,,,.,.,.,..

Transcription

1 Aquaculture feed ingredient component analysis and apparent digestibility coefficients of various feed ingredients :

2 ,,,.,.,.,..

3 (,,,,,,, +, ( ),, sand eel,, 1, 2),,,,,.,,,,,,,,,. 12 (, Isolate soy protein,,,, pea protein,,,,,, ),,,,,,,,,,,,,,. 4 (,,, ) (Reference diet). 14,,,,,,, +, ( ),, sand eel,,, Chromium oxide (Cr 2 O 3, Sigma-Aldrich).,,,, NFE,. 2.2 (Reference diet)

4 , Isolate soy protein,,,, pea protein,,,,,, Chromium oxide (Cr 2 O 3, Sigma-Aldrich).,,,, NFE,. 2.3, 4 (,, ) 10% 4 Chromium oxide (Cr 2 O 3, Sigma-Aldrich) 1%.,,,, NFE,.

5 ,,,,,,,,,,,, 14, 12, 4. 74%,,,,,,,, +, sand eel 63~69%,, 1, 2 46~59%. ISP (Isolate soy protein), Pea protein, 74~83%, RPC (rice protein concentration),,,,, 50~66%,,, 10~36%. 8.4~10.4%. 9.2~10.6%,,, 6.9~8.0%. 0.8~11.2%,., 0.8% 1.8%. 2.2~7.7%, 2.2%.,,,,,,,, Pea protein. C20:5n-3 C22:6n-3. C20:5n-3 C22:6n-3 3.4% 6.9%. 18:2n-6, n-3

6 6 -, C20:5n-3. C20:5n-3 C22:6n-3, C18:2n-6, C18:3n-3 C18:1n-9.,, 2~3%, 1.2~1.9%. 1%., 1.7ppm. 8.9ppm, 1.4ppm. 1.3ppm.., 1.18ppm,.,,, 0.03~0.07ppm., 2.58ppm, 1.42ppm.., 14.67ppm, 7.53ppm ppm, 0.264ppm, 0.146ppm, 0.1ppm (Reference diet) (Test diet).,,,,,,, +, ( ),, sand eel,, 1,

7 7-2 14, Chromium oxide (Cr 2 O 3, Sigma-Aldrich), 1.0%. 7: , 92.1% 93.7%,,, 78.1~78.3%.,,, sand eel 92.8~96.4%,, 61.1% 65.0%. 98.1%,,,,. NFE,, 91.1~94.9% NFE, 43.5%.,, (Reference diet) (Test diet)., Isolate soy protein,,,, pea protein,,,,,, 12, Chromium oxide (Cr 2 O 3, Sigma-Aldrich), 1.0%.

8 8-7:3, 100 g 40 g , 92.1%, 93.7%.,, 78.1~77.8%. pea protein,,, 78.1%, ISP. (70.9%) (76.1%). NFE (64.7%) (64.4%), (7%). (79.1%) (87.8%), (13.4%) (10.8%). 2.3.,,, 10% 6 (Cr 2 O 3 ) 1%. 70%,. 71~83%, 86%, (75.0%), (74.4%). (NFE) 64%,. 70%,., (58.0%).

9 ,.,,,,,.., pea protein C20:5n-3 C22:6n-3, C18:2n-6, C18:3n-3, C16:0, C18:1n-9. C16:0 C18:0. C16:0 80.9%, 82.1%, C18:0 78.8%, 76.4%. C18:1n-9 (89.9%), (78.6%). C18:2n-6 (94.2%) (93.6%). C18:3n-3 (95.6%), (92.4%) (94.6%). C20:5n-3 (96.0~97.4%) C22:6n-3 (96.0~97.1%).

10 10 - Ⅴ. -,

11 11 - SUMMARY Aquaculture feed ingredient component analysis and apparent digestibility coefficients of various feed ingredients 1. Aquaculture feed ingredient component analysis 1.1 Chemical composition of feed ingredients This study was designed to investigate proximate composition, amino acids, fatty acids, mineral, and heavy metal of various animal and plant feedstuff in aquatic feeds. Analysis of various ingredients showed that the highest crude protein was approximately 74.3% in sardine meal and the lowest protein was 46.6% in squid liver power1 and in 46.8% squid liver power2 and the range of protein content for other animal protein sources such as pollock meal, mackerel meal, anchovy meal, danish cod meal were 65-70%. In plant protein source, high amounts of variation were seen among different source. The minimum amount of crude protein was obtained approximately 10.6% in kelp meal and the maximum was for wheat gluten meal around 83.7%. Also, Pea protein, ISP, wheat gluten meal and RPC have high amount of protein 77.7%, 74.1%, 66.1% and 61.7%, respectively. Plant protein source such as wheat flour and rapeseed meal have shown lower Arg, Met and Lys compared to those of animal source. Regarding to fatty acid, pollack meal has highest EPA content while anchovy meal has highest content of DHA among animal source. Plant source were rich in C18:2n6. Wheat gluten meal, wheat flour and soybean meal have 63.9%, 59.2% and 57.5% of C18:2n6 respectively, while kelp meal, beer yeast and rapeseed meal has 7.2%, 17.5%, 26.6% of C18:2n6 respectively. The content of calcium, phosphorus and selenium were different among animal and plant protein source and most of the animal source had higher content of those elements. Among animal source meat meal and pollock meal had higher calcium and phosphorus content and higher content of selenium were obtained 6.9% in mackerel meal followed by 4.3% in squid liver power. In plant source, the amount calcium and phosphorus were not different and it was ranged between % among different ingredients. Comparison of heavy metal from different

12 12 - animal ingredients have shown that squid liver power1 and squid liver power2 had the highest proportion of cadmium. The highest content of Arsenic was 14.69ppm in sardine meal and cod meal has higher Hg content compare to other ingredients. Generally plant source had lower heavy metal compare to plant source. The highest content of cadmium was 1.28% in wheat flour and highest Hg was 0.054ppm in soybean meal. 2. Evaluation of apparent digestibility coefficients of various feed ingredients for flounder (Paralichthys olivaceus) 2.1 Apparent digestibility coefficients of animal feed ingredients in diets for olive flounder (Paralichthys olivaceus) Apparent digestibility coefficient dry matter (DM), crude protein (CP), crude lipid (CL), nitrogen free extract (NFE), gross energy (GE) in pollock meal, mackerel meal, anchovy meal, pollock meal, pollock meal, cod meal, danish cod meal, sardine meal+anchovy meal, sardine meal, tuna by-product meal, sand eel meal, meat meal, quid liver power1 and quid liver power2 were determined for flounder. A reference diet (RF) and test diets (consisting of 70% RF diet and 30% of the feedstuff) were used with 1.0% Cr 2 O 3 as an inert marker. The flounder, averaging 150.0g, was stocked in 400l fiber glass tanks at a density of 20 fish per tank. Feces were collected from triplicate groups of fish using a fecal collection column attached to the fish rearing tank. Apparent digestibility coefficients of flounder fed the reference diets of dry matter, crude protein, crude lipid, nitrogen free extract and energy were ranged 77.4%, 89.7%, 87.2%, 68.8% and 81.3% respectively. Apparent digestibility coefficients of flounder fed the test diets of dry matter, crude protein, crude lipid, nitrogen free extract and gross energy were highest for danish cod meal and sardine meal+anchovy meal. Statistics indicated that apparent dry matter digestibility of test diets for flounder ranged 57.1%-78.9% for animal products. Apparent protein and lipid digestibility of test diets ranged 73.1%- 91.9% and 58.8%-93.2% respectively, for animal products. Apparent nitrogen free extract and gross energy digestibility of test diets ranged 49.3%-76.1% and 54.9%- 83.5% respectively for animal products. Digestibility information could promote the use of substitutions in least-cost formulated diets for flounder.

