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Do formulated feeds for juvenile Panulirus ornatus lobsters require dietary cholesterol supplementation?
Aquaculture 307 (2010) 241–246
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Do formulated feeds for juvenile Panulirus ornatus lobsters require dietary cholesterol supplementation? Simon J. Irvin ⁎, Kevin C. Williams, Margaret C. Barclay, Simon J. Tabrett CSIRO Marine and Atmospheric Research, PO Box 120, Cleveland Qld. 4163, Australia
a r t i c l e
i n f o
Article history: Received 19 April 2010 Received in revised form 16 July 2010 Accepted 23 July 2010 Keywords: Phospholipids Feeds Crustacean Micro-nutrient deficiency
a b s t r a c t Mortality rates of wild spiny lobster seed Panulirus ornatus are typically high, with losses of 40–60% occurring within 30 days of capture. Mortality appears symptomatic of a micro-nutrient deficiency or captivity related stressors. Cholesterol is a micro-nutrient essential for healthy ecdysis, growth and survival in crustaceans. This paper reports a 7-week study examining the growth, survival and tissue cholesterol responses of juvenile P. ornatus lobsters fed a low cholesterol basal diet that was serially supplemented with cholesterol at 0.75 g kg− 1 increments from 1.25 to 4.25 g kg− 1 dry matter (DM). The basal diet contained 45 g kg− 1 phospholipid (from natural ingredients and supplemented soybean lecithin) and this was fed to all lobsters for 2 weeks prior to commencement of the experiment to reduce cholesterol reserves in the lobsters. A sixth diet comprising equal amounts of green-lipped mussel (Perna canaliculus) and whiting fillet (Silago ciliate) was included in the treatment array as a reference diet. One hundred and fifty lobsters were blocked by initial weight into five groups and then from within these groups they were equally distributed to 30 tanks (n = 5 tanks per treatment). There was no significant difference in survival, daily growth coefficient or tissue dry matter cholesterol content between lobsters fed the lowest and highest cholesterol diets: 55%, 349%, 243 mg 100 g− 1 and 70%, 456%, 253 mg 100 g− 1 respectively. However, there was a trend (P = 0.065) for lobster percentage weight gain to increase linearly with dietary cholesterol. There was a significant relationship between the starting weight of the lobsters and subsequent survival, with only 43% of the smallest lobsters (0.2 to 0.5 g block) surviving compared to 90% for the largest lobsters (2.71 to 3.40 g block). No specific dietary cholesterol requirement for small P. ornatus was determined. Feeds which contain practical marine proteins require no supplementary cholesterol, a significant cost saving in feed formulation. Feeds which contain high levels of plant proteins may require supplementary cholesterol to provide at least 4.0 g kg− 1 DM dietary cholesterol. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cholesterol is an essential and expensive micro-nutrient required by crustaceans. It forms the chemical framework for many hormones, including ecdysteroids that are critical for the initiation of metamorphosis and moulting in crustaceans (Teshima, 1997). Cholesterol also has an important, though poorly understood, interrelationship with dietary phospholipids (Teshima, 1997; Coutteau et al., 1997). Phospholipids act as surfactants for efficient emulsification of ingested lipid and thus assist the uptake of sterols (and other lipids) from the gut. They are also essential for the efficient transport of cholesterol from the digestive gland to target tissues during the moulting process (Conklin et al., 1983). Thus, dietary cholesterol requirements are likely to be influenced by the amount of phospholipid in the diet. ⁎ Corresponding author. Tel.: + 61 7 3826 7353; fax: + 61 7 3826 7222. E-mail addresses: [email protected] (S.J. Irvin), [email protected] (K.C. Williams). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.07.026
Cholesterol requirements have been estimated for numerous crustacean species including early work with the American clawed lobster (Homarus americanus) and more recently with crabs and penaeid shrimp (Castell et al., 1975; Conklin et al., 1983; Coutteau et al., 1997). Castell et al. (1975) found that a dietary dry matter (DM) cholesterol content of 5 g kg− 1 resulted in the best growth and survival of juvenile H. americanus lobsters whereas growth and survival were depressed with levels of 2 g kg− 1 or 20 g kg− 1. D'Abramo et al. (1984) found that a dietary cholesterol specification of 1.2–1.9 g kg− 1 was optimal for very small homarid lobsters (4th stage juveniles), although the slow growth of those lobsters of less than 1 g gain over 120 days is enough reason to question whether this was a reliable indication of the animal's cholesterol requirement. Kean et al. (1985) found that juvenile H. americanus lobsters fed a ‘zero’ cholesterol diet died, regardless of the amount of soybean lecithin (predominantly phosphatidylcholine) in the diet. Increasing the amount of cholesterol above 2.5 g kg− 1 or varying the amount of lecithin in the diet had no significant effect on lobster growth or survival.
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Culture of the fast growing and high value tropical spiny lobster Panulirus ornatus is an established industry in Vietnam and under development elsewhere in SE Asia (Williams, 2004). Survival of captured wild seed lobsters of a size of 2 g or less is typically low, 40 to 60% but can be 0%, with these deaths typically occurring during the first 30 days of nursery culture (Thuy and Ngoc, 2004). Inadequate nutrition from a diet comprised only of trash fish is likely to have contributed to this high mortality rate. Pelleted feed development studies at CSIRO with juvenile P. ornatus have shown growth and survival to be optimal with DM dietary protein and lipid specifications of 500–550 g kg− 1 and ~ 100 g kg− 1, respectively (Smith et al., 2003b, 2005; Barclay et al., 2006). However, during that work, lobsters fed a control diet of thawed New Zealand green-lipped (Perna canaliculus) or Tasmanian blue (Mytilus edulis) mussels grew poorly and mortalities increased over time, a symptom characteristic of a micro-nutrient deficiency. The Barclay et al. (2006) study eliminated dietary astaxanthin as a possible cause of the assumed micro-nutrient deficiency in the mussels as dietary astaxanthin supplementation of up to 75 mg kg− 1 had no effect on growth or survival of juvenile P. ornatus lobsters but did significantly increase carapace colouration and tissue astaxanthin levels. Cholesterol was initially not considered to be a possible limiting micro-nutrient for juvenile spiny lobster feeds because of its high content in natural ingredients. Cholesterol was the dominant sterol in New Zealand green-lipped and Tasmanian blue mussels collected from three different settlement sites but it was higher in green-lipped (~1.7 g kg− 1 DM) than blue (~ 0.9 g kg− 1 DM) mussel (Murphy et al., 2002). In the study of Barclay et al. (2006) where the feeding of sole diets of green-lipped and blue mussels were compared, growth of juvenile P. ornatus was better when fed green-lipped than blue mussels. If the mussel syndrome observed with P. ornatus was due to a cholesterol deficiency, the study of Barclay et al. (2006) suggests that the dietary DM cholesterol requirement must be more than 1.7 g kg− 1. To test this hypothesis, an experiment was designed to examine the responses of very small P. ornatus to supplementation of a low cholesterol basal diet with graded amounts of cholesterol in the presence of an adequate dietary supply of phospholipid. This paper reports the growth, survival and tissue cholesterol responses of the lobsters to dietary cholesterol supplementation. 2. Materials and methods 2.1. Overview and experimental design The experiment was carried out to study the growth and survival response of juvenile P. ornatus to diets containing a relatively narrow range of supplementary cholesterol. A standard growth assay was conducted with six treatments and five tank (n = 5) replicates. Because of the wide weight range of the lobsters available for the study, from 0.2 to 3.4 g, selected animals were weight-blocked into five groups of ascending weight. Within these weight-stratified blocks, lobsters were equally distributed to each of the six treatments in accordance with a balanced randomised block design. 2.2. Diet formulation and manufacture The treatments involved a reference diet of mussel and fish flesh and five formulated pelleted dry feeds that provided an increase in the dietary DM cholesterol content from a base level of 1.25 g kg− 1 in 0.75 g kg− 1 increments to 4.25 g kg− 1. The reference diet had an analysed cholesterol content of 3.1 g kg− 1 DM and consisted of equal parts by weight on a DM basis of chopped fish flesh (Silago ciliata) and green-lipped mussel meat (P. canaliculus). The fresh fish flesh and mussel meat were mixed together and fed to the lobsters as the fresh product. The formulations of the dry pelleted feeds (Table 1) were identical except that the intended supplements of cholesterol were
Table 1 Formulation and chemical composition of the cholesterol series of pelleted dry feeds and a reference diet of fresh natural items. Ingredient
C1.25 C2.0
Formulation (g kg− 1 as used) Fishmeal (de-fatted)a 260 Cholesterolb 0 c Starch 7.5 d Aqualipid-70 15 e Krill meal (spray dried) 150 100 Mussel (dry equiv)f Fish flesh (dry equiv.)f 100 Wheat gluteng 80 Wheat flour 240 h Other 37.5 Green-lipped mussel (dry)i i Fish flesh (dry) Transglutaminasej 10
260 0.75 6.75 15 150 100 100 80 240 37.5
C2.75
C3.5
C4.25
260 1.5 6.0 15 150 100 100 80 240 37.5
260 2.25 5.25 15 150 100 100 80 240 37.5
260 3.0 4.5 15 150 100 100 80 240 37.5
Reference
500 500 10
10
10
10
Analysed composition Dry matter (g kg− 1) 660 650 665 665 665 200 Crude protein (g kg− 1 DM) 595 597 598 599 595 −1 Ash (g kg DM) 98 97 97 97 97 109 109 103 109 109 Total lipid (g kg− 1 DM) Cholesterol (g kg− 1 DM) 1.25 1.92 2.76 3.43 4.09 3.10 Phospholipid (g kg−1 DM )k 52 44 44 42 46 Gross energy (MJ kg−1 DM) 20.2 20.1 20.2 20.2 20.3 a Peruvian N670 g kg− 1 CP, supplied by Ridley Aquafeeds Pty Ltd., Narangba, Qld., Australia. b Cholesterol, supplied by Sigma. c Corn starch, Janbak Industries Pty Ltd., Brisbane, Qld., Australia. d Soybean lecithin, 700 g kg− 1 lipid, supplied by Janbak Industries Pty Ltd., Brisbane, Qld., Australia. e Antarctic (Inual, Santiago, Chile), supplied by Ridley Aquafeeds Pty Ltd., Narangba, Qld., Australia. f Minced flesh of green-lipped mussel (P. canaliculus) or whiting fish (S. ciliata) prepared at CSIRO and used as the fresh product but reported in the formulation on a DM basis. g Vital gluten, 760 g kg− 1 CP, supplied by Janbak Industries Pty Ltd., Brisbane, Qld., Australia. h Provided in the diet (g/kg): canola oil (Crisco) 20; carophyll pink (10% astaxanthin), 1.1; choline chloride (70%), 0.2; ethoxyquin, 0.2; vitamin premix (Williams et al., 2004), 10; heat-stable vitamin C (Stay-C), 1; and trace mineral premix (Williams et al., 2004), 5. i Chopped flesh of green-lipped mussel (P. canaliculus) or whiting fish (S. ciliata) prepared at CSIRO. j TG activa transglutaminase, supplied by Kerry Ingredients Pty. Ltd., Brisbane, Qld., Australia. k Phospholipid calculated as 75% of polar lipid content as analysed by TLC-FID Iatroscan analysis (Nichols et al., 1994).
