indicated by a broken line) Values are given as means (Æ SE) values from the two replicate tanks Signi¢cant di¡erences between the di¡erent experimental groups are indicated by di¡erent letters (Tukey post hoc test, Po0.05) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 475 Aquaculture Research, 2011, 42, 469–479 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al 1.2 (A) Freshwater 1.1 Condition factor ab a a a a a ab a ab ab 1.0 ab ab b b ab ab b 0.9 b b b c 0.8 c c b b c c d Treatment period 0.7 May June July Aug Sept Month 1.2 * (B) Seawater Condition factor 1.1 a a a a a a a a * a a a 1.0 ab ab a a a b b b 0.9 0.8 b ab b c c b c c d Treatment period 0.7 May June July Aug Sept Month Figure Condition factor in Atlantic salmon post smolts fed di¡erent feeding regimes in freshwater (A) and seawater (B) Symbols: 100% ( & ), 50% (D), 25% ( } ) and 0% ( ) Values are given as means Æ SE (n 516^93 in each experimental group) Signi¢cant di¡erences between the di¡erent experimental groups (all groups in FWand SW tested simultaneously) are given by di¡erent letters (Tukey post hoc test, Po0.05) ÃA signi¢cant interaction between feeding regimes and salinity  tic cod, Gadus morhua (Jobling, MelÖy, Dos Santos & Christiansen 1994), and turbot, Scophthalmus maximus (S×ther & Jobling 1999), displayed a full weight recovery after restricted feeding similar to the present ¢ndings in freshwater (i.e FW25% and FW50%) Overall, the growth rates were higher in freshwater in line with previous ¢ndings where temporal di¡erences in the growth of Atlantic salmon smolts and post-smolts reared at di¡erent salinities are seen during summer (McCormick, Saunders & MacIntyre 1989; Duston 1994), but less so during autumn, winter and spring McCormick et al (1989) further indicated that ration was more important for 476 smolt growth than di¡erences in salinity, which is line with the present ¢ndings The CF of the control groups in June was similar to previous observations on cultured smolts (McCormick, Saunders, Henderson & Harmon 1987; Duston & Saunders, 1989; Handeland et al 2004) In other cases where smolts have been reared in freshwater beyond the peak of smolti¢cation, an increased CF has been reported (Eriksson & Lundquist 1982; Hoar 1988), and has been taken as a sign of desmolti¢cation, i.e., the loss of physiological adaptations to life in seawater Migrating smolts and post-smolts in nature may experience periods of restricted food r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 Aquaculture Research, 2011, 42, 469^479 E¡ect of feeding rations on growth and osmoregulation A K Imsland et al availability (Stefansson et al 2003), indicated by the presence of a large fraction of empty stomachs in samples of wild post-smolts (Levings, Hvidsten & Johnsen 1994; Andreassen, Martinussen, Hvidsten & Stefansson 2001) Arnesen,Toften, Agustsson, Stefansson, Handeland and Bj˛rnsson (2003) further demonstrated that reared post-smolts may take several weeks to resume feeding following transfer to seawater The present results suggest that the long-term consequences of such restricted feeding may involve a reduction in hypo-osmoregulatory ability An initial increase in gill NKA was seen in the SW groups Such an increase in NKA is generally accepted as an indicator of good smolt quality, and is correlated with seaward migration, hypo-osmoregulatory ability and high seawater survival rates in salmonids (McCormick et al 1987; Handeland, BjÖrnsson, Arnesen & Stefansson 2002) Consequently, the salmon smolts in the SW group can be considered to be fully smolti¢ed as NKA increased signi¢cantly in the initial rearing period, i.e 26 May to June The SWCTs demonstrated that starvation in FW led to a severe decline in the hypo-osmoregulatory capacity (measured as plasma Cl À ), with the most adverse e¡ect seen at the termination of the starvation period in July According to Jobling (1996), starvation results in a general decrease in the turnover of biochemical components in cells, a¡ecting both the synthesis and the breakdown of tissue, with growth re£ecting the di¡erence between these two processes The negative growth observed in the starved group indicates that tissue breakdown exceeds the synthesis of new cellular components This suggests that the observed increase in the plasma chloride levels may be caused by increased degradation of osmotic barriers, i.e external membranes or swelling (Olsen, Sundell, RingÖ, Myklebust, Hansen & Karlsen 2008) The initial increase in the plasma Cl À levels in the FW% group could possibly be linked to the reduced branchial NKA as measured in vitro for the FW% group in this period The explanation suggested for tilapia (Kˇltz & Jˇrss1991) and rainbow trout (Jˇrss et al.1987) is that a reduction in branchial NKA activity would be adaptive during periods of starvation in order to reduce ATP consumption A concurrent decline in NKA activity was seen in the SW-starvation groups with a rapid 50% increase in activity levels seen after re-feeding Stefansson et al (2009) reported loss of hypo-osmoregulatory ability in starved Atlantic salmon post-smolts in seawater, suggesting that this is a general physiological response of this species, independent of salinity, despite the absence of osmoregulatory disturbances within the time frame of the present study Studies of rainbow trout (Jˇrss, Bittorf, V˛lker & Wacke 1983; Jˇrss et al 1987) and tilapia (Jˇrss et al 1984; Kˇltz & Jˇrss1991) have shown a reduction in branchial NKA activity after several weeks of fasting, both in freshwater and in seawater, similar to the present ¢ndings Chloride cell numbers, structure and function were found to change with food deprivation (Kˇltz & Jˇrss 1991), suggesting that structural elements (fatty acids and/or amino acids) may become limiting after extended periods of food deprivation In the present study, starvation had a signi¢cant e¡ect on branchial NKA activity, with a more pronounced decline in NKA activity in the freshwater groups At the termination of the starving period, FW0% had a 64% lower (Po0.01) NKA activity compared with FW100% In a comparable trial with rainbow trout (40 g), a 44% reduction in NKA activity was found after weeks of starvation (Jˇrss et al 1987) The present ¢ndings are also in line with the ¢ndings with those of Virtanen and Soivio (1985), where an almost total loss of NKA activity was seen in starving Atlantic salmon smolts, concurrent with a signi¢cant reduction in circulating cortisol levels Partly based on own unpublished observations, on the e¡ects of a low-protein diet, Virtanen and Soivio (1985) suggested that the de¢ciency of some amino acid(s) may inhibit the pituitary^interrenal axis and thereby the production of key osmoregulatory hormones such as cortisol Considering the key role of cortisol in the di¡erentiation of chloride cells and the regulation of the NKA activity (McCormick 2001), this suggests a mechanism through which starvation may have a restraining e¡ect on the NKA enzyme Upon re-feeding in late July, the NKA activity increased in the starved group and the ¢nal NKA in September did not di¡er between the groups within each salinity regime However, a transient increase in NKA activity to levels above controls was observed in the SW0% and SW25% groups after weeks of re-feeding, whereas no di¡erences were seen at the conclusion of the experiment (September) It is possible that the transient increase in NKA is due to CG in the re-feeding period with increased ion in£ux over the intestine epithelium Sundell, Jutfelt, Agustsson, Olsen, Sandblom, Hansen & Bjornsson (2003) found that during the parr-smolt transformation in Atlantic salmon, the intestinal NKA increases in the anterior intestine and the paracellular permeability appears to decrease in the posterior intestine These events correspond with r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 477 Aquaculture Research, 2011, 42, 613^622 doi:10.1111/j.1365-2109.2010.02658.x The effects of dietary supplements of polyunsaturated fatty acid on pearl oyster, Pinctada margaritifera L., gonad composition and reproductive output Fariborz Ehteshami1,2, Annie Christianus1, Hossein Rameshi2, Sharr Azni Harmin1 & Che Roos Saad1 Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia (UPM), Serdang, Selangor, Malaysia Iranian Fisheries Research Organization (IFRO),West Fatemi,Tehran, Iran Correspondence: F Ehteshami, Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia E-mail: ehteshamei@yahoo.com Abstract Black-lip pearl oyster, Pinctada margaritifera broodstock was collected from the wild Egg production, hatching rate and larval development were compared between oysters induced to spawn within days after collection in the wild (T1), oysters fed a pure microalgae diet during 24 days before spawning (T2) and oysters fed the same microalgal diet in which 10% of the algae were replaced with mm polyunsaturated fatty acid (PUFA)-rich microspheres (T3) Administration of lipid microspheres resulted in larger sized eggs, a higher percentage of D-larvae and larger sized 48-h-old larvae (Po0.05) The total and neutral lipid contents of the gonad increased after oysters were fed with microalgae only or with supplementary diet The major neutral and polar fractions of saturated fatty acid (SFA) were 16C and 18C fatty acids, and not in£uenced by the diet (P40.05) The gonads of oysters fed supplementary PUFA contained more docosahexaenoic acid (DHA) and less monounsaturated fatty acids Higher level of DHA in gonads of T3 was associated with oogenesis and embryogenesis success The n-3/n-6 ratio in the neutral lipid fraction provides a good indication of the spawning condition and predicting egg size and hatching rate Keywords: Pinctada margaritifera, broodstock, microalgae, oogenesis, larvae, lipid Introduction Arti¢cial propagation of pearl oyster using oysters collected from natural stocks is a seasonal activity r 2010 Blackwell Publishing Ltd in temperate countries (Pouvreau, Gangnery,Tiapari, Lagarde, Garnier & Bodoy 2000) The reproductive season in north of the Persian Gulf commences in June when the water temperature passes 241C and ceases in September when the temperature declines (Ehteshami, Christianus & Rameshi 2007) Collection of broodstock and arti¢cial propagation is capital and labour intensive The survival and larval size are in£uenced by egg quality (Kraeuter, Castagna & Van Dessel 1982; Helm, Holland, Utting & East 1991) Temperature and light period also in£uence the reproductive output (Devauchelle & Mingant 1991); however, these factors can be kept optimum in the hatchery Helm et al (1991) showed that the percentage of D-stage larvae produced from eggs is correlated to the lipid content in the egg The lipid contents in eggs of Crassostrea virginica and Mercenaria mercenaria (L.) (Gallager & Mann 1986), Ostrea edulis (Lane1989), Pecten maximus (Marty, Delaunary, Moal & Samain 1992) and Pinctada margaritifera (Vahirua-lechat, Laure, Lecoz, Bianchini, Bellais & Moullac 2008) depended on the fatty acid content of the broodstock diet In some cases, spat growth and settlement were also in£uenced by egg quality (Helm, Holland & Stephenson 1973; Bayne, Gabbott & Widdows 1975; Kraeuter et al.1982) Wild-collected oysters with immature gonads should be conditioned before spawning, keeping them at optimal water temperature and photoperiod and feeding a high-quality microalgae diet The nutritional value of algae for bivalves is partially determined by the eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) content (Lane 1989; Hendriks, Van Duren & Herman 2003; 613 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al Milke, Bricelj & Parrish 2004) EPA and DHA are considered essential fatty acids as most of the bivalves, including P margaritifera, are unable to produce them from short-chain precursor fatty acids (Chu & Greaves 1991; Dealunay, Marty & Samain 1993;Vahirua-Lechat et al 2008) However, dietary requirements for DHA and EPAvary between species (Milke, Bricelj & Parrish 2006) Also, microalgae di¡er in composition, including their polyunsaturated fatty acid (PUFA) content (Napolitano, Ackman & Ratnayake 1990; Brown, Mccausland & Kowalski 1998; Leonardos & Lucas 2000) Most of the microalgae species contain moderate to high percentages of EPA (7^34% of total fatty acid) However, prymnesiophytes (e.g Pavlova spp and T Iso) and cryptomonads are relatively rich in DHA (0.2^11% of total fatty acid), whereas eustigmatophytes (Nannochloropsis spp.) and diatoms have the highest percentages of arachidonic acid (AA, 20:4n-6) (0^4% of total fatty acid) Chlorophytes (Dunaliella spp and Chlorella spp.) are de¢cient in both C20 and C22 PUFAs, although some species have small amounts of EPA (up to 3.2% of total fatty acid) (Brown, Je¡rey, Volkman & Dunstan 1997) Several attempts have been made to increase the quality and quantity of eggs released by conditioning broodstock feeding algae rich in PUFA, in particular DHA and EPA, or lipid microspheres or emulsions in the hatchery (e.g Chilean scallop Argopecten purpuratus (Caers, Coutteau, Cure, Morales, Gajardo & Sorgeloos 1999), European £at oyster O edulis (Lane 1989), and A purpuratus (Martinez, Brokordt, Aguilera, Soto & Guderley 2000) However, there has been no study on the e¡ects of supplementary PUFA on the gonad of pearl oysters broodstock The objective of this study was to determine the effect of PUFA-supplemented diet on the biochemical (proximate and fatty acid) composition of the gonad and reproductive performance (size and numbers of eggs, and size and percentage of D-larvae produced) of P margaritifera broodstock biopsy and macroscopic observations of gonad before transportation to the Mollusc Persian Gulf Research Center Hatchery The broodstock were thoroughly brushed free of fouling organisms, washed with freshwater and prepared according to Doroudi (2001) The 65 female P margaritifera broodstock were comprised of 15 ripe and 50 in stage of developing gonads based on the modi¢ed classi¢cations of O’Connor (2002) and Friedman (2004) (Table 1) The 15 ripe oysters were allocated to T1 Five out of 50 oysters in stage were randomly used for biochemical analyses at the starting point (T0) The remaining 45 oysters in stage were distributed randomly to T2,T3 and T4 Five out of15 oysters from T1,T2,T3 and T4 were used for biochemical analyses the same day the spawning was induced The remaining surviving oysters were used for spawning and egg collection Oysters of each treatment were kept in a rectangular ¢breglass tank that was ¢lled with 100 L of seawater and aerated moderately Four ripe male oysters (DVM 515 Æ cm) were added to the each tank at the spawning time These oysters were collected from Lavan Island according to criteria mentioned for female oysters, transferred to the hatchery, washed, prepared and directly used for spawning Oysters in T1were induced to spawn within days after harvest by thermal shock following the method of Doroudi (2001) Oysters in T2, T3 and T4 were kept under a similar condition for 24 days and fed with a di¡erent diet before spawning Oysters in T2 were fed with a ternary combination of microalgae using 50% of Isochrysis sp and equal cell numbers of diatoms, Chaetoceros muelleri and Chaetoceros Table Reproductive stages in the gonadal development of the genus Pinctada based on macroscopic observations (modi¢ed from O’Connor 2002 and Friedman 2004) Stage Description Methodology Experimental design Sixty-¢ve female P margaritifera broodstock (dorsoventral measurement, DVM 515 Æ cm) were collected from the natural stocks in Lavan Island, Persian Gulf (26148 05300 N, 53116 02100E), early in the reproductive season of 2007 Oysters were selected according to the size, external health appearance, 614 Aquaculture Research, 2011, 42, 613–622 Inactive, no evidence of gonad development The gonadal area is translucent and the digestive diverticula are visible Gonads are filling, discrete area of gonad distinguishable at the base of digestive diverticula Gonads are filling, cover approximately one-fourth of the cavity between the byssal gland and foot on one side of digestive diverticula without bulging appearance Gonads cover more than half of the cavity between the byssal gland and foot on both sides of digestive diverticula with bulging appearance Ripe, gonads are entirely filled, filling much of the cavity between the byssal gland and foot, around the retractor muscle and digestive diverticula, with a smooth texture and bulging appearance r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 