Effect of dietary supplementation of vitamins C and E on maternal performance and larval quality of the prawn Macrobrachium rosenbergii

Aquaculture 227 (2003) 131 – 146 www.elsevier.com/locate/aqua-online

Effect of dietary supplementation of vitamins C and E on maternal performance and larval quality of the prawn Macrobrachium rosenbergii Ronaldo O. Cavalli a,b,*, Frederico M.M. Batista c, Patrick Lavens b,d, Patrick Sorgeloos b, Hans J. Nelis e, Andre´ P. De Leenheer f a

Fundacßa˜o Universidade Federal do Rio Grande (FURG), Departamento de Oceanografia, C.P. 474, 96201-900 Rio Grande, RS, Brazil b Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium c UCTRA, Universidade do Algarve, Campus de Gambelas, 8000 Faro, Portugal d Inve Aquaculture, Hoogveld 91, 9200 Dendermonde, Belgium e Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium f Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium Accepted 29 May 2003

Abstract The effects of vitamin C (ascorbic acid, AA) and vitamin E (tocopherol) on the maternal performance and offspring quality of the freshwater prawn Macrobrachium rosenbergii were investigated. Prawn females were fed four diets containing different levels of 2-ascorbyl-Lpolyphosphate (ApP) and a-tocopherol acetate (a-TA) during 155 days. Three diets contained increasing AA levels (59, 121 and 918 Ag g 1 DW) and a basal level of a-TA (around 300 Ag g 1 DW), while a fourth diet contained comparatively higher a-TA levels (899 Ag g 1 DW) combined with 957 Ag AA g 1 DW. Higher dietary levels of AA and a-TA did not affect moulting, growth, or mortality rates of the broodstock. Also, breeding frequency and fecundity were not related to the dietary treatments. However, the contents of AA and a-tocopherol in the midgut gland, ovary, eggs, and newly hatched larvae increased along with higher dietary levels of these vitamins. Larvae from females fed higher levels of AA and a-TA tended to present an increased tolerance when exposed to increasing ammonia concentrations. The present results therefore suggest that broodstock diets containing around 60 Ag AA g 1 DW and 300 Ag a-TA g 1 DW are sufficient to ensure proper

* Corresponding author. Fundacßa˜o Universidade Federal do Rio Grande (FURG), Departamento de Oceanografia, C.P. 474, 96201-900 Rio Grande, RS, Brazil. Tel./fax: +55-53-236-1685. E-mail address: [email protected] (R.O. Cavalli). 0044-8486/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0044-8486(03)00499-X

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reproduction and offspring viability. However, feeding M. rosenbergii females higher dietary levels of both AA and a-TA (each around 900 Ag g 1 DW) might increase larval quality, as demonstrated in this study by the higher tolerance to the exposure to ammonia. D 2003 Elsevier B.V. All rights reserved. Keywords: Macrobrachium rosenbergii; Tocopherol; Ascorbic acid; Reproduction; Larval quality

1. Introduction During the past three decades, considerable attention has been paid to the effects of vitamins C (ascorbic acid, AA) and E (tocopherol) in broodstock performance, egg quality, and fry viability of several fish species (Watanabe and Takashima, 1977; Takeuchi et al., 1981; Soliman et al., 1986; Dabrowski and Blom, 1994; Mangor-Jensen et al., 1994; Sandnes et al., 1994). Observed effects of a deficiency of vitamin E include delay in ovarian development (Watanabe and Takashima, 1977), and decreased egg hatchability and fry survival (Takeuchi et al., 1981). The lack of dietary AA also resulted in lower egg hatching rates (Sandnes et al., 1994), egg strength (Mangor-Jensen et al., 1994), and poor fry survival (Soliman et al., 1986). In crustaceans, although a few studies suggested similar effects (Alava et al., 1993a,b; Cahu et al., 1995), knowledge on the vitamin metabolism and requirements specific to crustacean broodstock is yet much limited (Harrison, 1990, 1997). AA is known to take part in several biochemical reactions within the cells, all related to its ability to undergo reversible oxidation and reduction (Conklin, 1997). AA is involved in the biosynthesis of steroid hormones (Hilton et al., 1979) and collagen (Hunter et al., 1979; Lightner et al., 1979), and has also been shown to improve immune response (Li and Lovell, 1985), and tolerance to toxicants (Agrawal et al., 1978) and environmental stressors (Ishibashi et al., 1992; Merchie et al., 1995). Some crustaceans have a limited ability to synthesise AA (Lightner et al., 1979; Desjardins et al., 1985), but this is considered insufficient to meet metabolic requirements, as most species tested to date require a dietary source of AA (Conklin, 1997). As a result, requirements for AA in juvenile crustaceans are usually between 40 and 200 Ag g 1 dry weight (DW) of a chemically stable ascorbate compound (Conklin, 1997). Vitamin E, especially its most active form, a-tocopherol, is the major antioxidant present in cell membranes and thus protects cell and organelle membranes from oxidation by reacting with organic free radicals (Burton and Trabor, 1990). AA then promptly regenerates the resulting vitamin E radical (Packer et al., 1979). Based on the results of various studies, Conklin (1997) recommended that for ongrowing crustaceans, the level of vitamin E in diets should be 100 Ag g 1 DW. Previous studies have shown that higher levels of linoleic acid (18:2n 6) and n 3 highly unsaturated fatty acids (HUFA) increased fecundity, egg hatching efficiency and larval quality of Macrobrachium rosenbergii (Cavalli et al., 1999). Increased levels of HUFA may render membranes more susceptible to oxidation and may increase the requirement for antioxidants (Cowey et al., 1983). Cahu et al. (1993) demonstrated that the dietary supplementation of HUFA to Fenneropenaeus indicus broodstock induced a

