quaculture research, tập 42, số 11, 2011

Aquaculture Research, 2011, 42, 1577^1593 doi:10.1111/j.1365-2109.2010.02766.x REVIEW ARTICLE Marine wax ester digestion in salmonid fish: a review AndreÔ Sture Bogevik Institute of Marine Research, Matre Aquaculture Research Station, Bergen, Norway Correspondence: Present address: A S Bogevik, Institute of Marine Research, Austevoll Aquaculture Research Station, Bergen 5817, Norway Email: andreb@imr.no Abstract Alternative marine resources from lower trophic levels could partly cover the rapidly increasing needs for marine proteins and oils in the future The North Atlantic calanoid copepod, Calanus ¢nmarchicus, has a high level of lipids rich in n-3 fatty acids However, these animals have wax esters as the main lipid storage component rather than triacylglycerol (TAG) Although these esters are considered di⁄cult to digest by many ¢sh, is it well known that juvenile Atlantic salmon (Salmo salar) feed on zooplankton species It is therefore possible that the capacity to utilize these lipids should be well developed in salmonids Nonetheless, salmon hydrolyse wax esters slower than TAG and absorb fatty alcohols slower than fatty acids However, salmon have several adaptations to digest diets rich in wax esters These includes increased feed conversion, higher production of bile and higher activity of lipolytic enzymes in the midgut Atlantic salmon has been shown to feed and grow on diets with a medium amount of wax esters (30% of the lipid) with results comparable to ¢sh maintained on ¢sh oil diets Ingestion of higher level of wax esters (50% of the lipid) cause, however, poorer lipid digestibility and growth, so that optimal utilization of wax esters in Atlantic salmon is closer to 30% than 50% of the dietary lipid Keywords: Digestion, fatty acids, fatty alcohols, intestine, wax esters Introduction Aquaculture is the fastest growing food-producing sector accounting for almost 50% of global human ¢sh consumption and is perceived as having the great- r 2011 Blackwell Publishing Ltd est potential to meet the growing demand for aquatic food (FAO 2006a) World aquaculture has grown rapidly over the past 50 years from a production of o1 million tonnes in the early 1950s to more than 59 million tonnes in 2004 (FAO 2006a) The global production of salmonids (2004) is close to million tonnes (FAO 2006b) Forty per cent of the total global aquaculture production is dependent upon the use of feed either in the form of single dietary ingredients, homemade or industrially manufactured aquafeeds (FAO 2006a) Low-cost ¢sh species such as sardines, herrings or anchovies are commonly used as feed to produce higher valued species such as salmon, cod, tuna and grouper Many of these feed ¢sh are either highly exploited or over exploited (Naylor, Goldburg, Primavera, Kautsky, Beveridge, Clay, Folke, Lubchenco, Mooney & Troell 2000) Further growth in the industry is therefore dependent on the use of alternative and sustainable feed sources (Naylor et al 2000) Although terrestrial plant oils can be used as substitutes for ¢sh oil in salmonid diets, they not supply the n-3 highly unsaturated fatty acids (HUFA) normally found in ¢sh oils Plant oil-derived diets will therefore not increase the level of n-3 HUFA in salmonid £esh (Bell, McEvoy, Tocher, McGhee, Campbell & Sargent 2001; Bell, Henderson,Tocher, McGhee, Dick, Porter Smullen & Sargent 2002;Torstensen, FrÖyland & Lie 2004; Torstensen, FrƯyland, Ịrnsrud & Lie 2004; Fonseca-madrigal, Karalazos, Campbell, Bell & Tocher 2005) A dietary supply of these fatty acids is regarded as important for animals and humans to prevent fatty acid de¢ciency and development of several diseases including coronary heart diseases (Imazio, Forno, Quaglia & Trinchero 2003; Ruxton, Reed, Simpson & Millington 2004) In order to maintain a ‘healthy’ angle of marketing salmonid products, it is 1577 Wax ester digestion A S Bogevik Aquaculture Research, 2011, 42, 1577^1593 important to ¢nd alternative feed resources that would maintain a high level of marine-type HUFA to be channelled into human nutrition (Mo¡at & McGill 1993; Sargent & Tacon 1999; Pickova & MÖrkÖre 2007) Thus, other resources than terrestrial plants also have to be considered, including marine and animal byproducts At present, one alternative is to search for alternative and unexploited marine oil resources, such as harvest from lower trophic levels Krill and copepods constitute a huge biomass in the waters o¡ Norway, with an annual production in the magnitude of several hundred million tonnes (Dalpadado, Ellertsen, Melle & Skjoldal 1998; Madden, Beare, Heath, Fraser & Gallego 1999; Table 1) A harvest of only a small fraction of this, a few million tonnes, would cover the need for marine raw materials for the Norwegian ¢sh farming industry far into the future Several studies have showed that many krill species are suited as a replacement for ¢sh meal in formulated diets to salmonids (Storebakken 1988; Olsen, Suontama, Langmyhr, Mundheim, RingÖ, Melle, Malde & Hemre 2006; Suontama, Karlsen, Moren, Hemre, Melle, Langmyhr, Mundheim, RingÖ & Olsen 2007) Data on the use of zooplankton ¢sh-oil replacements in diets are, however, lacking Many species of krill (Meganyctiphanes norvegica, Euphausia superba) have lower lipid content (o40%) and are more suited as protein source, while small-sized species of krill (e.g Thysanoessa inermis) and calanoid copepods (e.g Calanus ¢nmarchicus) seem more desirable as lipid sources because they have higher levels of lipid (450%) during part of the season (Kattner & Krause 1987; S×ther & Mohr 1987; Falkpetersen, Hagen, Kattner, Clarke & Sargent 2000; Lee, Hagen & Kattner 2006) However, many of these animals, and a few other marine invertebrates and mesopelagic ¢sh species have wax esters as their main lipid storage component rather than triacylglycerols (TAG), the main storage lipid in most ¢sh species (Sargent, Lee & Nevenzel 1976; Falk-petersen, Sargent, Hopkins & Vaja 1982; Phleger, Nichols & Virtue 1997) Wax esters are more hydrophobic than TAG and poorly digested in most mammals, including humans, where the fatty alcohols are accumulated in the intestine This causes discomfort ranging from stomach cramps to rapid loose bowel movements in the form of oily diarrhoea (keriorrhoea) and absorption problems (steatorrhea) Other problems include loss of hair and skin damage (seborrhoea) (Hansen & Mead 1965; Berman, Harley & Spark 1981; Place 1992) However, in the marine environment, wax ester-rich krill and copepods are the principal food for many ¢sh species including herring, sardines, anchovies and young salmon (Place 1992) Provided that a sustainable harvesting regime is established, marine wax esters could contribute to a signi¢cant portion of the supply for marine HUFA for human consumption that would otherwise be inaccessible This review provides therefore new information in the ¢eld of using marine zooplankton with a high level of wax esters as lipid source in feeds to Atlantic salmon, as an alternative to ¢sh oils and vegetable oils Origin of wax esters Wax esters are esters of a long-chain fatty acid and a monohydric long-chain fatty alcohol (Cowey & Table Biomass and production (wet weight) of macrozooplankton standardized to an area of 3.1 million km2, corresponding to the total area of Norwegian and Barents Seas and eastern parts of the Greenland and Iceland Seas (reproduced from Suontama 2006) Species/group Biomass (mill tones) Euphausiids (krill) Euphausiids (krill) Amphipods Amphipods Calanus finmarchicus Calanus ssp Calanus ssp 91 161 201 49 22w 30–125 75 Production (mill tones) 242à 74à 88 120–500w 298w Original area (mill km2) 1.7 3.1 1.7 3.1 2.9 3.1 3.1 Source Dalpadado et al (1998) W Melle, unpubl obs Dalpadado et al., (1998) W Melle, unpubl obs Aksnes & Blindheim, (1996) Hassel & Melle, (1999) Holst, Couperus, Hammer, Jacobsen, Ja´kupsstovu, Krysov, Melle, Mork, Tangen Vilhja´lmsson and Smith (2000) ÃBased on P/B 51.5 (Sakshaug, Bjorge, Gulliksen, Loeng & Mehlum 1994) wBased on P/B (Sakshaug et al., 1994) ÃÃÃThe area represents main distributional area for the species 1578 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 Aquaculture Research, 2011, 42, 1577^1593 Wax ester digestion A S Bogevik der undesirable conditions (Nevenzel 1970; Sargent et al 1976) Figure General molecular structure of wax esters Wax esters in copepods Sargent 1977; Christie 2003; Fig 1) These are neutral lipids with low polarity and high hydrophobicity This property can be attributed to the fatty alcohol part that consists of a limiting number of chain lengths (C14 À 22) which are often saturated or monounsaturated However, the high level of unsaturated fatty acid in marine wax esters keeps it liquid at least down to 1C (Spark, 1982) Distribution and function of wax esters Phytoplankton contain little or no wax esters, so marine wax esters are primarily of animal origin Sargent et al (1976) showed the appearance of marine wax esters as a major lipid component in seven phyla, including Coelenerate, Ctenophora, Chaetognatha, Mollusca, Annelida, Arthropoda and Chordata Wax esters in copepods provide energy reserves during periods of starvation, for maintenance and reproduction (Lee & Puppione 1972; Sargent, Gatten & Henderson 1981; Falk-Petersen et al 2000; Lee et al 2006).Wax esters can also have a variety of other biological functions In vertical migrating copepods, wax esters are important constituents in osmotic regulation and buoyancy control through oxidation and biosynthesis of wax esters (Sargent et al 1976) In some myctophid ¢shes like the diurnal Cyclothone atraria, Hoplostethus atlanticus and Latimeria chalumnae, wax esters are important constituents in the swimbladder (Phleger 1998) In a few epipelagic ¢sh species (e.g Trichogaster cosby), wax esters are found in the roe as nourishment for larvae before initial feeding (Sand, Rahn & Schlenk 1973), while some deep-water ¢sh (e.g the deep-sea cod species Lotella phycis and Laemonema morosum) have been reported to have wax esters as energy stores in the muscle and other internal organs (Nevenzel 1970; Phleger et al 1997) In some whales (e.g Berardius bairdi), short chain TAG and wax esters are found in the head region and are involved in echo-location of zooplankton (Litch¢eld, Greenberg, Eaton & Ackman 1978) The high content of wax esters in blubber of whales (e.g Physeter catodon) appears to act as thermal insulation of the musculature and as energy supplies un- Copepods are the dominant zooplankton in most seas and have stores of wax esters, if present, in eggs and oil sacs (Lee et al 2006) All of the examined species belonging to the calanoid copepod families Calanidae, Euchaetidae, Lucicutiidae, Heterorabdidae and Augaptilidae have more than 20% of their lipid as wax esters and are found either in deep water or near-surface cold-temperature waters (Lee, Barnett & Hirota 1971; Sargent et al 1976) The wax esters of these marine copepods consist of fatty alcohols that are mostly saturated (14:0 and 16:0) and monounsaturated (16:1n7, 18:1n-9, 20:1n-9 and 22:1n-11) The fatty acid moieties have the same variety of chain lengths as fatty acids (C14 À 24) in TAG, with a high level of saturated (14:0 and16:0), monounsaturated (20:1n-9 and 22:1n11) and n-3 (18:4n-3, 20:5n-3 and 22:6n-3) fatty acids (Bauermeister & Sargent 1979a; Kattner 1989) The chain length of marine wax esters (total of alcohol plus acid) are thus in the range of C28 À 44, with C32, C34, C36 and C38 as the major components in deepwater zooplankton while upper-water zooplankton have C42 and C44 as additional components (Sargent et al 1976) This is assumed to be caused by lower levels of polyunsaturated fatty acid (PUFA) in their natural diets compared with photosynthetic algaes and diatoms occurring in the pelagic water column of Arctic waters (Benson, Lee & Nevenzel 1973) The copepod, C ¢nmarchicus (Fig 2), constitutes the largest zooplankton biomass in mixed polar and Atlantic waters and is distributed throughout the North Atlantic Ocean (Marshall & Orr 1955; Conover 1988) They are important prey organisms for larval stages of many commercial ¢sh stocks, including cod, haddock (Buckley & Lough 1987; Lynch, Lewis & Werner 2001), sardines, herring, anchovy (Marshall & Orr 1955; Dalpadado, Ellertsen, Melle & Dommasnes 2000), red¢sh (Runge & de Lafontain1996) and capelin (Astthorsson & Gislason 1997) Calanus ¢nmarchicus has a predominantly 1-year life cycle in the Norwegian Sea (Melle, Ellertsen & Skjoldal 2004) During development from egg to adult, C ¢nmarchicus passes through six naupliar stages and ¢ve copepodid stages (I^V) All these stages occur in the upper 100 m of the water column during spring and summer (Speirs, Gurney, Heath & Wood 2005) The copepod shows exponential r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 1579 Wax ester digestion A S Bogevik (a) Aquaculture Research, 2011, 42, 1577^1593 (b) Adult Copepodid stage Stage in development Lipid (% of dry weight) Wax esters (% of lipid) I 20 – 41 II 16 – 25 22 – 47 III – 29 28 – 69 IV 12 – 53 63 – 84 V 15 – 64 86 – 88 Female 16 – 50 69 – 81 Male 15 – 38 64 – 92 increases in total lipid and wax ester content throughout the development from nauplia to stage V copepodid (Fig 2) In general, the total lipid content increases from 8% of dry weight in copepodid I to a maximum of 64% in copepodid V, while the wax ester content increases from around 40% of lipid in copepodid I to almost 90% in copepodid V (Kattner & Krause 1987; Scott, Kwasniewski, Falk-petersen & Sargent 2000; Lee et al 2006) There is, however, some uncertainty in the data as results seem to vary with di¡erent studies (reviewed by Lee, Barnett et al 1971; Lee & Hirota 1973, Lee et al 2006) Kattner and Krause (1987) showed a variation of more than10% in the lipid level within copepodid stage between two close locations in the North Sea sampled during spring 1983^1984 This discrepancy could thus be due to sampling at di¡erent time and location or method of sampling and analysis At the end of the spring bloom, the predominant stages are copepodid IVor V (Lee, Nevenzel & Pa¡enh˛fer 1972) StageVanimals then descend to deep waters (600^1000 m) in late summer where they remain semi-torpid during the winter months (Speirs et al 2005) These individuals return to the surface as adults in the late winter season where the females spawn in conjunction with the phytoplankton bloom (Melle et al 2004) Biosynthesis of wax esters Fatty alcohols of C ¢nmarchicus are produced in two steps Firstly, fatty acids are synthesized by a de novo pathway resulting in fatty acids (Benson & Lee 1975; 1580 Figure Calanus ¢nmarchicus: (a) morphology (adapted from Sars 1903) and (b) variation in lipid and wax ester level among di¡erent stages caught in the North Atlantic (adapted from Kattner & Krause 1987 and Lee et al 2006) Bauermeister & Sargent 1979a; Sargent et al.1981) In the second step, the fatty acids are reduced to fatty alcohols with a strict preference for saturated and monounsaturated products (fatty acids) The main alcohols are therefore rich in 16:0 (10%), 20:1n-9 (23%) and 22:1n-11 (45%) (Fraser, Sargent & Gamble 1989; Kattner 1989; Albers, Kattner & Hagen 1996; Scott, Kwasniewski, Falk-petersen & Sargent 2002; Table 2) The ability to produce wax esters is unique to animals having the enzyme NADPH oxidoreductase (Sargent, Gatten & Mcintosh 1974) Although copepods can produce fatty acids for wax esters by the de novo pathway, they will also incorporate dietary fatty acids The composition will therefore vary with type of prey available at the time For example, diatoms (e.g Sceletonema costatum) and dino£agellates (e.g Gymnodinium breve) are regarded as important prey for C ¢nmarchicus (Marshall & Orr 1955) and will supply large amounts of 20:5n-3 and 22:6n-3, respectively, to be utilized as essential fattyacids and deposited in depot lipids (Lee, Nevenzel & Pa¡enh˛fer 1971; Sargent, Gatten, Corner & Kilvington 1977; Leblond & Chapman 2000; Table 2) Thus, C ¢nmarchicus feeding on these diets stores a high level of n-3 PUFA in the polar lipid fraction (60%), while saturated and monounsaturated fatty acids are more pronounced in TAG and wax esters (25^39%) (Fraser et al.1989) The synthesis of wax esters is initiated when food is in excess, and follows a seasonal pattern (Lee, Nevenzel et al 1971; Kattner 1989) The production of fatty alcohols and wax esters appears to decrease TAG synthesis (Sargent et al 1976) The fatty alcohols condense with fatty acyl CoA (dietary or endogenous r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 Aquaculture Research, 2011, 42, 1577^1593 Wax ester digestion A S Bogevik Table Fatty acid composition in two phytoplankton species (Skeletonema costatum and Gymnodinium breve) and in adult stages of Calanus ¢nmarchicus S costatumà G brevew C finmarchicusz Wax esters Saturated 14:0 16:0 18:0 Monounsaturated 18:1n-9 20:1n-9 22:1n-9 Total (n-3) 18:3n-3 18:4n-3 20:5n-3 22:6n-3 TAG FFA PL TAG PL TAG PL FA FAlc 17.5 9.8 7.7 15.9 13.5 2.4 26.7 20.4 6.3 15.0 0.5 Tr 8.9 Tr 0.1 25.2 10.1 Tr 54.0 14.5 35.6 2.7 19.4 7.8 45.0 4.2 28.2 4.6 29.4 7.5 32.9 39.6 18.8 21.4 16.5 – 32.9 Tr 4.5 35.1 Tr – 18.1 0.7 4.9 1.5 1.7 10.6 31.5 1.2 27.6 2.1 28.7 10.3 4.8 4.0 27.5 6.1 7.2 7.4 5.1 24.8 0.8 20.9 2.8 7.6 1.8 0.3 – 60.2 1.6 3.2 23.1 30.9 25.1 12.4 11.4 0.5 38.6 2.4 8.0 15.1 27.7 2.7 13.7 6.3 2.2 11.7 1.2 9.8 0.7 75.5 4.9 23.3 45.3 – – – – – 4.6 Values are given as percentage of fatty acids in TAG, free fatty acids (FFA) or phospholipids (PL) in phytoplankton and as percentage of fatty acids in TAG or PL, wax ester fatty acid (FA) or wax ester fatty alcohols (FAlc) in C ¢nmarchicus ÃLee et al (1971b) wLeblond and Chapman (2000) zFraser et al (1989) origin) by a reaction catalysed by a relatively nonspeci¢c ester synthetase to produce wax esters that could be stored (Bauermeister & Sargent 1979a; Sargent et al 1976) Calanus ¢nmarchicus in ¢sh feeds Fish oil has been the major lipid source in manufactured feed to carnivorous aquaculture The oils are normally produced from industrial ¢sh species like herring, sardine, sand-eel, menhaden, anchoveta and pout caught from ¢sheries in Northern Europe or Southern America The wax ester rich calanoid copepods are important constituents of the marine food chain in Northern Europe Depot lipids from ¢sh in this area have many similarities to what is found in C ¢nmarchicus including a high level of long-chained monounsaturated fatty acids found in the alcohol fraction of the wax esters (Ratnayake & Ackman 1979; Table 3) The coast of South America has other copepods with TAG containing fatty acids with a high level of polyunsaturated and saturated fatty acids that is re£ected in the oils from the ¢sh species in this area (Tocher 2003) Calanus Finmarchicus have traditionally been harvested by trawl and block frozen shortly after capture to prevent oxidation and loss of nutrients, which might be a problem when the animals are thawed However, new techniques in harvest and preservation are under development for production of products with minimal loss of nutrients Lipid are at the present extracted from thawed Calanus that are heated to 85^90 1C in a steam-heated vessel containing water/press liquid or in a scraped surface heat exchanger (e.g Contherm 6; Alfa Laval, RÖdovre, Denmark) Most of the liquid phase is removed in double screw press (e.g P13; Stord Bartz, Bergen, Norway) The press liquid is then heated to at least 90 1C in a scraped surface heat exchanger (e.g Contherm  6, Alfa Laval), and particulate matter is removed in a 100 mm wet sieve before the oil is extracted in an oil separator (e.g SA1; Westfalia Separator AG, Oelde, Germany) Low lipid diets are prepared by extrusion using similar standards and additives as used in commercial diets, followed by coating with the extracted Calanus oil (Olsen, Henderson, Sountama, Hemre, RingÖ, Melle & Tocher 2004; Bogevik, Tocher, Langmyhr,Waagbo & Olsen 2009) Harvests of C ¢nmarchicus could have a large variation in wax esters content of the lipid due to location of harvest, development stage and sex of the copepods as mentioned earlier Feed production is often based on replacing a certain percentage of the ¢sh oil with Calanus oil Thus, the ¢nal feed product composition could vary after the harvest product r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 1581 Wax ester digestion A S Bogevik Aquaculture Research, 2011, 42, 1577^1593 Table Neutral lipid content (% of lipid classes) and fatty acid composition (wt%) of Southern hemisphere ¢sh oil [(anchovy oil (AO)] and diets with Northern hemisphere ¢sh oil (FO) or elevated level of wax esters through inclusions of copepod oil (CO) extracted from Calanus ¢nmarchicus TAG Wax esters (WE) WE:TAG ratio Saturated 14:0 16:0 18:0 Monounsaturated 18:1n-9 20:1n-9 22:1n-9 Total (n-3) 18:3n-3 18:4n-3 20:5n-3 22:6n-3 AOà FO dietw FA FA – – 28.7 7.1 16.6 3.9 20.4 8.2 1.0 1.1 36.1 0.8 2.0 17.6 11.4 58.0 – – 20.7 4.6 13.6 1.6 46.8 8.6 10.1 14.2 26.4 2.5 4.3 7.6 10.5 Mixed dietw FA FAlc CO dietz FA 33.9 30.7 1:1 23.4 8.0 13.2 1.4 40.1 8.3 9.8 13.1 32.9 2.3 7.8 8.5 12.1 CO dietw FAlc FA 24.0 37.5 2:1 15.3 1.6 10.4 3.3 78.8 1.4 28.9 38.6 3.6 3.6 – – – 27.7 10.6 14.5 1.7 35.3 8.8 7.0 10.6 31.8 2.5 10.9 7.6 8.9 FAlc 11.2 47.7 4:1 17.2 1.7 14.6 0.9 80.1 3.8 29.2 40.3 1.3 1.3 – – – 27.3 9.8 14.6 1.3 31.8 6.7 7.6 10.2 37.5 2.7 12.9 9.2 10.8 14.1 1.3 9.9 2.9 80.3 1.4 29.4 42.0 3.4 3.4 – – – ÃHiggs,Balfry, Oakes, Rowshandeli, Skura & Deacon (2006) wBogevik et al (2009) zOlsen et al (2004) Olsen et al (2004) used feed where100% of the ¢sh oil was replaced by Calanus oil with a ¢nal product containing 37.5% wax esters of the lipid, while Bogevik et al (2009) also used feed that had a 100% replacement of the ¢sh oil resulting in ¢nal products that contained 47.7% wax esters of the lipid (Table 3) Standarization of time and location of harvest are thus important to avoid these di¡erences in the ¢nal product Fish oil diets from the Northern hemisphere have high levels of monounsaturated fatty acids (45^ 50%) These are seen in the form of fatty alcohols (80%) and to a lesser extent fatty acids (30^40%) in Calanus diets, the major being 22:1n-11 (Table 3) Furthermore, the Calanus diets have a higher level of 18:4n-3 and 20:5n-3, re£ected in a higher level of PUFA compared with the ¢sh oil diets (Table 3) Thus, the Calanus diets have su⁄cient amounts of essential fatty acids