I N C O R P O R AT I N G f i s h far m ing t e c h no l og y

July | August 2013 FOCUS | Probiotics

International Aquafeed is published six times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. Copyright 2013 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

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FOCUS | PROBIOTICS

Effect of probiotic, Hydroyeast Aquaculture as growth promoter for adult Nile tilapia
by FF Khalil, Ahmed ismail Mehrim and Montaha E M Hassan, Al-Mansoura University, Egypt ormones, antibiotics, ionophers and some salts compounds have been used as growth promoters and to some extent to prevent diseases. However, their inadequate applications show a negative effect on aquaculture production and environment (Gngora, 1998). Functional additives, like probiotics, are a new concept in aquaculture (Li and Gatlin III, 2004) where the additions of microorganisms in diets show a positive effect on growth caused by the best use of carbohydrates, protein, and energy (Irianto and Austin, 2002). It further diminishes mortality by disease, antagonism to pathogens, and better microbial intestinal balance in the environment (Holmstrm et al., 2003).
table 1: Details of the experimental treatments Details t1 t2 t3 t4 t5 t6 t7 t8 Basal ration (Br)+ 0 g Hydroyeast aquaculture/Kg diet (as a control) Basal ration (Br)+ 5 g Hydroyeast aquaculture/Kg diet Basal ration (Br)+ 10 g Hydroyeast aquaculture/Kg diet Basal ration (Br)+ 15 g Hydroyeast aquaculture/Kg diet Basal ration (Br)+ 0 g Hydroyeast aquaculture/Kg diet (as a control) Basal ration (Br)+ 0 g Hydroyeast aquaculture/Kg diet (as a control) Basal ration (Br)+ 5 g Hydroyeast aquaculture/Kg diet Basal ration (Br)+ 10 g Hydroyeast aquaculture/Kg diet Yellow corn rice bran Soybean meal (44%) Fish meal (65%) Salts Calcium carbonate Vegetable oil Premix Di-nitro bio (anti oxident) Bintonite (as banding agent) CFU/kg min. 5,000,000,000,000 22,500,000,000 22,500,000,000 22,500,000,000 22,500,000,000 nutrient composition Dry matter (DM) Crude protein (CP) ether extract (ee) ash Crude fibre nitrogen free extract (nFe) Gross energy (Kcal/100g DM(Ge)* Protein energy (P/e) ratio (mg CP/ Kcal Ge) 88.18 25.10 7.90 7.30 6.00 54.60 440.94 56.92 22.50 23.00 37.50 6.00 0.50 4.67 3.00 0.30 0.025 2.50

The use of probiotics for farm animals has increased considerably over the last 15 years. Once ingested, the probiotic microorganisms can modulate the balance and activities of the gastrointestinal microbiota, whose role is fundamental to gut homeostasis. The most important benefits of yeast and bacterial probiotics upon the gastrointestinal microbial ecosystem in ruminants and monogastric animals (equines, pigs, poultry, fish) were reported in the recent scientific literature (Chaucheyras-Durand and Durand, 2010). Nowadays, a number of preparations of probiotics are commercially available and have been introduced to fish, shrimp and molluscan farming as feed additives, or are incorporated in pond water (Wang et al., 2005).

Why tilapia?
Tilapias are the most successfully cultured fish in the world because of their fast growing and high efficiency to utilize the natural and artificial supplemented feeds (Ishak, 1980). Tilapias have become increasingly popular for farming as they are able to reproduce rapidly, easily bred in captivity, tolerate a wide range of environmental conditions, are highly resistant to diseases, and most important of all, have good flavour. Though the fish originated in Africa, Asian countries have become the leading producers of these fish (Rana, 1997). Tilapias are second only to carps as the most widely farmed freshwater fish in the world (FAO, 2010).

table 3: Ingredients and proximate chemical analysis (% on dry matter basis) of the experimental basal diet Ingedients %

table 2: Formula of the tested probiotic, Hydroyeast aquaculture Ingredients oligosaccharides enzymes amylase Protease Cellulase Pectinase Xylanse Phytase 3,750,000 500,000 200,000 100,000 10,000 3,000 Units/kg min. 50,000 ppm Yeats probiotics active live yeast Probiotics lactobacillius acidophlus Bifedobacterium longhum Bifedobactrium thermophylum Streptococcus faecium

