Journal of Experimental Marine Biology and Ecology 249 ...directory.umm.ac.id/Data...

LJournal of Experimental Marine Biology and Ecology249 (2000) 181–198

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Influence of dietary carbohydrate on the metabolism ofjuvenile Litopenaeus stylirostris

a , b a a b b*C. Rosas , G. Cuzon , G. Gaxiola , L. Arena , P. Lemaire , C. Soyez ,cA. Van Wormhoudt

a ´ ´Grupo de Biologıa Marina Experimental, Laboratorio de Ecofisiologıa, Fac. de Ciencias, UNAM, Apdo.Post. 69, Cd. del Carmen, Campeche, Mexico

bCentre Oceanologique du Pacifique (COP), IFREMER, BP 700, BP 7004, Taravao, Tahiti, FrenchPolynesia

c `Station de Biologie Marine du Museum National d’Histoire Naturelle et du College de France, BP 225,29900, Concarneau, France

Received 15 October 1999; received in revised form 15 January 2000; accepted 26 February 2000

Abstract

The effect of dietary carbohydrates (CBH) on glucose and glycogen, digestive enzymes,ammonia excretion and osmotic pressure and osmotic capacity of Litopenaeus stylirostrisjuveniles was studied. The increase of CBH, ranging between 1 and 33%, stimulates activities ofa-amylase and a-glucosidase in the hepatopancreas. High levels of glucose in hemolymph and ofglycogen in the hepatopancreas were reached at the highest level of dietary CBH; however, thekinetics of accumulation is different. Shrimps fed with low level of CBH needed 3 h to reachedglucose peak, whereas only 1 h is necessary for high CBH levels. A saturation curve was observedin glycogen level and a-amylase activity with maximum values in shrimp-fed diets containing21% CBH. This level could be used to be included as a maximum shrimp dietary CBH level.Pre-prandial glycogen levels were observed in shrimp fed a diet containing 1% CBH, indicating animportant gluconeogenesis, which affected the protein metabolism. The present results show that adiet containing 10% CBH may not be enough to cover the CBH requirement, which could besatisfied by dietary protein content. The low osmotic capacity observed in shrimp fed on a dietcontaining 10% CBH coincided with a relatively low post-prandial nitrogen excretion whichreflects a low concentration of amino acids circulating in hemolymph, which affected the osmoticpressure and the osmotic capacity. These results reflect the high plasticity of shrimp species to useprotein to obtain metabolic energy from food and its limited capacity for processing dietary CBH. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Carbohydrate metabolism; Penaeid shrimp; Enzymatic activity; Ammonia excretion; Osmoticcapability; Litopenaeus stylirostris juveniles

*Corresponding author. Tel.: 1 52-938-28-730; fax: 1 52-938-28-730.E-mail address: [email protected] (C. Rosas)

0022-0981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 00 )00184-2

182 C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 249 (2000) 181 –198

1. Introduction

A study on carbohydrates and the relation between this group of nutrients and proteinis justified by a growing consideration of minimizing the negative impact of feeds on theenvironment at the pond level. If carbohydrates can represent up to 50% of the diet (inweight), and a part of it is loss in the water enhancing the pollution, a reduction ofdietary carbohydrates is possible. Therefore, knowledge of the capacity of shrimp toutilize carbohydrates is important. Previous work (Andrews et al., 1972; Sick andAndrews, 1973; Deshimaru and Yone, 1978; Abdel-Rahman et al., 1979; Alava andPascual, 1987) investigated carbohydrate utilization relative to the sparing of dietaryprotein which can be achieved and consequently lead to a decrease in the amount ofnitrogen waste.

The blue shrimp Litopenaeus stylirostris is cultured in the South Pacific area and has agood potential to become an important shrimp species in the Americas (Cuzon andAquacop, 1998). Numerous research projects have studied the nutrition of juveniles of L.stylirostris, mainly addressing protein requirements (Fenucci et al., 1982; De La Lanzaet al., 1986; Kanazawa, 1993). Aquacop and Cuzon (1989) achieved average growthuntil they used multi-ingredient diets that satisfied the protein requirements through anative source of protein (from squid). The conclusions from many trials can besummarised as follows: (1) good quality protein is of paramount importance (forexample, a shrimp meal product from one place can be definitely superior to a productfrom an other place) and (2) in practical diets having the same protein level, (30%)squid and shrimp meal gave better growth results than soybean concentrate or fish meal.However, results from a variety of culture systems indicate that, on average, about 25%of nitrogen added as feed or other nutrient input is recovered by the target organism, theremainder affecting the water quality of the waste water (Hargreaves, 1998).

