Optimal dietary protein level and protein to energy ratio for hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles

Aquaculture 465 (2016) 28–36

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Optimal dietary protein level and protein to energy ratio for hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles Shuntian Jiang, Xiaoyi Wu ⁎, Yuan Luo, Mingjuan Wu, Senda Lu, Zibo Jin, Wei Yao Department of Aquaculture, Ocean College of Hainan University, Haikou 570228, China

a r t i c l e

i n f o

Article history: Received 26 June 2016 Received in revised form 21 August 2016 Accepted 23 August 2016 Available online 24 August 2016 Keywords: Hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) Growth Protein Energy

a b s t r a c t Two consecutive growth trials were undertaken to study optimal dietary protein level and protein to energy ratio of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles. In the first trial (trial 1), seven isolipidic and isoenergetic experimental diets were formulated to contain 38%, 42%, 46%, 50%, 54%, 58%, or 62% dietary crude protein (CP) level (dry-matter basis), being abbreviated as P38, P42, P46, P50, P54, P58 and P62, respectively. Each diet was given to triplicate groups (initial average body weight, 3.58 ± 0.05 g/fish) for 7 weeks. Weight gain (WG%) of fish fed with P38 and P42 were significantly lower (P b 0.05) than fish fed with P46, P50, P54, P58 or P62. Fish fed with P46 had significantly lower WG% than fish fed with P62. Fish fed with P38 exhibited significantly higher feed conversion ratio (FCR) than fish fed with P46, P50, P54, P58 or P62. Based on the quadratic regression model at 95% of maximum response of WG%, optimal dietary protein level of hybrid grouper juveniles was estimated to be 53.5% of dry matter. In trial 2, the other seven isonitrogenous (53.5% of dry matter) experimental diets were formulated to contain various dietary protein/energy ratios of 191, 173, 157, 145, 134, 124 and 116 mg/kcal (P/E191, P/E173, P/E157, P/E145, P/E134, P/E124 and P/E116), respectively. Each diet of trial 2 was given to triplicate groups (initial average body weight, 10.98 ± 0.15 g/fish) for 8 weeks. WG% of fish fed the diet with P/E191 was significantly lower than that of fish fed the diet with P/E173 or P/E157, and there were no significant differences in WG% values among fish fed diets with P/E191, P/E145, P/E134, P/E124 and P/E116. Values of DFI of fish fed P/E191 and P/E173 were significantly higher than those of fish fed P/E134, P/E124 and P/E116. Fish fed P/E191 exhibited significantly higher FCR than fish fed P/E157, P/E145, P/E134, P/E124 or P/E116, and fish fed P/E173 showed significantly higher FCR than fish fed P/ E124 or P/E116. IPF together with lipid contents of whole-body and liver were increased as dietary P/E ratio decreased. Results of trial 2 indicated that dietary P/E ratio of 157 mg/kcal was optimal for hybrid grouper juveniles. © 2016 Published by Elsevier B.V.

1. Introduction Protein, as the largest single cost factor in feeds (Islam and Tanaka, 2004; Hatlen et al., 2005), plays an important component in fish feed due to its necessity for maintaining fast growth of fish (NRC, 2011). High levels of good-quality protein generally result in rapid growth rates, especially for carnivorous fish (McGoogan and Gatlin, 1999), but when dietary protein level is increased excessively relative to energy, protein beyond that actively needed for growth can be broken down and used for energy, leading to not only the rising of feed cost but also more nitrogen loss to water environments (McGoogan and Gatlin, 2000; Webb and Gatlin, 2003; Wu and Gatlin, 2014), and the increasing of nitrogen in forms of ammonia in water might affect voluntary feed intake and growth of fish (Kaushik and Medale, 1994). Therefore, a formulated diet should provide individuals with adequate protein for ⁎ Corresponding author. E-mail address: [email protected] (X. Wu).

http://dx.doi.org/10.1016/j.aquaculture.2016.08.030 0044-8486/© 2016 Published by Elsevier B.V.

maximal growth and production, but excess protein should be avoided. A number of studies have been performed to optimize dietary protein requirements for some members of grouper family (Teng and Chua, 1978; Chen and Tsai, 1994; Shiau and Lan, 1996; Williams et al., 2004; Luo et al., 2004; Usman et al., 2005; Tuan and Williams, 2007; Shapawi et al., 2014; Wang et al., 2016), but, as with other animals, requirements may differ among fish species and age classes. Dietary protein to energy (P/E) ratio plays an important role in fish nutrition (Ellis and Reigh, 1991; Boujard and Médale, 1994) and can affect growth performance, feed efficiency and body composition of fish (Wang et al., 2016). Previous studies showed that dietary protein could be spared by adequate available dietary non-protein energy in grouper (E. malabaricus) (Shiau and Lan, 1996) as well as in other fish species (El-Sayed and Teshima, 1992; Grisdale-Helland and Helland, 1997; Nankervis et al., 2000; Morais et al., 2001). In cases where the dietary P/E ratio is unbalanced so that available non-protein energy is inadequate, fish may use dietary protein as an additional energy source to satisfy maintenance before growth (Cowey and Sargent, 1979; NRC,

