Preliminary assessment of dietary supplementation of Sangrovit® on red tilapia (Oreochromis niloticus) growth performance and health

Preliminary assessment of dietary supplementation of Sangrovit® on red tilapia (Oreochromis niloticus) growth performance and health

Aquaculture 294 (2009) 118–122 Contents lists available at ScienceDirect Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / ...

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Aquaculture 294 (2009) 118–122

Contents lists available at ScienceDirect

Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Preliminary assessment of dietary supplementation of Sangrovit® on red tilapia (Oreochromis niloticus) growth performance and health Mark D. Rawling ⁎, Daniel L. Merrifield, Simon J. Davies Aquaculture and Fish Nutrition Research Group, School of Biological Sciences, University of Plymouth, Plymouth, Devon, PL4 8AA, UK

a r t i c l e

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Article history: Received 15 January 2009 Received in revised form 6 May 2009 Accepted 13 May 2009 Keywords: Nile tilapia Sanguinarine Alkaloid Sangrovit® Growth

a b s t r a c t A preliminary study was conducted to evaluate the effect of graded dietary supplementation of Sangrovit®, a commercial product containing the isoquinoline alkaloid sanguinarine, on red tilapia (Oreochromis niloticus) growth performance, feed utilisation, hepatic function, haematological parameters and gut microbiota. Compared to the control group (1.03 g fish− 1 day− 1), significant elevations in mean daily feed intake (1.19–1.25) were observed in fish fed Sangrovit® diets during the 60-day feeding period. Consequently, the specific growth rate (3.94–4.05% day− 1) and weight gain (66.80–71.85 g fish− 1) were significantly higher in the Sangrovit®-fed groups. With the exception of total leukocyte levels, which were elevated in fish fed Sangrovit®-supplemented diets, haematological and immunological parameters remained unaffected. Hepatic alanine aminotransferase activity and hepatosomatic index also remained unaffected in all fish groups. Compared to the control group, the allochthonous lactic acid bacteria (LAB) populations were lower in fish fed diets containing Sangrovit® at 75 and 100 mg kg− 1. The present study demonstrates that Sangrovit® had a positive effect on tilapia growth performance with no apparent effects on carcass composition, hepatic function and health status. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Since the European Union ratified a ban in 2006 for the use of all sub-therapeutic antibiotics as growth-promoting agents (Regulation 1831/2003/EC), alternatives to traditional antibiotics and ionophore anticoccidials in aquaculture practices have received much attention (Shinn et al., 2003; Bricknell and Dalmo, 2005; Balcázar et al., 2006). Such measures may also help to facilitate consumer perceptions of bio-security and eco-friendly fish farming. Over the last decade research has focused on the application of using plant extracts (i.e. phytobiotics) such as aromatic plants (e.g., ginger, curcuma, coriander) and herbal products (e.g. roots, leaves, bark), essential oils (e.g., hydro-distilled volatile plant compounds) and oleoresins (extracts based on non aqueous solvents) to replace antibiotic growth promoters in terrestrial animal feeds (Bostsoglou et al., 2002; Jamroz and Kamel, 2002; Alcicek et al., 2003; Athanasiadou and Kyriazakis, 2004; Christaki et al., 2004; Mao et al., 2005; Kommera et al., 2006; Peeter et al., 2006; Yuan et al., 2006; Vieira et al., 2008). More recently such applications have begun to demonstrate positive effects in feeds for various freshwater fish species including rainbow trout (Oncorhynchus mykiss; Dügenci et al., 2003), Indian major carp (Catla catla; Dey and Chandra, 1995), Mozambique tilapia (Oreochromis mossambicus; Logambal et al., 2000) and Nile tilapia (Oreochromis niloticus; Yin et al., 2006; Francis et al., 2005; Ardó et al., 2008).

