Aquaculture nutrition, tập 19, số 1, 2013

Aquaculture Nutrition 2013 19; 1–14 doi: 10.1111/j.1365-2095.2011.00926.x 1,3 1,2,3 1,4 1,4 1,3 1,3 1,3 1,3 1,2,3 Animal Nutrition Institute, Sichuan Agricultural University, Ya’an, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Ya’an, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Sichuan, Ya’an, China; Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu, China This study investigated the effects of valine on growth, intestinal enzyme activities and microflora in juvenile Jian carp (Cyprinus carpio var Jian) A total of 1200 fish with an average initial weight of 9.67 ± 0.03 g were fed diets containing 5.3 (unsupplemented control), 8.7, 11.8, 14.9, 18.7 and 20.1 g valine kgÀ1 diet for 60 days Results indicated that the specific growth rate, feed efficiency, body protein and lipid content of fish were significantly improved by the dietary valine (P < 0.05) The hepatopancreas weight and activities of trypsin, amylase, lipase, chymotrypsin, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) took the similar trends Similarly, the optimum levels of dietary valine induced increases in the intestinal length, weight, folds height and activities of alkaline phosphatase, gamma-glutamyl transpeptidase and creatine kinase In contrast, the trends of muscle GOT activity and plasma ammonia content were opposite Intestinal Aeromonas, Escherichia coli, Lactobacillus and Bacillus were changed by dietary valine supplementations The dietary valine requirement for Jian carp (9.67–76.4 g) based on SGR was 13.7 g valine kgÀ1 diet (4.0 g valine 100 gÀ1 CP) Together, these results indicated that valine improved fish growth, digestive and absorptive ability KEY WORDS: Cyprinus carpio var Jian, growth, intestinal microflora, trypsin, valine Received 16 April 2011; accepted December 2011 Correspondence: Xiao-Qiu Zhou, Animal Nutrition Institute, Sichuan Agricultural University, Ya’an 625014, China E-mails: zhouxq@sicau edu.cn; xqzhouqq@tom.com ª 2012 Blackwell Publishing Ltd Fish, like other animals, not have a true protein requirement but require a well-balanced mixture of essential and non-essential amino acids (Wilson 2002) The essential amino acids for common carp (Cyprinus carpio) and chinook salmon (Oncorhynchus tshawytscha) are the same 10 amino acids that are required by the rat (Santiago & Lovell 1988) However, the complete 10 essential amino acid requirement have been established for only a limited number of cultured fish species (Abidi & Khan 2004) Valine was known as an essential amino acid for fish (Nose 1979) It has been demonstrated that dietary valine deficiency decreased the weight gain of common carp (Nose 1979) and Indian major carp (Cirrhinus mrigala) (Ahmed & Khan 2006) The weight gain of fish is associated with the accretion of protein (Bureau et al 2000) Sveier et al (2000) reported that protein deposition in fish is mainly associated with amino acid metabolism Fish have a remarkable capacity to utilize amino acids both as metabolic fuel and as precursors for protein, lipid and carbohydrate synthesis (Wood 1993) Glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) play an important role in ammonia detoxification in freshwater teleost (Hochachka & Somero 1973) Furthermore, fish excrete the majority of their nitrogenous waste as ammonia (Wood 2001) Plasma ammonia levels are the varied widely measurements of excretory processes for hagfish (Eptatretus stoutii) (Arillo et al 1991) The GOT and GPT activities and plasma ammonia content were affected by the levels of dietary protein in juvenile Jian carp (C carpio var Jian) (Liu et al 2009) However, the relationships between dietary valine and activities of GOT and GPT as well as plasma ammonia content (PAC) of fish are unclear Accordingly, further studies are required to address the effects of valine on amino acid metabolism in fish The intestine and pancreas are important for nutrient digestion and absorption of fish (Garcı´ a-Gasca et al 2006) Pedersen & Sissons (1984) reported that the growth and development of intestine and pancreas play an important role in digestion ability and absorption function of calf To our knowledge, few studies have been conducted to investigate the effects of valine on the growth of intestine and hepatopancreas of fish It has been demonstrated that the jejunal crypt depth in rat was affected by valine (Takada et al 2005) Furthermore, pancreatic protein content decreased in rat fed the valine-devoid diet (Sidransky et al 1960) Moreover, BCAA (valine, leucine and isoleucine) can provide nitrogen for the synthesis of glutamate in rat (Bixel et al 1997) and pig (Chen et al 2009) Lin & Zhou (2006) showed that glutamine can improve intestine protein content (IPC) in Jian carp These results indicated that valine may affect the growth of the intestine and pancreas of fish, which needs to be investigated Digestion and absorption of nutrients depend on the activities of the digestive and brush border enzymes (Klein et al 1998) The pancreas secretes a large number of digestive enzymes into the intestinal lumen, including lipase, amylase, trypsin and chymotrypsin (Hitoshi et al 2007) Moreover, Na+, K+-ATPase generates the Na+ gradient that drives amino acid and vitamin transport into cells and is critical for absorption of fluid from the intestine (Lingrel 2010) Gamma-glutamyl transpeptidase plays an essential role in the final hydrolysis and assimilation of dietary proteins (Douglas et al 1999) Furthermore, Villanueva et al (1997) reported that the absorption of nutrients such as lipid, glucose, calcium and inorganic phosphate was depended on the activity of alkaline phosphatase Additionally, creatine kinase plays a key role in the energy homeostasis (Wallimann et al 1998) Sidransky et al (1960) indicated that the acinar cells of the pancreas showed moderate loss of zymogen granules in rat fed with valine-devoid diet In human, valine can stimulate pancreatic enzyme secretion (Go et al 1970) Bixel et al (1997) reported that valine presented as vehicle molecule of nitrogen in the glutamate/glutamine cycle in rat Glutamine can increase the activities of intestinal lipase, protease, alkaline phosphatase and Na+,K+-ATPase in juvenile Jian carp (Lin & Zhou 2006) These data indicated that valine may influence the activities of digestive and brush border enzymes in fish, which needs to be investigated Microflora is associated with the gastrointestinal tract of fish and is sensitive to dietary changes (Ringø & Birkbeck 1999) Human intestinal microorganisms provide instructive signals for several aspects of intestinal development, including epithelial cell maturation (Hooper et al 2003) and angiogenesis (Stappenbeck et al 2002) Sugita et al (1996) indicated that the bacteria in marine crab digestive tract secreted amylase Amino acids can be used by microorganisms for construction of specific cell proteins or can undergo transformations leading to produce different metabolic substances (Ringø & Birkbeck 1999) Valine can be used as an effective source of nitrogen in the process of multiplication of the bacteria Desulfotomaculum ruminis (Szyman´ska et al 2002) These results indicated that valine may be related to the growth of fish intestinal microflora, which needs to be addressed Therefore, we hypothesize that valine could increase fish growth through increasing digestive and absorptive capacity and influencing the balance of intestinal microflora Hence, the purpose of this study was to investigate the effects of dietary valine on the growth, body composition, enzyme activities and intestinal microflora of fish, which could provide a part of theoretical evidence for the effect of valine on fish growth The optimum dietary valine requirement for the juvenile Jian carp was also evaluated The basal diet was formulated in Table Fish meal and gelatin were used as the main protein sources because they are limited in valine The dietary protein level was fixed at 340 g kgÀ1, reported optimum for the growth of Jian carp (Liu et al 2009) Crystalline amino acids (Jiangsu Nantong Eastern Amino Acid Co Ltd., Nantong, China) were used to simulate the amino acid profile of diets with 340 g kgÀ1 whole chicken egg protein, excluding the test amino acid valine, according to the method described by Abidi & Khan (2004) L-Valine was added to the experimental diets to provide graded concentrations of (unsupplemented control), 9, 12, 15, 18 and 21 g valine kgÀ1 diet Diets were made isonitrogenous by supplementation of glycine Zinc, iron, pyridoxine, pantothenic acid, inositol, thiamin and riboflavin were formulated to meet the nutrient requirements of Jian carp according to previous studies conducted in our laboratory (He et al 2009; Jiang et al 2009; Wen et al 2009; Li et al 2010; Ling et al 2010; Huang et al Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd Table Composition and nutrients content of basal diet À1 Ingredients g kg Fish meal Gelatin Crystal AA premix2 a-starch Corn starch Fish oil Soya bean oil Ca(H2PO4)2 Mineral premix3 Vitamin premix4 Ethoxyquin (300 g kgÀ1) Choline chloride (500 g kgÀ1) Cellulose