13 Apparent digestibility coefficients of vegetable feed ingredients in diets for olive flounder (Paralichthys olivaceus) A feeding experiment was conducted to determine apparent digestibility coefficients of dry matter, crude protein, crude lipid, nitrogen free extract, gross energy in wheat flour, ISP (Isolate soy protein), rice protein concentration, rapeseed meal, fermented soybean meal, Pea protein, corn gluten meal, fermented soybean meal, soybean protein concentration, soybean meal, wheat gluten meal, beer yeast and kelp meal were determined for flounder. Apparent digestibility coefficients (ADCs) were determined using a reference diet with chromic oxide indicator and test diets that combined 70% reference diet, by weight, and 30% of the test ingredient being evaluated. The flounder, averaging 150g, was stocked in 400L fiberglass tanks at a density of 20 fish per tank. Fecal samples were collected from three replicate groups of fish using fecal collection column attached to fish rearing tank. Apparent dry matter digestibility of flounder ranged 56.3%~75.8% for vegetable protein products. soybean protein concentration, and wheat gluten meal showed the higher dry matter digestibility among ingredient tested. Apparent protein and lipid digestibility of flounder ranged 72.6%~87.1% and 62.8%~93.0% respectively, for vegetable protein products. ISP (Isolate soy protein), Pea protein, soybean protein concentration, wheat gluten meal and beer yeast showed the higher protein digestibility among ingredients tested. The highest crude lipid digestibility was observed kelp meal among ingredient tested. Apparent nitrogen free extract and gross energy digestibility of test diets ranged 31.1%~73.1% and 55.3%~73.8% respectively for vegetable protein products. These data provide more precise information concerning nutrient and energy utilization of flounder and will allow ingredient substitutions in practical feed based on levels of available nutrients Effects of dietary carbohydrate source on nutrient digestibilities in flounder (Paralichthys olivaceus) The apparent digestibility coefficients (ADC) of dry matter, crude protein, crude lipid, nitrogen-free extract and energy in the diets contained squid liver oil (SLO), soybean oil (SO), linseed oil (LO) and lard (LD) as dietary lipid sources were determined to assess the nutritive value of these lipid sources. Chromic oxide was used as inert digestibility

14 14 - markers. The flounder (averaging) 150g, was stocked in 400L fiberglass tanks at a density of 20 fish per tank. Fecal samples were collected from three replicate groups of fish using fecal collection column attached to fish rearing tank. Apparent dry matter digestibility coefficient of flounder fed SLO diet was higher than that of fish fed SO, LO and LD diets. Apparent crude protein and crude lipid digestibility coefficients of flounder fed SLO diet was higher than that of fish fed SO, LO and LD diets. Apparent NFE digestibility coefficient of flounder fed SO diet was lower than those of fish fed SLO, LO and LD diets. Apparent gross energy digestibility of flounder fed D-BT diet was lower than that of fish fed the SLO diet. These results indicate that vegetable oils are well utilized by flounder, and particularly flounder (Paralichthys olivaceus) are able to efficiently utilize SLO compare to SO, LO and LD in the diets.

15 15 - CONTENTS Chapter 1 Introduction 18 Chapter 2 Present status of technical development in domestic and overseas countries 20 Chapter 3 Results 21 Section 1. Aquaculture feed ingredient component analysis 21 Section 2. Evaluation of apparent digestibility coefficients of various feed ingredients for flounder (Paralichthys olivaceus) 78 Chapter 4 Goal attainment of the present project and contribution to related field 136 Chapter 5 Application plan of results 137 Chapter 6 Information about scientific techniques of overseas countries 138 Chapter 7 References 139

16

17 %..,....,,,.,.,.

18 18 -

19 19-2,,. 1970, % , ,., turbot, flounder, platfish, cod, sea bass,.,.,,..

20 ,,,.,,,,,,,,,.,.,..,.

21 ,,. 10%, 65%,, 3-12%.,. 50%,.,,,,,,,, +, ( ),, sand eel,, 1, %, 55%, 2%, 5%,,..

22 22 -, Isolate soy protein,,,, pea protein,,,,,,.. 4., prostaglandin. linolenic acid(18:3n-3), linolenic acid linoleic acid, (turbot) EPA DHA n-3 (HUFA).,, linolenic acid n-3 HUFA,.,,,,,. (Caballero et al., 2002; Regost et al., 2003; Bell et al., 2004; Torstensen et al., 2005; Izquierdo et al., 2005).. EPA (eicosapentaenoic acid) DHA (docosahexaenoic acid) n-3 triacylglyceride. (Kinsella, 1987). polyunsaturated fatty acids, prostaglandin.,

23 23 -, (NRC, 1993). C18:2n-6 C18:3n-3 EPA DHA, EPA DHA n-3hufa EPA DHA (Kim and Lee, 2004). (Tacon, 2005),,.. (Regost et al., 2003; Bell et al., 2004; Izquierdo et al., 2005; Montero et al., 2005; Mourente et al., 2005a, b; Torstensen et al., 2005; Mourente and Bell, 2006; Richard et al., 2006; Piedecausa et al., 2007; Peng et al., 2008).,..,,,.

24 AOAC (1990) (N 6.25) Auto Kjeldahl System (Buchi B-324/435/412, Switzerland; Metrohm 8-719/806, Swizerland), ether, 105 dry oven , bomb calorimeter (Parr 1356, USA). 6N HCl 110 sand bath 24,, (L-8800, Hitachi, Column : Ion exchange, Injection Pump : Pressure Mpa, Flow Rate ml/min, Column Oven : Electrothermal cooling (30-70 ), Reaction Unit : Reaction Column (135, 50 ), Photometer : Wavelength 570 nm, 440 nm). Folch et al. (1957) (2:1) 14% BF 3 -methanol (Sigma, USA) methylation, capillary column (SP TM -2560, 100 m 0.25 mm i. d., film thickness 0.20 um, USA) gas chromatography (PerkinElmer, Clarus 600, USA). Carrier gas, Oven /min., injector 250, detector (FID) 260, 37 (PUFA 37 Component FAME Mix, USA) , (1:1) (Joninyvon, USA). (Ethos, Milestone, Itlay),.,, ,

25 25 - (1:1) (Analyticjena, Germany).

