Possibility of using basil (Ocimum basilicum) supplementation in Gilthead sea bream (Sparus aurata) diet

Egyptian Journal of Aquatic Research (2015) 41, 203–210

H O S T E D BY

National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com

FULL LENGTH ARTICLE

Possibility of using basil (Ocimum basilicum) supplementation in Gilthead sea bream (Sparus aurata) diet Ashraf Y. El-Dakar a, Shymaa M. Shalaby a, Bahig R. Nemetallah b, Norhan E. Saleh c,*, Eman M. Sakr c,d, Mohamed M. Toutou c a

Aquaculture Department, Faculty of Fish Resources, Suez University, Egypt Physiology Department, Faculty of Veterinary Medicine, Alexandria University, Egypt c Fish Nutrition Laboratory, Aquaculture Division, National Institute of Oceanography and Fisheries (NIOF), Egypt d Al-Farabi College for Dentistry and Nursing, Bio-Medical Sciences Department, Riyadh, Saudi Arabia b

Received 15 December 2014; revised 1 March 2015; accepted 1 March 2015 Available online 23 April 2015

KEYWORDS Growth; Feed utilization; Biometric studies; Enzymes

Abstract This study was carried out on sea bream (Sparus aurata) with initial fish weight of 1.9 ± 0.1 g to evaluate the effect of addition of basil (Ocimum basilicum), in different forms on; growth performance, feed utilization, body composition, biometric indices and some serum constituents. Four experimental diets were formulated; Diet 1: control diet (without basil, CTR); Diet 2: 2% dried basil leaves, (BL), Diet 3: 2% dried basil seeds, (BS) and Diet 4: 2% soaked and dried basil seeds, (BSS). Fish were fed experimental diets twice a day until visual apparent satiation, 7 days a week for 84 days. The results showed an improvement in fish growth and feed utilization indices when fed diets were supplemented either with BS or BSS respectively. Feeding of either BL or BS resulted in an enhancement in protein retention coinciding with depression in lipid content. Digestive enzyme (amylase and lipase) activities and serum total protein were elevated by the administration of basil in all forms to sea bream diet as compared to the control. ª 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Medicinal and aromatic plants can be used as feed additives; they can improve animal growth performance, play an * Corresponding author at: National Institute of Oceanography and Fisheries (NIOF), Qayed Bay, Anfoushy P.O., 21556 Alexandria, Egypt. Tel.: +20 3 4801138 (Office); fax: +20 3 4801174. E-mail address: [email protected] (N.E. Saleh). Peer review under responsibility of National Institute of Oceanography and Fisheries.

important role in improving the utilization efficiencies of fish diets and also can be considered as attractants. Furthermore, medicinal herbs may be optimal additives of artificial diets for aquatic animals from the stand point of their antimicrobial and antiseptic actions. Spices and natural herbs such as marjoram, basil, licorice root, black seed, peppermint, caraway and cumin have been shown to be beneficial by Sakr (2003), Shalaby et al. (2003), El-Dakar et al. (2004a,b,c), Ahmad and Abdel-Tawwab (2011) and Yılmaz et al. (2013).

http://dx.doi.org/10.1016/j.ejar.2015.03.001 1687-4285 ª 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

204 Common basil (Ocimum basilicum L.), a member of the Lamiaceae family is an annual herb which grows in several regions around the world. Basil is an essential oil crop which is cultivated commercially in many countries (Sajjadi, 2006) and has been extensively utilized in food as a flavoring agent, and in perfumery and medical industries (Telci et al., 2006). Bihari et al. (2011) reported that the phytochemical screening of O. basilicum revealed the presence of glycoside, gums, mucilage, proteins, amino acids, tannins, phenolic compound, triterpenoids, steroids, sterols, saponins, flavones and flavonoids in it. A total of 29 compounds representing 98.0– 99.7% of the oils are identified by Hussain et al. (2008) in O. basilicum. Essential oil composition and phenolics have been well reviewed by Makri and Kintzios (2008). Also, Tomar et al. (2010) reported that basil has exceptionally high content of B-carotene, and lutein-zeaxanthin. Basil leaves contain 0.17% oleanolic acid and a small amount of ursolic acid (List and Ho¨rhammer, 1977). The green leaves contain high concentration of vitamins, minerals and oils (Khare, 2007). Sakr (2003) reported that basil leaves are rich in ocimene, methyl chavecol and linalool which were predominantly effective compounds in the volatile oil and basil leaves meal may be acting on fish growth, feed and protein conversion, and nutrient retention efficiencies by its constitution of the volatile oil. Additionally, basil has shown antioxidant and antimicrobial activities due to its phenolic and aromatic compounds (Gutierrez et al., 2008). Accordingly, it was necessary to throw some more light on using basil in marine farmed fish diets and study its effects on fish performance. Nutrition of sea bream (Sparus aurata) is an area of research that has recently received considerable attention because of the influence of nutrition on fish growth and health as well as on cost of production. The present work was conducted to investigate the effects of using dried basil leaves, dried basil seeds and dried soaked basil seeds (to avoid negative effects of antinational factors in basil seeds), as growth promoters and feeding attractants; on growth, feed conversion, nutrient retention efficiencies, body composition, biometric measurement indices and some serum constituents for sea bream S. aurata fingerlings. Materials and methods Experimental fish and feeding regime Gilthead sea bream fingerlings were obtained from EL-Wafa Marine Fish Hatchery (Ismailia governorate) and transferred to Fish Nutrition Lab. NIOF, Anfoushy, Alexandria. Fish were acclimatized to the laboratory conditions for 20 days. Healthy fish were randomly distributed in 12 glass aquaria (100 L each) at a density of 16 fish per aquarium with an average weight of 1.9 ± 0.1 g/fish on triplicate groups per treatment. The aquaria were daily cleaned and excreta were siphoned. Light was about 12:12 h light: dark cycle throughout the day. Water quality parameters were monitored on a weekly basis throughout the experimental period using the standard APHA methodology (1995) by the mean of multi-purpose water meter (YSI 600 XL, Xylem Inc., USA). The parameters are; water temperature (20 C ± 2), salinity (37.5 ± 0.5 ppm), dissolved oxygen (7 ± 0.2 mg/l) and pH (7.9 ± 0.1). Fish were fed the test diets until visual apparent satiation, 7 days a week for

