Effect of different levels of dietary [alpha]-tocopherol on ovarian maturation and reproductive performance of broodstock Litopenaeus vannamei (Boone).

ABSTRACT Effects of 4 grade levels of dietary [alpha]-tocopherol (37.82, 115.64, 349.42 and 522.56 mg [kg.sup.-1] diet) on ovarian maturation and reproductive performance of broodstock shrimp, Litopenaeus vannamei were investigated. The [alpha]-tocopherol concentrations in hepatopancreas, ovaries, eggs and muscle of female shrimp increased significantly as dietary [alpha]-tocopherol levels increased. Survival, spermatophore weight, sperm count, gonadosomatic index (GSI), hepatosomatic index (HSI), fecundity and egg diameter were not significantly affected by dietary [alpha]-tocopherol levels. Average daily spawns, hatching rate and fertilization rate significantly increased as dietary [alpha]-tocopherol levels increased. Results of this study confirmed the importance of supplementation of [alpha]-tocopherol to broodstock diets fed to L. vannamei, and suggested that at least 350 mg [alpha]-tocopherol [kg.sup.-1] of diet was needed to achieve significantly better ovarian maturation and reproductive performance.

KEY WORDS: [alpha]-tocopherol, broodstock, Litopenaeus vannamei, maturation, reproductive performance

INTRODUCTION

In penaeid shrimp, broodstock nutrition is a key factor influencing sexual maturation, reproductive performance and offspring quality (Harrison 1990). To develop suitably performing formulated diets for shrimp broodstock, understanding nutrition-reproduction interactions and determining the specific nutrient requirements for successful maturation and spawning are necessary.

Vitamin E (tocopherols) is considered to be an essential dietary nutrient for crustaceans (Conklin 1997). Available data have shown that [alpha]-tocopherol, the most active form of vitamin E, is the predominant form in shrimp/or prawn tissue (Cavalli et al. 2001, Wouters et al. 2001), and DL-[alpha]-tocopherol acetate, a stable form of [alpha]-tocopherol, is the most commonly used vitamin E supplement in animal feeds (National Research Council 1983). The biological role of [alpha]-tocopherol is widely accepted to be a lipophilic antioxidant, protecting membranes of cells and organelles from oxidation by scavenging organic free radicals (Burton & Trahor 1990), an activity which may be especially important during embryonic and larval development. The importance of vitamin E for fish reproduction has long been recognized (Watanabe & Takashima 1977, Watanabe et al. 1985), but the importance of this vitamin in shrimp reproduction has only been recently demonstrated. Alava et al. (1993) reported that a [alpha]-tocopherol-deficient diet resulted in retarded gonadal maturation of Marsupenaeus japonicus. Chamberlain (1988) found a correlation between the percentage of abnormal sperm and dietary vitamin E deficiency in L. setiferus. In a trial using Fenneropenaeus indicus spawners, Cahu et al. (1995) demonstrated that hatching percentage increased when dietary [alpha]-tocopherol levels increased from 40-350 nag [kg.sup.-1]. In wild female broodstock L. vannamei, Wouters et al. (2001) detected an increase in [alpha]-tocopherol concentrations in the ovary prior to sexual maturation. All these results suggest that vitamin E plays a critical role in the shrimp maturation process.

Information about the effect of dietary [alpha]-tocopherol on ovarian maturation and reproduction of L. vannamei is lacking. In a previous study, we successively substituted a natural diet consisting of 50% bloodworm (Glycera chirori) and 50% oyster (Crassostrea rivularis) with a formulated diet for L. vannamei broodstock (Du et al. 2004a). In the current study, this previously successful formulated diet was used as the basal diet, and ROVIMIX E50 (Roche Sunve Vitamins Ltd, Shanghai, P.R. China) was added as a [alpha]-tocopherol source to investigate the effect of dietary [alpha]-tocopherol on the maturation and reproductive performance of L. vannamei. The effect of dietary levels of [alpha]-tocopherol on [alpha]-tocopherol concentrations in ovaries, hepatopancreas, eggs and muscle was also determined.