to cover requirements Although many ¢sh species have the ability to utilize dietary wax esters, the rate of intestinal hydrolysis appears to be lower than for TAG (Patton, Nevenzel & Benson 1975; Tocher & Sargent 1984) The fact that wax ester-rich animals are normal prey for wild Atlantic salmon (Salmo salar) (Rikardsen, Haugland, Bjorn, Finstad, Knudsen, Dempson, Holst, Hvidsten & Holm 2004) suggests an evolutionary adaptation to e¡ectively utilize these lipid sources 1582 The actual e⁄ciency of utilization has, however, not been the subject of extensive studies There is thus a general lack of knowledge on the utilization of wax ester-rich oils by farmed salmon Hydrolysis of wax esters in fish Bile salt-dependent lipase (BSDL) is believed to be the predominant lipase in ¢sh rather than the colipasedependent pancreatic lipase that is predominant in mammals (Patton et al 1975; Lie & Lambertsen 1985) Bogevik, Tocher, Waagbo and Olsen (2008b) showed that salmon lipases were more active in bile because desalting midgut extract reduced lipolytic activity by more than 70% Bile salt-dependent lipase is therefore most likely responsible for hydrolysis of TAG, as well as wax esters and sterol esters in salmonids The hydrolytic activity in the gut is, however, much lower for wax esters than for TAG (Patton et al 1975; Tocher & Sargent 1984; Olsen & RingÖ 1997) Consequently, TAG are hydrolysed faster than wax esters (Patton et al.1975) This is seen in both in vivo and in vitro studies, where BSDL has been reported to hydrolyze wax esters at rates of1^2 orders of magnitude slower than TAG and four- to ¢vefold slower than sterol esters in anchovy (Engraulis mordax) and rainbow trout r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 Aquaculture Research, 2011, 42, 1577^1593 (Patton et al 1975; Patton, Warner & Benson1977; Tocher & Sargent 1984) A recent study by Bogevik et al (2008b) showed that desalted midgut extract from salmon and rainbow trout hydrolysed wax esters fourfold faster than sterol esters, but approximately ¢vefold slower than TAG The higher activity against wax esters in this study could be due to di¡erent enzyme assay techniques Whether the slower hydrolysis of wax esters is due to the greater hydrophobicity of wax esters, associated with lower biliary emulsi¢cation or the speci¢city of the enzyme is still unclear Feeding higher portions of wax esters would, however, give slower release of lipolytic products than from TAG, which is more or less completely hydrolysed to fatty acids in salmon (Oxley, Bogevik, Henderson,WaagbÖ,Tocher & Olsen 2009) However, there is a di¡erence in the lipolytic activities between salmon and rainbow trout upon activation by bile In a previous study, Tocher and Sargent (1984) showed that rainbow trout intestinal lipase was inhibited at bile salts concentrations of around 10 mM (Tocher & Sargent 1984) This was also observed for rainbow trout midgut extract incubated with both TAG and sterol esters of Bogevik et al (2008b) However, this was not seen in salmon incubations Accordingly, there appear to be di¡erences in the bile salt activation of the luminal lipid enzyme between these two species The rate of hydrolysis is not only dependent on the substrate being TAG or wax esters but also on fatty acid/fatty alcohol unsaturation and chain length Analyses of wax esters in C ¢nmarchicus have shown that the long-chain monounsaturated fatty alcohols 20:1n-9 and 22:1n-11 are esteri¢ed predominantly to shorter chain fatty acids such as 14:0, whereas the medium-chain fatty alcohol 16:0 is esteri¢ed mostly to PUFA, particularly 18:4n-3 (Sargent & Henderson 1986) Several authors, reviewed by Olsen and RingÖ (1997), have stated that PUFA appear to be released at higher rates than monounsaturated followed by saturated fatty acids Furthermore, longer chain fatty acids seem to be hydrolysed at decreasing rates with increasing chain length However, Bogevik, Oxley and Olsen (2008) showed preferential hydrolysis of 16:0 compared with 20:1n-9 in ¢sh oil TAG hydrolysed by salmon lipases, indicating long-chained monounsaturated fatty acids were poor substrates for the lipase It follows that a preference of the salmon digestive lipase for ester linkages of unsaturated fatty acids would cause decreases in the proportions of 18:4n-3 and 16:0 alcohols in faecal lipid as observed in Olsen et al (2004), Bogevik et al (2009) Wax ester digestion A S Bogevik and Oxley et al (2009) At the same time, molecular species of wax esters containing 14:0 fatty acid esteri¢ed to 20:1n-9 and 22:1n-11 alcohols would be poor substrates for the lipase and would have low absorption rates In keeping with this, there was a substantial increase in the proportions of 14:0 fatty acid and 22:1n-11 alcohol in the faeces lipid relative to that of the diet (Olsen et al 2004; Bogevik et al 2009; Oxley et al 2009) Wax esters have a higher melting point than TAG and are intrinsically more hydrophobic, and therefore require proper emulsi¢cation for their hydrolysis by lipases The high dietary wax ester:TAG ratio (4:1) in diets with 100% replacement of ¢sh oil with Calanus oil (Bogevik et al 2009; Oxley et al 2009) could have limited the excess of the lipid for the lipases Comparably, 100% replacement of ¢sh oil with Calanus oil in Olsen et al (2004) resulted in a ratio of o2:1 between wax esters and TAG, due to a batch of C ¢nmarchicus with less wax esters (Table 3) This seemed su⁄cient for proper emulsion and comparable digestion to a ¢sh oil diet rich in TAG (Olsen et al 2004) Thus, the hydrophobic properties of wax esters seem to be dependent on a certain level of TAG to form an emulsion readily accessible for salmon lipases Absorption and metabolism of products from wax ester hydrolysis Luminal fatty acids are solubilized in bile to form mixed micelles before absorption (Fig 3) These are absorbed into the enterocytes mainly by passive diffusion at high luminal concentration, proceeding by carrier-mediated mechanisms, either facilitated diffusion or active transport, at low concentrations (Carlier, Bernard & Caselli 1991; Tso, Nauli & Lo 2004) Increased absorption with increased chain length and increased unsaturation (and decreased polarity) has been seen in trout enterocytes (Perez, Rodriguez & Henderson 1999; Oxley, Tocher, Torstensen & Olsen 2005) This was also observed in recent studies where absorption of 18:1n-9 was higher than 16:0 using enterocyte assay (Bogevik,Tocher,Waagbo & Olsen 2008a), and for apparent digestibility calculated from the comparison between dietary and faecal lipid content in salmon (Bogevik et al 2009; Oxley et al 2009) Wax ester-fed ¢sh seem to absorb fatty acids more readily than fatty alcohols This is supported by the elevated levels of 22:1n-11 alcohol in faeces of r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 1583 Wax ester digestion A S Bogevik Aquaculture Research, 2011, 42, 1577^1593 Lipid droplet Figure Sequential formation of mixed micelles from lipid droplets into the gastrointestinal (GI) lumen for absorption into di¡erent fates in the enterocytes Monoacylglycerols (MG), free fatty acids (FA) and free fatty alcohols (FAlc) are taken up in enterocytes from micelles in the GI lumen The biosynthesis of triacylglycerol are performed either through the 2MG-pathway or the glycerol-3-phosphate (G3P) pathway, with MG and FA as substrates respectively FAlc are converted to the corresponding FA and used in the biosynthesis of TAG See text for further explanations Modi¢ed from Rigler, Honkanen and Patton (1986) and Porter,Trevaskis and Charman (2007) herring, rainbow trout (Sargent, Mcintosh, Bauermeister & Blaxter1979), cod (Lie & Lambertsen1991) and salmon (Olsen et al 2004) fed wax esters from C ¢nmarchicus The rate-limiting step in wax ester assimilation could therefore be the absorption of free fatty alcohols, especially 22:1n-11 alcohol (Sargent et al 1979) Absorption of fatty alcohols has not been studied in detail However, the e⁄ciency of absorption seems to vary with species Rainbow trout, two-spot goby, scad and Atlantic cod fed C ¢nmarchicus have up to four times more fatty alcohols than fatty acids in their faecal fraction (Sargent et al 1979; Prahl, Eglinton, Corner & Ohara 1985; Olsen, Henderson & Pedersen 1991) The absorption appeared to be more e⁄cient in herring as less fatty alcohols than fatty acids were seen in the faecal fraction, suggesting that this species may be especially adapted to diets rich in wax esters (Sargent et al.1979) The absorption of fatty alcohols in Atlantic salmon has also been assumed to be rapid because fatty acids and fatty alcohols have been reported to be present in equal amounts in faecal materials (Olsen et al 2004) However, Bogevik et al (2008a) showed that the lipolytic products of wax esters were absorbed di¡erently in a dual-labelled fatty acid^fatty alcohol metabolism assay in sampled enterocytes For both substrates tested, (16:0 and 18:1n-9), there was a higher uptake of fatty acids than of fatty alcohols within a h incubation 1584 period Thus, passive absorption is favoured by high luminal fat concentration and decreased polarity (Tso et al 2004) The favoured absorption of fatty acids over the lesser polar fatty alcohols at low luminal fat concentration suggests that the transport is either facilitated or active, and to a less extent passive Furthermore, substrates delivered in ethanol solution in this study likely had properties that were di¡erent from fatty acids and fatty alcohols dissolved in micelles in vivo Increased fatty acid absorption could also be caused by non-luminal absorption, as fatty alcohols could exclusively be absorbed on the luminal side However, the fatty alcohol absorption was only one-third of the absorption of fatty acids indicating a highly favourable absorption of fatty acids Absorbed fatty alcohols are converted to corresponding fatty acids through two-step oxidation by fatty alcohol dehydrogenase and aldehyde dehydrogenase (Sund & Theorell 1963; Bauermeister & Sargent 1978; Bauermeister & Sargent 1979b) Normally, all fatty alcohol oxidation is assumed to occur in the intestine, but as fatty alcohols have been detected in other organs, e.g liver, these may be of importance when the intestinal metabolic capacity is overloaded (Patton & Benson 1975) Absorption of fatty alcohols appeared to be more rapid than their oxidation as they accumulated in the enterocytes before conversion (Bogevik et al 2008a) In vitro studies of entero- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 Aquaculture Research, 2011, 42, 1577^1593 cytes from salmon fed the Calanus diet tended to have a lower fatty alcohol accumulation and a larger fatty acid accumulation compared with enterocytes sampled from salmon fed the ¢sh oil diet (Bogevik et al 2008a,b; Bogevik, Oxley et al 2008) This indicates an up-regulation in the ability to oxidize fatty alcohols upon wax ester feeding Once inside the enterocytes, the metabolism of the fatty acids (including oxidized fatty alcohols) can proceed through several pathways depending on the metabolic status of the animal (Henderson 1996) In general, the bulk of absorbed fatty acids (alcohols) are esteri¢ed to TAG and incorporated into lipoproteins and thus