In recent years it has been demonstrated that the use of improper shrimp farmingmethods has damaged natural estuaries and bay systems (Lawrence, 1996). Thiscondition has primarily been caused by both the intensification (increased productionlevels per ha of pond water) of shrimp farming and the increase of ponds and area ofpond water in shrimp farm production. The shrimp farming industry is being constrainedby the need to maintain adequate water quality and acceptable levels of potentialpollutants in effluent discharges (Lawrence et al., 1998). Some researches have proposeddifferent strategies to minimize the pollutants in the effluent discharge for the benefit ofthe estuarine and coastal ecosystems. Diets with low protein content (Molina-Poveda,1998), the optimization of amino acid profile (Velasco et al., 1999), an optimumprotein /energy (P/E) ratio of the food (Pedrazzoli et al., 1998), improvement of the fishmeal quality (Ricque-Marie et al., 1998) an optimum feed management strategy

´(Cortes-Jacinto and Portillo, 1998; Velasco et al., 1999) and a deeper knowledge ofnutritional physiology and biochemistry (Ceccaldi, 1998) have been proposed.

Knowledge of the optimum levels of the other components of the diet (lipids andcarbohydrates) and their relation to the P/E ratio can be useful in reducing theproduction cost of the food and its effect as a pollutant. Although dietary carbohydratesare the most economical source of energy in food, little information about the utilization

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 249 (2000) 181 –198 183

of carbohydrate by shrimp is available (Pascual et al., 1983; Alava and Pascual, 1987;Shiau and Peng, 1992; Shiau, 1998). Some researchers have demonstrated that the typeand level of carbohydrates affect growth rate of M. japonicus (Deshimaru and Yone,1978; Abdel-Rahman et al., 1979), F. aztecus (Andrews et al., 1972) and Litopenaeusvannamei (Cousin, 1995). In P. monodon juveniles survival is affected by differentcarbohydrate levels and sucrose and glucose were better than trehalose in promotinggrowth of P. monodon (Pascual et al., 1983; Alava and Pascual, 1987)

The mechanisms responsible for the limited utilization of glucose by some species ofpenaeid shrimp are not yet fully understood. A negative physiological effect caused byglucose saturation due to a higher rate of absorption across the digestive tract is apossible explanation (Shiau, 1998). For this reason, many researchers have suggested theuse of more complex carbohydrates such as starch which undergoes enzymatichydrolysis before assimilation, in shrimp diets. Glucose from starch appears at gutabsorption sites at a rate slower than free glucose (Pascual et al., 1983; Alava andPascual, 1987; Shiau and Peng, 1992; Shiau, 1998). Although the activity of truea-amylases (EC 3.2.1.1, a-1,4-glucan 4-glucanhydrolases) from 40 crustacean species,including seven penaeid species, was recently recently reported (van Wormhoudt et al.,1995), no information about the carbohydrate-digesting enzymes of shrimp relative todifferent dietary carbohydrate levels is available.

Protein metabolism has been recognized as a key to the understanding of energyrequirements of shrimp because growth depends strictly on protein (Deshimaru andShigeno, 1972; Deshimaru and Yone, 1978; Lemos and Rodriguez, 1998). The highprotein requirement and the limited capacity of shrimp to store reserve substances likelipids and carbohydrates (Dall and Smith, 1986) could be related to the capacity ofshrimp to use proteins as a source of energy as well as for growth. In a recent study themaximum growth rate of L. vannamei juveniles was obtained when diets containing 50%protein and 1% carbohydrates were fed (Rosas et al., unpublished). The results of otherstudies showed that shrimp can change the metabolic substrates (measured as oxy-gen:nitrogen ratio) according to the physiological or nutritional requirements, passingfrom protein–lipid–carbohydate metabolism to protein metabolism, indicating thatshrimp are well adapted to use proteins as a source of energy (Rosas et al., 1995a,b,1999; Taboada et al., 1998). The limited use of dietary carbohydrates by shrimp,therefore, may be the consequence of metabolic adaptation to use the proteins as aprimary source of energy, because protein is the higher reserve substrate in shrimp,which can be converted to carbohydrates following the gluconeogenic pathway(Campbell, 1991).

Ammonia accounts for 60–100% of the products of nitrogen metabolism and itsformation may result directly from either deamination or transamination, both involvedin amino acidic gluconeogenesis (Claybrook, 1983). The ammonia excretion has beenused as a good indicator of the utilization of dietary protein for energy by shrimp (Dalland Smith, 1986; Lei et al., 1989; Taboada et al., 1998). The role of dietary proteins as asource of energy during post-absorptive processes that follow the ingestion of food hasbeen evaluated through the measurement of post-prandial nitrogen excretion (PPNE)(Rosas et al., 1995a,b). According to Claybrook (1983), both routine ammonia excretion


and PPNE indicate the use of dietary protein as a source of energy and can be used as anindirect measure of gluconeogenesis.