A.Y. El-Dakar et al. 84 days. Fish in each aquarium were counted and weighed (collectively) biweekly throughout the feeding trial. Total amount of feed consumed by the fishes in each aquarium, during the study period, was determined and feed consumed for each individual fish was calculated accordingly. Experimental diets Dried basil leaves and seeds were obtained from the local market. Four isonitrogenous (45% crude protein), isocaloric (477 kcal/100 g gross energy, GE) experimental diets were formulated to fulfill sea bream requirements at this age stage; Diet 1: control diet (without basil), (CTR); Diet 2: 2% dried basil leaves, (BL), Diet 3: 2% dried basil seeds, (BS) and Diet 4 2% soaked and dried basil seeds, (BSS) to partially replace wheat flour as shown in Table 1. All dry dietary ingredients were ground, sieved and blended together thoroughly then oil was added. The mixer was moistened; cold-pelleted with a laboratory mincer, and dried at 40 C for 24 h. then stored in plastic bags at 20 C until use. Data collection and sample analysis At the end of experiment, the fishes of each aquarium were weighed collectively and average final weight (g/fish) was calculated. Hematological analyses At the end of the experiment six fish from each aquarium were cut at the caudal peduncle and blood was collected in coded plastic tubes. Serum constituents Blood samples were collected and transferred to centrifuge tubes and allowed to clot at room temperature. Serum was then separated by centrifugation at 3000 rpm for 5 min. The serum was stored at -20 C until further analysis. Serum total protein (g/dl) was determined calorimetrically using kits supplied by El-Nasr Pharmaceutical Chemicals Co. (Egypt). Serum total lipid (g/dl); serum cholesterol (mg/dl); lipase and amylase activities were determined calorimetrically using commercial kit’s DIAMOND DIAGNOSTIC Co. (Egypt). Determination of some biometric indices After blood sample collection, all fish were scarified and soon the abdominal cavities of three fish from each aquarium were opened to remove viscera which are to be weighed at once. Total length, total weight, liver and viscera weights and gut length were recorded in order to calculate; hepatosomatic index (HSI), viscerosomatic index (VSI), condition factor (CF) and relative intestine length (RIL). Hepatosomatic index (HSI) was determined according to Busacker et al. (1990) as follows: HSI = 100 [liver weight (g)/ total body weight (g)]; Viscerosomatic index (VSI) was estimated according to Ricker (1979) as follows: VSI = 100 [viscera weight (g)/ total body weight (g)], CF was estimated according to Fulton

Basil supplementation in Gilthead sea bream diet Table 1

205

The composition and chemical analyses (% on dry matter basis) of the experimental diets.

Ingredients composition

Fish meal (68%) Soybean meal Yellow corn Wheat bran Wheat flour Premix1 Fish oil Sun flower oil Di-calcium phosphate Basil leaves (BL)2 Basil seed (BS)3 Basil soaked seed (BSS) Chemical composition% Dry matter (DM) Crud protein (CP) Ether extract Crude fiber Nitrogen free extract4 (NFE) Ash Gross energy5 (kcal/100 g DM) P:E ratio (mg CP: kcal)

Experimental diets composition (%) Control (CTR)

Basil leaves (BL)

Basil seed (BS)

Soaked and dried basil seed (BSS)

50 22 6 8 5.95 2 3 3 0.05 – – –

50 22 6 8 3.95 2 3 3 0.05 2 – –

50 22 6 8 3.95 2 3 3 0.05 – 2 –

50 22 6 8 3.95 2 3 3 0.05 – – 2

96.7 44.6 11.3 2.87 28.96 12.3 477.2 93.4

96.8 45.0 11.1 2.96 28.11 12.4 476.7 95.3

96.7 45.4 12.5 2.92 27.06 12.1 485.5 93.6

96.4 45. 7 11.6 2.94 28.14 11.9 481.4 94.3

1 Vit./min. Premix (mg kg 1); p-amino benzoic acid (9.48); D-biotin (0.38); inositol (379.20); niacin (37.92); Ca-pantothenate (56.88); pyridoxine–HCl (11.38); riboflavin (7.58); thiamine–HCl (3.79); L-ascorbyl-2-phosphate Mg (APM) (296.00); folic acid (0.76); cyanocobalamine (0.08); menadione (3.80), vitamin A-palmitate (17.85); a-tocopherol (18.96); calciferol (1.14). K2PO4 (2.011); Ca3(PO4)2 (2.736); MgSO4 7H2O (3.058); NaH2PO4 2H2O (0.795). 2 Chemical composition: 3.8% protein, 0.3% fat, 2.0% ash and 1.5% fiber. 3 Chemical composition: 10.5% protein, 18.4% fat, 5.4% ash and 3.6% fiber. 4 Calculated by difference. 5 Gross energy (GE) was calculated as 5.64, 9.44 and 4.11 kcal/100 g for protein, lipid and NFE, respectively (NRC, 1993).