distributed throughout the body (Hilditch, 1964; Lee & Puppione 1972; Benson et al 1973; Bauermeister & Sargent 1979b; Fig 3) Some fatty acids may be elongated/desaturated before transport but the extent of this modi¢cation is not known (Henderson 1996; Tocher 2003) However, liver TAG in salmonids have major similarities to dietary lipids indicating that these processes are not quantitatively important (Tocher 2003; Stubhaug,Tocher, Bell, Dick & Torstensen 2005) Smaller portions of fatty acids are esteri¢ed to other lipid classes or used in oxidative processes (Oxley et al 2005) The synthesis of TAG probably does not proceed through the monoacylglycerol acyltransferase pathways as digestion of wax esters will not produce 2-monoacylglycerol Rather, in these cases, TAG synthesis probably proceeds through the slower and more energetic consuming G3P pathway (Johnston 1977; Bauermeister & Sargent 1979b) Whether the small amount of glucose necessary for glycerogenesis in intestines stems from a small quantity of dietary glucose presented luminally to the intestine, through glyceroneogenesis from luminal amino acids or from blood glucose is not known However, the fatty alcohol incorporation into TAG proceeds at the same rate irrespective of whether the trout are maintained on wax ester or TAG-based diets suggesting that their capacity to metabolize fatty alcohol is not rate limiting (Bauermeister & Sargent 1979b) It was shown by Bogevik et al (2008a) that chain length and saturation (16:0 and 18:1n-9) and oxidative state (fatty acid/fatty alcohol) had only a minor in£uence on the degree of esteri¢cation to di¡erent lipid classes Both,16:0 and 18:1n-9 were to a minor extent chain-elongated/desaturated and were preferentially esteri¢ed into TAG The rate of incorporation of 18:1n-9 was higher than that of 16:0, which is in agreement with Oxley et al (2005) There was also more16:0 incorporated into phosphatidylcholine (PC) and phosphatidylserine than 18:1n- Wax ester digestion A S Bogevik A minor fraction of the fatty acids were oxidized, where dietary treatment of ¢sh oil with a higher level of monounsaturated fatty acids seem to improve oxidation of monounsaturated substrate compared with enterocytes from the Calanus oil-fed ¢sh (Bogevik et al 2008a) Factors regulating wax ester digestion in fish The release of su⁄cient amounts of bile and enzymes in the intestines of ¢sh is crucial for optimal digestion (Mankura, Kayama & Saito 1984; Tuchweber, Yousef, Ferland & Perea 1996; Tocher 2003) Bogevik et al (2009) elucidated the potential of salmon to adapt the digestive capacity following wax ester feeding One major ¢nding was that the gallbladder volume increased upon wax ester feeding (also seen in Oxley et al (2009) for salmon in freshwater and seawater), which agreed with previous reports from rainbow trout (Tocher & Sargent 1984) For both these studies was, the gallbladder volume at fasting in average 0.2 mL bile kg ¢sh À larger in salmon fed diets coated with Calanus oil compared with diets coated with ¢sh oil Salmon bile contains two major bile salts, taurocholate (420^450 mM) and taurochenodoxycholate (10^40 mM), in addition to phospholipids (mainly PC) and cholesterol (Bogevik et al 2009; Oxley et al 2009) The increased bile volume in wax ester-fed salmon was with unchanged bile composition compared with salmon fed on a ¢sh oil diet in Bogevik et al (2009), while there was a lower taurocholate concentration for the wax ester group in Oxley et al (2009) The reason for this discrepancy is unknown One possible explanation could be the slightly lower level of the bile precursor, cholesterol, in the Calanus diet in Oxley et al (2009) (6.5%) compared with in Bogevik et al (2009) (8.1%), or it is possible that the cholesterol production of bile is size-regulated as the ¢nal weight was larger in Bogevik et al (2009) (500 g) than in Oxley et al (2009) (300 g) The lipolytic activity in salmon midgut was increased upon feeding the Calanus oil-rich diet compared with a ¢sh oil diet (Bogevik et al 2009) This was especially seen by the higher hydrolysis of cholesterol esters and wax esters after h of incubation The increased enzyme activity was not due to increased bile volume as the assays were performed at a constant bile salt concentration of taurocholate In addition to regulation of bile and lipases, some birds have adapted a slower gastric emptying and re£ux of r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1577^1593 1585 Fish ontogeny in intensive or mesocosm hatchery D Zouiten et al (b) 75 Intensive Mesocosm Relative frequency (%) Relative frequency (%) (a) 90 Aquaculture Research, 2011, 42, 1723^1736 60 30 Intensive Mesocosm 50 25 24 25 Nunber of vertebrae 26 A B C D Skeletal developmental stage E Figure Relative frequency distributions of vertebrae count (a) and skeletal development stages (b) determined after double coloration (alcian blue/alizarin red) of skeletal structures, 37-day-old sea bass Dicentrarchus labrax, reared either with the intensive or the mesocosm technology (a) mm (b) mm Figure Mesocosm-reared (a) and intensive-reared (b) 37-day-old sea bass Dicentrarchus labrax, after double colouration (alcian blue/alizarin red) of skeletal structures distinguishing between ossi¢ed bones (in red) and cartilage (in blue) According to the morphological criteria described in Table 1, Fig 5(a) represents a larva at ossi¢cation stage E and Fig 5(b) represents a larva at ossi¢cation stage B soupe, Zambonino Infante & Cahu 2007) Larval growth di¡ered in the advantage of mesocosm and it can be inferred that the observed disparity ought to be e¡ectively related to the rearing technology which a¡ected larvae since early development Indeed, the di¡erence in growth was detectable as early as 16 DAH and was maintained until the end of the experiment Recently, similar results were reported for the growth of red porgy Pagrus pagrus in a study on the e¡ects of rearing techniques on larvae development (Roo, Socorro & Izquierdo 2010) However, in the present study, it is di⁄cult to determine precisely which of the variables (larval density, prey abundance, tank size, water turbidity, etc.), or their combi- 1730 nation, di¡ering between distinct experimental conditions, were causing the di¡erence in the growth observed between the treatments Prey abundance (Du¡y, Epifanio & Cope 1996; Clemmesen, Bˇhler, Carvalho, Case, Evans, Hauser, Hutchinson, Kjesbu, Mempel, Moksness, OtterÔ, Paulsen, Thorsen & SvÔsand 2003; Georgalas et al 2007), enclosure size (Clemmesen et al 2003), water turbidity and turbulence (Utne-Palm 2004) are known to a¡ect ¢sh larvae feeding and growth In both the experimental groups, trypsin and amylase secretion ratios presented patterns showing progressive increases with larval age and corresponding to the normal maturation of exocrine pancreas de- r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1723^1736 Aquaculture Research, 2011, 42, 1723^1736 Fish ontogeny in intensive or mesocosm hatchery D Zouiten et al 45 Compressed vertebra * Fusioned vertebrae * 2+ Deformities Prognathism Retracted maxillary Lordosis Scoliosis 40 Malformation rate (%) 35 30 25 20 15 10 Intensive Mesocosm Figure Incidence of the types of deformities in 37-dayold sea bass Dicentrarchus labrax larvae reared either with the intensive or the mesocosm technology Operculum deformities were not counted, ÃVertebral compression or fusion with no apparent body distortion; 21The incidence of ¢sh showing two or more deformities scribed in sea bass larvae (Zambonino Infante & Cahu 2001) Nonetheless, a higher trypsin secretion ratio was observed in DAH mesocosm-reared larvae compared with intensive-reared larvae Considering the feed distribution sequence, this di¡erence could be related to larvae feeding status at that time Indeed, DAH was before the onset of exogenous feeding in the intensive treatment, as Artemia distribution was not yet initiated, while in mesocosm enclosures, larvae were already in the presence of wild zooplankton that they consumed readily In ¢sh larvae, it was demonstrated that trypsin activity responds to both dietary rations and contents (Zambonino Infante et al 1996; PeÔres, Zambonino Infante & Cahu1998; Pedersen, Ueberschèr & Kurokawa 2003) Increased trypsin-like total proteolytic activity was also reported by Kolkovski (2001) in young (3^ 18 DAH) gilthead seabream larvae when visual (preys) or chemical (water from the Artemia rearing tank or even the supernatant of centrifuged liquid krill hydrolysate, containing FAA and other compounds found to be feed attractants) stimuli were introduced into the larval rearing water without any feed In mesocosm-reared larvae, a similar potential stimulation of pancreatic activity could also be assumed, especially as no di¡erence in trypsin secretion between treatments was observed, after initiation of Artemia or diet distribution The lower amylase secre- tion rate observed in 23- and 30-day-old larvae from intensive rearing indicated that pancreas maturation was less advanced in the larvae of this treatment, compared with mesocosm-reared larvae Development of intestinal structures has been described extensively in the larvae of several ¢sh species: during ontogenesis, the intestine becomes elongated, and the intestinal mucosa and fold height increase, leading to an increase in intestinal digestion and absorption area (Gatesoupe, Zambonino Infante, Cahu & Bergot 1999; Garc|Ô a Hernandez et al 2001; Ortiz-Delgado, Darias, Canỡavate, YuÔfera & Sarasquete 2003) This development is accompanied by an enhancement in enterocytes’ BBM enzymatic activities, wherein a drastic increase notably characterizes intestinal maturation (KvÔle et al 2007) In some species, including sea bass larvae (Zambonino Infante & Cahu 2001), this drastic increase in BBM enzymes is associated with a coexisting decrease in cytosolic enzyme activities of enterocytes Hence, intestinal maturation is often assessed by AP/leu^ala and AN/leu^ala ratios, which are considered to be good indicators of intestinal maturation (Zambonino Infante & Cahu 1994) The intestinal maturation indices obtained in this study were comparable with those reported in sea bass larvae by these previous authors or in other species like large yellow croaker (Pseudosciaena crocea) or thick-lipped grey mullet (Chelon labrosus) (Ma et al 2005; Zouiten et al 2008) The results revealed that the larvae from the intensive rearing acquired the adult mode of intestinal digestion between 23 and 30 DAH, which is in agreement with the normal intestinal maturation process of sea bass larvae reported by Zambonino Infante and Cahu (2001) Together with the enzymatic data, the well-developed mucosa shows that mesocosm-reared larvae have already achieved their intestinal maturation at 23 DAH, while intensivereared larvae still maintained a larval mode of digestion at this stage Histological observations of intestine as well as other digestive organs constitute a good indicator of the nutritional condition of larvae as it was shown in carp Cyprinus carpio fed experimental diets (Przybyl, Ostaszewska, Mazurkiewicz & Wegner 2006; Zhou, Zhao & Lin 2007) or sea bass subjected to partial starvation (Papadakis, Zaiss, Kyriakou, Georgiou, Divanach & Mylonas 2009) The well-developed fold observed as early as 23 DAH in mesocosm-reared larvae suggests that the phytoplankton and zooplankton naturally produced in the enclosures constitute a consistent trophic supply As usually observed in r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1723^1736 1731 Fish ontogeny in intensive or mesocosm hatchery D Zouiten et al mesocosm’s plankton assemblages under studied geo-climatic context, zooplankton was initially dominated by rotifers and then by copepods (Ben Khemis et al 2006), known as preferential preys for ¢sh larvae In addition, the phytoplankton in mesocosms may have triggered the maturation of both pancreatic and intestinal function, as it was shown in Cahu, Zambonino Infante, PeÔres, Quazuguel and Le Gall (1998), leading to a better e⁄ciency in assimilating the consumed feed in this treatment These conditions, met in the mesocosm, evidently should have allowed the larvae to attain a better nutritional status and should have contributed to enhancing their growth performance compared with larvae from the intensive treatment The di¡erence in growth between the larvae was obviously related to the di¡erence in the maturation of the digestive system Vice versa, the di¡erence in digestive maturation was evidently related to the di¡erence in growth Indeed, in ¢sh larvae, the stages of development are rather linked to larval size than larval age (Fuiman 1983; Osse, Van den Boogaart, Van Snick & Van der Sluys 1997; Vagner et al 2007) It would have been interesting to compare intensive and mesocosm larvae of similar sizes and check whether the delay in digestive maturation coincided exactly with the delay of growth observed in the intensive treatment Unfortunately, the sampling interval (about once a week) was too large and did not allow such a comparison Nevertheless, as larvae from both treatments were reared under similar thermal conditions, did not differ genetically, did not experience any trophic restrictions and received the same diets, the results of the present study pointed out overall that growth was faster and maturation of the digestive system occurred beforehand in mesocosm-reared larvae They also indicated that the delay in the digestive maturation of intensively reared larvae was compensated within a few days The skeleton is a complex metabolically active tissue that undergoes continuous remodelling throughout ¢sh life (Lall & Lewis-McCrea 2007) Osteological development including the formation of meristic characteristics and the emergence of major deformities are established during early larval ontogeny (Andrades et al 1996; Koumoundouros, Divanach & Kentouri 1999; Villeneuve et al 2006) Both meristic characteristics (Fowler 1970) and skeletal deformities (Koumoundouros, Divanach & Kentouri 2001; Koumoundouros et al 2002; Boglione et al 2003; Boglione, Marino, Giganti, Longobardi, De Marzi & Cataudella 2009) are known to be a¡ected by genetic 1732 Aquaculture Research, 2011, 42, 1723^1736 and environmental factors The results on meristic characteristics indicated that in both the groups, the number of vertebrae ranged between 24 and 26, with the dominant class corresponding to 25 vertebrae This is in agreement with commonly reported meristic counts in sea bass (Bouain1977) However, the loss of a vertebra was more frequently observed in the intensively reared larvae, while the appearance of an additional vertebra was more frequently observed in the mesocosm-reared larvae This is consistent with the study of Koumoundouros et al (2001) on the meristic characteristics of young Dentex dentex reared with different techniques and compared with wild juveniles The authors reported that the meristic characteristics of the normally reared ¢sh were not di¡erentiated from those of the wild, but they presented a higher variability In contrast to the present observation, it was shown that intensi¢cation increased the number of vertebrae in red porgy (Izquierdo et al 2010) Altogether, these results contribute to support the speci¢city of ¢sh species response to some factors that may potentially affect larval development Similar to the distributions of meristic characteristics at the end of the experiment, the results on both osteological development and incidence of malformation types showed di¡erent distributions between treatments Osteological development appeared to be more advanced in mesocosm- than in intensivereared larvae This is in agreement with the results of Roo et al (2010), who reported that red porgy larvae showed patterns di¡ering in the timing of the appearance and ossi¢cation of skeletal elements The rates and severity of skeletal malformations were lesser in the mesocosm, and in addition, fusion of vertebrae in the caudal region with no apparent body distortion represented the main type of malformation in this treatment In sea bream, it has been reported recently that this type of malformation might be a result of nutritionally induced accelerated skeletogenesis (Fernandez, Hontoria, Ortiz-Delgado, Kotzamanis, EsteÔvez, Zambonino-Infante & Gisbert 2008) Overall, the di¡erence in osteological development between the two groups is in agreement with several studies on the in£uence of rearing conditions or techniques on juvenile quality (Koumoundouros et al 2001, 2002; Sfakianakis et al 2006; Boglione et al 2009; Izquierdo et al 2010; Roo et al 2010) This di¡erence in skeletogenesis might be related to the diverse conditions of each rearing technique and tank size, larval density, light, water turbidity, current velocity, preys types, concentration, etc The experimental design of the present study does not allow isolating r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1723^1736 Aquaculture Research, 2011, 42, 1723^1736 Fish ontogeny in intensive or mesocosm hatchery D Zouiten et al clearly the speci¢c causative factors and their level of contribution to explain this di¡erence However, temperature, which is known to be a major factor a¡ecting larvae osteological development (Lewis, Lall & Witten 2004; Sfakianakis et al 2006), can be excluded as it did not di¡er between treatments Similarly, genetics cannot be considered as the larvae of the present study originated from the same egg batch Nevertheless, it is suggested that feeding conditions during the ¢rst days of larvae development were among the major factors a¡ecting vertebrae count distributions Indeed, several dietary factors are known to play a key role in bone metabolism and to a¡ect skeletal development (Cahu, Zambonino Infante & Takeuchi 2003; Lall & Lewis-McCrea 2007) Villeneuve et al (2006) showed that some nutrients, like phospholipids and vitamin A, a¡ect European sea bass morphogenesis and act particularly on the number of vertebrae, during the speci¢c window of time corresponding to 8^13 DAH Early larval nutrition (4^18 DAH) was also shown to exert a strong effect on skeletogenesis and further larval performance in sea bream (FernaŁndez et al 2008) Compared with intensive hatchery-reared larvae, larvae in the mesocosm were able to feed on wild plankton besides enriched added preys; hence, it can be inferred that the nutritional contribution of mesocosm wild zooplankton had key e¡ects on larval development at densities of 0.2^0.7 prey mL À These densities were low relative to distributed reared preys but were not negligible when compared with prey densities reported for the favourable growth of ¢sh larvae of several species in the wild or in the mesocosm (Du¡y et al 1996; Clemmesen et al 2003; Ben Khemis et al 2006) The skeletal system consists of bones and cartilage and serves multiple physiological functions, of which the most important is by far to support the structural integrity of the body for normal posture, development and locomotion (Lall & Lewis-McCrea 2007) Skeletal ossi¢cation during ontogenesis is a crucial event for later proper achievement of these functions In this study, skeletal development of 37-day mesocosmreared larvae was clearly more advanced As ¢sh larvae ossi¢cation is size dependent (Vagner et al 2007; KjƯrsvik, Olsen,Wold, Hoehne-Reitan, Cahu, Rainuzzo, Olsen, Òie & Olsen 2009), it is obviously related to their larger size, evidently a consequence of a comparatively better nutritional status Conversely, the more advanced skeletal development state might have contributed to ameliorate larvae nutritional status by improving their predation e⁄ciency Indeed, the development of locomotion is crucial both for pre- dator avoidance and for food capture e⁄ciency (van Snik, van den Boogaart & Osse 1997) Intensively reared red drum (Sciaenops ocellatus) larvae have been reported to present an altered mean routine swimming speed and time to response for a visual stimulus compared with wild larvae (Smith & Fuiman 2004) Similar to meristic characteristics, nutritional factors from wild zooplankton might also have contributed to the amelioration of ossi¢cation, especially thanks to the phospholipids and the highly unsaturated fatty acid content of wild zooplankton (Ruiz, Amat & Navarro 2008; van der Meeren, Olsen, Hamre & Fyhn 2008) Indeed, these dietary compounds have been shown to exert a bene¢cial e¡ect on the ossi¢cation process in sea bass larvae (Vagner et al 2007) and in Atlantic cod (Gadus morhua) larvae as well (KjÖrsvik et al 2009) Conclusion Digestive system maturation assessed both by enzymatic activities and by the histological structure of the intestine, and skeletal development, assessed by vertebra number, ossi¢cation level and typology of deformities, are pertinent indicators of ¢sh larval development In this study, these indicators showed that development occurs at an earlier stage in mesocosm-reared larvae The delay in digestive function maturation is compensated within a few days for intensive-reared larvae Mesocosm wild zooplankton, even at a low density, have major nutritional e¡ects on larval ontogenesis, markedly on skeletogenesis and development of meristic characteristics Acknowledgements This work was supported by the research funds of the INSTM and bene¢tted from cooperation with Ifremer, supported by the Embassy of France in Tunisia Particular thanks are due to the hatchery sta¡ of the INSTM for their help, to Pr Badreddine Sriha (Laboratoire d’anatomie et de cytologie pathologiques at CHU Fahret Hached ^ Sousse) for help with histology, as well as Dr JoseÔ Zambonino-Infante and Mrs Marie Madeleine le Gall (UMR 1067 Nutrition des Poissons at Ifremer) for their advice and support on biochemical analysis References Andrades J.A., Becerra J & Fernandez-Llebrez P (1996) Skeletal deformities in larval, juvenile and adult stages of r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1723^1736 1733 Fish ontogeny in intensive or mesocosm hatchery D Zouiten et al cultured gilthead seabream (Sparus aurata L.) 