Aquaculture Research, 2011, 42, 613^622 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al calcitrans Those in T3 were fed the same microalgal diet in which 10% of the algae were replaced with 2mm PUFA-rich microspheres to obtain the same total amount of particles supplied per tank Oysters in T2 and T3 were fed 2.5  104 particles per mL twice daily at 08:00 and 20:00 hours Oysters in T4 were kept unfed The enrichment emulsion was supplied by the Artemia Research Centre of Iran The emulsion contained 65% lipid on a wet weight basis, DHA/EPA ratio of 4, antioxidants, ethoxyquin (as preservative), emulsi¢ers and water Water temperature and salinity were maintained at 24 Æ 1C and 35 Æ 1g L À 1, respectively, and water was changed 100% each time before feeding Oysters were induced to spawn upon completion of the experimental period Eggs were collected from each individual female and suspended in 100 mL ¢ltered seawater Eggs of three subsamples of 0.5 mL were counted and measured under a stereomicroscope (fecundity) These procedures were repeated the following day for oysters not responding to the ¢rst thermal shock Egg diameter was used as an indicator of egg quality Fertilized eggs of the female broodstock of each treatment that responded to the ¢rst thermal shock were mixed and certain amounts of these eggs were randomly placed into three 100 L ¢breglass tanks Tanks were ¢lled with 80 L of mm ¢ltered and ultraviolet-treated seawater Water temperature and salinity were controlled at 29 Æ 1C and 35 Æ 1g L À respectively Seawater was not changed and gentle aeration was given Forty-eight hours after fertilization, all D-shape larvae were collected on a plankton net with a 35 mm mesh size and transferred to a 200 mL beaker The number of D-shape larvae was estimated based on ¢ve randomly selected subsamples of 0.5 mL to calculate the percentage of eggs developed into D-shape larvae (hatchability) Shell length [antero-posterior measurement (APM)] of 60 randomly selected D-larvae was measured under a stereomicroscope Analytical methods Five adult female oysters from each treatment that were not induced to spawn were transferred to the laboratory for biochemical analyses Gonads were dissected from the junction of the byssus gland and pearl sac and frozen in liquid nitrogen The frozen samples were then ground with a homogenizer for Aliquots of ground gonad was placed in a tube containing mL of chloroform^methanol (2:1 v/v) with 1% butylated hydroxytoluene (antioxidant) and C23:0 (internal standard) for lipid measurement Total lipids were extracted following the method of Folch, Lees and Sloane-Stanley (1957) The extract was shaken and equilibrated with one-fourth of its volume using a 0.7% w/v NaCl solution, when the mixture partitioned into two layers The lower layer was composed of chloroform^methanol^water in the proportion 86:14:1 (by volume) and contained virtually all of the lipids, while the upper phase consisted of the same solvents in the proportion of 3:48:47 (by volume) and contained much of the nonlipid contaminants The 2:1chloroform^methanol extracts were evaporated under vacuum conditions and recovered with three washings of 500 mL each of 98:2 chloroform^methanol The neutral (triglycerides, free sterols and sterol esters) and polar (phospholipids) lipids were eluted in a silica gel microcolumn (30 mm  mm) with 10 mL chloroform^methanol 98:2 and 10 mL methanol respectively The fractions were then collected in tapered vials Fatty acid methyl esters were analysed in a DANI1000 gas chromatograph with an auto sampler, a temperature programmable on column injector using an on-column injection Injections were performed on-column into a polar capillary column (30 mm  0.32 mm, 0.25 mm ¢lm layer thickness) connected to a pre-column The carrier gas used was hydrogen, at a pressure of 100 kPa and the detection mode FID The column temperature was programmed according to Vahirua-Lechat et al (2008) at 49 1C for min, then from 49 to 160 1C at a rate of 50 1C/min, 160 to 170 1C at a rate of 1C/min, 170 to 185 1C at a rate of 1C/min, 185 to 240 1C at a rate of 1C/min and a ¢nal plateau at 240 1C for 10 The injector was heated from 85 to 190 1C at 1C s À and retained at190 1C for 30 Fatty acid identi¢cation was based on their retention times with standard reference mixtures and designated the following formula: C:X (n-Y), where C is the number of carbon atoms, X is the number of double bonds and Y is the position of the ¢rst double bond counted from the CH3 terminal For carbohydrate determinations, 500 mL of homogenate was transferred to 1.7 mL micro-centrifuge tubes Carbohydrates were extracted as described by Acosta-Salmon (2004) The carbohydrate content was quanti¢ed following the phenol^sulphuric acid method of Dubois et al (1956) using D-glucose as standard Protein determination followed the method of Kjeldahl (1883) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 615 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al Statistical methods All statistical analyses were performed using SPSS 15.0 Data sets of egg numbers were normalized with log transformation Data of fatty acids were transformed to arcsine values before statistical analyses One-way analyses of variance and Tukey post hoc comparisons were carried out to determine the statistical di¡erences among treatments (Po0.05) Results Fecundity and hatchability After 24 days of broodstock conditioning and maturation period, the four test diets yielded di¡erences in gametogenesis and embryogenesis The spawning rates were 60%, 70%, 80% and 0% for T1,T2,T3 and T4 spawning treatments respectively One oyster died in each of treatment T2 and T3 before spawning throughout the study (Fig and Table 2) Considering surviving oysters,90% of oysters inT2 and T3 developed from stage at the beginning of the experiment to stage after 24 days of conditioning, while 50% of gonads in T4 remained in stage and 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Not Spawned Dead Spawned T1 T2 T3 T4 Figure Reproductive output of Pinctada margaritifera directly spawned (T1) and after 24 days (T2, T3 and T4) n 510 for all treatments Table E¡ect of diet on fecundity and embryonic development in Pinctada margaritifera Treatment Eggs per spawner (log) Egg size (lm) APM 48 h old larvae (lm) T1 T2 T3 6.40 (0.30)b 56.5(0.17)b 78.7 (2.5)b 25.7 (1.9)c 6.46 (0.28)ab 56.4 (0.17)b 78.6 (2.5)b 28.2 (2.0)b 7.30 (0.12)a 57.6 (0.5)a 80.1 (2.3)a 31.3 (1.9)a D-larvae (%) Values are means ( Æ standard error) Means in the same column with di¡erent superscripts are signi¢cantly di¡erent (Po0.05) n for eggs per spawner and egg size 60 for anteroposterior measurement 48-h-old larvae and 5 for D-larvae (%) 616 Aquaculture Research, 2011, 42, 613–622 the other half retrogressed or progressed to stage and respectively (Fig 2) Size of eggs in T3 was signi¢cantly larger than those in T2 and T1 (Po0.05) The increase in the number of eggs in T3 was statistically signi¢cant as compared with T1 but not with T2 There was no signi¢cant di¡erence between T1 and T2 in size and number of eggs (P40.05) T3 showed a better hatching rate and produced larger sized D-shape larvae compared with T1 and T2 (Po0.05) T2 showed better performance in hatching rate than T1; however, this di¡erence was not signi¢cant (P40.05) in relation to shell length (APM) of D-shape larvae (Table 2) Biochemical composition of gonad The female gonads showed a signi¢cant increase in total lipid from 90.8 mg g À dry weight at the start of the experiment (T0) to160.6,170 and190.4 mg g À dry weight in T1, T2 and T3 respectively (Po0.05) There was signi¢cant di¡erence between T3 and other treatments, except T2, in relation to total lipid (Po0.05) Considering the percentage of neutral lipid, an increasing trend was observed after 24 days of conditioning with microalgae only or microalgae with supplementary diet (Po0.05); however, T4 showed no development in neutral fatty acid (P40.05) The di¡erence in protein and carbohydrate contents of gonad of oysters in T1,T2 and T3 was not signi¢cant (P40.05); however, it was signi¢cantly greater than that in T0 (Po0.05) (Table 3) The levels of saturated fatty acid (SFA) and PUFA in gonad were not a¡ected after 24 days of conditioning with microalgae