(1904); CF = 100 * (TW/TL3) where; TW: Total fish weight (g); TL: Total fish length (cm). Relative intestine length (RIL) was determined according to Al-Hussini (1947) as follows: RIL = absolute intestine length (cm)/ TL (cm).

to a one way analysis of variance (Linear method) to test the effect of treatment inclusion on fish performance. Data was analyzed using SPSS program (1997), Version 16. Differences between means were compared using Duncan multiple range test at P < 0.05 level.

Chemical analyses of diets and fish Results Diets and whole-fish body from each treatment were analyzed according to the standard methods of AOAC (2000) for moisture, crude protein, crude fat and ash. Fish performance Growth rate and feed utilization efficiency indices calculated as mentioned by Ballestrazzi et al. (1994) are as follows: Weight gain = final weight (g) – initial weight (g); Specific growth rate (SGR) = 100 (ln W2 ln W1)/T; where W1 and W2 are the initial and final weight, respectively, and T is the number of days of feeding trial; Feed conversion ratio (FCR) = feed intake (g)/weight gain (g); Protein efficiency ratio (PER) = weight gain (g) /protein intake (g); Protein productive value (PPV) = 100 [protein gain (g)/protein fed (g)] and Energy utilization (EU;%)=100 · (energy gain (g)/energy intake (g)). Statistical Analyses Mean value and standard error (mean ± SE) for each parameter of all treatments was calculated. The results were subjected

Growth performance and feed utilization efficiency Initial weight, final weight, weight gain, specific growth rate and survival rate of sea bream fingerlings are presented in Table 2. The highest weight gain and specific growth rate values, at the end of the experiment, were recorded in the BS group to be 6.80 g and 1.41%/day, respectively and these values were significantly (P < 0.05) different comparing with either CTR or BL fish groups. Fish fed BSS diet showed insignificant growth indices comparing with all other groups. Best FCR and PER values were also recorded in fish fed BS diet (1.61 and 1.36 respectively). Fish fed basil in different forms showed superiority in all feed utilization indices comparing with the CTR fish group. No significant differences were recorded in the survival rate among groups (P > 0.05). Results of proximate analyses of fish fed the CTR and test diets are illustrated in Table 3. Moisture content in different fish groups ranged between 65.91% (BSS) and 68.81% (CTR). Fish fed BL and BS has significantly higher protein

206 Table 2

A.Y. El-Dakar et al. Effects of dietary BL, BS and BSS on growth performance and feed utilization indices of sea bream (mean ± SE). Experimental diets

Initial body weight (IW, g/fish) Final body weight (FW, g/fish) Total weight gain (TWG, g/fish) Specific growth rate (SGR, %/d) Feed intake (FI, g/fish) Protein intake (PI, %) Feed conversion ratio (FCR) Protein efficiency ratio (PER) Protein productive value (PPV) Energy utilization (EU, %) Survival rate (SR, %)

CTR

BL

BS

BSS

1.88 ± 0.1 7.41±0.05b 5.53±0.51b 1.25±0.01b 11.79±0.29a 5.08±0.13a 2.06±0.06a 1.09±0.03b 20.25±0.63b 11.16±0.33b 96 ± 2.08

1.88 ± 0.1 7.5±0.19b 5.6±0.19b 1.26±0.04b 10.20±0.49b 4.43±0.21b 1.76±0.03b 1.27±0.01ab 30.64±0.49a 9.30±0.16c 100 ± 0

1.89 ± 0.1 8.70±0.36a 6.80±0.37a 1.41±0.07a 11.33±0.36ab 4.96±0.16ab 1.61±0.07b 1.36±0.05a 27.67 ± 1.03a 14.74±0.47a 100 ± 0

1.89 ± 0.1 8.3±0.52ab 6.41±0.52ab 1.36±0.09ab 11.30±0.41ab 4.98±0.18ab 1.70±0.14b 1.29±0.1a 31.12 ± 2.26a 10.93±0.71b 98 ± 2.08

Means in the same rows having different letters are significantly (P < 0.05) different.

Table 3

Fish body composition (mean ± SE) (% wet matter) of sea bream fed BL, BS and BSC diets for 84 days. Body composition on wet weight (%)

Moisture Crude protein Lipid Ash

CTR

BL

BS

BSS

68.81 ± 0.51a 18.19 ± 0.44c 8.11 ± 0.26a 4.77 ± 0.06b

67.62 ± 0.41ab 22.55 ± 0.47a 5.21 ± 0.13b 4.34 ± 0.14c

66.90 ± 0.56bc 22.24 ± 0.27a 6.26 ± 0.19b 4.47 ± 0.08c

65.91 ± 0.28c 20.88 ± 0.43b 7.93 ± 0.23a 5.08 ± 0.10a

Means in the same rows having different letters are significantly (P < 0.05) different.