⁎ Corresponding author. School of Biological Sciences, University of Plymouth, Devon, UK. Tel.: +44 1752 232900; fax: +44 1752 232970. E-mail address: [email protected] (M.D. Rawling). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.05.005

Isoquinoline alkaloids represent one of the largest and most interesting groups of plant secondary metabolites as potential alternative growth promoters (Faddejeva and Belyaeva, 1997). Sanguinarine, an isoquinoline alkaloid that belongs to a group called benzo[c] phenanthridine alkaloids (QBA), has been reported to display a number of useful medicinal properties including antimicrobial (Simanek, 1985; Facchini, 2001), anti-inflammatory (Lenfeld et al., 1981) and immunomodulatory (Chaturvedi et al., 1997). Sanguinarine has also been reported to promote animal growth by increasing feed intake and decreasing amino acid degradation from decarboxylation (Lenfeld et al., 1981; Drsata et al., 1996; Kosina et al., 2004). Selected chirally alkaloids including sanguinarine are part of a commercial product: Sangrovit®, which has been investigated as a dietary supplement for swine and poultry (Tschirner et al., 2003, Vieira et al., 2008). To the authors' knowledge there is no literature to date concerning the use of alkaloid derivatives in diets for Nile tilapia. The aim of the current investigation was therefore to assess the dietary inclusion of Sangrovit® on the growth performance, feed utilisation, hepatic function, gut microbiota and haematological parameters of red tilapia (O. niloticus). 2. Materials and methods 2.1. Fish and experimental protocol The experiments were carried out at the Aquaculture and Fish Nutrition Research Aquarium, University of Plymouth, UK. Nile tilapia (O. niloticus) fry were obtained from the Institute of Aquaculture,

M.D. Rawling et al. / Aquaculture 294 (2009) 118–122

University of Stirling, UK. After 6 weeks of acclimation, 384 fish (6.8 ± 0.2 g) were randomly distributed into 10 × 150-l (32 fish per tank) fibreglass tanks containing aerated recirculated freshwater. Fish were fed to apparent satiation three times a day after the procedures of Riche et al. (2004) for 60 days. Fish were batch weighed on a weekly basis following a 24-h starvation period and reared at 28 ± 1 °C with a 12:12 h light:dark photoperiod. Water pH was maintained between 6.8 and 7.5, dissolved oxygen between 7.5 and 8 mg l− 1, ammonium between 0.04 and 0.08 mg l− 1, nitrite between 0.02 and 0.06 mg l− 1 and nitrate between 54 and 58 mg l− 1.

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weight (g), IW = initial weight (g), T = duration of feeding (days), WG = wet weight gain (g), FI = total feed intake after 60 days of feeding (g), PI = protein ingested (g) and FL = final length (cm). Twelve fish were sampled at the beginning of the trial and three fish per tank were sampled to determine carcass composition. Fish were dried in an air oven at 105 °C until a constant weight was achieved to determine moisture content. Dried fish were then pooled (three fish per sample) and ground for composition analysis according to AOAC (1995) protocols, all samples were analyzed in triplicate. 2.4. Blood parameters

2.2. Experimental diets Five isonitrogenous and isolipidic diets were formulated (Table 1). The basal diet served as a control diet (diet C). Four remaining diets consisted of the basal diet with varying inclusion levels of Sangrovit® (Phytobiotics Gmbh, Etville, Germany): 25 mg kg− 1 (diet 25S), 50 mg kg− 1 (diet 50S), 75 mg kg− 1 (diet 75S) and 100 mg kg− 1 (diet 100S). Each diet was produced by mechanically stirring the ingredients into a homogenous mixture using a Hobart food mixer (Hobart Food Equipment, Australia, model no: HL1400-10STDA mixer). Warm water was added to reach a consistency suitable for cold extrusion to form 1-mm pellets (PTM Extruder system, model P6, Plymouth, UK). Diets were dried in a hot air oven at 45 °C for 48 h. 2.3. Performance calculations and proximate analysis Growth performance and feed utilisation were assessed by net weight gain (NWG), specific growth rate (SGR), feed conversion ratio (FCR), apparent protein utilisation (APU), protein efficiency ratio (PER), condition factor (K) and mean daily feed intake (MDI). Calculations were made using the following formulae: WG (g fish− 1) = FW − IW; SGR (%) = 100((lnFW − lnIW) /T); MDI (g fish− 1 day− 1) = FI / T / No. of fish per tank; FCR = FI / WG; PER = WG / PI; K = FW / (FL3); APU (%) = 100 (increase in carcass protein (g) / protein fed (g)). Where FW = final