MATERIALS AND METHODS

Animals and Treatment

Pond-reared L. vannamei breeders were obtained form Dongfang Co. (Zhanjiang, P.R. China). They were held in maturation tanks for 3 wk to acclimate to the experimental conditions and fed the basal diet without any supplement of [alpha]-tocopherol acetate (Diet El). A unisex system as described by Browdy et al. (1996) was used: each of 4 maturation tanks was stocked with 15 females each and each of another 4 maturation tanks with 15 males. They were divided into four groups: E1-E4, each consisting of a male tank and a female tank. After acclimation, female shrimp were unilaterally eyestalk-ablated with a pair of flamed tweezers to enhance the rate of ovarian maturation. To identify each female within the same group, part of the telson was cut with the exception of five females and latter sampled to test gonadosomatic index (GSI) and hepatosomatic index (HSI). The duration of the postablation phase of the experiment was 50 days, during which, broodstock shrimp representing treatment E1-E4 were fed diets E1-E4, respectively. All treatments were randomly assigned to the maturation tanks.

The maturation tanks were rectangular-shaped cement tanks (2 x 3 [m.sup.2], 55 cm water depth) in which sand-filtered and UV-treated seawater was exchanged at a rate of 200% daily. The physicochemical parameters of the water were: mean temperature, 28.5 [+ or -] 1[degrees]C; mean salinity, 30 [+ or -] 0.5 mg [L.sup.-1]; mean pH, 8.2 [+ or -] 0.1; photoperiod, 12 hr light/12 hr dark; and light intensity, 200 1x. Under these conditions, the level of dissolved oxygen remained close to saturation; 6.2 g [m.sup.-3] and ammonia, nitrate and nitrite levels did not exceed 0.03, 0.01 and 0.25 mg [L.sup.-1], respectively.

Diets and Feeding

Ingredient composition of the basal diet is presented in Table 1. The alpha-tocopherol source used was Roche ROVIMIX E50, an alpha-tocopheryl acetate containing 500 mg [alpha]-tocopherol [g.sup.-1]. ROVIMIX E50 was added at different levels, compensated by appropriate additions of carboxy methylcellulose as filler (Table 2). Levels of [alpha]-tocopherol added in the dietary treatment were 0, 100, 300 and 500 mg/kg. To prepare the diets, all dried ingredients were smashed and sieved, and then thoroughly mixed with 35% (w/w) water. The resulting stiff dough was pelleted with a mincer with a 3-mm die, and dried at room temperature. The dried "spaghetti like" strands were then crumbled to about 5-mm long pieces and kept in sealed plastic bags at -20[degrees]C until use. Shrimp were fed at a daily rate of 5% of the tank biomass (wet weight), proportionately divided over 4 times daily (8:00, 11:00, 14:00 and 18:00). Uneaten food was collected daily.

Broodstock Maturation and Spawning

After unilateral-ablation, females were visually examined for ovarian maturation stages each day at 20:00 according to Wouters et al. (2001). When the majority of the females showed some degree of ovarian maturation, and just before the first mature female with intact telsons was ready to spawn, all females with intact telsons from each treatment were weighed and dissected to determine the gonadosomatic index (GSI = 100 x gonad weight / total body weight) and hepatosomatic index (HSI = 100 x hepatopancreas weight / total body weight).