Crystal AA premix5 165.0 45.0 195.2 230.0 224.2 15.2 19.2 24.4 10.0 10.0 0.5 À1 Nutrient contents g kg Crude protein Crude lipid n-3 n-6 Available phosphorus 327.9 62.1 10.0 10.0 6.0 1.3 20.0 40.0 Crude protein and crude fat were measured value Available phosphorus, n-3 and n-6 contents were calculated according to NRC (National Research Council) (1993) and Bell (1984) Crystal AA premix (g kgÀ1): arginine 12.18, isoleucine 13.66, leucine 22.57, lysine 18.19, methionine 8.35, cysteine 0.51, pheylalanine 15.03, tyrosine 12.17, threonine 12.90, tryptophan 3.95, histidine 6.41, L-glycin 69.30 Per kilogram of vitamin premix (g kgÀ1): retinyl acetate (500 000 IU gÀ1), 0.80 g; cholecalciferol (500 000 IU gÀ1), 0.48 g; DL-a-tocopherol acetate (500 g kgÀ1), 20.00g; menadione (500 g kgÀ1), 0.20 g; cyanocobalamin (100 g kgÀ1), 0.01 g; D-biotin (200 g kgÀ1), 0.50 g; folic acid (960 g kgÀ1), 0.52 g; thiamin nitrate (980 g kgÀ1), 0.10 g; ascorhyl acetate (920 g kgÀ1), 7.24 g; niacin (980 g kgÀ1), 2.85 g; meso-inositol (980 g kgÀ1), 52.86 g; calcium-D-pantothenate (980 g kgÀ1) 2.51 g; riboflavine (800 g kgÀ1), 0.63 g; pyridoxine hydrochloride (980 g kgÀ1), 0.76 g All ingredients were diluted with corn starch to kg Per kilogram of mineral premix (g kgÀ1): FeSO4·7H2O (197 g kgÀ1 Fe), 69.70 g; CuSO4·5H2O (250 g kgÀ1 Cu), 1.20 g; ZnSO4· 7H2O (225 g kgÀ1 Zn), 21.64 g; MnSO4·H2O (318 g kgÀ1 Mn), 4.09 g; KI (38 g kgÀ1 I), 2.90 g; NaSeO3 (10 g kgÀ1 Se), 2.50 g All ingredients were diluted with CaCO3 to kg Crystal AA premix (g kgÀ1) premix: L-valine was added to obtain graded level of valine, and the amount of corn starch and L-glycin were reduced to compensate, each mixture was made isonitrogenous Per kilogram of crystal AA premix composition from diet to was as follows (g kgÀ1): 0.0, 79.8, 159.6, 239.4, 319.3, 399.1 g valine, 244.6, 195.7, 146.7, 97.8, 48.9, 0.0 g L-glycin and 755.4, 724.5, 693.6, 662.7, 631.8, 600.9 g corn starch, respectively 2011; Tan et al 2011) The levels of other nutrients met the requirements for common carp according to the NRC (National Research Council) (1993) The pH of each diet was adjusted to 7.0 by gradually adding 6.0 M NaOH (Li et al 2009) After prepared completely, the diets were fan-dried at room temperature and stored at À20 °C until used (Shiau & Lo 2000) The valine concentrations in experimental diets were determined according to the Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd method described by Ahmed & Khan (2006) to be 5.5 (unsupplemented control), 9.7, 12.5, 15.6, 18.7 and 20.5 g valine kgÀ1 diet All experimental protocols were approved by the University of Sichuan Agricultural Animal Care Advisory Committee The juvenile Jian carp were obtained from the Tong Wei Hatchery (Sichuan, China) Fish were acclimated to the experimental conditions for one month A total of 1200 Jian carp, with a mean initial weight of 9.67 ± 0.03 g, were randomly distributed into each of 24 glass aquaria (90 L 30 W 40 H cm) connected to a closed recirculating water system with continuous aeration Each aquarium was randomly assigned to one of four replicates of the six dietary treatments The water change rate in each aquarium was maintained at 1.2 L minÀ1: water was drained through biofilters so as to reduce ammonia concentration and remove solid substances in the water The experimental units were maintained under a natural light and dark cycle Dissolved oxygen was higher than mg LÀ1 The water temperature and pH were 25 ± °C and 7.0 ± 0.3, respectively The fish were hand-fed with their respective diets to apparent satiation six times per day for the first 30 days and four times per day from 31st to 60th days This feeding rhythm was established in previous study performed by our laboratory (Xiao et al 2011) Uneaten feed was removed by siphoning after each meal Fish from each aquarium were weighed at the beginning and at the end of the feeding trial At the beginning of the experiment, 30 fish from the same population were randomly collected to determine the initial body composition At the end of the feeding trial, five fish from each aquarium were frozen for estimating the final protein, lipid and ash composition (AOAC (Association of Official Analytical Chemists) 1998) After 12 h of fasting, 15 fish from each aquarium were killed and the muscle, intestine and hepatopancreas were quickly collected and stored at À70 °C until analysis The intestines of another five fish from each aquarium were sampled to measure the height of intestinal folds according to Lin & Zhou (2006) Six hours after the last feeding, five fish from each aquarium were collected for obtaining blood samples from the caudal vein with heparinised syringes Prior to sampling, fish in each aquarium were fed solely and blood was withdrawn in order The blood samples were centrifuged at 4000 g for 15 (Liu et al 2009) Plasma was collected for ammonia determination using the method of Tantikitti & Chimsung (2001) The hepatopancreas, intestine and muscle samples were homogenized in 10 volumes (w/v) of ice-cold physiological saline solution and centrifuged at 6000 g for 15 at °C; then, the supernatant was stored for determining the activities of trypsin (Hummel 1959), chymotrypsin (Hummel 1959), amylase (Furne´ et al 2005), lipase (Furne´ et al 2005), alkaline phosphatase (Bessey et al 1946), creatinekinase (Tanzer & Gilvarg 1959), gamma glutamyl transpeptidase (Rosalki et al 1970) and Na+, K+-ATPase (Weng et al 2002) The protein content of the intestine and hepatopancreas was determined by the method of Bradford (1976) Three fish from each aquarium were collected for estimating the counts of intestinal microflora The activities of GOT and GPT in muscle and hepatopancreas were determined by the methods of Bergmeyer & Bernt (1974a,b) Data on initial body weight (IBW), final body weight (FBW), feed intake (FI), proximate composition of feed and carcass, hepatopancreas and intestinal weight, intestinal and body length, and hepatopancreas and intestinal protein were used to calculate the following parameters:  Percent weight gain (PWG) = 100 (g weight gain/g initial body weight)  Feed efficiency (FE) = (g weight gain/g feed intake)  Specific growth rate (SGR) = 100 [(ln(final weight) - ln(initial weight))/number of days]  Protein efficiency ratio (PER) = g weight gain/g protein intake  Protein retention value (PRV) = 100 (g fish protein gain/g protein intake)  Relative gut length (RGL) = 100 (cm intestine length/cm total body length)  Hepatosomatic index (HSI) = 100 (g wet hepatopancreas weight/g wet body weight)  Intestosomatic index (ISI) = 100 (g wet intestine weight/g wet body weight)  Intestine protein content (IPC) = 100 (g intestine protein/g wet intestine weight)  Hepatopancreas protein content (HPC) = 100 (g hepatopancreas protein/g wet hepatopancreas weight) The results are presented as the means ± SD All data were subjected to a one-way analysis of variance (ANOVA) Differences among the dietary treatments were determined using a Duncan’s multiple-range test at P < 0.05 level of significance through SPSS 17.0 for windows A quadratic regression model was used to determine the optimal level of dietary valine (Zeitoun et al 1976) The relationship between dietary valine and the growth performance, whole body composition, activities of digestive and absorptive enzymes were subjected to a quadratic regression model Table shows the final body weight, feed intake, FE, SGR and PER of juvenile Jian carp fed graded levels of valine Fish fed with the basal diet showed the lowest final body weight, feed intake, feed efficiency, SGR and PER (P < 0.05) The final body weight, SGR and FI significantly increased with the levels of dietary valine increased from 5.3 to 11.8 g kgÀ1 diet, and gradually decreased thereafter (P < 0.05) Furthermore, the SGR and FI showed significantly quadratic responses to the increasing levels of dietary valine as well as significant response to the mean valine intake per fish Feed efficiency significantly increased with increasing levels of dietary valine up to 11.8 g kgÀ1 diet, and gradually decreased thereafter (P < 0.05) A similar trend was observed for PER In addition, FE and PER were significantly quadratic responses to increasing dietary valine levels The dietary valine requirement estimated based on SGR was 13.7 g kgÀ1 diet, corresponding to 4.0 g 100 gÀ1 CP (Fig 1) As shown in Table 3, fish body moisture, protein and lipid content and PRV were significantly affected by dietary valine The body protein content and PRV were the lowest for fish fed with the basal diet (5.3 g valine kgÀ1 diet) (P < 0.05) The body lipid content was the lowest in fish fed with a diet containing 20.1 g valine kgÀ1 diet (P < 0.05) However, the moisture of fish carcasses was not influenced by the levels of dietary valine Moreover, regression analysis showed that fish body protein content, lipid and PRV were Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd Table Final body weight (FBW, g fishÀ1), feed intake (FI, g fishÀ1), feed efficiency (FE), specific growth rate (SGR), protein efficiency ratio (PER) of juvenile Jian carp (Cyprinus