made at the expense of equivalent amount of starch. To formulate a diet that was attractive to these small lobsters and as low as possible in cholesterol, it was necessary to de-fat the fish meal used in the diet. This involved de-fatting the fish meal with a hexane:ethanol solvent (2:1) to produce a low cholesterol protein source. Fishmeal and solvent were mixed in a beaker at a ratio of 4:1 and left for 10 min. The slurry was poured through a paper filter (Whatman No 4) to remove the solvent. The whole process was repeated. After the second extraction, the fishmeal residue was spread onto a tray and placed in a fume cupboard for 16 h. The extraction process reduced the DM cholesterol content of the fish meal from 3.72 to 1.29 g kg− 1. The DM cholesterol content of freeze-dried green-lipped mussel flesh and whiting flesh was 2.40 g kg− 1 and 3.49 g kg− 1 respectively, and when combined as the reference diet, had a DM cholesterol content of 3.1 g kg− 1. The diets were made at the CSIRO Marine Research Laboratory at Cleveland, Australia. For the formulated diets, fish flesh and greenlipped mussel meat were placed at − 20 °C until semi-frozen, then extruded through a 3 mm die plate of the meat grinder attachment of an A200 Hobart planetary dough mixer (Hobart Corporation, Troy, OH, USA) to form a homogenous mince. The dry ingredients were finely ground (b710 μm) using a mortar and pestle for small
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constituents or by hammer mill (Mikro Pulverizer, Metals Disintegration Coy, Summit, NJ, USA) for bulk ingredients. The fresh ingredients and transglutaminase binder were thoroughly mixed together using an industrial kitchen Kenwood KM800 planetary mixer (Kenwood Ltd, Havant Hants, UK) for 10 min before the cod liver oil and remaining dry ingredients were added followed by a further 10 min mixing to form a dough of approximately 40 to 50% moisture content. The dough was extruded through a 3 mm die plate of the meat grinder attachment of the A200 Hobart planetary dough mixer to form spaghetti like strands. The strands were placed in an airtight bag and set overnight in a refrigerator at 4 °C. The strands were stored at −20 °C until required for feeding. For the reference diet, equal parts (on a DM basis) of fresh finely chopped fish flesh and green-lipped mussel meat were mixed together and placed in an airtight bag and stored at − 20 °C until required for feeding. 2.3. Experiment management Lobsters used in the experiment were obtained as recently settled P. ornatus from Trinity inlet, Cairns, North Queensland (16° 55′ S, 145° 45′ E), hand collected and air freighted to CSIRO Cleveland. The experiment consisted of a 2-week body cholesterol depletion phase on the basal diet (C1.25) followed immediately with a 7-week growth assay. Prior to the experiment, lobsters were held for 3 weeks, during which time they were weaned from a diet of fish flesh and greenlipped mussel flesh to the low cholesterol base diet (C1.25). A total of 150 weight-stratified lobsters were distributed to 30 tanks arranged as five weight blocks of lobsters; each block comprised each of the six diet treatments. Each tank was stocked with five lobsters of matched weight. Tanks (120 L, circular polyethyhelene, 0.6 m diameter × 0.5 m deep) were installed within a light controlled laboratory (12:12 h dark–light cycle) and supplied with individual aeration and heated flowing seawater (0.6–1 L/min) enabling temperatures to be maintained between 28 and 30 °C. Sufficient clay bricks were placed in each tank to provide adequate and suitable shelter for all lobsters. Feed was offered three times daily nominally at 0800, 1700 and 2400 h, slightly to excess and with the major fraction provided at the 1700 h feed. All uneaten feed was removed daily by siphon cleaning and a daily record was kept of water temperature, observed moults and mortalities in each tank. Five lobsters representative of the replicate weight blocks were subjected to the same 2-week cholesterol depletion phase as for those used in the growth assay but were then sacrificed to determine the initial body cholesterol content. Lobsters were weighed at the start of the experiment, after 4 weeks and again at the conclusion of the 7-week growth experiment. With the exception of the two smallest blocks, losses due to mortality were replaced with a similarly sized tagged lobster to maintain stocking density; data from these lobsters were excluded from subsequent analyses. 2.4. Chemical analysis For the determination of whole body cholesterol content, a sample of five lobsters taken at the start of the experiment and lobsters in each tank at the end of the experiment was dried, weighed and sagitally sectioned and freeze-dried. The dried lobster biomass from each tank and samples of each diet were homogenised in a Knifetec grinder (Tekator, Sweden) so that all materials passed through a 1 mm sieve. Sub-samples of the homogenised lobster biomass and selected feed ingredients were analysed for DM and cholesterol content. The diets were analysed for DM, ash, crude protein (CP), total lipid, phospholipid, gross energy and cholesterol content by AOAC (1999) procedures (Table 1): DM from weight change following heating in a thermo-gravimetric analyser (Leco TGA-601, St Joseph, MI, USA) at 105 °C to a constant weight; ash from weight change following burning in a thermo-gravimetric analyser (Leco TGA-601)
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at 600 °C to constant weight; and CP (N × 6.25) by the Dumas combustion method, calibrated using aspartic acid. Total lipid was determined gravimetrically following a chloroform–methanol (2:1) extraction using the method of Folch et al. (1957). The oil class composition of the diet samples was determined by TLC-FID Iatroscan; polar lipid was dominant in all samples and comprised mostly phospholipids (approximately 75% of total polar lipid) since the TLC-FID Iastroscan method did not recover glycolipids (Nichols et al., 1994). Gross energy was determined by isothermal bomb calorimeter using a Leco AC200 Bomb Calorimeter (Leco Corp. St. Joseph, MI, USA). Sterols in the diet and tissue samples were extracted into hexane after direct saponification in 0.1 N KOH–ethanol (2:1) (Kovacs et al., 1979); the cholesterol content of the extracts was determined by gas chromatography using a Hewlett-Packard 5890 (Hewlett-Packard Company, PA, USA), fitted with a flame ionisation detector and a non-polar capillary column (HP-1, 50 m × 0.2 mm i.d., 0.11 μm film thickness, Hewlett-Packard). The sample was injected oncolumn at 90 °C with the oven temperature programmed to increase at 15 °C min− 1 to 250 °C followed by an increase of 4 °C min− 1 to 300 °C. The oven temperature was then held at 300 °C for a further 7 min. Sterols were identified by reference to the retention times of pure standards (Sigma) relative to that of the internal standard 5a-cholestane and quantified from their peak areas relative to that of the internal standard. 2.5. Calculations and statistical analysis Daily growth coefficient (DGC) and percentage weight gain (%WG) were derived as: 1=s We1 = s −Ws −1 DGC %d ¼ 100 d
!
