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Vitamin A in diets for Nile Tilapia 751 VITAMIN A IN DIETS FOR NILE TILAPIA Daniela Ferraz Bacconi Campeche1*; Rodrigo Ramos Catharino2; Helena Teixeira Godoy2; José Eurico Possebon Cyrino3 1 2 Embrapa Semi-Árido - BR 428, km 152, C.P. 23 - 56300-970 - Petrolina, PE - Brasil. UNICAMP/FEA - Depto. de Ciência de Alimentos, R. Monteiro Lobato, 80, C.P. 6121 - 13083-862 - Campinas, SP - Brasil. 3 USP/ESALQ - Depto. de Zootecnia, Setor de Piscicultura, Av. Pádua Dias, 11 - 13418-900 - Piracicaba ,
  Vitamin A in diets for Nile Tilapia 751 Sci. Agric. (Piracicaba, Braz.), v.66, n.6, p.751-756, November/December 2009 VITAMIN A IN DIETS FOR NILE TILAPIA Daniela Ferraz Bacconi Campeche 1 *;   Rodrigo Ramos Catharino 2 ; Helena Teixeira Godoy 2 ;José Eurico Possebon Cyrino 3 1  Embrapa Semi-Árido - BR 428, km 152, C.P. 23 - 56300-970 - Petrolina, PE - Brasil. 2 UNICAMP/FEA - Depto. de Ciência de Alimentos, R. Monteiro Lobato, 80, C.P. 6121 - 13083-862 - Campinas,SP - Brasil. 3 USP/ESALQ - Depto. de Zootecnia, Setor de Piscicultura, Av. Pádua Dias, 11 - 13418-900 - Piracicaba , SP - Brasil.*Corresponding author <daniela.campeche@cpatsa.embrapa.br> ABSTRACT:   Dietary vitamin supplementation decrease stress caused by high stocking density, and boosts immunological system of farmed fish. A studied was carried out to determine vitamin Arequirements of Nile tilapia ( Oreochromis niloticus ) in an all male group (13.8 ± 1.2 g) and a mixed sex population (9.8 ± 2.3 g). Fish stocked in 100-L plastic aquaria (26.0 ± 1.0ºC) were fed to near satiety,twice a day, seven days a week, during 75 days with vitamin A-free, semi-purified diets supplementedwith 0; 600; 1,200; 1,800; 2,400; 3,000; 3,600; 4,200; 4,800 and 5,400 International Units (IU) of retinyl palmitate (30% vitamin A) per kg of diet in a completely randomized experimental design, factorialarrangement 2 χ 10 (n = 4). Deficiency signs of vitamin A were observed in fish fed 0 to 1.200 IU vitaminA kg  –1 diet; moderate signs were observed in fish fed diets with 1.800 to 3.600 IU vitamin A kg  –1 diet;no interactions group*level (  p < 0.05) were detected. Dietary levels of vitamin A up to 5.400 IU kg  –1 influenced final weight and weight gain of fish (  p < 0.05), but did not influence feed consumption(  p > 0.05). A group effect was observed regarding all performance variables (  p < 0.0001). Quantificationof hepatic retinol (HPLC) detected vitamin A only in fish fed 5.400 IU retinol kg  –1 of diet, thereforecharacterizing that dietary retinol was used and stored. The quantity of 5.400 IU of retinol kg  –1 of dietis recommended for adequate nutrition of Nile tilapia.Key words: Oreochromis niloticus , retinol, nutrition VITAMINA A EM DIETAS PARA TILÁPIA DO NILO RESUMO:   A suplementação de vitaminas na dieta diminui o estresse e estimula o sistema imunológicocausado por altas densidades de estocagem dos peixes. Este trabalho determinou a exigência emvitamina A para a tilápia do Nilo em uma população monosexo masculina (13.8 ± 1.2 g) e em uma população srcinal (9.8 ± 2.3 g). Os peixes foram estocados em aquários plásticos de 100 L (26.0 ±1.0ºC) e alimentados “ad libitum”, duas vezes ao dia, sete dias da semana, durante 75 