Evaluation of the dietary essentiality of vitamins for Penaeus monodon

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Evaluation of the dietary essentiality of vitamins for Penaeus monodon H.R.V. REDDY, M. GANAPATHI NAIK & T.S. ANNAPPASWAMY University of Agricultural Sciences, College of Fisheries, Mathsyanagar, Mangalore, India Abstract The eect of deletion of individual water-soluble (thiamin, riboflavin, pyridoxine, cyanocobalamin, pantothenic acid, folic acid, niacin, biotin, choline, inositol and ascorbic acid) and fat-soluble vitamins (vitamins A, D, E and K) in semipurified diets on growth and survival of juvenile shrimp, Penaeus monodon, was studied in the laboratory for 8 weeks. Diets lacking riboflavin and vitamin K did not aect growth and survival of shrimp. However, deletion of inositol and choline in the diet resulted in poor growth. Maximum growth was observed in the control diet (C1) supplemented with all vitamins. Diets deficient in ascorbic acid, biotin, folic acid, niacin, thiamin and a-tocopherol resulted in poor appetite and poorer feed conversion eciency. All treatments except the control (C1) resulted in histopathological changes in the digestive gland cells. Detachment or destruction of the epithelial cells was observed in all treatments lacking individual vitamins but more severely in the treatment without a vitamin supplement, followed by inositol, choline and ascorbic acid deficient diets. KEY WORDS: KEY WORDS: Penaeus monodon, shrimp feed, vitamin require- ment Received 24 September 1997, accepted 14 May 1999 Correspondence: H.R.V. Reddy, University of Agricultural Sciences, College of Fisheries, Mathsyanagar, Mangalore–575 002, India Introduction To develop cost-eective, nutritionally balanced diets, prior knowledge of the essential nutrient requirements of the cultured species is a prerequisite. Although several contribu- tions exist concerning the protein, lipid and carbohydrate requirements of Penaeus monodon (New 1976; D’Abramo 1989; Kanazawa 1989), very few studies have been carried out on the vitamin requirements of this species (Catacutan & Cruz 1989; Conklin 1989; Shiau & Suen 1994). Existing feed formulations for this species use vitamin levels based on information derived from studies on related species. Numer- ous studies on vitamin nutrition have revealed that vitamins are essential for normal health and life functions, such as growth, development, metabolism, maintenance and repro- duction (New 1976; Conklin & Provasoli 1977; He et al. 1992). Widely divergent quantities of vitamins have been added into shrimp feeds using vitamin premixes. Specific problems, which conceivably might have been related to vitamin deficiencies, have not been noted. Thus, the objective of the study was to evaluate the relative dietary essentiality of water-soluble and fat-soluble vitamins for P. monodon. Materials and methods Post-larval Penaeus monodon were obtained from a commer- cial hatchery located at Mangalore and acclimatized to laboratory conditions for 30 days in FRP (fibreglass reinforced plastic) tanks (2000 L) and fed artificial pelleted feed (Table 1) for a period of 15 days prior to the start of the experiment. P. monodon juveniles (mean weight of 0.085 0.02 g) were stocked in circular FRP tanks (60 cm diameter, 75 cm high) at a rate of 25 individuals per tank (90 shrimp m –2 ) containing 200 L sea water in a recirculating system for 8 weeks. Each tank was provided with continuous aeration from an air-stone connected to an air pump. The water quality parameters were measured every week. Optimum temperature (28 1.5 °C), salinity (2.8–3.0 g L –1 ) and dissolved oxygen (6.0–6.5 mg L –1 ) were maintained during the experimental period. Ammonia and nitrite nitrogen levels remained below 0.1 mg L –1 . Fifteen diets were prepared each containing a feed base (Table 1) and deletion of one vitamin at a time (thiamin, riboflavin, pyridoxine, cyanocobalamin, pantothenic acid. folic acid, niacin, biotin, choline, inositol, ascorbic acid, A, D, E and K). In addition, two control diets containing either the complete vitamin mixture (C1) or no vitamin supplement (C2) were used. Each treatment consisted of two replicates. The shrimp were fed twice daily (morning and evening) at a rate of 267 Aquaculture Nutrition 1999 5 ; 267^275 . ............................................................................................. . ............................................................................................. Ó 1999 Blackwell Science Ltd