and We %WG ¼ 100 Ws where We and Ws are the weights of the lobsters at the end and start of the experiment, respectively, and d is the number of days on the experiment. Statistical packages for balanced (Balf) and regression analyses (Regn) prepared by the Queensland Department of Primary Industries Biometry branch were used for all statistical analyses. Differences between treatments in final weight and DGC were examined by a oneway ANCOVA using initial weight as the covariate. Response data for % WG, survival and tissue cholesterol were arcsine transformed and examined using a one-way ANOVA in accordance with the design of the experiment. Differences between treatments were tested for significance using Fisher's protected t-test (Snedecor and Cochran, 1989) wherein differences between means were examined only when the F-test of the ANOVA or ANCOVA was significant (P b 0.05). The relationship between %WG and dietary cholesterol level was also examined using regression analysis. 3. Results The block weight survival of lobsters (irrespective of dietary cholesterol level) is shown in Table 2. There was a significant relationship between starting size and survival rate. Survival ranged from 43% for the smallest block (0.2 to 0.50 g) to 90% for the largest block (2.71 to 3.40 g). Survival in the smallest block was significantly lower than for all other size classes, with the exception of the next largest block (0.51 to 0.90 g), which had a 63% survival. Because of the poor survival of the lobsters in the smallest block, all data for this replicate were deleted from all subsequent analyses.
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Table 2 Effect of block starting size of lobsters on subsequent survival during the 7-week experiment. Block weight (g)
Survival (%)
0.20–0.50 0.51–0.90 0.91–1.80 1.81–2.70 2.71–3.40
43d 63 cd 67bc 70ab 90a
a,b,c,d
Means with a common letter do not differ (P b 0.05).
The analysed total lipid, cholesterol and gross energy of the experimental diets are shown in Table 1. Cholesterol DM content ranged from 1.25 g kg−¹ in the non supplemental cholesterol diet to 4.1 g kg− ¹ in the highest cholesterol diet. Response traits of the lobsters to dietary cholesterol content are shown in Table 3. Survival was variable, lowest for lobsters in the lowest cholesterol diet (55%) and highest (85%) for lobsters fed the diet containing 3.4 g kg− 1 cholesterol; however there were no significant differences between the treatments. There were no significant treatment differences between the final weight or DGC. Percent weight gain was variable with lobsters fed the diet containing 4.1 g kg− 1 cholesterol achieving a significantly faster growth than those on the 2.0 g kg− 1 cholesterol diet. There was a trend (P = 0.065) for %WG to increase linearly with increasing dietary cholesterol content (Fig. 1). The tissue cholesterol level in the lobsters was not significantly different between treatments, ranging from 237 mg 100 g− 1 DM for lobsters sampled at the start of the experiment to 267 mg 100 g− 1 DM for lobsters at the end of the experiment that were fed the diet containing 3.4 g kg− 1 cholesterol. 4. Discussion Dietary cholesterol level had no significant effect on survival, although there was a trend towards lower survival with the lowest cholesterol diet. Across treatments, survival averaged 72.5%, which was typical of findings with other studies with very small spiny lobsters (Smith et al., 2003b; Johnston et al., 2003; Smith et al., 2005). However, percentage survival above 75% and as high as 100% has been observed in studies with western rock lobster Panulirus cygnus juveniles of 0.5–2 g starting weight (Glencross et al., 2001; Johnston et al., 2006). Lobster starting weight, and not dietary cholesterol level, had the greatest bearing on survival; the smaller the lobster the lower the chance of survival. Survival of the smallest block (0.2 to 0.5 g) of 43% was similar to that (53%) observed in a feed comparison study with similarly sized (0.3 g) P. ornatus (Pryimabodo, B. pers. comm.). Comparatively low survival of 41% was reported by Smith et al. Table 3 Performance1 of juvenile P. ornatus fed experimental feeds at 5 different cholesterol (chol) levels or a fresh ingredient reference diet. Treatment
Initial C1.25 C2.0 C2.75 C3.5 C4.25 Reference ± SEM 1
Start weight (g)
Final weight (g)
Weight gain (%)
Survival (%)
DGC (% d− 1)
Tissue chol (mg 100 g− 1)
2.3a 2.2a 2.3a 2.2a 2.1a 2.2a 0.12a
7.6a 6.9a 8.9a 7.8a 9.1a 7.4a 1.01
349ab 324b 395ab 369ab 456a 345ab 42.0
55a 80a 70a 85a 70a 75a 10.3
1.30a 1.20a 1.49a 1.39a 1.64a 1.32a 0.15
237 243a 242a 247a 267a 253a 243a 10.2
Data from the smallest weight block (0.2–0.5 g) were excluded from the analyses because of the low survival of lobsters in that replicate. a,b,c Within columns, means with a common superscript letter do not differ (P N 0.05).
Fig. 1. Relationship (P =0.065) between dietary dry matter cholesterol content (g kg− 1; X) and lobster weight gain percent (Y).
(2005) in a protein requirement study with larger juvenile P. ornatus (2.5 g ± 0.19 g). However this was concluded to be more likely related to a nutrient deficiency resulting from the sole feeding of green-lipped mussel. The cause of the high mortality for the smallest juveniles in our study has not been identified but most likely was due to these small lobsters being most vulnerable to cannibalism although other factors such as low nutritional reserves, poor intake of the offered food, etc. cannot be excluded. Compounding identification of causation factors is the tendency for companion lobsters to rapidly consume all, or sections of recently deceased lobsters, thus making accurate recording of moult frequency with small (b5 g) spiny lobsters difficult and impractical. Survival of seed P. ornatus lobsters held in individual cages during a 60-day growth study (Irvin and Williams, 2009) was significantly higher (P b 0.05) than those held communally (89 cf 72%, respectively) but growth rate was significantly higher with the communally housed lobsters (DGC of 1.0 cf 0.7% d− 1, respectively). These observations suggest that cannibalism is an important cause of juvenile lobster mortality but other factors, possibly nutritional, are also involved. Growth was high for all treatments: There was a trend (P = 0.065) for weight gain % to increase with increasing dietary cholesterol content (Fig. 1). Whether or not this was an artefact of the high growth observed for lobsters fed the highest cholesterol diet (C-4.25) or a true biological response is difficult to know. Removal of the smallest block of lobsters from the analysis because of low survival would have considerably weakened the statistical rigor of the experiment. The DGC for lobsters fed the highest cholesterol diet of 4.1 g kg− 1 DM was higher or similar (1.64%) to small P. ornatus lobsters fed the best performing diet in previous protein and astaxanthin requirement studies (Smith et al., 2005, Barclay et al., 2006) of 1.38% and 1.59% d− 1 respectively. The lobsters in the protein requirement study of Smith et al. (2005) were fed on diets varying only in the level of conventional full-fat fish meal and casein and contained 2.7 g kg− 1 DM dietary cholesterol. Lobsters fed a similar level of dietary cholesterol in our study also exceeded that (1.48% d− 1) of the best feed in the protein requirement study. As the major protein component of the experimental feeds in this study was de-fatted fish meal, it is clear that small P. ornatus have a low requirement for cholesterol (in the presence of 45 g kg− 1 DM phospholipid). Feeds which contain conventional proteins are likely to be over specified with respect to cholesterol and additional supplementation with cholesterol therefore unnecessary. An ingredient digestibility study with sub-adult P. ornatus by Irvin and Williams (2007) reported the potential for partial replacement of marine proteins with terrestrial plant proteins. It would be with such dietary formulations that addition of supplementary cholesterol should be considered to provide not less than 4.0 g kg− 1 DM dietary cholesterol. There was no significant difference in the whole body cholesterol content of lobsters fed different levels of dietary cholesterol. The role of the depletion diet was to reduce the cholesterol reserves of the lobsters prior to the experiment start and thus accelerate any effect dietary cholesterol level would have on growth and survival. The