carpio var Jian) fed diets containing graded levels of valine for 60 days1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 a FBW 50.35 ± 0.70 63.89 SGR 2.75 ± 0.02a 3.14 FI 54.61 ± 0.99a 68.50 FE 0.75 ± 0.008a 0.79 PER 1.24 ± 0.02a 1.65 Regressions YSGR = 1.682 + 0.246x À 0.009x2 YFI = 16.703 + 8.76x À 0.3098x2 YFE = 58.920 + 3.525x À 0.123x2 YPER = 0.001 + 0.284x À 0.010x2 11.8 ± ± ± ± ± b 1.73 0.04b 0.75b 0.017b 0.05b 76.38 3.44 78.67 0.85 2.03 14.9 ± ± ± ± ± d 3.09 0.07d 0.77e 0.039d 0.09d 18.7 74.32 3.40 77.13 0.84 1.97 R2 R2 R2 R2 = = = = ± ± ± ± ± d 1.86 0.04d 0.71d 0.024cd 0.06d 0.983 0.9843 0.948 0.973 69.10 3.27 72.65 0.82 1.81 20.1 ± ± ± ± ± c 1.54 0.04c 0.79c 0.021bcd 0.05c 63.95 3.15 67.48 0.80 1.66 P P P P < < < < ± ± ± ± ± 0.85b 0.02b 0.51b 0.016bc 0.03b 0.01 0.01 0.05 0.01 Values are mean ± SD of four replicate tanks (n = 4) Mean values with the different superscripts in the same row are significantly different (P < 0.05) Figure Quadratic regression analysis of specific growth rate (SGR) according to dietary valine levels (a) and against mean valine intake per fish (b)1 1Each point represents the mean of four groups of Jian carp with 50 fish per group The dietary valine requirement was 13.7 g kgÀ1 diet (4.0 g 100 gÀ1 CP) quadratic responses to the increasing levels of dietary valine The ash of fish carcasses was the lowest in Jian carp fed with the diet containing 20.1 g valine kgÀ1 diet (P < 0.05) Regression analysis showed that the body ash content was quadratic response to the increased levels of dietary valine Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd Plasma ammonia content (PAC) and the activities of GOT and GPT of juvenile Jian carp are shown in Table GOT and GPT activities in the hepatopancreas significantly increased with levels of dietary valine up to 11.8 g kgÀ1 diet, and gradually decreased thereafter (P < 0.05) Regression analysis showed that GOT and GPT activities in the hepatopancreas were quadratic responses to increasing levels of dietary valine In addition, GPT activities in muscle significantly increased with levels of dietary valine up to 11.8 g valine kgÀ1 diet, and gradually decreased thereafter (P < 0.05) However, the GOT activity in muscle was decreased with the increment levels of dietary valine (P < 0.05) Regression analysis showed that GOT and GPT activities in muscle were quadratic responses to the increasing levels of dietary valine, respectively Plasma ammonia content significantly decreased and the minimum value occurred in fish fed with diet containing 11.8 g valine kgÀ1 diet (P < 0.05), and increased thereafter Similarly, regression analysis showed that plasma ammonia content (PAC) was significantly quadratic response to increasing levels of dietary valine Hepatopancreas weight, hepatosomatic index, hepatopancreas protein content, intestinal length and weight, relative gut length, intestosomatic index and intestinal protein content of juvenile Jian carp fed with diets containing Table Body composition (g kgÀ1) and protein retention value (PRV,%) of juvenile Jian carp fed diets containing graded levels of valine (g kgÀ1 diet) for 60 days1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 a Moisture 707.6 ± 8.1 698.4 Protein 126.7 ± 6.3a 130.3 Lipid 120.5 ± 5.0b 126.4 Ash 28.7 ± 1.2b 28.6 PRV 38.6 ± 1.9a 39.7 Regressions Yprotein = 11.832 + 0.180x À 0.004x2 Ylipid = 11.197 + 0.243x À 0.013x2 Yash = 3.047 À 0.034x + 0.001x2 YPRV = 36.082 + 0.551x À 0.013x2 11.8 ± ± ± ± ± a 10.8 4.6ab 8.0b 1.7b 1.4ab 703.1 134.6 120.9 27.2 41.0 14.9 ± ± ± ± ± a 14.8 2.0b 7.7b 1.0ab 0.6b 18.7 704.6 134.8 119.5 27.1 42.1 R2 R2 R2 R2 = = = = ± ± ± ± ± a 12.0 5.3b 8.4b 1.4ab 1.6b 708.4 136.9 117.0 27.1 41.8 20.1 ± ± ± ± ± a 16.5 6.5b 6.0ab 0.7ab 2.0b 0.978 0.835 0.870 0.979 713.6 137.2 108.2 26.8 41.8 P P P P < = < < ± ± ± ± ± 16.2a 4.3b 5.4a 0.8a 1.3b 0.01 0.067 0.05 0.01 Values are mean ± SD of four replicate tanks, with five fish in each replicate Mean values with the different superscripts in the same row are significantly different (P < 0.05) Table The activities of glutamate oxaloacetate transaminase (GOT, U gÀ1 tissue) and glutamate pyruvate transaminase (GPT, U gÀ1 tissue) in muscle and hepatopancreas and plasma ammonia content (PAC, lmol LÀ1) of juvenile Jian carp fed diets containing graded levels of valine for 60 days1 Dietary valine levels (g kgÀ1diet) 5.3 8.7 GOT Muscle 4780 ± 161b 4637 ± 462b Hepatopancreas 6438 ± 125a 7437 ± 176b GPT Muscle 620 ± 47ab 773 ± 76c Hepatopancreas 6624 ± 146c 6739 ± 308cd PAC 268 ± 23ab 268 ± 23ab Regressions YGOT in muscle = 7453.519 À 603.711x + 26.026x2 YGOT in hepatopancreas = 3004.800 + 789.798x À 29.920x2 YGPT in muscle = À180.525 + 183.866x À 7.330x2 YGPT in hepatopancreas = 6379.605 + 131.803x À 11.382x2 YPAC = 384.561À29.656x + 1.604x2 11.8 14.9 18.7 20.1 3852 ± 317a 8275 ± 199c 3906 ± 277a 7918 ± 533c 5475 ± 184c 7900 ± 327c 5796 ± 436c 6385 ± 548a 1040 ± 79e 6928 ± 170d 235 ± 23a 937 ± 70d 5065 ± 326b 294 ± 30b 665 ± 55b 5051 ± 144b 428 ± 35c 567 ± 34a 4479 ± 258a 412 ± 55c R2 = 0.865 R2 = 0.812 R2 = 0.911 R² = 0.831 R2 = 0.911 P P P P P < = < = < 0.05 0.082 0.05 0.070 0.05 Values are mean ± SD of four replicate tanks, with five fish in each replicate Mean values with the different superscripts in the same row are significantly different (P < 0.05) graded levels of valine are presented in Table Hepatopancreas weight significantly increased and the maximum value occurred in fish fed with the diet containing 11.8 g valine kgÀ1 diet (P < 0.05), and then decreased A similar trend was observed in the intestinal weight Hepatosomatic index (HSI) and intestosomatic index (ISI) of fish decreased with higher levels of dietary valine (P < 0.05) Hepatopancreas and intestinal protein content were not influenced by the levels of dietary valine (P > 0.05) Regression analysis showed that the HW, IW and ISI were quadratic responses to the levels of dietary valine Intestinal length significantly increased and the maximum value occurred in fish fed with a diet containing 11.8 g valine kgÀ1 diet (P < 0.05), and then decreased thereafter A similar trend was observed in RGL Furthermore, regression analysis showed that the IL and RGL were quadratic response to the levels of dietary valine The folds height in all intestinal segments is given in Table Intestinal folds height in the proximal intestine (PI) was the highest in fish fed with a diet containing 11.8 g valine kgÀ1 diet (P < 0.05) Meanwhile, folds height in the mid (MI) and distal intestines (DI) showed the similar trends with that in PI The folds height in the PI was higher than that in the MI and DI Regression analysis suggested that the folds height in the PI, MI and DI were quadratic response to the levels of dietary valine Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd Table Hepatopancreas weight (HW, g fishÀ1), intestinal weight (IW, g fishÀ1), intestinal length (IL, cm fishÀ1), intestosomatic index (ISI), hepatosomatic index (HSI), relative gut length (RGL), hepatopancreas protein content (HPC) and intestinal protein content (IPC) of juvenile Jian carp fed diets containing graded levels of valine for 60 days Dietary valine levels (g kgÀ1 diet) 5.3 8.7 a HW 1.58 ± 0.09 1.88 HSI1 3.14 ± 0.18c 2.94 HPC2 3.47 ± 0.25a 3.55 IL1 19.23 ± 0.52a 21.23 RGL1 158.96 ± 4.32a 164.22 IW1 1.64 ± 0.06a 2.00 ISI1 3.25 ± 0.12d 3.12 IPC2 4.42 ± 0.12a 4.56 Regressions YHW = 0.307 + 0.290x À 0.011x2 YIL = 12.752 + 1.498x À 0.056x2 YRGL = 136.446 + 5.170x À 0.201x2 YIW = 0.659 + 0.231x À 0.009x2 YISI = 3.648 À 0.084x + 0.002x2 11.8 ± ± ± ± ± ± ± ± b 0.13 0.21b 0.21a 0.76c 5.89b 0.07c 0.11c 0.35a 2.31 3.02 3.81 23.13 171.26 2.25 2.95 4.76 14.9 ± ± ± ± ± ± ± ± e 0.13 0.17bc 0.24a 1.16e 8.61c 0.18e 0.23b 0.19a 18.7 2.13 2.88 3.83 22.44 167.31 2.14 2.89 4.60 R2 R2 R2 R2 R2 = = = = = ± ± ± ± ± ± ± ± d 0.16 0.24b 0.19a 0.73d 5.43bc 0.08d 0.11ab 0.37a 20.1 2.02 2.93 3.98 21.54 165.69 1.96 2.83 4.58 ± ± ± ± ± ± ± ± c 0.09 0.14b 0.36a 1.00c 7.66b 0.08c 0.12a 0.36a 1.63 2.55 3.77 20.28 157.12 1.84 2.88 4.60 0.869 0.953 0.839 0.969 0.979 P P P P P < < = < < ± ± ± ± ± ± ± ± 0.12a 0.19a 0.20a 1.12b 8.71a 0.07b 0.10ab 0.46a 0.05 0.05 0.065 0.01 0.01 Values are mean ± SD of four replicate tanks, with 15 fish in each replicate Mean values with the different superscripts in the same row are significantly different (P < 0.05) Values are mean ± SD of four replicate tanks, with five fish in each replicate Mean values with the different superscripts in the same row are significantly different (P < 0.05) Table Folds height (lm) in proximal intestine (PI), mid intestine (MI), distal intestine (DI) of juvenile Jian carp fed diets containing graded levels of valine for 60 days1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 b PI 1260 ± 115 1473 ± 106 MI 646 ± 62b 814 ± 66d DI 468 ± 45a 733 ± 69d Regressions YPI = 36.026 + 278.299x À 11.303x2 YMI = 238.712 + 106.207x À 4.800x2 YDI = À94.184 + 140.767x À 5.661x2 11.8 c 1773 ± 108 890 ± 71e 830 ± 59e 14.9 e 1670 ± 140 686 ± 61c 670 ± 57c 18.7 d R² = 0.819 R² = 0.899 R² = 0.899 20.1 c 1489 ± 131 510 ± 38a 550 ± 54b 902 ± 67a 480 ± 42a 476 ± 40a P = 