dias com dietasemi-purificada suplementada com 0; 600; 1.200; 1.800; 2.400; 3.000; 3.600; 4.200; 4.800 e 5.400 UI deretinyl palmitato (30% de vitamina A) por kg de dieta, em um delineamento experimental totalmente aoacaso e arranjo fatorial 2 χ 10 (n = 4). Deficiência nutricional severa foi observada em peixes alimentadoscom 0 a 1.200 UI vitamina A kg  –1 de dieta; sinais moderados foram encontrados em peixes alimentadoscom 1.800 a 3.600 UI vitamina A kg  –1 de dieta; interações grupo*nível (  p < 0,05) não foram detectadas.O aumento de nível de vitamina A influenciou no peso final e no ganho de peso dos peixes (  p < 0,05),mas não influenciou o consumo de ração (  p > 0,05). Foi observado efeito de grupo no desempenhodos peixes (  p < 0,0001). Foi detectado o retinol hepático através de HPLC somente no grupo alimentadocom 5.400 UI de retinol kg  –1 de dieta, caracterizando assim que o mesmo foi utilizado e armazenado. Aquantidade de 5.400 IU de retinol kg  –1 de dieta é a mínima recomendada para tilápia do Nilo.Palavras-chave: Oreochromis niloticus , retinol, nutrição INTRODUCTION Commercial production of tilapia requires the useof high quality, complete feed. Increasing vitaminsupplementation of complete diets decreases stresscaused by high stocking density, and boosts immuno-logical system of fish (Davis et al., 1998; Halver,1985; Toguyeni et al., 1997). On the other hand, in-adequate dietary vitamin supplementation can result indiseases outbreaks or reduced growth in a confinedfish population (Hepher, 1988; NRC, 1993; De Silva& Anderson, 1995; Harikumar et al., 1996; Goswami& Dutta, 1991; Taveekijaran, 1994; Thompson et al.,1995).Excess dietary fat-soluble vitamins are stored inhepatic lipid deposits (NRC, 1993; Ornsrud et al.,  Campeche et al. 752 Sci. Agric. (Piracicaba, Braz.), v.66, n.6, p.751-756, November/December 2009 2002); 90% of the stored vitamin A is found in the liver (Katuyama & Matsuno, 1988). Therefore, quantifyingvitamin A depots in hepatic tissue elicits establishingmetabolic and nutritional requirements (Hole & Tay-lor, 1996).Vitamin A requirements were determined for chan-nel catfish (1,000 to 2,000 International Units (IU) kg  – 1 of diet), salmonids (2,500 IU kg  –1 of diet), carp(4,000 to 20.000 IU kg  –1 of diet), Japanese flounder (10.000 IU kg  –1 of diet) and greasy grouper   Epinephelus tauvina (3,101 IU kg  –1 of diet) (NRC,1993; Hepher, 1998; Hernandez et al., 2007; Mohamedet al., 2003). Saleh et al. (1995) determined that vita-min A requirement of Nile tilapia is 5,000 IU kg  –1 diet.Hu et al. (2006) determined that vitamin A requirementof hybrid tilapia O. niloticus × O. aureus ranges on5,850 to 6,970 IU kg  –1 . This same author also regis-tered that tilapia can utilize β -carotene to fulfill the di-etary vitamin A requirements. However, Kubitza et al.