Aquaculture nutrition, tập 18, số 3, 2012

Aquaculture Nutrition 2012 18; 233248 doi: 10.1111/j.1365-2095.2011.00924.x 1,2 Department of Animal and Aquacultural Sciences, Aquaculture Protein Centre, CoE, Norwegian University of Life Sciences, As, Norway; Nofima, As, Norway Feed comprises the biggest cost in intensive fish farming and the quality of feed is therefore important A vast body of research has been carried out in order to investigate nutritional quality of alternative ingredients Effects of ingredients on physical quality are seldom included in these investigations Physical quality of feed varies with ingredient composition and processing condition and may interfere with feed intake, nutrient digestibility and therefore growth performance of the fish In this review, physical quality of extruded, high energy feed, and how ingredient composition and processing conditions affect the quality will be addressed Various pellet properties will be discussed and methods used to evaluate physical quality will be reviewed KEY WORDS: analytical methods, extrusion, extrusion conditions, feed ingredients, feed quality, fish feed, physical quality Received 16 May 2011; accepted 30 October 2011 Correspondence: Mette Sứrensen, Aquaculture Protein Centre, CoE, Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, As, Norway, and Nofima, As, Norway E-mail: mette sorensen@nofima.no The demand for compound aquafeeds was estimated to be 29.3 million tonnes in 2008 and is expected to grow along with increased global aquaculture production (FAO 2011) Since 1995 the compound fish feed production has grown at an average rate of 10.9 percent per year (FAO 2011) In order to relieve the pressure on fish meal in the steadily increasing aquaculture sector, alternative feed ingredients ê 2012 Blackwell Publishing Ltd from plant, microbial and other animal sources have been a prioritized field of research for many years Most studies evaluate the value of new ingredients in terms of nutritional quality with the main focus on digestibility, growth performance, health and feed intake Effects of ingredients on physical quality of the feed are, however, most often neglected in feeding experiments with fish Ingredient composition is the single most important variable affecting the physical quality of steam pelleted feeds (Behnke 1996), and is also important for the quality of extruded feeds (Refstie et al 2006; Sứrensen et al 2009, 2010, 2011; Glencross et al 2010, 2011a; Draganovic et al 2011; Kraugerud & Svihus 2011; Kraugerud et al 2011) The purpose of this review is to give an overview of some factors that influence physical quality of extruded fish feed and how physical quality of feed may affect feed intake and nutritional quality Methods used to evaluate extruded fish feed are also discussed Over the course of the past 30 years, extrusion processing has become the primary technique used for fish feed production, mainly because of the high physical and nutritional quality of the feed (Hilton et al 1981) The extrusion system consists of a barrel housing with one or two rotating screws (single or twin screw extruder) The system is also equipped with a preconditioner as well as an accompanying machine control system The preconditioner is a high speed mixing unit designed for the purpose of mixing water and steam into the blend of dry ingredients The overall goal with preconditioning is to supply the extruder barrel with an evenly moistened and preheated mix Preconditioning allows more efficient transfer of heat through friction in the extruder barrel, and also reduces the extruder barrel wear and energy consumption during feed production Moisture is necessary for gelatinization of starch and hydration of proteins During the extrusion cooking process, the blend of ingredients is turned into a melt employing a combination of high temperature (120130 C), high pressure (2030 bar) and shear forces This transformation of the ingredients is carried out in the extruder barrel The extruder barrel consists of extruder screws, heads and shear locks The screw is a long cylinder with helical flight wrapped around it Usually screw elements are mounted on a shaft with shearlocks or kneading elements mounted between the elements A huge variety of screw elements with different configurations exist, but normal practice is to configure the screw as a series of repeated conveying and mixing elements The conveying screw(s) generate the pressure necessary for the material to flow through the die, which causes the restriction in the outlet of the extruder The final dimensions of the pellets is shaped in a die, but is also affected by the energy input The two main sources of energy in an extrusion system are of mechanical or thermal origin Mechanical energy, reported as specific mechanical energy (SME), is generated by friction caused by the dough in the extruder barrel as it is moved forward by the rotating screw The SME is a product of torque and RPM divided by mass flow rate (Mercier et al 1989) Thus, heat generation is affected both by the choice of hardware and processing variables during the feed production Thermal energy, is for most expanded products added as steam in the preconditioner, in which the mash is usually heated to 8090 C in order to warm up and soften the ingredients Thermal energy is thus contributing approximately 2/3 and mechanical energy 1/3 of the energy needed to obtain an extrusion temperature of approximately 130 C The time that the feed mash is exposed to heating during preconditioning and extrusion, is normally less than five minutes The extrudate is shaped into pellets using a die at the outlet of the extruder in combination with rotating knives cutting the pellets at an appropriate length In order to increase the shelf life, the feed pellets are dried by reducing moisture content from approximately 300 g kg1 to 80 g kg1 For high energy pellets, additional oil is added using a vacuum coating system before the pellet is cooled and eventually bagged Physical quality of pelleted feeds is often defined as the ability of feed to be handled without creating an excessive amount of fines All feeds used in intensive aquaculture should be resistant to mechanical stress during transport, handling and in pneumatic feeding devices (Aarseth 2004; Aarseth et al 2006a) At the same time, the feed should have a texture and size that facilitate high feed intake (Hardy 1989) and efficient digestion by the fish (Lovell 1989; Baeverfjord et al 2006; Aas et al 2011b) Pellets that are too hard may cause digestive disturbances in the fish (Pillay & Kutty 2005) The latter authors have reported that overfeeding with hard pellets may result in swelling and rupture of the stomach This condition is associated with fermentation and gas formation in the stomach On the other hand, soft pellets or pellets with low water stability may cause oil separation in the stomach that potentially cause oil-belching in rainbow trout suffering from osmoregulatory stress, also associated with abdominal distension syndrome (Baeverfjord et al 2006; Aas et al 2011b) Bulk density of the pellet is important and can be adjusted during the production to control sinking velocity and buoyancy control Water stability and sinking velocity also have to be adjusted to the eating habits of the cultured fish species Some species are fed at the surface with floating feed, others are fed in the water column with use of slow sinking feed and some aquatic animals are bottom feeders Slow eating aquatic species such as shrimp and sea urchin need feeds that are water stable for hours without leaching nutrients Feed pellets used in the grow-out phase for Atlantic salmon may contain up to 40% oil These feeds should therefore be able to absorb and retain fat inside the pellet The different forces acting on pellets during conveying, handling and storage are usually defined as impact, compression and shear (Winowiski 1995) Impact forces shatter the pellet surface and any natural cleavage planes in the pellet Compression forces crush the pellet and also cause failure along cleavage planes Shear forces cause abrasion of the edges and surface of the pellet (Winowiski 1995) Several methods exist to determine the physical quality of pellets, however, many of the methods used for terrestrial animal feeds are not appropriate for fish feed Common methods used to analyze physical quality of extruded fish feed will be presented herein, whereas in depth overview of methods used in the feed and briquetting industry is given by other authors (Winowiski 1995; Thomas & Van der Poel 1996; Kaliyan & Morey 2009) Hardness or strength at rupture, defined as the maximum force needed to crush a pellet, is commonly determined Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd using a texture analyser (Thomas & Van der Poel 1996; Glencross et al 2010) This method assesses resistance to breaking when pellets are exposed to external pressure, and can be used to mimic the force on pellets during storage in bins or silos, crushing of pellets in a screw conveyor, and crushing of feed pellets between animal teeth (Kaliyan & Morey 2009) Despite the reliability of the test, there is a lack of standardization for analyzing the texture of feed pellets Most reports are of analysis of laying pellets, however, if the test is carried out with standing pellets the force needed to crush the pellet will increase (Sứrensen 2011, unpublished results) Results are therefore often not comparable The result of the test may also vary with the various probes and attachments used Hardness may be reported as shear force if a knife is used (Adamidou et al 2009; Glencross et al 2010) or pellet hardness if a flat ended probe is used (Aarseth et al 2006b; ỉverland et al 2009; Sứrensen et al 2009, 2010, 2011; Glencross et al 2010) Hardness of the pellet varies with degree of expansion, ingredients and processing conditions (Table 1; Figs & 2) Durability is the amount of fines produced from a sample of pellets after being subjected to mechanical or pneumatic agitation (Thomas & Van der Poel 1996; Kaliyan & Morey 2009) Pellet durability simulates forces on pellets taking place during filling of bins, during transportation from the feed factory to the farm, and during distribution in the feeding system at farms (Thomas & Van der Poel 1996; Sứrensen et al 2009) Pellets with high durability form fewer small particles and fines during bagging and storage and finally, show low degradation in pneumatic feeding devices when fed to fish (Aarseth et al 2006a; Sứrensen et al 2009; Aas et al 2011a) Different devices have been developed to assess durability of pellets, however, most of these cannot be used on oil-coated high energy extruded feed The tumbling box method is an accepted standard in the feed industry in North America (ASAE 1997) and is used to simulate formation of fines during mechanical handling This method uses 500 g of sifted pellets The pellets are placed in a box that revolves for a period of 10 at a speed of 100 rpm After testing, the pellets are screened on a mechanical sieve shaker with a sieve size of about 0.8 times the pellet diameter The tumbling box pellet durability index (PDI) is calculated as the mass of the pellets retained on the screen divided by the total mass of pellets Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd The Holmen durability tester has been developed to test effects of impact and shear forces during pneumatic conveying and is commonly used in Europe A sample size of 100 g of sifted pellets is conveyed with high air velocity through a tube with right angled bends for 30120 s, simulating the feed handling process of pellets subjected to impact and shear forces Fracture occurs when pellets strike the rightangle corners of the tester The Holmen PDI is calculated using the same procedure as for the tumbling box The LignoTester is another testing device used to simulate degradation caused by shear forces during pneumatic conveying This procedure uses a sample of 100 g of sifted pellets and blows them around a perforated chamber for 30 s Fines are removed as they are formed and pellets come out at the end of the cycle The remaining pellets are used to calculate the Ligno PDI The Norwegian fish feed industry has developed a new device, the DORIS Tester, to simulate the stresses that pellets are exposed to in pneumatic feeding devices This testing device is made of an Archimedes screw that feeds pellets into a vane and is simulating degradation by impact and shear (Aas et al 2011a) A sample size of 350 g is used in the procedure and fines, small fractures and whole pellets are screened and separated on a mechanical sieve shaker with a rack of sieves (Aas et al 2011a) The greatest limitation of the Holmen durability tester and Ligno tester when used on high energy salmon diets is leaking of oil as the pellets are circulated with the air Because of fat leaking, the collected feed samples will have an erroneously low weight Besides, oil in the testing devices will hamper proper collection of fine particles Holmen durability measurements on extruded fish feed are therefore most often reported for uncoated feed (Sứrensen et al 2009, 2010, 2011) The tumbling box durability reveals little differences in durability on extruded feed, even with the adjusted procedure adding hexagonal nuts to increase agitation (Table 1; Sứrensen et al 2010) Durability of uncoated pellets measured with the Holmen test, and use of the compression test to measure strength at rupture, are better methods than the tumbling box to evaluate differences in physical quality in extruded fish feed (Sứrensen et al 2010) The DORIS has shown a high correlation with Holmen durability, and is therefore the most appropriate method for coated high energy pellets (Sứrensen et al 2011) Water stability of feed is an important quality trait for slow eating aquatic animals when the feed has to be soaked Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd Lupin, Belara kernel meal Lupin, Wodjil protein concentrate Lupin, Myallie protein concentrate Pea protein concentrate, 350 g kg1 CP Pea protein concentrate, 500 g kg1 CP Starch source Lupin, Myallie kernel meal Soybean meal, defatted Soybean meal, defatted Full fat soybean meal, without KDF Full fat soybean meal, with KDF White flakes Lupin, Wodjil kernel meal 6 3 300 300 300 200 200 5, 5, 360 300, 300 300, 300 290 280 300 360, 300 690 200 5 Pellet size (mm) 640 750 700, 700 Marine ingredients Fish meal Fish meal Krill meal, partial deshelled Krill meal, whole krill meal Plant ingredients Soybean meal, defatted Inclusion level (g kg1) Protein source 0 NR, 23 20 19 69 75 81 82, 52 42 76, 78 19 15 NR NR, 18 12 43 16 36, 59 31 17 30 20, 40 Hardness (N) 40 NR 24 15 Expansion ratio (%) Table Physical quality of extruded feed for salmonids produced from various ingredients 86 74 Water stability index (%) 92 11 94 90 80 Holmen (%) Durability Tumbling box (%) 97 97 26 97 99 100 88 Ligno (%) 46 22 92 64 84 DORIS (%) ỉverland et al (2009) ỉverland et al (2009) Refstie et al (2006) Refstie et al (2006) Sứrensen et al (2009) Refstie et al (2006) and Glencross et al (2011b) Refstie et al (2006) and Glencross et al (2011b) Refstie et al (2006) Morken et al (2012) Morken et al (2012) Glencross et al (2011b) Sứrensen et al (2009) and Refstie et al (2006) ỉverland et al (2009) Hansen et al (2010) Sứrensen et al (2009) and Refstie et al (2006) Hansen et al (2010) Hansen et al (2010) Reference Sứrensen et al (2010) Sứrensen et al (2011) Tumbling box (%) 99 98 99 99 Holmen (%) 50 46 70 79 44 66 23 Sinking velocity is measured by dropping pellets one by one from a height of cm above the water surface into the centre of a transparent tube with a diameter of 300 mm and a height of 200 cm (Lekang et al 1991) The tube is filled with tap water of drinking quality, or water with a predefined salinity Because temperature and salinity both are factors that interfere with sinking velocity, they should be constant during the course of the test Therefore, water with defined salinity should be left for 24 h to achieve a constant temperature prior to the test The sinking speed is measured with a stop-watch over a distance of 150 cm between two fixed points 10 and 160 cm below the water surface, respectively Single pellets are randomly selected for sinking rate measurements, and sinking velocity is recorded as cm s1 NR = Not reported 38 20 150 160 40 43 27 11 27 20 28 33 33 21 10 6 240 160 160 130 150 150 Wheat Wheat Wheat Whole field beans Potato starch Gelatinized potato starch Wheat starch Pea starch Protein source Table (Continued) Inclusion level (g kg1) Pellet size (mm) Expansion ratio (%) Hardness (N) Water stability index (%) Durability Ligno (%) 63 44 23 75 Sứrensen et al (2010) Sứrensen et al (2011) Aas et al (2011b) Aas et al (2011b) Sứrensen et al (2010) Sứrensen et al (2010) Reference DORIS (%) in water for hours with minimum leaching of nutrients For fish that feed on a slow sinking pellet, water stability may be important to mimic the degradation pattern of feed in the gastrointestinal tract A procedure to determine water stability over time was described by Baeverfjord et al (2006) In brief the latter authors