26 | InternAtIonAl AquAFeed | July-August 2013

FOCUS | PROBIOTICS
table 4: effects of Hydroyeast aquaculture probiotic on growth performance of adult male O. niloticus

stock nutrition is recognised as a major facaDG Initial Final SGr tor that can aWG rGr (mg/ Sr (%) weight weight (%/d) fish/day) influence fish reproduction and subset1 81.0 117.6b 36.5c 45.1c 0.63c 0.64c 100.0 quent larval t2 82.4 137.2a 54.8ab 66.5a 0.94ab 0.88a 100.0 quality of t3 83.0 136.2a 48.9b 56.1b 0.84b 0.77b 100.0 many fish spet4 87.3 142.0a 58.9a 71.07a 1.01a 0.92a 100.0 cies (Izquierdo et al., 2001). Se 0.001 2.36 2.35 2.83 0.04 0.03 0.000 The developP-value 0.253 0.0004 0.0008 0.0008 0.0008 0.0008 0.526 ment of cost effective and table 5: effects of Hydroyeast aquaculture probiotic on growth performance of nutrient optiadult female O. niloticus mized brood aDG Initial Final SGr stock feeds for aWG rGr (mg/ Sr (%) weight weight (%/d) tilapia is both fish/day) pertinent and crucial. Yet, t5 75.4 105.3b 29.8b 39.6b 0.52b 0.57b 100.0 many studies t6 81.1 122.4a 41.3a 50.9a 0.71a 0.71a 100.0 have revealed the positive t7 83.0 126.1a 43.1a 51.9a 0.74a 0.72a 100.0 effects of t8 81.0 102.4b 21.4c 26.4c 0.37c 0.41c 100.0 probiotics on Se 0.002 1.43 1.42 1.73 0.02 0.02 0.000 growth perP-value 0.128 0.0001 0.0001 0.0001 0.0001 0.0001 0.466 formance in different O. niloticus stages such fry (Abdel-Tawwab et al., Food availability and quality are known to influence both fecundity and egg size in tilapia 2008; Lara-Flores et al., 2010; Abdelhamid et (Coward and Bromage, 2000). So, brood al., 2012; Abdel-Tawwab, 2012) and finger-

lings (Mehrim, 2009; Ghazalah et al., 2010; Khalafalla, 2010). However, no attempts were designed concerning the effects of probiotics on growth performance of adult fish. Therefore, the objectives of the present study were to evaluate the effects of graded levels of a new dietary probiotic Hydroyeast Aquaculture on both sexes of adult Nile tilapia, Oreochromis niloticus, concerning their growth performance, feed and nutrients utilization and carcass composition for eight weeks.

Experiment setup
This study was conducted in Fish Research Unit, Faculty of Agriculture, Mansoura University, Al-Dakahlia, Egypt. Both sexes of healthy adult Nile tilapia O. niloticus, with an average initial body weight (83.4 0.001 g) for adult males and (80.1 0.002 g) for adult females were purchased from Integrated Fish Farm at Al-Manzala, Al-Dakhalia, Egypt. Fish were stocked into rearing tanks for two weeks as an adaptation period, and fed on a basal diet during this period. Fish in both sexes (males and females), were distributed separately into eight experimental treatments (as three replicates per treatment) (Table 1). Fish in each treatment were stoked at 10 fish/ m3 per tank. Each tank (1 m3 in volume) was constructed with an upper irrigation open, an under drainage, and an air stone connected

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FOCUS | PROBIOTICS

table 6: effects of Hydroyeast aquaculture probiotic on feed and nutrients utilization of adult male O. niloticus Protein utilization treat. t1 t2 t3 t4 Se P-value Fl (g/fish) 123.1 117.7 129.3 129.9 5.22 0.364 FCr 3.4a 2.1b 2.7b 2.2b 0.16 0.002 Fe (%) 29.6c 46.9a 37.8b 45.4a 1.97 0.0009 PPV (%) 30.9a 29.0a 15.8b 18.8b 1.22 0.0001 Per 1.2c 1.8a 1.5b 1.8a 0.07 0.0008 eU (%) 15.7a 13.6b 6.6b 8.9c 0.64 0.0001