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modi¢es the expression of morphogenesis genes in European sea bass (Dicentrarchus labrax) British Journal of Nutrition 95, 677^687 von Cramon N., Ling E.N., Cotter D & Wilkins N.P (2005) Determination of body shape variation in Irish hatcheryreared and wild Atlantic salmon Journal of Fish Biology 66, 1471^1482 Zambonino Infante J.L & Cahu C (1994) Development and response to a diet change of some digestive enzymes in sea bass (Dicentrarchus labrax) larvae Fish Physiology and Biochemistry 12, 399^408 Zambonino Infante J.L & Cahu C (2001) Ontogeny of gastrointestinal tract of marine ¢sh larvae Comparative Biochemistry and Physiology C 130, 477^487 Zambonino Infante J.L., Cahu C.L., PeÔres A., Quazuguel P & Le Gall M.M (1996) Sea bass (Dicentrarchus labrax) larvae fed di¡erent Artemia rations: growth, pancreas enzymatic response and development of digestive functions Aquaculture 139, 129^138 Zar J.H (1999) Biostatistical Analysis, 4th edn Prentice Hall, Englewood Cli¡s, NJ, USA Zhou X.Q., Zhao C.R & Lin Y (2007) Compare the e¡ect of diet supplementation with uncoated or coated lysine on juvenile Jian Carp (Cyprinus carpio Var Jian) Aquaculture Nutrition 13, 457^461 Zouiten D., Ben Khemis I., Besbes R & Cahu C (2008) Ontogeny of the digestive tract of thick lipped grey mullet (Chelon labrosus) larvae reared in ‘‘mesocosms’’ Aquaculture 279, 166^172 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1723^1736 Aquaculture Research, 2011, 42, 1737^1740 doi:10.1111/j.1365-2109.2010.02752.x SHORT COMMUNICATION Protocol to enrich rotifers (Brachionus plicatilis) with iodine and selenium Ana Rita A Ribeiro1,2, Laura Ribeiro1, Maria T Dinis1 & Mari Moren2 CCMAR, Centre of Marine Sciences of Algarve, University of Algarve, Faro, Portugal NIFES, National Institute of Nutrition and Seafood Research, Nordnes, Bergen, Norway Correspondence: Ana Rita A Ribeiro, CCMAR, Centre of Marine Sciences of Algarve, University of Algarve, Faro, Portugal or NIFES, National Institute of Nutrition and Seafood Research, PO Box 2029 Nordnes, N-5817 Bergen, Norway E-mail: anarita.bio@gmail.com Fish production is the fastest growing food production sector of the world (FAO 2006) Iodine (I) and selenium (Se) are both essential for thyroid hormone production and hence crucial for normal ¢sh development (Leatherland 1994; Kohrle, Jakob, Contempre & Dumont 2005) Consequently, if one of these elements is missing in the water systems, it must be added to ¢sh diets Most larval ¢sh species are dependent on live prey at ¢rst feeding (Tocher, Mourente & Sargent 1997), and rotifers are one of the most widely used live preys (Dhert, Rombaut, Suantika & Sorgeloos 2001) Compared with wild copepods, the natural prey for marine ¢sh larvae, rotifers are low in I and Se Copepods contain between 50 and 350 mg of I per kg of dry weight (dry wt.) (Moren, Opstad, Van Der Meeren & Hamre 2006; Hamre, Srivastava, Ronnestad, Mangor-Jensen & Stoss 2008), whereas the I content in rotifers ranges between 0.52 and 7.9 mg kg À dry wt (Hamre, Mollan, S×le & Erstad 2008; Hamre, Srivastava et al 2008) Se levels range between and mg kg À dry wt in copepods and between 0.08 and1.4 mg kg À dry wt in rotifers (Solbakken, Berntessen, Norberg, Pittman & Hamre 2002; Hamre, Mollan et al 2008; Hamre, Srivastava et al 2008) As stated by FAO (FAO 2005), the expansion and the pro¢tability of aquaculture of marine and ornamental species are likely to be dependent on the use of recirculation systems In these systems, the water is often treated with ozone for quality control (Rueter & Johnson 1995; Krumins, Ebeling & Wheaton 2001) However, ozonation changes the physiochemical properties of the water, i.e bio-available I is removed and this can in turn lead to the r 2011 Blackwell Publishing Ltd development of thyroid lesions, e.g goitre (Bichsel & Von Gunten1999; Sherrill,Whitaker & Wong 2004) in particular if the live prey is not enriched with I (Ribeiro, Ribeiro, S×le, Hamre, Dinis & Moren 2009) While I de¢ciency has been shown to cause thyroid lesions in Senegalese sole juveniles (Ribeiro et al 2009), a de¢ciency in Se can lead to nerve cord and liver damage in adult rainbow trout (Bell, Pirie, Adron & Cowey 1986) or lethargy; loss of appetite and reduced muscle tone in salmon fry (Poston & Combs Jr 1979) Hence, a minimum of both minerals has to be delivered in a bio-available form to the fastdeveloping larvae The aim of the present study was to establish a protocol to enrich rotifers with I and Se up to levels found in copepods, which could be easily applied to the rotifer protocols used in commercial ¢sh larvae hatcheries Experiment1aimed to increase the I concentration in rotifers and was conducted at the University of Algarve, Faro, Portugal In this experiment, the rotifers (Brachionus plicatilis) were cultured in 120 L circular tanks, at a starting density of 200 rotifers mL À They were fed micro-algae on the ¢rst day and baker’s yeast during the following days (2 days) The tanks were continuously aerated (95% saturation) with water temperature and salinity at 26 1C and 24 g L À respectively Based on the levels of potassium iodide and potassium iodate used to enrich Artemia by Moren et al (2006), it was estimated that 260 mg of sodium iodide (NaI) had to be added for each million rotifer in the enrichment tank The I salt was mixed with a commercial enrichment product (CP) (DC DHA Selco, INVE Aquaculture NV, 1737 Protocol to enrich rotifers A R A Ribeiro et al Aquaculture Research, 2011, 42, 1737^1740 Dendermonde, Belgium) to simulate the procedures run daily at ¢sh hatcheries The I enrichment tests were conducted at the same water temperature and salinity as the culture and at a rotifer density of 200^300 ind mL À and the amount of CP was 250 mg L À water Two enrichments containing NaI plus CP (experimental enrichments) were prepared as well as two containing only CP (controls) by mixing them with 0.5 L of water for in a kitchen blender Two protocols, di¡ering in time used for enrichment, were tested In protocol A, half of the enrichment was given to the rotifers (two tanks, n 2) at h, and the other half at h before sample collection In protocol B, the total amount of enrichment was given to rotifers (n 2) h before sample collection Corresponding controls without I addition were run in parallel in two tanks (n 2) Experiment aimed to increase the level of bioavailable Se in the rotifers and was conducted at NIFES in Bergen, Norway The rotifers, B plicatilis, were obtained from Sagafjord Cod Hatchery (Stord, Norway) They were cultured at a density of 500 ind mL À in 200 L tanks with continuous aeration (95% saturation) with water temperature and salinity of 25 1C and 18^22 g L À respectively Rotifers were fed micro-algae and an amount of 0.021mL million rotifers day À of ¢sh oil (Rich S.A., Rich, Greece) The Se was given to the rotifers (n 3) h before sampling, without a commercial enrichment product The Se source tested was a selenized yeast (Selplex, ParanaŁ, Brazil), where most of the Se exists in the form of seleno-methionine (Encinar, SliwkaKaszynska, Polatajko, Vacchina & Szpunar 2003) Based on the levels of sodium selenite used in a previous study (Hamre, Mollan et al., 2008), a range of Se concentrations was tested (0.01, 0.02, 0.025, 0.04 and 0.08 g million rotifers À 1) to determine the optimal level of enrichment Rotifers were enriched in 15 L tanks continuously aerated (95% saturation) with water temperature and salinity of 26 1C and 24 g L À 1, respectively, and at a density of 200 ind mL À In both experiments, all sampled rotifers were collected by ¢ltering and thoroughly washed in distilled water Excess water was removed by a hygroscopic material and the rotifers were stored in 1.5 mL tubes at À 20 1C before analysis Both I and Se levels were determined using inductively coupled plasma mass spectroscopy Samples of rotifers of 100 mg dry wt were processed for I analyses using alkaline decomposition according to the protocol described by 1738 Julshamn, Dahl and Eckho¡ (2001), and those of Se were processed using an acid decomposition as described by Julshamn, Lundebye, Heggstad, Berntssen and Boe (2004) All statistical analyses were performed with STATISTICA software (version 7, Statsoft, Tulsa, OK, USA) Data from I enrichment were subjected to one-way ANOVA to test di¡erences in I content Fisher’s LSD was used to test for signi¢cant di¡erences between group means Di¡erences were considered signi¢cant at Po0.05 Data from Se enrichment were subjected to a linear regression analysis to establish the correlation between the Se amount in the enrichment and the content in the rotifers Results from Experiment showed that rotifers were enriched with I when NaI was added to the enrichments (Table 1) I content in rotifers from control enrichments were signi¢cantly lower (Po0.01) than the levels found in rotifers from the di¡erent protocols (A and B) In addition, these values were six to seven times higher than I levels found in control rotifers from previous experiments (Lie, Haaland, Hemre, Maage, Lied, Rosenlund, Sandnes & Olsen 1997; Hamre, Mollan et al 2008); however, the di¡erences observed might be due to seasonal or species variation, or due to di¡erent methods used for cultivation of rotifers The I levels in the enriched rotifers were 15 times higher than those of the control rotifers (Table 1) Signi¢cant di¡erences in I levels were observed between rotifers from the di¡erent protocols (Po0.01) Rotifers from protocol B contained 1.3 times more I compared with rotifers from protocol A (Table 1), suggesting that when using protocol A, the I ingested by rotifers had time to be evacuated by gut evacuation, and consequently less I was retained in the rotifers In addition, this level was within the range of concentrations found in copepods (Moren et al 2006), Table Iodine (I) concentration in rotifers, Brachionus plicatilis (mg kg À dry weight Æ SD), from protocols A, B compared with control rotifers Protocol mg kg À dry wt A B Control 58 Ỉ 3.2b 74.2 Ỉ 7.9c 5.35 Ỉ 0.9a Means Æ SD (n 2) Di¡erent superscripts within the same column represent signi¢cant di¡erences among treatments r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1737^1740 Aquaculture Research, 2011, 42, 1737^1740 showing that protocol B is the best to enrich rotifers and to later feed them to ¢sh larvae Results from Experiment showed that rotifers were enriched with Se when an organic source of Se was added to the enrichment tanks The rotifer concentrations of Se ranged from 35.9 (Ỉ 0.4 SD) to104.0 (Ỉ 0.4 SD) mg kg À dry wt., when enriched with 0.01^0.08 g selenized yeast per million rotifers respectively These values were more than 10 times higher than levels found in copepods (Hamre, Srivastava et al 2008) Nevertheless, the regression analyses showed a linear (R2 50.7438) relation between Se retention in rotifers and the amount of yeast used in the enrichment (Fig 1), indicating that this was a good predictor to determine the amount of Se that should be given to rotifers The Se levels obtained were higher than what has been observed in copepods (Hamre, Srivastava et al 2008) Hence, less Se should be given to rotifers in order to achieve a copepod level According to the linear regression, an amount of 0.003 g of selenized yeast should be given per million rotifers This amount was used in a ¢sh experiment byA R A Ribeiro, L Ribeiro, Ị S×le, K Hamre, M T Dinis and M Moren (unpubl data) and the rotifers reached concentrations between and mg of Se g À dry wt This, together with the results of the current