(1998) and Kubitza & Cyrino (1999) reported that Bra-zilian commercial feeds for omnivorous, tropical fishmay contain from 3,000 to 22,000 IU kg  –1 vitamin A.The aim of this study was to verify the use of vita-min A in diets for Nile tilapia, through the determina-tion of hepatic vitamin A storage capacity and doublecheck discrepant, reported dietary vitamin A require-ment of juvenile Nile tilapia fed semi-purified diets,through the evaluation of growth rate and deficiencysigns. MATERIAL AND METHODS Fish (19 per aquarium) were kept in 40 100-L plas-tic aquaria, supplied by a closed recirculation system.Aeration was provided continuously throughout theexperiment. Water pH, dissolved oxygen (OD) and tem- perature (26 ± 1.0°C) were monitored on a daily ba-sis. Fish were fed for 11 weeks with vitamin A-free,semi-purified diets supplemented with 0, 600, 1.200,1.800, 2.400, 3.000, 3.600, 4.200, 4.800 and 5.400IU of vitamin A kg  –1 diet (Table 1), in a completely ran-domized experimental design, factorial arrangement2 χ 10 (n = 4). Retinyl palmitate (Rovimix TO 500Roche ® ; 30% vitamin A) was used as dietary vitaminA source. The diet was formulated based on albuminand gelatin protein (Table 1). The mixture was extrudedthrough a mincer (ML-4.0 WEG- μ line); pellets werecollected, dried overnight in a forced air oven (55°C);grinded to 1 mm pellets, sized, hermetically packed andstored under refrigeration until use. Fish were fed tonear satiety twice a day (6:00 am and 6:00 pm).The trial was duplicated with (i) an all-male, sex-reversed Nile tilapia juvenile population (SR), (13.76± 1.21 g), and (ii) a mixed-sex population (NSR) (9.83 1 Units kg  –1 of diet: vit D 3 200,000 UI; vit E 12,000 mg; vit. K  3 2,400 mg; vit B 1 4,800 mg; vit B 2 4,800 mg; vit B 6 4,000 mg; vitB 12 4,800 mg; folic acid 1,200 mg; pantothenic acid 1,200 mg; vitC 48,000 mg; biotin 48 mg; niacin 24,000 mg; Fe 10.000 mg; Cu600 mg; Mn 4,000 mg; Zn 6,000 mg; I 20 mg; Co 2 mg; Se 20 mg. 2 Vit C (Lutavit C- Aquastab BASF ® ). 3 BHT= Butil hidroxitolueno. Table 1 - Formulation and proximate composition of theexperimental diets. stneidergnI )%(stnetnoC nimublA 01.23 nitaleG 07.7 hcratsnroC 31.44 lionaebyoS 00.6 esolulleC-a 00.6 etahpsohPmuiclaciB 00.3 ximlarenimdnaeerf -AnimatiV 05.0 CnimatiV 2 50.0 edirohcmuidoS 05.0 THB 3 20.0 noitisopmocetamixorP )%(rettamyrD 90.29 gklack(ygrenessorG 1– ) 1974 )%(nietorpedurC 07.43 )%(dipiledurC 56.2 )%(rebif edurC 38.3 )%(hsA 97.4 ± 2.30 g). Fish were acclimated to the aquaria and fedfor fifteen days prior to the beginning of the feedingtrials with the non-supplemented diet to simulate or in-duce deficiency (NRC, 1993).Apparent signs of vitamin A deficiency – e.g.exophthalmia, depigmentation, clouding of corneal epi-thelium, anorexia, warped gill operculum, reducedgrowth, poor feed efficiency, and high mortality(Tacon, 1992) – were recorded through visual obser-vations along the experimental period and at the endof the trial in all fish. The following growth data wererecorded at the beginning and ending the experimental period: initial and final weigh; weight gain [(finalweigh) - (initial weigh)]; feed consumption; feed con-version ratio [(feed consumption) ÷ (weight gain)]; andsurvival rate [100 × (final number of animals) ÷ (ini-tial number of animals)]. At the end of the trials, he- patic tissue was sampled from fish and stored in liq-uid nitrogen. High pressure liquid chromatography(HPLC) was utilized to quantify vitamin A in the he- patic tissue lipid depots (Landen-Júnior & Eitenmiller,1979).Data were submitted to ANOVA and regressionanalysis by the PROC GLM, SAS software (SAS In-  Vitamin A in diets for Nile Tilapia 753 