Table 4

Biometric parameters (mean ± SE) of sea bream fed BL, BS and BSS diets for 84 days. Experimental diets

Viscerosomatic index (VSI) Hepatosomatic index (HSI) Condition factor (CF) Relative intestine length (RIL)

CTR

BL

BS

BSS

4.7 ± 0.65b 1.4 ± 0.16 1.5 ± 0.04 0.89 ± 0.7b

5.1 ± 0.26ab 1.7 ± 0.28 1.4 ± 0.07 0.85 ± 0.9b

6.0 ± 0.36a 1.6 ± 0.09 1.5 ± 0.06 1.23 ± 0.1a

5.7 ± 0.23a 1.4 ± 0.19 1.4 ± 0.06 1.05 ± 0.1a

Means in the same rows having different letters are significantly (P < 0.05) different.

content (22.55 and 22.24%, respectively) when compared with fish fed either CTR or BSS diets (18.19 and 20.88%, respectively). Lipid and ash analyses indicate a significant decrease in their values in fish fed BL and BS diets when compared with CTR and BSS diets. Biometric measurements At the end of feeding trial hepatosomatic (HSI) and viscerosomatic (VSI) indices as well as condition factor (CF) and relative intestine length (RIL) of experimental fish groups were calculated and are summarized in Table 4. Fish fed BS diet has the highest values of VSI and RIL (6.0 and 1.23, respectively) and these values were significantly different with fish fed CTR diet (4.7 and 0.89, respectively). Significant variation was observed between BL and BS groups in RIL values. Neither of HSI nor CF values showed significant variations among treatments.

Serum constituents Table 5 illustrates that serum total protein concentration value in CTR and BS groups was equal (2.5 g/dl) and significantly lower (P < 0.05) comparing with other two fish groups. In contrast, serum total lipid values in CTR and BS were significantly higher relative to other groups (1.12 and 1.08 g/dl respectively). Cholesterol concentration in the CTR group was significantly (P < 0.05) the lowest among all groups. Serum lipase concentrations increased by including basil in different forms in fish diets relative to CTR meanwhile, serum amylase concentration was insignificantly different among groups (P > 0.05). Discussion Using of natural feed additive is becoming useful for fish feeding rather than classic chemical feed additives due to the

Basil supplementation in Gilthead sea bream diet Table 5

207

Serum constituents (mean ± SE) of sea bream fed BL, BS and BSS diets for 84 days. Experimental diets

Total protein (g/dl) Total lipid (g/dl) Cholesterol (mg/dl) Lipase (U/l) Amylase (U/l)

CTR

BL

BS

BSS

2.5 ± 0.06c 1.12 ± 0.04a 223.11 ± 0.05c 11.50 ± 0.5b 1.0 ± 0.1

3.7 ± 0.13a 0.69 ± 1.8c 274.22 ± 1.0a 15.06 ± 2.0a 1.2 ± 0.1

2.5±.04c 1.08 ± 0.6a 243.18 ± 0.7b 13.99 ± 1.0a 1.2 ± 0.2

3.2 ± 0.05b 1.03 ± 1.5b 254.53 ± 1.2ab 16.21 ± 2.0a 1.5 ± 0.1

Means in the same rows having different letters are significantly (P < 0.05) different.

accumulative effect of the chemical components induced deterrent effects on consumer health. The use of medicinal and aromatic plants in fish diets especially marines is still limited, this being accomplished only at experimental scale. In the present study an improvement in sea bream growth and feed utilization indices was recorded when fish fed diet containing basil seed when compared with either basil leaves or control diet but survival rates were insignificantly affected among groups. Superiority of using seeds rather than leaves in growth performance may be explained by that seeds contain much higher content of protein and lipid relative to leaves and also basil seeds contain active compounds such as planteose, mucilage, polysaccharides and fixed oil that consists of linoleic acid (50%), linolenic acid (22%), oleic acid (15%) as well as 8% unsaturated fatty acids (List and Ho¨rhammer, 1977). According to Amirkhani and Firouzbakhsh (2013), basil leaves extract improves growth and specific growth rate and lowers FCR of common carp (Cyprinus carpio) at 4% and 8% inclusion levels in fish diet, however, the survival was not affected (P > 0.05) by basil-supplemented diets. Also incorporation of dried basil leaves in Hybrid Tilapia, Oreochromis niloticus X Oreochromis aureus, fingerling diets improved growth rate significantly (P < 0.05) than the control diet especially at 2% dried basil leaves which achieved the best inclusion level (El-Dakar et al., 2008). Abbas (2010) reported that chicks fed diet supplemented with basil at 3 g/kg diet had the best FI, FCR, live body weight and feed efficiency. In European sea bass Dicentrarchus labrax species after the administration of 1% thyme in fish diet, an improvement in values of growth and feed utilization indicators (SGR, FCR, PER) was recorded (Ylmaz et al., 2012). In Japanese flounder Paralichthys olivaceus (Kim et al., 1998a), greasy grouper Epinephelus ustauvina (Sivaram et al., 2004), shrimp Penaeus indicus (Immanuel et al., 2004) and abalone Haliotis discus hannai (Lee et al., 2001), using herbs in diets promoted growth and feed efficiency. Kim et al. (1998b) suggested that unknown constituents in various medicinal herbs led to favorable results in Nile tilapia feeding trials. A wide range of herbs and aromatic plants were examined as prebiotic additives in Nile tilapia such as; fenugreek, eucalyptus, pepper, chamomile, thyme, marjoram leaves, caraway seeds, dried leaves of guava, camphor trees, garlic, black seed and fennel (Ahmad and AbdelTawwab, 2011; Diab et al., 2008; Khalafalla, 2009; Mostafa et al., 2009; Rattanachaikunsopon and Phumkhachorn, 2009; Zaki et al., 2012; Abdelhamid and Soliman, 2012) and results showed an improvement of growth performance. Also, Yılmaz et al. (2013) concluded that cumin can be used as a growth promoter and as an antimicrobial agent during first feeding of Oreochromis mossambicus. Neem Azadirachta indica dietary