experiment, shows that rotifers can be e¡ectively enriched with high levels of Se by using an organic source of Se The yeast used in the enrichment contains (Se) in the organic form of selenomethionine (Encinar et al 2003) Organic forms of Se have been shown to be more bio-available for ¢sh Protocol to enrich rotifers A R A Ribeiro et al (Wang, Lovell & Klesius 1997), and an Se supplementation can improve ¢sh immune response (Cotter, Craig & McLean 2008), growth (Abdel-Tawwab, Mousa, & Abbass 2007) and survival (Hamre, Mollan et al 2008) Therefore, the use of this yeast in the enrichment of rotifers is bene¢cial to marine ¢sh larvae in intensive culture Unlike I, Se is known to be toxic (NRC 1993), and consequently new experiments should be conducted in order to determine the requirement level of Se in ¢sh larvae In summary, rotifers can be enriched with I and Se, by using a h enrichment protocol with sodium (I) and a selenized yeast respectively Applying the same enrichment protocol in the culture of di¡erent ¢sh species allows the standardization of enrichment protocols and simpli¢es the evaluation of the e¡ects of nutrients This type of study is therefore essential, because it is an easy and practical protocol, applicable to any hatchery Acknowledgments We are indebted to the highly skilled technicians at the Centre of Marine Sciences of Algarve and at the National Institute of Nutrition and Seafood Research This work was supported by the DIGFISH project, POCI/CVT/58790/2004 (Fundac°aìo para a CieŒncia e Tecnologia ^ FCT, Portugal) Ribeiro A R A and Ribeiro L bene¢t from grants SFRH/BD/24803/2005 and SFRH/BPD/7148/2001 (Fundac°aìo para a CieŒncia e Tecnologia ^ FCT, Portugal) respectively References Figure Selenium (Se) concentration (mg kg À dry wt.) in rotifers Brachionus plicatilis sp as a function of level of selenized yeast given in the enrichment Five di¡erent levels of selenized yeast were tested: 0.01, 0.02, 0.025, 0.04 and 0.08 g million rotifers À 1) given to rotifers h before sample collection The regression equation was y 51530, 3x; r2 50.7438 Abdel-Tawwab M., Mousa M.A.A & Abbass F.E (2007) Growth performance and physiological response of African cat¢sh, Clarias gariepinus (B.) fed organic (Se) prior to the exposure to environmental copper toxicity Aquaculture 272, 335^345 Bell J.G., Pirie B.J.S., Adron J.W & Cowey C.B (1986) Some effects of selenium de¢ciency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri) BritishJournal of Nutrition 55, 305^311 Bichsel Y & Von Gunten U (1999) Determination of iodide and iodate by Ion chromatography with postcolumn reaction and UV/visible detection Analytical Chemistry 71, 34^38 Cotter P.A, Craig S.R & McLean E (2008) Hyperaccumulation of selenium in hybrid striped bass: a functional food for aquaculture? Aquaculture Nutrition 14, 215^222 r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1737^1740 1739 Protocol to enrich rotifers A R A Ribeiro et al Aquaculture Research, 2011, 42, 1737^1740 Dhert P., Rombaut G., Suantika G & Sorgeloos P (2001) Advancement of rotifer culture and manipulation techniques in Europe Aquaculture 200,129^146 Encinar J.R., Sliwka-Kaszynska M., Polatajko A., Vacchina V & Szpunar J (2003) Methodological advances for selenium speciation analysis in yeast Analytica Chimica Acta 500,171^183 FAO (2005) Regional Review on Aquaculture Development, Western European Region, FAO, Rome, Italy FAO (2006) The State ofWorld Fisheries and Aquaculture FAO, Rome, Italy Hamre K., Mollan T.A., S×le Ị & Erstad B (2008) Rotifers enriched with iodine and selenium increase survival in Atlantic cod (Gadus morhua) larvae Aquaculture 284, 190^195 Hamre K., Srivastava A., Ronnestad I., Mangor-Jensen A & Stoss J (2008) Several micronutrients in the rotifer Brachionus sp may not ful¢l the nutritional requirements of marine ¢sh larvae Aquaculture Nutrition 14, 51^60 Julshamn K., Dahl L & Eckho¡ K (2001) Determination of iodine in seafood by inductively coupled plasma/ mass spectrometry Journal of AOAC International 84, 1976^1982 Julshamn K., Lundebye A.K., Heggstad K., Berntssen M.H.G & Boe B (2004) Norwegian monitoring programme on the inorganic and organic contaminants in ¢sh caught in the Barents Sea, Norwegian Sea and North Sea, 1994 to 2001 Food Additives and Contaminants: Part A 21, 365^376 Kohrle J., Jakob F., Contempre B & Dumont J.E (2005) Selenium, the thyroid, and the endocrine system Endocrine Reviews 26, 944^984 Krumins V., Ebeling J & Wheaton F (2001) Part-day ozonation for nitrogen and organic carbon control in recirculating aquaculture systems Aquacultural Engineering 24, 231^241 Leatherland J.F (1994) Re£ections on the thyroidology of ¢shes from molecules to humankind Guelph Ichthyology Reviews 2, 1^67 Lie O., Haaland H., Hemre G.-I., Maage A., Lied E., Rosenlund G., Sandnes K & OlsenY (1997) Nutritional composition of rotifers following a change in diet from yeast and 1740 emulsi¢ed oil to microalgae Aquaculture International 5, 427^438 Moren M., Opstad I., Van Der Meeren T & Hamre K (2006) Iodine enrichment of Artemia and enhanced levels of iodine in Atlantic halibut larvae (Hippoglossus hippoglossus L.) fed the enriched Artemia Aquaculture Nutrition 12, 97^102 NRC (1993) Nutrient Requirements of Fish, pp.1^116 National Research Council, National Academy Press, Washington, DC, USA Poston H.A & Combs G.F Jr (1979) Interrelationships between requirements for dietary selenium, vitamin E and L-ascorbic acid byAtlantic salmon (Salmo salar) fed a semipuri¢ed diet Fish Health News 84, 6^7 Ribeiro A.R.A., Ribeiro L., S×le Ị., Hamre K., Dinis M.T & Moren M (2009) Iodine enriched rotifers and Artemia prevents goitre in Senegalese sole (Solea senegalensis) larvae reared in a recirculating system Aquaculture Nutrition (in press), doi:10.1111/j.1365-2095.2009.00740.x Rueter J & Johnson R (1995) The use of ozone to improve solids removal during disinfection Aquacultural Engineering 14,123^141 Sherrill J., Whitaker B.R & Wong G.T.F (2004) E¡ects of ozonation on the speciation of dissolved iodine in arti¢cial seawater Journal of Zoo andWildlife Medicine 35, 347^355 Solbakken J.S., Berntessen M.H.G., Norberg B., Pittman K & Hamre K (2002) Di¡erent iodine and thyroid hormone levels between Atlantic halibut larvae fed wild zooplankton or Artemia from ¢rst exogenous feeding until post metamorphosis Journal of Fish Biology 61,1345^1362 Tocher D.R., Mourente G & Sargent J.R (1997) The use of silages prepared from ¢sh neural tissue as enrichers for rotifers (Brachionus plicatilis) and Artemia in the nutrition of larval marine ¢sh Aquaculture 148, 213^231 Wang C., Lovell R.T & Klesius P.H (1997) Response to Edwardsiella ictaluri challenge by channel Cat¢sh fed organic and inorganic sources of selenium Journal of Aquatic Animal Health 9, 172^179 Keywords: rotifers, enrichment, iodine, selenium r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1737^1740 Aquaculture Research, 2011, 42, 1741^1744 doi:10.1111/j.1365-2109.2010.02763.x SHORT COMMUNICATION Triploid induction of black tiger shrimp, Penaeus monodon (Fabricius) using cold shock Andrew T Wood, Gregory J Coman, Andrew R Foote & Melony J Sellars CSIRO Food Futures National Research Flagship, CSIRO Marine and Atmospheric Research, Cleveland, Qld, Australia Correspondence: A T Wood, CSIRO Food Futures National Research Flagship, CSIRO Marine and Atmospheric Research, PO Box 120, Cleveland, Qld 4163, Australia E-mail: andrew.wood@csiro.au Introduction Recent successes in commercial scale domestication and selective breeding programmes of Penaeus monodon have created a high-value genetic resource due to their improved growth and survival over progeny of wild broodstock, increasing overall harvest yield (Preston, Coman, Sellars, Cowley, Dixon, Li & Murphy 2009) The increased harvest yield of these stocks has created a demand within the Australian shrimp farming industry for access to elite domesticated seed stock for commercial grow-out Prevention of unlicensed breeding is a high priority for these producers in order to protect the investment in the creation of these elite domesticated stocks The development of a commercial genetic protection strategy for P monodon, such as reproductive sterility, would be of high value to producers and could increase the availability of high performance domesticated stocks throughout the global market Such technology may also be transferable to other penaeid species Triploid induction has been shown to retard gonadal development in Fenneropenaeus chinensis and Marsupenaeus japonicus, likely resulting in reproductive sterility of these penaeid species (Li, Xiang, Zhang, Zhou, Zhang & Wu 2003; Xiang, Li, Zhang, Zhang, Yu, Zhou & Wu 2006; Sellars, Wood, Dixon, Dierens & Coman 2009) Triploid induction has also been trialled with Litopenaeus vannamei and Fenneropenaeus indicus (AQUACOP, Ledu & Diter 1993; Dumas & Ramos 1999; Garnica-rivera, Arredondo-Figueroa & de los Angeles Barriga-Sosa 2004; de Almeida Aloise, de Assis Maia-Lima, de Oliveira, de Melo Cabral & Molina 2010) Triploid shrimp have been r 2011 Blackwell Publishing Ltd induced by the inhibition of polar body (PB) extrusion during meiosis (either stopping PB or PB 2; or PB and PB extrusion) (Li, Xiang, Zhou, Wu & Zhang 2003; Li, Zhang, Yu, Liu, Zhang, Zhou & Xiang 2006; Sellars, Degnan & Preston 2006) Inductions have been successful using a variety of shock agents including temperature (heat and cold; Dumas & Ramos 1999; Li, Xiang, Zhou et al 2003; de Almeida Aloise et al 2010) and chemicals (6-dimethylaminopurine and cytochalasin B; Bao, Zhang, Wang & Dai 1994; Norris, Coman, Sellars & Preston 2005; Sellars et al 2006) There are few reports of direct comparisons of induction agents, however, it has been found that cold shock is more successful than heat shock at inducing triploids in L vannamei (de Almeida Aloise et al 2010) To date there have been no reported attempts of triploid induction in P monodon and conferred traits are unknown This paper reports on the viability of cold shock for producing triploid P monodon through assumed prevention of PB extrusion Materials and methods Wild P monodon broodstock were sourced from a population o¡ the coast of Innisfail (17153 S, 146101 0E), Queensland, Australia and transported to the CSIRO Cleveland Marine and Atmospheric Research Laboratories Broodstock were maintained in 10 000 L sand substrate tanks receiving L À of 29 Ỉ 1C seawater under a photoperiod of 14-h light:10-h dark Broodstock were fed to satiation on a diet of squid (Nototodarus sp.), green-lipped mussels (Perna sp.), ox liver and polychaetes (Diopatra sp.) twice daily Female 1741 Triploid induction of black tiger shrimp A T Wood et al Aquaculture Research, 2011, 42, 1741^1744 broodstock were unilaterally eyestalk ablated days before the commencement of spawning experiments Ovarian maturation was examined daily by shining a torch beam through the dorsal exoskeleton Ripe females (ready to spawn) (Tan-Fermin & Pudadera 1989) were selected and placed into 100 L £owthrough cylindrical spawning tanks maintained with 0.5 L À seawater at 29 ặ 1C Tanks were Âtted with an automatic spawning detection system (ASDS; Coman, Norris, Pendrey & Preston 2003) and four glass trays to