Sci. Agric. (Piracicaba, Braz.), v.66, n.6, p.751-756, November/December 2009 stitute, 2001), for linear and quadratic effects of thetreatments on final weight (FW), weight gain (WG),feed conversion rate (FCR) and survival (S). RESULTS AND DISCUSSION Deficiency signs The same clinical deficiency signs were observedfor animals of all groups fed vitamin-A deficient diet. Normally colored livers with dark-colored gal bladders,a characteristic sign of clinical stress (Halver, 1989;Roberts, 1981; Post, 1987; Steffens, 1989; Tacon,1992; Plumb, 1999) was also recorded (Table 2).Vitamin A deficiency signs in O. niloticus include:abnormal swimming behavior; internal hemorrhages; protruded, blind eyes; anemia; hemorrhage in the baseof fins and in the skin (Saleh et al., 1995). In advanceddeficiency condition Saleh et al. (1991) also observedwidespread depigmentation and edemas in the abdo-men, sometimes associated with ascites; reduction of mucus secretion and dry, hard mucous tissue. Lesionsobserved post-mortem appeared as ascites, clubbedgills and hemorrhagic kidneys. Hemorrhagic, amor- phous, granulomatous spleen; necrotic, granulomatous,amorphous liver were conspicuously found in the present work in fish receiving less than 1,200 IU vi-tamin A kg  –1 diet. Spleen severe conditions were alsoregistered to a lesser extent in fish fed diets contain-ing 1,800~2,400 IU vitamin A kg  –1 diet.Cherry salmon Oncorhynchus massou fed vitaminA-deficient diets for 15 weeks presented clinical signssimilar to those described above (Taveekijaran et al.,1994). Similar observations were reported for catfish  Heteropneustes fossilis , greasy grouper   Epinephelustauvina , Atlantic halibut  Hippoglossus hippoglossus L .and sunshine bass juvenile  Morone chrysops × M. saxatilis (Harikumar et al., 1996, Mohamed et al.,2003; Moren et al., 2004; Hemre et al., 2004). Theuse of advanced juveniles, which may have adequate body reserves of vitamin A, may explain the low inci-dence of ocular problems, opposing to observationsof Poston et al. (1977) with rainbow trout. Growth parameters Weight gain (WG), feed conversion ratio (FCR),survival (S) and feed consumption rate (FCR) data are presented in Table 3. A linear effect (  p < = 0.01) wasdetected for FW and FCR, but no effect was detectedregarding WG (  p > = 0.05) (Figure 1 and 2). This mayhave resulted from differences in fish initial weight.Several research reports are in accord to these results.Hu et al. (2006) reported that hybrid tilapia fed dietssupplemented with 50,000 IU vitamin A kg  –1 present better weight gain (601%) and better feed conversionratio (1.00). Saleh et al. (1995) also observed that Niletilapia juveniles fed diets supplemented with 5,000 IUvitamin A kg  –1 presented better weight gain (23.9 g), better feed consumption rate (60.2 g), and better feed Table 2 -   Percent incidence of  c linical signs of vitamin A deficiency recorded in all Nile tilapia juveniles fed diets varyingvitamin A levels. teiD )%(dedrocerAnimativf osngisycneicif eD 0 006 002,1 008,1 004,2 000,3 006,3 002,4 008,4 004,5 gkAtivgm--------------------------------------- 1– --------------------------------------- gnirudsngislacinilC tnemirepxeeht doirep RS RSN RS RSN RS RSN RS RSN RS RSN RS RSN RS RSN RS RSN RS RSN RS RSN