0.077 P < 0.05 P < 0.05 Values are means ± SD of four replicate groups, with five fish in each group Mean values with the different superscripts in the same row are significantly different (P < 0.05) The activities of trypsin, lipase, chymotrypsin and amylase in the intestine were enhanced with the increment levels of dietary valine (Table 7) Trypsin and lipase activities were the highest in Jian carp fed with a diet containing 11.8 g valine kgÀ1 (P < 0.05) A similar trend was observed in amylase activity The chymotrypsin activity in the intestine was significantly lower in fish fed with diets containing 5.3 and 20.1 g valine kgÀ1 diet (P < 0.05) Regression analysis showed that the trypsin, chymotrypsin, lipase and Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd amylase in the intestine were quadratic response to the levels of dietary valine Trypsin and lipase activities in the hepatopancreas were the highest in Jian carp fed with the diet containing 11.8 g valine kgÀ1 diet (P < 0.05) Amylase and chymotrypsin activities in the hepatopancreas were the highest in Jian carp fed with the diet containing 14.9 g valine kgÀ1 diet (P < 0.05) Regression analysis showed that the trypsin, lipase and amylase activities in the hepatopancreas were quadratic response to the levels of dietary valine (Table 8) Activities of alkaline phosphatase, Na+, K+-ATPase, c-GT and CK in the intestine are presented in Table Table Activities of tryspin (U gÀ1 tissue), chymotrypsin (U gÀ1 tissue), lipase (U gÀ1 tissue) and amylase (U gÀ1 tissue) in whole intestine of juvenile Jian carp fed diets containing graded levels of valine for 60 days1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 a 11.8 c Trypsin 1.03 ± 0.06 1.49 ± 0.07 Chymotrypsin 3.19 ± 0.31a 3.83 ± 0.24b Lipase 3072 ± 280b 3795 ± 343c Amylase 1369 ± 36a 1417 ± 29b Regressions Yintestine lipase = À1608.316 + 1067.603x À 42.746x2 Yintestine trypsin = 0.050 + 0.243x À 0.010x2 Yintestine amylase = 1217.251 + 35.420x À 1.390x2 Yintestine chymotrypsin = 1.741 + 0.351x À 0.014x2 1.65 3.89 5692 1429 14.9 ± ± ± ± d 0.09 0.27b 297e 23bc 1.46 3.83 4427 1461 R2 R2 R2 R2 = = = = 18.7 ± ± ± ± c 0.12 0.26b 221d 28c 1.19 3.67 3885 1373 20.1 ± ± ± ± b 0.06 0.32b 221c 20a 0.799 0.917 0.814 0.883 1.14 3.08 2259 1377 P P P P = < = < ± ± ± ± 0.09b 0.23a 221a 33a 0.090 0.05 0.080 0.05 Values are mean ± SD of four replicate tanks, with fish in each replicate Mean values with the different superscripts in the same column are significantly different (P < 0.05) Table Activities of tryspin (U gÀ1 tissue), chymotrypsin (U gÀ1 tissue), lipase (U gÀ1 tissue) and amylase (U gÀ1 tissue) in hepatopancreas of juvenile Jian carp fed diets containing graded levels of valine for 60 days1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 Trypsin 0.12 ± 0.01a 0.52 ± 0.02c Chymotrypsin 2.97 ± 0.19a 3.08 ± 0.23a Lipase 5873 ± 533c 6324 ± 560cd Amylase 1101 ± 49a 1163 ± 50bc Regressions Yhepatopancreas trypsin = À0.793 + 0.219x À 0.008x2 Yhepatopancreas lipase = 1601.738 + 999.059x À 43.255x2 Yhepatopancreas amylase = 873.015 + 52.240x À 2.020x2 11.8 0.76 4.03 8222 1190 14.9 ± ± ± ± 0.04e 0.27c 533e 43cd 0.59 5.42 6505 1237 18.7 ± ± ± ± 0.05d 0.36d 485d 28d R2 = 0.917 R2 = 0.847 R2 = 0.862 0.49 4.17 5150 1129 20.1 ± ± ± ± 0.03c 0.15c 454b 43ab 0.40 3.44 4246 1109 ± ± ± ± 0.01b 0.31b 221a 34a P < 0.05 P = 0.060 P = 0.051 Values are mean ± SD of four replicate tanks, with five fish in each replicate Mean values with the different superscripts in the same column are significantly different (P < 0.05) The activities of alkaline phosphatase in the PI, MI and DI were the highest in Jian carp fed with the diet containing 11.8 g valine kgÀ1 diet (P < 0.05) A similar trend was observed in c-GT activity Regression analysis showed that alkaline phosphatase and c-GT activities in the PI, MI and DI were quadratic response to the levels of dietary valine Activity of Na+, K+-ATPase in the PI was not influenced by the levels of dietary valine (P > 0.05) Na+, K+-ATPase activity in the MI was the highest in Jian carp fed with the diet containing 14.9 g valine kgÀ1 diet In the DI, the Na+, K+-ATPase activity was the highest in fish fed with a diet containing 11.8 g valine kgÀ1 diet (P < 0.05) In the MI and DI, Na+, K+-ATPase activity showed a quadratic response to increasing levels of dietary valine The activity of CK in the PI, MI and DI was the highest in fish fed with a diet containing 14.9 g valine kgÀ1 diet Regression analysis showed that the CK activity in the MI was significantly quadratic response to the increasing levels of dietary valine The counts of Lactobacillus, Bacillus, Escherichia coli and Aeromonas in the intestine are presented in Table 10 Lactobacillus populations increased with increasing levels of valine Bacillus significantly improved with higher levels of dietary valine up to 14.9 g valine kgÀ1 diet, and then decreased E coli in the intestine were the lowest in fish fed with diets containing 11.8 and 14.9 g valine kgÀ1 diet (P < 0.05) When the level of dietary valine was 20.1 g valine kgÀ1 diet, intestinal Aeromonas was significantly higher than that of fish fed with diets containing 18.7 g valine kgÀ1 diet (P < 0.05) Regression analysis showed that the populations of intestinal Lactobacillus, E coli and Aeromonas were quadratic responses to increasing levels of dietary valine The importance of dietary valine for normal growth of Jian carp was demonstrated in the present study In this study, a Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd Table Activities of alkaline phosphatase (AKP, mmol of nitrophenol released per gram tissue per hour), Na+,K+-ATPase (lmol of phosphorus released per gram tissue per hour), gamma-glutamyl transpeptidase (c-GT, mmol of 5-amino-2-nitrobenzoate released per gram tissue per minute) and creatinekinase (CK, lmol of phosphorus released per gram tissue per hour) in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of juvenile Jian carp fed diets containing graded of valine for 60 days1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 11.8 14.9 18.7 20.1 1004 ± 84c 1196 ± 101e 116 ± 6.1d 925 ± 101c 929 ± 73d 92.2 ± 8.0c 710 ± 67b 770 ± 60c 90.0 ± 7.0c 630 ± 53b 581 ± 39b 80.0 ± 8.6b 17.2 ± 0.7d 19.9 ± 1.9d 41.9 ± 2.5d 16.8 ± 1.0cd 18.9 ± 1.6d 37.3 ± 2.6c 16.2 ± 1.6cd 16.8 ± 1.6c 31.6 ± 2.8b 15.6 ± 1.2c 16.9 ± 1.0c 29.7 ± 2.6b 405 ± 31a 333 ± 24d 352 ± 15d 433 ± 23a 419 ± 27e 337 ± 17d 404 ± 35a 419 ± 26e 209 ± 17c 405 ± 31a 306 ± 24c 156 ± 11b 206 ± 7.5c 401 ± 26c 189 ± 16c 247 ± 10d 388 ± 23c 203 ± 10d 186 ± 9.1b 315 ± 24b 106 ± 6.3a 147 ± 11a 287 ± 20a 114 ± 8.5a AKP PI 472 ± 36a 631 ± 40b MI 485 ± 5.6a 810 ± 66c DI 68.7 ± 6.9a 92.7 ± 8.0c c-GT PI 9.30 ± 0.71a 12.3 ± 0.9b MI 7.88 ± 0.52a 12.4 ± 1.1b a DI 26.7 ± 2.2 36.3 ± 2.5c Na+, K+-ATPase PI 409 ± 24a 410 ± 15a MI 170 ± 9.6a 216 ± 18b a DI 104 ± 8.5 212 ± 18c CK PI 185 ± 13b 177 ± 10b MI 389 ± 15c 393 ± 17c b DI 144 ± 12 139 ± 9.8b Regressions YAKP in PI = À424.066 + 199.609x À 7.347x2 YAKP in MI = À618.081 + 258.657x À 9.915x2 YAKP in DI = 5.336 + 15.252x À 0.578x2 Yc-GT in PI = À1.817 + 2.441x À 0.079x2 Yc-GT in MI = À8.837 + 3.714x À 0.123x2 Yc-GT in DI = 2.914 + 5.767x À 0.224x2 YNa+,K+-ATPase in MI = À158.188 + 67.137x À 2.072x2 YNa+,K+-ATPase in DI = À352.592 + 103.885x À 3.915x2 YCK in MI = 299.460 + 21.726x À 1.108x2 R2 R2 R2 R2 R2 R2 R2 R2 R2 = = = = = = = = = 0.860 0.885 0.773 0.937 0.914 0.932 0.816 0.939 0.974 P P P P P P P P P = < = < < < = < < 0.052 0.05 0.108 0.05 0.05 0.05 0.079 0.05 0.01 Values are mean ± SD of four replicate tanks, with five fish in each replicate.Mean values with the different superscripts in the same row are significantly different (P < 0.05) Table 10 Effects of diets containing graded levels of valine (g kgÀ1 diet) on Lactobacillus (LB, log CFU gÀ1 intestine content), E coli (EC, log CFU gÀ1 intestine content), Bacillus (log CFU gÀ1 intestine content) and Aeromonas (log CFU gÀ1 intestine content) in whole intestine of juvenile Jian carp1 Dietary valine levels (g kgÀ1 diet) 5.3 8.7 a LB 7.10 ± 0.10 7.14 Bacillus 6.07 ± 0.09a 6.36 EC 7.59 ± 0.03c 7.48 Aeromonas 9.25 ± 0.09a 9.15 Regressions YLactobacillus = 7.371 À 0.084x + 0.006x2 YE coli = 8.136 À 0.127x + 0.005x2 YAeromonas = 9.926 À 0.156x + 0.007x2 11.8 ± ± ± ± a 0.08 0.13bc 0.03b 0.06a 7.16 6.41 7.41 9.13 14.9 ± ± ± ± a 0.03 0.07c 0.03a 0.17a 7.69 7.37 7.40 9.12 18.7 ± ± ± ± b 0.07 0.13d 0.03a 0.03a R2 = 0.918 R2 = 0.944 R2 = 0.770 7.79 6.32 7.62 9.27 20.1 ± ± ± ± b 0.12 0.08bc 0.02c 0.11a 8.36±0.02c 6.20 ± 0.07ab 7.80 ± 0.04d 9.79 ± 0.05b P < 0.05 P < 0.05 P = 0.110 Values are mean ± SD of four replicate tanks, with three fish in each replicate Mean values with the different superscripts in the same row are significantly different (P < 0.05) reduced weight gain was observed in Jian carp fed with the valine-insufficient diet Similar observations have been reported in Indian major carp (Ahmed & Khan 2006) A Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd reduction in feed intake was regarded as the primary factor responsible for the depressed growth observed in Atlantic salmon fry (Rollin et al 