essential amino acid digestibility of different ingredients in striped catfish (pangasianodon hypophthalmus) diet

Reference diet contained fishmeal 26%, soybean meal 48%, wheat flour 20%, squid oil 2%, premix 1% and chrome oxide 1% whereas experimental diets were formulated as 70% reference diet plu

CANTHO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

LY MINH QUAN

ESSENTIAL AMINO ACID DIGESTIBILITY OF DIFFERENT

INGREDIENTS IN STRIPED CATFISH (Pangasianodon

hypophthalmus) DIET

A thesis submitted in a partial fulfillment of the requirements for

the degree of Bachelor of science in Aquaculture

Cantho, December 2014

ESSENTIAL AMINO ACID DIGESTIBILITY

OF DIFFERENT INGREDIENTS IN STRIPED CATFISH

(Pangasianodon hypophthalmus) DIET

Ly Minh Quan, Tran Thi Thanh Hien Department of Nutrition and Aquatic Product Processing College of Aquaculture and Fisheries, Can Tho University

ABSTRACT

Striped catfish (Pangasianodon hypophthalmus) is a dominated aquaculture species in Mekong Delta, Vietnam which its production accounted for 1.2 million tons in 2013 Feed used for striped catfish aquaculture was estimated up to 2 million tons Balancing the amino acid profile of the diet with free amino acids can be conducted as a cost-effective strategy to reduce not only fish meal incorporation level but also protein level of the diet, improve fish growth performance The experiment was conducted to determine the digestibility of amino acid in different common ingredients used for catfish fingerlings (90 g/fish) diets which contributed to formulate diets on basis of total amino acids to reduce variability catfish‘s requirement Reference diet contained fishmeal (26%), soybean meal (48%), wheat flour (20%), squid oil (2%), premix (1%) and chrome oxide (1%) whereas experimental diets were formulated as 70% reference diet plus 30% of each common ingredients (fishmeal, defatted soybean meal, rice bran meal, defatted rice bran meal, wheat bran meal) The apparent coefficients digestibility (ADC) was calculated for the EAA (essential amino acids) in each ingredients and feces There are no significantly different of digestibility of EAA between fishmeal and defatted soybean However, digestibility of EAA in wheat bran, rice bran and defatted rice bran diets were significantly lower than fishmeal (P <0.05)

Keyword: striped catfish, apparent digestibility, essential amino acids

Title: Essential amino acid digestibility of different ingredients in strip catfish (Pangasianodon hypophthalmus) diet

1 Introduction

Striped catfish (Pangasianodon hypophthalmus) is a dominated aquaculture species

in Mekong Delta, Vietnam The production has rapidly increased since the first

successful of inducing spawning in catfish (Khanh et al., 1996), accounted for 1.2

million tons in 2013 with an estimated export income of U.S $ 1.76 billion (MARD, 2013) Amount of feed used for this industry was estimated up to 2 million tons of commercial pellet feed In striped catfish production, feeding cost accounted for high portion of production cost, up to 80% (Muzinic, 2005) Many studies on nutritional requirement of striped catfish have been done e.g growth and protein requirement of

fingerlings of three catfish of the Mekong River (Hung et al., 2002), striped catfish methionine requirement (Hien et al., 2009), lysine requirement (Hien, 2009), digestibility of feed ingredients for striped catfish (Hien et al., 2010), replacement of

fishmeal by soybean meal in diets of striped catfish (Hien and Phong, 2011), protein and energy requirements of striped catfish using a bio-energetic factorial approach

(Glencross et al., 2010)

Balancing the amino acid profile of the diet with free amino acids can be conducted

as a cost effective strategy to reduce not only fish meal incorporation level but also

factorial method – the requirement at ration level (RRL) method – in which a diet that may or may not nutritionally complete in fed at graded levels, thus achieving graded intake of amino acids to estimate requirements for maintenance and maximum growth or protein gain This approach has been successfully applied in

Atlantic salmon (Helland et al., 2010; Grisdale-Helland et al., 2013) and tilapia (Lemme et al., 2013)

Lacking of information on the amino acids requirement of striped catfish is therefore the main constrain on utilization of supplemental amino acids and thereby development of more sustainable diets for catfish The main objective of this study is

to evaluate digestibility of amino acid in different common ingredients (rice bran, fishmeal, and defatted soybean meal and wheat flour) which contributed to formulate diets on basis of total amino acids to reduce variability in meeting amino acid catfish’s requirement