26 26 -

27 27 -

28 28 -

29 29 -

30 30 -. Table 1, Fig. 1 Fig %,,,,,,,, +, sand eel 63~69%,, 1, 2 46~59%.,,,,. ISP (Isolate soy protein), Pea protein, 74~83%, RPC (rice protein concentration),,,,, 50~66%,,, 10~36% (Table 2, Fig. 3, 4).

31 31 - Table 1. (%) (%) (%) (%) (%) (kcal/g) sand eel

32 32 - Fig. 1.

33 33 - Fig. 2.

34 34 - Table 2. (%) (%) (%) (%) (%) (kcal/g) ISP RPC Pea protein ISP: Isolate soy protein 2 RPC: rice protein concentration

35 35 - Fig. 3.

36 36 - Fig. 4.

37 37 - Fig. 5.

38 38 - Fig. 6.

39 39 - Table 3, 4, 5 6, Fig 7, 8, 9, 10, 11, 12, 13, ~7.1%. 6.8~7.8%,,, 4.5~3.7%. 0.3~9.4%,., 0.3% 0.2%. 1.2~7.3%, (1.6%) (1.9%).,,,, (Fig, 7),,,,, Pea protein (Fig 12).,.

40 40 - Table 3. (% in ingredient) Arg His Ile Leu Lys Met Cys Phe Tyr Thr Val Asp Ser Glu Gly Ala

41 41 - Table 4. < > (% in ingredient) + sand eel Arg His Ile Leu Lys Met Cys Phe Tyr Thr Val Asp Ser Glu Gly Ala

42 42 - Table 5. (% in ingredient) ISP 1 RPC 2 Pea protein Arg His Ile Leu Lys Met Cys Phe Tyr Thr Val Asp Ser Glu Gly Ala ISP: Isolate soy protein 2 RPC: rice protein concentration

43 43 - Table 6. (% in ingredient) Arg His Ile Leu Lys Met Cys Phe Tyr Thr Val Asp Ser Glu Gly Ala

44 44 - Fig. 7. argininc (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

45 45 - Fig. 8. histidine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

46 46 - Fig. 9. isoleucine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

47 47 - Fig. 10. leucine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

48 48 - Fig. 11. lysine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

49 49 - Fig. 12. methionine + cysteine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

50 50 - Fig. 13. phenylalanine + tyrosine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

51 51 - Fig. 14. threonine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

52 52 - Fig. 15. valine (% in protein) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

53 53 -, Table 7, 8, 9, 10 11, C18:2n-6, C18:3n-3, C20:5n-3, C22:6n-3 Fig. 16, 17, C20:5n-3 C22:6n-3. C20:5n-3 C22:6n-3 3.4% 6.9%. 18:2n-6, n-3, C20:5n-3. C20:5n-3 C22:6n-3, C18:2n-6, C18:3n-3 C18:1n-9. (Caballero et al., 2002; Regost et al., 2003; Bell et al., 2004; Torstensen et al., 2005; Izquierdo et al., 2005).. EPA (eicosapentaenoic acid) DHA (docosahexaenoic acid) n-3 triacylglyceride. (Kinsella, 1987). polyunsaturated fatty acids, prostaglandin. n-3,,.

54 54 - Table 7. (% of total fatty acid) C14: C16: C16: C17: C18: C18:1n C18:2n C18:3n C20: C18:3n C22: C22:1n C20:4n C23: C22: C20:5n C22:3n C22:4n C22:5n C22:6n

55 55 - Table 8. (% of total fatty acid) sand eel + C14: C16: C16: C17: C18: C18:1n C18:2n C18:3n C20: C18:3n C22: C22:1n C20:4n C23:0 0.7 C22:2 0.1 C20:5n C22:3n3 C22:4n C22:5n C22:6n

56 56 - Table 9. (% of total fatty acid) Pea ISP 1 RPC 2 protein C14: C16: C16: C17: C18: C18:1n C18:2n C20: C18:3n C18:3n C22:0 1.9 C20:3n6 0.4 C20:4n6 7.6 C20:5n3 C22:4n6 1 ISP: Isolate soy protein 2 RPC: rice protein concentration

57 57 - Table 10. (% of total fatty acid) C14: C16: C16: C17: C18: C18:1n C18:2n C20:0 2.3 C18:3n C18:3n C22:0 0.3 C20:3n6 C20:4n6 5.0 C20:5n3 2.8 C22:4n6

58 58 - Table 11. (% of total fatty acid) C14: C16: C16: C17: C18: C18:1n C18:2n C20: C18:3n C18:3n C22: C20:3n C20:3n3 2.5 C20:4n6 C20:5n C22:6n3 5.7

59 59 - Fig. 16., C18:2n-6 (% of total fatty acid) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

60 60 - Fig. 17., C18:3n-3 (% of total fatty acid) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

61 61 - Fig. 18., C20:5n-3 (% of total fatty acid) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

62 62 - Fig. 19., C22:6n-3 % of total fatty acid) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

63 63 -,, Table 12, 13, 14.,, 2~3%, 1.2~1.9%. 1% ( 12-14)., 1.7ppm.,,, 99%,.,,,,.,,. 0.4~0.5%, 86~88%.,,,,.. glutathione peroxidase glutathione, E..,,,, glutathione peroxidase.,.

64 64 - Table 12. Ca (%) P (%) Se (ppm) ( ) sand eel

65 Ca (%) P (%) Se (ppm) ISP RPC Pea protein Wheat gluten Beer yeast kelp meal ISP: Isolate soy protein RPC: rice protein concentration

66 Ca (%) P (%) Se (ppm)

67 67 - Fig. 20., (Ca) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

68 68 - Fig. 21., (P) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

69 69 - Fig. 22., (Se) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

70 70 -,,,, Table 15, 16, 17,, Pig 23, 24, 25, ppm, 1.4ppm. 1.3ppm.., 1.18ppm,.,,, 0.03~0.07ppm., 2.58ppm, 1.42ppm.., 14.67ppm, 7.53ppm ppm, 0.264ppm, 0.146ppm, 0.1ppm.

71 71 - Table 15. Cd (ppm) Pb (ppm) Cr (ppm) As (ppm) Hg(ppm) ( ) sand eel

72 Cd (ppm) Pb (ppm) Cr (ppm) As (ppm) Hg(ppm) ISP RPC Pea protein Wheat gluten Beer yeast kelp meal ISP: Isolate soy protein 2 RPC: rice protein concentration

73 Pb (ppm) Cd (ppm) Cr (ppm) As (ppm) Hg (ppm)

74 74 - Fig. 23., (Cd) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

75 75 - Fig. 24., (Cr) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

76 76 - Fig. 25., (As) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

77 77 - Fig. 26., (Hg) 1., 2., 3., 4., 5., 6., 7., 8. +, 9., 10., 11. Sand eel, 12., 13., 14., 15., 16. ISP (Isolate soy protein), 17. RPC (Rice protein concentration), 18., 19., 20. Pea protein, 21., 22., 23., 24., 25., 26., 27., 28., 29., 30.

