Assessing the use of a dietary probiotic/prebiotic as an enhancer of spinefoot rabbitfish Siganus rivulatus survival and growth

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1 Aquaculture Nutrition ; Assessing the use of a dietary probiotic/prebiotic as an enhancer of spinefoot rabbitfish Siganus rivulatus survival and growth A.Y. EL-DAKAR 1, S.M. SHALABY 2 & I.P. SAOUD 3 1 Fish Resources and Aquaculture Department. Suez Canal University, El-Arish, Egypt; 2 Aquaculture Division, National Institute of Oceanography and Fisheries, Al-Anfoshy, Alexandria, Egypt; 3 Department of Biology, American University of Beirut, Beirut, Lebanon Abstract The use of prebiotics and probiotics as feed supplements that improve efficiency of intestinal bacteria is becoming de rigueur in animal husbandry in many regions worldwide. We tested the effects of a commercial probiotic (Biogen Ò ) containing allicin, high unit hydrolytic enzyme, Bacillus subtilis spores and ginseng extracts on survival, growth, carcass composition and feed cost/benefit in rabbitfish Siganus rivulatus. Fifteen net cages ( cm; L W H) were stocked with 10 juvenile rabbitfish (10.3 g per fish) each and placed in a large rectangular tank and offered feed at 4% body weight daily. Cages were offered one of five isonitrogenous and isocaloric diets containing 0, 1, 2, 3 and 4 g kg )1 probiotic at three replicates per treatment for 98 days. Fish in all cages were weighed at 2-week intervals and feed regimen was adjusted accordingly. Rabbitfish offered the control diet exhibited lower growth and feed utilization than all experimental treatments. There was no effect of probiotic inclusion level on survival but growth was better at all inclusion levels than in the control. No significant differences (P > 0.05) in growth were observed among fish groups fed various levels of the probiotic. Carcass composition was not affected by dietary probiotic inclusion. Ultimately, when all variables are considered, Biogen Ò inclusion to diets appears to reduce feed cost per unit growth of rabbitfish. KEY WORDS: cost/benefit analysis, growth, probiotic, rabbitfish, Siganus rivulatus Received 6 December 2006, accepted 27 March 2007 Correspondence: I. Patrick Saoud, Department of Biology, American University of Beirut, Bliss St., Beirut, Lebanon. is08@aub.edu.lb Introduction Rabbitfish aquaculture is established in various regions around the Indo-Pacific and has good potential in the Middle East (Stephanou & Georgiou 1999; Pillay & Kutty 2005). It is a herbivorous fish and can potentially be reared using diets without fishmeal. However, a perceived disadvantage of feeds without fishmeal is a reduction in digestibility and assimilation by the fish. Various studies showed that farmed fish performance might be ameliorated by using feed additives such as aromatic plant extracts including spices, digestive enzymes and probiotics. Spice and natural herbs such as marjoram, basil, licorice root, black seed and peppermint have been shown to be beneficial by Abd Elmonem et al. (2002), Sakr (2003), Shalaby et al. (2003) and El-Dakar et al. (2004a,b,c). Another class of feed additives to receive attention in aquaculture recently has been probiotics (live microbes that may improve intestinal microbial balance) (Gatesoupe 1999; Jrianto & Austin 2002). Evidence of the beneficial effects of probiotics gave birth to the concept of prebiotics (Teitelbaum & Walker 2002) which are defined by Gibson & Roberfroid (1995) as Ôa nondigestible food ingredient which beneficially affects the organism by selectively stimulating the growth of and/or activating the metabolism of one or a limited number of health promoting bacteria in the intestinal tract, thus improving the host s intestinal balanceõ. Examples of prebiotic use in aquaculture are limited and some information on prebiotic use in general is offered by Szilagyi (2002) and Teitelbaum & Walker (2002). Although scientists cited above have demonstrated beneficial effects of using probiotics or sometimes prebiotics as fish feed additives, information on the interaction among prebiotic spices, digestive enzymes and probiotics in fish diets is very scarce. In the present study various inclusion levels of a feed additive that contains a probiotic bacteria, digestive... Ó 2007 Blackwell Publishing Ltd 407