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tissue cholesterol of lobsters fed the cholesterol depletion diet of 1.25 g kg− 1 DM for 9 weeks (243 mg 100 g− 1 DM) was not significantly different from that of lobsters fed the highest cholesterol diet of 4.1 g kg− 1 for 7 weeks (253 mg 100 g− 1 DM) and only slightly higher than the amount of cholesterol in the initial lobsters (237 mg 100 g− 1 DM). Therefore the depletion diet, which contained no supplemental cholesterol and with a large component of the protein from de-fatted fish meal, had sufficient cholesterol to maintain tissue cholesterol levels at near maximum capacity. Considerably more work has been done with other crustaceans to elucidate their dietary cholesterol requirements. For penaeid shrimp, estimates typically range from 2.5 to 15 g kg− 1 (DM) of the diet (Teshima, 1997) while work at this lab with Penaeus monodon (Smith et al., 2003a) has shown that a dietary DM specification of 1.7 g kg− 1 cholesterol was adequate in the presence of 17 g kg− 1 dietary phospholipid. Gong et al. (2000) characterized the dietary cholesterol requirement of Litopenaeus vannamei to be 3.5 g kg− 1 in the absence of dietary phospholipid and to decrease to 1.3 to 1.4 g kg− 1 and to 0.5 g kg− 1 as the dietary phospholipid content increased from 15 to 30 g kg− 1 and to 50 g kg− 1, respectively. With juvenile mud crabs Scylla serrata, Sheen (2000) found that a dietary cholesterol level of 5.1 g kg− 1 optimized growth and survival of the crabs when they were fed a purified diet devoid of soy lecithin (but containing 60 g kg− 1 of a 2:1 cod liver oil and corn oil mixture); diets with cholesterol concentrations of b2 or N11 g kg− 1 depressed growth. However, Holme et al. (2006) was unable to determine a specific dietary cholesterol requirement for S. serrata megalopa as the low endogenous cholesterol level present in the base diet (1.4 g kg− 1 DM) was sufficient to support acceptable survival in development to the first crab stage. In contrast, a study by Sheen (2000) with juvenile mud crabs (S. serrata) reported an optimal dietary cholesterol requirement of 5.1 g kg− 1. In that study, crabs fed 5.0 g kg− 1 dietary cholesterol grew significantly faster than those fed 2.1 g kg− 1; however there was no difference in tissue cholesterol content of the crabs after 84 days. This suggests that tissue cholesterol content alone is not a good indicator of the efficiency of cholesterol usage in crustaceans. It is possible that the mud crab diets, which contained no supplemental lecithin, caused a reduced efficiency in the transport and utilisation of cholesterol. The interaction between phospholipid and cholesterol is little understood but widely reported for different crustacean species (Lester et al., 1975). More recently, a study by Holme et al. (2006) with megalopa mud crab S. serrata, found that cholesterol supplementation was only beneficial when soy lecithin (predominantly phosphatidylcholine) inclusion was 20 g kg− ¹ or less. Lecithin appears to have a sparing effect on cholesterol, resulting in a reduction in the cholesterol requirement, most likely due to an improved efficiency in the digestibility and uptake of cholesterol. In our study, all feeds contained an inclusion level of 15 g kg− 1 soy lecithin (~10 g kg− 1 phosphatidylcholine) and a total dry matter phospholipid content of 45 g kg− 1. It is reasonable thus to infer that this had a sparing effect on the cholesterol requirement, contributing to the non-determination of a specific cholesterol requirement for this species. This finding is broadly supported in a recent review of the nutrient requirements of larvae of the mud crab S. serrata (Holme et al. 2009), in which the cholesterol requirements of a variety of juvenile crustaceans were compared. The review concluded that there is an important interaction between cholesterol and phospholipid, and that the determined cholesterol requirement will depend on the phospholipid level and the presence of other dietary factors. Our study used wild caught lobsters from a wide size range and required blocking animals by weight. The majority of nutrient requirement studies reported with crustaceans use hatchery reared stock, and thus have the advantage of using animals with a narrow size range and known age, genetic and nutritional history. Currently, this is not possible with P. ornatus as economic hatchery seed production has not yet been developed as the journey from phyllosma
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to puerulus stage is long (5 to 8 or more months) and difficult. Therefore the age, health and remaining energy reserve of these settled lobsters are likely to be highly variable. This may explain why the variability within treatments was also high. Interest in commercial closure of the life cycle of P. ornatus has recently intensified with experimental numbers of puerulus being produced by a number of research and commercial operators. The production of commercial numbers of seed lobsters with a known genetic and nutritional background will allow and make necessary the evaluation or reevaluation of nutrient requirements. 5. Conclusions No specific dietary cholesterol requirement was determined from the results of this study. However, in the absence of some better definition of a dietary requirement, a dietary specification of 4.0 g kg− 1 DM cholesterol is recommended for juvenile spiny lobsters. Feeds which contain practical marine proteins are unlikely to require any supplementary cholesterol, a significant cost saving in feed formulation. Feeds which contain high levels of plant proteins may require supplementary cholesterol to provide a total dietary level of not less than 4.0 g kg− 1 DM cholesterol. Acknowledgements The Authors thank internal referees Brett Glencross and Greg Coman at CSIRO Marine and Atmospheric research for their constructive comments, which have considerably improved this paper. Many thanks also to Dr Clive Jones and his team from QDPIF in Cairns for collecting and transporting the juvenile lobsters used for the research. This work was supported by a grant from the Australian Centre for International Agricultural Research as part of an international collaborative project (FIS/2001/058) on “Sustainable tropical spiny lobster aquaculture in Vietnam and Australia. References AOAC International, 1999. Official Methods of Analysis, 16th edn. Association of Official Analytical Chemists, Arlington, VA, USA. Barclay, M.C., Irvin, S.J., Williams, K.C., Smith, D.M., 2006. Comparison of diets for the tropical lobster Panulirus ornatus: astaxanthin supplemented feeds and mussel flesh. Aquac. Nutr. 12, 117–125. Castell, J.D., Mason, E.G., Covey, J.F., 1975. Cholesterol requirements in the juvenile lobster Homarus americanus. J. Fish. Res. Board Can. 32, 1431–1435. Conklin, D.E., D'Abramo, L.R., Norman-Boudreau, K., 1983. Lobster nutrition. In: McVey, J.P., Moore, J.R. (Eds.), CRC Handbook of Mariculture Vol. 1, Crustacean Aquaculture. CRC Press Inc., Boca Raton, USA, pp. 413–423. Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P., 1997. Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155, 149–164. D'Abramo, L.R., Bordner, C.E., Conklin, D.E., Baum, N.A., 1984. Sterol requirement of juvenile lobsters, Homarus spp. Aquaculture 42, 13–25. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem. 226, 497–509. Glencross, B., Smith, M., Curnow, J., Smith, D., Williams, K., 2001. The dietary protein and lipid requirements of post-puerulus western rock lobster, Panulirus cygnus. Aquaculture 199, 119–129. Gong, H., Lawrence, A.L., Jiang, D.H., Castille, F.L., Gatlin III, 2000. Lipid nutrition of juvenile Litopneaus vannamei: 1. Dietary cholesterol and de-oiled soy lecithin requirements and their interaction. Aquaculture 190, 305–324. Holme, M.H., Zeng, C., Southgate, P.C., 2006. The effect of supplemental dietary cholesterol on growth, development and survival of mud crab, Scylla serrata, megalopa fed semi-purified diets. Aquaculture 261, 1328–1334. Holme, M.H., Zeng, C., Southgate, P.C., 2009. A review of recent progress toward development of a formulated microbound diet for mud crab, Scylla serrata, larvae and their nutritional requirements. Aquaculture 286, 165–175. Irvin, S.J., Williams, K.C., 2007. Apparent digestibility of selected marine and terrestrial ingredients for the tropical spiny lobster Panulirus ornatus. Aquaculture 269, 456–463. Irvin, S.J., Williams, K.C., 2009. Comparison of the growth and survival of Panulirus ornatus seed lobsters held in individual or communal cages. In: Williams, K.C. (Ed.), Spiny lobster aquaculture in the Asia-Pacific Region. : ACIAR Proceedings No. 132. Australian Centre for International Agricultural Research, Canberra, Australia, pp. 89–96.
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Johnston, D.J., Calvert, K.A., Crear, B.J., Carter, C.G., 2003. Dietary carbohydrate/lipid ratios and nutritional condition in juvenile southern rock lobster, Jasus edwardsii. Aquaculture 220, 667–682. Johnston, D., Melville-Smith, R., Hendriks, B., Maguire, G.B., Phillips, B., 2006. Stocking density and shelter type for the optimal growth and survival of western rock lobster Panulirus cygnus (George). Aquaculture 260, 114–127. Kean, J.C., Castell, J.D., Boghen, A.G., D'Abramo, L.R., Conklin, D.E., 1985. A re-evaluation of the lecithin and cholesterol requirements of juvenile lobster (Homarus americanus) using crab protein-based diets. Aquaculture 47, 143–149. Lester, R., Carey, M.C., Little, J.M., Cooperatein, L.A., Dowd, S.R., 1975. Crustacean intestinal detergent promotes sterol solubilisation. Science 26, 1098–1100. Kovacs, M.P., Anderson, W.E., Ackman, R.G., 1979. A simple method for the determination of cholesterol and some plant sterols in fishery-based food products. J. Food Sci. 44, 1299–1305. Murphy, H.J., Mooney, G.D., Mann, N.J., Nichols, P.D., Sinclair, A.J., 2002. Lipid, FA and sterol composition of New Zealend green lipped mussel (Perna canaliculus) and Tasmanian blue mussel (Mytilus edulis). Lipids 37, 587–595. Nichols, P.D., Nichols, D.S., Bakes, M.J., 1994. Developments with marine oil products in Australia. AOCS Int. News on Fats. AOCS Int. News Fats, Oils Relat. Mater. 5, 254–261. Sheen, S.-S., 2000. Dietary cholesterol requirement of juvenile mud crab Scylla serrata. Aquaculture 189, 277–285.