During the postablation period, the remaining females were examined daily and those with mature ovaries were transferred to tanks containing the corresponding group of males to mate. Each mature female with an attached spermatophore was placed into an individual 120-L spawning tank. In the next morning, spawns were examined and shrimp were returned to their respective maturation tanks. Fecundity (number of eggs per spawn) was estimated by counting three 50-mL samples of water in the spawning tank after vigorous mixing to ensure a homogenous suspension of eggs. Fertilization rate, based on the presence of a double membrane and/or embryonic development in eggs, was determined from examination of three 50-mL samples from each spawn. A sample of 10,000 eggs, from each spawn was individually incubated at 29[degrees]C to estimate the hatching rate (% nauplii/fertilized eggs). Hatching rate was calculated by counting the number of nauplii per spawn sample through collection by positive phototropism. Egg diameter was estimated by measuring 50 eggs from each spawn with a light microscope and micrometer. About 100 mg of eggs were sieved from each spawn and immediately rinsed in freshwater and stored at -70[degrees]C. Egg samples from the same group of females were pooled to obtain sufficient tissue for [alpha]-tocopherol analysis. At the end of the experiment, five stage II females from each group were dissected. Hepatopancreata, ovaries and muscle of females from each dietary treatment were pooled and stored at -70[degrees]C for latter [alpha]-tocopherol analysis. Spermatophore quality was based on sperm count and spermatophore weight according to Alfaro & Lozano (1993). At the end of the experimental period, five males with mature spermatophores from each treatment group were selected, and their spermatophores were extruded by gently pressing around the coxae of the fifth pair of pereopods. Both sides of the compound spermatophore were weighed to the nearest 1 mg. The quantities of sperm were determined by homogenizing the compound spermatophore in a calcium-free solution and by counting the sperm cells under light microscopy.

Biochemical Analysis

Triplicate biochemical analysis of the basal diet was conducted according to the following standard procedures (AOAC 1990). Moisture was determined by oven drying to constant weight at 105[degrees]C. Crude protein (N x 6.25) was derived from Kjeldahl nitrogen analysis. Ash was determined as the residue after muffle furnace ignition at 550[degrees]C for 6 h. Total lipid content was determined after Soxhlet extraction with petroleum ether at 60[degrees]C for 8 h.

Tissue [alpha]-tocopherol content was determined triplicately with HPLC (HP 1100) using methods modified slightly from those of Alava et al. (1993) and Huo et al. (1999). The analytical condition of HPLC was as follows: detection at UV-284 nm; column temperature, 35[degrees]C; eluent, methanol-water (96: 4, v/v); flow speed, 1.0 mL/min, [alpha]-tocopherol was extracted form samples by the method of Huo et al. (1996). Alpha-tocopherol was extracted from 0.2 g of freeze-dried samples by homogenization in 2 mL of methanol containing butylhydroxytoluene (1 mg/mL). The homogenates were clarified by centrifugation and passed through a 0.45 [micro]m-pore-size syringe filter (SIGMA). Twenty microliters of the filtrate were introduced into the HPLC injection port. Alpha-tocopherol (95%, SIGMA) was used as standard.

Statistical Analyses

An individually marked female within the same treatment group was considered to be an experimental unit for statistical analysis. This is the most commonly used statistical procedure applied in shrimp reproduction trials (Wouters et al. 2002). Data of GSI, HSI, fecundity, daily spawns per female, egg diameter, fertilization rate and hatching rate from each group were subjected to 1-way ANOVA and subsequent Duncan's multiple-range test to determine difference in means. Prior to analysis, Levene's test for homogeneity of variances was used to verify the assumptions for further analysis. There was no need to transform data. A regression analysis was used to determine if a relationship existed between either fecundity, egg diameter, fertilization rate or hatching rate and the spawn order. No relationships were detected, and spawn order was not considered to be a factor to include in the evaluation. An alpha level for all tests was set at 0.05. Statistical analysis was performed using Systat package (Systat Software Inc. 1996).

RESULTS

Average initial weight of female and male broodstock was 51.4 [+ or -] 3.56 g and 48.02 [+ or -] 5.39 g, respectively. Survival of male and female broodstock within the different groups is presented in Table 3. A slight loss, probably induced by manipulation, occurred in all groups during the experiment; therefore, number of spawns was calculated based on per female per day.