2006) Ahmed & Khan (2006) Figure Quadratic regression analysis of feed intake (FI) according to dietary valine levels (a) and against mean valine intake per fish (b)1 1Each point represents the mean of four groups of Jian carp with 50 fish per group demonstrated that a depressed growth rate in Indian major carp (C mrigala) fed a diet containing less than the optimum amount of valine was due to loss of appetite and poor feed efficiency This study also showed that the deficiency of dietary valine caused a decrease in FI Moreover, regression analysis revealed that FI was significantly quadratic response to the increasing levels of dietary valine and significant response to the mean valine intake per fish (Fig 2) Additionally, correlation analysis indicated that the FI was positively related to the SGR (r = +0.999, P < 0.01), PER (r = +0.998, P < 0.01) and FE (r = +0.999, P < 0.01) (Fig 3) These results indicated that the increased SGR, PER and FE in fish fed diet with valine supplementation may be partly associated with the increased FI Bureau et al (2000) found that fish weight gain is associated with the accretion of protein, fat etc In the present study, fish fed with the basal diet (5.3 g valine kgÀ1 diet) showed the lowest body protein and PRV These data indicated that valine enhanced the protein utilization of fish Similar observations were reported in Indian major carp (Ahmed & Khan 2006) Yoshizawa (2004) reported that BCAA (valine, leucine and isoleucine) played an important role in protein synthesis in mammals In addition, fish body lipid content were significantly lower when dietary valine exceeding 14.9 g valine kgÀ1 diet (P < 0.05) A similar trend was found in Indian major carp, Labeo rohita (Hamilton) fry (Abidi & Khan 2004) Based on the quadratic regression analysis for SGR, the valine requirement of juvenile Jian carp was estimated to Figure Linear regression analysis between feed intake (FI) and SGR (a), feed efficiency (FE, b) and protein efficiency ratio (PER, c) of juvenile Jian carp fed diets containing graded levels of valine for 60 days1 1Each point represents the mean of four groups of Jian carp with 50 fish per group be 13.7 g valine kgÀ1 diet (4.0 g valine 100 gÀ1 CP) which was higher than that for common carp (NRC 1993) It may attribute to the difference in fish species Jian carp is the new variety of C carpio in China (Sun et al 1995) and grows 30% faster than the common carp (Dong & Yuan 2002) It is consistent with the studies that Jian carp had higher nutrient requirements than common carp, such as lysine (Zhou et al 2008), pyridoxine (He et al 2009), inositol (Jiang et al 2009), thiamine (Huang et al 2011) and zinc (Tan et al 2011) Lim et al (2001) reported that reduction in the rate of proteolysis and amino acid catabolism resulted in a decrease in ammonia production of mudskippers (periophthalmodon schlosseri and boleophthalmus boddaerti) In this study, the plasma ammonia content was lower for fish fed with optimum levels of dietary valine It suggested that the optimal valine supplementation reduced the production of ammonia, supporting a higher protein efficiency ratio in this group GOT and GPT play an important role in protein and amino acid catabolism (Balogun & Fetuga 1980; Segner & Verreth 1995) In the present study, GOT and Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd experiment, day 55 The feed was provided three times a day in 3-h intervals On day 48, all fish from each tank were weighed and 30 randomly selected fish were distributed back to each tank to decrease density and enhance their growth Sampling of fish was carried out periodically to monitor fish growth The average water temperature was 22.6 ± 0.8 °C (Kwasek et al., in press) The system used was semi-recirculated system with a refreshment rate of 3.3 L minÀ1 per 440 L The city water used was filtered through activated charcoal filters and additionally treated with sodium thiosulphate to keep chlorine level >0.1 mg LÀ1 The outflow water returning to the system was additionally treated with filtration unit (Aquatic Life Support Filtration Unit, Aquanetics System Pak, San Diego, CA, USA) Each tank received a flow rate of 0.3 L minÀ1 The photoperiod was 13 h light: 11 h dark For histological analyses, five fish were sampled from each aquarium before first feeding on the 48 day of the study (15 fish from each experimental group) The fish were anesthetized with MS-222 (Tricaine methanesulphonate; Sigma-Aldrich, Munich, Germany) and preserved in 4% buffered formaldehyde and Bouin’s solution The samples were subjected to a standard histological procedure: whole fish were embedded in paraffin, cut into transverse sections of lm thickness using microtome (Leica RM 2265; Leica Microsystems, Nussloch, Germany) and stained with alcian blue periodic acid-Schiff’s reagent (AB/PAS) pH 2.5, 1.0, 0.5 Periodic acid-Schiff’s reagent was used to stain for glycogen and mucins (carbohydrate compounds) with diastase addition for non-specific staining (PAS/D) (Pearse 1985) Immunohistochemical methods were used to detect gastrin/ CCK, lymphocytes T (CD3), proliferating cell nuclear antigen (PCNA) and oligotransporter (PepT1) Proliferating cells were identified using antibodies directed against PCNA (Ostaszewska et al 2008) Procedure of immunohistochemical staining for PepT1, and gastrin/CCK was described by Ostaszewska et al (2010a) The CD3positive T cells were detected according to Bakke-McKellep et al (2007) Histological analyses were performed to evaluate morphological and morphometric changes in fish hepatocytes and enterocytes Metabolic activity of liver was measured using hepatocyte surface area, number of proliferating hepatocyte nuclei and overall relative volume (in per cent) of cytoplasm regions containing glycogen and lipids Measurements of intestinal fold height, number of goblet cells, gastrin/CCK-positive cells and PCNA-positive nuclei were taken in liver sections and longitudinal sections of intestine of five fish from each experimental group (25-folds fish replicates), using the Nikon ECLIPSE 90i microscope at 4009 magnification, connected with a digital camera Nikon DS5-U1, and a computer image analysis system NIS – Elements AR (Nikon Corporation, Tokyo, Japan) The samples stained with fluorescein isothiocyanate were viewed using the confocal microscope Olympus FV-1000 equipped with FV10-ASW ver 1.4 software (Olympus, Warsaw, Poland) Statistical analysis was performed using one-way ANOVA followed by Duncan multiple test (Statistica 9.0) to evaluate the differences in final body mass and the results of histological and immunohistochemical analysis among experimental groups The results in graphs (Statistica 9.0) and in tables show arithmetic means for each group with significant differences marked (P 0.05) The weight of fish was not different among dietary treatments at days 32 and 48 of the experiment (P > 0.05) After 55 days, however, the weight of juvenile yellow perch fed the LG diet was greater (1.35 ± 0.11 g) than the C (P < 0.05) group but not different from FL and BO groups (1.31 ± 0.03; 1.16 ± 0.10 g, respectively) (P > 0.05) (Fig 2) The results of histological analyses revealed that fish fed BO, C and LG showed correct morphology of posterior intestine The nuclei were located in basal part of enterocytes, and supranuclear regions showed distinct absorptive vacuoles The height of intestinal folds did not significantly differ among the groups (Table 2) Goblet cells secreting acidic (AB/PAS pH 2.5 positive) or sulphate (AB/PAS pH 1.0 positive) glycoproteins were the most abundant between posterior intestine enterocytes of perch fed C diet (Fig 1A), while the least abundant in fish fed FL diet (Fig 1B) (Table 2) Lamina propria in the folds of posterior intestine of LG fed fish was significantly thinner compared to the other groups (Table 2) Endocrine gastrin/CCK-positive cells were observed in the gastric glands (Fig 2A), pyloric caeca (Fig 2B) and in the anterior intestine of fish from all experimental groups However, gastrin/CCK-positive cells were the most numerous in fish fed LG diet (Fig 2C), while the least in fish fed BO (Fig 2D) but the differences were insignificant (Table 2) The CD3-positive cells (T lymphocytes) were observed in posterior intestine of fish from all experimental groups They were present in lamina propria in the folds of basal enterocytes, and they also infiltrated intestinal epithelium Aquaculture Nutrition 19; 100–109 ª 2012 Blackwell Publishing Ltd Table Results of the growth and morphometric data of the intestine and liver of yellow perch juveniles fed commercial Bio Oregon (BO) and experimental diets (C, FL, LG) (mean ± SD, n = 3) Dietary treatment Morphometric data BO Body mass (g) Day 32 Day 48 Day 55 Height of posterior intestine mucosal folds (lm) Number of goblet cells in 100 lm posterior intestine mucosal folds Width of lamina propria in posterior intestine (lm) Number of Gas-positive cells in 25 folds of anterior intestine Number of CD3-positive cells in 100 lm of posterior intestine fold Hepatocyte area (lm2) Number of proliferating cell nuclear antigen-positive