Smith, D.M., Tabrett, S.J., Barclay, M.C., 2003a. Cholesterol requirements of subadult black tiger shrimp Penaeus monodon (Fabricius). Aquac. Res. 32, 399–405. Smith, D.M., Williams, K.C., Irvin, S., Barclay, M., Tabrett, S., 2003b. Development of a pelleted feed for juvenile tropical spiny lobster (Panulirus ornatus): response to dietary protein and lipid. Aquac. Nutr. 9, 231–237. Smith, D.M., Williams, K.C., Irvin, S.J., 2005. Response of the tropical spiny lobster Panulirus ornatus to protein content of pelleted feed and to a diet of mussel flesh. Aquac. Nutr. 11, 209–217. Snedecor, G.W., Cochran, G.W., 1989. Statistical Methods, 8th edn. Iowa State University Press, Ames, IA, USA. 503 p. Teshima, S.-I., 1997. Phospholipids and sterols. In: D'Abramo, L.R., Conklin, D.E., Akiyama, D.M. (Eds.), Crustacean nutrition, advances in world aquaculture. World Aquaculture Society, 6. Baton Rouge, LA, USA, pp. 85–107. Thuy, N.B.T., Ngoc, N.B., 2004. Current status and exploitation of wild spiny lobsters in Vietnamese waters. In: Williams, K.C. (Ed.), Spiny Lobster ecology and exploitation in the South China Sea Region. ACIAR No. 120. Australian Centre for International Agricultural Research, Canberra, Australia, pp. 13–16. Williams, K.C., 2004. Spiny lobster ecology and exploitation in the South China Sea Region. Spiny Lobster Ecology and Exploitation in the South China Sea Region. ACIAR No. 120. Australian Centre for International Agricultural Research, Canberra, Australia. 73pp.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Do formulated feeds for juvenile Panulirus ornatus lobsters require dietary cholesterol supplementation? Simon J. Irvin ⁎, Kevin C. Williams, Margaret C. Barclay, Simon J. Tabrett CSIRO Marine and Atmospheric Research, PO Box 120, Cleveland Qld. 4163, Australia
a r t i c l e
i n f o
Article history: Received 19 April 2010 Received in revised form 16 July 2010 Accepted 23 July 2010 Keywords: Phospholipids Feeds Crustacean Micro-nutrient deficiency
a b s t r a c t Mortality rates of wild spiny lobster seed Panulirus ornatus are typically high, with losses of 40–60% occurring within 30 days of capture. Mortality appears symptomatic of a micro-nutrient deficiency or captivity related stressors. Cholesterol is a micro-nutrient essential for healthy ecdysis, growth and survival in crustaceans. This paper reports a 7-week study examining the growth, survival and tissue cholesterol responses of juvenile P. ornatus lobsters fed a low cholesterol basal diet that was serially supplemented with cholesterol at 0.75 g kg− 1 increments from 1.25 to 4.25 g kg− 1 dry matter (DM). The basal diet contained 45 g kg− 1 phospholipid (from natural ingredients and supplemented soybean lecithin) and this was fed to all lobsters for 2 weeks prior to commencement of the experiment to reduce cholesterol reserves in the lobsters. A sixth diet comprising equal amounts of green-lipped mussel (Perna canaliculus) and whiting fillet (Silago ciliate) was included in the treatment array as a reference diet. One hundred and fifty lobsters were blocked by initial weight into five groups and then from within these groups they were equally distributed to 30 tanks (n = 5 tanks per treatment). There was no significant difference in survival, daily growth coefficient or tissue dry matter cholesterol content between lobsters fed the lowest and highest cholesterol diets: 55%, 349%, 243 mg 100 g− 1 and 70%, 456%, 253 mg 100 g− 1 respectively. However, there was a trend (P = 0.065) for lobster percentage weight gain to increase linearly with dietary cholesterol. There was a significant relationship between the starting weight of the lobsters and subsequent survival, with only 43% of the smallest lobsters (0.2 to 0.5 g block) surviving compared to 90% for the largest lobsters (2.71 to 3.40 g block). No specific dietary cholesterol requirement for small P. ornatus was determined. Feeds which contain practical marine proteins require no supplementary cholesterol, a significant cost saving in feed formulation. Feeds which contain high levels of plant proteins may require supplementary cholesterol to provide at least 4.0 g kg− 1 DM dietary cholesterol. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Cholesterol is an essential and expensive micro-nutrient required by crustaceans. It forms the chemical framework for many hormones, including ecdysteroids that are critical for the initiation of metamorphosis and moulting in crustaceans (Teshima, 1997). Cholesterol also has an important, though poorly understood, interrelationship with dietary phospholipids (Teshima, 1997; Coutteau et al., 1997). Phospholipids act as surfactants for efficient emulsification of ingested lipid and thus assist the uptake of sterols (and other lipids) from the gut. They are also essential for the efficient transport of cholesterol from the digestive gland to target tissues during the moulting process (Conklin et al., 1983). Thus, dietary cholesterol requirements are likely to be influenced by the amount of phospholipid in the diet. ⁎ Corresponding author. Tel.: + 61 7 3826 7353; fax: + 61 7 3826 7222. E-mail addresses: [email protected] (S.J. Irvin), [email protected] (K.C. Williams). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.07.026
Cholesterol requirements have been estimated for numerous crustacean species including early work with the American clawed lobster (Homarus americanus) and more recently with crabs and penaeid shrimp (Castell et al., 1975; Conklin et al., 1983; Coutteau et al., 1997). Castell et al. (1975) found that a dietary dry matter (DM) cholesterol content of 5 g kg− 1 resulted in the best growth and survival of juvenile H. americanus lobsters whereas growth and survival were depressed with levels of 2 g kg− 1 or 20 g kg− 1. D'Abramo et al. (1984) found that a dietary cholesterol specification of 1.2–1.9 g kg− 1 was optimal for very small homarid lobsters (4th stage juveniles), although the slow growth of those lobsters of less than 1 g gain over 120 days is enough reason to question whether this was a reliable indication of the animal's cholesterol requirement. Kean et al. (1985) found that juvenile H. americanus lobsters fed a ‘zero’ cholesterol diet died, regardless of the amount of soybean lecithin (predominantly phosphatidylcholine) in the diet. Increasing the amount of cholesterol above 2.5 g kg− 1 or varying the amount of lecithin in the diet had no significant effect on lobster growth or survival.