Recently several authors showed that the osmotic capacity (Lignot et al., 1999) andmetabolites, like hemolymph glucose (Racotta and Palacios, 1998) and digestive glandglycogen (Rosas et al., 1995b), are directly related to protein and carbohydratemetabolism. Coupled with ammonia excretion, these indices can be useful in developingan understanding of carbohydrate metabolism, relative to physiological adaptation. Inthis study the effects of dietary carbohydrate level on adaptation of digestive enzymes,as related to digestion, glucose hemolymph level, hepatopancreatic glycogen, ammoniaexcretion and osmotic pressure of Litpanaeus stylirostris juveniles are evaluated.

2. Material and methods

2.1. Animals and zootechnical measurements

Live animals (9.4560.15 g wet weight) were obtained from the ‘Centre´Oceanologique du Pacifique’ (Ifremer) located in Tahiti. One hundred shrimp were

reared for 15–18 days at a density of eight shrimp per 100-l tank, in a flow-through seawater system (35 ppt). The light–dark photoperiod was 12 h/12 h and water temperaturewas 28618C. The shrimp were fed ad libitum 3 times a day (08, 12 and 20 h). Uneatenfood particles were removed regularly. Four tanks were randomly assigned to eachcarbohydrate level.

2.2. Diets

The juveniles were fed formulated semi-purified diets, prepared with different levelsof carbohydrates: 1, 10, 21 and 33%. The ingredient composition of the experimentaldiets are presented in Table 1. The experimental diets were prepared by throughlymixing the dry ingredients with oil and then adding water until a stiff dough resulted.The dough was then passed through a mincer with a die, and the resulting spaghetti-likestrings were air dried at 608C. After drying, the diets were broken up, sieved to aconvenient pellet size, and stored at 2 48C.

2.3. Biochemical analysis

The specific enzymatic activities of a-amylase, a-glucosidase and total proteases ofthe midgut gland were evaluated after 15–18 days of acclimation to the different diets.Five digestive glands per dietary treatment were collected. These were individuallyhomogenized (Ultraturrax) for 30 s with distilled water. The homogenates werecentrifuged at 20 000 3 g for 30 min at 48C and the supernatants collected. Proteinconcentration was determined by the method of Bradford (1976) using reagents fromBiorad (Hercules, CA) and bovine serum albumin (BSA) as the standard. a-Glucosidase


Table 1Ingredient composition of experimental diets

Ingredients Carbohydrate level (% dry weight)

1 10 21 33

Wheat flour 5 15 35 53.5Fish meal 30 30 30 30

aSPFC 90% 10 10 5 5Soybean meal 38.5 33.5 18.5Gluten 5Krill paste 2 2 2 2Alginate Na 2.5 2.5 2.5 2.5Fish oil 3 3 3 3Lecithin 2 2 2 2

bRovimix 1 1 1 1cMinerals 1 1 1 1

100 100 100 100

%CP 48 43 34 26%CBH 1 10 21 33% lipid 7 7 7 7MJ/kg 13 14 14.5 15

a ˆSoluble fish protein concentrate, 90% protein (Sopropeche, Boulogne s /mer, France).b Retinyl palmitate (vitamin A), 8 000 000 IU; cholecalciferol (vitamin D ), 196 000 IU; a-tocopherol3

acetate (vitamin E), 10 000 mg/kg; vitamin K , 800 mg/kg; ascorbyl phosphate (vitamin C), 15 000 mg/kg;3

thiamine (vitamin B ), 700 mg/kg; riboflavin (vitamin B ), 2000 mg/kg; pyridoxin (vitamin B ), 1000 mg/kg;1 2 6

niacin (vitamin PP), 10 000 mg/kg; calcium pantothenate, 5000 mg/kg; cyanocobalamin (vitamin B ), 5012

ˆmg/kg; folic acid, 250 mg/kg; biotin, 30 mg/kg; inositol, 30 000 mg/kg (Hofmann La Roche, Basle,Switzerland).

c Disodium phosphate and monopotassium phosphate in equal amounts.

(EC 3.2.1.20) was assayed spectrophotometrically using p-nitrophenyl-a-D-glucopy-ranoside (Sigma, St. Louis, MO) as the substrate. The standard reaction mixture (1 mMsubstrate in 50 mM sodium phosphate buffer, pH 6, containing an aliquot of enzymesolution) was incubated at 378C for 30 min. The reaction was stopped with Na CO 1 M2 3

(0.1 vol) (Thirunavukkarasu and Pries, 1983) and the absorbance was read at 410 nm.One unit (U) of enzyme activity is defined as the amount of enzyme capable of

21 21hydrolysing 1 mmol of substrate per min (´ 5 18 000 l mol cm ). Amylase activitywas measured according to a modified Bernfield’s method (1955) using 1% glycogen assubstrate diluted in a 2.5 nM MnCl , 10 mM Na Cl, 10 mM, pH 7, phosphate buffer.2