supplementation had little effect on Tilapia zillii species growth performance, compared to control variant (Jegede and Fagbenro, 2008). Positive effects of phyto-additives on growth and feed utilization can be explained in two ways; first that olfactory feed ingredients like spices (basil, caraway, etc.) enhance growth through their ability to act as feeding enhancers (El Dakar et al., 2007; Ahmad and Abdel-Tawwab, 2011). The second explanation may be because it contains vital compounds like aromatic oils, essential fatty acids, vitamins, minerals, etc. (Azeez, 2008), and these compounds may have important effects on growth. Daniel et al. (2011) reported the presence of high concentrations of potassium, K, (28770 mg/kg), calcium, Ca, (17460 mg/kg) and appreciable concentrations of sodium, Na, (280 mg/kg) and magnesium, Mg, (266 mg/kg) in basil O. basilicum. It is therefore concluded that, basil contains bioactive compounds and minerals that could enhance the curative process of health. Also research in animals indicates that some phyto-additives stimulate digestion with an enhanced bile acid concentration and stimulated the pancreas and increased secretion of digestive enzyme activities (lipases, amylases and proteases) (Bhosale et al., 2010; Srinivasan, 2005) and this explanation coincided with the present results where serum amylase and lipase concentrations were increased by administration of basil in fish diets. Essa et al. (2010) showed a significant improvement of the growth and the digestive enzyme (amylase, protease and lipase) activities by the administration of probiotics to Nile tilapia diet as compared to the control. Zolfaghari and Firouzbakhsh (2013) stated that adding basil aqueous extract to rainbow trout diet may improve the positive physiological characteristics. Body chemical composition, in the present study; indicates a significant increase in fish protein content coinciding with a significant decrease in lipid content either in fish fed basil leaves (BL) or seeds (BS). Our results were parallel with Abdelhamid and Soliman (2012) results where they reported that using dried leaves of guava and camphor trees in tilapia diets significantly improved fish carcass composition (protein, ether extract, energy content). Al Basali and Mohamad (2010) recorded an increase of total protein content in common carp fed four herbal extracts namely; basil (O. basilicum), cinnamon (Cinnamomum zelanicum), walnut (Juglans regia) and peppermint (Mentha piperita). In general, adding spices and medicinal herbs such as garlic, onion, marjoram, caraway, basil, anise, fennel, licorice, black seeds and fenugreek to fish diets resulted in improvement of protein digestibility and energy retention (Sakr, 2003; El-Dakar et al., 2004a,b; El-Dakar, 2004 and ElDakar et al., 2008).

208 The present results show insignificant differences in HSI values among treatments meanwhile; considerable increase in VSI and RIL as fish fed basil seed. Intestine length is considered to be an indicator of diet (Kramer & Bryant, 1995) and, particularly in fishes, can be used for interspecific dietary comparisons (Karachle & Stergiou, 2010). Mohammad et al. (2012) recorded that, HSI of O. niloticus did not display any significant difference among the treatments by various S. cerevisiae levels, whereas, VSI fingerlings were significantly (P < 0.05) affected. However, Seung-Cheol et al. (2007) reported that when red sea bream, Pagrus major, was fed mixture of four herbs namely; Massa medicata, Crataegi fructus, Artemisia capillaries and Cnidium officinale no significant differences in HSI and VSI values were recorded comparing with the CTR group. Cholesterol is an essential structural component of animal cell membranes that is required to maintain both membrane structural integrity and fluidity. In the present study, an elevation in serum cholesterol levels in fish fed basil in different forms comparing with fish fed control diet was observed indicating that using of basil improved diet quality. Nakagawa et al. (1984) stated that serum cholesterol levels were lowered by various external factors as impaired diet quality in yellowtail. Also, in red sea bream reared in high density conditions, the level of plasma cholesterol was significantly lowered (Tanaka and Inoue, 2005). Gatlin (2007) demonstrated that a reduction in plasma total cholesterol level is linked to impaired disease resistance. As serum protein level is an important indicator of humoral defense system of fish the increased levels of total serum protein, in the present study, when basil was used in different forms, indicates better immunological status in fish. Dugenci et al. (2003) reported that plasma protein level was high in fish fed with 1% ginger added diet compared with the control group and concluded that plasmatic protein increased in the fish fed with plant extracts. Sado et al. (2014) reported that total plasmatic protein in juvenile pacu Piaractus mesopotamicus showed a significant effect by using mannan oligosaccharides (MOS) levels. Also, an increase in total plasmatic protein was observed in rainbow trout, Oncorhynchus mykiss fed with prebiotics for seven days (Siwick et al., 1994) and kutum, Rutillus frisii kutum fries fed with 3% dietary commercial prebiotic for 56 days (Yousefian et al., 2012). Also, plasma total protein content increased in all experimental groups compared to the control group in mixed use of thyme and fennel oils in rainbow trout (Gulec et al., 2013). Conclusion The present investigation showed a significant improvement of fish growth, feed utilization and digestive enzyme (amylase and lipase) activities by the administration of basil seeds to sea bream diet as compared to the control. The beneficial effects of using basil on fish growth appear to be associated with higher protein retention and lower lipid content. The results indicate that basil has some ability to activate the immune system of sea bream by increasing serum total protein. Basil seed supplementation to fish feeds appears to have beneficial effects. More research is necessary to evaluate basil supplementation in sea bream diet according to its digestibility, amino acid profile and content of anti-nutritional factors.