cell nuclei per mm2 of liver tissue Values with different letter are significantly different (P C (À) 0.69 0.79 1.16 213.95 5.61 ± ± ± ± ± 0.07a 0.09a 0.10ab 47.86a 1.02b 0.69 0.83 1.12 186.02 7.18 FL (free Lys) ± ± ± ± ± 0.01a 0.02a 0.06a 45.38a 0.65a 0.76 0.90 1.31 205.52 3.72 ± ± ± ± ± 0.02a 0.09a 0.03ab 63.93a 0.04c LG (dipeptide) 0.79 0.91 1.35 197.28 5.30 ± ± ± ± ± 0.04a 0.06a 0.11b 48.19a 0.87b 12.59 ± 3.75a 24.17 ± 3.46a 12.48 ± 3.43a 31.80 ± 5.85a 12.76 ± 2.89a 27.51 ± 6.15a 9.47 ± 2.67b 33.95 ± 5.13a 25.28 ± 5.86ab 21.39 ± 2.91b 30.56 ± 3.77a 11.23 ± 3.43c 135.54 ± 18.18a 546.67 ± 145.95a 111.53 ± 13.93ab 615.24 ± 160.14a 105.76 ± 16.47b 613.33 ± 126.79a 0.05) (a) (b) (a) (b) (c) (d) Figure Longitudinal section of the posterior intestine of yellow perch: (a) fed C diet – numerous goblet cells (arrows); (b) fed FL diet – scarce goblet cells (arrow) Alcian blue periodic acidSchiff’s staining Scale bars: 10 lm Figure Gastrin/cholecystokinin (CCK)positive cells (arrows) in: (a) gastric glands, scale bar 10 lm, and (b) pyloric caeca, scale bar 100 lm Longitudinal section of intestine of fish fed: (c) LG diet – numerous gastrin/CCK-positive cells, (d) Bio Oregon diet – scarce gastrin/ CCK-positive cells Gastrin/CCK staining Scale bars: 10 lm Aquaculture Nutrition 19; 100–109 ª 2012 Blackwell Publishing Ltd 107.20 ± 14.16ab 506.67 ± 132.46a (a) (b) Figure Longitudinal section of posterior intestine of yellow perch: (a) fed FL diet – numerous CD3-positive cells (arrows), (b) fed LG diet – scarce CD3positive cells (arrows) Scale bars: 10 lm Lymphocytes were the most abundant in FL group (Fig 3A) (Table 2) and scarce in LG group (Fig 3B) (Table 2) in which they were observed only in the folds of basal enterocytes In groups FL and C, the CD3-positive cells were found in submucosa, in the folds of basal enterocytes and between epithelial cells Immunopositive reaction indicating the presence of PepT1 oligopeptide transporter was observed in apical part of the brush border of anterior intestine of all fish The brush border of anterior intestine was the most immunopositive in fish fed LG diet (Fig 4A), while the weakest reaction occurred in group fed C diet (Fig 4B) In the (a) remaining experimental groups, the reaction showed intermediate level All fish showed normal structure of liver Hepatocytes were located around sinuses with thin endothelium Histological sections revealed bile ducts and large blood vessels Bile ducts were lined with cubical epithelium and surrounded by connective tissue The largest hepatocytes were observed in fish fed BO diet (Fig 5A), while the smallest in FL group (Fig 5B), the differences being significant (Table 2) Relative volume of lipids in hepatocytes cytoplasm was higher in fish fed BO and FL than in those fed C and LG (Fig 6) Fish fed LG diet relative glycogen and lipid volume were similar (Fig 6) (b) Figure Strong PepT1 immunopositive reaction (arrows) in the brush border of anterior intestine of fish fed LG diet (a) and weak reaction (arrows) in the brush border of fish fed C diet (b) Scale bars 30 lm (a) (b) Figure Cross-section of the liver of yellow perch juveniles fed: (a) Bio Oregon diet – the largest hepatoctes and (b) FL diet – the smallest hepatocytes Scale bars: 10 lm (alcian blue periodic acid-Schiff’s staining) Aquaculture Nutrition 19; 100–109 ª 2012 Blackwell Publishing Ltd 0.9 Glycogen 0.8 Lipids A A 0.7 Relative volume (%) 0.6 Figure The average relative volume (% ± SD, n = 3) of intracellular glycogen and lipid deposition in the hepatocytes Values with different letters are significantly different (P 0.05) B a B 0.5 b 0.4 0.3 c c 0.2 0.1 0.0 BO Number of hepatocyte PCNA-positive nuclei did not significantly differ among the experimental groups; however, they were the most abundant in fish fed C and FL diets (Fig 7A,B) (Table 2) Fish fed LG and BO showed well-developed exocrine pancreas The acinar cells contained abundant proenzyme granules On the other hand, fish fed FL and C diets showed shrunk acinar cells with less proenzyme granules Moreover, in fish fed C diet, local necrotic areas were observed in pancreatic tissue The growth rate of yellow perch in this study was comparable to a similar size fish reported earlier in our laboratory (Rinchard et al 2008) Growth of yellow perch of initial size 0.17 g differed significantly among different commercial diets between 0.47 and 1.4 g The results of this study revealed no adverse effects of WG in yellow perch diet on morphology of fish digestive tract and confirmed earlier observations that WG supplemented with essential amino acids is a good protein source (a) Figure Cross-section of the liver of yellow perch juveniles: (a) fed Bio Oregon, (b) fed C showing immunohistochemical detection of proliferating cell nuclear antigen-positive nuclei (arrows) Scale bars: 10 lm Aquaculture Nutrition 19; 100–109 ª 2012 Blackwell Publishing Ltd C Diets FL LG for fish (Rodehutscord et al 1995; Ostaszewska et al 2010a) Earlier studies showed in fish fed high-plant-protein-based diets pathological changes in intestinal morphology such as goblet cell hypertrophy and hyperplasia, decreases in the number of absorptive vacuoles, increased cellularity of the lamina propria, decreased mucosal folds and infiltration of inflammatory cells in lamina propria or epithelium Such disturbances were observed in Atlantic salmon (Storebakken et al 2000; Krogdahl et al 2003) and in rainbow trout (Refstie et al 2000; Ostaszewska et al 2005) Enteritis in salmonid fishes was not induced by antinutritional factors, such as oligosaccharides and phytates (Sørensen et al 2011) Wheat gluten contains, similarly as soybean, some fibres and anti-nutrients, such as protease inhibitors and phytin (Francis et al 2001) but despite these disadvantages, no significant adverse effects on yellow perch digestive tract were observed This is in accordance with the results of other studies in which a moderate addition of vegetal protein did not disturb morphology of fish intestine (Olsen et al 2007; Ostaszewska et al 2010a) Similarly, even a high level of vegetal protein (25–66%) consisting of SBM, (b) WG meal and corn gluten meal did not alter gut histology in turbot (Psetta maxima) juveniles (Bonaldo et al 2011) Histological analysis of intestinal epithelium morphology of yellow perch revealed that no pathological changes occurred in fish, except for the group fed C diet which showed considerably more goblet cells This indicates alterations in the mucosa structure, disturbances of intracellular digestion and, in consequence, metabolic disorders Hyperplasia and hypertrophy of goblet cells in Atlantic cod (Gadus morhua) were also noted by Olsen et al (2007) in fish fed 100% vegetal protein (WG and soy protein concentrate) Number of goblet cells increased also in the intestine of common carp (Cyprinus carpio) (Ura´n et al 2008a) and trout (van den Ingh et al 1991) fed soybean-containing diets The results of our recent studies of rainbow trout (Ostaszewska et al 2010a) and carp (Ostaszewska et al 2010b) showed that wheat-gluten-based diet deficient in lysine caused an increase in abundance of goblet cells secreting acidic glycoproteins: sialomucins or sulphate mucins Gastrin and CCK are structurally related peptides with common C-terminal pentapeptide amide, the highly conserved sequence contained within both peptides displays unavoidable cross-reactivity with all gastrin/CCK-like peptides (Himick & Peter 1994) Many authors suggest that in fish, gastrin and CCK-8 are located in the same neuroendocrine cells of pyloric part of stomach and in pyloric caeca (Garcı´ a Herna´ndez et al 1994; Reinecke et al 1997) However, Bosi et al (2004) using double-immunofluorescent staining detected both gastrin and CCK-8 in numerous neuroendocrine cells of trout pyloric caeca and anterior intestine Gastrin alone was present in the stomach and CCK-8 exclusively in central part of the intestine The presence of gastrin-positive neuroendocrine cells in pylorus and anterior intestine of yellow perch was reported by Reifel et al (1983) In the present study, gastrin/CCK-positive neuroendocrine cells were found mainly in gastric glands and less numerous also in pylorus portion, pyloric caeca and in the anterior intestine Similarly as in mammals, also in fish gastrin occurs in fundic exocrine cells that produce HCl and pepsinogen and are also called oxyntopeptic cells (Bjenning & Holmgren 1988) In northern snakehead (Channa argus) and yellow catfish (Pelteobagrus fulvidraco), gastrin immunoreactive neuroendocrine cells were observed only in the stomach (Pan et al 2000) On the other hand, in Japanese flounder (Paralichthys olivaceus) (Kurokawa et al 2003), gastrin immunoreactive cells occurred in the intestine Similar to yellow perch, location of gastrin/CCK-positive cells was observed in brown trout (Salmo trutta) (Bosi et al 2004) The results of present study confirmed that release of CCK and gastrin is stimulated by the ingestion of a meal and its chemical composition in most single-stomached species (Liddle 2000) Fish intestinal epithelium contained similar number of gastrin/CCK