S. Jiang et al. / Aquaculture 465 (2016) 28–36

1993), thus, decreasing growth and production (Schlosser et al., 2005; Hammer et al., 2006). Conversely, excessive non-protein energy can reduce feed intake (Lovell, 1979), produce fatty fish (Reinitz et al., 1978; Fu et al., 2001) and inhibit the utilization of other nutrients (Winfree and Stickney, 1981). Therefore, balancing the protein to energy ratio of aquaculture feed is essential for efficient protein deposition (Cuzon and Guillaume, 1997). Winfree and Stickney (1981) reported that maintaining the balance of protein and energy ratio in feed can be conducive to the use of feed energy. The optimum dietary P/E ratio (mg protein/ kcal) has been determined for several other grouper species (a review, Williams, 2009): C. altivelis, 122 (a fish size of 11–30 g, 71% dietary CP and 16% dietary CL), 110 (a fish size of 11–30 g, 69% dietary CP and 26% dietary CL), or 105 (a fish size of 136–320 g, 56% dietary CP and 22% dietary CL); E. coioides, 159 (a fish size of 11–24 g, 57% dietary CP and 10% dietary CL) or 156 (a fish size of 11–24 g, 53% dietary CP and 10% dietary CL); E. malabaricus, 110 (a fish size of 17–84 g, 55% dietary CP and 12.5% dietary CL), 99 (a fish size of 4–13 g, 46.4% dietary CP and 8.1% dietary CL), 141 (a fish size of 9–61 g, 51% dietary CP and 7.9% dietary CL), or 119 (a fish size of 11–49 g, 51% dietary CP and 17% dietary CL), which indicated that optimum dietary P/E ratio may vary not only among species and age classes but also with the variations in dietary CP or CL levels. Hybrid grouper between the brown-marbled grouper (Epinephelus fuscoguttatus) and giant grouper (Epinephelus lanceolatus) has been largely cultured in China due to its increasing market demand in recent years, but up to date, there has little information on nutrition of this species. Jiang et al. (2015) reported that growth of juvenile hybrid grouper was not negatively affected when reducing dietary protein level from 55% to 45%, and results from the other study (Rahimnejad et al., 2015) recommended 50% of dietary crude protein level, 14% dietary crude lipid level as well as 23.9 mg protein kJ−1 of dietary P/E ratio for efficient growth of juvenile hybrid grouper. Both studies above only designed four different dietary protein levels (40%, 45%, 50% and 55%), which are not enough for using the quadratic regression model to establish optimal dietary protein requirement, and also, the ranges of dietary energy levels designed in these two studies (3.60 kcal/g, 4.19–5.16 kcal/g) were too narrow for determining the optimal dietary P/E ratio for this fish. Therefore, one of the aims in this study was to determine optimal dietary protein requirement for hybrid grouper, and the other aim was to establish optimal dietary P/E ratio at the optimal dietary protein level. 2. Materials and methods 2.1. Experimental diets and designs Two consecutive trials were designed. In trial 1, seven isolipidic (7% of dry matter) and isoenergetic (360 kcal per 100 g dry matter) experimental diets (Table 1) were formulated to contain 38%, 42%, 46%, 50%, 54%, 58%, or 62% dietary crude protein (CP) level (dry-matter basis), being abbreviated as P38, P42, P46, P50, P54, P58 and P62, respectively. 7% of dietary crude lipid level designed in this study was based on the results from a previous study (Jiang et al., 2015). Since digestible energy coefficients for the ingredients used are not available for hybrid grouper, gross energy was calculated using physiological fuel values of 4.0, 4.0 and 9.0 kcal/g (16.7, 16.7 and 37.7 kJ/g) for carbohydrate, protein and lipid, respectively (Garling and Wilson, 1977; Lee and Putnam, 1973). Amino acids composition of experimental diets in trial 1 was shown in Table 2. In trial 2, seven isonitrogenous experimental diets (Table 3) were formulated to contain various dietary protein/energy ratios of 191, 173, 157, 145, 134, 124 and 116 mg/kcal (P/E191, P/E173, P/E157, P/E145, P/E134, P/E124 and P/E116), respectively, and based on the results of trial 1, dietary crude protein level in trial 2 was designed at 53.5% of dry matter. Fishmeal was well ground, and all dry ingredients were carefully weighed and mixed in a Hobart mixer (A-200 T Mixer Bench Model unit, Resell Food Equipment Ltd., Ottawa, Canada) for 30 min, where

29

Table 1 Formulations and analyzed composition of experimental diets (dry-matter basis) in trial 1. Dietary crude protein levels % 38 Peruvian fishmeal (Anchovy)a Casein Chile fish oil (Salmon)b Vitamin mixturec Mineral mixtured Soya bean lecithin Corn starch Carboxymethyl cellulose Cellulose Analyzed composition of dietse Dry matter % Crude protein % Crude lipid % Ash % (Crude fiber + NFE)f Gross energy (kcal/g)g Crude protein/gross energy (mg/kcal)

42

46

50

54

58

62

15.17 21.03 26.89 32.74 38.60 44.46 50.32 31 4.97 1 0.5 1 36.25 1 9.11

31 4.57 1 0.5 1 32.25 1 7.65

31 4.17 1 0.5 1 28.25 1 6.19

31 3.77 1 0.5 1 24.25 1 4.73

31 3.38 1 0.5 1 20.25 1 3.27

31 2.98 1 0.5 1 16.25 1 1.81

31 2.58 1 0.5 1 12.25 1 0.35

89.9 37.2 6.9 4.4 51.1 3.56 104

89.4 42.2 6.6 5.1 46.3 3.57 118

89.7 45.8 6.8 5.7 42.7 3.57 128

92.9 49.4 6.6 7.1 39.2 3.54 140

94.3 53.7 6.9 8.4 34.6 3.58 150

94.7 57.8 6.3 8.1 31 3.53 164

94.4 61.9 7.0 8.3 26.3 3.60 172

a Yongsheng Feed Corporation, Binzhou, China; proximate composition (% dry matter): moisture, 8.9; crude protein, 73.6; crude lipid, 7.6. b High Fortune (Fujian) Bio-Tech Co. Ltd., Fuzhou, China. c Vitamin mixture (mg/g mixture): thiamin hydrochloride, 2.5; riboflavin, 10; calcium pantothenate, 25; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; ascorbic acid, 50; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 20; retinol acetate, 1; cholecalciferol, 0.0025; biotin, 0.25; vitamin B12, 0.05. All ingredients were diluted with alpha-cellulose to 1 g (from Lin and Shiau, 2003). d Mineral mixture (mg/g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4·2H2O, 135.8; MgSO4·7H2O, 132; Na2HPO4·2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO4·7H2O, 3; CoCl2·6H2O, 1; MnSO4·H2O, 0.8; KI, 0.15; AlCl3·6H2O, 0.15; CuCl2, 0.1 (from Lin and Shiau, 2003). e Values represent means of duplicate samples. f [Crude fiber + nitrogen free extract (NFE)] = 100 − (% ash + % protein + % lipid). g By calculation: Fuel values for carbohydrate, protein and lipid were 4.0, 4.0 and 9.0 kcal/g (16.7, 16.7 and 37.7 kJ/g), respectively.