used 10 g samples of pellets that were placed into circular wire netting baskets with mm mesh size and a diameter of cm The test is carried out in triplicate Baskets with feed samples were placed in 600 mL beakers and 300 mL of tap water was added The beakers were then incubated in a water bath at 23 C and subjected to 100 shakings per for 30, 60, 120 and 240 min, respectively When terminating the incubation, the baskets were gently dried with paper tissues and weighed before the baskets were placed in a heating cabinet at 105 C for 18 h After drying, the baskets were again weighed to determine the residual dry matter in each basket The Water stability was calculated as the difference in DM weight before and after incubation in water divided by DM weight of the feed before incubation Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd Bulk density is an important property that determines floatability or sinking velocity of pellets (Chevanan et al 2007, 2009), and is directly related to the degree of expansion during extrusion (Glencross et al 2011b) A floating pellet is more expanded and has a lower bulk density compared to a sinking pellet Bulk density of pellet needs to be adjusted according to feeding management practices and feeding habits of the target species, and usually a bulk density greater than 525 g L1 is needed for sinking pellets in seawater 35 g L1 (Glencross et al 2011b) Bulk density is analyzed by filling up a measuring cylinder of known volume Pellets are carefully poured into a tared cylinder until a pile of feed has developed on top A scraper is used to remove the excess feed by pulling once gently over the edge of the cylinder The content of the full cylinder is then weighed on a balance In order to standardize the procedure, pellets should be poured from a funnel, preloaded with feed, and the cylinder should not be tapped prior to weighing (Aarseth et al 2006b) Each measurement should be carried out in triplicate, and bulk density for each replicate is calculated as mass of the sample to the unit volume of the sample (g L1) Volumetric displacement methods can also be used to measure specific density of the pellets with improved accuracy (Draganovic et al 2011) Fat absorption capacity of the pellets during top dressing of oil in vacuum coating systems is another pellet property that is highly correlated with expansion ratio (Sứrensen et al 2010) Expansion of the pellets is correlated to oil absorption capacity, however, the bulk density needs to be optimized in order to ensure sinking pellets in case of salmonids Because energy content in feed is highly correlated to growth performance and feed utilization in Atlantic salmon (Hillestad & Johnsen 1994) the commercial grow out diets used in Atlantic salmon production in Norway contain up to 400 g kg1 fat A method used to analyze fat absorption capacity is described in Sứrensen et al (2011) Oil in surplus is added to the vacuum coater When all the oil is distributed onto the feed, the pressure is returned back to atmospheric pressure, and the oil is retained inside the pellets Surplus oil is removed by gently compressing the pellets between of 45 layers of oil-absorptive linings Oil absorption is determined as weight increase of sample (g) divided by initial weight of sample (g) times 100 Oil leaking can be determined as the loss of oil from pellets Leaking of oil from high energy diets is a problem because of lowered energy content and different nutritional profile Besides, an oil layer in the pipeline used for pneumatic conveying may act as a sticky layer leading to build-up of small feed particles If a layer of oil and small particles is allowed to build up, this will eventually result in blocked pipes Oil leaking from the pellet should therefore be avoided Research has shown that oil leaking is affected by choice of ingredients and processing conditions (ỉverland et al 2007; Sứrensen et al 2011) Sứrensen et al (2010) found that oil leaking was not associated with oil level in the feeds, but was related to feeds with low absorption capacity Neither was oil leaking correlated with expansion of the pellet or other physical quality parameters Most likely oil leaking is related to microstructure of the pellet Different procedures have been used to assess oil leaking ỉverland et al (2007) placed kg coated feed in plastic buckets equipped with an absorbing plastic coated lining in the bottom The bucket was closed with a lid and stored at room temperature (2022 C) After one week of storage the feed was emptied from the bucket, and the fat runoff from the pellets was weighed in order to measure fat leaking Another and faster method is to incubate smaller amounts of coated pellets, 100 g, at 40 C in a heating cabinet for 24 h (Sứrensen et al 2010) The pellets are placed on an absorptive lining in a plastic box After incubation, all pellets and dust are removed from the box, and the new weight of the box and blotting paper is weighed on a balance No investigation has been carried out to compare the two methods reported by ỉverland et al (2007) and Sứrensen et al (2010) Modern aqua feeds are made from a range of ingredients that are combined to meet the nutritional requirements The feed mash undergoes significant changes during the course of the conditioning and extrusion process as it is heated, kneaded and sheared This process may affect the nutrient digestibility, growth rate and feed conversion efficiency of the feed A temperature higher than 100 C is needed in order to flash off steam and expand the feed as it leaves the die Effects of extrusion temperature on digestibility and utilization of fish meal based diets by rainbow trout were examined in two different experiments (Sứrensen et al 2002, 2005) In the experiment of Sứrensen et al (2002) a fishmeal-based diet was extruded with a twinscrew extruder at three temperatures (100, 125 and 150 C) In another experiment (Sứrensen et al 2005), a fishmealbased diet was extruded using a single screw extruder at two temperatures (100 and 140 C) The two studies showed that extrusion processing with temperatures in the range from 100 to 150 C did not affect digestibility of protein, individual amino acids or energy for fish meal based diets Neither was the feed conversion or net accumulation efficiency of protein and energy significantly different in trout fed diets extruded at 100 and 140 C The growth rate was, however, improved at the highest extrusion temperature in the latter study In line with these results, Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd Barrows et al (2007) found no significant effect of extrusion temperature on apparent digestibility of protein, organic matter, lipid, energy or carbohydrate in diets containing soybean meal for rainbow trout Increased retention time in the extruder negatively affected feed intake and weight gain, whereas high temperature (127 C) resulted in improved feed conversion rate (FCR) compared to the lower temperature of 93 C (Barrows et al 2007) In contrast to Barrows et al (2007) improved digestibility of most major nutrients and amino acids was reported in Atlantic salmon fed diets with defatted soybean meal when the extrusion temperature was increased from 110 to 150 C during processing (Morken et al 2012) The latter investigation was carried out using higher extrusion temperatures compared to Barrows et al (2007) and this may explain the differences in effects on nutrient digestibility These studies with fish meal as protein source either alone (Sứrensen et al 2002, 2005) or in combination with soy proteins (Barrows et al 2007; Morken et al 2012) showed that feed extruded at the highest temperature was better utilized either due to higher availability of nutrients, higher utilization of the nutrients, or a favourable feed structure that stimulated feed intake Damage to proteins during heat processing is a function of temperature, time, moisture and the presence of reducing substances (Papadopoulos 1989) The relatively high moisture content (250300 g kg1) combined with short duration of exposure (0.52 min) implies that extrusion is not detrimental to the nutritional value of the feed as long as the temperature does not exceed 150 C Moisture content is of critical importance in order to maintain nutritional quality during heating In order to prevent losses of essential nutrients, a moisture content of 250 300 g kg1 during wet extrusion of diets for fish and pets has been recommended (Rokey 1994) Improved growth performance of shrimp fed diets extruded at high moisture contents in comparison to dry extrusion conditions emphasizes