Means in the same column having different small letters are significantly differ (P 0.05); SE = Standard Error table 7: effects of Hydroyeast aquaculture probiotic on feed and nutrients utilization of adult female O. niloticus Protein utilization treat. t5 t6 t7 t8 Se P-value Fl (g/fish) 113.0c 120.5b 122.6a 113.2c 0.62 0.0001 FCr 3.7b 2.9c 2.8c 5.3a 0.12 0.0001 Fe (%) 26.4b 34.3a 35.1a 18.8c 1.17 0.0001 PPV (%) 13.6b 25.6a 15.0b 5.2c 0.67 0.0001 Per 1.1b 1.3a 1.4a 0.8c 0.04 0.0001 eU (%) 11.2b 15.4a 11.1b 4.9c 0.41 0.0001

Means in the same column having different small letters are significantly differ (P 0.05); SE = Standard Error

sexes of adult O. niloticus such as average total weight gain (AWG), average daily gain (ADG), relative growth rate % (RGR), specific growth rate %/day (SGR) and survival rate % (SR) were calculated. Feed conversion ratio (FCR), feed efficiency % (FE), protein efficiency ratio (PER), protein productive value % (PPV) and energy utilization % (EU) were calculated according to the following equations: AWG (g/fish) = [Average final weight (g) - Average initial weight (g)]. ADG (g/fish/day) = [AWG (g) / experimental period in days (d)]. RGR = 100 [AWG (g)/Average initial weight (g)]. SGR (%/day) = 100 [In final body weight - In initial body weight] / experimental period in days (d). FCR = Feed Intake, (g)/Live weight gain (g). FE = 100 [Live weight gain (g)/Feed Intake, (g)]. PER = Live weight gain (g)/protein intake (g). PPV (%) = 100 [Final fish body protein content (g) - Initial fish body protein content (g)]/crude protein intake (g). EU (%) = Retained energy x 100/consumed feed energy SR = 100 [Total number of fish at the end of the experimental/Total number of fish at the start of the experiment]. * GE (Kcal/100 g DM) = CP x 5.64 + EE x 9.44 + NFE x 4.11 calculated according to NRC (1993).

to an electric compressor. Fresh underground water was used to change one third of the water in each tank every day.

Diet
The tested probiotic Hydroyeast Aquaculture formula shown in Table 2, was produced by Agranco Corp, USA. The commercial diet, as basal ration (BR), used in the present study contains 25 percent crude protein, it was purchased from Al-Manzala manufacture for fish feed, Integrated Fish Farm at Al-Manzala, Dakhalia, Egypt. The ingredients and proximate chemical analysis of this commercial diet according to the manufacture's formula, is shown in Table 3. The diet was ground to add the tested probiotic (Hydroyeast Aquaculture) at levels of 0, 5, 10 and 15 g/Kg diet, referred to

treatments No. T1, T2, T3 and T4, respectively, for males and T5, T6, T7 and T8 treatments for females (Table 1) and then all diets were repelleted. The experimental diets were introduced by hand twice daily at 9 a.m and 15 p.m at 3% of the fish biomass at each tank. The feed quantity was adjusted bi-weekly according to the actual body weight changes.

Statistical analysis
The obtained data for males or for females were statistically analysed using general liner models (GLM) procedure according to SAS (2001) for users guide. The differences between means of treatments were compared for the significance (P 0.05) using Duncan's multiple rang test (Duncan, 1955), as described by Bailey (1995).

Fish sampling and performance parameters

At the start and at the end of the experiment, fish samples were collected and kept frozen till the proximate analysis of the whole fish body according to AOAC (2000). Energy content in experimental fish was calculated according to NRC (1993), being 5.64 and 9.44 kcal/g for CP and EE, respectively. Growth performance parameters of both
28 | InternAtIonAl AquAFeed | July-August 2013

Results
Growth performance parameters Male Growth performance parameters of adult males O. niloticus illustrated in Table 4 revealed that T4 (15 g Hydroyeast Aquaculture/Kg diet) was the best treatment followed by T2 (5 g Hydroyeast Aquaculture/Kg diet) and