Enzymatic activity was expressed as mg of maltose liberated during 10 min at 378C.Proteolytic activity was measured using 1% azo-casein as substrate diluted in a sodiumphosphate 10 mM, pH 7, buffer at 378C for 30 min. Total proteases is defined by mg

21 21hydrolysed azo-casein min mg of protein.Glycogen content of the midgut gland was determined from a sample of five shrimp

from each treatment, at time 0 (after 12 h fasting) and 1, 2, 3, 6 and 7 h after feeding.Glycogen was extracted following the method reported by Dubois et al. (1956). Eachmidgut gland was first homogenized in trichloroacetic acid (TCA, 5%) for 2 min at16 000 rpm, and centrifuged (3000 rpm for 5 min). This procedure was performed two


times. One ml of TCA was pipetted into tubes and mixed with 5 vol of 95% ethanol.The tubes were then placed in an oven at 37–408C for 3 h. After precipitation occurred,the tubes were centrifuged at 3000 rpm for 15 min. The precipitate containing theglycogen was dissolved by addition of 0.5 ml of boiling water and 5 ml of concentratedsulfuric acid and phenol (5%). The contents of the tubes were transferred to acolorimeter tube and read at 490 nm in a microplate spectrophotometer.

The level of glucose in the hemolymph of the same shrimp used for the glycogendetermination was measured. Before the midgut gland was excised, 100–300 ml ofhemolymph were extracted with a syringe inserted at the base of the fifth pereiopod afterthe shrimp had been blot dried. A sub-sample was obtained with another syringecontaining a 12.5% solution of sodium citrate, which was used to prevent clotting (Rosaset al., 1995b) The glucose concentration in the hemolymph was measured with acommercial kit for medical diagnosis (Merckotest 3306). Glucose content of the extractof the midgut gland was measured with a commercial kit (Biotrol Diagnostic), using asreactive a phosphate 100 mM buffer at pH 7.3 which contained phenol 16 mM,4-aminoantipyrine 0.25 mM, glucose oxidase at 20 000 U/ l and perioxidase 1000 U/ l.The absorbance was measured at 500 nm.

2.4. Physiological analysis

Osmotic pressure of the hemolymph of the same shrimp used for the glycogen andglucose determinations, was measured with a Wescor 5500 vapor pressure osmometerthat required 8 ml of sample per titration.

Osmotic capability (OC) of shrimp was measured for 15 intermolt shrimp selectedfrom each treatment and exposed to a rapid change in salinity from 35 to 5 ppt. Theseshrimp were held in 75-l tanks containing water at a salinity of 35 ppt for 12 h prior tothe experiment. During this time the shrimp were fed with the experimental diets. Thenthese shrimp were transferred to a similar tank containing water at 5 ppt and held therefor 2 h. Fifty shrimp from each treatment were maintained in 35 ppt and served as acontrol group. After a 2-h period, hemolymph samples were collected similar to theprocedure followed to collect samples for pre- and post-prandial glucose and osmoticpressure of the hemolymphs. OC is defined as the difference between the osmoticpressure of hemolymph and of the external medium, at a given salinity. The osmoticpressure of the water was measured in a Wescor 5500 vapor pressure osmometerrequiring 8 ml of sample per titration.

Ammonia excretion was measured using the indophenol blue technique. Fifteenshrimp from each dietary treatment were starved for 12 h. Groups of five shrimp eachwere placed in 30-l chambers which were connected to a water recirculating system(Charmantier et al., 1994). To prevent handling stress, the shrimp were conditioned inthe chambers for 12 h before any reading was recorded. Ammonia excretion wascalculated as the product of the difference in the water ammonia concentration at the

21input and output of each chamber, timing the water flux (1 l min ). The readings werecorrected relative to a control chamber that contained no shrimp. The first reading wasconsidered to be the rate of ammonia excretion under fasting conditions. Thereafter, 2 gof food per five animals were added to each chamber, and the amount of of ammonia


excretion after 1, 2, 3, 4, 6 and 7 h was determined. The shrimp were then sacrificed,dried (608C) and weighed until a constant weight was attained.

The ingestion rate was measured in the chambers used to determine ammoniaexcretion. At the end of the experiments, all remaining food was recovered, carefullyseparated and dried (608C) until a constant weight was attained. At the same time, acontrol experiment was conducted to evaluate loss of weight due to the natural leachingof the food in the chambers. Two grams of dry food were placed in a similar chamberwithout animals. After 7 h, the remaining food was recovered, dried, and weighed. Theamount of ingested food was determined as the original amount minus the remainingamount plus the amount lost due the leaching. The ingested food was expressed as

21 21J day g wet weight .