the significance of moisture during processing (Obaldo et al 2000) Low moisture content in combination with heating of fish proteins to temperatures higher than 100 C, increased cross-linking between amino acids (Opstvedt et al 1984; Finley 1989) causing reduced digestibility of nearly all amino acids, especially cysteine (Andorsdottir 1985; Ljứkjel et al 2000) Reduced digestibility of cysteine was also shown in rainbow trout when water addition to Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd the extruder was restricted, compared to when the diet was produced at elevated moisture contents (Sứrensen et al 2002) Cysteine reacts readily during heat treatment to form disulphide bonds between cysteine units (Bender 1978) The reduction in cysteine digestibility in heat treated proteins has been explained by the formation of SS bonds, assumed to be resistant to proteolytic cleavage (Friedman 1982) In contradiction, Aslaksen et al (2006) reported no effect on protein digestibility or absorption of disulphide products in mink, despite an increased content of disulphide bonds in soybean meal extruded diets These results suggest that the content of disulphide bonds and cross linked amino acids probably need to exceed a threshold level before negative effects are noted on protein digestibility Production methods used to produce high quality pellets are challenging and investigation of the physical quality of commercial fish feed pellets, i.e pellet hardness, durability, sinking velocity and water absorption, has shown that the quality varies (Chen et al 1999) Although extrusion technology is used with the purpose of producing feed with high physical quality, recent research has shown that physical quality varies with diet formulation (Glencross et al 2010, 2011a; Draganovic et al 2011; Kraugerud & Svihus 2011; Kraugerud et al 2011), extruder configuration (Sứrensen et al 2009, 2010), and processing parameters (Kraugerud & Svihus 2011; Kraugerud et al 2011; Morken et al 2012; Sứrensen et al 2011) All variables including feed composition and processing conditions that interfere with expansion of the product influence pellet structure and durability An inverse relationship between expansion of the pellet and physical quality reported as hardness (Fig 1a) or durability (Fig 1b) has been reported in a number of studies (Aarseth et al 2006b; Hansen & Storebakken 2007; Sứrensen et al 2009, 2010, 2011; Glencross et al 2011b; Kraugerud & Svihus 2011; Kraugerud et al 2011; Morken et al 2012) Screw configuration directly influence the cooking and transformation of the feed dough inside the extruder barrel (Barres et al 1990), due to changes in residence time (Olkku et al 1980), degree of filling, energy input to the material (Erdemir et al 1992; Yam et al 1994; Sứrensen et al 2011), and shear rate (Gogoi et al 1996) Changing the configuration of the screws can therefore be used to manipulate expansion, bulk density, hardness and durability towards targeted values (Gogoi et al 1996; Sứrensen et al (a) y = 0.8167x + 49.583 R = 0.9381 35 30 25 140 120 Hardness (N) Expansion (%) (a) Expansion versus hardness 40 20 15 10 100 80 60 40 20 25 30 35 Standard Production Low High 20 40 Hardness (N) Wheat starch Expansion versus holmen durability 35 30 25 20 15 10 Whole bean Dehulled bean Starch rich ingredients Wheat Oats (b) 140 y = 0.0187x + 2.6246x 58.894 R = 0.9052 50 60 70 80 90 120 100 Holmen durability (%) Figure Relationship between expansion ratio and hardness (a) and expansion ratio and durability (b) (Hansen & Storebakken 2007) 2010) Sứrensen et al (2010) investigated the effect of three different screw configurations that all generated unique SMEs Screw configuration had a significant impact on the strength and Holmen durability of the feed, however, there were no correlation between SME and these quality characteristics when different carbohydrate sources were used Although variation was observed in Holmen durability and strength, the same ranking was noted among carbohydrate sources for three different screw configurations Screw speed (RPM) in the extruder is directly correlated with SME Greater expansion ratio is reported with increasing RPM (Sứrensen et al 2011) Optimizing pellet quality by manipulation of RPM to change SME was also investigated by Kraugerud et al (2011) and Kraugerud & Svihus (2011) A total of 11 diets in which fish meal was partly replaced with either plant protein ingredients or starch rich ingredients were produced at four different SMEs (Fig 2a,b) Different SMEs were obtained by adjusting moisture and RPM of the extruder Feed containing different ingredients showed great variation in physical quality, such as hardness (Fig 2a,b) and responded differently to changes in RPM Differences in torque were also recorded among the diets suggesting that the diets formed different viscosities during the extrusion process The recorded SME therefore varied and the measured responses had moderate correlation to SME Torque is associated with friction, or resistance to Hardness (N) Expansion (%) (b) 40 Peas 100 Standard Production Low High 80 60 40 20 Fish meal Corn gluten Soybean meal Sunflower Lupin Rapeseed Protein rich ingredients Figure Differences in hardness (a, b) in pellets processed from either starch rich ingredients (a) or plant protein ingredients (b) at different SMEs achieved mainly by adjusting screw speed (RPM) and moisture level The study was divided into three feed production trials: (1) feed production at standardized parameters, (2) feed production at commercial parameters, (3) feed produced at two different SME levels The standardized processing conditions aimed to study how diets with different ingredients responded to identical treatment The flow rate of the extruder was kept constant and, the screw speed was set to 400 rpm and total moisture to 308 g kg1 For feed produced at commercial parameters the extrusion process was optimized to give a bulk density of 460 g L1 and a pellet diameter of mm The targeted bulk density was obtained by adjusting (releasing) steam pressure from section four in the extruder The High (45.4) and low (36.5) SME treatments were carried out by adjusting moisture and screw speed (Kraugerud et al 2011) flow, and is affected by RPM, fill and viscosity of the material in the screw channel (Harper 1989) This study clearly demonstrated the difficulty of producing pellets with predictable physical quality The overall conclusion from the experiment was that extrusion processing parameters needed to obtain targeted physical qualities need to be based on specific knowledge of each ingredient Temperature during extrusion has an impact on the gelatinization of starch and denaturing of proteins Elevated Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd temperature in front of the die reduces the viscosity and increases the steam pressure of the melt, which in turn directly influence expansion of the extruded high energy pellets (Aarseth et al 2006b; Morken et al 2012) The latter authors showed that pellets became more brittle due to increased expansion and less resistant to mechanical stresses with increasing extruder temperature Functional properties of ingredients may be defined as the ingredients ability to form pellets with defined physical quality in terms of durability, hardness, density, oil absorption capacity and oil leaking Physical quality of pellets depends on the bonding between particles and varies with ingredient composition, particle size distribution of the ingredients, conditioning, cooling and drying (Behnke 1996) Rumpf (1962) suggested that binding with or without solid bridges is the main mechanism of bonding of particles Bonds without a solid bridge may be important when particles are brought close together, whereas bonds with solid bridge are reported to be important in pellets (Kaliyan & Morey 2010) These bonds can, for example, be formed by diffusion of molecules between particles at the contact point, crystallization of particles from different ingredients, chemical reactions, and solidification of melted components (Kaliyan & Morey 2010) Water added as a liquid or steam is activating natural binders such as soluble carbohydrates, starch, proteins and minerals Moreover, moisture itself is a film binder forming bonds between particles by weak forces such as hydrogen bonds and van der Waals forces (Pietsch 2002) Inherent binding characteristics differ among ingredients depending on chemical constituents and functional properties (Behnke 1996; Cavalcanti & Behnke 2005a,b; Lundblad et al 2009; Sứrensen et al 2009, 2010, 