Alpha-tocopherol concentrations in hepatopancreata, ovaries, eggs and muscle of female L. vannamei, as well as in the experimental diets are presented in Table 4. The actual [alpha]-tocopherol concentrations in the experimental diets were higher than the supplemented amounts because the ingredients of the basal diet contained 37.82 mg [kg.sup.-1] [alpha]-tocopherol. In the present study, female shrimp sampled to determine [alpha]-tocopherol content were at maturation stage II, with mean GSI values of 1.45 [+ or -] 0.07 for group E1, 1.52 [+ or -] 0.05 for group E2, 1.60 [+ or -] 0.04 for group E3 and 1.63 [+ or -] 0.07 for group E4, respectively. Results showed that the [alpha]-to-copherol content of tissue was significantly affected by dietary [alpha]-tocopherol levels. Concentrations of [alpha]-tocopherol in the ovaries and eggs were higher than those in the hepatopancreata. Levels of [alpha]-tocopherol in the ovaries of shrimp in groups E3 and E4 (186.43 mg [kg.sup.-1] and 207.37 mg [kg.sup.-1]) were significantly higher than those of shrimp in groups E2 (93.77 mg [kg.sup.-1]) and E1 (75.04 mg [kg.sup.-1]). Alpha-tocopherol concentrations in the hepatopancreata and eggs increased significantly as levels of dietary [alpha]-tocopherol increased, and generally values for each of the groups differed significantly from one another. Supplementation of [alpha]-tocopherol acetate also increased the [alpha]-tocopherol content in muscle. The [alpha]-tocopherol concentration in muscle of shrimp from group E2 (50.6 mg [kg.sup.-1]) was significantly higher than that of shrimp from group El (24.5 mg [kg.sup.-1]), but was significantly lower than that of either group E3 (89.3 mg [kg.sup.-1]) or group E4 (104.6 mg [kg.sup.-1]).

Ovarian maturation and reproductive performance of shrimp representing the different treatments are presented in Table 3. For male broodstock L. vannamei, spermatophore weight and sperm count were not significantly different. As dietary [alpha]-tocopherol levels increased, GSI and HSI values of female broodstock did not differ significantly. Average daily spawns per female were significantly affected by dietary [alpha]-tocopherol levels. Females in treatment E3 gave the highest daily spawns, significantly higher than those in treatments E2 and El. Daily spawns of females in treatments E3 and E4 were not significantly different.

Fecundity and egg diameter were not significantly different among the dietary treatments. Supplementation of [alpha]-tocopherol acetate to the basal diet significantly increased the fertilization rates for treatments E3 (59.6%) and group E4 (61.45%) relative to treatments E2 (51.3%) and E1 (45.4%). The hatching rates for treatments E1 (31.2%) and E2 (34.2%) were significantly lower than those for treatments E3 and E4.

DISCUSSION

The current study showed that [alpha]-tocopherol content in the basal diet (37.82 mg [kg.sup.-1]) was sufficient to maintain normal survival, successive maturation and spawns of L. vannamei; however, to achieve significantly higher daily spawns, fertilization rates and hatching rates, supplementation of [alpha]-tocopherol to broodstock diets is essential.