2.5. Statistical analysis

The effect of different dietary levels of carbohydrate on the digestive enzymes,glucose content of midgut gland extract, glucose hemolymph level, digestive glandglycogen, ammonia excretion rates, osmotic pressure, ingestion rate and osmoticcapability was analyzed separately using ANOVA. Homogeneity of variances wasverified with the Cochran’s test. Means for each treatment were compared usingDuncan’s multiple range test, only after ANOVA indicated significant differences amongall the dietary treatments (Zar, 1974)

3. Results

3.1. Ingestion rate and survival

A significant reduction in ingestion rate was recorded as dietary CBH increased withcomparatively high values for animals fed diets containing 1% CBH (1.4 J /day per gwet weight (ww)) and low values in shrimp fed diets containing 21 and 33% CBH (0.5and 0.45 J /day per g ww, respectively) (P , 0.05) (Fig. 1). Survival ranged from 96 to100% and was unaffected by CBH level (P . 0.05).

3.2. Biochemical analysis

The specific activities of the a-amylase and a-glucosidase were significantly affectedby the level of dietary carbohydrates (P , 0.05) (Table 2). For both carbohydrases, asthe level carbohydrate increased, the specific activities increased. A saturation curve ofa-amylase activity was observed, with the maximum activity in shrimp fed dietscontaining 21 and 33% CBH (P . 0.05). The maximum values of a-glucosidase wereobtained at dietary levels of 21 and 33% (Table 2). Total proteases did not significantlydiffer among diets (P . 0.05). Glucose content in the midgut gland extract for the 1%dietary carbohydrate treatment was significantly lower than that of each of the otherthree treatments (P , 0.05) (Table 2)

No significant differences in pre-prandial glucose level were observed among the


Fig. 1. Effect of dietary carbohydrate level on ingestion rate (J /day per g ww) of L. stylirostris juveniles.Mean6S.E; different letters indicate statistical differences (P , 0.05).

shrimp fed diets with the different levels of CBH, with values ranging from 13.7mg/100 ml (10% CBH) to 21.9 mg/100 ml (21% CBH). The pre-prandial level ofglycogen in the digestive gland ranged between 4.3 and 6.9 mg/g of tissue, with lowvalues in animals fed the diet containing 1% CBH and higher values in animals fed dietscontaining 21 and 33% CBH (Table 2) (P , 0.05). Increases in mean post-prandialglucose and glycogen concentration (Table 3) were observed in response to differentdietary CBH levels. In all cases, glucose and glycogen levels notably increased at 1–3 hafter feeding, and returned to the pre-feeding rate 6–7 h later (Fig. 2). The maximumpost-prandial glucose level was found in shrimp fed the diet containing 10% CBH (73

Table 2Effect of carbohydrate levels (%) on the specific activity of total proteases: a-amylase and a-glucosidase

aenzymes of the hepatopancreas of juveniles of Litopenaeus stylirostris

Low carbohydrate High carbohydrate

A B C D

Diet (%):bCP 48 43 32 26cCL 7 7 7 7

dCBH 1 10 21 33

P/E ratio 40 33 26 18(mg/kJ)Total proteases 2.0760.29* 1.9960.22* 2.5660.13* 2.3260.22*

21Glucose (mg ml ) 52613* 110617** 129623** 13765**homogenizedin hepatopancreasa-Amylase 0.4460.09* 0.7060.06*,** 0.8760.12** 0.9060.05**a-Glucosidase 264.367.46* 256.35616.92* 366.74630.42** 433.48639.60**Total protein 23.765.33* 41.0364.96** 43.1568.9** 38.01613.33**

a Values mean6S.E.b Protein concentration (%).c Lipid concentration (%).d Carbohydrate concentration (%).Entries with differing numbers of asterisks are significantly different (P , 0.05).


Table 3Effect of carbohydrate levels (%) on growth rate (mg/day), survival, glucose in hemolymph, and glycogen in

adigestive gland of Litopenaeus stylirostris

Low carbohydrate High carbohydrate

A B C D

Diet (%):bCP 48 43 32 26cCL 7 7 7 7

dCBH 1 10 21 33P/E ratio (mg/kJ) 40 33 26 18

Pre-prandialPlasma glucose level 18.460.4* 13.760.4* 21.960.8** 17.760.5*(mg/100 ml)Digestive gland glycogen 4.360.1* 6.060.2** 7.160.2** 6.960.2**(mg/g of tissue)Number of animals 6 6 5 6

Post-prandial at the peakPlasma glucose level 38.162.7* 73.064.0** 55.162.2*** 37.762.3*(mg/100 ml)Time to reach the peak (h) 3 3 2 1Digestive gland glycogen 6.460.1* 9.360.5** 13.360.7*** 11.760.4***(mg/g of tissue)Time to reach the peak (h) 1 3 2 2Number of animals 6 5 5 5