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Culture of the fast growing and high value tropical spiny lobster Panulirus ornatus is an established industry in Vietnam and under development elsewhere in SE Asia (Williams, 2004). Survival of captured wild seed lobsters of a size of 2 g or less is typically low, 40 to 60% but can be 0%, with these deaths typically occurring during the first 30 days of nursery culture (Thuy and Ngoc, 2004). Inadequate nutrition from a diet comprised only of trash fish is likely to have contributed to this high mortality rate. Pelleted feed development studies at CSIRO with juvenile P. ornatus have shown growth and survival to be optimal with DM dietary protein and lipid specifications of 500–550 g kg− 1 and ~ 100 g kg− 1, respectively (Smith et al., 2003b, 2005; Barclay et al., 2006). However, during that work, lobsters fed a control diet of thawed New Zealand green-lipped (Perna canaliculus) or Tasmanian blue (Mytilus edulis) mussels grew poorly and mortalities increased over time, a symptom characteristic of a micro-nutrient deficiency. The Barclay et al. (2006) study eliminated dietary astaxanthin as a possible cause of the assumed micro-nutrient deficiency in the mussels as dietary astaxanthin supplementation of up to 75 mg kg− 1 had no effect on growth or survival of juvenile P. ornatus lobsters but did significantly increase carapace colouration and tissue astaxanthin levels. Cholesterol was initially not considered to be a possible limiting micro-nutrient for juvenile spiny lobster feeds because of its high content in natural ingredients. Cholesterol was the dominant sterol in New Zealand green-lipped and Tasmanian blue mussels collected from three different settlement sites but it was higher in green-lipped (~1.7 g kg− 1 DM) than blue (~ 0.9 g kg− 1 DM) mussel (Murphy et al., 2002). In the study of Barclay et al. (2006) where the feeding of sole diets of green-lipped and blue mussels were compared, growth of juvenile P. ornatus was better when fed green-lipped than blue mussels. If the mussel syndrome observed with P. ornatus was due to a cholesterol deficiency, the study of Barclay et al. (2006) suggests that the dietary DM cholesterol requirement must be more than 1.7 g kg− 1. To test this hypothesis, an experiment was designed to examine the responses of very small P. ornatus to supplementation of a low cholesterol basal diet with graded amounts of cholesterol in the presence of an adequate dietary supply of phospholipid. This paper reports the growth, survival and tissue cholesterol responses of the lobsters to dietary cholesterol supplementation. 2. Materials and methods 2.1. Overview and experimental design The experiment was carried out to study the growth and survival response of juvenile P. ornatus to diets containing a relatively narrow range of supplementary cholesterol. A standard growth assay was conducted with six treatments and five tank (n = 5) replicates. Because of the wide weight range of the lobsters available for the study, from 0.2 to 3.4 g, selected animals were weight-blocked into five groups of ascending weight. Within these weight-stratified blocks, lobsters were equally distributed to each of the six treatments in accordance with a balanced randomised block design. 2.2. Diet formulation and manufacture The treatments involved a reference diet of mussel and fish flesh and five formulated pelleted dry feeds that provided an increase in the dietary DM cholesterol content from a base level of 1.25 g kg− 1 in 0.75 g kg− 1 increments to 4.25 g kg− 1. The reference diet had an analysed cholesterol content of 3.1 g kg− 1 DM and consisted of equal parts by weight on a DM basis of chopped fish flesh (Silago ciliata) and green-lipped mussel meat (P. canaliculus). The fresh fish flesh and mussel meat were mixed together and fed to the lobsters as the fresh product. The formulations of the dry pelleted feeds (Table 1) were identical except that the intended supplements of cholesterol were
Table 1 Formulation and chemical composition of the cholesterol series of pelleted dry feeds and a reference diet of fresh natural items. Ingredient
C1.25 C2.0
Formulation (g kg− 1 as used) Fishmeal (de-fatted)a 260 Cholesterolb 0 c Starch 7.5 d Aqualipid-70 15 e Krill meal (spray dried) 150 100 Mussel (dry equiv)f Fish flesh (dry equiv.)f 100 Wheat gluteng 80 Wheat flour 240 h Other 37.5 Green-lipped mussel (dry)i i Fish flesh (dry) Transglutaminasej 10
260 0.75 6.75 15 150 100 100 80 240 37.5
C2.75
C3.5
C4.25
260 1.5 6.0 15 150 100 100 80 240 37.5
260 2.25 5.25 15 150 100 100 80 240 37.5
260 3.0 4.5 15 150 100 100 80 240 37.5
Reference
500 500 10
10
10
10
Analysed composition Dry matter (g kg− 1) 660 650 665 665 665 200 Crude protein (g kg− 1 DM) 595 597 598 599 595 −1 Ash (g kg DM) 98 97 97 97 97 109 109 103 109 109 Total lipid (g kg− 1 DM) Cholesterol (g kg− 1 DM) 1.25 1.92 2.76 3.43 4.09 3.10 Phospholipid (g kg−1 DM )k 52 44 44 42 46 Gross energy (MJ kg−1 DM) 20.2 20.1 20.2 20.2 20.3 a Peruvian N670 g kg− 1 CP, supplied by Ridley Aquafeeds Pty Ltd., Narangba, Qld., Australia. b Cholesterol, supplied by Sigma. c Corn starch, Janbak Industries Pty Ltd., Brisbane, Qld., Australia. d Soybean lecithin, 700 g kg− 1 lipid, supplied by Janbak Industries Pty Ltd., Brisbane, Qld., Australia. e Antarctic (Inual, Santiago, Chile), supplied by Ridley Aquafeeds Pty Ltd., Narangba, Qld., Australia. f Minced flesh of green-lipped mussel (P. canaliculus) or whiting fish (S. ciliata) prepared at CSIRO and used as the fresh product but reported in the formulation on a DM basis. g Vital gluten, 760 g kg− 1 CP, supplied by Janbak Industries Pty Ltd., Brisbane, Qld., Australia. h Provided in the diet (g/kg): canola oil (Crisco) 20; carophyll pink (10% astaxanthin), 1.1; choline chloride (70%), 0.2; ethoxyquin, 0.2; vitamin premix (Williams et al., 2004), 10; heat-stable vitamin C (Stay-C), 1; and trace mineral premix (Williams et al., 2004), 5. i Chopped flesh of green-lipped mussel (P. canaliculus) or whiting fish (S. ciliata) prepared at CSIRO. j TG activa transglutaminase, supplied by Kerry Ingredients Pty. Ltd., Brisbane, Qld., Australia. k Phospholipid calculated as 75% of polar lipid content as analysed by TLC-FID Iatroscan analysis (Nichols et al., 1994).
made at the expense of equivalent amount of starch. To formulate a diet that was attractive to these small lobsters and as low as possible in cholesterol, it was necessary to de-fat the fish meal used in the diet. This involved de-fatting the fish meal with a hexane:ethanol solvent (2:1) to produce a low cholesterol protein source. Fishmeal and solvent were mixed in a beaker at a ratio of 4:1 and left for 10 min. The slurry was poured through a paper filter (Whatman No 4) to remove the solvent. The whole process was repeated. After the second extraction, the fishmeal residue was spread onto a tray and placed in a fume cupboard for 16 h. The extraction process reduced the DM cholesterol content of the fish meal from 3.72 to 1.29 g kg− 1. The DM cholesterol content of freeze-dried green-lipped mussel flesh and whiting flesh was 2.40 g kg− 1 and 3.49 g kg− 1 respectively, and when combined as the reference diet, had a DM cholesterol content of 3.1 g kg− 1. The diets were made at the CSIRO Marine Research Laboratory at Cleveland, Australia. For the formulated diets, fish flesh and greenlipped mussel meat were placed at − 20 °C until semi-frozen, then extruded through a 3 mm die plate of the meat grinder attachment of an A200 Hobart planetary dough mixer (Hobart Corporation, Troy, OH, USA) to form a homogenous mince. The dry ingredients were finely ground (b710 μm) using a mortar and pestle for small
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constituents or by hammer mill (Mikro Pulverizer, Metals Disintegration Coy, Summit, NJ, USA) for bulk ingredients. The fresh ingredients and transglutaminase binder were thoroughly mixed together using an industrial kitchen Kenwood KM800 planetary mixer (Kenwood Ltd, Havant Hants, UK) for 10 min before the cod liver oil and remaining dry ingredients were added followed by a further 10 min mixing to form a dough of approximately 40 to 50% moisture content. The dough was extruded through a 3 mm die plate of the meat grinder attachment of the A200 Hobart planetary dough mixer to form spaghetti like strands. The strands were placed in an airtight bag and set overnight in a refrigerator at 4 °C. The strands were stored at −20 °C until required for feeding. For the reference diet, equal parts (on a DM basis) of fresh finely chopped fish flesh and green-lipped mussel meat were mixed together and placed in an airtight bag and stored at − 20 °C until required for feeding. 2.3. Experiment management Lobsters used in the experiment were obtained as recently settled P. ornatus from Trinity inlet, Cairns, North Queensland (16° 55′ S, 145° 45′ E), hand collected and air freighted to CSIRO Cleveland. The experiment consisted of a 2-week body cholesterol depletion phase on the basal diet (C1.25) followed immediately with a 7-week growth assay. Prior to the experiment, lobsters were held for 3 weeks, during which time they were weaned from a diet of fish flesh and greenlipped mussel flesh to the low cholesterol base diet (C1.25). A total of 150 weight-stratified lobsters were distributed to 30 tanks arranged as five weight blocks of lobsters; each block comprised each of the six diet treatments. Each tank was stocked with five lobsters of matched weight. Tanks (120 L, circular polyethyhelene, 0.6 m diameter × 0.5 m deep) were installed within a light controlled laboratory (12:12 h dark–light cycle) and supplied with individual aeration and heated flowing seawater (0.6–1 L/min) enabling temperatures to be maintained between 28 and 30 °C. Sufficient clay bricks were placed in each tank to provide adequate and suitable shelter for all lobsters. Feed was offered three times daily nominally at 0800, 1700 and 2400 h, slightly to excess and with the major fraction provided at the 1700 h feed. All uneaten feed was removed daily by siphon cleaning and a daily record was kept of water temperature, observed moults and mortalities in each tank. Five lobsters representative of the replicate weight blocks were subjected to the same 2-week cholesterol depletion phase as for those used in the growth assay but were then sacrificed to determine the initial body cholesterol content. Lobsters were weighed at the start of the experiment, after 4 weeks and again at the conclusion of the 7-week growth experiment. With the exception of the two smallest blocks, losses due to mortality were replaced with a similarly sized tagged lobster to maintain stocking density; data from these lobsters were excluded from subsequent analyses. 2.4. Chemical analysis For the determination of whole body cholesterol content, a sample of five lobsters taken at the start of the experiment and lobsters in each tank at the end of the experiment was dried, weighed and sagitally sectioned and freeze-dried. The dried lobster biomass from each tank and samples of each diet were homogenised in a Knifetec grinder (Tekator, Sweden) so that all materials passed through a 1 mm sieve. Sub-samples of the homogenised lobster biomass and selected feed ingredients were analysed for DM and cholesterol content. The diets were analysed for DM, ash, crude protein (CP), total lipid, phospholipid, gross energy and cholesterol content by AOAC (1999) procedures (Table 1): DM from weight change following heating in a thermo-gravimetric analyser (Leco TGA-601, St Joseph, MI, USA) at 105 °C to a constant weight; ash from weight change following burning in a thermo-gravimetric analyser (Leco TGA-601)
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at 600 °C to constant weight; and CP (N × 6.25) by the Dumas combustion method, calibrated using aspartic acid. Total lipid was determined gravimetrically following a chloroform–methanol (2:1) extraction using the method of Folch et al. (1957). The oil class composition of the diet samples was determined by TLC-FID Iatroscan; polar lipid was dominant in all samples and comprised mostly phospholipids (approximately 75% of total polar lipid) since the TLC-FID Iastroscan method did not recover glycolipids (Nichols et al., 1994). Gross energy was determined by isothermal bomb calorimeter using a Leco AC200 Bomb Calorimeter (Leco Corp. St. Joseph, MI, USA). Sterols in the diet and tissue samples were extracted into hexane after direct saponification in 0.1 N KOH–ethanol (2:1) (Kovacs et al., 1979); the cholesterol content of the extracts was determined by gas chromatography using a Hewlett-Packard 5890 (Hewlett-Packard Company, PA, USA), fitted with a flame ionisation detector and a non-polar capillary column (HP-1, 50 m × 0.2 mm i.d., 0.11 μm film thickness, Hewlett-Packard). The sample was injected oncolumn at 90 °C with the oven temperature programmed to increase at 15 °C min− 1 to 250 °C followed by an increase of 4 °C min− 1 to 300 °C. The oven temperature was then held at 300 °C for a further 7 min. Sterols were identified by reference to the retention times of pure standards (Sigma) relative to that of the internal standard 5a-cholestane and quantified from their peak areas relative to that of the internal standard. 2.5. Calculations and statistical analysis Daily growth coefficient (DGC) and percentage weight gain (%WG) were derived as: 1=s We1 = s −Ws −1 DGC %d ¼ 100 d
!