78 ~70%.., (Hajen et al., 1993a; Windell et al., 1978; Smith, 1971; Cho and Slinger, 1979; Choubert et al., 1982; Spyridakis et al., 1989)., 7:3,.,,. 1.. (Reference diet) (Test diet) (Table 1).,,,,,,, +, ( ),, sand eel,, 1, 2 14, Chromium oxide (Cr 2 O 3, Sigma-Aldrich), 1.0%. 7:3, 100 g 40 g

79 (Fig 1.). Table 2.. (Reference diet) (Test diet) (Table 1)., Isolate soy protein,,,, pea protein,,,,,, 12, Chromium oxide (Cr 2 O 3, Sigma-Aldrich), 1.0%. 7:3, 100 g 40 g (Fig. 1). Table 3..,,, 10% 6 (Cr 2 O 3 ) 1% (Table 4)., 100 g 40 g

80 80 - Figure 1.

81 81 - Table 1. Composition of the reference and test diets Ingredients Reference diet Test diet Fish meal (mackerel+anchovy, 1:1) 60.0 Wheat flour 19.0 α-potato starch 10.0 Squid liver oil 50 Mineral mix Vitamin mix Vitamin C (50%) 0.5 Vitamin E (25%) 0.2 Choline salt 0.3 Chromium oxide 1.0 Reference diet 70.0 Test ingredients 30.0 Total MgSO 4.7H 2 O, 80.0; NaH 2 PO 4.2H 2 O, 370.0; KCl, 130.0; Ferriccitrate, 40.0; ZnSO 4.7H 2 O, 20.0; Ca-lactate, 356.5; CuCl, 0.2; AlCl 3.6H 2 O, 0.15; Na 2 Se 2 O 3, 0.01; MnSO 4.H 2 O, 2.0; CoCl 2.6H 2 O, DL-a tocopheryl acetate, 18.8; thiamin hydrochloride, 2.7; riboflavin, 9.1; pyridoxine hydrochloride, 1.8; niacin, 36.4; Ca- D -pantothenate, 12.7; myo-inositol, 181.8; D -biotin, 0.27; folicacid, 0.68; p-aminobezoicacid, 18.2; menadione, 1.8; retinylacetate, 0.73; cholecalficerol, 0.003; cyanocobalamin,

82 82 - Table 2. Proximate composition of the reference and animal protein source test diets (% dry matter). Diets Crude protein Crude lipid Ash Gross energy Reference diet (R) % R +30% test ingredient sand eel

83 83 - Table 3. Proximate composition of the reference and vegetable source test diets (% dry matter). Diets Crude protein Crude lipid Ash Gross energy Reference diet (R) ISP (Isolate soy protein) % R +30% test ingredient Pea protein

84 84 - Table 4. Ingredient of experimental diets for lipid source digestibility Diets Ingredients (%) Fish meal (mackerel+anchovy, 1:1) SLO SO LO LD Corn gluten meal Dehulled soybean Wheat flour a-potato-starch Squid liver oil 10.0 Soybean oil 10.0 Linseed oil 10.0 Lard 10.0 Vitamin premix Mineral premix Stay-C (50%) Vitamin E (25%) Cr 2 O Proximate composition (%, dry matter basis) Crude protein Crude lipid Ash

85 85-2., FRP. Figure. 2. ( : 150 ± 8.0 g) 14, L L/min C. Figure 2.

86 ,., (10:00 hr) C. (freezer dryer). Figure 3.

87 87-3.., (Cr 2 O 3 ) (Hanley, 1987), Cho and Slinger (1979) Sugiura et al. (1998). (Cr 2 O 3 ). ADC of dry matter (%) = (100 - (dietary Cr 2 O 3 /feces Cr 2 O 3 ) 100), ADC of nutrients or energy (%) = ( 1 - dietary Cr 2O 3 feces Cr 2 O 3 feces nutrient or energy dietary nutrient or energy ) 100. ADC of test ingredient (%) = 100/30 (ADC in test diet 0.7 ADC in reference diet).

88 88 -. AOAC (1990) (N 6.25) Auto Kjeldahl System (Buchi B-324/435/412, Switzerland; Metrohm 8-719/806, Swizerland), ether, 105 dry oven , bomb calorimeter (Parr 1356, USA). 6N HCl 110 sand bath 24,, (L-8800, Hitachi, Column : Ion exchange, Injection Pump : Pressure Mpa, Flow Rate ml/min, Column Oven : Electrothermal cooling (30-70 ), Reaction Unit : Reaction Column (135, 50 ), Photometer : Wavelength 570 nm, 440 nm). Folch et al. (1957) (2:1) 14% BF 3 -methanol (Sigma, USA) methylation, capillary column (SP TM -2560, 100 m 0.25 mm i. d., film thickness 0.20 um, USA) gas chromatography (PerkinElmer, Clarus 600, USA). Carrier gas, Oven /min., injector 250, detector (FID) 260, 37 (PUFA 37 Component FAME Mix, USA). SPSS (Version 21.0) One-way ANOVA-test, Duncan's multiple test (p < 0.05).

89 reference,,, NFE Table 5. Reference,,,, 79.6%, 90.5%, 88.0%, NFE 69.9%, 80.7%. reference.,,, Table 6., 92.1% 93.7%,,, 78.1~78.3%.,,, sand eel 92.8~96.4%,, 61.1% 65.0%. 98.1%,,,,. NFE,, 91.1~94.9% NFE, 43.5%.,,.

90 90 - Table 5. Apparent digestibility coefficients (%) for dry matter, crude protein, lipid, nitrogen-free extract and energy of the olive flounder fed the diets containing various protein ingredients Dry matter Crude protein Crude lipid NFE Energy Ref ±0.61 a 81.9±0.36 a 78.1±0.60 a 68.4±1.20 ab 70.0±0.63 a ±0.84 cd 84.8±0.59 c 88.6±0.97 de 72.5±0.97 de 74.3±0.34 bc ±0.46 b 88.5±0.24 e 84.9±0.31 c 66.0±1.33 a 78.0±0.67 de ±0.71 b 83.0±1.02 b 78.3±0.62 a 70.0±0.92 bcd 72.3±1.12 b ±0.72 c 88.6±0.26 ef 78.3±0.79 a 69.0±0.92 abc 75.5±0.81 cd ±0.37 f 92.2±0.18 g 87.3±0.33 d 74.7±0.52 e 84.4±0.35 f ±0.06 e 91.8±0.09 g 89.3±0.24 e 70.7±0.35 bcd 83.8±0.56 f ±0.62 e 92.4±0.18 g 91.0±0.64 f 79.8±1.52 f 85.0±0.88 f ±0.12 cd 89.3±0.06 ef 77.8±0.20 a 67.8±0.17 ab 77.8±0.36 de ±0.22 d 89.8±0.19 f 81.5±0.16 b 69.9±0.70 bcd 79.7±0.16 e ±0.68 f 91.4±0.41 g 88.7±0.56 e 79.1±0.56 f 82.8±1.95 f ±0.38 cd 88.9±0.18 ef 84.2±0.32 c 70.4±0.64 bcd 79.7±0.82 e ±0.49 d 87.3±0.22 d 84.0±0.12 c 72.2±0.83 cde 76.1±0.88 cd ±0.29 cd 89.5±0.14 ef 89.3±0.16 e 67.9±0.37 ab 79.3±0.32 e 1., 2., 3., 4., 5., 6., 7., 8. +, 9. ( ), 10., 11. sand eel, 12., 13. 1, 14. 2)