2 Material and methods

2.1 Test ingredients

The common ingredients used in this study included fishmeal, defatted soybean meal, rice bran meal, defatted rice bran meal, wheat bran meal (Table 1) Reference diet was formulated as fishmeal (26%), soybean meal (48%), wheat flour (20%), squid oil (2%), premix (1%) and chrome oxide (1%)

Table 1 Chemical compositions ingredients (%) (DM-dry matter, CP-crude protein, CL- crude lipid,

NFE-nitrogen free extract, CF-crude fibre, GE-gross energy, FM-fishmeal, SM-soybean meal, WB-Wheat bran, RB-rice bran)

DM (%) CP (%) CL (%) NFE (%) Ash (%) CF (%) GE KJ/g

Data were calculated basing on dry matter

The experiment included six different treatments with three replications Each treatment was set up by completely random design Five experimental diets were formulated by 70% reference diets and 30% of the feedstuff (fishmeal, defatted soybean meal, rice bran, defatted rice bran and wheat bran) and 1% Cr2O3 was used

as marker Formulation and chemical composition of experimental diets was presented in Table 2

Table 2 Formulation and chemical composition of experimental diets (FM-fishmeal,

SM-soybean meal, WB-Wheat bran, RB-rice bran)

Ingredients Reference FM Defatted SM RB Defatted RB WB

Data were calculated basing on dry matter DM: dry matter, CP: crude protein, CL: crude lipid, NFE:- -nitrogen free extract, CF: crude fibre, GE: gross energy

2.2 Experimental design

Striped catfish juveniles (90g initial body weight) obtained from hatchery in An Giang province were acclimated in the 10m3 tanks for 2 weeks Fish were then stocked in 18 experiment tanks (170 L) at density of 30 fish/tankand fed with the reference and test diets for 6 weeks Feces was collected by settling over a 20-h daily period Samples were pooled per each tank and stored at -20°C until being freeze-dried for 96 hours

The feces samples was collected at 2:00 PM every day before feeding until getting enough the amount of feces (10g of dried weight) After 20 hours, feces was deposited to glass (Duran, Germany) (250mL) through a pipe in the bottom of experimental composite tank

In this experiment, fish was fed 2-3% of body weight The water quality parameters were checked every day The temperature was maintained from 26 to 29oC and dissolved oxygen was above 6 mg/L while pH was recorded, 7 – 8.5

2.4 Analytical procedures

The chemical compositions of feed and feces were analyzed following the Association of Official Analytical Chemists (2000) Dry matter was determined by

drying sample in oven at 105oC until stable weight; crude protein was analyzed following Kjeldahl method; crude fat was determined by Soxhlet method after ethyl ether extraction

Gross energy was analyzed by Parr 6100 calorimeter and Cr2O3 concentration was measured by a spectrophotometer involving perchloric acid digestion (Furukawa and Tsukahara, 1966)

Amino acids composition was examined by liquid chromatography mass spectrometry (LCMSMS) Samples was digested in HCL 6M for 14 hours at 110oC before derived and injected into LCMSMS

Apparent digestibility coefficient (ADC)

Where:

ADCdiet: apparent digestibility of test diet

ADCingr: apparent digestibility of ingredient

ADCref: apparent digestibility of reference diet

A: % indicator in feeds

B: % indicator in feces

Apparent digestibility coefficient nutrient (ADCNu)

Where:

A’: % nutrient in feeds

B’: % nutrient in feces

ADCNu-diet: apparent digestibility coefficient nutrient of test diet

ADCNu-ingr: apparent digestibility coefficient nutrient of ingredient

ADCNu-ref: apparent digestibility coefficient nutrient of reference diet

Dref: % nutrient of reference diet

Dingredient: % nutrient of ingredient

3 Result and discussion

3.1 Digestibility of experimental diets

Table 4 The ADC (apparent digestibility coefficient) (%) of experimental diets

ADC diet (%) ADC Protein (%) ADC Energy (%) ADC Lipid (%)

Defatted SM 79.0 ± 0.01 a 89.7 ± 0.01 a 85.7 ± 0.01 a 94.0 ± 0.01 a

Defatted RB 75.3+0.02 c 85.7 ± 0.01 b 81.0 ± 0.01 b 90.7 ± 0.01 ba

The values showed mean ± standard deviation Means in the same column with different letters are significantly different (P<0.05) FM: fishmeal, SM: soybean meal, WB: Wheat bran, RB: rice bran There was no significant difference in apparent digestibility coefficient (ADC) between fishmeal, defatted soybean meal and reference diets The ADC diet, ADC protein, ADC lipid and ADC energy of fishmeal, defatted soybean meal and reference diets were significantly higher than other diets The ADC energy and ADC lipid of wheat bran diet was significantly lower than other diets These result showed that diets with the majority component of fishmeal and soybean are advantages in nutrition values compare to others In addition, it indicated the potentially replacement of fishmeal by soybean meal in diets of striped catfish (Hien, 2011) There are many researches about digestibility on several species with different materials The nutritional value of an ingredient is based not only on its chemical composition but also on the amount of the nutrients or energy the fish can absorb and utilize Digestibility shows the fraction of the nutrient or energy in the ingested feedstuff that is not excreted in the feces (Lazard, 1993)