after oil was gradually added, while mixing constantly. Then, 30– 50 mL of water was slowly blended into the mixture for each 100 g of dry matter. The diets were produced in a noodle-like shape of 3-mm in diameter using a twin-screw extruder (Institute of Chemical Table 2 Amino acid compositions (%) of experimental diets (dry-matter basis) in trial 1.a AA/∑AA

Dietary crude protein levels % 38

42

46

50

54

58

62

EAA Lysine Arginine Methionine Threonine Isoleucine Leucine Phenylalanine Valine Histidine ∑EAA

2.52 1.46 0.93 1.52 1.81 3.49 1.46 2.01 0.93 16.12

2.66 1.71 1.03 1.71 2.00 3.83 1.59 2.21 1.02 17.75

2.94 2.00 1.17 1.94 2.17 4.17 1.72 2.38 1.10 19.58

3.38 2.16 1.22 2.08 2.36 4.54 1.87 2.60 1.23 21.43

3.68 2.48 1.43 2.30 2.53 4.82 1.97 2.76 1.31 23.26

4.11 2.78 1.54 2.47 2.75 5.16 2.15 2.96 1.42 25.33

4.16 2.94 1.47 2.71 2.64 5.65 2.29 2.96 1.28 26.10

NEAA Aspartic acid Serine Glutamic acid Glycine Alanine Cystine Tyrosine Proline ∑NEAA ∑AA

2.79 1.70 9.28 1.05 1.46 0.29 1.58 3.11 21.26 37.38

3.23 1.89 10.13 1.28 1.68 0.33 1.73 3.35 23.62 41.37

3.64 2.09 10.83 1.51 1.96 0.35 1.94 3.46 25.78 45.36

3.97 2.27 11.75 1.65 2.14 0.38 2.02 3.71 27.88 49.31

4.36 2.42 12.36 1.92 2.45 0.43 2.29 3.84 30.06 53.32

4.75 2.53 12.82 2.19 2.70 0.53 2.44 3.96 31.93 57.26

4.95 2.65 14.02 2.21 2.85 0.48 2.49 5.47 35.14 61.24

a

Values represent means of duplicate samples.

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S. Jiang et al. / Aquaculture 465 (2016) 28–36

Table 3 Formulations and analyzed composition of experimental diets (dry-matter basis) in trial 2. Dietary protein to energy ratio (mg protein/kcal) 191 Peruvian fishmeal (Anchovy)a Casein Chile fish oil (Salmon)b Vitamin mixturec Mineral mixtured Soya bean lecithin Corn starch Carboxymethyl cellulose Cellulose Analyzed composition of dietse Dry matter % Crude protein % Crude lipid % Ash % (Crude fiber + NFE)f Gross energy (kcal/g)g Crude protein/gross energy (mg/kcal)

173

157

145

134

124

116

47.54 47.54 47.54 47.54 47.54 47.54 47.54 22 2.33 1 0.5 1 0.75 1 23.88

22 4.33 1 0.5 1 3.75 1 18.88

22 7.33 1 0.5 1 4.5 1 15.13

22 10.33 1 0.5 1 5.25 1 11.38

22 13.33 1 0.5 1 6 1 7.63

22 16.33 1 0.5 1 6.75 1 3.88

22 19.33 1 0.5 1 7.5 1 0.13

90.1 53.7 6.7 8.4 33.9 2.78 193

91 53.5 8.1 8.1 32.7 3.02 177

93.4 53.6 11.1 8.2 29.5 3.32 161

92.9 53.1 14.7 8.2 26.5 3.66 145

81.3 54.1 17.0 8.3 23.1 3.93 138

86.8 53.2 20.3 8.3 20.8 4.23 126

84.9 54.0 23.1 8.7 17.2 4.54 119

a

Yongsheng Feed Corporation, Binzhou, China; proximate composition (% dry matter): moisture, 8.9; crude protein, 73.6; crude lipid, 7.6. b High Fortune (Fujian) Bio-Tech Co. Ltd., Fuzhou, China. c Vitamin mixture (mg/g mixture): thiamin hydrochloride, 2.5; riboflavin, 10; calcium pantothenate, 25; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; ascorbic acid, 50; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 20; retinol acetate, 1; cholecalciferol, 0.0025; biotin, 0.25; vitamin B12, 0.05. All ingredients were diluted with alpha-cellulose to 1 g (from Lin and Shiau, 2003). d Mineral mixture (mg/g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4·2H2O, 135.8; MgSO4·7H2O, 132; Na2HPO4·2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO4·7H2O, 3; CoCl2·6H2O, 1; MnSO4·H2O, 0.8; KI, 0.15; AlCl3·6H2O, 0.15; CuCl2, 0.1 (from Lin and Shiau, 2003). e Values represent means of duplicate samples. f [Crude fiber + nitrogen free extract (NFE)] = 100 − (% ash + % protein + % lipid). g By calculation: Fuel values for carbohydrate, protein and lipid were 4.0, 4.0 and 9.0 kcal/g (16.7, 16.7 and 37.7 kJ/g), respectively.