FOCUS | PROBIOTICS
table 8: effects of Hydroyeast aquaculture probiotic on carcass composition of adult male O. niloticus % on dry matter basis treat. DM 25.3 t1 t2 t3 t4 Se P- value 24.8a 20.6b 18.2c 17.9c 0.19 0.0001 CP 52.2 58.9a 58.1a 55.4b 55.5b 0.55 0.003 ee 30.7 25.2a 23.8b 24.3ab 24.8ab 0.37 0.123 ash 16.9 15.9c 18.1b 20.3a 19.7a 0.35 0.0001 eC 585.1 570.4a 552.9b 541.8c 547.5bc 2.21 0.0001 t5 t6 t7 t8 Se P- value treat. DM 24.3 20.9b 22.4a 17.1d 18.4c 0.09 0.0001 at the start of the experiment at the end of the experiment table 9: effects of Hydroyeast aquaculture probiotic on carcass composition of adult female O. niloticus % on dry matter basis CP 59.2 53.9c 60.2a 55.7b 55.6bc 0.50 0.0001 ee 23.6 26.8a 24.1b 25.7a 25.7a 0.44 0.015 ash 17.1 19.1a 15.7b 18.5a 18.6a 0.29 0.0001 eC 557.5 557.7b 566.9a 557.6b 559.9b 2.54 0.070 at the start of the experiment at the start of the experiment

Means in the same column having different small letters are significantly differ (P 0.05). DM: Dry matter (%); CP: Crude protein (%); EE: Ether extract (%); EC: Energy content (Kcal/100 g), calculated according to NRC (1993); SE: Standard Error

Means in the same column having different small letters are significantly differ (P 0.05). DM: Dry matter (%); CP: Crude protein (%); EE: Ether extract (%); EC: Energy content (Kcal/100 g), calculated according to NRC (1993); SE: Standard Error

T3 (10 g Hydroyeast Aquaculture/Kg diet), which were gave significantly (P 0.05) final body weight, AWG, RGR, ADG and SGR than the control (T1). But, no significant (P 0.05) differences between T2 and T3 for final weight, AWG and ADG, as well as in SR among all treatments. Female Data of growth performance parameters of adult females O. niloticus revealed that T7 (10 g Hydroyeast Aquaculture/Kg diet) was the best treatment followed by T6 (5 g Hydroyeast Aquaculture/Kg diet), which were gave significantly (P 0.05) increased final body weight, AWG, RGR, ADG and SGR than T8 (15 g Hydroyeast Aquaculture/ Kg diet) and the control (T5). However, no significant (P 0.05) effects in SR among all treatments (Table 5).

and females within all treatments concerning, feed and nutrients utilization parameters may be due to the differences in sexes, metabolism, physiological responses and sexual behaviours of fish during this stage of life.

Fish carcass composition

Feed and nutrients utilization

Male Results of feed nutrients utilization parameters of adult males O. niloticus were shown in Table 6, whereas T4 gave the highest significantly (P 0.05) increased FE, PER and the best FCR followed by T2 compared with the control (T1) and T3. In contrast, PPV or EU increased significantly (P 0.05) in T1 followed by T2 compared with T3 and T4. However, no significant (P 0.05) differences in FI among all treatments. Female Adult females' O. niloticus fed 10 g Hydroyeast Aquaculture/kg diet (T7) showed a significant (P 0.05) increase in FI, FE, PER and the best FCR followed by fish fed 5 g Hydroyeast Aquaculture/kg diet (T6) compared with the control (T1). However, treatment 6 gave significantly (P 0.05) increase of PPV and EU among all treatments (Table 7). Generally, the differences between males

Male Proximate chemical analysis of the whole adult male O. niloticus body at the start and at the end of the experiment is summarized in Table 8. These data indicated that there were significant (P 0.05) increases gets fish into shape of DM and EC content in the control group (T1) compared with the dietary inclusion of Hydroyeast Aquaculture (T2, T3 and T4), but CP content was increased significantly (P 0.05) in T1 or T2 than the T3 and T4. However, Reduces deformities in larvae an unclear trend LARVIVA ProStart is the first early weaning was observed in diet with a unique probiotic approved by the EE, where the European Food Safety Authorities for its increasing in EE documented effect in reducing the occurrence content was not of vertebral deformities in fish larvae and fry. significant in T1 compared with T3 and T4 and www.biomar.com significant as compared with T2. In contrast, of
July-August 2013 | InternAtIonAl AquAFeed | 29

these results ash content increased significantly in T3 and T4 compared with T2 and the control T1. Generally, proximate chemical analysis of the whole fish body at the start, revealed higher DM, EE and EC than in the end of the experiment, but CP and ash were lower at the start than at the end of the experiment. Female Adult female O. niloticus fed the 5 g Hydroyeast Aquaculture/kg diet (T6)