neuroendocrine cells, and no significant differences among experimental groups were found Slightly lower density of these cells occurred in fish fed commercial diet containing FM as main protein source However, in the rat, food restriction induced a reduction in pancreatic function (decrease in amylase secretion) by a mechanism that evidently involves a decrease in CCK release and a down regulation of the CCK receptors (Chowdhury & Rayford 2001) These data suggest that CCK plays an important physiological role in adaptation to food deficiency and thereby to a lowering of body size in rats and possibly in other mammals The highest number of gastrin/CCK-positive cells was observed in the intestine of fish fed diet supplemented with LG, which confirms observations made by Cuber et al (1990), who found out that oligopeptides induced CCK secretion in rat duodenum and polypeptides and single amino acids reduced the level of CCK Lymphocyte-like cells were observed between enterocytes of common carp (Huttenhuis et al 2005), sea bass (Dicentrarchus labrax) (Abelli et al 1997), rainbow trout (McMillan & Secombes 1997) and some other species of Teleostei In yellow perch, the CD3-positive cells were observed intercellularly at the base of the posterior intestine epithelium, in lamina propria and submucosa The highest number of CD3-positive cells was found in posterior intestine of fish fed FL diet Alterations in small intestine induced by WG include atrophy of villi, hyperplasia in crypts and infiltration of inflammatory cells in lamina propria and intestinal epithelium (Sollid 2004) The results of present study not support this observation because no inflammation occurred in the intestine of fish fed gluten-based diets However, an increase in density of CD3-positive cells was noted in the basal region of epithelium lining the lamina propria, and migration of these cells towards apical part of epithelial cells resulted rather from lysine deficiency in C diet, and the presence of free amino acids in FL diet Yellow perch did not show enteritis No symptoms such as reduced intestinal fold height, lack of supranuclear vacuoles in enterocytes, enlargement of central stroma within the mucosal folds, connective tissue hypertrophy or deep infiltration of inflammatory cells into lamina propria and submucosa (Lilleeng et al 2009) were observed Posterior Aquaculture Nutrition 19; 100–109 ª 2012 Blackwell Publishing Ltd intestine enteritis was observed in Atlantic salmon fed diets containing soybean saponins (Knudsen et al 2007); however, in purified WG used in this experiment with perch, saponins were not present Absorption of nutrients from vertebrate intestinal lumen involves peptide transporters that bind them to brush border and transfer into the cells across cell membranes (Chen et al 2005; Verri et al 2003) Among these proteins, the PepT1 plays a key role in absorption of protein hydrolysis products such as di- and tripeptides Short peptides, di- and tri-peptides are transported by the oligotransporter PepT1 in the intestinal folds of Teleostei (Clements & Raubenheimer 2006; Verri et al 2003) The results obtained for zebrafish (Danio rerio) (Verri et al 2003), cod (Rønnestad et al 2007), rainbow trout (Ostaszewska et al 2010a) and common carp (Ostaszewska et al 2010b) revealed that PepT1 transporter protein is active mainly in the brush border of anterior and middle intestine Similar location of PepT1 transporter in the yellow perch intestine was observed in the present study Ostaszewska et al (2010b) found that feeding common carp wheat-gluten-based diet supplemented with LG stimulated both expression of PepT1 gene and the level of transporter protein itself in the anterior intestine brush border On the other hand, deficiency of lysine in fish diet adversely affected both fish growth and PepT1 gene expression (Ostaszewska et al 2010b) Similarly, the results of present study also showed a positive effect of Lys-Gly supplementation on immunofluorescent expression of PepT1 protein in anterior intestine of yellow perch Weaker immunopositive reaction was observed in fish fed lysine-deficient diet On the contrary, the results obtained by Bakke et al (2010), revealed no significant effect of various protein sources in diet on expression of PepT1 oligotransporter gene The livers of yellow perch showed correct structure and no changes indicating fatty degeneration (staetosis) Fish fed higher-plant-protein diets showed an increase in hepatic lipid content (Kjær et al 2009) The results of our study on common carp also revealed that feeding fish wheat-glutenbased diets supplemented with dipeptide or free amino acids, similarly as lysine-deficient diet, may induce higher hepatic lipid level (Ostaszewska et al 2010b) On the contrary, in the present study, the highest lipid content in cytoplasm of hepatocytes was observed in group fed commercial diet (BO) The largest hepatocyte size of fish fed BO diet indicates excessive fat content in this feed (almost three times higher than in formulated diets) which probably was the cause of increased hepatic lipid content Aquaculture Nutrition 19; 100–109 ª 2012 Blackwell Publishing Ltd in this group of fish Elevated hepatic lipid level was observed also in FL group No significant differences in the number of PCNA-positive hepatocyte nuclei were observed among experimental groups of fish However, slightly elevated hepatocyte proliferation rate occurred in fish fed C and FL diets Similar increase in hepatocyte proliferation rate was observed in rat fed free amino acids compared to animals given wheybased diet (Poullain et al 1989) The higher number of PCNA-positive cell nuclei in liver of fish fed C and FL diets compared to the other groups indicates rather damage repair than a proliferative response to dietary components (Chung et al 2001) The results of present study revealed a positive effect of LG dipeptide supplement on fish body mass, health status, digestive tract morphology, function and hepatic performance The present work was supported by the USDA Special Grant # 600006883 and the Ministry of Science and Higher Education N31103032/2256 Abdelghany, A.E (2003) Partial and complete replacement of fish meal with gambusia meal in diets for red tilapia ‘Oreochromis niloticus O mossambicus’ Aquacult Nutr., 9, 145–154 Abelli, L., Picchietti, S., Romano, N., Mastrolia, L & Scapigliati, G (1997) Immunohistochemistry of gut-associated lymphoid tissue of the sea bass Dicentrarchus labrax (L.) 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Fish Shellfish Immunol., 25, 751–760 Ura´n, P.A., Aydin, R., Schrama, J.W., Verreth, A.J & Rombout, J.H.W.M (2008b) Soybean meal-induced uptake block in Atlantic salmon Salmo salar distal enetrocytes J Fish Biol., 73, 2571– 2579 Verri, T., Kottra, G., Romano, A., Tiso, N., Peric, M., Maffia, M., Boll, M., Argenton, F., Daniel, H & Storelli, C (2003) Molecular and functional characterisation of the zebrafish (Danio rerio) PEPT1-type peptide transporter FEBS Lett., 549, 115–122 Vigna, S.R (2000) Evolution of the cholecystokinin and gastrin peptides and receptors Am Zool., 40, 287–295 Volkoff, H & Peter, R.E (2006) Feeding behavior of fish and its control Zebrafish, 3, 131–140 Volkoff, H., Canosa, L.F., Unniappan, S., Cerd′a-Reverter, J.M., Bernier, N.J., Kelly, S.P & Peter, R.E (2005) Neuropeptides and the control of food intake in fish Gen Comp Endocrinol., 142, 3–19 Aquaculture Nutrition doi: 10.1111/j.1365-2095.2012.00949.x 2013 19; 110–116 Departamento de Produccio´n Animal, Facultad de Veterinaria, Universidad de Leo´n, Leo´n, Spain The aim of this study was to evaluate the effects of practical diets with different levels of vitamin C on survival and growth of juvenile crayfish (Pacifastacus leniusculus) An 80-day trial was conducted with stage juveniles from the onset of exogenous feeding Four practical diets differing in the content of L-ascorbyl-2-monophosphate-Na were prepared: 0, 0.2, 0.4 or 0.8 g kgÀ1 diet Each diet was tested on grouped or individually isolated crayfish Diets had no significant effects on survival of crayfish held in groups (average: 73.1%) neither on the isolated ones (average: 90%) Crayfish fed the diet with 0.2 g kgÀ1 of L-ascorbyl-2monophosphate-Na had the highest growth (groups: 13.64 mm carapace length (CL), 542.4 mg weight; isolated: 17.05 mm CL, 1213.2 mg weight) and the lowest feed conversion ratio (average of grouped and isolated: 0.99) This study provides the first data on vitamin C requirements of freshwater crayfish A level of 0.2 g L-ascorbyl-2-monophosphate-Na kgÀ1 diet (0.07 g ascorbic acid equivalent) can be recommended for juvenile P leniusculus during the first period of intensive rearing KEY WORDS: astacid crayfish, juvenile rearing, Pacifastacus leniusculus, practical diet, vitamin C Received 20 July 2011, accepted February 2012 Correspondence: Jesu´s D Celada, Departamento de Produccio´n Animal, Facultad de Veterinaria, Universidad de Leo´n, Campus de Vegazana s/n, 24071 Leo´n, Spain E-mail: jdcelv@unileon.es There are more than 540 recognized species of freshwater crayfish in the world (Hobbs 1988) Several of