The increase in [alpha]-tocopherol concentrations in eggs, hepatopancreata and ovaries of female L. vannamei as dietary [alpha]-tocopherol levels increased was similar to the results obtained by Cahu et al. (1995). In their study, [alpha]-tocopherol concentration in eggs of Fen. indicus increased from 177 mg [kg.sup.-1] to 587 mg [kg.sup.-1] as dietary [alpha]-tocopherol levels increased from 82 mg [kg.sup.-1] to 354 mg [kg.sup.-1]. The beneficial action of [alpha]-tocopherol is most probably related to its antioxidant properties. The [alpha]-tocopherol accumulated in the egg yolk acts as a natural antioxidant (Harrison 1997), and may be especially important for the embryonic and larval development. This positive effect has been verified in the study of Cahu et al. (1995), who showed that hatching rate of Fen. indicus increased significantly, from 28% to 55%, when egg [alpha]-tocopherol concentration increased from 177 mg [kg.sup.-1] to 587 mg [kg.sup.-1]. In the present study, elevation of [alpha]-tocopherol content in ovaries and eggs relative to dietary supplementation might also suggest its positive effect on ovarian maturation and embryonic development. Wouters et al. (2001) found that [alpha]-tocopherol concentrations in ovaries of wild L. vannamei increased as sexual maturation proceeded, and suggested that this vitamin has a critical role in advancing the shrimp maturation process. Similarly, Cavalli et al. (2001) detected a sharp increase of [alpha]-tocopherol in the ovaries of wild Macrobrachium rosenbergii during the initial gonadal development from stage I to III. They also suggested the dietary importance of this vitamin to serve as a major antioxidant agent during ovarian maturation. For penaeid shrimp, hepatopancreas and muscle tissues are the main storage organs for [alpha]-tocopherol (Cahu et al. 1995). The lower concentration assayed in muscle of females of groups El and E2 might indicate that this vitamin was transported from muscle to ovary during vitellogenesis, as was reported in Fen. indicus (Cahu et al. 1995) and Mar. japonicus (Alava et al. 1993).

Little information on the effect of broodstock nutrition on reproductive performance of male broodstock shrimp is available. In the present study, lack of a significant change in spermatopbore weight and sperm count as levels of dietary [alpha]-tocopberol increased suggests that further studies need to be conducted to evaluate the effect of higher dietary [alpha]-tocopherol levels on the spermatophore quality of male broodstock L. wmnamei. GSI and HSI indicate the accumulation of nutrients in the ovaries and hepatopancreata of female L. vannamei. Similar to a previous study conducted by Du et al. (2004b), the period between eyestalk ablation and the first maturation of females with intact telsons was only 6 days, and the similar values of GSI and HS1 might be attributed to the relatively short period of time. In the present study, females fed diets containing 349.42 and 522.56 mg [kg.sup.-1] [alpha]-tocopherol gave significantly higher daily spawns, suggesting that at least 349.42 mg [kg.sup.-1] of [alpha]-tocopherol in broodstock diet was needed to significantly enhance the ovarian maturation of female L. vannamei. Alava et al. (1993) also demonstrated the positive effect of [alpha]-tocopherol on ovarian maturation, and showed that 482 mg [kg.sup.-1] [alpha]-tocopherol in the broodstock diet was necessary to cause ovarian maturation of Mar. japonicus.

Alpha-tocopherol is also an important factor to improve the fertilization and hatching performance of shrimp eggs. In the study conducted by Cahu et al. (1995), hatching rate increased significantly, from 28% to 55%, when dietary [alpha]-tocopherol levels increased from 82 mg [kg.sup.-1] to 354 mg [kg.sup.-1]. The positive effect of high dietary [alpha]-tocopherol content on the improvement of egg hatchability was concluded. Similarly, egg hatching rate was also positively correlated with [alpha]-tocopherol concentration of eggs in the studies conducted by Cahu et al. (1991, 1993). Results of this study showed that diets containing [alpha]-tocopherol levels of at least 349.42 mg [kg.sup.-1] and fed to L. vannamei broodstock could significantly increase the fertilization rate and hatching rate of eggs.

ACKNOWLEDGMENTS

The authors thank Mr. Wenlin Chen for kindly offering shrimp breeders and experimental facilities. Sincere thanks are also given to Mr. Qicun Zbou and Kaimin Cheng for their help in feed manufacturing. This work was funded by Zhanjiang Scientific Project (No 2004-1-5) and Science Innovative Project form Chinese Academy of Science (KSCX-2-1-04-04).