and fry

FOCUS | PROBIOTICS showed significant (P 0.05) increase in DM, CP and EC contents among all treatments. However, both the EE and ash contents recorded the same trend, whereas increased insignificantly in the control group (T5) compared with T7 and T8 and significantly increased compared with T6. In general, unclear trend was recorded in proximate chemical analysis of the whole adult female' O. niloticus body at the start and at the end of the experimental period, which there were higher DM and CP than in the end of the experiment, but EE and ash were lower at start than at the end of the experiment. Meanwhile, no any remarkable changes were observed in EC content at the start and the end of the experimental period (Table 9). (2008) concluded that, super Biobuds can improve body gain, survival and enhance resistance to challenge infection. Yet, Abdelhamid and Elkatan (2006) found that dietary supplementation of Biobuds slightly improved body weight gain but reduced the survival rate of tilapia fingerlings. El-Haroun et al. (2006) and El-Haroun (2007) reported that Biogen dietary supplementation improved growth performance and feed utilization, carcass protein and fat percentages as well as economical profit in Nile tilapia and catfish culture, respectively. In this respect, Mehrim (2009) reported that dietary probiotic (Biogen) had significantly (P 0.05) increased all growth performance parameters of O. niloticus compared with the control group. Yet, Marzouk et al. (2008) found that probiotics (B. subtillis and Saccharomyces cerevisae) revealed significant improvement in growth parameters of O. niloticus. However, Shelby et al. (2006) noted that the probiotic used with juvenile channel catfish diet had lack effect on specific growth promoting. Also, He et al. (2009) found that supplementation of dietary DVAQUA showed no effects on growth performance, feed conversion and survival rate of the hybrid tilapia. The reasons for the differences between fish species have not been elucidated, but might be due to the differences in aquaculture and physiological conditions, composition of the probiotic and the type of basal ingredients in diets. In this context, many studies concluded a positive effect of using viable microorganisms in probiotic mixtures into diets of fish (Pangrahi et al., 2005; Barnes et al., 2006; AboState et al., 2009). According to the results of the present study and those obtained by others; it seems that probiotics may stimulate appetite and improve nutrition by the production of vitamins, detoxification of compounds in the diet, and by breakdown of indigestible components (Irianto and Austin, 2002). Also, Varley (2008) cited also that probiotics show real benefits in the synergistic effects with the beneficial bacteria in making inroads into improving gut health.

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Discussion
The positive effects in the present study of Hydroyeast Aquaculture probiotic on adult males and females O niloticus growth performance and feed utilization, was found by Eid and Mohamed (2008), where they proved that Biogen and Prmifer improved the growth performance, feed conversion, protein efficiency ratio and apparent protein digestibility for monosex tilapia fingerlings compared to fish fed the control diet. Moreover, El-Ashram et al.

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Probiotics and FCR

Probiotics improve feed conversion efficiency and live weight gains (Saenz de Rodriguez et al., 2009). So, the supplementation of commercial live yeast, S. cerevisiae, improved growth and feed utilization (AbdelTawwab et al., 2008). Yet, similar results were obtained when S. cerevisiae was added to fish diet for Israeli carp (Noh et al., 1994) and Nile tilapia (Lara-Flores et al., 2003). Moreover, Mehrim (2009) found similar positive effects of Biogen on growth performance, feed conversion ratio and carcass composition of O. niloticus. Rawling et al. (2009) reported that daily feed intake was significantly

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FOCUS | PROBIOTICS reported that increasing dietary Betafin (betaine) level caused a significant improve of O. niloticus body composition. On the other side, the results in the present study are in close agreement with those of EL-Haroun et al. (2006), Mohamed et al. (2007), and Eid and Mohamed (2008) for tilapia and EL-Haroun, (2007) for catfish. In addition, Mehrim (2009) found positive effects of inclusion of Biogen at a level of 3 g/kg on carcass composition of mono-sex O. niloticus fingerlings. These positive effects in carcass composition of experimental fish may be due to the dietary probiotic Biogen, which caused the good growth performance of treated fish compared with the control group, as present findings of adult males and females O. niloticus growth performance (Tables 4 and 5), respectively. From the forgoing results, it could be concluded that Hydroyeast Aquaculture probiotic is useful at levels 15 g/kg diet (T4) and 10 g /kg diet (T7) for enhancing production performance of adult males and females Nile tilapia O. niloticus respectively, so may be using of this probiotic led to economic efficiency especially, for fish farming and hatcheries. This article was originally published on