3.2 Digestibility of ingredients

The ADC of ingredients are presented in the table 5

Table 5 The ADC (%) of fishmeal (FM), Defatted soybean (SB), Wheat bran (WB), Rice bran (RB)

and defatted RB

ADC Ingredient (%) ADC Protein (%) ADC Energy (%) ADC Lipid (%)

Defatted SM 79.33 ± 0.01 a 91.33 ± 0.03 a 87.33 ± 0.03 a 86.33 ± 0.06 a

Defatted RB 66.67 ± 0.05 b 64.00 ± 0.05 b 71.00 ± 0.06 c 64.67 ± 0.07 b The values showed mean ± standard deviation

Means in the same column with different letters are significantly different (P≤0.05)

ADC ingredients of fishmeal and defatted soybean meal were significantly higher than the others According to Hien (2004), the higher the protein level in ingredient, the higher digestibility ingredient This was proved by the function of protein, stimulating the digestion and increasing the trypsin and lipase (protein digestive enzyme)

In addition, ADC protein has the huge influence to the environment, ingredients with low ADC proteins can increase the waste from fish In this experiments, the ADC protein of fishmeal and defatted soybean were significantly higher than others The

reason for this was the fiber in rice bran and wheat bran is usually high According to Hien (2009), the fiber concentration in feed decreasing the ADC of feed

Every digestion and metabolism action in fish are always related to energy, the ability

of provide energy of an ingredient is very important in determining the nutrition value

of feedstuff The ADC energy of fishmeal and defatted soybean meal were significantly higher than rice bran and defatted rice bran while wheat bran had the lowest value

3.3 Amino acids composition

Amino acids composition of ingredients were presented in Table 6

Table 6 Amino acids composition of ingredients (% amino acid in crude protein) (FM-fishmeal,

SB-soybean, WB-wheat bran, RB-rice bran)

Values were calculated basing on dry matter of the sample (MET-methionine, CYS-cysteine, LYS-lysine, THR-threonine, ARG-arginine, ILE-isoleucine, LEU-leucine, VAL-valine, HIS-histidine, PHE-phenylalanine, GLY-glycine, SER-Serine, PRO-proline, ALA-Alanine, ASP-aspartic, GLU-glutamic)

The level of methionine and lysine in fishmeal is higher than other ingredients According to Hien (2004), these amino acids are limited in plant ingredients, and usually not enough for the growth of fish The concentration of cysteine and phenylalanine are also important due to its rules replacing methionine and tyrosine demand The amino acid compositionsof fishmeal were optimal compared to other ingredients Fishmeal usually used in diet to increase the efficiency and digestibility

of protein, and make the feed more attractive (Mile and Chapman, 2010) In addition, the concentration of digestive amino acids (lysine, methionine, and leucine) were

high which is based for almost aquaculture feed production (Keller, 1990) The

disadvantages of fishmeal are the price and the source that are fluctuation Amino acid composition of defatted soybean meal showed the most potential ingredient to

replace or reduce the ratio of fishmeal in feed diet due to its rational price and stable source (Hertrampf and Piedad-Pascual, 2000) However, the level of methionine and lysine in soybean meal were much lower compared to fishmeal The amount of methionine and cysteine in the soybean meal was not satisfied for fish growth (NRC, 1993) Although these value are still very few compare to fishmeal, defatted soybean can replace fishmeal at 70% with methionine and lysine additional in feed production for striped catfish at juvenile stage (Phong, 2011) The amino acids of other plant ingredients were very low compared to defatted soybean meal and fishmeal in this experiment In the context of animal feeding, protein generally refer to crude protein The requirement for dietary protein has two components (Lazard et al., 1993): (i) a need for indispensable amino acids that the fish cannot synthesize and (ii) a supply of either dispensable amino acids or sufficient amino nitrogen to enable fish to synthesize them The concept of balance amino acids is basic to protein requirement, meaning the minimum amount needed to meet requirements for amino acids and to achieve maximum growth An absolute requirement for 10 amino acids (arginine, histamine, isoleucine, leonine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) has been demonstrated in all fish species examined The requirements for maintenance and for gain above maintenance for individual amino acids in fish have been determined using diets with gradient levels of a deficient amino acid (Rodehutscord et al., 1997; Hauler and Carter, 2001; Abboudi et al.,