At the end of the growth trial five fish per tank were anaesthetized with tricane methanesulfate (MS222) at 150 mg l− 1. Blood was sampled from the caudal vein using a 25-gauge needle and 1-ml syringe. Subsamples of whole blood were left to clot for a period of 12 h (at 4 °C) and then centrifuged at 3600 ×g for 6 min to recover serum. Serum was removed and stored at − 80 °C until analysis of glucose content and lysozyme activity. Haematocrit was determined by the microhaematocrit method as described by Brown (1988) and reported as percentage packed cell volume (% PCV). Leukocyte and erythrocyte counts were determined by diluting whole blood in Dacies solution (1/50 dilution) and enumeration in a haemacytometer. Haemoglobin was determined based on Drabkin's cyanide–ferricyanide solution (Sigma-Aldrich Ltd, Dorset, UK). Serum glucose was determined by the glucose oxidase–peroxidase test (Trinder, 1969). Serum glucose samples were incubated at room temperature (~20 °C) for 15 min; the resulting supernatant was cooled in cold water (2–4 °C) and measured spectrophotometrically at 505 nm. Serum lysozyme assay was based upon the lysis of Micrococcus lysodeikticus (Ellis, 1990). Briefly, 50 μl of serum was added to 950 μl of M. lysodeikticus; at a concentration of 200 mg ml− 1 in 0.05 M Na2HPO4 (pH 6.2). After mixing, the reduction in turbidity was measured between 0.5 and 4.5 min at 530 nm at 22 °C. One unit of lysozyme activity was defined as a decrease in absorbance of 0.001 unit min− 1. 2.5. Hepatosomatic index, viscerosomatic index and liver alanine aminotransferase

Table 1 Formulation of experimental diets.

Ingredients Herring meal LT92a Corn starchb Lysamine pea proteinc Glutalys (maize)d Fish oile Sunflower oil Vitamin/mineral premixf Barox plus liquid (antioxidant) Sangrovit®g (mg kg− 1)

C

25S

50S

75S

100S

300.00 365.01 164.74 100.00 30.00 17.75 20.00 0.50 –

300.00 365.01 164.74 100.00 30.00 17.75 20.00 0.50 25

300.00 365.01 164.74 100.00 30.00 17.75 20.00 0.50 50

300.00 365.01 164.74 100.00 30.00 17.75 20.00 0.50 75

300.00 365.01 164.74 100.00 30.00 17.75 20.00 0.50 100

94.5 38.1 8.8 7.0 18.7

92.5 37.6 8.8 6.7 19.5

94.6 39.0 8.5 6.6 18.8

93.3 39.6 8.6 6.7 20.6

Proximate analysis (% dry matter basis) Dry matter (%) 93.7 Crude protein (%) 37.8 Crude lipid (%) 8.6 Ash (%) 6.9 Gross energy (MJ kg− 1) 20.4