higher in red tilapia (O. niloticus) fed Sangrovit (Phytobiotics Gmbh, Etville, Germany) supplemented diets compared to control and that feed utilization was not significantly affected suggesting that improved growth was likely to be due to improved appetite of fish fed diets containing Sangrovit. The improved fish growth and feed utilization may possibly be due to improved nutrient digestibility. In this regard, Tovar et al. (2002), Lara-Flores et al. (2003), and Wach et al . (2006) found that the addition of live yeast improved diet and protein digestibility, which may explain the better growth and feed efficiency seen with yeast supplements. Also, De Schrijver and Ollevier (2000) reported a positive effect on apparent protein digestion when supplementing turbot feeds with the bacteria Vibrio proteolyticus.

Effect of yeast on wholebody composition

Growth of fish and feed conversion

together with carcass composition are generally affected by species, genetic strain, sex, stage of reproductive cycle, etc, leading to different nutritional requirements (Jauncey, 1998). In this respect, yeast supplementation significantly affected the whole-fish body composition (Abdel-Tawwab et al., 2008). These results suggest that yeast supplementation plays a role in enhancing feed intake with a subsequent enhancement of fish body composition, as well as yeast supplements significantly affected ash content of O. niloticus (Abdel-Tawwab, 2012). On the other hand, changes in protein and lipid content in fish body could be linked with changes in their synthesis, deposition rate in muscle and/ or different growth rate (Abdel-Tawwab et al., 2006). In this topic, Khattab et al. (2004) reported that crude protein, total lipids and ash were significantly (P < 0.01) affected by protein level and increasing stocking density rate of tilapia fish. Yet, Abdelhamid et al. (2007)

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FOCUS | PROBIOTICS

Effects of Sangrovit on the performances of tra catfish (Pangasius hypophthalmus) cultured in earthen ponds

by Dr Nguyen Nhu Tri and Prof Le Thanh Hung, Nong Lam University, Vietnam

ra catfish (Pangasius hypophthalmus) is a dominant cultured species in Vietnam in terms of production. Its aquaculture production in 2011 was approximate 1.2 million metric tons. The main culture system for tra catfish is an intensive model in earthen ponds with a very high density and yield. In this model, feed cost comprises the main part of production cost. The cost feed increased recently, leading to a reduction in profit for farmers. In order to improve the profit of tra catfish farming, feed cost should be reduced. One of the measures to reduce feed cost is through feed additives which improve nutrient absorption and utilization. Sangrovit is an all-natural feed additive that has been utilized in many animal species. The main active ingredients of Sangrovit include bitter constituents, which help to enhance the absorption of feed and promote digestion. The special active substances included in Sangrovit, belonging to the group of Benzophenanthridine and Protopine Alkaloids, have the additional effects of improving hormonal and chemostatic regulating mechanisms in feed uptake, nutrient absorption and availability. Therefore, it is feasible to utilize its characteristics in tra catfish intensive culture system in Vietnam. The purpose of this study was to evaluate the effects of Sangrovit on the final mean weight, FCR, survival rate and non-specific immunity parameters of Pangasius hypophthalmus fingerling cultured in hapa installed in an earthen pond. The present study consisted of two experiments conducted at Experimental Station, Nong Lam University, Ho Chi Minh City, Vietnam.

Figure 1: Experimental hapa

table 1: tested Sangrovit concentration Control 1 2 50g/ton of feed 3 75g/ton of feed 4 100g/ton of feed

0g/ton of feed 25g/ton of feed

Effect of Sangrovit used as feed additives for Pangasius hypophthalmus feed

Pangasius hypophthalmus fingerlings (9,8 0,1 g) were randomly stocked into 15 1 m3 cages (1x1x1.3 m) installed in a 600 m2 earthen pond at an initial density of 40 fish per hapa (Figure 1) for four different levels of Sangrovit supplementation (Table 1) with three replicates per treatment to determine the optimal dose in the feed. The utilized feed was 2 mm extruded floating feed with the following specifications: 30 percent protein, 5 percent fat, ME min 2,800 Kcal and 6 percent maximum fibre. Fish were fed to satiation twice a day. This experiment was conducted for 16
34 | InternAtIonAl AquAFeed | July-August 2013

table 2: Controlled water quality variables temp Maximum average Minimum 31.2C 29.3C 27.1C D.o. 8.2 ppm 4.9 ppm 1.6 ppm pH 6.79 6.78 6.71

weeks. Fish were weighed every four weeks to monitor growth and survival rates. At the end of the first experiment, fish were harvested, counted and group weighed to determine final mean weight, FCR and survival rate. Water quality variables were measured as follow: Dissolved oxygen, temperature and pH were measured twice a day (7 am and 4 pm) using YSI-550 digital oxygen/temperature meter