collect spawned embryos (Sellars et al 2006) Chilled seawater was maintained in a 150 L water bath at a temperature below 8.5 1C A total of 71 induction treatments (excluding controls) were completed across four spawnings at temperatures ranging from 6.5 to 13.8 1C and over three treatment durations (2, and min) Detection of the spawning by the ASDS was taken as time zero The female was removed from the tank immediately post spawning and embryos were left to settle into the glass trays for between and The glass trays were then removed from the tank and the embryos concentrated by siphoning water from above settled embryos Treatment beakers (425 mL) were ¢lled with varying volumes of chilled seawater, the volume estimated so the desired shock temperature would be reached when the embryos were added A control beaker containing 27 1C seawater was also used In an aim to prevent the extrusion of PB the initial timing for induction was 30 s post spawning, this was decided as the complete extrusion of PB in P monodon occurs between 10 and 15 post spawning (Pongtippatee-taweepreda, Chavadej, Plodpai, Pratoomchart, Sobhon, Weerachatyanukul & Withyachumnarnkul 2004) The induction shock commenced at 30 s with the addition of 120 mL of embryos to beakers containing cold seawater, this was completed by post spawning The shock temperature of each treatment beaker was measured and recorded using calibrated thermometers After the desired treatment duration a 120 mL aliquot of embryos was removed from the treatment beaker and poured into a beaker containing 270 mL of 27 1C seawater to stop the cold shock, this process was also carried out for the controls The embryos were then left to hatch for 20^24 h at 28 1C Approximately 50^60 nauplii from each treatment and control were separated from unhatched embryos and sampled into microcentrifuge tubes, excess seawater was removed and remaining nauplii frozen in liquid nitrogen Samples were stored at À 80 1C until analysis Triploid rates of each sample were determined by 1742 measuring DNA content by £ow cytometry To each sample 500 mL of MPBS propidium iodide stain (MPBS: 11.0 g L À NaCl,0.2 g L À KCl,1.15 g L À Na2HPO4 containing 0.1% triton X-100, 0.2 mg mL À Rnase A, 0.02 mg mL À PI) and mL of a1:100 dilution of internal DNA standard gluteraldehyde-¢xed chicken red blood cells (Robinson 1993) was added Nauplii were aspirated using a 25 G needle and mL syringe to create a cell suspension and ¢ltered through a 63 mm mesh screen to remove debris Samples were analysed using a Cell Lab Quanta SC MPL £ow cytometer (Brea, CA, USA) The triploid rate of each sample was determined using FCS Express software (Los Angeles, CA, USA) The percentage of triploids in each sample was calculated using methods adapted from those used by Foote, Sellars, Coman and Merritt (2010) to determine tetraploid induction rates To determine a true triploid cell count the number of expected S-phase diploid cells were deducted from the number of cells in the triploid peak This was calculated by determining the average percentage of S-phase diploid cells contained in control samples and using this value to calculate the expected number of S-phase diploid cells in each treatment sample Once the true triploid cell count was determined, the proportion of triploids for each sample could be determined by dividing the triploid cell count by the triploid plus diploid cell count Initially, the e¡ects of temperature, treatment duration and spawning on triploid induction rates were analysed by multiple regression (SAS Institute Software, Cary, NC, USA, 1999) Subsequently, the relationship between treatment duration and triploid induction rates was analysed using a simple regression model for each individual spawning using data from all temperature treatments Results and discussion The success of inductions was highly variable between spawnings, however, triploid nauplii were produced at all temperatures trialled, ranging from 6.5 to 13.8 1C (Fig 1) A multiple regression analysis using values from all four spawnings determined that temperature did not have a signi¢cant impact (P40.05) on triploid induction rates within the trialled temperature range Regression analyses for individual spawnings determined that the duration of shock application had a signi¢cant impact (Po0.05) on induction rates in spawnings two, three and four, with longer shock durations producing a higher proportion of triploid nauplii Duration of shock application had no r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1741^1744 Aquaculture Research, 2011, 42, 1741^1744 Triploid induction of black tiger shrimp A T Wood et al Figure Triploid induction rates (%) of Penaeus monodon nauplii from four spawnings when subjected to di¡erent shock temperatures (1C) at durations of 2, and signi¢cant impact (P40.05) on triploid induction rates in spawning one, possibly due to the low induction rates overall within this spawning Triploid nauplii induction rates ranged from 0% to12.4% for 2-min duration, 0% to 44.5% for 4-min duration and 0% to 76.7% for 6-min duration across all four spawnings The most e¡ective individual treatment produced 76.7% triploid nauplii in spawning four at a temperature of 9.0 1C for a duration of These results demonstrate that high triploid induction rates are possible in P monodon using cold shock at the reported treatment and duration times Triploid inductions of up to100% have been reported in another tropical penaeid species, L vannamei, using a cold shock temperature of 10 1C (Dumas & Ramos 1999; de Almeida Aloise et al 2010) This induction temperature is comparable with the most successful individual induction of this study at 9.0 1C The high variability in triploid induction e⁄ciency between spawnings found in this present study has also been reported in F chinensis and M japonicus (Li, Xiang, Zhou et al 2003; Norris et al 2005) As timing of the induction shock is critical to preventing polar body extrusion, variability in optimal timing of the application of the shock likely contributes to variability in induction rates between spawnings The asynchronous spawning of embryos by penaeid shrimp contributes largely to the variability of optimal timing of induction shocks between embryos within the same spawning Broadening the duration of the induction shock provides one means to increase induction rates, through increasing the opportunity for the shock to be applied to more of the r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1741^1744 1743 Triploid induction of black tiger shrimp A T Wood et al Aquaculture Research, 2011, 42, 1741^1744 embryos from the spawning at the optimal time for induction However, this may prove detrimental as it has been reported that as the duration of treatment increases, hatch rate decreases when inducing triploid L vannamei using cold shock (de Almeida Aloise et al 2010) Other factors, such as the temperature of spawning tank water (Li, Xiang, Zhou et al 2003) and variation in identifying the time of spawning (Norris et al 2005) also impact on induction e⁄ciency, and so contribute to inter-spawning induction variability Results from the present study indicate triploid P monodon nauplii are able to be produced using cold shock; however, further research is required to evaluate and re¢ne the cold shock protocols It is also evident that to improve the reliability of triploid inductions further research is required to increase synchronicity of embryo development and improve the accuracy of spawning detection Acknowledgments We thank Michael Anderson and David Blyth for their assistance with broodstock shrimp rearing This study was supported by the Seafood Cooperative Research Centre References AQUACOP, Ledu C & Diter A (1993) Induction of polyploidy nauplii in Penaeus indicus Aquaculture 111, 315 Bao Z., Zhang Q.,Wang H & Dai J (1994) Cytochalasin B induced triploidy in Penaeus chinensis Acta Oceanologica Sinica 13, 261^267 Coman F.E., Norris B.J., Pendrey R.C & Preston N.P (2003) A simple spawning detection and alarm system for penaeid shrimp Aquaculture Research 34, 1359^1360 de Almeida Aloise D., de Assis Maia-Lima F., de Oliveira R., de Melo Cabral T & MolinaW (2010) Ploidy manipulation and polyploid detection in the white shrimp Litopenaeus vannamei (Boone 1931) (Decapoda, Penaeidae) Marine Biotechnology, doi 10.1007/s10126-010-9266-2 Dumas S & Ramos R (1999) Triploidy induction in the paci¢c white shrimp Litopenaeus vannamei (Boone) Aquaculture Research 30, 621^624 Foote A., Sellars M., Coman G & Merritt D (2010) Cytological defects during embryogenesis in heat-induced tetraploid Kuruma shrimp Penaeus japonicus Arthropod Structure and Development 39, 268^275 1744 Garnica-Rivera C., Arredondo-Figueroa J.L & de los Angeles Barriga-Sosa I (2004) Optimization of triploidy induction in the Paci¢c white shrimp, Litopenaeus vannamei Journal of Applied Aquaculture 16, 85^94 Li F., Xiang J., Zhang X., Zhou L., Zhang C & Wu C (2003) Gonad development characteristics and sex ratio in triploid Chinese shrimp (Fenneropenaeus chinensis) Marine Biotechnology 5, 528^535 Li F., Xiang J., Zhou L.,Wu C & Zhang X (2003) Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis Aquaculture 219, 221^231 Li F., Zhang C., Yu K., Liu X., Zhang X., Zhou L & Xiang J (2006) Larval metamorphosis and morphological characteristic analysis of triploid shrimp Fenneropenaeus chinensis (Osbeck, 1765) Aquaculture Research 37, 1180^1186 Norris B.J., Coman F.E., Sellars M.J & Preston N.P (2005) Triploid induction in Penaeus japonicus (Bate) with 6-dimethylaminopurine Aquaculture Research 36, 202^206 Pongtippatee-Taweepreda P., Chavadej J., Plodpai P., Pratoomchart B., Sobhon P.,Weerachatyanukul W & Withyachumnarnkul B (2004) Egg activation in the black tiger shrimp Penaeus monodon Aquaculture 234, 183^198 Preston N.P., Coman G.J., Sellars M.J., Cowley J.A., DixonT.J., LiY & Murphy B.S (2009) Advances in Penaeus monodon breeding and genetics In: The Rising Tide, Proceedings of the Special Session on Sustainable Shrimp Farming,World Aquaculture 2009 ( ed by C.L Browdy & D.E Jory), pp.1^11 The World Aquaculture Society, Baton Rouge, LA, USA Robinson J.P (ed.) (1993) Handbook of Flow Cytometry Methods.Wiley-Liss, NewYork, NY, USA Sellars M., Degnan B & Preston N (2006) Production of triploid Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) nauplii through inhibition of polar body I, or polar body I and II extrusion using 6-dimethylaminopurine Aquaculture 256, 337^345 Sellars M.J.,Wood A.T., Dixon T.J., Dierens L.M & Coman G.J (2009) A comparison of heterozygosity, sex ratio and production traits in two classes of triploid Penaeus (Marsupenaeus) japonicus (Kuruma shrimp): polar body I vs II triploids Aquaculture 296, 207^212 Tan-Fermin J & Pudadera R.A (1989) Ovarian maturation stages of the wild giant tiger prawn, Penaeus monodon Fabricius Aquaculture 77, 229^242 Xiang J., Li F., Zhang C., Zhang X., Yu K., Zhou L & Wu C (2006) Evaluation of induced triploid shrimp Penaeus (Fenneropenaeus) chinensis cultured under laboratory conditions Aquaculture 259, 108^115 Keywords: penaeid, triploid, polyploidy, Penaeus monodon, cold shock r 2011 Blackwell Publishing Ltd, Aquaculture Research, 42, 1741^1744