Indian Journal of Experimental Biology Vol. 42, January 2004, pp. 58-62 Evaluation of dietary essentiality of vitamins for Penaeus monodon (Fabricius) Ashwin Rai & H R V Reddy University of Agricultural Sciences, Col lege of Fisheries, Mathsyanagar, Mangalore 575 002 India Received 22 May 2003; revised 29 Augus1 2003 The effect of exclusion of individual water-soluble (thi amine, riboflavin, pyridoxine, cyanocobalamin. pantothenic acid, folic ac id, ni acin, biotin, choline, inositol, ascorbic acid) and fat-soluble vitamins (vit. A, D, K and E) in semi-purified diets on growth and survival of juvenile shrimp, P. monodon was studied in the laboratory for 8 weeks. Diets Jacking riboflavin and vitamin K did not affect growth a nd survival of shrimp. However, deletion of inositol and choli ne resulted in poor growth. Maximum growth was observed in the control diet (CI) which was supplemented with all vitamins. Di et deficient in ascorbic acid, biotin, folic acid, niacin, thiamine and a.-tocopherol resulted in poor appetite and poorer feed conversion efficiency. All treatments except the control (Cl ) resulted in histological changes in the digestive gland cells. Detachment or destruction of the epithelial cells was observed in all treatments lacking individual vitamins but more severely in the treatment without a vitamin supplement fo llowed by inositol, choline and ascorbic acid deficient diets. Keywords: Histology digestive gland, Penaeus rnon odon, Mid-gut gland, Vitamins To develop cost effective, nutritionally balanced diets prior knowledge of the essential nutrient requirements of the cultured species is a pre requisite. Although several contributions exist concerning the protein, lipid and carbohydrate requirements of Penaeus monodon 1 ' 4, very few studies have been canied out on the vitamin requirements of this species5-7. In fact, existing feed formulations for this species use vitamin levels based on information derived from studies on related species. Numerous studies on vitamin nutrition have revealed that vitamins are essential for normal health and life functions such as growth, development, metabolism, maintenance and reproduction 7-9. Widely divergent quantities of vitamins have been added into shrimp feeds using vitamin premixes. Specific problems, which conceivably may have been related to vitamin deficiencies, have not been noted. Thus, the objective of the study was to evaluate the relative dietary essentiality of water-soluble and fatsoluble vitamins for P. monodon. Materials and Methods Post larvae of Penaeus monodon (mean weight 40±15 mg) were obtained from a commercial hatchery located at Mangalore and acclimatized to laboratory conditions for 30 days in Fiberglass Reinforced Plastic tanks (2000 1) and fed artificial *Correspondent author : Phone: 91-0824-2249256 Fax: 91-0824-2248366 E-mail : winrai@yahoo.com pelleted feed (Table 1) for 15 days prior to the start of the experiment. Juveniles (mean weight of 0.085 ± 0.02 g) were stocked in circular Fiberglass Reinforced Plastic tanks (0.6 m diam. x0.75 m h) at a rate of 25 individuals per tank containing 2000 1 sea water in a recirculatory system for 8 weeks. Each tank was provided with continuous aeration from an airstone connected to an air pump. Optimum temperature (28 ± l.5 C), salinity (28-30ppt) and dissolved oxygen (6.0-6.5 mgll) were maintained. Ammonia and nitrite-nitrogen levels remained below 0. I mg/1. Fifteen diets were prepared each containing a feed base (Table 1) and exclusion of one vitamin at a time (thiamine, riboflavin, pyridoxine, cyanocobalamin, pantothenic acid, folic acid, niacin, biotin, choline, inositol, ascorbic acid, vitamin A, D, E and K). In addition, two control diets containing either the complete vitamin mixture (Cl) or no vitamin supplement (C2) were used. Each treatment consi sted of two replicates. The shrimp were fed twice daily (morning and evening) at a rate of 10% body weight Faeces and uneaten food were removed and mortality recorded daily. Shrimps were weighed once in two weeks before feeding. The shrimp were netted, blotted with a dry gauze cloth and weighed individually. When the shrimp showed clear vitamin deficiency symptoms (4 111 week) each group was divided into two subgroups of equal number and mean body weight and one group was used for a recovery test usmg complete vitamin mixture.