2 408 A. Y. El-Dakar et al. enzymes and prebiotic spices were used to evaluate the effect on siganid survival, growth, feed conversion, nutrient utilization, body composition and economics. Materials and methods Experimental system The present work was performed at the Mariculture Research Center, Faculty of Environmental Agricultural Sciences, Suez Canal University, El-Arish, North-Sinai, Egypt. Experimental fish were collected from the Mediterranean Sea coast at El-Arish and immediately transported in 50 L plastic buckets to a rectangular fibreglass tank filled with seawater. Fish were quarantined for 1 week in the rearing tank, and then sorted by weight removing all large and small individuals. The feeding trial included 15 net cages ( cm; L W H) placed in a large fibreglass tank using three replicate cages per treatment. Ten juvenile fish (average weight 10.3 g) were randomly stocked into each experimental cage. During the first 3 days of the experiment, dead fish were replaced with individuals of the same size. Approximately 35% of the water was exchanged daily and aeration was provided using submerged air diffusers and a reciprocating air blower. Salinity, temperature, ph, dissolved oxygen and photoperiod were measured daily and remained at approximately 34 g kg )1, 27 C, 8.9, 7 mg kg )1 and 12 : 12 L : D, respectively. Ammonia and nitrite nitrogen were measured periodically and remained below 0.2 mg L )1 due to daily water exchange. Diet preparation and feeding Experimental diets were prepared on site using locally available ingredients (Table 1). The recipe was designed to contain 30% crude protein and 7.7% lipids. Ingredients were thoroughly mixed then boiling water was added to the mixture. The mash was pelletized using a meat mincer with a 1.5-mm die. Pellets were dried at 40 C in an oven and stored at )20 C. The feed additive used was a commercial natural enhancer mix (NEM), Biogen Ò, ChinaWay Corporation, Taiwan, produced in Taiwan, which contains allicin (0.247 lg g )1 ), high unit hydrolytic enzyme (3690 lg g )1 ), Bacillus subtilis spores ( cells g )1 ) and ginseng extracts. Five experimental diets were produced, a control and four containing the NEM at 0, 1, 2, 3 and 4 g kg )1. Each diet was offered to three randomly chosen cages at 4% of biomass daily, 6 days a week, for 98 days. Daily ration was divided into three equal portions supplied at 9.00, and h. Table 1 Ingredients and proximate analysis of experimental diets Ingredients (g kg )1 ) All offered feed was consumed by the fish within 20 min. Fish were weighed at 2-week intervals and ration was adjusted according to new fish weight per cage. Sample collection and analysis Fish meal Soybean meal Wheat milling by-product Wheat bran Corn starch Biogen Ò Sunflower oil Fish oil Vitamin and mineral premix 1 Chemical composition Dry matter (g kg )1 ) (g kg )1 ) DM basis Crude protein Ether extract Crude fibre Nitrogen free extract Ash Calculated energy value GE (kj g )1 diet) DE (kj g )1 diet) P/E ratio (mg CP kj )1 DE) Eco Vit, Egyptian Veterinary products and feed additives Co., Demyatta, Egypt. The vitamin and premix provided the following per kg of experimental diet: IU, 0.7 g, IU, 2 mg, 2.5 mg, 2 mg, 10 mg, 3 mg, 5 mg, 2 mg, 2 mg, 5.5 mg, 200 g, 90 g, 40 g, 2.5 g, 48 g, 3.6 g, 23.5 g, 8 g, 450 mg, 200 mg and 20 mg of vitamin A, vitamin C (Stay C Ò, 35% active), vitamin D 3, vitamin E, vitamin B 2, vitamin K 3, nicotineamide, vitamin B 6, vitamin B 12, vitamin B 1, folic acid, Ca-D-pantothenate, calcium, phosphate, sodium, copper, magnesium, manganese, zinc, iron, cobalt, iodine and selenium, respectively. Fish and feed samples were taken at the beginning of the experiment and stored at )20 C pending composition analysis. At the end of the experiment, all fish were individually weighed and total length was measured. Five fish were randomly taken from each cage, killed and dissected. Livers and viscera were weighed to estimate hepatosomatic index (HSI). Viscera comprised of liver, digestive tract and intraperitoneal fat. Samples of carcass and viscera from each cage were pooled and stored at )20 C for subsequent proximate analysis. Chemical analyses of feed, viscera and fish were performed according to the methods described by AOAC (1990). Gross energy content of experimental diets and fish...