Transcript of Evaluation of the dietary essentiality of vitamins for Penaeus monodon

Evaluation of the dietary essentiality of vitamins for Penaeusmonodon

H.R.V. REDDY, M. GANAPATHI NAIK & T.S. ANNAPPASWAMYUniversity of Agricultural Sciences, College of Fisheries, Mathsyanagar, Mangalore, India

Abstract

The e�ect of deletion of individual water-soluble (thiamin,

ribo¯avin, pyridoxine, cyanocobalamin, pantothenic acid,

folic acid, niacin, biotin, choline, inositol and ascorbic acid)

and fat-soluble vitamins (vitamins A, D, E and K) in

semipuri®ed diets on growth and survival of juvenile shrimp,

Penaeus monodon, was studied in the laboratory for 8 weeks.

Diets lacking ribo¯avin and vitamin K did not a�ect growth

and survival of shrimp. However, deletion of inositol and

choline in the diet resulted in poor growth. Maximum growth

was observed in the control diet (C1) supplemented with all

vitamins. Diets de®cient in ascorbic acid, biotin, folic acid,

niacin, thiamin and a-tocopherol resulted in poor appetite

and poorer feed conversion e�ciency. All treatments except

the control (C1) resulted in histopathological changes in the

digestive gland cells. Detachment or destruction of the

epithelial cells was observed in all treatments lacking

individual vitamins but more severely in the treatment

without a vitamin supplement, followed by inositol, choline

and ascorbic acid de®cient diets.

KEY WORDS:KEY WORDS: Penaeus monodon, shrimp feed, vitamin require-

ment

Received 24 September 1997, accepted 14 May 1999

Correspondence: H.R.V. Reddy, University of Agricultural Sciences,

College of Fisheries, Mathsyanagar, Mangalore±575 002, India

Introduction

To develop cost-e�ective, nutritionally balanced diets, prior

knowledge of the essential nutrient requirements of the

cultured species is a prerequisite. Although several contribu-

tions exist concerning the protein, lipid and carbohydrate

requirements of Penaeus monodon (New 1976; D'Abramo

1989; Kanazawa 1989), very few studies have been carried

out on the vitamin requirements of this species (Catacutan &

Cruz 1989; Conklin 1989; Shiau & Suen 1994). Existing feed

formulations for this species use vitamin levels based on

information derived from studies on related species. Numer-

ous studies on vitamin nutrition have revealed that vitamins

are essential for normal health and life functions, such as

growth, development, metabolism, maintenance and repro-

duction (New 1976; Conklin & Provasoli 1977; He et al.

1992). Widely divergent quantities of vitamins have been

added into shrimp feeds using vitamin premixes. Speci®c

problems, which conceivably might have been related to

vitamin de®ciencies, have not been noted. Thus, the objective

of the study was to evaluate the relative dietary essentiality of

water-soluble and fat-soluble vitamins for P. monodon.

Materials and methods

Post-larval Penaeus monodon were obtained from a commer-

cial hatchery located at Mangalore and acclimatized to

laboratory conditions for 30 days inFRP(®breglass reinforced

plastic) tanks (2000 L) and fed arti®cial pelleted feed (Table 1)

for a period of 15 days prior to the start of the experiment.

P. monodon juveniles (mean weight of 0.085 � 0.02 g) were

stocked in circular FRP tanks (60 cm diameter, 75 cm high) at

a rate of 25 individuals per tank (90 shrimp m±2) containing

200 L sea water in a recirculating system for 8 weeks. Each

tank was provided with continuous aeration from an air-stone

connected to an air pump. The water quality parameters

were measured every week. Optimum temperature

(28 � 1.5 °C), salinity (2.8±3.0 g L±1) and dissolved oxygen

(6.0±6.5 mg L±1) were maintained during the experimental

period. Ammonia and nitrite nitrogen levels remained below

0.1 mg L±1.