FOCUS | PROBIOTICS and Fisher-Scientific portable pH meter. Total ammonia nitrogen and nitrite were measured three times a week by spectrometric method. Water in the pond was exchanged regularly to maintain good quality for the entire experimental period. The parameters of the water quality were maintained at the levels shown in the Table 2.
table 3: Final mean weight, SGr (add SGr), FCr and survival rate of experimented fish Parameter Initial Wt (g) SGr (%/day) FCr Control 9.77 0.10a 2.31 0.04a 1.70 0.02a 1 9.78 0.02a 84.25 9.15ab 2.41 0.11ab 1.57 0.08b 92.50 2.50a 2 9.72 0.01a 87.19 3.23ab 2.46 0.04b 1.60 0.01ab 90.83 2.88a 3 9.74 0.01a 92.96 3.97b 2.51 0.04b 1.51 0.05b 94.17 5.20a 4 9.82 0.01 a 89.29 3.45b 2.47 0.11b 1.49 0.11b 90.83 1.44a

Final Wt (g) 75.86 3.45a

Survival rate (%) 93.33 5.20a

Sampling and analysis

Initially, fish were weighed and randomly distributed into hapa. To minimize the fish not being stressed whilebeing weighed, MS222 was used to anaesthetize the fish. At the end of the trial, fish in each hapa were weighed. Growth performances were monitored using specific growth rates (SGR) as follows: Specific Growth Rate (SGR) In which: - W2 : Mean weight at the end of the experiment - W1 : Mean weight at the beginning of the experiment - T2- T1 : Duration of the experiment 112 days (16 weeks)
table 4: lysozyme activity of experimented fish time 0 hr 24 hrs 72 hrs Control 128.3 35.3a 146.7 38.8a 161.7 34.0a 1 126.7 30.6a 154.2 47.9a 217.5 23.8ab 2 145.8 36.4a 157.5 18.0a 313.3 52.8ab 3 135.0 32.5a 185.0 2.50a 381.7 25.7b 4 147.5 24.6a 175.0 55.2a 339.2 121.6b

table 5: White blood cell density (x103 cell/mm3) of experimented fish time 0 hr 24 hrs 72 hrs Control 83.83 3.05a 93.61 30.18a 9.96 4.77a 1 97.02 20.44a 15.20 3.56a 2 95.78 3.89a 12.80 1.92a 3 105.58 11.88a 13.10 4.35a 4 98.34 32.44a 14.93 2.29a

102.57 27.39a 118.70 35.35a 125.75 23.06a 116.17 30.99a

Feed efficiency
In the study, feed efficiency was monitored using the feed conversion ratio (FCR) and protein efficiency ratio (PER) as follows

FCR = Total feed intake/ Total weight gain (W2 - W1) PER = (W2 - W1) / Protein intake Statistical analyses were performed using Minitab software version 16.0. Data collected from the experiment were analyzed using one-way analysis of variance

to determine if significant differences (P<0.05) in final mean weight, FCR, survival rate and non-specific immunity parameters between treatments. Tukey multiple comparison test was utilized to determine differences among treatment means.