91 91 - Figure 1. Apparent digestibility coefficients (%) for dry matter of the grower flounder fed the diets containing various animal protein ingredients.

92 92 - Figure 2. Apparent digestibility coefficients (%) for crude protein of the grower flounder fed the diets containing various animal protein ingredients.

93 93 - Figure 3. Apparent digestibility coefficients (%) for crude lipid of the grower flounder fed the diets containing various animal protein ingredients.

94 94 - Table 4. Apparent digestibility coefficients (%) for nitrogen-free extract of the grower flounder fed the diets containing various animal protein ingredients.

95 95 - Table 5. Apparent digestibility coefficients (%) for gross energy of the grower flounder fed the diets containing various animal protein ingredients.

96 96 - Table 6. Apparent digestibility coefficients (%) for dry matter, crude protein, lipid, nitrogen-free extract and energy of the test ingredients in grower flounder Dry matter Crude protein Crude lipid NFE Energy ±0.60 a 61.1±1.42 a 55.0±2.05 a 43.5±3.12 a 64.8±3.80 abc ±0.36 de 70.8±2.09 b 90.0±0.71 de 59.3±2.36 bc 78.7±1.80 cd ±0.31 bc 83.4±1.45 cd 77.9±1.67 c 71.6±4.11 de 56.8±5.74 a ±0.62 a 65.0±3.86 a 55.6±2.68 a 52.6±4.19 ab 70.2±4.36 abc ±0.79 a 83.7±1.55 cd 55.8±3.04 a 63.3±4.14 cd 66.9±7.98 abc ±0.33 cde 95.5±0.98 e 86.0±1.03 d 92.9±2.24 g 86.1±1.72 d ±0.03 f 94.2±0.54 e 92.3±0.33 e 91.1±3.14 g 72.6±2.55 bc ±0.17 f 96.4±1.23 e 98.1±1.72 f 94.9±3.13 g 88.4±4.35 d ±0.20 a 85.9±0.80 d 54.2±0.99 a 71.0±2.25 de 63.1±1.89 ab ±2.79 bcd 87.7±0.50 d 66.5±1.17 b 77.3±1.45 ef 70.0±3.55 abc ±0.56 de 92.8±1.29 e 90.4±2.17 de 87.6±7.47 fg 88.0±6.29 d ±0.32 b 84.4±1.00 d 75.3±1.69 c 77.2±3.08 ef 71.5±4.05 bc ±0.16 b 79.4±1.28 c 74.6±1.02 c 65.3±3.88 cd 77.5±3.87 cd ±0.16 e 86.7±0.29 d 92.5±0.73 e 75.9±4.84 ef 63.4±1.39 ab 1., 2., 3., 4., 5., 6., 7., 8. +, 9. ( ), 10., 11. sand eel, 12., 13. 1, 14. 2)

97 97 - Fig. 6. Apparent digestibility coefficients (%) for crude protein of the test ingredients in flounder.

98 98 - Table 7. Apparent digestibility coefficients (%) for crude lipid of the grower flounder fed the diets containing various animal protein ingredients.

99 99 - Table 8. Apparent digestibility coefficients (%) for nitrogen-free extract of the grower flounder fed the diets containing various animal protein ingredients.

100 100 - Table 9. Apparent digestibility coefficients (%) for gross energy of the grower flounder fed the diets containing various animal protein ingredients.

101 reference,,, NFE Table 10. Reference,,,, 79.6%, 90.5%, 88.0%, NFE 69.9%, 80.7%. reference.,,, NFE Table 11., 92.1%, 93.7%.,, 78.1~77.8%. pea protein,,, 78.1%, ISP. (70.9%) (76.1%). NFE (64.7%) (64.4%), (7%). (79.1%) (87.8%), (13.4%) (10.8%).

102 102 - Table 10. Apparent digestibility coefficients (%) for dry matter, crude protein, lipid, nitrogen-free extract and energy of the olive flounder fed the diets containing various protein ingredients Dry matter Crude protein Crude lipid NFE Energy Ref ±0.63 e 84.6±0.39 d 65.7±0.10 a 72.6±0.91 e 67.7±0.90 cd ±0.15 e 86.8±0.06 ef 73.1±0.23 b 57.9±0.19 c 69.1±1.18 de ±1.23 a 73.2±1.26 a 74.1±0.72 b 52.9±0.72 ab 64.8±1.48 bc ±0.74 b 84.9±0.32 de 74.7±0.59 b 56.7±1.04 bc 68.3±0.84 d ±0.74 bcd 81.1±0.37 c 68.5±1.18 a 63.3±0.72 d 67.4±0.31 cd ±0.29 de 88.3±0.20 f 68.2±0.49 a 55.8±0.53 abc 69.7±0.33 de ±0.49 bc 78.8±0.45 b 65.8±0.39 a 65.8±0.32 d 59.4±0.53 a ±2.64 f 86.8±1.26 ef 77.9±2.97 c 75.3±2.32 e 75.9±2.49 f ±0.82 bcd 87.0±0.31 f 69.1±1.03 a 56.5±2.51 abc 69.6±0.66 de ±0.33 f 88.2±0.53 f 68.9±0.65 a 72.0±2.42 e 75.8±0.40 f ±0.91 cde 87.8±0.29 f 82.8±0.25 d 52.2±0.70 a 71.9±0.61 e ±1.45 a 77.3±0.83 b 84.4±0.52 d 56.4±1.43 abc 62.8±1.19 b 1., 2. Isolate soy protein, 3., 4., 5., 6. pea protein, 7., 8., 9., 10., 11., 12.

103 103 - Fig. 10. Apparent digestibility coefficients (%) for dry matter of the grower flounder fed the diets containing various vegetable protein ingredients.

104 104 - Fig. 11. Apparent digestibility coefficients (%) for crude protein of the grower flounder fed the diets containing various vegetable protein ingredients.