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decrease of a-tocopherol concentrations in female tissues and eggs. Therefore, it is of interest to assess whether increasing dietary levels of antioxidants would improve broodstock performance when accompanied by high dietary levels of HUFA. Accordingly, in the present study, we investigated the possibility of improving broodstock performance and offspring quality of M. rosenbergii through the increase of dietary AA levels in a HUFArich diet. Furthermore, possible synergistic effects of vitamins C and E were evaluated through the supplementation of a comparatively higher dietary dose of a-tocopherol acetate (a-TA).

2. Materials and methods 2.1. Source of animals Adults of the freshwater prawn M. rosenbergii were obtained from pond sources in Thailand. Only active individuals in the intermoult period and with no body injury or damage were selected. They were conditioned in 20-l plastic bags within insulated containers at around 20 jC for transportation to Belgium. Upon arrival, prawns were acclimated to laboratory conditions in a freshwater recirculation system for 10 days. During this period, temperature was gradually increased from 20 to 27– 28 jC and prawns were fed a shrimp maturation diet (Inve Technologies, Belgium). At the start of the experimental period, all females were sexually mature and males were either of the blue-claw (BC) or orange-claw (OC) morphotypes (Cohen et al., 1981). The initial weight and total length of females and males were 23.6F3.8 g and 13.3F0.7 cm, and 62.9F9.1 g and 16.4F0.8 cm, respectively. 2.2. Experimental set-up Twenty female prawns were divided into four feeding groups and randomly distributed over three 190-l independent recirculation units composed of a holding tank and a biofilter. Perforated PVC plates divided the holding tanks into individual compartments, each housing one female. One BC male was maintained in each experimental unit to stimulate female maturation (Nagabhushanam et al., 1989). Additional BC and OC males were maintained in a separate recirculation unit under similar conditions. Around 20% of the water of each unit was exchanged daily when excess feed and faeces were siphoned out. Levels of NH4+ – N, NO2 – N and NO3 – N were periodically verified. The photoperiod was set at 12-h light at an intensity of 600 lx at the water surface. 2.3. Diet preparation and feeding The composition and manufacturing of the experimental diets were based on Cavalli et al. (1999). Diets had a similar basal composition (Table 1), but different concentrations of AA and a-tocopherol acetate (Table 2). The sources of AA and vitamin E were 2-ascorbylL-polyphosphate (ApP; Stay C, 35% activity; Roche, Belgium) and DL-a-tocopherol acetate (a-TA; 91% activity; Federa, Belgium), respectively. The various concentrations

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Table 1 Composition of the basal diet Ingredients a

Casein Lobster mealb Soy protein isolatec Squid mealb Shrimp mealb Crab mealb Threoninea Valinea Lysinea Histidinea Argininea Leucinea Iso-leucinea Soybean oild Fish oil E50e Cholesterola Soy lecithinf Attractant (protein extract)g a-Starcha Kappa-carrageenana Astaxanthinh Choline chloridea Mineral mixi Vitamin mixj BHTk BHAk Vitamin C (2-ascorbyl-L-polyphosphate)h Vitamin E (DL-a-tocopherol acetate)k Cellulosea