Engineering, South China University of Technology, Guangzhou, China) and then pelletized. All diets were air dried at 25 °C for about 24 h, sieved and then packaged and stored frozen (−20 °C). 2.2. Experimental procedures Hybrid grouper juveniles were obtained from a commercial hatchery (Changjiang, Hainan, China). In trial 1, experimental fish were acclimated with the diet containing 50% crude protein and 7% crude lipid for 16 days prior to the trial, and then, groups of 22 fish (average initial weight of 3.58 ± 0.05 g/fish) were randomly distributed into 21 small floating cages (L 120 cm × W 70 cm × H 50 cm) which were labeled and located in seven connective 6-m3 indoor concrete ponds (L 3 m × W 2 m × H 1 m) with 3 cages occurring in each pond. All ponds

received flowing sea water (salinity: 33.1 g/L) from the same reservoir at a rate of 3 L/min. During the experimental period, each dietary treatment had three replicates, and each replicate cage was in different ponds. Throughout the growth trial, dissolved oxygen content in the tanks was measured every other day with a portable meter (HATCH HQ30d, Hatch Lange GMBH; http://www.hach-lange.es) and values ranged at 5.9–6.3 mg/L. Ammonia (0–0.20 mg/L) was measured with a portable spectrophotometer (HATCH DR 2800, Hatch Lange GMBH; http://www.hach-lange.es). Water temperature was daily registered using maximum-minimum thermometers and maintained at 27–28 ° C. Fish were exposed to a 12 h: 12 h light: dark cycle and fed each dietary treatment twice daily (0800 h and 1600 h) to apparent satiation until pellets were first seen to sink to bottom of the pond. Feed intake was recorded daily, and experimental ponds and cages were cleaned once a week after experimental fish were weighed. The growth trial was continued for 7 weeks. In trial 2, groups of 22 fish (average initial weight of 10.98 ± 0.15 g/fish) were randomly distributed into 21 small floating cages, and other procedures of the growth trial were as the same as in trial 1. Fish in trial 2 were fed the experimental diets for 8 weeks. 2.3. Sampling and analysis At the beginning of trial 1 or trial 2, 10 fish were sampled and stored at −20 °C for analysis of initial whole-body protein content. At the end of trial 1 or trial 2, two fish per cage were collected for whole-body composition analysis. Three fish per cage were individually weighed and dissected to obtain liver, intestine and intraperitoneal fat (IPF) weights for computing body condition indices including hepatosomatic index (HSI) ((liver wt./live wt.) ∗ 100) and IPF ratio ((IPF wt. / live wt.) ∗ 100), respectively. Intraperitoneal fat was obtained by removing and weighing the fat from the abdominal cavity as well as that adhering to the intestine of the fish. Condition factor (CF) was also computed as (body weight × 100) / (body length)3. Muscle and liver samples for compositional analysis also were taken from these three fish. Livers for glycogen analysis in trial 2 were quickly dissected from another two randomly selected fish which were removed from each replicate cage, and dissected livers were wrapped in aluminum foil, frozen in liquid nitrogen, and stored at −80 °C until analyzed. Crude protein (N × 6.25) was determined by the Kjeldahl method after acid digestion using an auto Kjeldahl System (1030-Auto-analyzer, Tecator, Sweden). Crude lipid was determined by ether extraction using a Soxtec System HT (Soxtec System HT6, Haineng SOX406, Shandong, China). Dry matter was determined by heating at 125 °C for 3 h, and ash was quantified after heating at 650 °C for 3 h (AOAC, 1990). Glycogen contents in liver were analyzed according to Hassidh and Abraham (1957). The amino acid levels of the diets and muscle were determined using the L-8900 amino acid analyzer (Hitachi, Japan) after acid hydrolysis in 10 mL of 6 N HCl for 24 h at 110 °C in glass tubes under nitrogen (Unnikrishnan and Paulraj, 2010).

Table 4 Growth performance and feed utilization of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary crude protein levels for 7 weeks in trail 1.1 Dietary crude protein levels %

WG (%)2 DFI (g 100 g fish−1 day−1)3 FCR4 PER5 Survival (%) 1 2 3 4 5

38

42

46

50

54

58

62

746 ± 27c 3.73 ± 0.09a 1.16 ± 0.03a 2.32 ± 0.06 98.5 ± 1.52

801 ± 34c 3.28 ± 0.01ab 1.01 ± 0.01ab 2.35 ± 0.02 98.5 ± 1.52

1017 ± 42b 3.26 ± 0.02ab 0.98 ± 0.02b 2.24 ± 0.05 98.5 ± 1.52

1123 ± 41ab 3.17 ± 0.16b 0.92 ± 0.05bc 2.22 ± 0.22 100 ± 0.00

1126 ± 37ab 2.98 ± 0.16b 0.86 ± 0.05bc 2.18 ± 0.13 100 ± 0.00

1142 ± 39ab 2.76 ± 0.09b 0.79 ± 0.03c 2.19 ± 0.08 100 ± 0.00

1218 ± 48a 2.81 ± 0.07b 0.80 ± 0.02c 2.02 ± 0.06 100 ± 0.00

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different. Weight gain: 100 × (final mean weight − initial mean weight) / initial mean weight. Daily feed intake = 100 × feed offered / average total weight / days. Feed conversion ratio: g dry feed / g weight gain. Protein efficiency ratio: g weight gain / g protein fed.

S. Jiang et al. / Aquaculture 465 (2016) 28–36

31

95%Ymax=1136

X=53.5

Fig. 1. Relationship of weight gain (WG, %) with dietary protein levels of hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) juveniles.

2.4. Statistic analysis Experimental data obtained for response parameters and presented as means ± SEM (standard error of the mean) of three replications were tested by subjecting the data to one-way analysis of variance (ANOVA) and Tukey's test using SPSS 18.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USAV). The quadratic regression model at 95% of maximum response (Zehra and Khan, 2014) was used to estimate optimum dietary protein requirement based on WG%.

3. Results 3.1. Trial 1 3.1.1. Growth performance and feed utilization Weight gain (WG%) of hybrid grouper fed with P38 or P42 was significantly lower (P b 0.05) than that of fish fed with P46, P50, P54, P58 or P62 (Table 4), and fish fed with P46 had significantly lower WG% than fish fed with P62 while among fish fed P50, P54, P58 and P62, WG% did not display significant differences (P N 0.05). Based on the second polynomial regression analysis of WG% at 95% of maximum response, optimal dietary protein requirement for hybrid grouper was estimated to be 53.5% of dry matter (Fig. 1). Daily feed intake (DFI) of fish fed with P38 was significantly higher than that of fish fed with P50, P54, P58 and P62. Fish fed P38 exhibited significantly higher FCR than fish fed P46, P50, P58 and P62 while fish P42 and P46 fed showed significantly higher FCR than fish fed P58 and P62. Protein efficiency ratios (PERs) of fish fed different dietary CP levels were not significantly different. Survival rates of fish (N 98%) were not affected by different experimental treatments.