105 105 - Fig. 12. Apparent digestibility coefficients (%) for crude lipid of the grower flounder fed the diets containing various vegetable protein ingredients.

106 106 - Fig. 13. Apparent digestibility coefficients (%) for nitrogen-free extract of the grower flounder fed the diets containing various vegetable protein ingredients.

107 107 - Fig. 14. Apparent digestibility coefficients (%) for gross energy of the grower flounder fed the diets containing various vegetable protein ingredients.

108 108 - Table 11. Apparent digestibility coefficients (%) for dry matter, crude protein, lipid, nitrogen-free extract and energy of the test ingredients in grower flounder Dry matter Crude protein Crude lipid NFE Energy ±0.60 a 70.1±1.21 d 13.9±3.25 a 37.2±3.80 cd 79.1±3.51 d ±0.36 de 77.4±0.73 ef 38.4±0.70 b 42.0±4.03 de 30.1±1.73 b ±0.31 bc 32.2±4.73 a 41.9±2.68 b 27.8±6.59 bc 13.4±3.21 a ±0.62 a 71.3±1.66 de 43.7±2.15 b 39.4±4.44 cde 26.0±4.75 ab ±0.79 a 58.6±0.85 c 23.2±3.67 a 36.2±2.02 cd 47.9±2.52 c ±0.33 cde 82.8±0.79 f 22.0±2.04 a 44.1±2.19 de 23.1±3.78 ab ±0.03 f 50.8±1.87 b 14.2±1.55 a 9.73±2.59 a 56.2±2.33 c ±0.17 f 77.5±3.74 ef 54.3±9.76 c 64.7±7.01 f 87.8±7.60 d ±0.20 a 78.1±0.63 f 25.1±3.23 a 43.7±1.21 de 25.3±9.04 ab ±2.79 bcd 82.2±1.66 f 24.5±2.63 a 64.4±3.00 f 76.9±9.03 d ±0.56 de 80.9±1.47 f 70.9±1.29 d 51.3±3.17 e 10.8±3.34 a ±0.32 b 45.9±2.16 b 76.1±0.81 d 19.4±4.13 ab 24.9±2.51 ab 1., 2. Isolate soy protein, 3., 4., 5., 6. pea protein, 7., 8., 9., 10., 11., 12.

109 109 - Fig. 15. Apparent digestibility coefficients (%) for crude protein of the grower flounder fed the diets containing various vegetable protein ingredients.

110 110 - Fig. 16. Apparent digestibility coefficients (%) for crude lipid of the grower flounder fed the diets containing various vegetable protein ingredients.

111 111 - Fig. 17. Apparent digestibility coefficients (%) for nitrogen-free extract of the grower flounder fed the diets containing various vegetable protein ingredients.

112 112 - Fig. 18. Apparent digestibility coefficients (%) for gross energy of the grower flounder fed the diets containing various vegetable protein ingredients.

113 113 -.,,, NFE Table %,. 71~83%, 86%, (75.0%), (74.4%). (NFE) 64%,. 70%,., (58.0%).

114 114 - Table 12. Apparent nutrients digestibility (%) of flounder fed different dietary lipid sources Diets Dry matter Crude protein Crude lipid NFE Gross energy SLO 70.7±0.65 b 83.8±0.40 c 86.4±0.31 c 62.5±0.96 b 70.7±1.56 b SO 63.5±1.77 a 78.9±1.03 b 84.2±0.79 b 54.6±2.18 a 63.3±3.73 ab LO 63.9±0.50 a 73.6±0.40 a 75.0±0.41 a 64.7±0.56 b 63.3±0.82 ab LD 60.3±1.31 a 71.8±1.63 a 74.4±0.72 a 61.9±0.67 b 58.0±2.03 a

115 115 - Fig. 19. Apparent dry matter digestibility (%) of flounder fed different dietary lipid sources

116 116 - Figure 20. Apparent crude protein digestibility (%) of flounder fed different dietary lipid sources

117 117 - Fig. 21. Apparent crude lipid digestibility (%) of flounder fed different dietary lipid sources

118 118 - Fig. 22. Apparent NFE digestibility (%) of flounder fed different dietary lipid sources

119 119 - Fig. 23. Apparent gross energy digestibility (%) of flounder fed different dietary lipid sources

120 ( ) Table 7.. Table 8.,. Table 9.,,,,,. Table 13.. Table 14., pea protein...,.,..

121 121 - Table 13. Amino acids composition (% in protein) of the experimental diets Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Val , 2., 3., 4., 5., 6., 7.,8. +, 9., 10., 11.Sand eel, 12., 13., 14.

122 122 - Table 14. Amino acid composition (% in protein) of the fecal in grower flounder fed the different animal protein source Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Val 1 5.9±0.19 ns 2.6±0.07 abcd 4.8±0.09 ab 8.3±0.13 b 7.5±0.13 de 5.5±0.36 ab 8.5±0.09 ab 5.7±0.09 ab 6.0±0.15 ns 2 5.7± ±0.07 cd 4.9±0.15 ab 8.1±0.26 ab 7.4±0.27 cde 5.7±0.23 ab 8.5±0.26 ab 5.5±0.23 ab 5.1± ± ±0.05 cd 4.5±0.01 ab 7.5±0.65 ab 6.7±0.55 abcd 6.8±1.05 b 9.1±0.80 b 5.3±0.50 ab 5.7± ± ±0.06 abc 4.9±0.07 ab 8.3±0.10 b 8.0±0.20 e 5.4±0.26 ab 8.6±0.20 ab 5.5±0.12 ab 5.8± ± ±0.32 ab 4.2±0.51 a 7.4±0.97 ab 6.5±0.84 abcd 5.1±0.81 a 7.5±1.01 ab 5.9±0.26 b 5.2± ± ±0.06 abcd 4.6±0.19 ab 8.0±0.12 ab 6.7±0.38 abcd 5.5±0.07 ab 8.4±0.22 ab 5.3±0.18 ab 5.8± ± ±0.32 a 4.1±0.55 a 6.8±0.89 a 5.8±0.77 a 5.0±0.52 a 8.3±0.47 a 5.0±0.65 a 5.2± ± ±0.06 d 4.7±0.24 ab 7.8±0.35 ab 6.7±0.06 abcd 5.7±0.47 ab 8.4±0.37 ab 5.5±0.29 ab 5.9± ± ±0.06 cd 5.0±0.12 ab 8.5±0.33 b 7.0±0.19 bcde 6.3±0.44 ab 8.8±0.31 ab 5.6±0.12 ab 6.0± ± ±0.09 abcd 4.7±0.10 ab 7.8±0.09 ab 6.0±0.01 ab 6.4±0.43 ab 8.5±0.22 ab 5.4±0.13 ab 6.0± ± ±0.03 abcd 4.9±0.23 ab 8.2±0.18 ab 7.3±0.15 cde 5.5±0.27 ab 8.7±0.25 ab 5.7±0.15 ab 6.0± ± ±0.07 abcd 6.2±1.70 b 7.6±0.19 ab 6.3±0.15 abc 5.6±0.29 ab 7.8±0.23 ab 5.2±0.12 ab 5.7± ± ±0.18 abcd 4.8±0.21 ab 8.8±0.24 b 6.6±0.20 abcd 5.3±0.28 ab 8.1±0.61 ab 5.4±5.05 ab 5.9± ± ±0.09 bcd 4.9±0.06 ab 8.2±0.10 ab 6.0±0.06 ab 6.2±0.44 ab 8.7±0.19 ab 5.1±0.13 ab 6.0± , 2., 3., 4., 5., 6., 7.,8. +, 9., 10., 11.Sand eel, 12., 13., 14.