2011; Glencross et al 2011a,b; Kraugerud & Svihus 2011; Kraugerud et al 2011), and is associated with water absorption capacity of ingredients (Hemmingsen et al 2008) Small particle size distribution in the mash also improves physical pellet quality because smaller particles more readily absorb moisture compared to large particles and are easier to agglomerate (Hemmingsen et al 2008; Kaliyan & Morey 2009) Based on the inherent binding capability and effects on pellet quality, different ingredients were assigned a pelletability index (MacMahon & Payne 1991) Carbohydrates and proteins are structure forming materials (Guy 2001) that may improve the quality of pellets, whereas fat is a Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd lubricant reducing pellet quality (Guy 2001; Cavalcanti & Behnke 2005a,b; Morken et al 2012) Although extruder systems are flexible and can produce pellets with high quality from ingredients with low pelletability index, feed constituents such as starch, protein, fibre and fat affect physical properties such as strength, durability and expansion ratio Because modern extruded fish feed is made from a mixture of ingredients containing chemical components with different functionality, it is challenging to produce pellets with predictable physical quality Variation in physical quality parameters of extruded feeds containing marine ingredients, plant protein ingredients or different starch sources is shown in Table Starch is added to feed for carnivorous fish primarily as a partly digestible binder and to facilitate expansion of the feed Kraugerud et al (2011) reported a moderate but positive correlation between amount of added purified wheat starch and expansion, durability and hardness No correlation was, however, found among total starch level (added purified starch and starch derived from the ingredients) and these parameters These findings suggest that starch is not the most important binder in extruded feed and are supported by previous research with pelleted broiler feeds (Zimonja & Svihus 2009) Starch sources with good binding and expansion properties are needed because starch is kept to a minimum in diets for salmonids due to the low capacity of carnivorous fish to digest and metabolize starch (Hemre et al 2002) The functional properties of starch are activated by gelatinization, a process that is mediated by free water and elevated temperature in the system Use of pregelatinized starch improved pellet quality both in steam pelleting (Wood 1987; Zimonja & Svihus 2009) and extrusion (Sứrensen et al 2010) compared to native starch Therefore it has been generally accepted that high gelatinization of starch in extruded pellets improves pellet quality However, a recent study comparing effects of different feed processing techniques (steam pelleting at low and high temperature, expander conditioning prior to pelleting and extrusion processing) on nutritional and physical quality of feed, Lundblad et al (2011) showed that the extruded feed had the highest starch gelatinization and the lowest PDI The authors concluded that this was mainly because the extruded diets were 25% more expanded compared to the pelleted diets The greatest improvements in extruded compared to steam pelleted diets are therefore higher water stability and possibilities to adjust bulk density and fat absorption capacity The functional properties differ among starches due to differences in amylose-amylopectin ratio, size and shape of the starch granule and other chemical components associated with the granule A high amylose content increases the visco-elastic behaviour and flow properties of melted starch (Chinnaswamy 1993; Kokini 1993) Wheat of food-grade quality is extensively used by the feed industry due to high functional and nutritional properties However, recent research has shown that replacing wheat with potato starch improved durability and hardness of the extruded fish feed, whereas pea starch improved durability but not hardness of extruded fish meal based diets (Table 1) Greater oil leaking was observed when potato starch (Sứrensen 2011, unpublished results) and pea starch (Sứrensen et al 2011) replaced wheat in fish meal based diets Feed additives that affect pH in the diet may change the binding properties of starch In a recent study Morken et al (2012) demonstrated that formic acid added to soybased diets most likely reduced the hydrophilic properties of starch resulting in greater expansion and reduced binding strength with increasing extrusion temperature (Fig 3) Based on these findings the authors suggested that use of acidic feed additives should be used with great care in diets with poor inherent binding properties Dietary fibre is made up by a mixture of soluble and insoluble components that are indigestible for carnivore fish and should be kept at a minimum in diets for these species Dietary fiber can be classified as water soluble or water insoluble, and the solubility is related to the hydrophilic and hydrophobic properties of the repeating monomeric unit and bonds of the fibre The source and amount of fibre affect quality of the pellets (Hsieh et al 1989; Lue et al 1990; Hansen & Storebakken 2007; Kraugerud et al 2011) measured as viscosity, bulking effect, water holding 18 Holmen durability index Holmen durability index (%) Expansion ratio 16 80 14 12 60 10 40 20 Expansion ratio (%) 100 0 + 110 C + 130 C + 150 C Figure Holmen durability and expansion ratio in diets with soybean meal supplemented with (+) or without () sodium diformate extruded at 100, 130 or 150 C Increasing extrusion temperature gave higher expansion and reduced Holmen durability, in particular for the diets added sodium diformate (Morken et al 2012) capacity and ability to form gel Water soluble fibres may improve the structural integrity of pellets by increasing the viscosity and thereby embedding coarser particles into a network (Thomas et al 1997) Recent research have shown increased durability and pellet hardness with increasing cellulose inclusion in the diet (Hansen & Storebakken 2007; Kraugerud et al 2011) The latter authors also suggested that the effect of fibre was an indirect effect associated with reduction in expansion mediated through either disruption of cell walls, or through a reduction of die swell as the extrudate leave the extruder (Guy 2001) Water insoluble fibres may prevent bonding between particles due to resilience characteristics (Thomas et al 1998) Water insoluble fibres that entangle and fold between particles may result in weak spots and more fragmentation of the pellet Refstie et al (2006) reported that the diets containing lupin kernel meals had greater durability and were harder compared to lupin protein concentrates The differences between the kernel meals and the concentrates are mainly that concentrates contain less fibre than the kernel meal (Glencross et al 2005) The functionality of protein as a binder is associated with the structure of the protein, which is mainly determined by the amino acid composition and processing history Proteins in native or undenatured state have better binding properties than heated or denatured proteins Wood (1987) found that inclusion of raw and unprocessed protein, rather than denatured protein, improved pellet hardness and pellet durability Improved hardness was also observed for extruded feed when untoasted rather than toasted, soybean meal replaced fish meal in the diet (Sứrensen et al 2009) Native proteins have a higher solubility than denatured proteins, and solubility of main feed constituents appears to be an important factor for the quality of the final product Heat treated proteins are already unfolded and form less soluble aggregates Several investigations have reported improved hardness and durability of pellets when fish meal was replaced with less heat treated plant proteins such as soybean meal, pea protein concentrate, sunflower meal, lupin, rapeseed meal and wheat gluten (Table 1; Fig 2; Draganovic et al 2011) However, Draganovic et al (2011) reported that although wheat gluten and soy protein concentrated was positively associated with hardness of the pellets, fish meal had unique functional properties not present in the plant ingredients used in the experiment Fish meal contains mainly fibrous proteins that has been through several steps of heating prior to processing into fish feed, whereas plant proteins are dominated by globular proteins When protein is exposed Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd sh per tank were sampled for the analyses of plasma biochemical parameters Blood was collected from the