3.1.2. Body condition indices Values of hepatosomatic index (HSI) of fish decreased as dietary CP levels increased (Table 5). Fish fed with P38 had significantly higher HSI than fish fed other experimental dietary CP levels, and fish fed with P42 or P46 showed significantly higher HSI than fish fed with P50, P54, P58 or P62. Values of intraperitoneal fat ratio (IPF) and condition factor (CF) of fish were not significantly influenced by dietary CP levels. 3.1.3. Whole-body composition and amino acids in muscle tissue Dietary protein levels had no significant influences on whole-body moisture, protein, lipid and ash contents of experimental fish (Table 6). Muscle amino acids compositions were shown in Table 7. Except for serine, contents of other amino acids in muscle showed no significant differences among fish fed different dietary CP levels. Muscle serine content of fish fed P38 was significantly lower than that of fish fed P58 or P62, and serine content of fish fed P42 was significantly lower than that of fish fed P62. 3.2. Trial 2 3.2.1. Growth performance and feed utilization WG% of hybrid grouper fed with P/E191 was significantly lower than that of fish fed with P/E173 or P/E157 but did not differ from WG% of fish fed other experimental P/E ratios (Table 8), and values of WG% were not significantly different among fish fed with P/E173, P/E157, P/E145, P/ E134, P/E124 and P/E116. Values of DFI in fish fed with P/E191 and P/ E173 were significantly higher than those of fish fed with P/E134, P/ E124 and P/E116. Fish fed with P/E191 exhibited significantly higher FCR than fish fed with P/E157, P/E145, P/E134, P/E124 or P/E116, and fish fed with P/E173 showed significantly higher FCR than fish fed

Table 5 Body condition indices of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary crude protein levels for 7 weeks in trail 1.1 Dietary crude protein levels %

HSI2 IPF3 CF4 1 2 3 4

38

42

46

50

54

58

62

5.63 ± 0.20a 3.28 ± 0.08 2.00 ± 0.08

4.33 ± 0.33b 3.04 ± 0.07 2.00 ± 0.07

3.93 ± 0.10bc 3.14 ± 0.18 2.02 ± 0.03

3.26 ± 0.11c 3.16 ± 0.20 2.00 ± 0.04

2.30 ± 0.06d 2.79 ± 0.14 1.92 ± 0.02

2.07 ± 0.12d 2.63 ± 0.14 1.90 ± 0.06

1.81 ± 0.11d 3.01 ± 0.05 1.93 ± 0.07

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different. Hepatosomatic index = 100 × (hepatosomatic weight / whole body weight). Intraperitoneal fat ratio = 100 × (intraperitoneal fat weight / whole body weight). Condition factor = 100 × (live weight / length3).

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S. Jiang et al. / Aquaculture 465 (2016) 28–36

Table 6 Whole-body compositions (fresh-wt. basis) of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary crude protein levels for 7 weeks in trail 1. Dietary crude protein levels %

Moisture % Protein % Lipid % Ash %

38

42

46

50

54

58

62

69.31 ± 0.04 18.43 ± 0.22 7.49 ± 0.14 4.21 ± 0.05

70.07 ± 0.23 18.32 ± 0.06 6.24 ± 0.40 4.70 ± 0.08

69.39 ± 0.25 18.29 ± 0.25 7.00 ± 0.23 4.04 ± 0.14

69.30 ± 0.20 18.27 ± 0.34 6.42 ± 0.39 4.28 ± 0.06

69.31 ± 0.28 18.89 ± 0.17 6.72 ± 0.35 4.48 ± 0.08

69.45 ± 0.29 18.95 ± 0.13 6.42 ± 0.49 4.49 ± 0.14

69.35 ± 0.41 18.56 ± 0.31 6.54 ± 0.44 4.46 ± 0.25

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different.

with P/E124 or P/E116. There were no significant differences in PERs of fish fed different dietary P/E ratios. Protein productive value (PPV) of fish fed with P/E191 was significantly lower than that of fish fed with P/E157 or P/E145, and PPV of fish fed with P/E173 was significantly lower than that of fish fed with P/E157. Survival rates of fish were not affected by different experimental treatments.

P/E ratios. Whole-body lipid content was increased with decreasing dietary P/E ratios. Fish fed with P/E191 had significantly lower wholebody lipid than fish fed with other experimental dietary P/E ratios, and fish fed with P/E124 and P/E116 showed significantly higher whole-body lipid than fish fed with other dietary P/E ratios.

3.2.2. Body condition indices and hepatic lipid and glycogen contents There were no significant differences in values of HSI of fish fed different dietary P/E ratios (Table 9). IPF ratio was increased as dietary P/E ratios decreased. Values of IPF ratio of fish fed with P/E191 were significantly lower than those of fish fed with P/E145, P/E134, P/E124 and P/ E116 but showed no significant differences compared to those of fish fed with P/E173 or P/E157. Fish fed with P/E116 displayed significantly higher IPF ratio than fish fed with P/E191, P/E173, P/E157 and P/E145 but did not differ from fish fed with P/E124. Liver lipid content was increased as dietary P/E ratio decreased, and fish fed with P/E191, P/ E173 and P/E157 showed significantly lower liver lipid contents than fish fed with P/E145, P/E134, P/E124 and P/E116. Hepatic glycogen contents were not influenced by different dietary treatments.