Fifteen diets were prepared each containing a feed base

(Table 1) and deletion of one vitamin at a time (thiamin,

ribo¯avin, pyridoxine, cyanocobalamin, pantothenic acid.

folic acid, niacin, biotin, choline, inositol, ascorbic acid, A, D,

E and K). In addition, two control diets containing either the

complete vitamin mixture (C1) or no vitamin supplement (C2)

were used. Each treatment consisted of two replicates. The

shrimp were fed twice daily (morning and evening) at a rate of

267

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Ó 1999 Blackwell Science Ltd

10% body weight per day. Faeces and uneaten food were

removed and mortality recorded daily. Shrimp were weighed

every 2 weeks before feeding. The shrimp were netted, blotted

with a dry gauze cloth and weighed individually. When the

shrimp showed clear vitamin de®ciency symptoms (week 4)

each group was divided into two subgroup of equal number

and mean body weight and one group was used for a recovery

test and fed the C1 diet supplemented with complete vitamins

for remainder of the study period.

Histology of the digestive gland

Upon termination of the experiment, three individuals in the

intermoult stage were taken from each treatment and ®xed in

Bouin's solution for 24 h. The ®xed tissues were dehydrated

in ascending concentrations of alcohol, cleared in toluene,

embedded in para�n wax and sectioned with a rotary

microtome at 6 lm. Sectioned tissues were stained with

haematoxylin and eosin (H&E).

Growth data were analysed for statistical signi®cance by

one-way ANOVAANOVA and the Duncan's multiple range test

(Gomez & Gomaz 1976).

Results

The response of shrimp in terms of percentage weight gain,

feed conversion ratio and survival are presented in Table 2

and growth and relative percentage weight gain in Figs 1

and 2.

Thiamin

In the thiamin-de®cient group the percentage weight gain

decreased gradually after week 4 and shrimp showed

anorexia. The relative percentage weight gain was 75% of

that of the control group and survival was 92%. In the

recovery test, weight gain and food consumption improved.

Pyridoxine

The shrimp fed the pyridoxine-de®cient diet showed de-

pressed growth after week 5 of feeding. The relative

percentage weight gain was 74% of that of the control

group, feed e�ciency decreased after week 4 and survival was

92%. In the recovery test, the feed e�ciency and growth

improved gradually.

Niacin

Niacin de®ciency resulted in poorer feed e�ciency and

shrimp showed inappetence. Prolongation of niacin de®cien-

cy caused blackening of the gills in some shrimp at the end of

week 8. The survival was 86%. The relative percentage

weight gain was 74% of that of the control group. In the

recovery test, growth and feed e�ciency improved.

Choline

Deletion of choline resulted in decreased growth and a�ected

food consumption after week 4. The shrimp exhibited

anorexia after 3 weeks of feeding, which resulted in passive

activity of the shrimp. The relative percentage weight gain

was 52% of that of the control group and survival was 80%.

Table 1 Composition of basal diet

Ingredient g kg)1

Casein (vitamin free) 500Dextrin 200Cellulose 100Cod liver oil 50Amino acid mixture1 30Mineral mixture2 100Vitamin mixture3 20

1Amino acid mixture: according to Shiau & Jan (1992).2Mineral mixture: according to Shiau & Chou (1991).3Providing the following amounts of vitamins (mg k g^1 diet): ascorbicacid 30; biotin 5; calciferol 5; choline 1000; cyanocobalamin 5; folic acid20; inositol 1250; menadione 10, niacin 200; pantothenic acid 150;pyridoxine 50; ribo£avin 50; thiamin 50; a-tocopherol 400; vitamin A 660.