3 Dietary enhancer for rabbitfish 409 samples were calculated by using factors of 23.62, 39.5 and kj g )1 for protein, lipid and carbohydrate, respectively (NRC 1993). Digestible energy content was estimated from standard physiological fuel values as 16.72, and kj g )1 for protein, carbohydrate and lipid, respectively (Garling & Wilson 1976). Specific growth rate (SGR), feed conversion ratio (FCR), protein retention (PR), energy retention (ER), condition index (CI) and yield were calculated as: SGR ¼ 100 ðln FBW ln IBWÞ=t where FBW is final body weight (g), IBW is initial body weight (g) and t is time in days. FCR ¼ weight of feed offered to fish (g)=weight gain of fish (g) PR ¼½protein retained (g)=protein fed (g)š100 ER ¼ energy retained in fish (J)=energy retained in fish (J) 100 CI ¼ 100 ½body weight (g)=length 3 ðcmþš % Yield ¼ 100 ½gutted weight (g)=total weight (g)š Statistical analysis All statistical analyses were performed according to Sandecor & Cochran (1982) using MStat-C software (MStat-C 1988). Data were analysed using two-way analysis of variance to determine significant differences among treatment means. Differences among individual means were subjected to Duncan s multiple-range test. Results and discussion Dietary NEM supplement to fish feed appeared to provide beneficial effects on growth of rabbitfish, although not on survival. Results indicated that fish offered diets containing NEM exhibited greater growth than those offered the control diet (Table 2). There were no significant differences in growth (P > 0.05) among fish offered diets with various inclusion levels of NEM. Final body weight, SGR and FCR of fish offered feeds with NEM inclusion were superior to those of fish offered feed without NEM inclusion. However, the level of inclusion of NEM in the feed had little effect on growth or FCR. We thus assume that an inclusion of 1 g kg )1 is at or above minimum requirements. Similar results were observed by El-Haroun et al. (2006) who supplemented Biogen Ò in diets of Nile tilapia at concentrations much higher than those used in the present experiment but observed results comparable to those of present work. Improvement in growth and feed conversion of siganids might be attributed to the presence of any of the compounds in the NEM or to an interaction among these compounds. In addition to growth, the FCR, PR and ER of fish offered the NEM supplemented feed were significantly better (P < 0.05) than those offered the control diet. However, no significant differences (P > 0.05) were observed among fish groups offered feed with various levels of NEM inclusion. These results might be due to positive effect of Bacillus subtilis and Table 2 Performance of rabbitfish offered diets with various inclusion levels of natural enhancer mix Item PSE Initial weight (g per fish) Final weight (g per fish) 55.2 c 61.9 ab 64.0 a 62.3 ab 60.6 b 0.53 Weight gain (g) 44.8 c 51.5 b 53.8 a 52.0 ab 50.5 ab 0.54 SGR (% day )1 ) 1.7 b 1.8 a 1.9 a 1.8 a 1.9 a 0.08 Total length (cm per fish) Survival (%) 90 b 95 a 85 c 95 a 85 c 0.78 FCR 3.6 b 2.6 a 2.6 a 2.7 a 2.7 a 0.19 PR% 17.4 b 23.5 a 24.0 a 25.2 a 26.1 a 0.52 ER% 11.2 c 15.7 a 15.6 a 14.8 ab 14.7 a 0.40 % Yield HSI 2.9 c 3.9 a 3.9 a 3.4 b 3.2 bc 0.19 Condition index 2.3 b 2.5 a 2.5 a 2.3 ab 2.2 b 0.16 Values in the same row with the same superscript are not significantly different (P > 0.05). PSE, pooled standard error. % Yield ¼ 100 (gutted weight/body weight). HSI (hepatosomatic index) ¼ 100 (liver weight/body weight). Condition index ¼ 100 [body weight (g)/length 3 (cm)]....