4. Discussion

3.3. Whole-body compositions Whole-body moisture content of fish fed with P/E191, P/E173 or P/ E157 was significantly higher than that of fish fed with P/E145 P/E134, P/E124 or P/E116 (Table 10), and fish fed with P/E145 showed significantly higher whole-body moisture content than fish fed with P/E116. Whole-body protein content was not significantly affected by dietary

4.1. Growth performance Results based on the quadratic regression model at 95% of maximum response of WG% obtained in trial 1 indicated that optimal dietary protein level for hybrid grouper was estimated to be 53.5% of dry matter. It has been shown that juvenile groupers, irrespective of species, require high protein (N 50% DM) (Williams, 2009), such as 56% in humpback grouper (Usman et al., 2005), 55% in while Malabar grouper (Tuan and Williams, 2007) and 50.83% in spotted grouper (Wang et al., 2016), respectively. Dietary proteins ingested by fish are first digested into free amino acids which are then used as the substrates for protein synthesis or partially used as energy sources. Although amino acid absorption capacity of fish may be upregulated to compensate for the amino acids deficit caused by dietary composition (Santigosa et al., 2011a, 2011b), this compensation mechanism often is not enough when dietary protein was much lower than the requirement (GarcíaMeilán et al., 2013). This possibly explained that fish fed P38, P42 and P46 had significantly lower WG% than fish fed P62 but there were no significant differences in WG% among fish fed P50, P54, P58 and P62.

Table 7 Muscle amino acids compositions of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary crude protein levels for 7 weeks in trail 1.1 AA/∑ AA

Dietary crude protein levels % 38

42

46

50

54

58

62

EAA Lysine Arginine Methionine Threonine Isoleucine Leucine Phenylalanine Valine Histidine ∑EAA

2.04 ± 0.05 1.43 ± 0.00 0.68 ± 0.01 1.09 ± 0.00 1.13 ± 0.01 2.05 ± 0.00 0.80 ± 0.00 1.06 ± 0.00 0.53 ± 0.00 10.81 ± 0.07

2.11 ± 0.03 1.49 ± 0.02 0.68 ± 0.03 1.10 ± 0.02 1.13 ± 0.03 2.15 ± 0.06 0.83 ± 0.02 1.02 ± 0.07 0.49 ± 0.06 10.99 ± 0.16

2.06 ± 0.03 1.51 ± 0.04 0.67 ± 0.03 1.12 ± 0.01 1.14 ± 0.02 2.19 ± 0.08 0.83 ± 0.02 1.03 ± 0.06 0.50 ± 0.06 11.05 ± 0.06

2.00 ± 0.12 1.47 ± 0.00 0.67 ± 0.03 1.15 ± 0.03 1.12 ± 0.03 2.12 ± 0.02 0.81 ± 0.01 1.05 ± 0.03 0.51 ± 0.03 10.90 ± 0.18

1.98 ± 0.05 1.47 ± 0.01 0.63 ± 0.02 1.19 ± 0.04 1.09 ± 0.02 2.15 ± 0.04 0.83 ± 0.02 0.97 ± 0.05 0.46 ± 0.04 10.77 ± 0.07

1.88 ± 0.06 1.48 ± 0.01 0.63 ± 0.04 1.19 ± 0.03 1.01 ± 0.03 2.16 ± 0.01 0.83 ± 0.00 1.01 ± 0.05 0.47 ± 0.03 10.77 ± 0.16

1.99 ± 0.01 1.50 ± 0.02 0.68 ± 0.02 1.14 ± 0.01 1.16 ± 0.01 2.14 ± 0.02 0.83 ± 0.01 1.10 ± 0.01 0.55 ± 0.00 11.09 ± 0.11

NEAA Aspartic acid Serine Glutamic acid Glycine Alanine Cystine Tyrosine Proline ∑NEAA ∑AA

2.47 ± 0.01 0.89 ± 0.01c 4.69 ± 0.02 1.12 ± 0.12 1.45 ± 0.00 0.24 ± 0.01 0.82 ± 0.00 1.01 ± 0.01 12.70 ± 0.02 23.52 ± 0.06

2.48 ± 0.03 0.89 ± 0.02bc 4.64 ± 0.1 1.13 ± 0.03 1.47 ± 0.01 0.24 ± 0.03 0.87 ± 0.03 1.15 ± 0.17 12.85 ± 0.08 23.84 ± 0.20

2.47 ± 0.03 0.91 ± 0.02abc 4.63 ± 0.16 1.13 ± 0.02 1.49 ± 0.02 0.22 ± 0.03 0.89 ± 0.04 1.19 ± 0.18 12.93 ± 0.11 23.98 ± 0.16

2.39 ± 0.07 0.92 ± 0.01abc 4.82 ± 0.05 1.13 ± 0.01 1.44 ± 0.01 0.23 ± 0.03 0.86 ± 0.00 1.11 ± 0.14 12.88 ± 0.07 23.78 ± 0.12

2.35 ± 0.05 0.95 ± 0.03abc 4.94 ± 0.12 1.07 ± 0.01 1.43 ± 0.00 0.18 ± 0.04 0.91 ± 0.04 1.23 ± 0.14 13.07 ± 0.23 23.84 ± 0.15

2.37 ± 0.08 0.98 ± 0.00ab 5.01 ± 0.04 1.09 ± 0.03 1.47 ± 0.02 0.17 ± 0.03 0.88 ± 0.00 1.24 ± 0.14 13.21 ± 0.05 23.98 ± 0.17

2.51 ± 0.02 0.99 ± 0.01a 4.90 ± 0.04 1.16 ± 0.02 1.48 ± 0.02 0.22 ± 0.00 0.86 ± 0.02 0.98 ± 0.01 13.07 ± 0.13 24.17 ± 0.24

1

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different.