Percentage of diet 15 14 8.75 5 2.5 2.5 0.8 0.65 0.9 0.45 1 0.4 0.16 2.1 2.33 0.6 1 3.6 23 5 1.25 1 4 2 0.005 0.005 0.0145 – 0.29 0.0055 – 0.055 1.655 – 1.98

a

Sigma, USA. Rieber & Son, Norway. c Protein Technologies International, Belgium. d Vandemoortele, Belgium. e Inve Aquaculture, Belgium. f Emulpur N, Lucas Meyer, Germany. g Primex, Norway. h Roche. i Kanazawa et al. (1977). j Modified from Kanazawa et al. (1977), no Na-ascorbate or a-tocopherol added. k Federa. b

of AA and a-TA were balanced by proportional amounts of cellulose. Diets containing 59, 121 and 918 Ag AA g 1 DW were referred to as ‘Low’, ‘Medium’ and ‘High’, respectively. A fourth experimental diet (named ‘Extra’), with a concentration of AA similar to diet ‘High’ (957 Ag AA g 1 DW) but a comparatively higher level of a-TA (899 Ag g 1 DW), was also evaluated. Levels of a-,g-, and y-tocopherol were not significantly different between diets. Diets were air-dried for 24 h at room temperature, conditioned in vacuum-sealed bags and stored at 20 jC until use.

R.O. Cavalli et al. / Aquaculture 227 (2003) 131–146 Table 2 Mean (FS.D.) content of ascorbic acid (Ag AA g (n=3)

AA a-Tocopherol acetate a-Tocopherol g-Tocopherol y-Tocopherol

1

DW) and tocopherols (Ag g

1

135

DW) in the experimental diets

Low

Medium

High

Extra

59.1F6.2c 261.9F19.4b 31.4F1.3a 16.2F1.8a 5.1F0.4a

121.6F5.0b 246.3F30.4b 29.5F3.2a 15.8F1.1a 4.9F0.1a

918.8F48.3a 314.0F10.2b 32.7F4.0a 18.1F2.4a 5.2F0.8a

957.4F27.5a 898.9F48.9a 33.1F0.9a 16.4F0.4a 5.1F0.2a

Within rows, values with different superscripts are significantly different (Tukey; p<0.05).

The water stability of the diets was determined as the percentage of total dry matter remaining after immersing three replicates of 1 g pellets of known moisture content in deionised water at 28 jC for 15 and 60 min. Results were expressed as insoluble dry matter (% IDM). Additionally, to estimate vitamin losses during exposure to water and storage, the contents of AA and tocopherols in the diets were determined after 15 min of immersion in water, and 25 weeks after processing, respectively. Female prawns were fed the respective experimental diet once daily around 18.00 h at approximately 3% body weight per day. Males, however, were fed the same diet as during acclimation. Exuviae were left in the tanks to allow for consumption by the prawns. 2.4. Experimental procedures The experiment lasted 155 days. Every morning, the occurrence of moults and mortality was recorded. After moulting, females with developed ovaries were transferred to the male tank and placed with a single BC male for 5 h. Only males with hard carapace and intact claws were selected for mating. The choice of male for mating followed a rotation scheme so that no male effect would be inflicted. One week after moulting, manual removal of the egg clutch from the abdomen was carried out. Female somatic weight and egg clutch weights were measured after blotting the surface water. Three aliquots of eggs from each clutch were sampled to estimate the number of eggs per gram. Also, three clutches containing around 100 eggs were brought into three 200-ml conical-shaped bottles filled with 6xsalinity water at 28 jC. A point aeration was installed in the bottom of each cone to keep the eggs in suspension in the incubation medium and to provide oxygen to the developing embryos. Newly hatched larvae were reared for 8 days utilising a recirculated clear-water system comprising a series of aerated 20-l tanks. Standard conditions were applied to the culture of larvae: temperature 28 jC, salinity 12– 14x , density of 50 larvae/l, photoperiod of 12h light per day at an intensity of 750 lx, and feeding to satiation solely on freshly hatched Artemia franciscana nauplii (GSL strain). 2.5. Estimation of larval quality Larval quality was evaluated as the tolerance of newly hatched and 8-day-old larvae to a short-term (24 h) ammonia stress test (Cavalli et al., 2000). Groups of 30 newly hatched