Table 2 Relative weight gain, feed conversion and survival over 8-

week period of Penaeus monodon fed individual vitamin-de®cient

diets compared with control (data in each column with same

superscript letter are not signi®cantly di�erent)

Deficient vitamin Weight gain (%)Feed conversionratio Survival (%)

Control (C1) 540a 2.37 þ 0.30 90Ascorbic acid 400b 3.28 þ 0.11 80Biotin 380b 3.21 þ 0.30 90Calciferol 420b 2.63 þ 0.41 90Choline 280c 3.02 þ 0.12 80Cyanocobalamin (B12) 380a 2.77 þ 0.69 90Folic acid 360b 3.12 þ 0.20 94Inositol 260c 3.15 þ 0.17 84Menadione 530a 2.79 þ 0.09 90Niacin 400b 3.00 þ 0.41 86Pantothenic acid 400b 3.16 þ 0.27 81Pyridoxine (B6) 400a 2.87 þ 0.21 92Riboflavin (B2) 520a 2.56 þ 0.17 95Thiamin (B1) 410a 3.00 þ 0.18 92a-Tocopherol 370b 3.01 þ 0.38 85Vitamin A acetate 480a 2.56 þ 0.52 92Control (C2) 120d 3.82 þ 0.38 60

H.R.V. Reddy et al.

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In the recovery test, the growth and food consumption

improved rapidly.

Ascorbic acid

The shrimp fed an ascorbic acid de®cient diet showed a general

decline activity, leading to poor food intake and anorexia.

Prolonged de®ciency resulted in blackening of gills and lesions

in the abdominal region. The relative percentage weight gain

was 74% of that of the control group. In recovery test, growth

and food e�ciency recovered gradually after week 6.

Riboflavin

The shrimp in this treatment were not a�ected by deletion of

ribo¯avin. The relative percentage weight gain was 96% and

survival was 95%. In the recovery test, the weight gain and

food consumption did not show any improvement.

Inositol

The percentage weight gain of the shrimp fed the inositol-

de®cient diet decreased after week 4. The shrimp showed

anorexia and food consumption decreased gradually after

week 3. The relative percentage weight gain was only 44% of

that of the control group with survival of 84%. In the

recovery test, the shrimp showed improved growth and food

consumption.

Folic acid

Growth and food consumption decreased after week 4 of

feeding the folic acid-de®cient diet. The relative percentage

weight gain was 63% of that of the control group. The

shrimp showed gradual improvement in weight gain and

food consumption in the recovery test.

Cyanocobalamin

Percentage weight gain showed a decreasing trend beginning

at week 4. The relative percentage weight gain was 70% of

that of the control group. In the recovery test, weight gain

and food consumption gradually recovered.

Pantothenic acid

Deletion of pantothenic acid from the diet a�ected the growth

and survival of shrimp at week 5. Shrimp showed inappetence.

Prolonged de®ciency caused partial moulting in the abdomi-

nal region of the shrimp. The relative percentage weight gain

was 74% of that of the control group. The shrimp showed

better growth and food consumption in the recovery test.

Biotin

Percentage weight gain and food consumption was reduced

from week 4. Relative percentage weight gain was 67% of

that of the control and survival was 90%. In the recovery

test, weight gain and food consumption improved.

Vitamin A acetate

The response of the shrimp in terms of percentage weight

gain and survival decreased after week 5. The relative

percentage weight gain was 89% of that of the control.

Shrimp in the recovery test showed improved growth and

food consumption.

Calciferol

Shrimp fed on the vitamin D de®cient diets showed poor

response to growth and food conversion e�ciency beginning

at week 4. The relative percentage weight gain was 90% of

that of the control. In the recovery test, shrimp showed

improved growth and food consumption.

a-Tocopherol

Shrimp exhibited poor growth and inappetence after week 4.

The relative percentage weight gain was only 57 of the

control group accompanied by 85% survival. In the recovery

test, both growth and food consumption increased.