A.Y. El-Dakar et al. References A.O.A.C. Association of Official Analytical Chemists, 2000. International Official Methods of Analysis, 17th ed. Arlington, Virginia, USA. A.P.H.A. American Public Health Association, 1995. Standard Methods for Examination of Water and Waste Water, 18th edition. Washington D.C, USA, 769. Abbas, R.J., 2010. Effect of using fenugreek, parsley and sweet basil seeds as feed additives on the performance of broiler chickens. Int. J. Poult. Sci. 9 (3), 278–282. Abdelhamid, A.M., Soliman, A.A.A., 2012. Possibility of using dried leaves of guava and camphor trees in tilapia diets. J. Arabian Aquacult. Soc. 7 (1). Ahmad, M.H., Abdel-Tawwab, M., 2011. The use of caraway seed meal as a feed additive in fish diets: growth performance, feed utilization, and whole-body composition of Nile tilapia, Oreochromis niloticus (L.) fingerlings. Aquaculture 314 (1–4), 110–114. Al Basali, H., Mohamad, S., 2010. Immune response of common carp (Cyprinus carpio) fed with herbal immunostimulants diets. J. Anim. Vet. Adv. 9 (13), 1839–1847. http://dx.doi.org/ 10.3923/javaa.2010.1839.1847. Al-Hussini, A.H., 1947. In: The Feeding Habits and the Morphology of the Alimentary Tract of Some Teleosts Living in the Neighborhood of the Marine Biological Station, Hurghada, Red Sea, vol. 5. Publications of the Marine Biological Station, Hurghada (Red Sea), pp. 1–61, ISSN 0370–0534. Amirkhani, N., Firouzbakhsh, F., 2013. Protective effects of basil (Ocimum basilicum) ethanolic extract supplementation diets against experimental Aeromonas hydrophila infection in common carp (Cyprinuscarpio). Aquac. Res. 2013, 1–9. http://dx.doi.org/10.1111/ are.12217. Azeez, S., 2008. Cumin. In: Parthasarathy, V.A., Chempakam, B., Zachariah, T.J. (Eds.), Chemistry of Spices. CAB Int., Oxford Shire, pp. 242–259. Ballestrazzi, R., Lanari, D., Agaro, E.D., Mono, A., 1994. The effect of dietary protein levels and source on growth, body composition, total amino acid and reactive phosphate excretion of growing sea bass. Aquaculture 127, 197–206. Bhosale, S.V., Bhilave, M.P., Nadaf, S.B., 2010. Formulation of fish feed using ingredients from plant sources. Res. J. Agric. Sci. 1 (3), 284–287. Bihari, G.C., Manaswini, B., Prabhat, J., Kumar, T.S., 2011. Pharmacognostical and phytochemical investigation of various tulsi plants available in south eastern odisha. Int. J. Res. Pharm. Biomed. Sci. 2 (2), 605–610. Busacker, G.P., Adelman, I.R., Goolish, E.M., 1990. Growth. In: Schreck, C.B., Moyle, P.B. (Eds.), Methods for Fish Biology. American Fisheries Society, Bethesda, Maryland, pp. 363–387. Daniel, V.N., Daniang, I.E., Nimyel, N.D., 2011. Phytochemical analysis and mineral elements composition of Ocimum basilicum obtained in Jos Metropolis, Plateau State, Nigeria. Int. J. Eng. Technol. IJET-IJENS 11 (06), 135–137. Diab, A.S., Aly, S.M., John, G., AbdeHadi, Y., Mohammed, M.F., 2008. Effect of garlic, black seed and Biogen as immunostimulants on the growth and survival of Nile tilapia, Oreochromis niloticus (Teleostei: Cichlidae) and their response to artificial infection with Pseudomonas fluorescens. Afr. J. Aquat. Sci. 33 (1), 63–68. Dugenci, S.K., Arda, N., Candan, A., 2003. Some medicinal plants as immunostimulant for fish. J. Ethnopharmacol. 88 (1), 99–106. El-Dakar, A.Y., 2004. Growth response of hybrid tilapia, Oreochromis niloticus, Oreochromis auraus to diets supplemented to different levels of caraway seeds. Agric. Sci Mansoura Univ. 29, 6083–6094. El Dakar, A.Y., Shalaby, S.M., Saoud, I.P., 2007. Assessing the use of a dietary probiotic/prebiotic as an enhancer of spinefoot rabbitfish