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FOCUS | PROBIOTICS

Figure 2: impact of Sangrovit supplementation on the Specific Growth Rate

Figure 3: impact of Sangrovit supplementation on the FCR

Figure 4: Lysozyme activity of experimented fish

Figure 5: White blood cell density of experimented fish

Results and discussion

Data presented in Table 3 shows that final mean weight of the first three treatments was not significantly different (P>0.05). However, final mean weight of treatments 3 and 4 was significantly higher than that of the control. The survival rate of all treatments was not significantly different. The highest SGR was obtained in treatment 3, followed by treatments 4 and 2 and significantly different from the control. It means that Sangrovit supplemented to the feed at levels from 50-100 g/ton of feed are able to enhance growth rate of tra catfish (Figure 2). Data are expressed as Mean SD. Data with the same superscript in the same row are not significant differences (P>0.05) One of the most important parameters to consider in tra catfish farming is FCR. Data from Table 3 showed that FCR of the control treatment was significantly higher than those belonged to treatments 1, 3 and 4. Sangrovit supplementation to the feed at levels of 75 and 100 g/

ton helps to reduce FCR remarkably (Figure 3). That means the Sangrovit supplementation in P. hypophthalmus feed has improved the feed utilization by enhancing the secretion of internal enzymes in digestive tract.

Effect of Sangrovit on health improvement

After the first experiment was completed, 15 fish from each hapa were randomly assigned to a 100-L fibreglass tank of a wet lab for a challenge test with Edwardsiella ictaluri to evaluate the effects of Sangrovit on health status of experimented fish.

ozyme activity. Lysozyme activity of all five treatments is presented in Table 4 and Figure 4. Data are expressed as Mean SD. Data with the same superscript in the same row are not significant differences (P>0.05) Data in Table 4 and Figure show that serum lysozyme activity of all treatments
table 6: Survival rate of tra catfish at 14 days post challenge treatment Control 1 2 3 4 Survival rate (%) 11.11 3.85a 17.78 3.85ab 13.33 6.67ab 22.22 3.85b 20.00 6.67ab

Lysozyme activity
When the first experiment was completed, five fish from each hapa were challenged by dipping in a low-density Edwardsiella ictaluri solution (1.28 x 105 CFU/mL) for one hour in order to stimulate the activity of immune system. Blood samples were taken right before challenging, 24 hours and 72 hours after challenging to measure lys-

was not significantly different at 0 hours and 24 hours post challenge with Edwardsiella ictaluri. However, this parameter was significantly higher in treatments 3 and 4 as compared to the control at 72 hours post challenge. The supplementation of Sangrovit at levels of 75 and 100 g/ton of feed helped to boost immune system of tra catfish through increasing lysozyme activity

36 | InternAtIonAl AquAFeed | July-August 2013

FOCUS | PROBIOTICS blood cell density was not significantly different among treatments at 0 hours, 24 hours and 72 hours post challenge. However, this parameter tended to be higher in Sangrovit supplemented treatments as compared to the control at 24 hours and 72 hours post challenge. Sangrovit might enhance the production of white blood cell to fight against infected bacteria. Figure 6: Survival rate of the tra catfish during 14 days to a level that significantly different from the control. fish were isolated and classified by IDS 14 GNR test kit of Nam Khoa company. The results of the second experiment were presented in Table 6 showed that the mortality of tra catfish started from day four and stopped at day 11-post challenge. The survival rate of tra catfish in treatment 3 was significantly higher than that of the control. Sangrovit supplementation at a level of 75 g/ton of feed could enhance survival rate of tra catfish when challenged by Edwardsiella ictaluri.

Conclusions
Notably Sangrovit inclusion to commercial feed at levels of 75 g/ton enhanced growth rate, SGR and lysozyme activity of Pangasius hypophthalmus, and it also reduced FCR significantly. Further, a positive effect on the test fish challenged with Edwardsiella ictaluri was detected. The measured influence can be attributed to the known effect on nutrient absorption and on the anti-inflammatory mode of action of Sangrovit, which is particularly focused on the intestinal tract. Thus feed conversion and digestibility and the health status of fish was improved. Therefore, Sangrovit is recommended to be used in the feed at this level to increase return on investment of Pangasius hypophthalmus farming industry. FEATURE

Survival

White blood cell density

White blood cell density was determined at the same time frame as lysozyme activity measurement and presented in Table 5 and Figure 5. Data are expressed as Mean SD. Data with the same superscript in the same row are not significant differences (P>0.05) Data in Table 5 shows that white

Experimented fish were dipped in an Edwardsiella ictaluri solution (3.39 x 105 CFU/ml) for 1 hour and then returned to the tank. Mortalities were monitored for 14 days. During this period, water temperature was maintained at 26 C, an optimal temperature for bacteria growth. Dead or moribund fish were retrieved from the tanks. They were necropsied, and bacterial laboratory diagnosis was made from the moribund and freshly dead fish. Bacteria from moribund and freshly dead

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