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SHAOBO DU, (1) * CHAOQUN HU, (1) SHIQING CAI, (1) QI SHEN (1) AND SHIXUAN ZHENG (2)

(1) The Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology Chinese Academy of Sciences, 510301, Guangzhou, People's Republic of China; (2) Yuehai Feed Limited, 524017, Zhanjiang, People's Republic of China

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Ingredients and proximate biochemical composition of the basal diet

Ingredients                                           Weight
                                               (g [kg.sup.-1] diet)

Fish meal (CIPSSA, Puerto Montt. Peru)                340
Oyster (Crassostrea rivularis) meal                   200
Bloodworm (Glycera chirori) meal                      100
Alpha starch ([section])                              206.714
shrimp head meal (50% CP) ([section])                  50
Cod liver oil ([section])                              40
Soybean lecithin ([section])                           15
Ca[([H.sub.2]P[O.sub.3]).sub.2] x [H.sub.2]O           10
Cholesterol                                             5
Sodium alginate                                        20
Vitamin C (Stay c-35) ([dagger])                        2.286
Vitamin mix *                                          10
Proximate biochemical composition                       (%)
Moisture                                         6.01 [+ or -] 0.18
Ash ([paragraph])                               15.58 [+ or -] 0.25
Crude protein ([paragraph])                     49.66 [+ or -] 0.82
Total lipid ([paragraph])                       12.14 [+ or -] 0.49

([section]) Alpha starch, shrimp head meal, soybean lecithin and cod
liver oil were supplied by Yuehai Feed Co Ltd, China.

([dagger]) Stay C-35 (Roche Sunve Vitamins Ltd., Shanghai), mainly
monophosphate, containing 350 mg L-ascorbic acid [g.sup.-1].

* Vitamin composition (mg/IU [kg.sup.-1] diet): vitamin A palmitate
100 000 IU; vitamin [D.sub.3] 10 000 IU; biotin 7 mg; ascorbic acid
800 mg; HCl 100 mg; riboflavin 120 mg; thiamin HCl 50 mg; folic acid
13 mg; cyanoco-balamin 1 mg; menadione bisulfite 60 mg; choline
chloride 2,000 mg.

([paragraph]) Ash, protein and lipid content was calculated based
on dry matter.

TABLE 2.
Levels of [alpha]-tocopherol acetate supplemented to the
experimental diets

                                          Diets

Variable Ingredients              E1    E2    E3     E4

Carboxymethylcellulose
  (mg [kg.sup.-1] diet)         1000   800   400      0
[alpha]-tocopherol acetate
  (mg [kg.sup.-1] diet) *          0   200   600   1000
[alpha]-tocopherol equivalent
  (mg [kg.sup.-1] diet)            0   100   300    500

* ROVIMIX E-50 (Roche Sunve Vitamins Ltd., Shanghai), containing 500
mg [alpha]-tocopherol [g.sup.-1].

TABLE 3.
Maturation and reproductive performance of broodstock L. vannamei
fed diets supplemented with different levels of [alpha]-tocopherol.
Values are mean [+ or -] S.D. (n), except for survival.

    Dietary Treatment                     E1

Survival (females)                       90%
Survival (males)                         86.7%
Spermatophore weight (g)      0.066 [+ or -] 0.019 (5)
Sperm count (x [10.sup.6])    12.43 [+ or -] 4.94 (5)
GSI (1)                        2.36 [+ or -] 0.81 (5)
HSI (2)                        3.20 [+ or -] 0.57 (5)
Daily spawns per female *     0.030 [+ or -] 0.013 (a) (7)
Fecundity (x [10.sup.3])     128.84 [+ or -] 20.96 (10)
Egg diameter ([micro]m)      266.54 [+ or -] 6.21 (50)
Fertilization rate (%) *      45.37 [+ or -] 4.17 (a) (10)
Hatching rate (%) *           31.15 [+ or -] 2.76 (a) (10)