123 123 - Table 15. Apparent amino acid availabilities (%) of the animal protein ingredients in grower flounder Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Val ±0.35 a 85.1±0.15 a 78.2±0.68 a 80.5±0.26 a 82.6±0.21 a 78.6±1.21 a 78.4±0.32 a 79.2±0.29 a 78.4±0.43 a ±0.60 b 89.8±0.06 bc 84.9±0.75 cd 86.0±0.49 bc 87.7±0.42 b 82.6±0.67 abcd 83.9±0.60 ab 84.8±0.27 b 84.2±0.45 b ±1.88 de 90.7±0.40 cde 87.7±0.38 de 89.0±0.40 de 91.1±0.38 ef 79.6±4.53 ab 84.0±1.91 ab 86.6±1.50 bc 87.4±0.12 d ±1.14 a 86.0±0.78 a 79.9±1.65 ab 81.8±0.92 a 83.1±0.69 a 80.0±1.71 ab 79.4±1.25 ab 80.4±1.13 a 80.2±0.97 a ±1.15 bcd 90.4±0.97 cd 86.2±2.02 cd 87.4±1.23 cd 89.3±1.05 cd 86.0±1.66 def 86.2±1.42 ab 86.9±1.47 bcd 86.3±1.18 cd ±0.35 f 93.5±0.27 g 91.0±0.75 e 91.9±0.27 f 93.5±0.47 gh 91.1±0.12 f 90.4±0.40 b 91.3±0.45 f 91.0±0.47 e ±1.02 ef 93.2±0.98 fg 91.1±2.04 e 92.0±1.07 f 93.6±0.86 h 90.5±0.93 ef 76.4±12.46 a 90.8±1.18 ef 90.6±1.21 e ±0.57 f 93.2±0.12 fg 91.2±1.11 e 92.1±0.55 f 93.7±0.19 h 90.0±0.86 ef 90.4±0.58 b 91.2±0.60 f 90.9±0.55 e ±0.18 def 91.0±0.15 cde 86.9±0.44 cde 88.5±0.38 de 91.0±0.17 ef 85.5±0.95 cde 86.9±0.35 ab 88.5±0.29 cde 87.3±0.32 d ±0.17 def 92.2±0.19 efg 88.6±0.26 de 90.0±0.15 e 92.1±0.10 fg 80.5±1.11 abc 88.1±0.22 ab 89.2±0.43 def 88.2±0.13 d ±0.12 cde 91.5±0.26 de 86.7±1.05 cde 88.5±0.35 de 90.1±0.27 de 87.1±0.83 def 86.7±0.28 ab 87.8±0.22 cd 87.0±0.49 d ±0.24 def 91.5±0.23 de 82.6±5.23 bc 88.9±0.31 de 90.8±0.24 ef 84.3±1.01 bcd 86.9±0.47 ab 87.6±0.31 cd 87.4±0.37 d ±0.50 bc 88.7±0.72 b 84.6±1.37 cd 85.6±0.09 bc 88.1±0.48 bc 83.1±1.35 abcd 85.4±1.40 ab 85.0±0.23 b 84.5±0.62 bc ±0.44 def 91.7±0.09 def 87.7±0.15 de 89.0±0.32 de 91.8±0.22 f 86.8±0.75 def 87.4±0.28 ab 89.3±0.49 def 87.6±0.09 d 1., 2., 3., 4., 5., 6., 7.,8. +, 9., 10., 11.Sand eel, 12., 13., 14.

124 124 - Table 16. Amino acids composition (% in protein) of the experimental diets Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Val , 2. Isolate soy protein, 3., 4., 5., 6. pea protein, 7., 8., 9., 10., 11., 12.

125 125 - Table 17. Amino acid composition (% in protein) of the fecal in grower flounder fed the different vegetable protein source Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Val 1 5.4±0.33 b 2.7±0.03 bc 4.8±0.15 ab 8.3±0.13 a 7.1±0.07 c 6.2±0.52 ab 8.6±0.09 abc 5.5±0.12 bc 6.3±0.17 b 2 5.4±0.33 b 2.7±.010 bc 4.9±0.17 b 8.8±0.21 a 6.9±0.38 c 5.5±0.41 ab 9.0±0.36 c 5.6±0.12 c 6.2±0.32 ab 3 5.2±0.29 b 2.5±0.10 b 4.4±0.09 ab 7.8±0.10 a 5.4±0.25 ab 8.0±0.58 c 8.9±0.17 bc 5.4±0.24 bc 6.2±0.12 ab 4 5.2±0.22 b 2.7±0.01 bc 4.7±0.12 ab 8.1±0.03 a 6.7±0.03 c 5.7±0.32 ab 8.4±0.18 abc 5.5±0.03 bc 6.2±0.12 b 5 5.3±0.03 b 2.6±0.01 bc 4.8±0.09 ab 8.3±0.09 a 6.5±0.20 c 5.1±0.67 a 8.4±0.15 ab 5.3±0.12 bc 5.8±0.06 ab 6 5.3±0.13 b 2.6±0.06 bc 4.8±0.12 b 8.2±0.15 a 6.5±0.13 c 5.8±0.59 ab 8.6±0.18 abc 5.5±0.07 bc 6.0±0.20 ab 7 4.8±0.13 ab 2.6±0.13 bc 4.4±0.03 ab 7.9±0.07 a 6.2±0.21 bc 5.6±0.23 ab 8.2±0.15 a 5.1±0.15 ab 5.5±0.15 ab 8 5.3±0.03 b 2.6±0.03 bc 4.8±0.12 b 8.4±0.10 a 6.7±0.06 c 5.5±0.26 ab 8.4±0.12 abc 5.4±0.03 bc 6.0±0.13 ab 9 5.3±0.09 b 2.5±0.01 b 4.8±0.03 b 8.3±0.07 a 6.5±0.06 c 5.6±0.32 ab 8.5±0.13 abc 5.5±0.06 bc 6.0±0.06 ab ±0.54 a 2.2±0.15 a 4.3±0.32 a 11.2±1.39 b 4.7±0.94 a 5.0±0.35 a 8.9±0.18 bc 4.7±0.32 a 5.3±0.41 a ±0.09 b 2.7±0.07 bc 4.6±0.18 ab 7.8±0.12 a 6.9±0.23 c 5.0±1.01 a 8.1±0.09 a 5.7±0.09 c 6.3±0.56 b ±0.10 b 2.8±0.10 c 4.5±0.25 ab 7.4±0.10 a 6.9±0.05 c 6.9±0.10 bc 8.1±0.10 a 5.6±0.05 bc 6.0±0.10 ab 1., 2. Isolate soy protein, 3., 4., 5., 6. pea protein, 7., 8., 9., 10., 11., 12.