revilelaP 31.0 31.0 25.0 93.0 31.0 elacstuohtiwnikS 31.0 31.0 31.0 tcarataC 62.0 31.0 31.0 31.0 laretalegarromeH nif 31.0 mlathpoxE 31.0 retf asngislacinilC tnemirepxeeht doirep seticsA 31.0 31.0 ,suotamolunarG neelpscitorcen 56.0 56.0 36.2 56.0 01.2 72 17.1 44.1 44.1 93.0 56.0 31.0 31.0 31.0 ,suotamalunarG revilsuohprom 49.3 00.5 49.3 49.3 01.2 56.0 31.0 31.0 31.0 yendiksuohpromA 36.2 36.2 49.3 49.3 SR = sex-reverted; NSR = non sex-reverted  Campeche et al. 754 Sci. Agric. (Piracicaba, Braz.), v.66, n.6, p.751-756, November/December 2009 conversion ratio (2.5), than fish fed diets containing0, 10,000 or 40,000 IU vitamin A kg  –1 . Mohamed etal. (2003) observed that diets supplemented with 3,764mg vitamin A kg  –1 for greasy grouper led to a better weight gain (420.94%), better feed conversion ratio(1.42) and better protein efficiency ratio (2.08). Sun-shine bass fed diets supplemented with 509 – 40,516µg vit A kg  –1 had no difference in weight gain (269-285%) or feed efficiency (0.88-0.89) (Hemre et al.,2004). On the other hand, Hernandez et al. (2007) ob-served that Japanese flounder   Paralicthys olivaceous fed fish meal-based diets supplemented with 0.00 IUvitamin A kg  –1 presented better specific growth rate(4.9%). Also, Atlantic halibut fed diets supplementedwith 0-250 mg of retinal kg  –1 had no differences infinal weight or mortality (Moren et al., 2004).Survival rate obeyed to a quadratic effect (  p < =0.001) (Figure 3). Saleh et al. (1995) reported thatgroups of Nile tilapia receiving 5,000 and 10,000 IUvitamin A kg  –1 diet had 93% survival ratio. Thus in-creasing dietary vitamin A levels up to 10,000 IU kg  –1 do not significantly reduce survival rate of Nile tila- pia. Mortality rate of rainbow trout juveniles was notinfluenced when feeding on either vitamin A-free dietor diets supplemented with 10,000 IU of retinyl palmi-tate kg  –1 for a maximum 20 weeks (Poston et al.,1977). This data for rainbow trout corroborates re-sults for Japanese flounder, greasy grouper, hybrid ti-lapia, and sunshine bass (Hernandez et al., 2007;Mohamed et al., 2003; Hemre et al., 2004; Hu et al.,2006). Table 3 - Growth performance of the juveniles fed diets varying vitamin A levels. SR = sex-reversed; NSR = non sex-reversed, IW= initial weight; FW = final weight; WG = weight gain; FC = food consumption; FCR = food conversion rate. ecnamrof rephtworG puorG WI WF GW CF RCF --------------------------------------------------g------------------------------------------------- RS 67.31 07.34 49.92 28.33 21.1 RSN 38.9 30.72 22.71 54.52 94.1 levelAnimatiV gkUI( 1– )0 41.11 16.13 64.02 64.23 85.1 006 46.01 90.03 54.91 26.92 35.1 0021 49.11 92.33 53.12 38.82 83.1 0081 07.11 48.23 41.12 40.62 82.1 0042 39.21 27.63 97.32 61.82 02.1 0003 37.21 42.73 15.42 01.92 02.1 0063 65.21 85.83 20.62 58.03 32.1 0024 79.01 20.73 50.62 09.92 71.1 0084 47.11 94.73 57.52 43.92 61.1 0045 55.11 18.83 62.72 40.23 02.1 Figure 2 -   Food conversion rate (CR) of the animals (FCSR: sex-reverted; FCNSR: non sex-reversed in response to thelevels of vitamin A inclusion.Figure 1 - Final weight (FW) of the animals (SR: sex-reverted; NSR: non sex-reversed) in response to the levels of vitamin A inclusion.
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