    Dietary Treatment                     E2

Survival (females)                       80%
Survival (males)                         93.3%
Spermatophore weight (g)      0.071 [+ or -] 0.020 (5)
Sperm count (x [10.sup.6])    14.16 [+ or -] 5.36 (5)
GSI (1)                        2.22 [+ or -] 0.44 (5)
HS 1 (2)                       3.37 [+ or -] 0.45 (5)
Daily spawns per female *     0.042 [+ or -] 0.015 (b) (8)
Fecundity (x [10.sup.3])     130.15 [+ or -] 22.80 (15)
Egg diameter ([micro]m)      268.31 [+ or -] 5.76 (50)
Fertilization rate (%) *      51.26 [+ or -] 5.96 (a) (15)
Hatching rate (%) *           34.22 [+ or -] 6.22 (a) (15)

    Dietary Treatment                     E3

Survival (females)                       90%
Survival (males)                         80%
Spermatophore weight (g)      0.075 [+ or -] 0.021 (5)
Sperm count (x [10.sup.6])    13.22 [+ or -] 42.7 (5)
GSI (1)                        2.52 [+ or -] 0.48 (5)
HS 1 (2)                       3.62 [+ or -] 0.74 (5)
Daily spawns per female *     0.074 [+ or -] 0.030 (c) (9)
Fecundity (x [10.sup.3])     130.91 [+ or -] 29.26 (29)
Egg diameter ([micro]m)      265.87 [+ or -] 4.30 (50)
Fertilization rate (%) *      59.59 [+ or -] 7.05 (b) (29)
Hatching rate (%) *           55.20 [+ or -] 6.25 (b) (29)

    Dietary Treatment                     E4

Survival (females)                       90%
Survival (males)                         86.7%
Spermatophore weight (g)      0.073 [+ or -] 0.024 (5)
Sperm count (x [10.sup.6])    14.53 [+ or -] 4.11 (5)
GSI (1)                        3.12 [+ or -] 0.77 (5)
HS 1 (2)                       3.60 [+ or -] 0.50 (5)
Daily spawns per female *     0.071 [+ or -] 0.021 (c) (9)
Fecundity (x [10.sup.3])     123.25 [+ or -] 31.17 (29)
Egg diameter ([micro]m)      267.83 [+ or -] 2.34 (50)
Fertilization rate (%) *      61.09 [+ or -] 4.25 (b) (29)
Hatching rate (%) *           53.15 [+ or -] 10.69 (b) (29)

* Values in the same row with different superscripts are
significantly different (P < 0.05).

(1) GSI, gonadosomatic index; (2) HSI, hepatosomatic index.

TABLE 4.
[alpha]-tocopherol concentrations (mg [kg.sup.-1]) in experimental
diets, and in eggs, ovaries, hepatopancreata and muscle of female
L. vannamei fed the different diets

Dietary Treatment              E1                         E2

Experimental
  diets             37.82 [+ or -] 1.45        115.64 [+ or -] 3.82
Hepatopancreas      10.00 [+ or -] 1.24 (a)     47.90 [+ or -] 6.53 (b)
Ovary               75.04 [+ or -] 5.31 (a)     93.77 [+ or -] 3.69 (a)
Egg                 56.60 [+ or -] 1.24 (a)    163.80 [+ or -] 7.2 (b)
Muscle              24.51 [+ or -] 2.9 (a)      50.58 [+ or -] 1.86 (b)

Dietary Treatment               E3                         E4

Experimental
  diets             349.42 [+ or -] 5.83       522.56 [+ or -] 10.41
Hepatopancreas      112.65 [+ or -] 6.56 (c)   156.65 [+ or -] 4.43 (d)
Ovary               186.43 [+ or -] 7.14 (b)   207.37 [+ or -] 7.48 (b)
Egg                 280.42 [+ or -] 7.00 (c)   391.01 [+ or -] 5.34 (d)
Muscle               89.27 [+ or -] 7.6 (c)    104.55 [+ or -] 4.6 (c)

[alpha]-tocopherol concentrations were based on dry weight. Values are
means [+ or -] SD (n = 3). Within the same row, values with different
superscripts are significantly different (P < 0.05).