them, mostly belonging to the family Astacidae, have attracted a strong interest for aquaculture purposes However, juvenile astacid culture under controlled conditions remains largely unsuccessful During three decades, the main constraints include poor survival and growth rates during the first months of independent life As feeding is a decisive factor in this critical period, a wide variety of natural feeds (mainly fresh or frozen animals and vegetables) and dry diets formulated for other aquatic species either alone or supplemented with natural feeds have been tested to improve survival and growth rates of astacid juveniles from the onset of exogenous feeding (Gonza´lez et al 2010) Recent advances (Gonza´lez et al 2008, 2009, 2012) have provided improvements on feeding schedules, opening the possibility of approaching a new step: a specific practical diet for juvenile astacid crayfish Thus, Carral et al (2011) prepared an extruded diet, which was tested as the sole feed from the onset of exogenous feeding The good results obtained showed its feasibility to be used as a basis for further nutritional studies on juvenile crayfish Vitamins are essential for early stages of crustaceans, and their absence leads to rapid death of animals (Cahu 2004) The water-soluble vitamin C (ascorbic acid) is known to take part in reactions within the cells, related to its ability to undergo reversible oxidation and reduction (Conklin 1997) Ascorbic acid is also involved in the biosynthesis of steroid hormones and collagen, and has been shown to improve immune response and tolerance to toxicants and environmental stressors (Waagbø 2010) Its inclusion in formulated diets is considered necessary, although involves problems linked to losses of bioactivity Ascorbic acid is the most sensitive vitamin to degradation during feed processing and storage (He & Lawrence 1993) Moreover, in decapod crustaceans, which are slow feeders that tear and reduce the feed particles before ingestion, ª 2012 Blackwell Publishing Ltd leaching of water-soluble vitamins is a significant problem (Conklin 1997) Thus, suggested dietary levels of vitamin C in crustaceans depend on its form Simple ascorbic acid results in the level of apparent feed requirement being much higher than the biological requirement (Conklin 1997), and up to 10 g kgÀ1 diet have been recommended for crustacean larvae (Cahu 2004) However, provision of any of the ascorbic acid stabilized forms, as phosphate moieties, at much lower levels is recommended for crustacean diets (Conklin 1997) Most knowledge about vitamin C requirements in crustaceans has been derived from studies of earlier stages of cultured species as Penaeus (Litopenaeus) vannamei (He & Lawrence 1993; Niu et al 2009), Penaeus monodon (Hsu & Shiau 1998), Penaeus indicus (Reddy et al 2001), Macrobrachium rosenbergii (D’Abramo et al 1994) or Marsupenaeus japonicus (Moe et al 2004, 2005) These studies showed that vitamin C is an essential nutrient for the tested species Regarding astacid crayfish, there is very little knowledge on nutrition, and vitamins requirements are totally unknown The aim of this study was to evaluate the effects of practical diets with different levels of vitamin C (phosphate form) on survival and growth of juvenile signal crayfish from the onset of exogenous feeding In late November, egg-bearing females (Pacifastacus leniusculus Dana) from a crayfish farm were transferred to the laboratory After egg development, hatching and first moult, stage juveniles were obtained under controlled conditions An 80-day experiment was conducted with stage juveniles from the start of first feeding (average: 5.31 ± 0.04 mm carapace length (CL) and 30.35 ± 0.4 mg weight) A total of 1240 juveniles were housed in fibreglass tanks in groups at a stocking density of 100 mÀ2, and to avoid possible influence of intraspecific behavioural interactions such as cannibalism, individual isolation of crayfish was also carried out Thus, four feeding treatments were tested on groups (three tanks for each treatment, n = 300) and individually isolated animals (10 replicates for each treatment) Grouped crayfish were kept in 12 tanks with m2 surface and 200 L water Each tank was provided with shelters: four groups of four jointed sections of PVC pipe (4 cm long 20 mm diameter) and three pieces of undu- Aquaculture Nutrition 19; 110–116 ª 2012 Blackwell Publishing Ltd lating fibre cement (54 20.5 cm) Isolated crayfish were kept individually in 40 tanks (0.15 m2, 25 L water) with one group of four jointed sections of PVC pipe (4 cm long 20 mm diameter) as shelter Aerated artesian well water was supplied in open system (flow throughout), and each tank had its own water inlet (flow rate: 1.5 L minÀ1 for the tanks of 200 L and 0.25 L minÀ1 for the tanks of 25 L) and outlet, provided with a 250-lm mesh filter outlet to avoid the escape of crayfish and feed The parameters of incoming water quality were pH 8.1, hardness 5.2°dH (German degrees, calcium 32.3 mg LÀ1), total dissolved solids 112.5 mg LÀ1 and total suspended solids 36.7 mg LÀ1 Throughout the trial, dissolved oxygen content was measured in the tanks every other day, and values were around mg LÀ1 (range was 6.2–8.3) Ammonia and nitrites were measured once a week from water samples taken from inside the tanks; values were always as follows: ammonia [...]... ration levels for Atlantic salmon, Salmo salar Aquaculture, 2 61, 215– 224 Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd Aquaculture Nutrition 2013 19; 35–44 doi: 10.1111/j.1365-2095.2011.00921.x 1 2, 2 2 1 1 1 The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan; Animal Nutrition, Faculty of Fisheries, Kagoshima University,... lysine requirement of juvenile Jian carp (Cyprinus carpio var Jian) Aquacult Nutr., 14, 381–386 Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd Aquaculture Nutrition 2013 19; 15–34 1 1 3 1 1 doi: 10.1111/j.1365-2095.2011.00927.x 2 2 4 Faculty of Biosciences and Aquaculture, University of Nordland, Bodø, Norway; 2 Department of Biological and Environmental Sciences,... Biosciences and Aquaculture, University of Nordland, Bodø, Norway Johnsen, C.A., Hagen, Ø & Bendiksen, E.A˚ (2011) Long-term effects of high-energy, low-fishmeal feeds on growth and flesh characteristics of Atlantic salmon (Salmo salar L.) Aquaculture, 312, 109–116 Johnston, I.A (1999) Muscle development and growth: potential implications for flesh quality in fish Aquaculture, 177, 99–115 Aquaculture. .. in the size range of 40–60 lm were most abundant in the 1+ group, but shifted towards 1100 g, being higher for the 0+ group (Fig 5e) During the Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd – – – – – – 79* 1.2 5.5* 1.9 612 9.4 85.3 3.8 58 1.5 5.5 1.5 ± ± ± ± 30 ± 6* 14.8 ± 1.0 1.08 ± 0.10* 4... and chymotrypsin (Hitoshi et al 2007) In this study, there are significant correlation between trypsin, lipase and amylase activities in intestine and hepatopancreas (rtrypsin = +0.9 01, P < 0.05; Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell Publishing Ltd rlipase = +0.914, P < 0.05; ramylase = +0.983, P < 0.01) These data indicated that the increase of intestine digestive enzyme activities... apparent satiation Pellet size and dietary composition changed in accordance with commercial practice (dietary protein; 470 – > 350 g kg 1, and dietary lipid; 220 g kgÀ1 –> 350 g kgÀ1) as the fish grew larger, resulting in a concomitant increase in Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd dietary energy (gross energy; 22.3 –> 25.6 MJ kgÀ1) Dietary astaxanthin content... digestive enzyme activity Aquacult Res., 41, 861–870 Dong, Z.J & Yuan, X.H (2002) The utilizations of heterosis in common carp in China Aquacult Asia, 12, 14–15 Douglas, S.E., Gallant, J.W & Bullerwell, C.E (1999) Molecular investigation of aminopeptidase N expression in the winter flounder, Pleuronectes americanus J Appl Ichthyol., 15, 80–86 Aquaculture Nutrition, 19; 1–14 ª 2012 Blackwell... Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd was constant towards the final sampling (Fig 7) At the 3000 and 4300 g sampling points, fillets were firmest in the 0+ group (P = 0.033 and 0.015, respectively) and not influenced by body weight when analysis was performed on standardised muscle blocks However, firmness was strongly dependent on body weight (r2 = 0.880, P < 0.0 01,. .. differences between smolt groups (0+ CG vs 1+ CG) are indicated with asterisks; *0.01 < P < 0.05 or ***P < 0.001 Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd tions, explaining 13.5% and 22.6% of the total variation respectively (linear, P < 0.0 01, n = 320) The a*-value increased in fish from both smolt groups during the experiment (Tables 2 & 3), but dropped in the 1+... at the initial (40 g) sampling point, being higher in the 0+ (768 g kgÀ1) than 1+ group (732 g kgÀ1), and decreased to Aquaculture Nutrition, 19; 15–34 ª 2012 Blackwell Publishing Ltd 670 g kgÀ1 and 649 g kgÀ1 towards the final sampling, respectively (P < 0.0 01, Fig 8) With the exception of the 1100 and 2000 g sampling points, water remained highest in the 0+ group Reduced meal frequency