S. Jiang et al. / Aquaculture 465 (2016) 28–36

33

Table 8 Growth performance and feed utilization of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary protein/energy ratios for 8 weeks in trail 2.1 Dietary protein to energy ratio (mg protein/kcal) 191 2

WG (%) DFI (g 100 g fish−1 day−1)3 FCR4 PER5 PPV6 Survival (%) 1 2 3 4 5 6

173 b

809 ± 6 1.26 ± 0.01a 0.79 ± 0.004a 2.35 ± 0.01 41.9 ± 0.6c 100 ± 0.00

157 a

935 ± 24 1.26 ± 0.05a 0.75 ± 0.004ab 2.38 ± 0.09 42.7 ± 0.4bc 95.6 ± 4.43

145 a

134 ab

955 ± 2 1.14 ± 0.02ab 0.71 ± 0.010bc 2.64 ± 0.04 46.8 ± 1.2a 100 ± 0.00

124 ab

884 ± 27 1.16 ± 0.01ab 0.72 ± 0.01bc 2.62 ± 0.04 46.4 ± 0.8ab 100 ± 0.00

116 ab

860 ± 23 1.12 ± 0.02b 0.70 ± 0.013bc 2.63 ± 0.05 45.6 ± 0.2abc 100 ± 0.00

879 ± 12 1.12 ± 0.04b 0.70 ± 0.016c 2.62 ± 0.10 45.1 ± 1.2abc 97.8 ± 2.23

879 ± 17ab 1.12 ± 0.01b 0.70 ± 0.006c 2.64 ± 0.02 44.4 ± 0.8abc 100 ± 0.00

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different. Weight gain: 100 × (final mean weight − initial mean weight) / initial mean weight. Daily feed intake: 100 × feed offered / average total weight / days. Feed conversion ratio: g dry feed / g weight gain. Protein efficiency ratio: g weight gain / g protein fed. Protein productive value: 100 × g protein gain / g protein fed.

Similar results were also reported in other studies (Deng et al., 2011; García-Meilán et al., 2013; Jiang et al., 2015). In trial 2, results indicated that the optimal dietary P/E ratio for maximizing growth and feed efficiency of hybrid grouper juveniles appeared to be 157 mg/kcal, which disagreed with the study (Rahimnejad et al., 2015) in which 100 mg/kcal of protein to energy ratio and 50% dietary protein of dry matter were recommended for hybrid grouper, respectively. The divergence might be related to differences in dietary gross energy and protein levels of these two studies. The higher level of dietary gross energy (5.0 kcal/g) and the lower dietary CP level (50%) in the study of Rahimnejad et al. (2015) in comparison to that of diet (3.40 kcal/g gross energy and 53.5% CP) of this study resulted in the lower dietary P/E ratio. Results obtained in this study were similar to that of our previous study (Jiang et al., 2015) which showed that fish fed dietary P/E ratio of 154 mg/kcal (3.60 kcal/g gross energy and 55% CP) had the best growth performance. In this trial, all experimental diets had same crude protein content, so the higher dietary P/E ratio, the lower the gross energy of the diet. The significantly lower WG% of fish fed with P/E191 compared to fish fed with P/E173 or P/E157 was possibly attributed to the lower dietary gross energy level which might induce more protein metabolized to energy in fish fed with P/E 191 compared to fish fed with P/E173 or P/E157. Reduction in dietary energy was also reported to decrease fish growth in the study of Nankervis et al. (2000). Carbohydrate was used in trial 1 to replace protein to achieve protein gradation, and lipid was used to adjust the dietary energy level in trial 2, so higher levels of carbohydrate and lipid were included in the low-protein diets of trial 1 and in the low P/E diets of trial 2, respectively. Similar to results from the other study (Shiau and Lan, 1996), the high dietary non-protein energy content negatively affected growth performance. 4.2. Feed utilization In trial 1, feed conversion improved with increasing dietary protein levels, and fish fed low CP levels (P38, P42, P46) showed higher FCRs

than fish fed high CP levels (P50, P54, P58 and P62), agreeing with the results reported in other studies (Tuan and Williams, 2007; García-Meilán et al., 2013; Shapawi et al., 2014; Pinto et al., 2015; Jiang et al., 2015; Rahimnejad et al., 2015). This may be due to the inadequacy of dietary amino acids in the low CP contained diets. Protein efficiency ratio obtained in this trial decreased almost linearly with increasing dietary protein content up to 54% and then reached a plateau. Generally protein efficiency ratio decreases with increasing dietary protein level as has been noted for other species (Dabrowski, 1977; Jauncey, 1982; Siddiqui et al., 1988; Akand et al., 1989; Chen and Tsai, 1994; Wang et al., 2016). Daily feed intake showed a descending linear trend, i.e., the higher the dietary protein level, the lower the feed intake of hybrid grouper, which was in accordance with other studies (Gaylord and Gatlin, 2001; Fortes-Silva et al., 2011; Coutinho et al., 2012; Guo et al., 2012; Sá et al., 2014; Pinto et al., 2015; Rahimnejad et al., 2015; Wang et al., 2016). This can be explained by the fact that when fish are fed protein levels below optimum requirement, they need to consume more feeds to make up for protein required for growth and metabolism, and at high and optimum protein levels, less feed is required to maintain a balance between the energy for growth and metabolism (Rahimnejad et al., 2015; Pinto et al., 2015). In trial 2, results of the higher FCRs of fish fed with relatively high P/E ratios (191 and 173) compared to fish fed with relatively low P/E ratios (157, 145, 134, 124 and 116) was in line with the results observed in other studies (Hassan et al., 1995; Jantrarotai et al., 1998; Yamamoto et al., 2000; Wang et al., 2006; Letelier-Gordo et al., 2015; Rahimnejad et al., 2015). The significantly higher DFI of fish fed P/E191 and P/E173 compared to fish fed P/E134, P/E124 and P/E116 indicated that feed intake would rise when dietary energy was at a low value. Usually, as in most animals, fish regulate feed intake by the digestible energy composition of diets (Kaushik and Medale, 1994; Bureau et al., 2002; Vivas et al., 2006), and when an essential nutrient is dietary-deficient, this regulation is usually observed to meet this particular nutrient need. A reduction in feed intake was also notable in other fish species when fed a high-energy diet (Wang et al., 2006; Rueda-López et al., 2011). Reduced consumption of the relatively low P/E ratio diets with improved growth

Table 9 Hepatosomatic index, intraperitoneal fat ratio and hepatic lipid, glycogen contents (fresh-wt. basis) of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary protein/energy ratios for 8 weeks in trail 2.1 Dietary protein to energy ratio (mg protein/kcal)