Glucose level 7 h after feedingPlasma (mg/100 ml) 19.160.6* 20.161.2* 20.060.4* 32.061.3**Glycogen level 7 h after feedingDigestive gland (mg/g of tissue) 4.560.1* 5.060.2* 2.760.1** 5.560.1***Number of animals 5 5 6 6

a Values mean6S.E.b Protein concentration (%).c Lipid concentration (%).d Carbohydrate concentration (%).Entries with differing numbers of asterisks are significantly different: P , 0.05.

mg/100 ml), followed by shrimp fed diets containing 21% CBH (55.1 mg/100 ml) and1 and 33% CBH (mean value of 37.9 mg/100 ml) (P , 0.05) (Table 3). The maximumpost-prandial level of glycogen increased as levels of dietary carbohydrate increasedwith low values in animals fed the diet containing 1% CBH (6.4 mg/g of tissue) andsignificantly higher levels in shrimp fed diets containing 21% CBH (13.3 mg/g oftissue) and 33% CBH (11.7 mg/g of tissue) (P , 0.05) (Table 3).

The time to reach the peak level of glucose in the hemolymph and glycogen in thedigestive gland varied according to the dietary CBH levels (Table 3, Fig. 2). The time toreach the peak of glucose hemolymph level was inversely related to dietary carbohydratelevels, 3 h in animals fed low CBH levels (1 and 10%), 2 h in animals fed dietscontaining 21% CBH and 1 h in animals fed diets containing 33% CBH (Table 3). Thetime to reach the peak of digestive gland glycogen was 1 h in animals fed diets


Fig. 2. Effect of feeding on midgut gland glycogen concentration (mg/g tissue) and hemolymph glucose levels(mg/100 ml) in L. stylirostris juveniles fed with different carbohydrate levels (%). Mean6S.E.

Fig. 3. Post-prandial glycogen concentration (mg/g tissue) and a-amylase activity (IU/mg protein) of L.stylirostris juveniles fed diets containing different levels of carbohydrates (%). Mean6S.E.


containing 1% CBH, 3 h in animals fed diets containing 10% CBH and 2 h in animalsfed diets containing 21 and 33% CBH (Table 3).

A saturation curve was observed in post-prandial glycogen concentration and a-amylase activity in relation to dietary CBH levels (Fig. 3). Significantly greater glycogenaccumulation and enzymatic activity was observed in animals fed diets containing 21and 33% CBH (P , 0.05).

3.3. Physiological analysis

Ammonia excretion was affected by dietary carbohydrate levels (Fig. 4). For alldietary treatments a higher rate of ammonia excretion was observed for animals afterfeeding (P , 0.05). Post-prandial nitrogen excretion (PPNE) resulted in an increase of92% (1% CBH), 46% (10% CBH), 33% (21% CBH) and 84% (33% CBH) relative tothe rate obtained in fasting animals (Fig. 4) (P , 0.05). The maximum pre- andpost-prandial ammonia excretion was observed in shrimp fed diets containing 1% CBHfollowed by that observed for shrimp fed diets containing 10, 21 and 33% CBH(P , 0.05) (Fig. 4).

Osmotic pressure was affected by dietary carbohydrate levels and nutritional conditionof the shrimp (Fig. 5a). In shrimp fed diets containing 10 and 33% CBH, thepre-prandial osmotic pressures were significantly higher than those observed in feedinganimals (P , 0.05). In shrimp fed diets containing 1% CBH post-prandial osmoticpressure was higher than that observed in fasting animals (P , 0.05). In animals feddiets containing 21% CBH no differences were observed in pre- and post-prandialosmotic pressure (P . 0.05). The lowest osmotic pressure was observed in feedinganimals fed diets containing 10% CBH and the highest in feeding animals fed dietscontaining 1% CBH (Fig. 5a) (P , 0.05).

No significant differences were observed in the osmotic capability (OC) obtainedbetween shrimp fed diets containing 1, 21 and 33% CBH (P . 0.05). The OCdetermined for animals fed diets containing 10% CBH was significantly lower than that

Fig. 4. Pre and post-prandial nitrogen ammonia excretion of L. stylirostris juveniles fed with different levelsof dietary carbohydrates. Mean6S.E. Different letters or symbols (*) indicate statistical differences (P , 0.05).


Fig. 5. Effect of dietary carbohydrate on pre- and post-prandial osmotic pressure (mOsm/kg; A) and osmoticcapability (mOsm/kg; B) after a change in salinity of L. stylirostris juveniles.

observed for animals fed diets containing each of the other carbohydrate levels(P , 0.05) (Fig. 5b).

4. Discussion

The concept that CBH can substitute for the proteins as an energy source in shrimpfood, thereby reducing the costs associated with feed production, is already documented(Cruz-Suarez et al., 1994). However, many authors have demonstrated that marineshrimp have severe restrictions for the utilization of dietary CBH (Bages and Sloane,1981; Alava and Pascual, 1987; Shiau and Peng, 1992).