Each ingredient component is expressed as g kg− 1 per diet. Dietary codes: C = control group; 25S = 25 mg kg− 1; 50S = 50 mg kg− 1; 75S = 75 mg kg− 1; 100S = 100 mg kg− 1. a Herring meal: United fish products, Aberdeen, Scotland, UK. b Corn starch: Sigma-Aldrich Ltd, UK. c Lysamine pea protein: Roquette Frêres, France. d Glutalys (maize): Roquette Frêres, France. e Epanoil: Sevenseas, UK, Ltd. f Premier nutrition vitamin/mineral premix contains: 121 g kg calcium, Vit A 1.000 μg kg, Vit D3 0.100 µg kg, Vit E (as alpha tocopherol acetate) 7.0 g kg, Copper (as cupric sulphate) 250.0 mg kg, Magnesium 15.6 g kg, Phosphorous 5.2 g kg. g Sangrovit®: source: selected poppy seeds, selected chirally structured alkaloids of botanical origin e.g. sanguinarine, phytobiotics feed additives Gmbh, Etville, Germany.

At the end of the trial three fish per tank were euthanized with tricane methanesulfate (MS222) at 150 mg l− 1 followed by destruction of the brain. Subsequently liver samples were removed, immediately frozen in liquid nitrogen and stored at −80 °C until analysis of enzymatic activities. The same three fish were used to record vicera and whole body weight to determine the hepatosomatic (HSI) and viscerosomatic (VSI) indexes. Calculations were made on a wt./wt. basis using the following formulae: HSI = (LW / BW)× 100; VSI: (VW / BW) × 100. Where LW = liver weight, VW = viscera weight, BW = body weight. In order to measure alanine aminotransferase activity (ALAT EC 2.6.1.2) liver samples were homogenized (dilution 1/10) in ice cold buffer (30 mM HEPES, 0.25 mM saccharose, 0.5 mM EDTA, 5 mM K2HPO4, 1 mM dithiothreitol, pH 7.4) after Enes et al. (2008). After 30 min centrifugation (1500 ×g) the resultant supernatant was removed and stored at −80 °C for enzymatic analysis. The ALAT activity was assayed using a commercial kit (ALAT/GPT, ref: G8255, Sigma-Aldrich). Enzyme activity was measured at 340 nm at 28 °C (pH 7.4). Liver protein concentration was determined according to Bradford (1976) using bovine serum albumin (Sigma-Aldrich) as a standard. Enzyme activity was expressed per mg of hepatic protein and one unit of enzyme activity was defined as the amount of enzyme that catalyzed the hydrolysis of 1 µmol of substrate per minute at assay temperature. 2.6. Enumeration of intestinal microbiota Six fish per tank were sampled in order to enumerate the intestinal microbiota. The gastrointestinal tract in its entirety was removed

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Table 2 Growth performance, hepatosomatic (HSI) and viscerosomatic (VI) indexes of red tilapia after 10 weeks of feeding on experimental diets. Parameters

C

25S

50S

75S

100S

P-value

Initial body weight (g fish− 1) Final body weight (g fish− 1) Weight gain (g fish− 1) Mean daily feed intake (g fish− 1day− 1) Condition factor (K) Apparent protein utilisation (%) Protein efficiency ratio (PER) Specific growth rate (% day− 1) Feed conversion ratio (g g− 1) VSI (%) HSI (%)

6.80 ± 0.10 57.80 ± 1.98a 51.00 ± 1.84a 1.03 ± 0.03a 1.82 ± 0.18 49.15 ± 1.84 2.59 ± 0.09 3.54 ± 0.06a 1.03 ± 0.05 8.34 ± 1.03 1.61 ± 0.28

6.77 ± 0.12 78.65 ± 9.12b 71.85 ± 8.98b 1.22 ± 0.12b 2.11 ± 0.30 50.75 ± 6.31 2.88 ± 0.36 4.05 ± 0.20b 1.11 ± 0.09 8.36 ± 1.33 1.48 ± 0.45

6.87 ± 0.06 74.70 ± 3.39b 67.85 ± 3.32b 1.25 ± 0.01b 2.01 ± 0.13 43.91 ± 2.16 2.59 ± 0.13 3.98 ± 0.08b 1.08 ± 0.03 9.03 ± 1.28 1.83 ± 0.20