and enrichment lipid However, unfed oysters in T4 showed an increase and decrease in levels of SFA and PUFA of neutral lipids respectively (Po0.05) There was no signi¢cant (P40.05) di¡erence in relation to monounsaturated fatty acids 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Stage Stage Stage Stage T1 T2 T3 T4 Figure Reproductive stages of female Pinctada margaritifera at the end of the conditioning period r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 Aquaculture Research, 2011, 42, 613^622 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al Table E¡ect of diet on lipid, protein and carbohydrate (mg g À 1dry weight) and neutral lipid (NL, in % of total lipid) of female gonad of Pinctada margaritifera (n 5) T0 Lipid NL Protein Carbohydrate T1 90.8 63.0 109.7 15.2 (13.6)d (1.2)b (16.5)b (4.0)b T2 160.6 70.3 200.4 25.5 (10.0)b (2.5)a (26.2)a (4.7)a T3 170.0 69.2 207.0 28.3 (10.1)ab (2.4)a (31.5)a (4.5)a T4 190.4 71.2 206.5 27.5 (10.0)a (2.4)a (18)a (5.4)a 114 64.3 122.4 16.3 (15.2)c (3.5)b (19.6)b (5.0)b Values are means (Æ Standard error) Homogenous treatments were shown with same alphabetic numbers in each line Table Categories and ratios (% of total fatty acids) of fatty acids in the neutral lipids of female gonads of Pinctada margaritifera T0 Mean Æ SD SSFA SMUFA SDMA Sn-9 Sn-7 SPUFA Sn-4 Sn-6 Sn-3 n-3/n-6 DHA/EPA 22:5/AA AA/EPA PUFA/SFA 33.5 15.0 0.4 6.5 8.5 50.4 0.4 7.0 42.6 6.1 4.2 1.0 0.3 1.5 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 2.0 0.3 0.2 0.1 0.2 2.5 0.2 0.1 2.1 0.4 0.5 0.1 0.1 0.2 T1 Mean Æ SD 33.0 14.9 0.9 6.5 8.4 50.6 0.2 6.9 43.1 6.2 4.4 0.9 0.3 1.5 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ T2 Mean Æ SD 1.3 0.3 0.4 0.2 0.1 2.3 0.1 0.1 2.1 0.4 0.5 0.1 0.0 0.1 32.2 15.0 0.6 6.8 8.2 51.6 0.1 6.9 44.2 6.4 4.5 1.3 0.3 1.6 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 1.2 0.4 0.3 0.2 0.2 2.4 0.1 0.3 1.9 0.6 0.6 0.2 0.0 0.1 T3 Mean Æ SD 31.2 13.3 0.5 5.7 7.6 54.4 0.2 6.4 47.2 7.3 4.8 1.2 0.3 1.8 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 1.3 0.3 0.3 0.1 0.2 2.4 0.1 0.4 1.8 0.8 0.6 0.1 0.0 0.2 T4 Mean Æ SD 37.2 15.9 0.4 6.5 9.4 45.6 0.3 6.4 38.7 6.0 4.1 1.1 0.3 1.2 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 3.4 0.6 0.2 0.3 0.3 3.3 0.2 0.4 2.7 0.9 0.6 0.1 0.1 0.2 SSFA, total of saturated fatty acids; SMUFA, total of monounsaturated fatty acids; SPUFA, total of polyunsaturated; SDMA, total of dimethyl acetal; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; AA, arachidonic acid (MUFA) of neutral lipid among T0, T1 and T2, but T3 and T4 were signi¢cantly di¡erent as compared with other treatments and with each other (Po0.05) (Table 4) In the polar fraction, MUFA in oysters from T4 di¡ered signi¢cantly (Po0.05) when compared with other treatments (Table 5) SFA including 14:0, 16:0 and 18:0 in neutral lipid of the gonad of oysters from T4 were signi¢cantly more than other treatments (Po0.05) (Fig 3) The major SFA both in neutral and polar fractions of lipid were 16:0 and 18:0, indicating that their levels were not under the in£uence of diet (Figs and 4) Hatchery conditioning of oysters with combinations of microalgae (T2) had no e¡ect on the levels of 16:1n-7, 18:1n7 and 20:1n-9 compared with oysters from T0 and T1 However, a signi¢cant increase was observed for 18:1n-9 in oysters from T2 (Figs and 4) Feeding on supplementary diet (T3) increased the level of DHA compared with T0, T1 and T4 (Po0.05) but was not e¡ective to increase the level of EPA of neutral lipid (P40.05) The ratio of DHA/EPAwas not altered signi¢cantly even in oysters kept unfed for 24 days (P40.05) Supplementary feeding was e¡ective on increasing the ratio of n-3/n-6 as compared with other treatments in the neutral fraction of lipid but not signi¢cant in the polar fraction The n-3/n-6 and 22:5n/AA ratios, irrespective of the conditioning treatment, were consistently (Po0.05) higher in the neutral lipids than in the phospholipids Conversely, the ratios of AA/EPA, DHA/EPA and PUFA/SFA were higher in phospholipids (Tables and 5) Discussion Pearl oysters such as P margaritifera show a di¡erent reproductive behaviour in tropical and temperate areas (Tranter 1958; Saucedo, Rodr|¤ guez-jaramillo, Aldana-avile¤s, Monsalvo-spencer, Reynoso-granados,Villarreal & Monteforte 2001) It is not di⁄cult to ¢nd ripe gonads throughout the year in tropical r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 617 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al Aquaculture Research, 2011, 42, 613–622 Table Categories and ratios (% of total fatty acids) of fatty acids in the polar lipids of female gonads of Pinctada margaritifera T0 Mean Æ SD SSFA SMUFA SDMA Sn-9 Sn-7 SPUFA Sn-4 Sn-6 Sn-3 n-3/n-6 DHA/EPA 22:5/AA AA/EPA PUFA/SFA 24.8 8.9 3.2 5.5 3.4 60.3 2.0 11.2 43.2 3.9 4.5 0.5 0.8 2.4 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ T1 Mean Æ SD 1.8 0.6 0.5 0.3 0.3 5.0 0.5 1.3 2.9 0.8 1.2 0.1 0.0 0.4 23.3 9.0 7.5 5.6 3.4 58.5 1.0 11.8 40.7 3.4 5.0 0.5 1.0 2.5 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ T2 Mean Æ SD 1.9 0.3 0.9 0.2 0.1 5.1 0.3 1.3 3.2 0.7 1.9 0.0 0.1 0.5 22.4 9.9 4.5 6.7 3.2 61.9 0.5 12.0 43.9 3.7 4.7 0.6 0.9 2.8 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 1.6 0.6 0.7 0.4 0.2 4.9 0.3 1.3 3.0 0.7 1.5 0.1 0.1 0.5 T3 Mean Æ SD 21.1 9.7 4.3 6.1 3.6 63.5 0.6 11.8 45.7 3.9 4.8 0.6 0.9 3.0 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 2.0 0.6 0.8 0.3 0.3 5.2 0.3 1.5 3.1 0.9 1.3 0.1 0.0 0.6 T4 Mean Æ SD 27.5 10.5 3.7 6.0 4.5 56.3 1.0 10.3 40.2 3.9 4.7 0.5 0.8 2.1 Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ Æ 2.5 0.6 0.7 0.3 0.3 6.3 0.5 1.4 4.1 1.1 2.0 0.1 0.1 0.5 SSFA, total of saturated fatty acids;SMUFA, total of monounsaturated fatty acids;SPUFA, total of polyunsaturated; SDMA, total of dimethyl acetal; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; AA, arachidonic acid T0 T1 T2 T3 T4 40 Fatty acid composition (%) 35 30 25 20 15 Figure Neutral fatty acids compositions in female gonads of Pinctada margaritifera Expressed as the percentage of total fatty acids of the fraction (mean Æ SD, n 5) 10 countries However, there are one or a few peaks in gonad development in temperate waters including Persian Gulf (Ehteshami et al 2007) Moreover, pearl oysters reproduction behaviour is another limiting factor Once a male oyster releases the sperm due to some stimulating factor such as an increase in water temperature, the majority of other oysters in the same community will release the sperm or egg (Southgate & Lucas 2008); consequently, ¢nding ripe oysters ready to spawn would not be easy even during the reproductive season and would need precise monitoring of the developmental phase of the gonads Results of this study showed that oysters in early gonadal development stage could be used for arti¢cial propagation after keeping in desirable condi- 618 tions of temperature and feeding for a certain period of time (in this study, 24 days) to compensate the shortage of ripe broodstock High response of broodstock to inducing for egg release (even more than oysters induced to spawn after collection in the wild) and a low mortality rate (one out of10) before spawning are quite acceptable for arti¢cial propagation There is no comprehensive de¢nition of egg quality in aquaculture; however, it is possible to de¢ne and quantify with predictors and validators criteria (Cannuel & Beninge 2005) Egg number and size are two of the most common criteria being used to assess the success rate of propagation activities (Gallager & Mann1986; Doroudi 2001; Hendriks et al 2003) Both of these criteria were improved or at least not r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 Aquaculture Research, 2011, 42, 613^622 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al T0 T1 T2 T3 T4 40 Fatty acid composition (%) 35 Figure Polar fatty acids compositions in female gonads of Pinctada margaritifera Expressed as the percentage of total fatty acids of the fraction (mean Æ SD, n 5) 30 25 20 15 10 decreased after hatchery feeding and conditioning Results of this study con¢rm the theory that there is a positive relationship between broodstock diet, lipid content and egg size Oysters whose gonad developed from stage to in the hatchery through conditioning (T2 and T3) showed better hatching