2006, 2007; Rollin et al., 2006; Hauler et al., 2007; Peres and Oliva-Teles, 2008; Bodin et al., 2008; Bodin et al., 2009)

3.4 ADC of EAA (essential amino acids) in experimental diets

The ADC of methionine of fishmeal, defatted soybean meal, wheat bran and reference diet was not significantly difference The digestibility of lysine in fishmeal and soybean meal was significantly higher than others This is because of concentration of lysine in fishmeal and soybean was higher than other plant ingredients The digestibility of EAAs in fishmeal diet were mostly higher than other plant diets While the digestibility of amino acids in diet with addition of wheat bran, rice bran and defatted rice bran were significantly lower than reference diet This means the decreasing fishmeal level in diet may lead to decrease amino acids digestibility

Table 7 ADC (%) of EAA in experimental diet (FM-fishmeal, SM-soybean, WB-wheat bran,

RB-ricebran)

FM Defatted

SM WB RB Defatted RB References MET 91.6 ± 0.01 a 89.6 ± 0.01 a 88.6 ± 0.01 a 84.3 ± 0.02 b 84.7 ± 0.03 b 88.6 ± 0.01 a

LYS 95.7 ± 0.01 a 95.7 ± 0.01 a 93.3 ± 0.01 b 92.7 ± 0.01 b 92.7 ± 0.01 b 94.0 ± 0.01 b

THR 93.7 ± 0.01 a 91.3 ± 0.01 ab 89.0 ± 0.01 b 85.3 ± 0.01 c 86.0 ± 0.01 c 90.7 ± 0.01 b

ARG 94.7 ± 0.01 ab 96.1 ± 0.01 a 94.3 ± 0.01 b 94.1 ± 0.01 b 93.3 ± 0.01 b 94.7 ± 0.01 ab

ILE 92.0 ± 0.01 a 90.3 ± 0.01 ab 89.1 ± 0.01 bc 85.1 ± 0.02 d 87.7 ± 0.01 c 88.7 ± 0.01 bc

LEU 93.0 ± 0.01 a 92.3 ± 0.01 ab 91.1 ± 0.01 bc 88.3 ± 0.01 d 89.7 ± 0.01 cd 90.3 ± 0.01 c

VAL 92.0 ± 0.01 a 90.0 ± 0.01 b 87.7 ± 0.01 c 84.7 ± 0.01 d 85.3 ± 0.01 d 89.3 ± 0.01 bc

HIS 93.7 ± 0.01 a 94.0 ± 0.02 a 93.0 ± 0.02 ab 92.0 ± 0.01 b 91.7 ± 0.01 b 93.7 ± 0.01 a

PHE 89.3 ± 0.01 ab 89.3 ± 0.01 ab 90.7 ± 0.02 a 85.3 ± 0.02 c 87.0 ± 0.02 bc 87.3 ± 0.01 abc The values showed mean ± standard deviation

Means in the same row with different letters are significantly different (P≤0.05) (MET-methionine, CYS-cysteine, LYS-lysine, THR-threonine, ARG-arginine, ILE-isoleucine, LEU-leucine, VAL-valine, HIS-histidine, PHE-phenylalanine, GLY-glycine, SER-Serine, PRO-proline, ALA-Alanine, ASP-aspartic, GLU-glutamic)

Generally, fish cannot store free amino acids Amino acid was absorbed to synthesize protein, unless it will metabolize to another free amino acid or burning as energy for metabolism (Lazard, 1993) In this case, if the EAA becomes non-essential amino acid, it will be a large waste Therefore, the imbalance of amino acid in feeds will lead to waste a lot of EAAs The efficiency of using protein will be reduced either lacking or excessing any amino acids

3.5 ADC of EAA of ingredients

The ADC of essential amino acids in ingredient were shown in Table 8

Table 8 ADC (%) of EAA (FM-fishmeal, SB-soybean, WB-wheat bran, RB-ricebran)

The values showed mean ± standard deviation

Means in the same row with different letters are significantly different (P<0.05) (MET-methionine, CYS-cysteine, LYS-lysine, THR-threonine, ARG-arginine, ILE-isoleucine, LEU-leucine, VAL-valine, HIS-histidine, PHE-phenylalanine, GLY-glycine, SER-Serine, PRO-proline, ALA-Alanine, ASP-aspartic, GLU-glutamic)