6.83 ± 0.12 73.60 ± 2.12b 66.80 ± 1.98b 1.19 ± 0.01b 1.95 ± 0.19 48.15 ± 1.49 2.59 ± 0.08 3.94 ± 0.05b 1.08 ± 0.03 9.25 ± 1.21 1.56 ± 0.32

6.87 ± 0.15 74.65 ± 7.99b 67.70 ± 8.06b 1.24 ± 0.06b 1.91 ± 0.08 42.81 ± 4.96 2.59 ± 0.31 3.96 ± 0.18b 1.11 ± 0.07 9.08 ± 1.19 1.80 ± 0.37

0.675 0.043 0.049 0.050 0.145 0.312 0.653 0.047 0.690 0.557 0.329

Values expressed as means ± standard deviation (n = 2). Dietary codes: C = control group; 25S = 25 mg kg− 1; 50S = 50 mg kg− 1; 75S = 75 mg kg− 1; 100S = 100 mg kg− 1. ab Significant differences between groups are indicated by difference in superscript letters.

aseptically. Autochthonous and allochthonous microbial populations were isolated from the posterior intestinal tract as described by Merrifield et al. (2009). As inter-fish variation has been reported previously (Spanggaard et al., 2000; Liu et al., 2008) the material from three fish was pooled in order to reduce variation (Hovda et al., 2007; Merrifield et al., 2009), thus, yielding four samples per treatment. Samples were appropriately diluted with PBS and 100 µl was spread onto duplicate tryptone soy agar (TSA, Oxoid, Basingstoke, UK) plates to determine total aerobic heterotrophic populations and de Man, Rogosa, Sharpe plates (MRS, Oxoid UK) for lactic acid bacterial (LAB) populations. The plates were incubated at 28 °C in a humidity controlled incubator after Hu et al. (2007) and inspected regularly for up to 4 weeks. Colony forming units (CFU) g− 1 were calculated by counting colonies from statistically viable plates (i.e. plates containing 30–300 colonies) after Al-Harbi and Uddin (2004). 3. Statistical analysis All data are presented as means ± standard deviation (SD). Data was transformed where necessary and statistical analysis was conducted using SPSS statistics version 15 for windows (SPSS Inc., Chicago, Il, USA) and accepted at the P b 0.05 level. Data were analyzed using a one-way ANOVA. Significant differences between control and treatment groups were determined using post-hoc Fisher's LSD test. 4. Results and discussion 4.1. Growth, feed utilisation and carcass composition Growth performance and feed utilisation of tilapia after 60 days of feeding on experimental diets are presented in Table 2. A high growth

performance was observed in all groups; fish biomass increased by over 700% with FCR ≤ 1.0 and SGR N 3.5. SGR improved significantly from 3.54 ± 0.06% day− 1 in the control fed fish (C) to between 3.94 ± 0.05 and 4.05 ± 0.20% day− 1 in the Sangrovit® fed fish. Mean weight gain of all Sangrovit® fed fish (66.80–71.85 g fish− 1) was significantly greater than control fed fish (50.9 g fish− 1). Although there is no comparable data regarding Sangrovit® in fish studies, Vieira et al. (2008) reported a significant increase in growth performance of broilers fed Sangrovit® supplemented diets (25–50 mg kg− 1) for 42 days (P b 0.05). Even though the results seem to indicate a stimulatory effect of Sangrovit® on fish growth, gaps exist in the understanding of the mechanisms of action of Sangrovit® in fish and terrestrial farm animals. Sangrovit® is composed of a number of individual compounds including sanguinarine that are from alkaloid origin. Preliminary trials have demonstrated that sanguinarine as a pure extract fed to broilers (15–100 mg kg− 1) had no significant effect on growth (Kosina et al., 2004; Psotova et al., 2006). Further experimental results might clarify whether specific components possess higher potency as appetite enhancers in fish. Additionally future research in this area should concentrate on understanding the physiological mechanisms by which dietary Sangrovit® improves growth in tilapia. Previously, Drsata et al. (1996) identified that sanguinarine through the inhibition of the enzyme aromatic amino acid decarboxylase (AAD) plays an important role in governing the stimulus to feed (Udenfriend, 1964; Blundell et al., 1973; Fletcher 1988; Dourish et al., 1989; Stallone and Nicoladis, 1989). In the present study after 60 days of feeding on the experimental diets mean daily feed intake of all Sangrovit® fed groups was significantly higher (1.19 ± 0.01–1.25 ± 0.01 g fish− 1 day− 1) than control fed fish (1.03 ± 0.03). This suggests that the finding of Drsata et al. (1996) may also be applicable to fish;