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larvae were exposed to increasing concentrations of total ammonia (TAN; NH4++NH3) in 40-ml Pasteur tubes filled with water at 6xsalinity and no aeration, while 8-day-old larvae (n=25) were exposed to different TAN concentrations in glass cones containing 800 ml of water (12xsalinity) and provided with gentle aeration. The solution of ammonia was obtained by dissolving reagent grade ammonium chloride (NH4Cl). No feed was offered during exposure. Larvae not responding to mechanical stimuli were considered dead. If mortality in the control was higher than 10%, the test was not considered for analysis. 2.6. Sampling of tissues and biochemical analyses At the end of the experimental period, four stage V females (Chang and Shih, 1995) from each treatment were food-deprived for 24 h and sacrificed. Midgut gland, ovary, and tail muscles were dissected out, weighed and frozen until analysis. The gonado-somatic (GSI) and midgut-somatic indices (MSI) were estimated as the percentage of ovary and midgut gland to total body weight, respectively. Experimental diets, female tissues, eggs, newly hatched and 8-day-old larvae were sampled for the determination of AA and tocopherol levels. Only larvae from the 4th and 5th spawns were collected for vitamin analysis. Eight-day-old larvae were starved for at least 2 h before sampling. Samples for tocopherols were maintained at 20 jC, while those for AA were kept at 80 jC until analysis. AA and tocopherol levels were analytically determined as described by Nelis et al. (1997) and Huo et al. (1999), respectively. 2.7. Statistical analysis One-way analysis of variance (ANOVA) was used initially to detect differences between dietary treatments. Analyses were followed by Tukey’s HSD test ( p<0.05). The homogeneity of the variances of means was confirmed by the tests of Cochran, Hartley and Barlett (Sokal and Rohlf, 1995). Percentage data were transformed to arcsine prior to analysis, although non-transformed values are presented here. Comparisons between LC50 were obtained by examining the 95% confidence limits for overlap (APHA, 1989).

3. Results There were no significant differences in measured water quality variables neither within female tanks, nor between female and male tanks. Overall temperature was 29.2F0.6 and 29.2F0.3 jC in the female and male tanks, respectively. NH4+ – N, NO2 –N and NO3 –N levels were below 0.2, 0.06 and 120 mg l 1, respectively. The experimental diets presented good water stability (Fig. 1). After 15- and 60-min immersion, around 93% and 91% of the dry matter was retained, respectively. No differences were evident between the different diets. As regards vitamin retention, from 25.2% to 33.6% of the initial AA levels were lost after 15 min in water (Fig. 2). Losses of

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Fig. 1. Stability of the experimental diets, expressed as the percentage of insoluble dry matter (% IDM), after 15and 60-min immersion in deionised water at 28 jC.

a-T due to water exposure were much lower and ranged from 2.0% to 7.3%. Losses due to storage were negligible for both AA and a-T. Growth and mortality rates, and the duration of the intermoult period were not related to the dietary treatments (Table 3). GSI and MSI of females at the end of the experimental period were also not affected by the different dietary treatments. Egg wet and dry weight, and volume tended to increase with higher dietary supplementation of AA and vitamin E (Table 4). Fecundity, expressed as the efficiency

Fig. 2. Retention of ascorbic acid and a-tocopherol in the experimental diets after 25 weeks stored at (‘‘Storage’’) and 15 min immersed in deionised water at 28 jC (‘‘Leaching’’).

20 jC

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Table 3 Survival, growth, duration of the intermoult period, gonado-somatic (GSI) and midgut-somatic (MSI) indices of M. rosenbergii females fed diets with different levels of vitamins C and E for 155 days Diets

Survival (%) Growth rate (mg day 1) Intermoult period (days) GSI (%) MSI (%)

Low

Medium

High

Extra

80 87.3F52.3 (4) 28.8F4.1 (16) 7.05F1.47 (4) 3.57F0.46 (4)

100 56.6F16.3 (5) 25.4F2.1 (14) 7.46F0.35 (4) 4.43F0.97 (4)

80 91.6F31.0 (4) 29.8F5.5 (10) 8.26F1.71 (4) 3.70F0.69 (4)

100 38.4F26.4 (5) 28.7F4.7 (15) 6.53F1.27 (4) 4.13F0.15 (4)

Values are meanFS.D. (n), except for survival. Means were not significantly different ( p>0.05).