The ADC of methionine and lysine were not significant difference between fishmeal, defatted soybean and wheat bran ADC of methionine and lysine in rice bran and defatted rice bran were significantly lower than fishmeal and defatted soybean meal There was no significant difference between ingredients in digestibility of arginine

MET 94.3 ± 0.01 a 91.3 ± 0.05 a 84.0 ± 0.02 a 58.3 ± 0.19 b 61.3 ± 0.18 b LYS 98.3 ± 0.01 a 98.3 ± 0.03 a 87.3 ± 0.06 ab 81.0 ± 0.14 b 82.7 ± 0.07 b THR 97.7 ± 0.01 a 92.3 ± 0.03 a 80.7 ± 0.12 a 47.0 ± 0.08 b 56.7 ± 0.13 b ARG 94.7 ± 0.01 97.3 ± 0.02 93.7 ± 0.05 90.0 ± 0.07 88.3 ± 0.07 ILE 95.7 ± 0.02 a 92.7 ± 0.03 a 87.7 ± 0.09 a 56.0 ± 0.18 b 59.7 ± 0.08 a LEU 96.3 ± 0.02 a 95.0 ± 0.02 a 90.3 ± 0.06 ab 70.0 ± 0.11 b 82.3 ± 0.03 c VAL 95.0 ± 0.02 a 91.7 ± 0.03 a 78.7 ± 0.06 b 55.7 ± 0.12 c 66.3 ± 0.05 c HIS 94.7 ± 0.01 a 95.0 ± 0.02 a 92.0 ± 0.06 ab 83.0 ± 0.10 b 81.7 ± 0.04 b PHE 92.3 ± 0.01 ab 91.7 ± 0.04 ab Not measured 69.7 ± 0.17 b 84.7 ± 0.11 b

The digestibility of threonine, isoleucine, leucine, valine, histidine and phenylalanine were found no significant difference between fishmeal, defatted soybean and wheat bran (excepting valine) ADC of essential amino acid in rice bran and defatted rice bran was much lower than others However, the ADC of isoleucine in defatted rice bran was significantly higher than rice bran

The digestibility of EAA of different ingredients is very important in feed formulation that containing fully balanced EAA Deficiency of dietary amino acids

in fish generally includes poor growth, high feed conversion ratio and low appetite

(Lazard et al., 1993) e.g deficiency of methionine in trout caused development of bilateral lens cataracts and suffer poor growth and survival (Poston et al., 1977, cited

from Lazard, 1993) Tryptophan deficiency caused scoliosis in sockeye salmon

(Havler and Shanks, 1960, cited from Lazard, 1993) and rainbow trout (Shanks et al.,

1962; Kloppel and Post, 1975, cited from Lazard, 1993), but apparently not in the

catfish (Wilson et al., 1978, cited from Lazard, 1993) Moreover, tryptophan

deficiency also caused abnormal calcium deposits in the kidney and the body plates surrounding the notochord and sheath (Klopped and Post, 1975, cited from Lazard, 1993) Lysine deficiency in rainbow trout caused caudal fin rot (loss much of the fin) (Ketola, 1979, cited from Lazard, 1993)

Protein is the main organic component in fish, occupies 60-75% fish body weight (Halver, 1988) Although the structure of protein is very complex, it is digested into amino acids when fish consumed The ADC of EAAs in fishmeal was no significant difference compared to defatted soybean in this experiment, ranged from 91 to 98%, indicating a high protein quality of these ingredients That shows the potential to replacing fishmeal in diet by defatted soybean meal

However, the amino acids digestibility will be decreased when feed contain high fibre In some case, feed contain too high starch can also reduce the absorption of EAA because of reduction of trypsin and pepsin activities That is the reason while the ADC of EAA in rice bran, defatted rice bran were significantly lower than others ingredients The lower digestibility of EAA in rice bran has been reported for tilapia

(Guimaraes et al., 2008) and rainbow trout (Gaylord et al., 2010) The reasons could

be related to high content of fibre, lipids, and anti-nutritional compounds (Guimaraes

et al., 2008; Beyer et al., 1983)

4 CONCLUSION

The ADC of EAA in fishmeal was significantly higher than other plant ingredients There was no significant difference between fishmeal and defatted soybean in term

of EAAs digestibility

Increasing the ratio of fishmeal in diet, the ADC of EAA was slightly increased compared to reference diet, whereas no significant reduction in EAAs ADC of defatted soybean meal Moreover, the EAAs ADC of other treatments (wheat bran, rice bran and defatted rice bran) was slightly decreased compared to reference diet