4 410 A. Y. El-Dakar et al. spices (garlic and gingers) present in the NEM. Bacillus subtilis have been shown to produce digestive enzymes such as amylase, protease and lipase which enrich the concentration of intestinal digestive enzymes (Lee & Lee 1990). The bacteria could also have improved digestive activity via synthesis of vitamins and cofactors or via enzymatic improvement (Gatesoupe 1999). Gullian et al. (2004) demonstrated a significant growth increase in shrimp inoculated with Bacillus sp. whilst El-Dakar & Goher (2004) found that enhanced growth was generally obtained in shrimp fed diets with B. subtilis inclusion. Alternatively, improvement in growth and feed conversion efficiency may be attributed to the beneficial effect of garlic and ginger supplementation or to improved digestion due to the hydrolytic enzyme. El-Dakar et al. (2005) reported beneficial effects of spice supplementation to shrimp diets. These findings are in agreement with those obtained by Abd Elmonem et al. (2002), Sakr (2003), Shalaby et al. (2003), El-Dakar et al. (2004a,b,c) and Shalaby (2004). 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 2004; El-Dakar et al. 2004a,b), and in enhancement in growth and feed conversion (Abd Elmonem et al. 2002; Shalaby et al. 2003; El-Dakar et al. 2004a,b,c; Shalaby 2004). Spice supplementation to fish feeds appears to have beneficial effects and warrants further investigation. Biogen Ò supplementation to diets resulted in reduced FCR and improved PR and ER. El-Haroun et al. (2006) found similar effects of Biogen Ò supplementation on feed utilization by Nile tilapia. Although results among experiments can only be compared when experimental protocols are identical and the only variable is fish species, results of the present experiment and those of El-Haroun et al. (2006) suggest that Biogen Ò supplementation to feeds can reduce fish production cost by improving feed utilization. Although growth rate, FCR, PR and ER were improved by NEM supplementation, the level at which NEM was added to the diet had no effect. On the other hand, per cent yield was not affected by dietary NEM supplementation. Although experimental fish grew faster than controls, the ratio of body mass to guts remained similar. This fact is perplexing as liver weight varied with level of NEM supplementation and livers were part of viscera. The only explanation we have is that gut weight decreased when liver weight increased. Liver weight as a proportion of body weight was lowest in the control, increased with low additions of NEM, but then decreased again as NEM levels in the diet were Table 3 Composition of carcass and viscera of fish fed different levels of tested natural enhancer mixture Item PSE Carcass composition Moisture (g kg )1 ) 668 b 666 bc 676 a 650 d 663 c 2.7 Crude protein 169 bc 166 c 169 b 183 a 171 bc 2.4 Ether extract 80 a 85 a 81 a 72 b 68 b 2.3 Ash 65 b 71 a 65 b 71 a 63 b 1.9 Viscera composition (g k g )1 on DM basis) Dry mater 780 d 831 c 857 b 863 b 884 a 3.7 Crude protein* 114 a 110 a 67 c 100 ab 84 bc 5.0 Ether extract* Ash* 77 c 120 a 112 ab 887 c 962 bc 4.6 Values in the same row with the same superscript are not significantly different (*P > 0.05). PSE, pooled standard error. increased. We have no biological explanation for such results and attribute them to chance. The CI of the fish offered low levels of NEM was greater than CI of controls which had a CI equal to that of fish offered diets with high levels of NEM. These results reflect the small differences in weight but no differences in length of the harvested fish. However, these results also mirror the HSI results, indicating that although CI is generally thought of as a health indicator, a large CI as in the present work could be a reflection of an enlarged liver. More research is needed before HSI can be correlated to CI of the fish. Data of the carcass composition of rabbitfish offered diets with various levels of NEM as