of egg production by female weight (eggs g 1), was not affected by the different dietary treatments (Table 4). Breeding frequency, egg hatchability, initial larval size, and survival and size after 8 days of culture were also not related to the treatments. The concentrations of AA in the female tissues, eggs, newly hatched and 8-day-old larvae are shown in Fig. 3. The contents of AA in the MG, ovary, eggs and newly hatched larvae from females fed diets Low and Medium were significantly lower than those fed diets High and Extra. On the other hand, no differences were found among the AA concentration of muscle tissues and 8-day-old larvae from the different treatments. Irrespective of dietary treatment, higher AA levels were present in the ovary, followed by eggs and 8-day-old larvae. Table 4 Reproductive performance and offspring quality of M. rosenbergii females fed diets with different concentrations of vitamins C and E Diets Low

Medium

High

Extra

Fecundity 1423.2F218.4 (16) 1554.0F194.2 (16) 1458.5F332.4 (12) 1446.8F216.1 (16) (eggs g 1 female) Breeding frequency 4.0F1.4 (5) 3.6F0.9 (5) 2.8F1.5 (5) 3.4F0.5 (5) (spawns/female) Egg wet weight (Ag) 81.0F4.0b (17) 81.2F5.4b (18) 81.9F6.5ab (12) 85.4F4.5a (17) 34.5F2.6b (18) 34.8F3.8ab (12) 36.4F2.2a (17) Egg dry weight (Ag) 34.0F2.1b (16) 3 b b a Egg volume (mm ) 0.071F0.005 (15) 0.073F0.004 (18) 0.077F0.007 (12) 0.078F0.004a (17) Hatching rate (%) 86.2F7.4 (15) 86.6F8.4 (16) 83.4F11.1 (12) 89.6F7.6 (16) Newly hatched larvae 0.028F0.002 (14) 0.029F0.003 (16) 0.030F0.005 (11) 0.031F0.003 (13) dry weight (mg) Newly hatched larvae 1.988F0.191 (12) 1.995F0.178 (15) 2.045F0.208 (11) 2.009F0.180 (13) length (mm) 8-day-old larvae dry 0.238F0.055 (13) 0.227F0.056 (15) 0.268F0.072 (12) 0.236F0.045 (14) weight (mg) 8-day-old larvae 3.964F0.355 (13) 4.105F0.408 (10) 3.990F0.434 (11) 4.183F0.441 (12) length (mm) Survival after 8 82.9F12.3 (14) 86.2F12.3 (11) 83.0F10.9 (11) 87.1F8.4 (13) days (%) Values represent meanFS.D. (n). Means within rows with different superscripts are significantly different.

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Fig. 3. Concentration of ascorbic acid (Ag g 1 DW) in the muscle tissue, midgut gland (MG), ovary, eggs, newly hatched and 8 day-old larvae of M. rosenbergii females fed diets with different levels of vitamins C and E. Within tissues, different superscript letters indicate significant differences ( p<0.05).

The concentrations of the various forms of tocopherol in the different tissues, and in the eggs and larvae are summarised in Tables 5 and 6, respectively. Significant amounts of aTA were detected in the MG, ovary, egg and newly hatched larvae, but not in muscle and 8-day-old larvae. The amounts of a-TA were significantly higher in the tissues and eggs/ larvae of females fed the diet with higher a-TA content. The levels of a-T followed a Table 5 Content of tocopherols (Ag g 1 DW) of the tissues of M. rosenbergii females fed diets with different concentrations of vitamins C and E Diets Low

Medium

High

Extra

Midgut gland a-Tocopherol acetate a-Tocopherol g-Tocopherol y-Tocopherol

202.8F67.4b 163.4F19.2b 12.8F1.6 3.8F0.9

176.5F25.7b 224.7F16.5b 14.4F0.2 4.2F0.1

209.4F48.8b 136.5F22.3b 11.0F1.0 3.1F0.8

499.3F72.1a 758.3F164.8a 11.6F7.4 1.7F1.5

Ovary a-Tocopherol acetate a-Tocopherol g-Tocopherol y-Tocopherol

42.6F11.4b 328.1F62.3b 25.9F0.2ab 2.9F0.4a

33.9F6.1b 405.8F82.2b 23.3F6.8ab 2.2F0.3ab

53.1F6.1b 320.9F104.2b 34.7F8.5a 2.6F0.3ab

115.7F12.9a 778.0F122.1a 10.9F0.7b 1.5F0.1b

Muscle tissue a-Tocopherol acetate a-Tocopherol g-Tocopherol y-Tocopherol

n.d. 142.1F30.0 13.2F3.3a 1.0F0.1

n.d. 149.3F19.5 10.4F1.6a 1.2F0.1

n.d. 144.1F4.7 11.9F0.2a 1.0F0.2

n.d. 184.9F13.7 4.3F0.3b 1.3F0.1

Values are meansFS.D. (n=3). Within rows, different superscripts represent significant differences ( p<0.05). n.d.=not detected.