performance than wild ones (T1) However, Cannuel and Beninge (2005) found that there is no relationship between conditioning treatment and oocyte quality predictor or subsequent validator They assumed that winter reserve accumulation is more important than trophic conditioning for successful gametogenesis The higher percentage of D-larvae produced could be due to larger egg size of treatments and that allow more space for accumulation of lipid and other endogenous reserves, which in turn is very essential for providing energy for the very early stage of D-larvae The importance of endogenous reserves laid down in the eggs for providing energy during embryogenesis and growth before larvae are able to feed on exogenous sources has been quoted (Gallager & Mann 1986; Whyte, Bourne & Ginther 1991; Labarta, FernaŁndezreiriz & Pe¤rez-Camacho 1999) The increase was observed in total lipids, and neutral lipid in developed gonad oysters (T1, T2 and T3) compared with early developing gonad oysters (T0) could be acquired directly from feeding and perhaps indirectly the transfer from the digestive gland The role of transfer of lipid from the digestive gland to the gonad was more obvious in oysters that were kept unfed for 24 days (T4), but their total lipid content was still more than that in early developing oysters (T0) The role of the digestive gland at gametogenesis of oysters has been studied (Napolitano & Ackman 1993; Soudant, Marty, Moal, Robert, Que¤re¤, Le Coz & Samain 1996; Saucedo, Racotta, Villarreal & Monteforte 2002) The increase in the total protein of T4 in comparison to T0 was not signi¢cant, and so it is more doubtful to discuss the role of muscle in transferring of protein to the gonad The importance of adductor muscle in storing of protein and its role at reproductive cycles of many bivalves has been reported (Barber & Blake 1985; Soudant, Marty et al 1996; Racotta, Ramirez, Avila & Ibarra 1998) Nevertheless, the result of this study is not su⁄cient to assess the contribution of muscle and digestive gland in providing food elements for gametogenesis The uniformity of the relative proportions of SFA, PUFA and MUFA in neutral and polar lipids of female gonad of some bivalves has been reported (Soudant, Marty et al 1996; Acosta-Salmon 2004; Vahirua-Lechat et al 2008) This is probably due to a metabolic necessity to maintain equilibrium between the di¡erent categories of fatty acids (Soudant, Marty et al 1996) Results of this study also generally support the ¢ndings of these researchers; however, the increase in the level of SFA and the decrease in the level of PUFA of unfed oysters may suggest interruption or r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 619 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al malfunctioning of the mechanisms that are responsible for elongation of short-chain fatty acids to long ones due to a long starvation period Moreover, the decrease in the relative proportion of MUFA in the neutral lipid of oysters that were fed extra PUFA was coupled with an increased level of DHA, implicating the ability of broodstock to digest emulsi¢ed droplets These observations were more evident in the neutral lipid than in the polar lipid of gonads, suggesting that the phospholipid storage of gonad is before the neutral lipid and relates to the early gametogenesis Helm et al (1991) and Utting and Millican (1997) also concluded that the PUFA content of eggs is dependent on a two-phase process controlled at ¢rst by the environmental conditions during the fattening of broodstock and early gametogenesis (phospholipid PUFAs) and then by the conditions during the later stages of oocyte development (neutral lipid PUFAs) The observed constancy in MUFAs including16:1n7, 18:1n-7 and 20:1n-9 is probably due to wild oysters in T0 and T1 captured in early summer when there is enough varieties of phytoplankton with di¡erent kinds of fatty acids Moreover, oysters in T2 and T3 were fed on ternary combination of microalgae, and so the shortages of some fatty acids in one microalga were compensated with the other two microalgae Soudant, Marty et al (1996) used three di¡erent kinds of microalgae to feed broodstock of P maximus in separate treatments and concluded that the MUFA composition of neutral lipids re£ected very closely to that of the dietary microalgae The e¡ect of extra DHA via PUFA supplementation resulted in more DHA content of neutral lipid of gonads in T3 compared with T0 and T1 However, it appears that microalgae, especially Isochrysis, had enough DHA; therefore, no signi¢cant di¡erence between T2 and T3 was observed High level of DHA in Isochrysis has been reported previously (Dealunay et al 1993; Soudant, Marty et al 1996; Mart|¤ nez-fernaŁndez, Acosta-salmo¤n & Southgate 2006) Comparatively DHA/EPA ratios in neutral and polar lipids show that DHA was incorporated selectively in the polar lipids of gonads Similar results were reported in P margaritifera (Acosta-Salmon 2004; Vahirua-Lechat et al 2008) and P maximus (Soudant, Marty et al 1996) DHA plays an essential role at the structural and functional levels of cellular membranes involved in oogenesis and embryogenesis (Uki, Kemayama & Watanabe 1985; Whyte et al 1991; Soudant, Marty et al 1996; Soudant,Van Ryckeghem, Marty, Moal, Samain & Sorgeloos 1999) The greater egg number, size and better hatching rate produced 620 Aquaculture Research, 2011, 42, 613–622 by broodstock fed with supplementary food may be associated with higher contents of DHA The importance of DHA at fecundity and hatching rate has been reported in ¢sh (Watanabe 1985), shrimp (Xu, Ji, Castell & O’Dor1994) and bivalves (Soudant, Marty et al 1996; Hendriks et al 2003) Soudant, Moal, Marty and Samain (1996) showed that the DHA/EPA ratio varied considerably according to the diet after 11 weeks of conditioning of the P maximus by using three microalgal diets However, in this study, the n-3/n-6 ratio in the neutral lipid fraction was more representative of di¡erences in the conditioning methods of oysters for spawning, egg size and number and hatching rate The level of AA in the phospholipid fraction was obviously more than its level in neutral lipid in all treatments; moreover, the AA/EPA ratio in polar lipid was more than 2.5 times that in neutral lipid Soudant, Marty et al (1996) reported similar results with P maximus and suggested that AA plays a speci¢c role in membrane lipid metabolism during gametogenesis in female Conclusion There are few reports describing the means by which pearl oysters are conditioned and induced to maturation, and most are focused on studies of rearing larvae obtained from matured wild pearl oysters This study reveals new information on the role of supplementary PUFAs in conditioning and improving the reproductive output of P margaritifera Broodstock in early stages of gametogenesis could be transferred to the hatchery and successfully utilized in arti¢cial propagation after proper feeding with a mixture of microalgae and supplementary PUFA Also, this study, for the ¢rst time, discussed the relationship between polar and neutral fatty acids of gonad with predictor criteria of oogenesis of blacklip pearl oyster Results of this study show that supplementary PUFA with a DHA/EPA ratio of can replace microalgae by 10% to produce larger eggs with a higher hatching rate and larger P margaritifera D-shape larvae These ¢ndings are useful for the improvement of broodstock management protocols for P margaritifera References Acosta-Salmon H (2004) Broodstock management and egg quality of the pearl oysters P margaritifera and P fucata PhD thesis, James Cook University,Townsville,162pp r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 Aquaculture Research, 2011, 42, 613^622 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al Barber B.J & Blake N.J (1985) Intra-organ biochemical transformation associated with oogenesis in the bay scallop, Argopecten irradians concentricus (Say), as indicated by 14C incorporation Biological Bulletin 168, 39^49 Bayne B.L., Gabbott P.A & Widdows J (1975) Some e¡ects of stress in the adult on the eggs and larvae of Mytilus edulis L Journal of the Marine Biological Association of the United Kingdom 55, 675^689 Brown M.R., Je¡rey S.W.,Volkman J.K & Dunstan G.A (1997) Nutritional