and We %WG ¼ 100 Ws where We and Ws are the weights of the lobsters at the end and start of the experiment, respectively, and d is the number of days on the experiment. Statistical packages for balanced (Balf) and regression analyses (Regn) prepared by the Queensland Department of Primary Industries Biometry branch were used for all statistical analyses. Differences between treatments in final weight and DGC were examined by a oneway ANCOVA using initial weight as the covariate. Response data for % WG, survival and tissue cholesterol were arcsine transformed and examined using a one-way ANOVA in accordance with the design of the experiment. Differences between treatments were tested for significance using Fisher's protected t-test (Snedecor and Cochran, 1989) wherein differences between means were examined only when the F-test of the ANOVA or ANCOVA was significant (P b 0.05). The relationship between %WG and dietary cholesterol level was also examined using regression analysis. 3. Results The block weight survival of lobsters (irrespective of dietary cholesterol level) is shown in Table 2. There was a significant relationship between starting size and survival rate. Survival ranged from 43% for the smallest block (0.2 to 0.50 g) to 90% for the largest block (2.71 to 3.40 g). Survival in the smallest block was significantly lower than for all other size classes, with the exception of the next largest block (0.51 to 0.90 g), which had a 63% survival. Because of the poor survival of the lobsters in the smallest block, all data for this replicate were deleted from all subsequent analyses.
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Table 2 Effect of block starting size of lobsters on subsequent survival during the 7-week experiment. Block weight (g)
Survival (%)
0.20–0.50 0.51–0.90 0.91–1.80 1.81–2.70 2.71–3.40
43d 63 cd 67bc 70ab 90a
a,b,c,d
Means with a common letter do not differ (P b 0.05).
The analysed total lipid, cholesterol and gross energy of the experimental diets are shown in Table 1. Cholesterol DM content ranged from 1.25 g kg−¹ in the non supplemental cholesterol diet to 4.1 g kg− ¹ in the highest cholesterol diet. Response traits of the lobsters to dietary cholesterol content are shown in Table 3. Survival was variable, lowest for lobsters in the lowest cholesterol diet (55%) and highest (85%) for lobsters fed the diet containing 3.4 g kg− 1 cholesterol; however there were no significant differences between the treatments. There were no significant treatment differences between the final weight or DGC. Percent weight gain was variable with lobsters fed the diet containing 4.1 g kg− 1 cholesterol achieving a significantly faster growth than those on the 2.0 g kg− 1 cholesterol diet. There was a trend (P = 0.065) for %WG to increase linearly with increasing dietary cholesterol content (Fig. 1). The tissue cholesterol level in the lobsters was not significantly different between treatments, ranging from 237 mg 100 g− 1 DM for lobsters sampled at the start of the experiment to 267 mg 100 g− 1 DM for lobsters at the end of the experiment that were fed the diet containing 3.4 g kg− 1 cholesterol. 4. Discussion Dietary cholesterol level had no significant effect on survival, although there was a trend towards lower survival with the lowest cholesterol diet. Across treatments, survival averaged 72.5%, which was typical of findings with other studies with very small spiny lobsters (Smith et al., 2003b; Johnston et al., 2003; Smith et al., 2005). However, percentage survival above 75% and as high as 100% has been observed in studies with western rock lobster Panulirus cygnus juveniles of 0.5–2 g starting weight (Glencross et al., 2001; Johnston et al., 2006). Lobster starting weight, and not dietary cholesterol level, had the greatest bearing on survival; the smaller the lobster the lower the chance of survival. Survival of the smallest block (0.2 to 0.5 g) of 43% was similar to that (53%) observed in a feed comparison study with similarly sized (0.3 g) P. ornatus (Pryimabodo, B. pers. comm.). Comparatively low survival of 41% was reported by Smith et al. Table 3 Performance1 of juvenile P. ornatus fed experimental feeds at 5 different cholesterol (chol) levels or a fresh ingredient reference diet. Treatment
Initial C1.25 C2.0 C2.75 C3.5 C4.25 Reference ± SEM 1
Start weight (g)
Final weight (g)
Weight gain (%)
Survival (%)
DGC (% d− 1)
Tissue chol (mg 100 g− 1)
2.3a 2.2a 2.3a 2.2a 2.1a 2.2a 0.12a
7.6a 6.9a 8.9a 7.8a 9.1a 7.4a 1.01
349ab 324b 395ab 369ab 456a 345ab 42.0
55a 80a 70a 85a 70a 75a 10.3
1.30a 1.20a 1.49a 1.39a 1.64a 1.32a 0.15
237 243a 242a 247a 267a 253a 243a 10.2
Data from the smallest weight block (0.2–0.5 g) were excluded from the analyses because of the low survival of lobsters in that replicate. a,b,c Within columns, means with a common superscript letter do not differ (P N 0.05).
Fig. 1. Relationship (P =0.065) between dietary dry matter cholesterol content (g kg− 1; X) and lobster weight gain percent (Y).