a natural growth promoter are given in Table 3. Although significant differences in moisture level and crude protein proportion are apparent among treatments, they do not follow a trend and thus suggest that variations among results are probably due to inherent variation associated with using wild undomesticated gene stock in research. However, results suggest a positive correlation between NEM supplementation and lipid concentration of the carcass. Similar results were observed when spices were used in graded levels in fish diets, e.g. black seed and roquette seed by-products (Abd Elmonem et al. 2002); licorice root meal (Shalaby et al. 2003); fennel seed meal (El-Dakar et al. 2004c). Conversely, El-Haroun et al. (2006) found no effect of NEM supplementation to tilapia feed on any of the carcass proximate composition parameters. Viscera composition was affected by graded levels of NEM supplementation to rabbitfish diets. Moisture levels in the viscera decreased with an increase in NEM levels in the diets. On the other hand, viscera fat content did not change as NEM level increased. It is noteworthy to state that viscera of aquacultured rabbitfish tend to be very high in lipids...

5 Dietary enhancer for rabbitfish 411 Table 4 Cost benefit analysis of fish fed different levels of test natural enhancer mixture Item MSE Cost per kg diet Incidence cost a 5.4 b 5.4 b 5.7 b 5.8 b 0.20 Change in IC Profit index b 1.9 a 1.9 a 1.8 a 1.7 a 0.05 Change in PI Values in the same row with the same superscript are not significantly different (P > 0.05). MSE, mean standard error. Cost of fishmeal, soybean meal, wheat milling by-product, wheat bran, corn starch, Biogen Ò, sunflower oil, fish oil, vitamin and minerals premix per kg were 4.8, 2.2, 1.0, 0.75, 0.7, 35.0, 5.0, 5.0 and 8.5 LE, respectively. 1 Incidence cost ¼ feed cost consumed to produce 1 kg weight gain fish. 2 Profit index ¼ value of fish/cost of feed consumed, 1 kg fresh fish equals 10 LE (Egyptian Lira). compared with viscera of wild rabbitfish of the same size and age (I. Saoud, unpublished data). This may be due to the fact that rabbitfish is an algaevore in nature while under culture conditions it is offered feed with high protein and lipid content. We believe that rabbitfish should be offered a vegetable diet with low levels of oil supplementation. Incidence cost values of rabbitfish offered diets with 0, 1, 2, 3 and 4 g kg )1 NEM supplement were 5.81, 4.28, 4.35, 4.66 and 4.96 LE kg )1 gain, respectively (Table 4). These results indicated that dietary NEM supplementation would decrease feed costs by 73%, 73%, 77% and 78% for fish offered diets with 1, 2, 3 and 4 g kg )1 NEM, respectively. Profit index thus improved with NEM supplementation to the basal diet, but level of supplementation did not confer any additional economic benefit. Similar results were observed by El-Haroun et al. (2006) using the same brand of NEM feed supplement (Biogen Ò ) in the feed of tilapia Oreochromis niloticus. We conclude from the present research that dietary supplements can impart beneficial effects on rabbitfish growth and that translates into financial benefits for farmers by decreasing feed cost per unit growth of fish. Probiotics also help in disease resistance of tilapia (Shelby et al. 2006) and could be beneficial for rabbitfish. Future research will identify the benefits of the various probiotics and prebiotics within the NEM we used, and interactions amongst them. Rabbitfish are an algaevorous family of fish but that does not necessarily mean they can be reared on artificial diets with no fishmeal inclusion. However, the supplementation of NEM to vegetarian diets may improve digestibility and assimilation, thus making the diets suitable for siganid aquaculture. Research into the physiological effects of probiotic and prebiotic use is warranted. References Abd Elmonem, A., Shalaby, S.M.M. & El-Dakar, A.Y. 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