properties of microalgae for mariculture Aquaculture 151, 315^331 Brown M.R., McCausland M.A & Kowalski K.N (1998) The nutritional value of four Australian microalgal strains fed to Paci¢c oyster 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A study using oocyte quality predictors and validators in Crassostrea gigas Aquatic Living Resources 18, 35^43 Chu F.E & Greaves J (1991) Metabolism of palmitic, linoleic, and linolenic acids in adult oysters Crassostrea Virginica Marine Biology 110, 229^236 Dealunay F.Y., Marty J.M & Samain J.F (1993) The e¡ect of monospeci¢c algal diets on growth and fatty acid composition of Pecten maximus Journal of Experimental Marine Biology and Ecology 173,163^179 Devauchelle N & Mingant C (1991) Review of the reproductive physiology of the scallop, Pecten maximus, applicable to intensive aquaculture Aquatic Living Resources 4, 41^51 Doroudi S.M (2001) Development and culture of black-lip pearl oyster, Pinctada margaritifera (Linnaeus) larvae PhD thesis, James Cook University,Townsville, 153pp Dubois M., Gilles K.A., Hamilton J.K., Rebers P.A & Smith F (1956) Colorimetric method for determination of sugars and related substances Analytical Chemistry 28,350^356 Ehteshami F., Christianus A & Rameshi H (2007) Pinctada margaritifera propagation and larval rearing up to spat in North coast of Persian Gulf 8th Asian Fisheries Forum, India,170pp Folch J., Lees M & Sloane-Stanley G.H (1957) A simple method for the isolation and puri¢cation of total lipids from animal tissues Journal of Biological Chemistry 226, 497^509 Friedman K.J (2004) Mother-of-Pearl Shell (Pinctada maxima): Stock Evaluation for Management and Future Harvesting in Western Australia Department of Fisheries, Perth, Western Australia Gallager S.M & Mann R (1986) Growth and survival of larvae of Mercenaria mercenaria (L.) and Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs Aquaculture 56,105^121 Helm M.M., Holland D.L & Stephenson R.R (1973) The e¡ect of supplementary algal feeding of hatchery breeding stock of Ostrea edulis (L.) on larval vigour Journal of the Marine Biological Association of the United Kingdom 53, 673^684 Helm M.M., Holland D.L., Utting S.D & East J (1991) Fatty acid composition of early non-feeding larvae of the European £at oyster, Ostrea edulis Journal of the Marine Biological Association of the United Kingdom 7, 691^705 Hendriks I.E.,Van Duren L.A & Herman P.M.J (2003) E¡ect of dietary polyunsaturated fatty acids on reproductive output and larval growth of bivalves Journal of Experimental Marine Biology and Ecology 296,199^213 Kjeldahl J.Z (1883) A new method for the determination of nitrogen in organic bodies Analytical Chemistry 22, 366 Kraeuter J.N., Castagna M & Van Dessel R (1982) Egg size and larval survival of Mercenaria mercenaria (L.) and Argopecten irradians (Lamarck) Journal of Experimental Marine Biology and Ecology 56, 3^8 Labarta U., FernaŁndez-Reiriz M.J & Pe¤rez-Camacho A (1999) Energy, biochemical substrates and growth in the larval development, metamorphosis and postlarvae of Ostrea edulis (L.) Journal of Experimental Marine Biology and Ecology 238, 225^242 Lane A (1989) The e¡ect of a microencapsulated fatty acid diet on larval production in the European £at oyster Ostrea edulis In: Aquaculture ^ A Biotechnology in Progress (ed by N De Pauw, E Jaspers, H Ackefors & N Wikins), pp 657–664 European Aquaculture Society, Bredene, Belgium Leonardos N & Lucas I.A.N (2000) The use of larval fatty acids as an index of growth in Mytilus edulis L larvae Aquaculture 184,155^166 Martinez G., Brokordt K., Aguilera C., Soto V & Guderley H (2000) E¡ect of diet and temperature upon muscle metabolic capacities and biochemical composition of gonad and muscle of Argopecten purpuratus Lamarck 1819 Journal of Experimental Marine Biology and Ecology 247, 29^49 Mart|¤ nez-FernaŁndez E., Acosta-Salmo¤n H & Southgate P.C (2006) The nutritional value of seven species of tropical microalgae for black-lip pearl oyster (Pinctada margaritifera, L.) larvae Aquaculture 257, 491^503 Marty Y., Delaunary F., Moal J & Samain J.F (1992) Changes in fatty acid composition of Pecten maximus (L.) during larval development Journal of Experimental Marine Biology and Ecology 163, 221^234 Milke L.M., BriceljV.M & Parrish C.C (2004) Growth of postlarval sea scallops, Placopecten magellanicus, on microalgal diets, with emphasis on the nutritional role of lipids and fatty acids Aquaculture 234, 293^317 Milke L.M., Bricelj V.M & Parrish C.C (2006) Comparison of early life history stages of the bay scallop, Argopecten irra- r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 621 The e¡ects of dietary supplements of PUFA on pearl oyster F Ehteshami et al dians: e¡ects of microalgal diets on growth and biochemical composition Aquaculture 260, 272^289 Napolitano G.E & Ackman R.G (1993) Fatty acid dynamics in sea scallops Placopecten magellanicus (Gmelin) from Georges Bank, Nova Scotia Journal of Shell¢sh Research 12, 267^277 Napolitano G.E., Ackman R.G & Ratnayake W.M.N (1990) Fatty acid composition of three cultured algal species (Isochrysis galbana, Chaetoceros gracilis and Chaetoceros calcitrans) used as food for bivalve larvae Journal of World Aquaculture Society 21,122^130 O’Connor W.A (2002) Latitudinal variation in reproductive behavior in the pearl oyster, Pinctada albina sugillata Aquaculture 209, 333^345 Pouvreau S., GangneryA.,Tiapari J., Lagarde F., Garnier M & Bodoy A (2000) Gametogenic cycle and reproductive effort of the tropical blacklip pearl oyster, Pinctada margaritifera (Bivalvia: Pteriidae), cultivated in Takapoto atoll (French Polynesia) Aquatic Living Resources 13, 37^48 Racotta I.S., Ramirez J.L., Avila S & Ibarra A.M (1998) Biochemical composition of gonad and muscle in the catarina scallop, Argopecten ventricosus, after reproductive conditioning under two feeding systems Aquaculture 163,111^122 Saucedo P., Rodr|¤ guez-Jaramillo C., Aldana-Avile¤s C., Monsalvo-Spencer P., Reynoso-Granados T., Villarreal H & Monteforte M (2001) Gonadic conditioning of the cala¢a mother-of-pearl oyster, Pinctada mazatlanica (Hanley, 1856), under two temperature regimes Aquaculture 195, 103^119 Saucedo P., Racotta I.S.,Villarreal H & Monteforte M (2002) Seasonal changes in the histological and biochemical pro¢le of the gonad, digestive gland and muscle of the cala¢a mother-of-pearl oyster, Pinctada mazatlanica (Hanley 1856) associated with gametogenesis Journal of Shell¢sh Research 21,127^135 Soudant P., MartyY., Moal J., Robert R., Que¤re¤ C., Le Coz J.R & Samain J.F (1996) E¡ect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process Aquaculture 143, 361^378 622 Aquaculture Research, 2011, 42, 613–622 Soudant P., Moal J., Marty Y & Samain J.F (1996) Impact of the quality of dietary fatty acids on metabolism and the composition of polar lipid classes in female gonads of Pecten maximus (L.) 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F., LeCoz J.R., Bianchini J.P., Bellais M & Moullac G.L (2008) Changes in fatty acid and sterol composition during oogenesis in the pearl oyster Pinctada margaritifera Aquaculture Research 39,1739^1746 Watanabe T (1985) Importance of the study of broodstock nutrition for further development of aquaculture In: Nutrition and Feeding in Fish (ed by C Cowey, A.M Mackie & L Bell), pp 395^414 Academic Press, London, UK Whyte J.N.C., Bourne N & Ginther N.G (1991) Depletion of nutrient reserves during embryogenesis in the scallop Patinopecten yessoensis (Jay) Journal of Experimental Marine Biology and Ecology 149, 67^79 Xu X.L., Ji W.J., Castell J.D & O’Dor R.K (1994) In£uence of dietary lipid sources on fecundity, egg hatchability and fatty acid composition of Chinese prawn (Penaeus chinensis) broodstock Aquaculture 119, 359^370 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 613^622 Aquaculture Research, 2011, 42, 623^627 doi:10.1111/j.1365-2109.2010.02653.x SHORT COMMUNICATION Effect of various protein sources in formulated diets on the growth and body composition of juvenile sea cucumber Apostichopus japonicus (Selenka) Joo-Young Seo, Il-Shik Shin & Sang-Min Lee Faculty of Marine Bioscience and Technology, Gangneung-Wonju National University, Gangneung, South Korea Correspondence: S-M Lee, Faculty of Marine Bioscience