A significant induction of a-amylase and a-glucosidase activities related to anincrease in dietary CBH levels was found for juvenile L. stylirostris. There is evidencefor an adaptation of these digestive enzymes to the dietary level of wheat flour. Similarresults have been reported for the amylasic activity in Homarus americanus (Hoyle,


1973) and Palaemon serratus (van Wormhoudt et al., 1980). In L. vannamei a regulationat the level of transcription for amylase was reported when animals were fed dietscontaining a constant level of starch (Le Moullac et al., 1996). For L. stylirostris theregulation of enzymatic activity needs to be studied.

The a-glucosidase participates in the final intracellular digestion of the dietarycarbohydrates in the B cells (blister-like cells) of the midgut gland of shrimps (LeChevalier and Van Wormhoudt, 1998). The main function of shrimp a-glucosidase is tocomplete the breakdown of maltose and oligosaccharides liberated by amylo-hydrolysis(a- [1–4]-malto-oligosaccharide residues obtained after the action of a-amylase).Glucose is the final product of this process and it is evident that the glucose content ofthe extract of the midgut gland (13765 mg/ml), and the activity of dietary a-glucosidase increased according to CBH increment. This enzyme has a specific activitymore pronounced for linear chains of glucose, which can explain the similar di-gestibilities observed for starches in L. vannamei and L. stylirostris (Cousin, 1995).Cousin et al. (1996) showed that native starch or precooked starch were similarlydigested by juvenile shrimp and without any difference in contribution to growth.

Intermediary metabolic enzymes may provide additional information about howshrimp can utilize glucose. Activities measured confirmed (i) the low entry of glucoseinto the citric acid cycle; (ii) the influence of dietary CBH on the rate of conversion ofglucose to glycogen; and (iii) the role of PEPCK and gluconeogenesis at low dietaryCBH levels. Hexokinase of crab was quickly saturated because of its low K value, andm

no specific phosphatase was present (Loret, 1990), but this response remains to beevaluated in shrimps fed diets containing different levels of CBH. The interest lies inidentifying the routes of glucose metabolism via phosphorylation, via pentose phos-phates or via the synthesis of acetylglucosamine and the activities of enzymes related tosuch pathways.

Glucose and glycogen levels of L. stylirostris reached different levels according to thetype of diet. In both cases levels were higher after feeding, showing that CBHmetabolism is affected by dietary CBH levels. Higher levels occurred between 1 and 3 hafter feeding, depending on the CBH level. In all cases, initial CBH values were reachedbetween 6 and 7 h after feeding (Fig. 2). Similar results have been reported by Rosas etal. (1995b) for L. setiferus fed with squid, for L. vannamei and M. japonicus fed dietscontaining different sources of CBH (Abdel-Rahman et al., 1979; Cousin, 1995) and M.japonicus fed corn starch (Shiau and Peng, 1992). Here, the onset of the glucosehemolymph level was affected by the dietary CBH level. Shrimp fed with low CBHlevel (1 and 10% CBH) needed 3 h to reach the glucose peak in comparison to 2 h forshrimp fed a diet containing 20% CBH and only 1 h in shrimp fed a diet containing 30%CBH (Fig. 2). Abdel-Rahman et al. (1979) reported that serum glucose levels in M.japonicus increased rapidly after administration of glucose, resulting in a physiologicallyabnormal elevation of serum glucose concentration. The abnormal level impairedutilization of CBH as an energy source and inhibited the absorption of amino acids inthe intestine.

Once the food is digested in the gut, chyme and fine particles are digested in thelumen and absorbed by diffusion to the inner portions of the digestive gland tubules,thus initiating the accumulation of glycogen (Al-Mohanna and Nott, 1987). The


glycogen accumulation after feeding was affected by the dietary CBH, showing asaturation curve with the low values observed in shrimp fed with low CBH diets and ahigher values observed in animals fed between 21 and 33% CBH (Fig. 3). A similarsaturation curve was observed in a-amylase activity indicating a limited capacity of L.stylirostris to store and process dietary CBH, which is around 20% of dietary CBH. Asimilar saturation curve of post-prandial glycogen concentration has been obtained in ourlaboratory, with the maximum glycogen concentration occurring in L. setiferus and L.vannamei juveniles fed diets containing 23% CBH (Rosas et al., unpublished). Takinginto account these results the highest possible level of dietary CBH level should beconsidered the level at which the maximum capacity to store and process dietary CBHoccurs.