(2005) in a protein requirement study with larger juvenile P. ornatus (2.5 g ± 0.19 g). However this was concluded to be more likely related to a nutrient deficiency resulting from the sole feeding of green-lipped mussel. The cause of the high mortality for the smallest juveniles in our study has not been identified but most likely was due to these small lobsters being most vulnerable to cannibalism although other factors such as low nutritional reserves, poor intake of the offered food, etc. cannot be excluded. Compounding identification of causation factors is the tendency for companion lobsters to rapidly consume all, or sections of recently deceased lobsters, thus making accurate recording of moult frequency with small (b5 g) spiny lobsters difficult and impractical. Survival of seed P. ornatus lobsters held in individual cages during a 60-day growth study (Irvin and Williams, 2009) was significantly higher (P b 0.05) than those held communally (89 cf 72%, respectively) but growth rate was significantly higher with the communally housed lobsters (DGC of 1.0 cf 0.7% d− 1, respectively). These observations suggest that cannibalism is an important cause of juvenile lobster mortality but other factors, possibly nutritional, are also involved. Growth was high for all treatments: There was a trend (P = 0.065) for weight gain % to increase with increasing dietary cholesterol content (Fig. 1). Whether or not this was an artefact of the high growth observed for lobsters fed the highest cholesterol diet (C-4.25) or a true biological response is difficult to know. Removal of the smallest block of lobsters from the analysis because of low survival would have considerably weakened the statistical rigor of the experiment. The DGC for lobsters fed the highest cholesterol diet of 4.1 g kg− 1 DM was higher or similar (1.64%) to small P. ornatus lobsters fed the best performing diet in previous protein and astaxanthin requirement studies (Smith et al., 2005, Barclay et al., 2006) of 1.38% and 1.59% d− 1 respectively. The lobsters in the protein requirement study of Smith et al. (2005) were fed on diets varying only in the level of conventional full-fat fish meal and casein and contained 2.7 g kg− 1 DM dietary cholesterol. Lobsters fed a similar level of dietary cholesterol in our study also exceeded that (1.48% d− 1) of the best feed in the protein requirement study. As the major protein component of the experimental feeds in this study was de-fatted fish meal, it is clear that small P. ornatus have a low requirement for cholesterol (in the presence of 45 g kg− 1 DM phospholipid). Feeds which contain conventional proteins are likely to be over specified with respect to cholesterol and additional supplementation with cholesterol therefore unnecessary. An ingredient digestibility study with sub-adult P. ornatus by Irvin and Williams (2007) reported the potential for partial replacement of marine proteins with terrestrial plant proteins. It would be with such dietary formulations that addition of supplementary cholesterol should be considered to provide not less than 4.0 g kg− 1 DM dietary cholesterol. There was no significant difference in the whole body cholesterol content of lobsters fed different levels of dietary cholesterol. The role of the depletion diet was to reduce the cholesterol reserves of the lobsters prior to the experiment start and thus accelerate any effect dietary cholesterol level would have on growth and survival. The
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tissue cholesterol of lobsters fed the cholesterol depletion diet of 1.25 g kg− 1 DM for 9 weeks (243 mg 100 g− 1 DM) was not significantly different from that of lobsters fed the highest cholesterol diet of 4.1 g kg− 1 for 7 weeks (253 mg 100 g− 1 DM) and only slightly higher than the amount of cholesterol in the initial lobsters (237 mg 100 g− 1 DM). Therefore the depletion diet, which contained no supplemental cholesterol and with a large component of the protein from de-fatted fish meal, had sufficient cholesterol to maintain tissue cholesterol levels at near maximum capacity. Considerably more work has been done with other crustaceans to elucidate their dietary cholesterol requirements. For penaeid shrimp, estimates typically range from 2.5 to 15 g kg− 1 (DM) of the diet (Teshima, 1997) while work at this lab with Penaeus monodon (Smith et al., 2003a) has shown that a dietary DM specification of 1.7 g kg− 1 cholesterol was adequate in the presence of 17 g kg− 1 dietary phospholipid. Gong et al. (2000) characterized the dietary cholesterol requirement of Litopenaeus vannamei to be 3.5 g kg− 1 in the absence of dietary phospholipid and to decrease to 1.3 to 1.4 g kg− 1 and to 0.5 g kg− 1 as the dietary phospholipid content increased from 15 to 30 g kg− 1 and to 50 g kg− 1, respectively. With juvenile mud crabs Scylla serrata, Sheen (2000) found that a dietary cholesterol level of 5.1 g kg− 1 optimized growth and survival of the crabs when they were fed a purified diet devoid of soy lecithin (but containing 60 g kg− 1 of a 2:1 cod liver oil and corn oil mixture); diets with cholesterol concentrations of b2 or N11 g kg− 1 depressed growth. However, Holme et al. (2006) was unable to determine a specific dietary cholesterol requirement for S. serrata megalopa as the low endogenous cholesterol level present in the base diet (1.4 g kg− 1 DM) was sufficient to support acceptable survival in development to the first crab stage. In contrast, a study by Sheen (2000) with juvenile mud crabs (S. serrata) reported an optimal dietary cholesterol requirement of 5.1 g kg− 1. In that study, crabs fed 5.0 g kg− 1 dietary cholesterol grew significantly faster than those fed 2.1 g kg− 1; however there was no difference in tissue cholesterol content of the crabs after 84 days. This suggests that tissue cholesterol content alone is not a good indicator of the efficiency of cholesterol usage in crustaceans. It is possible that the mud crab diets, which contained no supplemental lecithin, caused a reduced efficiency in the transport and utilisation of cholesterol. The interaction between phospholipid and cholesterol is little understood but widely reported for different crustacean species (Lester et al., 1975). More recently, a study by Holme et al. (2006) with megalopa mud crab S. serrata, found that cholesterol supplementation was only beneficial when soy lecithin (predominantly phosphatidylcholine) inclusion was 20 g kg− ¹ or less. Lecithin appears to have a sparing effect on cholesterol, resulting in a reduction in the cholesterol requirement, most likely due to an improved efficiency in the digestibility and uptake of cholesterol. In our study, all feeds contained an inclusion level of 15 g kg− 1 soy lecithin (~10 g kg− 1 phosphatidylcholine) and a total dry matter phospholipid content of 45 g kg− 1. It is reasonable thus to infer that this had a sparing effect on the cholesterol requirement, contributing to the non-determination of a specific cholesterol requirement for this species. This finding is broadly supported in a recent review of the nutrient requirements of larvae of the mud crab S. serrata (Holme et al. 2009), in which the cholesterol requirements of a variety of juvenile crustaceans were compared. The review concluded that there is an important interaction between cholesterol and phospholipid, and that the determined cholesterol requirement will depend on the phospholipid level and the presence of other dietary factors. Our study used wild caught lobsters from a wide size range and required blocking animals by weight. The majority of nutrient requirement studies reported with crustaceans use hatchery reared stock, and thus have the advantage of using animals with a narrow size range and known age, genetic and nutritional history. Currently, this is not possible with P. ornatus as economic hatchery seed production has not yet been developed as the journey from phyllosma
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to puerulus stage is long (5 to 8 or more months) and difficult. Therefore the age, health and remaining energy reserve of these settled lobsters are likely to be highly variable. This may explain why the variability within treatments was also high. Interest in commercial closure of the life cycle of P. ornatus has recently intensified with experimental numbers of puerulus being produced by a number of research and commercial operators. The production of commercial numbers of seed lobsters with a known genetic and nutritional background will allow and make necessary the evaluation or reevaluation of nutrient requirements. 5. Conclusions No specific dietary cholesterol requirement was determined from the results of this study. However, in the absence of some better definition of a dietary requirement, a dietary specification of 4.0 g kg− 1 DM cholesterol is recommended for juvenile spiny lobsters. Feeds which contain practical marine proteins are unlikely to require any supplementary cholesterol, a significant cost saving in feed formulation. Feeds which contain high levels of plant proteins may require supplementary cholesterol to provide a total dietary level of not less than 4.0 g kg− 1 DM cholesterol. Acknowledgements The Authors thank internal referees Brett Glencross and Greg Coman at CSIRO Marine and Atmospheric research for their constructive comments, which have considerably improved this paper. Many thanks also to Dr Clive Jones and his team from QDPIF in Cairns for collecting and transporting the juvenile lobsters used for the research. This work was supported by a grant from the Australian Centre for International Agricultural Research as part of an international collaborative project (FIS/2001/058) on “Sustainable tropical spiny lobster aquaculture in Vietnam and Australia. References AOAC International, 1999. Official Methods of Analysis, 16th edn. Association of Official Analytical Chemists, Arlington, VA, USA. Barclay, M.C., Irvin, S.J., Williams, K.C., Smith, D.M., 2006. Comparison of diets for the tropical lobster Panulirus ornatus: astaxanthin supplemented feeds and mussel flesh. Aquac. Nutr. 12, 117–125. Castell, J.D., Mason, E.G., Covey, J.F., 1975. Cholesterol requirements in the juvenile lobster Homarus americanus. J. Fish. Res. Board Can. 32, 1431–1435. Conklin, D.E., D'Abramo, L.R., Norman-Boudreau, K., 1983. Lobster nutrition. In: McVey, J.P., Moore, J.R. (Eds.), CRC Handbook of Mariculture Vol. 1, Crustacean Aquaculture. CRC Press Inc., Boca Raton, USA, pp. 413–423. Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P., 1997. Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155, 149–164. D'Abramo, L.R., Bordner, C.E., Conklin, D.E., Baum, N.A., 1984. Sterol requirement of juvenile lobsters, Homarus spp. Aquaculture 42, 13–25. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem. 226, 497–509. Glencross, B., Smith, M., Curnow, J., Smith, D., Williams, K., 2001. The dietary protein and lipid requirements of post-puerulus western rock lobster, Panulirus cygnus. Aquaculture 199, 119–129. Gong, H., Lawrence, A.L., Jiang, D.H., Castille, F.L., Gatlin III, 2000. Lipid nutrition of juvenile Litopneaus vannamei: 1. Dietary cholesterol and de-oiled soy lecithin requirements and their interaction. Aquaculture 190, 305–324. Holme, M.H., Zeng, C., Southgate, P.C., 2006. The effect of supplemental dietary cholesterol on growth, development and survival of mud crab, Scylla serrata, megalopa fed semi-purified diets. Aquaculture 261, 1328–1334. Holme, M.H., Zeng, C., Southgate, P.C., 2009. A review of recent progress toward development of a formulated microbound diet for mud crab, Scylla serrata, larvae and their nutritional requirements. Aquaculture 286, 165–175. Irvin, S.J., Williams, K.C., 2007. Apparent digestibility of selected marine and terrestrial ingredients for the tropical spiny lobster Panulirus ornatus. Aquaculture 269, 456–463. Irvin, S.J., Williams, K.C., 2009. Comparison of the growth and survival of Panulirus ornatus seed lobsters held in individual or communal cages. In: Williams, K.C. (Ed.), Spiny lobster aquaculture in the Asia-Pacific Region. : ACIAR Proceedings No. 132. Australian Centre for International Agricultural Research, Canberra, Australia, pp. 89–96.
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