HSI2 IPF3 Lipid % Glycogen % 1 2 3

191

173

157

145

134

124

116

2.14 ± 0.09 1.82 ± 0.09d 4.51 ± 0.09b 12.48 ± 0.35

2.15 ± 0.05 2.10 ± 0.16cd 4.72 ± 0.16b 10.98 ± 0.52

2.28 ± 0.16 2.47 ± 0.07cd 5.10 ± 0.29b 11.24 ± 0.24

2.26 ± 0.04 2.97 ± 0.03c 6.47 ± 0.14a 11.76 ± 0.50

2.52 ± 0.17 3.97 ± 0.03b 6.35 ± 0.19a 12.18 ± 0.58

2.48 ± 0.13 4.77 ± 0.35ab 6.70 ± 0.28a 10.59 ± 0.68

2.54 ± 0.10 5.42 ± 0.26a 7.08 ± 0.09a 11.86 ± 1.20

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different. Hepatosomatic index = 100 × (hepatosomatic weight / whole body weight). Intraperitoneal fat ratio = 100 × (intraperitoneal fat weight / whole body weight).

34

S. Jiang et al. / Aquaculture 465 (2016) 28–36

Table 10 Whole-body composition (fresh-wt. basis) of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) juveniles fed different dietary protein/energy ratios for 8 weeks in trail 2.1 Dietary protein to energy ratio (mg protein/kcal) 191 Moisture % Protein % Lipid % Ash % 1

71.67 ± 0.28 17.49 ± 0.29 5.11 ± 0.40d 3.65 ± 0.07

173 a

71.30 ± 0.15 16.91 ± 0.08 6.65 ± 0.10c 3.69 ± 0.16

157 a

70.48 ± 0.38 17.45 ± 0.21 6.96 ± 0.32c 4.04 ± 0.14

145 a

69.16 ± 0.17 17.43 ± 0.17 8.48 ± 0.19b 4.28 ± 0.06

134 b

68.27 ± 0.25 17.08 ± 0.32 9.51 ± 0.31b 4.48 ± 0.08

124 bc

116 bc

67.94 ± 0.29 16.91 ± 0.14 10.98 ± 0.24a 4.49 ± 0.14

67.14 ± 0.23c 16.61 ± 0.36 11.61 ± 0.20a 4.46 ± 0.25

Values are means (±SEM) of three replicate cages, and values within the same row with different letters are significantly (P b 0.05) different.

rates yielded improved feed efficiency. In this trial, the increments in dietary energy or decrements in P/E ratio resulted in increased PER and PPV, indicating greater growth per unit protein consumed, a trend common to a variety of teleost fish species (Hillestad and Johnsen, 1994; Catacutan and Coloso, 1995; Dias et al., 1998; Company et al., 1999; Vergara et al., 1999; Nankervis et al., 2000). A significant protein sparing effect of both lipid and carbohydrate energy was also reported in other studies (Einen and Roem, 1997; Refstie et al., 2001; Kim and Lee, 2005; Grisdale-Helland et al., 2008; Song et al., 2009; Chatzifotis et al., 2010; Ding et al., 2010).

5. Conclusions

4.3. Body conditions

Acknowledgments

In trial 1, HSI was decreased with increasing dietary CP level, which accorded with previous studies (Li et al., 2010; Jiang et al., 2015). This might be ascribed to the fact that dietary corn starch in this trial decreased as dietary CP increased. Dietary digestible carbohydrate often has a positive relation to hepatic glycogen or HSI (Rawles and Gatlin, 1998; Gaylord and Gatlin, 2000; Wu et al., 2015; Luo et al., 2016). In trial 2, the improvement in the IPF ratio with the decrement of dietary P/E ratio was ascribed to the increasing of dietary available energy, which was in line with other studies (Nematipour et al., 1992; Gaylord and Gatlin, 2001).

This study was supported by the National Natural Science Fund (no.: 31260641) and Hainan International Science and Technology Cooperation Projects (no.: ZDYF2016222). Authors wish to appreciate the editor and anonymous reviewers for their valuable suggestions on our manuscript.

4.4. Whole-body, muscle as well as liver compositions In trial 1, contents of whole-body protein, lipid and ash were not influenced by dietary protein levels, agreeing with reports of Li et al. (2010) and Jiang et al. (2015). In red spotted grouper (Wang et al., 2016), it was reported that whole-body protein content was increased with dietary CP increasing, but in malabar grouper (Tuan and Williams, 2007), the whole body protein content decreased linearly as dietary protein increased. Further studies are needed to focus on effects of different dietary CP levels on the synthesis and deposition rate of protein which could influence the composition of fish (Smith, 1981; Abdel-Tawwab and Ahmad, 2009). The increasing muscle serine content with increasing dietary CP level may be attributed to the increment in dietary serine content from dietary CP. In trial 2, carcass lipid and P/E ratios were inversely related, and increased dietary energy or lipid resulted in increased carcass lipid content, which was in accordance with other studies (Catacutan and Coloso, 1995; Nankervis et al., 2000). Increased carcass lipid suggested that the additional dietary energy from lipid was at least partially stored as body lipid. Such trends have been reported for many other species (Hillestad and Johnsen, 1994; Dias et al., 1998; Company et al., 1999; Vergara et al., 1999). Carcass moisture was inversely related to body fat as in other fishes (Winfree and Stickney, 1981; Zeitler et al., 1984; Parazo, 1990; Catacutan and Coloso, 1995). As with whole-body lipid, decreasing dietary P/E ratio at the same protein level also resulted in greater accumulation of liver lipid. The positive correlation noted between dietary energy and liver lipid content was also observed in other fish species (Nematipour et al., 1992; Ali and Jauncey, 2005).

This study demonstrated that based on the second polynomial regression analysis of WG% at 95% of maximum response, optimal dietary protein requirement for hybrid grouper was estimated to be 53.5% of dry matter, and in terms of weight gain and body composition (fish quality), optimal dietary protein to energy ratio for hybrid grouper was 157 mg/kcal. Lower P/E ratios not only resulted in lower weight gain but also produced higher lipid accumulation in the abdominal cavity.

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