RAI & REDDY: EVALUATION OF DIETARY ESSENTIALITY OF VITAMINS FOR P. MONO DON (FABRICIUS) 59 Ingredient Table 1 -Composition of basal diet Casein-Vitamin free Dextrin Cellulose Cod liver oi l Vitamin mixture* (see below) Amino acid mixture 1 Mineral mixture 2 Composition of vitamin mixture Vitamin mg/g Vitamin g 100 g -I 50 20 io 5 2 3 10 Thiamin 5.0 Folic acid Riboflavin 5.0 Vitamin A acetate(20,000 IU) Pyridoxine 5.0 Vitamin D (40,000 IU/g) Pantothenic acid 15.0 Vitamin E acetate (250 IU/g) Niacin 20.0 Menadione (Vit. K) Biotin 0.5 Choline Inositol 125.0 Ascorbic acid cyanocobalamin 1 Amino acid mixture: according to Shiau and Jan 25 _ 2 Mineral mixture: according to Shiau and Chou 26 *Vitamin mixture: according to Shiau and Suen 7 _ mg/g 2.0 6.0 0.5 40.0 1.0 100.0 3.0 0.5 Histology of digestive gland- In general, the impact of nutritional changes is profound on the midgut gland. Upon termination of the experiment, three individuals in the intermolt stage were taken from each treatment and fixed in Bouin's solution for 24 hr. The fixed tissues were dehydrated in ascending concentrations of alcohol, cleared in toluene, embedded in paraffin wax and sectioned with a rotary microtome at 6!lm. Sectioned tissues were stained with haemotoxyline and eosin (H & E). Growth data were analyzed for statistical significance by one-way ANOVA and the Duncan's multiple range test 10. Results The responses of shrimp in terms of percent weight gain, feed conversion ratio and survival are presented in Table 2. Water soluble vitamins-thiamin: In the thiamin deficient group, the percentage weight gain decreased gradually after 4th week. The shrimp showed anorexia. The relative percentage weight gain was 75 and survival was 92%. In the recovery test, weight gain and food consumption improved. Pyridoxine : The shrimp fed the pyridoxine deficient diet showed suppressed growth after the 5 th week. The relative weight gain was 74%, feed efficiency decreased after the 4th week and survival Table 2-Relative weight gain, feed conversion (mean±se) and survival over 8 week period of P. monodon fed individual vitamin excluded diets compared to control Excluded vitamin Weight Feed conversion Survival gain ratio (%) (%) Control (C!-contains all vitamins) 540 a 2.37 ± 0.30 90 Thiamine (8 l) 4 10 a 3.00 ± 0.18 92 Riboflavin (B2) 520 a 2.56 ± 0.17 95 Pyridoxine (B6) 400 a 2.87 ± 0.21 92 Cyanocobalamin (B 12) 380 a 2.77 ± 0.69 90 Pantothenic acid 400 b 3.16 ± 0.27 81 Folic acid 360 b 3.12 ± 0.20 94 Niacin 400 b 3.00 ± 0.41 86 Biotin 380b 3.21 ± 0.30 90 Choline 280 c 3.02 ± 0.12 80 Inositol 260 c 3. 15±0.17 84 Ascorbic acid 400b 3.28 ± 0.11 80 Vitamin A acetate 480 a 2.56 ± 0.56 92 Calciferol 420 b 2.63 ± 0.41 90 a-tocopherol 370 b 3.01 ± 0.38 85 Menadione 530 a 2.79 ± 0.09 90 Control (C2-contains no vitamins) 120 d 3.82 ± 0.38 60 Figures in each column with same superscript are not significantly different at 5% level was 92%. In the recovery test, the feed efficiency and growth improved gradually. Niacin: Niacin deficiency resulted in poorer feed efficiency and shrimp showed inappetence. Prolongation of niacin deficiency caused blackening of the gills in some shrimp at the end of 8th week. The survival was 86%. The relative weight gain was 74% and in the recovery test growth and feed efficiency improved. Choline: Exclusion of choline resulted in decreased growth and affected the food consumption after the 4 th week. The shrimp exhibited anorexia after three weeks of feeding which resulted in passive activity of the shrimp. The relative weight gain was 52% and survival was 80%. In the recovery test the growth and food consumption improved rapidly. Ascorbic acid: The shrimp fed on ascorbic acid deficient diet showed a general decline in activity leading to poor food intake and anorexia. Prolonged deficiency resulted in blackening of gills and lesions in the abdominal region. The relative weight gain was 74%. In the recovery test growth and food efficiency recovered gradually after the 6th week. Riboflavin: The shrii;tlp in this treatment were not affected by exclusion of riboflavin. The relative