Table 3 Haematological and immunological parameters of red tilapia after 10 weeks of feeding on experimental diets. Parameter

C

25S

50S

75S

100S

P-values

Haematocrit (% PCV) Haemoglobin (g dl− 1) RBC (106 µl) WBC (104 µl) MCV (fL) MCH(pg) MCHC (g dl− 1) Lysozyme activity (U ml− 1) Serum glucose (g dl− 1)

30.15 ± 2.7 7.46 ± 0.9 1.49 ± 0.2 1.61 ± 0.3a 208.31 ± 46.9 51.35 ± 11.4 24.80 ± 2.8 693.78 ± 122.8 112.90 ± 13.7

32.30 ± 3.2 7.26 ± 1.6 1.42 ± 0.3 3.93 ± 1.1c 233.81 ± 45.4 51.94 ± 12.2 22.55 ± 4.3 532.00 ± 180.1 138.90 ± 23.2

32.10 ± 3.4 7.68 ± 1.5 1.43 ± 0.2 4.45 ± 1.5c 227.09 ± 36.3 54.31 ± 12.7 23.40 ± 4.1 620.00 ± 193.6 144.20 ± 40.5

33.90 ± 3.1 7.98 ± 1.5 1.55 ± 0.2 4.25 ± 2.1c 221.51 ± 29.4 52.78 ± 13.7 23.63 ± 4.4 675.55 ± 142.1 123.10 ± 26.0

30.60 ± 5.4 7.17 ± 1.5 1.57 ± 0.3 3.35 ± 2.3b 196.59 ± 30.6 46.27 ± 9.4 24.06 ± 2.7 638.22 ± 144.3 132.90 ± 33.5

0.227 0.713 0.540 0.001 0.213 0.653 0.741 0.197 0.164

Values expressed as means ± standard deviation (n = 10). Dietary codes: C = control group; 25S = 25 mg kg− 1; 50S = 50 mg kg− 1; 75S = 75 mg kg− 1; 100S = 100 mg kg− 1. a–c Significant differences between groups are indicated by different superscript letters. RBC—Red Blood Cells. WBC—White Blood Cells. MCV—Mean Corpuscular Volume. MCH—Mean Corpuscular Haemoglobin. MCHC—Mean Corpuscular Haemoglobin Concentration.

M.D. Rawling et al. / Aquaculture 294 (2009) 118–122 Table 4 Red tilapia gastrointestinal total viable (TVC) and lactic acid bacteria (LAB) levels. Treatment

Sample

TVC

LAB count

C

D M D M D M D M D M

1.29 × 108 1.55 × 106 3.15 × 108 4.86 × 106 2.51 × 108 2.12 × 106 7.51 × 107 2.58 × 107 1.24 × 108 3.04 × 106

3.57 × 107a – 4.86 × 106a – 2.33 × 106ab – 4.29 × 105b – 1.35 × 106b –

25S 50S 75S 100S

Values expressed as mean CFU g− 1 for mucosal lining (M) and digesta (D) (n = 4). Dietary codes: C = control group; 25S = 0.025 g kg− 1; 50S = 0.050 g kg− 1; 75S = 0.075 g kg− 1; 100S = 0.100 g kg− 1. a–b Significant differences between groups are indicated by different superscript letters.