and Technology, Gangneung-Wonju National University, Gangneung 210-702, South Korea E-mail: smlee@gwnu.ac.kr The sea cucumber (Apostichopus japonicus, Selenka) is an important ¢shery product in Korea, China and Japan (Sloan 1984) The demand for this species has increased in response to an increased interest in healthy human foods However, landings of wild sea cucumber have declined continuously due to overexploitation (Conand 2004; Uthicke 2004) Land-based intensive culture of sea cucumbers has recently been established in Korea and China Algae powder is commonly used as an important feed ingredient for sea cucumber (Sui 1989; Battaglene, Seymour & Ramofa¢a 1999) Although algae powder yields good growth, it is di⁄cult to ensure a constant supply, both qualitatively and quantitatively The nutritional composition changes with the season (Lobban & Harrison 1994), the shelf-life is limited, a¡ecting quality, and labour expenses for harvesting are high (Lawrence, Olave, Otaiza, Lawrence & Bustos 1997; Fernandez & Pergent 1998) In ¢sh nutrition, protein is the most expensive dietary ingredient, with a direct impact on growth performance (Lovell 1989) Recently, several studies on nutrient requirements (Seo, Choi, Kim, Cho, Park & Lee 2008; Choi, Seo & Lee 2009) and feed ingredients (Yuan, Yang, Zhou, Mao, Zhang & Liu 2006; Slater, Je¡s & Carton 2009; Liu, Dong, Tian, Wang & Gao 2009) for sea cucumber have been carried out Nevertheless, information on possible dietary protein sources for A japonicus is still limited Therefore, this study investigated the e¡ect of various dietary protein sources on the growth and body composition of A japonicus r 2010 Blackwell Publishing Ltd Seven isonitrogenous (30% crude protein) and isolipidic (7% lipid) experimental diets were prepared to contain 41% ¢sh meal (FM), 59% soybean meal (SBM), 55% wheat £our with 29% ¢sh meal (WF), 50% sesame seed meal with 10% ¢sh meal (SSM), 55% soybean^curd residues with 25% ¢sh meal (SCR), 50% Sargassum thunbergii powder with 32% ¢sh meal (ST) and 50% mixture of the six protein sources (MIX) (Table 1) For each diet, moist pellets were prepared using a pellet machine The pellets were ground to a powder (180 mm mesh) after drying overnight at 60 1C All diets were stored at À 30 1C until used The proximate and essential amino acid compositions of the experimental diets are shown in Table Juvenile sea cucumbers were obtained from a private hatchery (Wando, Korea) and acclimated to the experimental condition for weeks After the conditioning period, groups of 50 sea cucumbers with an average individual body weight of 0.8 Æ 0.05 g (mean SE) were randomly distributed between 21 rectangular plastic tanks, each holding 40 L of water All treatments were executed in triplicate All tanks were fed once per day (17:00 hours) at 5% body weight per day for weeks Composite sand-¢ltered seawater was supplied at a £ow rate of 1L À and aeration was provided continuously into each tank Water temperature was maintained at 10.4 Æ 2.1 1C The natural photoperiod was followed during the experiment Sea cucumbers in each tank were collectively weighed at the start, and after and weeks The sea cucumbers were starved for 48 h before sampling 623 Dietary protein sources forApostichopus japonicus J-Y Seo et al Aquaculture Research, 2011, 42, 623^627 Table Ingredient composition (%) of the experimental diets Diets Ingredients FM SBM Fish meal Soybean meal Wheat flour Sesame seed meal Soybean–curd residue Sargassum thunbergii Dextrin Squid liver oil Soybean oil Vitamin premixà Mineral premixw Sodium alginate Choline chloride 41.0 WF SSM SCR ST MIX 29.0 10.0 25.0 32.0 24.3 8.3 8.3 8.3 8.3 8.3 21.2 2.0 2.3 1.5 2.0 5.0 0.2 59.0 55.0 50.0 55.0 46.0 4.3 1.5 2.0 5.0 0.2 26.0 4.0 2.3 1.5 2.0 5.0 0.2 3.0 2.0 2.3 1.5 2.0 5.0 0.2 31.0 0.3 1.5 2.0 5.0 0.2 10.0 1.0 0.3 1.5 2.0 5.0 0.2 50.0 5.0 2.0 2.3 1.5 2.0 5.0 0.2 ÃVitamin premix contained the following amount, which was diluted in cellulose (g kg À mix): L -ascorbic acid, 200.0; DL-a-tocopheryl acetate, 20.0; thiamin hydrochloride, 5.0; ribo£avin, 8.0; pyridoxine hydrochloride, 2.0; niacin, 40.0; Ca-D-pantothenate, 12.0; myo-inositol, 200.0; D-biotin, 0.4; folic acid, 1.5; p-amino benzoic acid, 20.0; menadione, 4.0; retinyl acetate, 1.5; cholecalciferol, 0.003; cyanocobalamin, 0.003 wMineral premix contained the following ingredients (g kg À mix): NaCl,7.0; MgSO4 Á 7H2O,105.0; NaH2PO4 Á 2H2O,175.0; KH2PO4, 224.0; CaH4(PO4) Á H2O, 140.0; ferric citrate, 17.5; ZnSO4 Á 7H2O, 2.8; Ca lactate, 21.8; CuCl, 0.2; AlCl3 Á 6H2O, 0.11; KIO3, 0.02; Na2Se2O3, 0.007; MnSO Á H2O, 1.4; CoCl2 Á 6H2O, 0.07 Table Proximate and essential amino acid composition of the experimental diets FM Nutrient contents (% dry matter) Dry matter 99.0 Crude protein 29.8 Crude lipid 7.5 Ash 8.3 Carbohydrate 53.4 Gross energy (MJ kg À 1) 18.8 Essential amino acid composition (% of protein) Arg 6.3 His 3.9 Ile 4.8 Leu 8.5 Lys 8.9 Met1Cys 2.9 Phe1Tyr 6.5 Thr 4.7 Val 5.3 Total 51.8 SBM WF SSM SCR ST MIX 99.6 30.9 7.2 5.5 55.5 19.2 92.8 32.3 6.9 6.4 54.5 18.8 99.8 29.7 6.9 6.6 54.4 18.4 94.7 31.8 7.1 7.0 54.1 18.4 91.7 30.5 6.8 30.1 32.6 14.2 94.0 32.3 6.6 10.6 50.5 18.4 7.3 2.7 5.0 8.7 7.0 0.8 7.9 4.2 5.3 48.9 5.8 4.1 4.6 8.3 7.0 2.5 6.7 4.3 5.3 48.6 9.3 3.0 4.4 8.1 4.4 1.8 7.4 4.0 5.6 48.0 6.1 4.0 4.9 8.7 7.7 2.1 7.1 4.9 5.7 51.2 6.1 3.2 4.8 8.2 8.1 2.5 6.9 4.7 5.4 49.9 6.6 3.7 4.9 8.5 7.1 2.3 6.8 4.6 5.6 50.1 One hundred sea cucumbers at the beginning and all surviving sea cucumbers per tank at the end of the feeding trial were collected and stored at À 75 1C for chemical analyses Proximate composition was analysed according to the standard methods (AOAC 1990) The amino acid composition of protein sources and the experimental diets were analysed 624 using an automatic amino acid analyser (L-8800, Hitachi,Tokyo, Japan) Data were subjected to one-way ANOVA to test the e¡ects of dietary protein sources on growth performance Duncan’s multiple range test (Duncan 1995) was used to rank di¡erent treatments signi¢cantly (Po0.05) All statistical analyses were carried out r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 623^627 Aquaculture Research, 2011, 42, 623^627 Dietary protein sources for Apostichopus japonicus J-Y Seo et al Table Growth and survival of sea cucumber fed the experimental diets containing di¡erent protein sources for weeks Diets IMWà FM SBM WF SSM SCR ST MIX 0.8 0.8 0.8 0.8 0.8 0.8 0.8 Æ Æ Æ Æ Æ Æ Æ FMWw 0.01 0.01 0.01 0.02 0.01 0.02 0.01 1.3 1.5 1.2 1.2 1.4 1.6 1.5 Æ Æ Æ Æ Æ Æ Æ Survival (%) 0.05ab 0.08c 0.03ab 0.08a 0.08bc 0.05c 0.08c Æ Æ Æ Æ Æ Æ Æ 85 85 82 87 75 85 74 WGz 1.3 7.4 1.8 5.8 1.8 1.8 8.7 54.7 86.0 51.0 45.3 74.0 95.7 81.3 SGR‰ Æ Æ Æ Æ Æ Æ Æ 7.51ab 7.37c 3.78ab 8.69a 10.60bc 7.31c 5.36c 0.78 1.11 0.74 0.66 0.98 1.20 1.06 Æ Æ Æ Æ Æ Æ Æ 0.08ab 0.07c 0.05ab 0.11a 0.10bc 0.07c 0.05c Values (mean Æ SE of three replications) in the same column not having a common superscript are signi¢cantly di¡erent (Po0.05) ÃInitial mean weight (g sea cucumber À 1) wFinal mean weight (g sea cucumber À 1) zWeight gain (%) (¢nal weight À initial weight)  100/initial weight ‰Speci¢c growth rate (% day À 1) (ln ¢nal weight À ln initial weight)  100/days Table Proximate and essential amino acid composition in whole body of sea cucumber fed the experimental diets containing di¡erent protein sources for weeks FM SBM Proximate composition (% dry matter) Crude protein 33 Æ 0.6 32 Æ Crude lipid 2.0 Æ 0.80 1.3 Æ Ash 45 Æ 0.4 46 Æ Essential amino acid composition (% protein) Arg 7.5 Æ 0.12 7.6 Æ His 1.9 Æ 0.03 1.9 Æ Ile 4.1 Æ 0.56 4.0 Æ Leu 6.6 Æ 0.01 6.6 Æ Lys 6.4 Æ 0.07ab 6.4 Æ Met1Cys 1.9 Æ 0.03 1.9 Æ Phe1Tyr 7.4 Æ 0.10 7.5 Æ Thr 5.6 Æ 0.03ab 5.5 Æ Val 5.1 Æ 0.07 5.1 Æ Total 46.5 46.5 WF SSM SCR ST MIX 1.0 0.45 0.6 33 Æ 0.8 1.3 Æ 0.32 46 Æ 0.3 32 Æ 0.5 2.1 Æ 0.11 46 Æ 0.7 32 Æ 0.3 2.1 Æ 0.30 45 Æ 0.5 33 Æ 0.2 2.0 Æ 0.53 45 Æ 1.1 32 Æ 0.6 1.6 Æ 0.37 46 Æ 0.5 0.15 0.01 0.03 0.06 0.06ab 0.06 0.09 0.01ab 0.07 7.6 Æ 1.9 Æ 4.0 Æ 6.5 Æ 6.3 Æ 1.9 Æ 7.4 Æ 5.6 Æ 5.2 Æ 46.4 7.4 Æ 1.8 Æ 4.0 Æ 6.4 Æ 6.1 Æ 1.7 Æ 7.1 Æ 5.4 Æ 5.0 Æ 44.9 7.4 Æ 1.9 Æ 4.1 Æ 6.7 Æ 6.5 Æ 1.9 Æ 7.5 Æ 5.7 Æ 5.1 Æ 46.8 7.5 Æ 1.9 Æ 4.1 Æ 6.7 Æ 6.5 Æ 1.9 Æ 7.5 Æ 5.6 Æ 5.2 Æ 46.9 7.6 Æ 2.0 Æ 4.1 Æ 6.6 Æ 6.3 Æ 1.1 Æ 7.4 Æ 5.6 Æ 4.8 Æ 45.5 0.07 0.01 0.06 0.10 0.07ab 