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Table 6 Content of tocopherols (Ag g 1 DW) of eggs, newly hatched and 8-day-old larvae from M. rosenbergii females fed diets with different concentrations of vitamins C and E Diets Low

Medium

High

Extra

Eggs a-Tocopherol acetate a-Tocopherol g-Tocopherol

63.0F10.3b (15) 389.9F117.8b (15) 18.6F2.9a (15)

55.7F8.9b (17) 379.9F51.8b (17) 15.3F3.1b (17)

60.3F13.8b (12) 392.5F77.5b (12) 15.1F3.5b (12)

178.5F43.7a (16) 913.3F257.4a (16) 10.4F3.7c (16)

Newly hatched larvae a-Tocopherol acetate a-Tocopherol g-Tocopherol

49.6F7.2b (2) 342.9F17.0b (2) 23.4F4.8a (2)

49.4F9.8b (6) 379.8F79.7b (6) 13.5F3.0b (6)

47.6F11.2b (3) 551.9F68.3ab (3) 13.0F5.3b (3)

203.1F63.3a (6) 996.3F226.4a (6) 8.7F2.1b (6)

8-day-old larvae a-Tocopherol acetate a-Tocopherol g-Tocopherol

n.d. (2) 257.2F25.3 (2) 0.3F0.1 (2)

n.d. (3) 414.3F85.1 (4) 0.5F0.9 (3)

n.d. (2) 291.1F4.9 (2) 1.6F0.2 (2)

n.d. (4) 399.9F177.1 (4) 0.6F0.7 (4)

Values are means F S.D. (n). Within rows, different superscripts represent significant differences ( p<0.05). n.d.=not detected.

similar trend as a-TA, being significantly higher in the MG, ovary, eggs and newly hatched larvae of females fed diet Extra. No differences in terms of a-T content were found among treatments for muscle tissues and 8-day-old larvae. The highest levels of a-T were detected in the eggs and newly hatched larvae, followed by the MG and ovary. g-T levels in the MG did not vary among dietary treatments. However, the contents of g-T in the ovary and muscle were significantly lower in the treatment Extra. Eggs and newly hatched larvae from females fed diet Low had significantly higher g-T contents. Levels of y-T in the female tissues were comparatively lower than the other forms of tocopherol. No y-T was detected in the eggs and larvae. The tolerance of newly hatched and 8-day-old larvae to ammonia exposure tended to increase with increasing levels of AA (Table 7). Higher dietary levels of a-TA did not affect the tolerance to ammonia of newly hatched larvae, but it positively augmented the ammonia tolerance of 8-day-old larvae.

Table 7 Tolerance to the exposure to total ammonia (LC50, 24 h) of newly hatched larvae and 8-day-old larvae originating from M. rosenbergii females fed diets with different levels of vitamins C and E Diets Low Newly hatched larvae (ppm TAN) 8-day-old larvae (ppm TAN)

Medium b

42.4F10.9 (11) 132.9F25.8b (14)

High ab

45.0F7.3

(13)

135.2F25.5b (15)

Extra a

52.3F8.5 (11)

51.8F13.1a (15)

155.1F10.9ab (12)

164.6F29.4a (14)

Values are means F S.D. (n). Within rows, different superscripts represent significant differences ( p<0.05).