The lack of differences in pre-prandial glucose and glycogen levels betweentreatments, indicates that shrimp can produce and store CBH from dietary carbohydratesand the gluconeogenesis pathway. The present results suggest that shrimp can use thegluconeogenesis pathway according to CBH availability, with a higher gluconeogenicactivity in shrimp fed diets containing comparatively low amounts of CBH, and low orno gluconeogenic activity in shrimp fed diets containing more than 20% CBH. Anintermediate gluconeogenic activity could be expected in shrimp fed with intermediatelevels of CBH. Realizing that the protein is the source for gluconeogenic formation ofCBH (Campbell, 1991), the shrimp protein metabolism should be affected by dietscontaining different CBH levels following gluconeogenesis

The osmotic capability (OC) has already been evaluated in studies with Homarusamericanus (Young-Lai et al., 1991), M. japonicus (Charmantier-Daures et al., 1988)and L. vannamei (Charmantier et al., 1994) to monitor the physiological condition ofcrustaceans exposed to different experimental conditions (dissolved ammonia, salinityand dissolved oxygen). In the present study the OC was used to determine if there areany relationships among levels of dietary CBH levels, glucose hemolymph level andprotein–carbohydrate metabolism. The differences observed between shrimp fed withdifferent CBH levels could be related to the effect of dietary glucose level onCBH-protein requirements associated with salinity tolerance. The highest level ofpost-prandial plasmatic glucose of shrimp fed a diet containing 10% CBH, indicates ahigher production of glucose derived from reserves of glycogen in muscle and midgutgland. If carbohydrates from the gluconeogenesis pathway are related to dietary protein

´content (Claybrook, 1983; Gomez et al., 1988) levels of hemolymph glucose are thoughtto have been derived from proteins, reducing the amounts of amino acids forosmoregulation. The present results show a dietary level of 10% CBH was insufficient tomeet energy needs which could be satisfied by dietary protein. The low OC observed inshrimp fed a diet containing 10% CBH coincided with a relatively low PPAE whichreflects a low concentration of amino acids circulating in hemolymph (Rosas et al.,1998). In contrast shrimp fed a diet containing a low level of CBH (1%) had enoughprotein (48%) to maintain an adequate level of glucids and free amino acids (FAA) forosmoregulation. The high OC observed in shrimp fed with that diet coincided with ahigh PPNE, which reflects a high amino acid catabolism. In L. stylirostris, the pool ofamino acids could act as a buffer between the catabolism of body proteins and therequirements for energy for intermediate metabolism and osmotic pressure which is


apparently related to the CBH and protein requirements (McFarland and Lee, 1963;Claybrook, 1983; Mayzaud and Conover, 1988).

Depending on physiological condition the amino acids may be of alimentary ormuscular origin (Regnault, 1986; Rosas et al., 1995a) and are reflected in the pre- andpost-prandial ammonia excretion. Low values were found in fasting shrimp and fedshrimp produced high values (Fig. 4).

In this study, fasting (12 h) caused a 50% reduction in the protein metabolism of L.stylirostris juveniles, showing a high sensitivity to food deprivation. This type ofresponse may be related to the limited capacity of shrimp to store reserve substances,which has been observed previously in L. setiferus, F. duorarum, L. schmitti and F.notialis (Rosas et al., 1995a,b, 1999). In fed shrimp an increase in post-prandial nitrogenexcretion reflects an increase in the amino acids of alimentary origin, which in turnrelate to the role of dietary protein in energy metabolism. In the case of fed L.stylirostris juveniles, the regulating capacity of the ammonia excretion depends on thedigested food protein which is inversely related to the level of dietary CBH (Fig. 4).This type of dependence has been observed in other shrimp species reflecting the highplasticity of shrimp species to use protein as a source of metabolic energy (Rosas et al.,1996).

The level of ammonia excretion demonstrates that protein catabolism in this shrimpspecies is comparatively high (Fig. 4) (Claybrook, 1983; Pierce and Crawford, 1996).Ammonia excretion is not regulated by shrimp but directly related to the level of dietaryprotein, as shown in this and other studies with shrimp (Gauquelin, 1996) and for lobster(Capuzzo, 1980). The ammonia produced reflects the catabolism for essential aminoacids for energy (Barclay et al., 1983). Catabolism of essential amino acids (EAA)yields a higher metabolizable energy than that derived from glucose dextrins. Realizingthat crustaceans are ammoniotelic and use less energy than birds and mammals duringmechanical and biochemical transformations of food (called apparent heat increment orspecific dynamic action) (Cowey and Forster, 1971), we can expect shrimp to be betteradapted to use proteins as a source of energy than carbohydrates, which have anapparent limit of 20% in diet in L. stylirostris.

Acknowledgements

´This study was partially financed by the Direccion General de Asuntos del Personal´Academico of the National Autonomous University of Mexico with the grant to Carlos

Rosas and Gabriela Gaxiola (Project N214596), and CONACYT (Project 3293P-9607).´We also acknowledge financial support by ANUIES-Mexico and ECOS-France pro-

grammes. Thanks are given to two reviewers for improvement in the manuscript. [SS]

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