60 INDIAN J EXP BIOL, JANUARY 2004 weight gain was 96% and survival was 95%. In the recovery test the weight gain and food consumption did not show any improvement. Inositol : The shrimp showed anorexia and food consumption decreased gradually after the third week. The percent weight gain of the shrimp fed the inositol deficient diet decreased after the 4th week. The relative weight gain was only 44% with survival of 84%. In the recovery test the shrimp showed improved growth and food consumption. Folic acid : Growth and food consumption decreased after the 4th week of feeding the folic acid deficient diet. The relative weight gain was 63 %. The shrimp showed gradual improvement in weight gain and food consumption in the recovery test. Cyanocobalamin: Percentage weight gain showed a decreasing trend beginning at the 4th week. The relative weight gain was 70%. In the recovery test weight gain and food consumption gradually recovered. Pantothenic acid : Exclusion of pantothenic acid affected the growth and survival of shrimp at the 5th week. Shrimp showed inappetence. Prolonged deficiency caused partial molting in the abdominal region of the shrimp. The relative weight gain was 74%. The shrimp showed better growth and food consumption in the recovery test. Biotin : Percentage weight gain and food consumption was reduced from the 4th week. Relative weight gain was 67% and survival was 90%. In the recovery test weight gain and food consumption improved. Fat soluble vitamins- Vitamin A acetate: The response of the shrimp in terms of percent weight gain and survival decreased after the 5th week. The relative weight gain was 89%. Shrimp in the recovery test showed improved growth and food consumption. Calciferol: Shrimp fed on the vitamin D deficient diets showed poor growth and food conversion efficiency beginning at the 4th week. The relative weight gain was 90%. In the recovery test shrimp showed improved growth and food consumption. a-tocopherol : Shrimp exhibited poor growth and inappetence after the 4th week. The relative weight gain was only 57% accompanied by 80% mortality. In the recovery test both growth and food consumption increased. Menadione : Shrimp growth and foods consumption was not affected by feeding a vitamin K deficient diet. The relative weight gain was 98% with a survival of 90%. Controls-C1: The weight gain was 540 % with 90% survival. The FCR was better than all the other treatments. C2 : The growth and survival were poor. The weight gain was 120% with a survival rate of 60%. Relative weight gain-the effects of each vitamin deficiency on relative weight gain expressed as percentage of weight gain in the control group. Deficiency of inositol, choline, tocopherol, pantothenic acid, ascorbic acid, folic acid, biotin, thiamin had the most significant effects (P < 0.05) on relative weight gain. On the other hand, the weight gain in shrimp fed riboflavin and vitamin K deficient diets was almo.st equivalent to that of the control group. Feed efficiency - The effect of exclusion of individual vitamins on feed conversion ratio is shown in Table 2. The feed efficiency of each deficient group showed an almost similar tendency to relative weight gain. The food conversion ratio (FCR) were best in shrimp fed diets supplemented with the complete vitamin mixture and poorest in shrimp fed diets deficient in ascorbic acid, tocopherol, biotin, choline, folic acid, niacin, pantothenic acid, thi amine and the negative control (C2). Histology-The digestive gland cells of P. monodon juveniles fed diet (C1) had numerous cells and showed normal cellular layers (Fig. la), while those shrimp fed specific vitamin deficient diets exhibited histological changes (Fig. l b). The shrimp fed the negative control diet (C2) lacking all vitamins had complete loss or destruction of epithelial cells (Fig. lc). Discussion In the present study reduced growth (89% of control group) in vitamin A deficient treatment demonstrates that vitamin A is an essential nutrient for P. monodon. Its physiological functions are involved in regeneration of light sensiti ve compounds in the retina, transportation of calcium across some membranes, reproduction and integrity of cellular and subcellular membranes 1 1 ' 12 The utilization of ~ carotene by vertebrates as a precursor to vitamin A is well understood 1 1. 12 The dietary essentiality of vitamin A for P. vannamei has been documented 9 Result of the present study revealed that vitamin D is essential in diets for P. monodon. In vertebrates, vitamin D functions as a precursor to 1,2, 5- dihydroxy-cholecalciferol, which stimulates calcium absorption from the intestine and regulates its movement to maintain physiologically normal levels