caudal vein with a heparinized syringe and transferred into a heparinized tube Plasma was recovered after centrifugation (6000 g, 10 min) and immediately stored at )70 C until analysis All samples were pooled by tank for analysis Analysis of dry matter (105 C, 24 h), crude protein (Kjeldahl nitrogen ã 6.25), crude lipid (ether extraction by Soxhlet method) and ash (550 C, 18 h) in experimental diets and whole-body samples were performed following standard laboratory procedures (AOAC 1995) Crude bre content in diets was determined by an automatic analyzer (M6-1020; Tecator, Hoeganaes, Sweden) Nitrogen-free extract (NFE) in diets was estimated as the weight dierence using crude protein, crude lipid, crude bre and ash content data The gross energy was calculated assuming 23.7, 39.5 and 17.2 kJ g)1 as estimates of gross energy content of protein, lipid and NFE, respectively (Young et al 2005) The plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase, alkaline phosphatase (ALP), superoxide dismutase (SOD), total antioxidant capacity (TAC), glutathione peroxidase (GSH-Px), catalase and lysozyme activities, and glucose, total protein, creatinine, blood urea nitrogen (BUN), triglycerides, total cholesterol and malondialdehyde contents were determined by using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) Liver fat was determined according to Folch et al (1957) All percentage data were arcsine-transformed before analysis Data were subjected to one-way analysis of variance (ANOVA) Tukeyếs multiple range tests (P < 0.05) Correlation analyses between the dietary lysozyme content in diets and growth or plasma biochemical parameters were performed by tting the data into a curve linear model selecting the model giving the best t Statistical analysis was performed using the SPSS 16.0 (SPSS Inc., Chicago, IL, USA) for Windows Part of the data of the control group used for comparison in this study have been previously reported in another study (Deng et al in press), because the two experiments using the same control treatment were performed at the same time In this study, the feed eciency ratio ranged from 0.45 to 0.52, which is relatively lower compared with that observed in most studies with rainbow trout (Gaylord & Barrows 2009; Barrows et al 2010; Nang Thu et al 2011), but is comparable with that reported by Refstie et al (2000) and Aksnes et al (2006) for the same species The present experimental diets were cold-pelleted to avoid lysozyme inactivation, whereas the total lipid content (about 180 g kg)1 diet) in diets was relatively higher compared with that in most aquatic feeds These may reduce the feed pellet water stability, which is an important internal physical property (Chen & Jenn 1992) Previous studies with rainbow trout showed that sh fed diet with low water stability had markedly higher feed intake as compared with sh fed diet with high water stability (Terjesen et al 2008; Aas et al 2009), whereas the apparent digestibility of dry matter and nutrients in diet with low water stability was markedly lower as compared with that of diet with high water stability (Aas et al 2009) Further, low feed pellet water durability may also lead to the leaching loss of nutrients in diet and the diculty of uneaten feed collection Thus, the low feed eciency in this study may be partly related to the low feed pellet water stability, which was conrmed by previous study (Chen & Jenn 1992) In addition, the soybean meal content in the present experimental diets was 600 g kg)1 diet, which was relatively high for the maximum growth of rainbow trout (Yang et al 2010) The high soybean meal content was probably another possible reason to cause poor feed eciency In this study, dietary lysozyme supplementation generally increased the survival of sh although no signicant dierent was observed (Table 2) Similarly, the addition of lysozyme at 450600 mg kg)1 in diets signicantly increased the growth performance, feed utilization, and protein and lipid retentions (Table 2) This observation is in line with that of Zhang et al (2008), who reported that 500 mg kg)1 dietary lysozyme inclusion markedly increased the weight gain and feed eciency in broiler chickens Similarly, the growth parameters and feed utilization were increased by the incorporation of 100300 mg kg)1 lysozyme in broiler chickens diets (Cheng et al 2009; Lu et al 2009; Ding 2010), 70 mg kg)1 lysozyme in duck diets (Gu & Zhang 2008), 100 mg kg)1 lysozyme in meat rabbit diets (Guo et al 2010) and 300500 mg kg)1 lysozyme in weaned piglet diets (Wang et al 2008; Lu et al 2010; Wu et al 2010) These results indicated that dietary lysozyme supplementation has the growth-stimulating action, but the mechanism by which this occurs is not well known In chick, the antimicrobial activity of lysozyme could result in better intestinal health and improved digestion and absorption (Liu et al 2010) and thereby improved the growth performance (Zhang et al 2008; Cheng et al 2009) Aquaculture Nutrition 18; 332339 ể 2011 Blackwell Publishing Ltd Table Growth performance, feed utilization and liver characteristics of rainbow trout fed diets with different lysozyme levels for 10 weeks1 Dietary lysozyme level (mg kg)1) 150 300 450 600 Pooled SD Regression analysis ANOVA F-value P-value Initial weight (g) Final weight (g) 7.56 41.9a 7.78 48.5b 7.78 48.5b 7.78 49.3b 7.89 50.8b 0.44 3.74 0.171 6.145 0.948 0.009 Feed intake (g per fish) SGR (% per day)2 76.7a 81.1b 78.4ab 79.2ab 79.6ab 1.75 6.973 0.006 2.14a 2.29ab 2.29ab 2.31b 2.33b 0.08 5.390 0.014 FER3 0.45a 0.50ab 0.52b 0.52b 0.54b 0.04 5.941 0.010 PER4 1.04a 1.17ab 1.19ab 1.22b 1.26b 0.09 6.048 0.010 Protein retention (%)5 Lipid retention (%)6 18.6a 22.4ab 22.6ab 22.9b 22.8b 0.54 5.735 0.018 31.8a 38.3b 38.2b 38.2b 37.5b 0.88 7.065 0.010 Survival (%) 91.1 95.6 97.8 97.8 7.51 0.460 0.764 Liver characteristics Total lipid (g kg)1) HSI (%)7 38.8 1.49 38.3 1.46 37.3 1.44 38.0 1.41 0.25 0.04 1.574 1.434 0.255 0.292 100 38.8 1.45 Equation R2 y = 41.97 + 7.823 (1 ) e)0.0101x) y = 2E-07x3 ) 2E-04x2 + 0.0431x + 76.923 y = 2.14 + 0.171 (1 ) e)0.0114x) y = 0.447 + 0.086 (1 ) e)0.00656x) y = 1.043 + 0.214 (1 ) e)0.00478x) y = )2.6E-05x2 + 0.021x + 18.948 y = )5.2E-05x2 + 0.039x + 32.420 y = 3E-07x3 ) 3E-04x2 + 0.0819x + 91.511 0.667 0.001 0.514 0.041 0.667 0.001 0.696 0.05), but n-6/n-3 ratios increased with increasing dietary ARA levels Total saturated fatty acid (SFA) content in liver was the lowest for sh fed the diet containing the lowest dietary ARA level and shown no signicant dierence among other treatments (P > 0.05) For several enzymes involved in hepatic intermediary metabolism, SDH activity increased with increasing dietary Table Effect of dietary ARA level on growth performance and feed utilization of Synechogobius hasta Dietary ARA level (g kg)1 total fatty acids) 0.6 IBW FBW WG SGR Survival FI FCR 7.83 19.18 144.9 1.60 78.3 20.8 1.83 8.6 0.35 0.80 0.9a 0.01a 7.6 1.7a 0.08b 7.88 23.43 197.7 1.95 80.0 23.9 1.54 16.7 0.29 0.47 16.8b 0.10b 8.7 1.1b 0.06a 7.93 24.09 204.0 1.98 81.7 24.6 1.52 32.7 0.48 0.65 10.0b 0.06b 5.8 0.5b 0.04a 7.88 22.76 189.2 1.89 78.3 23.4 1.57 64.8 0.28 0.71 18.8b 0.12b 5.8 1.9b 0.11a 7.82 21.53 176.6 1.81 83.3 23.1 1.70 0.45 1.05 29.4ab 0.19b 10.4 0.9ab 0.21ab Values are means SD of three replicate tanks, and values with the different letters within the same row are significantly different at P < 0.05; IBW (g fish)1), initial mean body weight; FBW (g fish)1), final mean body weight; WG (weight gain, %) = (final mean body weight ) initial mean body weight)/initial mean body weight ã 100; SGR (specific growth rate, % day)1) = 100 ã (ln (final mean body weight) ) ln (initial mean body weight))/days; Survival = 100 ã (final fish number)/(initial fish number); FI (g fish)1): feed intake; FCR (feed conversion ratio) = g feed intake/(g final fish weight ) g initial fish weight + g dead fish) Aquaculture Nutrition 18; 340348 ể 2011 Blackwell Publishing Ltd similar trend and declined with increasing dietary ARA levels (P < 0.05) The highest HL activity was observed in sh fed the diet containing 8.6 g ARA kg)1 total FAs Dietary ARA levels showed no signicant eect on MDH activity (P > 0.05) Eect of dietary ARA levels on several antioxidant enzymatic activities and MDA levels in liver of S hasta was shown in Table SOD activity increased with increasing dietary ARA levels GPx and CAT activities and MDA levels in liver showed similar trend They tended to increase with increasing dietary ARA levels from 1.0 to 32.7 g ARA kg)1 total FAs and then declined with further increase to 64.8 g ARA kg)1 total FAs Y = 1.8933 Y = 0.0236 X + 1.6399 (R2 = 0.806) Xopt = 10.74 Figure Relationship between SGR and dietary ARA level for Synechogobius hasta juveniles based on broken-line regression analysis, where Xopt represents the optimal dietary ARA level for the maximum SGR of S hasta ARA levels from 1.0 to 16.7 g kg)1 total FAs (P < 0.05) (Table 5) and then declined with further increase to 64.8 g ARA kg)1 total FAs LDH and LPL activities showed In the present study, growth performance (WG and SGR) responded to dietary ARA level, in agreement with several other studies showing a positive eect of dietary ARA on growth in sh species (Estevez et al 1997; Harel et al 2001) The best growth obtained in sh fed the diet with 8.6 to 32.7 g ARA kg)1 may be attributable to the best FCR obtained in the group By contrast, Fountoulaki et al (2003) Table Effect of dietary ARA level on hepatic lipid content and hepatic main fatty acid composition (g kg)1 total fatty acids) of juvenile Synechogobius hasta Dietary ARA level (g kg)1 total fatty acids) 0.6 Lipid P Saturates P Monoenes 18:2n-6 20:4n-6 n-6 PUFA 18:3n-3 20:5n-3 22:6n-3 n-3 PUFA P P n-6/ n-3 ARA/EPA 8.87 317.0 234.3 33.7 24.3 66.0 13.7 110.7 200.7 325.0 0.20 0.22 8.6 0.42c 2.6a 18.5 2.5b 4.5a 4.0a 1.5 3.1b 18.0 15.7 0.00a 0.04a 16.7 7.80 329.3 223.0 29.7 30.7 68.7 13.0 108.3 198.7 320.0 0.21 0.28 0.44c 9.1ab 5.3 1.5ab 3.2b 4.9a 1.7 3.5ab 7.2 5.3 0.02ab 0.04ab 32.7 6.57 339.7 221.3 28.3 34.7 74.0 12.0 103.7 193.0 311.0 0.24 0.34 0.21b 8.7ab 10.0 4.9ab 3.5b 7.2ab 2.6 5.0ab 20.0 20.7 0.01bc 0.05b 64.8 5.63 331.3 219.3 27.3 43.0 80.7 10.3 103.0 201.3 314.7 0.26 0.42 0.32ab 8.1ab 7.6 2.3a 3.0c 6.0b 1.5 5.6ab 8.1 13.8 0.03c 0.05c 5.27 332.0 217.7 26.0 47.7 81.3 10.0 101.0 202.0 313.0 0.26 0.47 0.23a 12.2b 5.9 3.5a 1.2c 4.7b 2.0 2.6a 6.2 3.5 0.01c 0.02c Values are means SD of three replicate tanks and values within the same row with different letters are significantly different (P < 0.05) Table Effect of dietary ARA level on several hepatic enzymatic activities of juvenile Synechogobius hasta Dietary ARA level (g kg)1 total fatty acids) 0.6 SDH LDH MDH LPL HL 4.33 0.73 6.04 1.71 1.41 8.6 0.54a 0.04c 0.27 0.20a 0.37a 5.87 0.58 5.91 7.08 6.15 16.7 0.32b 0.05b 0.66 0.98c 0.46c 7.37 0.48 5.93 5.98 4.52 32.7 0.68c 0.05a 0.45 0.42bc 0.65b 6.75 0.49 5.78 5.81 4.68 64.8 0.47c 0.02a 0.52 0.73b 0.31b 4.36 0.55 6.28 4.84 4.38 0.30a 0.03ab 0.57 0.66b 0.79b Values are means SD of three replicate tanks, and values within the same row with different letters are significantly different (P < 0.05); Units: SDH, LDH, MDH, LPL and HL: U mg)1 protein Aquaculture Nutrition 18; 340348 ể 2011 Blackwell Publishing Ltd Table Effect of dietary ARA level on several antioxidant enzymatic activities in liver of juvenile Synechogobius hasta Dietary ARA level (g kg)1 total fatty acids) 0.6 GPx SOD CAT MDA 14.91 7.57 11.93 4.88 8.6 0.12a 0.72a 1.26a 0.74a 15.29 10.31 14.89 6.09 16.7 0.87ab 0.19b 0.91b 0.46a 16.16 10.04 15.00 9.16 32.7 0.80abc 1.50b 1.43b 0.63b 17.54 12.55 18.11 11.15 64.8 1.47c 1.28c 1.45c 0.62c 17.14 15.46 16.44 10.98 1.26bc 1.13d 1.08bc 1.74c Values are means SD of three replicate tanks, and values within the same row with different letters are significantly different (P < 0.05); Unit: GPx, SOD and CAT: U mg)1 protein; MDA: nmol mg)1 protein observed that the increase in dietary ARA levels did not dierentiate growth and feed eciency in sh In addition, dietary ARA/EPA ratios may be another reason for the explanation of the growth performance among the treatments Several researchers pointed out the importance of the n-3 and n-6 FA balance in the diet (Glencross et al 2002; Tan et al 2009) Recommendations for dietary ARA/EPA ratios in other sh species range from 3:1 for striped bass (Harel et al 2001), 1:1 for sea bass and 0.1:1 for halibut and turbot (Sargent et al 1999) In the present study, broken-line regression analysis of SGR against dietary ARA level indicated that optimal dietary ARA requirement for juvenile S hasta was 10.74 g kg)1 total FAs In the present study, increasing dietary ARA concentration resulted in a concomitant increase in tissue ARA concentrations, similar to other reports (Bransden et al 2004, 2005; Villalta et al 2005) However, our study also indicated that sh fed the lowest ARA diets (0.6 g kg)1 total FAs) showed a certain amount of ARA content (24.3 g kg)1 total FAs) in liver Similarly, Willey et al (2003) pointed out that relatively high levels of ARA were found within the tissue of sh fed undetectable levels of ARA in the diet, reecting its conserved nature and implying an essential physiological role On the other hand, a certain amount of ARA was observed in sh fed the lowest ARA diet, which may be attributable to the use of trash sh (with non-determined ARA content) within the acclimation period ARA has been shown to be highly conserved and metabolically prioritized during periods of starvation in turbot larvae (Rainuzzo et al 1994) Despite almost constant EPA in diets, tissue EPA concentrations generally declined as more ARA was provided in the diet, in agreement with several reports (Tocher & Sargent 1986; Bransden et al 2004, 2005; Lund et al 2007) While catabolism for energy is one possible explanation, more likely the reductions in EPA can be explained by the competitive interaction of EPA and ARA (Tocher & Sargent 1986; Villalta et al 2005) and a higher anity of ARA to the cell enzymatic binding site competing for the FAs (Bell et al Aquaculture Nutrition 18; 340348 ể 2011 Blackwell Publishing Ltd 1995; Whelan 1996) In contrast to several other studies (Bransden et al 2005; Lund et al 2007), the present study suggested that hepatic DHA content remained relatively constant with increasing dietary ARA level, denoting the importance of DHA as a structural component of the cell membrane in S hasta This preferential conservation of DHA has also been described with the same sh species in our recent studies (Luo et al 2008) In the present study, dietary ARA levels signicantly inuenced several enzymatic activities involved in hepatic intermediary metabolism in S hasta However, currently, no information is available on the eects of dietary ARA levels on these enzymes in sh, and there are very few studies concerning the nutritional regulation of these enzymes in sh, making it dicult to make any comparative analysis LDH is the key enzyme for the glycolytic pathway The reduction in LDH activity with increasing dietary ARA levels (P < 0.05) will reduce energy availability via glucose metabolism Tan et al (2009) also reported reduced LDH activity in sh fed the diet containing declining ALA/LNA ratios In the present study, SDH activity increased with increasing dietary ARA levels from 0.6 to 16.7 g kg)1 total FAs (P < 0.05) and then declined with further increase to 64.8 g kg)1 total FAs, but MDH activity showed no signicant dierences among the treatments SDH and MDH are primary enzymes in the oxidative catabolism of carbohydrate SDH has been used eectively as a marker of mitochondrial abundance and activity to identify any possible physiological disturbance in shes (Lehninger et al 1993) As of yet, we have no information on the eects of dietary ARA levels on MDH and SDH activities in sh In the present study, LPL activity declined with dietary ARA levels LPL is considered as a key enzyme in the lipid deposition and metabolism of many tissues It participates in the cellular uptake of plasma chylomicron and very low-density lipoprotein in various extrahepatic tissues (Nilsson-Ehle et al 1980) The strong reduction in LPL activities is inversely related to lipid deposition and may correspond to a form of liver protection, limiting lipid uptake from plasma lipoproteins (Ibarz et al 2007) The reduction in hepatic lipid content with increasing dietary ARA addition coincided with the reduction in hepatic LPL activity The highest HL activity was observed in sh fed the diet containing 8.6 g kg)1 total FAs HL is primarily synthesized in liver and involved in chylomicron-remnant and high-density lipoprotein metabolism (Santamarina-Fojo et al 1998) Tan et al (2009) reported that hepatic HL activity tended to increase with increasing dietary ALA/LNA ratio Tan et al (2009) also suggested that the changes in the activities of enzymes might reect also a change in the quantity of the enzyme present Accordingly, it appears worth exploring the underlying molecular mechanisms in the nutritional control of hepatic intermediary metabolism related to dietary ARA level in sh In the present study, SOD activity increased with increasing dietary ARA levels Hepatic GPx and CAT activities and MDA levels