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4. Discussion Although vitamin losses during processing, storage and leaching may be of significance, these are usually not considered. In crustaceans, which are slow feeders that reduce the food particles before ingestion, losses due to leaching might reduce the actual amount of vitamin available to the prawn (Conklin, 1997). In this respect, the experimental diets used in this study presented no losses of vitamins due to prolonged storage. Furthermore, though considerable amounts of AA were found to have leached after immersion in water for 15-min, the actual losses were probably minimised because female prawns were seen to handle the pellets soon after they were offered. The essentiality of AA for the reproduction of crustaceans was first evidenced by Guary et al. (1975) when they demonstrated its accumulation in the ovary of wild Palaemon serratus. This finding was confirmed when diets deficient in AA resulted in high mortality of eyestalk-ablated Marsupenaeus japonicus females (Alava et al., 1993a), retarded the ovarian development of M. japonicus (Alava et al., 1993b), and decreased the hatching rate of F. indicus eggs (Cahu et al., 1995). The present results, however, indicate no effect of the increasing AA dietary levels on survival, moulting and growth of prawn females. Also, AA levels had no influence in most of the reproductive performance parameters evaluated (breeding frequency, fecundity, and egg hatching rate and larval survival). Egg size, however, varied considerably with respect to dietary treatment. Elevated levels of both AA and a-T resulted in larger eggs. According to Katre (1977), possible causes for differences in egg size are the duration of vitellogenesis, during which yolk reserves are transferred to the oocytes, and the number of oocytes competing for this yolk. However, none of these possibilities seem to apply here as neither the intermoult period, which to some extent may reflect the duration of vitellogenesis, nor fecundity differed among treatments. Another possible explanation could be related to the amount of yolk reserves available during vitellogenesis. It is reasonable to suppose that the dietary supplementation of vitamins C and E enhanced the synthesis of egg yolk precursors in the MG by protecting the hepatic tissues from lipid peroxidation and damage to cell membranes. A similar phenomenon has been demonstrated in laying hens supplemented with vitamin E (Bollengier-Lee et al., 1998). Nevertheless, the impact of differences in egg size on larval quality could not be demonstrated here. M. rosenbergii females fed different diets also produced different sized eggs (De Caluwe´ et al., 1995; Cavalli et al., 1999), but again no advantages in terms of larval survival or performance were observed. The relatively extended periods of embryonic and larval development, which may mask any possible differences in egg size, may be responsible for these results. Therefore, more studies are needed to establish the exact relationship between dietary AA and a-T concentrations in eggs and their performance. In an earlier study, Guary and Guary (1975) concluded that the eggs of P. serratus were able to synthesise AA during the early stages of development. Alternatively, Cavalli et al. (2001) reasoned that if eggs were able to synthesise AA, then there would be no need for females to accumulate considerable amounts of AA into their gonads, as claimed for P. serratus (Guary et al., 1975). Therefore, from a metabolic viewpoint, it seems more reasonable that the AA would be consumed during embryonic development, as previously suggested for M. rosenbergii (Cavalli et al., 2001). In fact, the present results indicate a 20% to 40% decrease in AA levels in newly hatched larvae compared to the eggs collected

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7 days after spawning. This is in accord with the previous findings that around 65% of the initial AA content of rainbow trout eggs are utilised during embryonic development (Sato et al., 1987). Feeding increasing AA levels to M. japonicus females resulted in higher contents of this vitamin in the MG, ovary and hemolymph, but not in the muscle tissue (Alava et al., 1993a,b). Similarly, muscle AA levels in M. rosenbergii were not affected by the dietary treatments, but the contents of AA in the MG and ovary were significantly higher at the dietary level of 950 Ag AA g 1 DW. The levels of 2-ascorbyl-L-polyphosphate in the broodstock diets also affected the content of AA in the eggs. Moreover, the high levels of AA in the eggs were carried over to the larvae, which in turn resulted in an increased larval tolerance to ammonia. In this respect, Mazik et al. (1987) also observed that catfish (Ictalurus punctatus) fed a diet rich in AA was less susceptible to the toxicity of ammonia. An additive effect of vitamin E on larval tolerance to ammonia was also observed. The exposure of crustaceans to ammonia results in an increased cellular uptake and use of acylglycerols (Racotta and Herna´ndez-Herrera, 2000). Therefore, higher dietary levels of vitamin E, the major antioxidant present in cell membranes, could potentially enhance the use of these lipids by protecting them from peroxidation and, as a result, larvae would be better equipped to cope with higher levels of ambient ammonia. Aside from the tolerance to ammonia, no further differences in terms of larval performance were detected. Blom and Dabrowski (1996) considered that a high concentration of AA in the eggs of rainbow trout only had positive effects on the early life stages of the offspring and therefore concluded that the dietary intake by the larvae was more important to the post-hatching performance. This is supported by De Caluwe´ et al. (1995) who found that the resistance of M. rosenbergii post-larvae to a salinity stress was affected by the larval feeding regime, regardless of the dietary history of the broodstock. Deficiency of vitamin E has been shown to retard the ovarian development of M. japonicus (Alava et al., 1993b). In fish, higher dietary levels of vitamin E resulted in an increased percentage of buoyant eggs (considered an indirect parameter of normal egg development), improved hatching rates, and a higher production of viable larvae of red sea bream (Watanabe et al., 1985). Similarly, Cahu et al. (1991a) found a linear correlation between the hatching rate of F. indicus eggs and a-T concentration. The levels of a-T in the eggs increase along with the dietary provision of a-TA (Watanabe et al., 1985; Cahu et al., 1991a, 1995; De Caluwe´ et al., 1995). The present study also indicated that, within the range tested, the higher the dietary a-T content, the higher its incorporation into the eggs. Watanabe et al. (1985) indicated that the incorporation of dietary vitamin E into the eggs occurred together with lipids. Cavalli et al. (2001) found a high correlation between the deposition of lipids and a-T in the ovary of M. rosenbergii and concluded that this was in line with the antioxidative function of this vitamin. In comparison to other tissues, the muscles had relatively lower concentrations of AA and a-T, but as the muscle mass comprises around 40% of the live weight of female prawns, it represents the most important storage site of these vitamins in M. rosenbergii. In the present study, levels of a-T in the muscles of females fed diets containing relatively lower a-TA content were not depleted in comparison to the muscle tissues of females fed 950 Ag a-T g 1 DW. Hence, the transfer of a-T from muscles to other tissues does not seem to occur under the dietary vitamin E levels employed in the present study.