RAI & REDDY: EVALUATION OF DIETARY ESSENTIALITY OF VITAMINS FOR P. MONODON(FABRICIUS) 61 Fig.!a-Normal cell profile of shrimp fed a complete vitamin supplemented diet. b: Damaged cell of shrimp fed vitamin deficient diet. c: Severely damaged cells with extensive necrosis of epithelial cells of shrimps fed diets without vitamin supplement. in the blood 11 12 In crustaceans, vitamin D functions in the mfneralization of the exoskeleton even though marine crustaceans can absorb significant amounts of dissolved calcium through the gills. Significantly suppressed growth and poor appetite were observed in shrimp fed a vitamin E deficient diet. Vitamin E is important in protecting polyunsaturated fatty acids in biomembranes from oxidation and is required in preventing varied diseases in animals 1 u 2. Vitamin E was required by Daphnia magna for normal growth and reproductive activity 13. Thus vitamin E may be also essential in preventing the oxidation of polyunsaturated fatty acids in feeds as well as shrimp tissues. The presence and absence of vitamin K supplement had no significant effect on shrimp growth and survival. Vitamin K is required by vertebrates for normal blood clotting mechanisms to operate. However, there is no evidence to support this function in crustaceans. Vitamin K was not essential to Daphnia magna has been demonstrated 14 The nonessentiality of vitamin K in the diet of P. vanamei is well proved 9. The results of the present study demonstrate that among the water soluble vitamins choline, inositol and pantothenic acid deficiency most severely retarded the growth of the shrimp followed by biotin, niacin, pyridoxine and cyanocobalamine deficiency. Besides reduced growth, poorest survival was observed in shrimp fed niacin, choline, inositol and ascorbic acid deficient diets. Similarly, the essentiality of choline and inositol in the diet of P. japonicus has been proved 15. Information on vitamin deficiency syndromes in shrimp is rather scarce. The only well documented vitamin deficiency disease in penaeid shrimp is the black death syndrome related to ascorbic acid deficiency 16 Ascorbic acid is shown to be required by P.. 17 18 p lifi.. 16 d p.. 19. Japonzcus ',. ca z emzenszs an. merguzenszs. Prolonged deficiency of niacin and ascorbic acid resulted in formation of black lesions on the body in some test individuals; whereas, deficiency of pantothenic acid resulted in partial molting in a few test individuals. Similar observations were made in P. indicui 0. As reported in the present study, depressed growth in P. japonicus fed pyridoxine and thiamine deficient diets noticed 21 Exclusion of riboflavin from the feed did not affect the growth of P. monodon in the present study. However, exclusion of riboflavin led to increased growth in juvenile fresh water prawn Macrobrachium
62 INDIAN 1 EXP BIOL, JANUARY 2004 rosenbergip 2 Increased growth in P. monodon fed riboflavin deficient diets has been documented 6 In the present study, normal cellular structures were observed in the mid-gut gland of shrimp fed the control (Cl) diet supplemented with all vitamins. Detachment of epithelial cells was observed in other treatments but more severely in treatments fed the negative control (C2) (no vitamin supplement) and diets deficient in inositol, choline, pantothenic acid and ascorbic acid. Similar observations were made in case of P. monodon fed on diets deficient in water soluble vitamins 6 ' 23 ' 24 '. The results of the present study demonstrated that the fat soluble vitamins A, D, E and 10 water soluble vitamins (thiamine, riboflavin, pyridoxine, cyanocobalamine, pantothenic acid, folic acid, niacin, biotin, choline, inositol, ascorbic acid) are essential for P. monodon. No evidence for essentiality of vitamin K or riboflavin in P. monodon was observed. 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