Basil supplementation in Gilthead sea bream diet Siganus rivulatus survival and growth. Aquac. Nutr. 13 (6), 407– 412. El-Dakar, A.Y., Hassanien, G.D., Gad, S.S., Sakr, S.E., 2008. Use of dried basil leaves as a feeding attractant for hybrid tilapia, Oreochromis niloticus · Oreochromis aureus, fingerlings. Mediterr. Aquacult. J. 1 (1), 35–44. El-Dakar, A.Y., Hassanien, G.D.I., Gad, S.S., Sakr, S.E., 2004a. Use of medical and aromatic plants in fish diets: I. Effect of dried marjoram leaves on performance of hybrid tilapia Oreochromis niloticus,ÆOreochromis auraus, fingerlings. J. Egypt. Acad. Soc. Environ. Dev. (B. Aquacult.) 5, 67–83. El-Dakar, A.Y., Hassanien, G.D.I., Gad, S.S., Sakr, S.E., 2004b. Effect of dried basil leaves on performance of hybrid tilapia Oreochromis niloticus · Oreochromis auraus, fingerlings. In: 3rd Inter. Conf. on Animal Production and Health in Semi-Arid Areas. Suez Canal University, pp. 265–277. El-Dakar, A.Y., Shalaby, S.M.M., Abd Elmonem, A.I., Wahbi, O.M., 2004c. Enhancement of performance using fennel seeds meal as feed additive for Nile tilapia Oreochromis niloticus. J. Egypt. Acad. Soc. Environ. Dev. (B. Aquacult.) 5, 43–67. Essa, M.A., El-Serafy, S.S., El-Ezabi, M.M., Daboor, S.M., Esmael, N.A., Lall, S.P., 2010. Effect of different dietary probiotics on growth, feed utilization and digestive enzymes activities of Nile Tilapia, Oreochromis niloticus. J. Arabian Aquacult. Soc. 5 (2), 143–161. Fulton, T.W., 1904. The rate of growth of fishes. Fish. Bd. Scotland 22, 141–211. Gatlin, D.M., 2007. Dietary Supplements for the Health and Quality of Cultured Fish by CAB International 2007. Printed and bound in the UK by Cromwell Press, Trowbridge, ISSN: 9781845931995. Gulec, A.K., Danabas, D., Ural, M., Seker, E., Arslan, A., Serdar, O., 2013. Effect of mixed use of thyme and fennel oils on biochemical properties and electrolytes in rainbow trout as a response to Yersinia ruckeri infection. Acta Vet. BRNO 82, 297–302. http:// dx.doi.org/10.2754/avb201382030297. Gutierrez, J., Barry-Ryan, C., Bourke, P., 2008. The antimicrobial efficacy of plant essential oil combinations and interactions with food ingredients. Int. J. Food Microbiol. 124, 91–97. Hussain, A.I., Farooq Anwar, F., Sherazi, S.T., Przybylski, R., 2008. Chemical composition, antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depend on seasonal variations. Food Chem. 108 (3), 986–995. Immanuel, G., Vicncybai, V.C., Sivaram, V., Palavesam, A., Marian, M.P., 2004. Effect of butanolic extracts from terrestrial herbs and seaweed on the survival, growth and pathogen (Vibrio parahaemolyticus) load on shrimp Penaes indicus juveniles. Aquaculture 236, 53–65, J. Egypt. Acad. Soc. Environ. Dev. (B. Aquacult.), 5:43–67. Jegede, T., Fagbenro, O., 2008. Dietary neem (Azadirachta indica) leaf meal as reproduction inhibitor in red belly tilapia, Tilapia zillii. In: 8th International Symposium on Tilapia in Aquaculture, pp. 365– 373. Karachle, P.K., Stergiou, K.I., 2010. Intestine morphometrics: compilation and analysis of bibliographic data. Acta Ichthyologica et Piscatoria 40 (1), 45–54, ISSN 0137–1592. Khalafalla, M.E., 2009. Utilization of some medical plants as feed additives for Nile Tilapia, Oreochromis niloticus, feeds. Mediterr. Aquacult. J. 2 (2), 10–19. Khare, C.P., 2007. Indian medicinal plants, an illustrated dictionary. Springer India (P) Ltd., New Delhi, pp. 442–444. Kim, D.S., Kim, J.H., Jeong, C.H., Lee, S.Y., Lee, S.M., Moon, Y.B., 1998a. Utilization of obosan (dietary herbs). I. Effects on survival, feed conversion ratio and condition factor in olive flounder Paralichthys olivaceus. J. Aquacult. 7, 159–164. Kim, D.S., Noh, C.H., Jung, S.W., Jo, J.Y., 1998b. Effect of Obosan supplemented diet on growth, feed conversion ratio and body composition of Nile tilapia, Oreochromis niloticus. J. Aquacult. 11, 83–90.