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Our results confirm the findings of Cahu et al. (1991a) that crustaceans can utilise a-TA and store it as free a-T. However, considerable quantities of a-TA were detected in the MG, ovary, eggs and newly hatched larvae. Huo et al. (1999) also found a-TA in the eggs of M. rosenbergii and initially suspected an artefact. However, the authors were able to confirm the identity of a-TA after converting it to a-T by saponification. Nevertheless, an explanation to this biological phenomenon is still lacking. The present results clearly suggest that a diet containing 60 Ag AA g 1 DW is sufficient to ensure proper reproduction and offspring viability of M. rosenbergii. A quantitative requirement equivalent to 104 mg AA kg 1 of diet was estimated for M. rosenbergii juveniles using survival as the measured response to the different dietary levels of ascorbyl-2-monophosphate and ascorbyl-6-polyphosphate (D’Abramo et al., 1994). It might be possible that, as demonstrated in salmonid fishes (Sato et al., 1978; Waagbo et al., 1989), the requirements for AA in adult animals are reduced with age and/or size as a result of lower needs for biochemical functions, more efficient endogenous reuse, or an increased storage capacity (Waagbo et al., 1989). However, contrasting results showing improvements in the reproductive performance with higher AA levels have also been reported. Blom and Dabrowski (1995) estimated that the dietary AA levels needed to sustain the reproductive success of rainbow trout should be over 400 Ag g 1 DW, or eight times higher than the recommended dietary levels for ongrowing fish (National Research Council, 1993). Similarly, for F. indicus, dietary AA levels over 600 Ag g 1 DW resulted in the production of eggs of superior quality (Cahu et al., 1991b, 1995), which contrasts with the usual recommendation of 40 to 200 Ag g 1 DW for ongrowing crustaceans (Conklin, 1997). From the present results of this study, we conclude that diets containing 60 Ag AA g 1 DW and 300 Ag a-TA g 1 DW are sufficient to ensure proper reproduction and offspring viability. An improvement in larval quality might be achieved by utilising higher AA and a-TA inclusion levels particularly under adverse rearing conditions. Nevertheless, more research is still needed to establish actual dietary requirements for crustacean broodstock.

Acknowledgements The authors are indebted to Mr. Nopadol Phuwapanish, Department of Fisheries, Thailand, for arranging for the capture and transportation of prawns. Most sincere thanks are also due to Petra Rigole, Laboratory of Pharmaceutical Microbiology, for her unconditional support in the analysis of vitamins. We also acknowledge two anonymous referees for their suggestions on a previous version of this manuscript. Funds for this study were partially provided by the Brazilian Council for Science and Technology (CNPq). R.O. Cavalli is a research fellow of this agency.

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