Menadione

Shrimp growth and food consumption was not a�ected by

feeding a vitamin K-de®cient diet. The relative percentage

weight gain was 98% of that of the control group, with

survival of 90%.

Relative weight gain

The e�ects of each vitamin de®ciency on relative weight gain

expressed as percentage of weight gain in the control group

are shown in Fig. 2. De®ciency of inositol, choline, tocop-

herol, pantothenic acid, ascorbic acid, folic acid, biotin,

thiamin had the most signi®cant e�ects (P > 0.05) on

relative weight gain. The weight gain in shrimp fed ribo¯a-

vin-de®cient and vitamin K-de®cient diets was almost equiv-

alent to that of the control group.

Evaluation of vitamins in Penaeus monodon diets

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Figure 1 Percentage weight gain of Penaeus monodon juveniles fed single vitamin de®cient diets. (a) Cyanocobalamin; (b) folic acid; (c) niacin;

(d) choline; (e) thiamin; (f) ribo¯avin; (g) pyridoxine; (h) ascorbic acid; (i) inositol; (j) biotin; (k) pantothenic acid; (l) vitamin A; (m) vitamin D;

(n) vitamin E; (o) vitamin K; (p) control diets.

H.R.V. Reddy et al.

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Figure 1 (Contd.)

Evaluation of vitamins in Penaeus monodon diets

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Feed efficiency

The e�ect of deletion of individual vitamins on feed

conversion ratio is shown in Table 2. The feed e�ciency of

each de®cient group showed an almost similar tendency to

relative weight gain. The FCR values were best in shrimp fed

diets supplemented with the complete vitamin mixture and

poorest in shrimp fed diets de®cient in ascorbic acid,

tocopherol, biotin, choline, folic acid, niacin, pantothenic

acid, thiamin and the negative control (C2).

Histopathology

The digestive gland cells of P. monodon juveniles fed diet

(C1) had numerous cells and showed normal cellular layers

(Fig. 3) while, those shrimp fed speci®c vitamin de®cient

diets exhibited histopathological changes (Fig. 4). The

shrimp fed the negative control diet (C2) lacking all

vitamin had complete loss or destruction of epithelial cells

(Fig. 5).

Discussion

In the present study, reduced growth (89% of that of the

control group) in the vitamin A-de®cient treatment demon-

strates that vitamin A is an essential nutrient for P. monodon.

Its physiological functions are involved in regeneration of

light-sensitive compounds in the retina, transportation of

calcium across some membranes, reproduction and integrity

of cellular and subcellular membranes (National Research

Council 1983, 1987). A few studies (National Research

Council 1983, 1987) have demonstrated that b-carotene can

be utilized by vertebrates as a precursor to vitamin A. He

et al. (1992) also demonstrated the dietary essentiality of

vitamin A for P. vannamei.

Results of the present study revealed that vitamin D is in

diets essential for P. monodon. In vertebrates, vitamin D

functions as a precursor to 1,25-dihydroxy-cholecalciferol,

which stimulates calcium absorption from the intestine and

Figure 2 Relative weight gain of Penaeus monodon juveniles fed

single-vitamin de®cient diets.

Figure 3 Mid-gut cells of Penaeus

monodon fed the complete vitamin sup-

plement diet (C1) showing normal cells.

H.R.V. Reddy et al.

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regulates its movement to maintain physiologically normal

levels in the blood (National Research Council 1983, 1987).

In crustaceans, vitamin D functions in the mineralization of

the exoskeleton, even though marine crustaceans can absorb

signi®cant amounts of dissolved calcium through the gills

(He et al. 1992).

Signi®cantly depressed growth and poor appetite were

observed in shrimp fed a vitamin E-de®cient diet. Vitamin E

is important in protecting polyunsaturated fatty acids in

biomembranes from oxidation and is required in prevention

of various animal diseases (National Research Council 1983,

1987). Vichover & Cohen (1938) demonstrated that vitamin

E was required by Daphnia magna for normal growth and

reproductive activity.