0.03 0.09 0.03ab 0.03 0.19 0.03 0.07 0.09 0.09a 0.12 0.10 0.09a 0.09 0.03 0.01 0.07 0.10 0.07b 0.12 0.15 0.03b 0.15 0.12 0.01 0.03 0.03 0.06b 0.03 0.06 0.03ab 0.01 0.17 0.03 0.20 0.03 0.07ab 0.56 0.07 0.09ab 0.20 Values (means Æ SE of three replications) in the same row not having a common superscript are signi¢cantly di¡erent (Po0.05) using SPSS program version 12.0 (SPSS Michigan Avenue, Chicago, IL, USA) The growth and survival per treatment after weeks are shown in Table Animals responded differently to the di¡erent diets used Survival ranged from 74 to 87% and was not signi¢cantly di¡erent among treatments Weight gain and speci¢c growth rate (SGR) were signi¢cantly a¡ected by the dietary protein source (Po0.05) The highest weight gain and SGR were observed with the ST diet A similar (P40.05) weight gain and SGR were obtained with the SBM, SCR and MIX diets The lowest weight gain and SGR were observed in sea cucumbers fed the SSM diet There were no signi¢cant di¡erences in the ¢nal body weight, weight gain and SGR among sea cucumbers fed the FM,WF and SSM diets The nutrient composition, taste, texture and palatability of feed ingredients can in£uence feed intake and growth The results in this study showed that Sargassum thunbergii, soybean meal, soybean^curd residue and our mixture of several ingredients are useful ingredients to formulate sea cucumber diets A similar ¢nding was reported for abalones (Haliotis asinine and Haliotis fulgens) and snail (Semisulcospira coreana) (Guzman & Viana 1998; Bautista-Teruel, Fermin & Koshio 2003) Generally, carnivorous species require a high proportion of ¢sh meal in the diet for good growth and feed utilization (Jang, Kim & Lee 2005; Radford, Marsden, Davison & Je¡s 2007) Omnivorous and herbivorous species can still show good growth when fed diets with less or no ¢sh meal (Lee,Yun & Hur 1998; Hwang, Kim, Park, Bang, r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 623^627 625 Dietary protein sources forApostichopus japonicus J-Y Seo et al Kim, Jang & Lee 2003) Sea cucumbers forage sediments rich in organic matter including bacteria, protozoa, diatoms and plant detritus (Yingst 1976; Moriarty 1982; Zhang, Sun & Wu 1995; Kang, Kwon & Kim 2003) In the present study, it was not possible to measure the daily feed intake because it was di⁄cult to collect uneaten food from the tanks Analyses of the fatty acid and ¢bre content of the di¡erent diets and body compositions of sea cucumbers at harvest and direct monitoring of feed intake can help to ¢ne-tune diet compositions for sea cucumbers The whole-body amino acid composition was not signi¢cantly a¡ected by the experimental diets (Table 4) No signi¢cant di¡erences were observed in the amino acid composition between sea cucumbers fed the ¢sh meal diet and other diets The Met1Cys content of sea cucumbers fed the SBM diet, which contained the lowest Met1Cys content (Table 2), was comparable with that of animals fed other diets This suggests that sea cucumbers may ingest more of the SBM diet to compensate the lack of Met1Cys Dabrowski, Arslan, Terjesen and Zhang (2007) reported that the feed intake of midas cichlids fed an amino acid imbalanced diet was signi¢cantly increased compared with that of ¢sh fed an amino-acid-balanced diet Moisture, crude protein, crude lipid and ash contents of sea cucumbers were not signi¢cantly di¡erent among the dietary treatments This was in accordance with the results reported by Senaratna, Evans, Southam and Tsvetnenko (2005) and Tidwell, Goyle, Bright and Yasharian (2005) In conclusion, the results of this study indicate that soybean meal, Sargassum thunbergii powder or a mixture of ¢sh meal, soybean, wheat £our, sesame seed meal, soybean^curd residues and S thunbergii could be good protein sources in sea cucumber feeds From an economic point of view, soybean meal with a high protein content, a well-balanced amino acid pro¢le and a steady market supply might become a more reliable protein source than S thunbergii, especially when the demand for, and the production of, A japonicas continues to increase and outstrips the supply of S thunbergii Acknowledgments This work was supported by funds from the Ministry for Food, Agriculture, Forestry and Fisheries in Korea 626 Aquaculture Research, 2011, 42, 623^627 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480^489 Aquaculture Research, 2011, 42, 480^489 shrimp Litopenaeus vannamei (Boone, 1931) Aquaculture 219, 655^670 Hardy R.W (2010) Utilization of plant proteins in Âsh diets: eĂects of global demand and supplies of Âshmeal Aquaculture Research 41,770^776 Hernandez C., Olvera-Novoa M.A., Aguilar-Vejar... hatchery-reared Salmo salar and comparison with wild smolts Aquaculture 45, 97^109 ZauggW.S & McLain L.R (1982) A simpliÂed preparation for adenosine triphosphate determintaion in gill tissue Canadian Journal of Fisheries and Aquatic Sciences 39, 215^217 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 479 Aquaculture Research, 2011, 42, 480^489 doi:10.1111/j.1365-2109.2010.02642.x Effects... Q.C., Tan B.P., Chi S.Y & Dong X.H (2009) Apparent digestibility of selected feed ingredients for white shrimp Litopenaeus vannamei, boone Aquaculture Research 41,78^86 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 480^489 489 Aquaculture Research, 2011, 42, 490^498 doi:10.1111/j.1365-2109.2010.02644.x Role of organic fertilizers in walleye (Sander vitreus) production in plastic-lined culture... R.C., Clouse C.P & Harding L.M (1993) Pond production of Ângerling walleye, Stizostedion vitreum, in the Northern Great Plains Journal of Applied Aquaculture 2, 33^58 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 490^498 Aquaculture Research, 2011, 42, 499^505 doi:10.1111/j.1365-2109.2010.02645.x Cellulase-producing bacteria of Aeromonas are dominant and indigenous in the gut of Ctenopharyngodon... with the following thermo-proÂles: initial denaturation at 95 1C for 5 min; 30 cycles of 95 1C for 1min, 50 1C for 1min, and 72 1C for 2 min; and a Ânal r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 499^505 Aquaculture Research, 2011, 42, 499^505 Indigenous cellulase-producing bacteria in grass carp Y Jiang et al extension step at 72 1C for 10 min PuriÂed PCR s ampliÂcation products were... refer to the name of clone screened from cellulose^Congo red agar The scale bar indicates 0.2% sequence variation r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 499^505 501 Indigenous cellulase-producing bacteria in grass carp Y Jiang et al 98 0.2 Aquaculture Research, 2011, 42, 499^505 CD7 AF427152(Aeromonas sp PAR3) CD8 AY910844(Aeromonas salmonicida subsp a) 67 CD27 X74683(Aeromonas sobria)... gut tissue of C idella using the genomic DNA-based 16S rRNA gene library in this research The genomic DNA from bacteria isolated directly from the gut was r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 499^505 Aquaculture Research, 2011, 42, 499^505 Indigenous cellulase-producing bacteria in grass carp Y Jiang et al Table 1 Relative abundance and aliation of bacteria detected in the empty... of a feed is an important factor to consider in determining the utilization of the feed (Akiyama, Coelho, Lawrence & Robison 1989; Yang, r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 480^489 Aquaculture Research, 2011, 42, 480^489 EĂects of spray-dried blood cell meal on L vannamei H Niu et al Diet 0 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 80 EAA retention (%) 70 60 aaaa 50 40 30 aaaa a aaa... Biology 206, 3425^3435 Jrss K., Bittorf Th., Vlker Th & Wacke R (1983) InÊuence of nutritional biochemical sea water adaptation of rainbow r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 469^479 Aquaculture Research, 2011, 42, 469^479 EĂect of feeding rations on growth and osmoregulation A K Imsland et al trout (Salmo gairdneri Richardson) Comparative Biochemistry and Physiology 75B,713^717... 93.9 2.0 72.9 5.8 136.8 94.8 23.5 Essential amino acid for shrimp (Akiyama et al 1991; Forster et al 2003) Tryptophan was not determined r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 480^489 481 Aquaculture Research, 2011, 42, 480489 EĂects of spray-dried blood cell meal on L vannamei H Niu et al were formulated to be isonitrogenous (approximately 326 g kg 1 crude protein) and isolipidic