but this would require further research to confirm. The dietary inclusion of Sangrovit® had no effect on apparent protein utilisation and protein efficiency ratio, suggesting utilisation of available dietary protein was similar across all experimental fish. Despite increased growth of all fish fed Sangrovit®, the supplementation of Sangrovit® had no effect on proximate composition (data not shown). Additionally hepatic somatic (HSI) and visceral indices (VI) were not affected. The use of plant products in practical diets for fish is a very topical concept in aquaculture (for a review see Gatlin et al., 2007) and future studies should therefore look at using plant extracts as potential growth promoters as well as alternative protein sources.

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topic of great interest regarding fish gut microbiota (for reviews see Ringø and Gatesoupe, 1998; Balcázar et al., 2006; Gatesoupe, 2008); certain LAB species have demonstrated positive effects on fish health and growth as probiotics (Balcázar et al., 2006; Balcázar et al., 2007; Panigrahi et al., 2007; Merrifield et al., in press) and yet some LAB species are known to cause disease (Eldar et al., 1999; Gatesoupe, 2008). These findings suggest that dietary inclusion of Sangrovit® may affect the intestinal LAB populations of tilapia; further work should be conducted with molecular based analysis to identify specifically which species are affected. 5. Conclusion The present study demonstrated that low levels of Sangrovit® (25– 100 mg kg− 1) had a positive effect on tilapia growth performance with no apparent effects towards carcass composition, hepatic function or health status. The fact that mean daily feed intake was significantly higher in fish fed Sangrovit® supplemented diets compared to control and that feed utilisation was not significantly affected suggests that improved growth was likely to be due to improved appetite of fish fed diets containing Sangrovit®. As a result the practical implications of Sangrovit® inclusion in aquafeeds include the potential for increasing feed intake with no detrimental effect toward efficiency of feed utilisation of cultured fish. This would allow for faster fish growth leading to improved production time; however, research is required with different important aquaculture species and longer time scales to fully evaluate the value of including Sangrovit® at industrial farming levels.

4.2. Blood parameters There are a number of specific haematological parameters recognised as valuable tools for monitoring fish health and physiological responses to environmental stress (Bhaskar and Rao, 1984; Schuett et al., 1997; Jawad et al., 2004). Svobodova et al. (1991) suggested that icthyo-haematology is useful in the assessment of feed composition, nutritional status in relation to environmental conditions affecting fish. In the present study haematocrit, haemoglobin and erythrocyte levels were not affected by any of the experimental diets (Table 3). Serum lysozyme activity and serum glucose, often used as indicators of stress (Wendelaar Bonga,1997), also remained unaffected by the inclusion of Sangrovit® at all dietary levels. Compared to control fed fish (1.61 × 104 µl− 1) total leukocyte levels were significantly elevated in fish fed Sangrovit® (3.35–4.45 × 104 µl− 1). 4.3. Hepatic function Alanine aminotransferase (ALAT) is an important aminotransferase found in the livers of fish (Cowey and Walton, 1989; Fynn-Aikins et al., 1995). In the current study, liver ALAT activity of fish fed Sangrovit® was unaffected compared to control fed fish (data not shown). The present study suggests that the inclusion of Sangrovit® had no apparent effect on hepatic function. 4.4. Culturable intestinal microbiota The intestinal microbiota has been shown to be sensitive to dietary ingredients (Ringø et al., 2006a,b; Ringø et al., 2008; Merrifield et al., in press). Therefore, the present study included a preliminary investigation of the aerobic heterotrophic autochthonous and allochthonous populations found within the posterior intestine (Table 4). There were no significant differences of total viable populations between the groups. Despite this the allochthonous LAB populations of fish fed diets containing Sangrovit® at 75 and 100 mg kg− 1 were significantly lower than the control group. Autochthonous LAB levels were statistically too low to enumerate. LAB are currently a

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