The presence or absence of a vitamin K supplement had no

signi®cant e�ect on shrimp growth and survival. Vitamin K

is required by vertebrates for normal blood clotting mech-

anisms to operate. However, there is no evidence to support

this function in crustaceans. Schechter (1950) demonstrated

that vitamin K was not essential to D. magna. He et al.

(1992) also demonstrated the non-essentiality of vitamin K in

the diet of P. vannamei.

Results of the present study demonstrated that among the

water-soluble vitamins choline, inositol and pantothenic acid

severely retarded the growth of the shrimp followed by

biotin, niacin, pyridoxine and cyanocobalamin. In addition

to reduced growth, poorest survival was observed in diets

de®cient in niacin, choline, inositol and ascorbic acid.

Figure 4 Digestive gland cells of Pen-

aeus monodon fed diets de®cient in either

choline, inositol, ascorbic acid or vita-

min E. Note degeneration of epithelial

cells.

Figure 5 Mid-gut cells of Penaeus

monodon fed diet lacking in vitamins

(C2). Note disrupted basal membrane

and cells.

Evaluation of vitamins in Penaeus monodon diets

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273

Similarly Kanazawa et al. (1976) reported the essentiality of

choline and inositol in the diet of Penaeus japonicus.

However, Deshimaru & Kuroki (1979) reported the non-

essentiality of choline in the diet of P. japonicus.

Information on the vitamin de®ciency syndrome in shrimp

is rather scarce. The only well-documented vitamin de®ciency

disease in penaeid shrimp is the black death syndrome related

to ascorbic acid de®ciency (Lightner et al. 1977). Prolonged

de®ciency of niacin and ascorbic acid resulted in formation of

black lesions on the body in some individuals whereas

de®ciency of pantothenic acid resulted in partial moulting in

few individuals. Similar observations were made by Gopal &

Raj (1993) in Penaeus indicus. However, Shiau & Suen (1994)

did not come across such incidence in P. monodon fed niacin-

de®cient diets. Ascorbic acid is shown to be required by

P. japonicus (Deshimaru & Kuroki 1976; Guary et al. 1976),

Penaeus californiensis (Lightner et al. 1977) and Penaeus

merguiensis (AQUACOP 1978). As reported in the present

study Deshimaru & Kuroki (1979) also observed depressed

growth in P. japonicus fed pyridoxine and thiamin de®cient

diets.

Deletion of ribo¯avin from the feed did not a�ect the

growth of P. monodon in the present study. However, Heinen

(1994) reported that deletion of ribo¯avin led to increased

growth in juvenile fresh-water prawn Macrobrachium rosen-

bergii. Catacutan & Cruz (1989) also reported increased

growth in P. monodon fed ribo¯avin de®cient diets.

In the present study, normal cellular structures were

observed in the mid-gut gland fed control (C1) diet supple-

mented with all vitamins. Detachment of epithelial cells was

observed in other treatments but more severely in treatments

fed diet de®cient in control (C2) (no vitamin supplement),

inositol, choline, pantothenic acid and ascorbic acid. Storch

et al. (1984), Vogt et al. (1985) and Catacutan & Cruz (1989)

also made similar observation in P. monodon fed diets

de®cient in water-soluble vitamins.

Results of this study demonstrate that fat-soluble vitamins,

A, D and E and 10 water-soluble vitamins (thiamin,

ribo¯avin, pyridoxine, cyanocobalamin, pantothenic acid,

folic acid, niacin, biotin, choline, inositol, ascorbic acid) are

essential for P. monodon. No evidence for the vitamin K and

ribo¯avin requirement by P. monodon was observed.

Acknowledgements

This research was supported by the Indian Council of

Agricultural Research grant. The authors are also indebted

to S.L. Shanbhogue, Director of Instruction (F) for the

facilities.

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