209 Kramer, D.L., Bryant, M.J., 1995. Intestine length in the fishes of a tropical stream: 1. Ontogenetic allometry. Environ. Biol. Fishes 42, 115–127, ISSN 0378–1909. Lee, S.M., Park, C.S., Kim, D.S., 2001. Effects of dietary herbs on growth and body composition of juvenile abalone, Haliotis discus hannai. J. Korean Fish. Soc. 34, 570–575. List, P.H., Ho¨rhammer, L., 1977. Hagers Handbuch der Pharmazeutischen Praxis, fourth ed. Springer Verlag BerlinHeidelberg, Germany, Band VI A. Makri, O., Kintzios, S., 2008. Ocimum spp. (basil): botany, cultivation, pharmaceutical properties, and biotechnology. J. Herbs Spices Med. Plants 13, 123–150. Mohammad, A.R., Mohammad, Z., Vahid, Y., Mohammad, M.S., 2012. Effect of different levels of dietary supplementation of Saccharomyces cerevisiae on growth performance, feed utilization and body biochemic al composition of Nile Tilapia (Oreochromis niloticus) fingerlings. J. Persian Gulf (Mar. Sci.) 3 (9), 15–24. Mostafa, A.A.Z.M., Ahmad, M.H., Mousallamy, A., Samir, A., 2009. Effect of using dried fenugreek seeds as natural feed additives on growth performance, feed utilization, whole-body composition and entropathogenic Aeromonas hydrophila – challenge of monosex Nile Tilapia O. niloticus (L) fingerlings. Aust. J. Basic Appl. Sci. 3 (2), 1234–1245. Nakagawa, H., Kumai, H., Nakamura, M., Nanba, K., Kasahara, S., 1984. Diagnostic studies on disease of sardine-fed yellowtail. Bull. Jpn. Soc. Sci. Fish. 50 (5), 775–782. NRC (National Research Council), 1993. Nutrient Requirements of Fish. National Academy Press, Washington, DC. Rattanachaikunsopon, P., Phumkhachorn, P., 2009. Protective effect of clove oil-supplemented fish diets on experimental Lactococcus garvieae infection in tilapia. Biosci. Biotech. Biochem. 73, 2085– 2089. Ricker, W.E., 1979. Growth rates and models in fish physiology. In: Hoar, W.S., Randall, D.J., Brett, J.R. (Eds.), vol. 8. Academic Press, New York, pp. 678–738. Sado, R.Y., Bicudo, A.J.A., Cyrino, J.E.P., 2014. Hematology of juvenile pacu, Piaractus mesopotamicus (Holmberg, 1887) fed graded levels of mannan oligosaccharides (MOS). Lat. Am. J Aquat. Res. 42 (1), 30–39, http://dx.doi.org/103856/vol42-issue1fulltext-3. Sajjadi, S.E., 2006. Analysis of the essential oils of two cultivated basil (Ocimum basilicum L.) from Iran. Daru 14 (3), 128–130. Sakr, S.E., 2003. Studies on the feeding attractants for fish (MSc. thesis). Faculty of Environmental & Agriculture Science, Suez Canal University, El-Arish, Egypt. Seung-Cheol, J.I., Osamu, T., Gwan-Sik, J., Si-Woo, L., Katsuya, I., Manabu, S., Kenji, T., 2007. Dietary medicinal herbs improve growth and some non-specific immunity of red sea bream Pagrus major. Fish. Sci. 73, 63–69. Shalaby, S.M.M., AbdElmonem, A.I., El-Dakar, A.Y., 2003. Enhancement of growth performance, feed and nutrient utilization, of Nile tilapia, Oreochromis niloticus, using of licorice roots (Erksous) as a feed attractive. J. Egypt. Acad. Soc. Environ. Dev. (B. Aquacult.) 4, 119–142. Sivaram, V., Babu, M.M., Immanuel, G., Murugadass, S., Citarasu, T., Marian, M.P., 2004. Growth and immune response of juvenile greasy groupers (Epinephelu stauvina) fed with herbal antibacterial active principle supplemented diets against Vibrio harveyi infections. Aquaculture 237, 9–20. Siwick, A.K., Anderson, D.P., Rumsey, G.L., 1994. Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Vet. Immunol. Immunop. 41, 125–139. SPSS, 1997. Statistical package for the social sciences, Versions16. SPSS in Ch, Chi-USA. Srinivasan, K., 2005. Spices as influencers of body metabolism: an overview of three decades of research. Food Res. Int. 38 (1), 77–86.

210 Tanaka, S., Inoue, M., 2005. Effectiveness of low rearing density against red sea bream iridoviral disease in red sea bream Pagrus major. Fish Pathol. 40, 181–186. Telci, I., Bayram, E., Yilmaz, G., Avci, B., 2006. Variability in essential oil composition of Turkish basils (Ocimum basilicum L.). Biochem. Syst. Ecol. 34, 489–497. Tomar, U.S., Daniel, V., Shrivastava, K., Panwar, M.S., Pant, P., 2010. Comparative evaluation and antimicrobial activity of Ocimum basilicum I Linn (Labiatae). J. Global Pharma Technol. 2 (5), 49–53. Ylmaz, S., Ergu¨n, S., C¸elik, E.S ß ., 2012. Effects of herbal supplements on growth performance of sea bass (Dicentrarchus labrax): change in body composition and some blood parameters. J. BioSci. Biotech. 1 (3), 217–222. Yılmaz, S., Ergu¨n, S., Soytasß, N., 2013. Dietary supplementation of cumin (Cuminum cyminum) preventing streptococcal disease during

A.Y. El-Dakar et al. first-feeding of Mozambique tilapia (Oreochromis mossambicus). J. BioSci. Biotech. 2 (2), 117–124. Yousefian, M., Hedayatifard, M., Fahimi, S., Shikholeslami, M., Irani, M., mirinia, C., Mousavi, S.E., 2012. Effect of prebiotic supplementation on growth performance and serum biochemica parameters of kutum (Rutillus frisiikutum) fries. Asian J. Anim. Vet. Adv. 7, 684–692. Zaki, M.A., Labib, E.M., Nour, A.M., Tonsy, H.D., Mahmoud, S.H., 2012. Effect of some medicinal plants diet on mono sex Nile tilapia (Oreochromis niloticus), growth performance, feed utilization and physiological parameters. Asia-Pac. Chem., Biol. Environ. Eng. Soc., 220–227 Zolfaghari, A., Firouzbakhsh, F., 2013. The effect of Basil (Ocimum basilicum) aqueous extract on growth changes, hematology and serum biochemical parameters in rainbow trout (Oncorhynchus mykiss). J. Vet. Res. 68 (4), 397–404.