in liver also tended to increase with increasing dietary ARA levels from 0.6 to 32.7 g kg)1 total FAs and then declined with further increase to 64.8 g kg)1 total FAs SOD, GPx and CAT are three common antioxidant enzymes, and they constitute the rst line of antioxidant enzymatic defence SOD dismutates superoxide anion to H2O2; CAT catalyses the breakdown of hydrogen peroxide to water and molecular oxygen GPx, a selenium-dependent enzymes, decomposes peroxides using the peptide glutathione (GSH) as their cosubstrate (Halliwell & Gutteridge 1996; Tovar-Ramirez et al (2010) MDA, which is an index of lipid peroxidation, can intensively react with various cellular components, seriously damaging enzymes and membranes and inducing the decrease of membranous electric resistance and uidity, which leads to the destruction of the membrane structure and physiological integrity (Chandran et al 2005) Lipid peroxidation, specically HUFA oxidation, is highly deleterious, resulting in damage to cellular biomembranes Increased antioxidant enzymatic activities and MDA levels obtained in the present study were not expected because the liver fat content decreased with the increased dietary ARA supplement and because the total absolute highly unsaturated FAs content should be lower in the higher ARA group So far, the activities of the antioxidant enzymes have been measured in several sh (Peters et al 1994; Otto & Moon 1996; Peters & Livingstone 1996) However, these studies focused on the role of the enzymes in pollutant detoxication (Peters et al 1994) or developmental aspects (Otto & Moon 1996; Peters & Livingstone 1996) rather than the eects of dietary nutrients, and accordingly, it is very dicult to make a comparative analysis In the present study, we cannot explain the reason for increased antioxidant enzyme activities and MDA levels by dietary ARA although we speculate that the sh in this study were under a mild oxidative stress at the high levels of ARA However, the generalization on the inuence of dietary ARA levels on antioxidant status cannot be made because the above speculation needs to be reconrmed under dierent oxidative stress conditions In conclusion, optimal dietary ARA requirement for juvenile S hasta was 10.74 g kg)1 total FAs, based on the broken-line regression analysis of SGR against dietary ARA levels Decient or excessive dietary amounts of ARA could reduce growth performance, change hepatic intermediary metabolism and antioxidant responses in S hasta, which might have deleterious consequences for sh This study is one of the few to date that has examined the eect of dietary ARA on these parameters in juvenile sh, and there are still questions in this aspect that are yet to be answered by future research in this very interesting eld This study was supported by the Open Fund of Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture (grant no BZ2009-04), partly by New Century Excellent Talents in University, Ministry of Education, China (grant no NCET08-0782), and by the Fundamental Research Funds for the Central Universities of China (grant no 52204-10078) We are very grateful to three anonymous reviewers for their constructive suggestions and comments Aebi, H (1984) Catalase in vitro Methods Enzymol., 105, 121126 AOAC (Association of Ocial Analytical Chemists) (1995) Ocial Methods of Analysts, 16th edn AOAC, Arlington, VA Bai, S.C & Lee, K.J (1998) Dierent levels of dietary DL a-tocopheryl acetate aect the vitamin E status of juvenile Korean rocksh, Sebastes schlegeli Aquaculture, 161, 405414 Ballart, X., Siches, M., Peinado Onsurbe, J., Lopez-Tejero, D., Llobera, M., Ramirez, I & Robert, M.Q (2003) Isoproterenol increases active lipoprotein lipase in adipocyte medium and in rat plasma Biochimie, 85, 971982 Bell, J.G & Sargent, J.R (2003) Arachidonic acid in aquaculture feeds: current status and future opportunities Aquaculture, 218, 491499 Bell, J.G., Castell, J.D., Tocher, D.R., MacDonald, F.M & Sargent, J.R (1995) Eects of dierent dietary arachidonic acid: docosahexaenoic acid ratios on phospholipid fatty acid compositions and prostaglandin production in juvenile turbot (Scophthalmus maximus) Fish Physiol Biochem., 14, 139151 Bessonart, M., Izquierdo, M.S., Salhi, M., Hernandez-Cruz, C.M., Gonzalez, M.M & Fernandez Palacios, H (1999) Eect of dietary arachidonic acid levels on growth and fatty acid Aquaculture Nutrition 18; 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waterborne zinc exposure on metal accumulation, enzymatic activities and histology of Synechogobius hasta Ecotoxicol Environ Saf., doi: 10.1016/j.ecoenv 2011.06.018 Aquaculture Nutrition 18; 340348 ể 2011 Blackwell Publishing Ltd [...]... growth, nutrient retention and slaughter quality Aquaculture, 124, 109116 Hilton, J.W., Cho, C.Y & Slinger, S.J (1981) Effect of extrusion processing and steam pelleting diets on pellet durability, pellet water absorption and the physiological response of rainbow trout (Salmo gairdneri) Aquaculture, 25, 185194 Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd Honorato, C.A.,... settlement faecal collection methods Aquaculture, 245, 211220 Glencross, B.D., Booth, M & Allan, G.L (2007) A feed is only as good as its ingredients: a review of ingredient evaluation for aquaculture feeds Aquaculture Nutr., 13, 1734 Glencross, B., Hawkins, W., Maas, R., Karopoulos, M & Hauler, R (2010) Evaluation of the influence of different species and cultivars of lupin kernel meal on the extrusion process,... out routinely, in particular when Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd new ingredients are investigated or if processing conditions differs among the feeds It is concluded that standardized and suitable methods are needed to evaluate the physical quality of extruded fish feed This work was financially supported by Aquaculture Protein Centre, CoE (Project No 14949... oats starch on physical pellet quality and nutritional value for broilers Anim Feed Sci Technol., 149, 287297 Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00891.x 2012 18; 249257 1 1 2 1 1 3 2 Institut National des Sciences et Technologies de la Mer, Salammbo, Tunisia; University of Namur (FUNDP), Unit of... fed graded levels of dietary lipids with or without ethoxyquin Aquaculture, 2 03, 8599 Kjứrsvik, E., Olsen, C., Wold, P.-A., Hoehne-Reitan, K., Cahu, C.L., Rainuzzo, J., Olsen, A.I., Oie, G & Olsen, Y (2009) Comparison of dietary phospholipids and neutral lipids on skeletal development and fatty acid composition in Atlantic cod (Gadus morhua) Aquaculture, 294, 246255 Koven, W.M., Kolkovski, S., Tandler,... Aquaculture Nutrition 18; 249257 ể 2011 Blackwell Publishing Ltd compositions of Eurasian perch Perca uviatilis Aquac Int., 9, 437449 Zakes, Z., Szkudlarek, M., Wozniak, M., Karpinski, A & Demska-Zakes, K (2001) Eect of dietary protein:fat ratios on metabolism, body composition and growth of juvenile pikeperch, Stizostedion lucioperca (L.) Czech J Anim Sci., 46, 2733 Aquaculture Nutrition 2012. .. Muthukumarappan, K., Rosentrater, K.A & Julson, J.L (2007) Effect of die dimensions on extrusion processing parameters and properties of DDGS-based aquaculture feeds Cereal Chem., 84, 389398 Chevanan, N., Muthukumarappan, K & Rosentrater, K.A (2009) Extrusion studies of aquaculture feed using distillers dried grains with solubles and whey Food Bioprocess Technol., 2, 177185 Chinnaswamy, R (1993) Basis of cereal... Edwards, R.H & McCarthy, K.L (1992) Effect of screw configuration on mechanical energy-transfer in twin-screw extrusion of rice flour Lebensm Wiss Technol., 25, 502508 FAO (2011) Aquaculture development 5 Use of wild fish as feed in aquaculture FAO Technical Guidelines for Responsible Fisheries No 5, Suppl 5 FAO, Rome, p 79 Finley, J.W (1989) Effects of processing on proteins: an overview In: Protein Quality... trypsin inhibitors by thiols J Sci Food Agric., 33, 165172 Glencross, B., Evans, D., Dods, K., McCafferty, P., Hawkins, W., Maas, R & Sipsas, S (2005) Evaluation of the digestible value of lupin and soybean protein concentrates and isolates when fed to rainbow trout, Oncorhynchus mykiss, using either stripping or settlement faecal collection methods Aquaculture, 245, 211220 Glencross, B.D., Booth,... of starch by extrusion processing is referred to as gelatinization, and is reported to range between 73100% in extruded fish feed (Hansen et al 2010; Kraugerud et al 2011) Aquaculture Nutrition 18; 233248 ê 2012 Blackwell Publishing Ltd A few studies have investigated the effects of physical quality on feed intake and nutrient utilization in fish (Hilton et al 1981; Obaldo et al 1999; Baeverfjord