126 126 - Table 18. Apparent amino acid availabilities (%) of the vegetable protein ingredients in grower flounder Arg His Ile Leu Lys Met + Cys Phe + Tyr Thr Val ±0.38 d 88.3±0.49 de 81.3±0.23 cd 83.5±0.35 d 85.4±0.28 bc 78.2±2.10 bc 81.5±0.45 c 83.0±0.53 cdef 80.7±0.82 b ±0.12 ef 87.0±0.60 cd 82.3±0.55 cd 83.2±0.38 d 85.9±0.73 bc 76.1±1.79 b 81.7±0.73 c 81.9±0.43 def 80.9±0.98 b ±1.49 b 76.8±1.02 a 73.1±1.07 b 71.4±0.88 b 75.7±1.11 b 44.5±2.93 a 66.0±0.93 a 62.7±1.58 a 69.7±0.81 a ±0.33 ef 88.4±0.12 de 80.9±0.73 c 83.8±0.32 de 86.5±0.26 bc 74.2±1.19 b 81.6±0.09 c 83.7±0.33 def 80.7±0.73 b ±0.32 cd 84.5±0.39 b 81.6±0.74 cd 79.9±0.60 c 83.5±0.78 bc 74.1±3.06 b 78.1±0.57 b 76.8±1.13 b 80.6±0.44 b ±0.21 fg 89.2±0.30 de 85.0±0.48 d 86.9±0.31 fg 89.2±0.27 c 80.2±2.01 bc 85.6±0.32 e 84.8±0.15 ef 84.2±0.58 b ±0.52 c 85.1±0.41 bc 84.1±0.33 bc 88.2±0.22 g 80.4±0.31 bc 79.8±0.75 bc 82.9±0.15 cd 80.3±0.61 c 83.6±0.35 b ±1.37 ef 88.4±1.38 de 81.6±1.98 bc 84.0±1.68 de 86.4±1.44 bc 84.4±1.23 cd 83.0±1.80 cd 83.8±1.82 def 81.4±1.92 b ±0.20 e 87.3±0.41 cde 81.9±0.45 bc 82.3±0.52 cd 85.6±0.50 bc 77.0±1.79 b 82.0±0.64 c 81.2±0.73 cd 80.8±0.58 b ±1.25 g 93.0±0.58 f 90.2±0.93 e 85.0±1.63 def 91.8±1.78 c 87.3±1.17 d 88.2±0.06 f 88.1±1.05 g 90.1±0.96 c ±0.43 efg 89.6±0.72 e 84.3±1.11 bc 86.4±0.71 efg 88.3±0.84 c 86.6±2.37 d 84.6±0.63 de 85.2±0.44 fg 83.5±2.03 b ±1.46 a 75.9±1.29 a 69.6±2.50 a 67.8±1.31 a 59.8±12.48 a 41.4±1.67 a 64.0±1.42 a 65.5±1.63 a 70.4±1.31 a 1., 2. Isolate soy protein, 3., 4., 5., 6. pea protein, 7., 8., 9., 10., 11., 12.

127 127 - Table19. C20:5n-3 C22:6n-3, C18:2n-6, C18:3n-3, C16:0, C18:1n-9. Table 18. C16:0 C18:0. C16:0 80.9%, 82.1%, C18:0 78.8%, 76.4%. C18:1n-9 (89.9%), (78.6%). C18:2n-6 (94.2%) (93.6%). C18:3n-3 (95.6%), (92.4%) (94.6%). C20:5n-3 (96.0~97.4%) C22:6n-3 (96.0~97.1%).

128 128 - Table 19. Major fatty acids composition (% of total fatty acids) of the experimental diets Diets (% of total fatty acids) FA SLO SO LO LD C14: C16: C18: C18:1n C18:2n C18:3n C20:4n C20:5n C22:6n

129 129 - Table 20. Major fatty acids composition (% of total fatty acids) of the fecal in grower flounder fed the different lipid source Diets SLO SO LO LD C14:0 8.4±0.01 c 1.6±0.12 a 1.7±0.07 a 2.7±0.07 b C16:0 33.1±0.47 c 23.6±0.88 b 14.7±0.21 a 33.1±0.13 c C18:0 7.6±0.23 b 7.8±0.14 b 5.4±0.06 a 15.4±0.35 c C18:1n9 10.8±0.37 a 27.6±0.12 c 22.8±0.24 b 28.4±0.31 c C18:2n6 10.2±1.53 b 18.9±0.53 c 22.3±0.35 d 7.1±0.21 a C18:3n3 1.5±0.44 a 1.6±0.04 a 11.0±0.35 b 0.7±0.18 a C20:4n6 1.2±0.03 a 0.3±0.05 a 0.5±0.27 a 0.2±0.10 b C20:5n3 2.4±1.07 ns 1.1± ± ±0.08 C22:6n3 4.8±0.11 c 3.7±0.14 b 2.7±0.23 a 2.5±0.08 a

130 130 - Table 21. Apparent digestibility coefficients (ADC, % weight) of selected fatty acids in flounder fed the experimental diets. Diets SLO SO LO LD C14:0 84.2±0.33 b 90.2±0.58 c 80.4±1.10 a 82.1±0.50 ab C16:0 80.9±0.41 b 86.6±0.43 c 82.1±0.58 b 75.9±0.25 a C18:0 78.8±0.81 b 80.9±1.10 c 76.4±0.69 b 58.8±1.75 a C18:1n9 89.9±0.49 d 84.8±0.61 c 73.6±0.52 a 78.6±0.25 b C18:2n6 90.4±1.59 b 94.2±0.22 c 79.6±0.34 a 93.6±0.35 c C18:3n3 80.8±5.58 a 95.6±0.12 b 92.4±0.18 b 94.6±0.50 b C20:4n6 93.0±0.24 ns 96.1± ± ±0.35 C20:5n3 97.4±1.19 ns 97.2± ± ±0.01 C22:6n3 96.0±0.07 ns 97.1± ± ±0.15

131 131 - Figure 24. Apparent C18:2n-6 digestibility of flounder fed different dietary lipid sources

132 132 - Figure 25. Apparent C18:3n-3 digestibility of flounder fed different dietary lipid sources

133 133 - Figure 26. Apparent C20:5n-3 digestibility of flounder fed different dietary lipid sources

134 134 - Figure 27. Apparent C22:6n-3 digestibility of flounder fed different dietary lipid sources

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