Effect of Using Baker's Yeast and Exogenous ...

Journal of Agricultural Science and Technology B 2 (2012) 15-28 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250

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Effect of Using Baker’s Yeast and Exogenous Digestive Enzymes as Growth Promoters on Growth, Feed Utilization and Hematological Indices of Nile tilapia, Oreochromis niloticus Fingerlings Ashraf Mohamed Abdelsamee Goda1, Hafez Abdel-Hamid Hassan Mabrouk1, Mohamed Abd El-Hamid Wafa1 and Tarek Mohamed El-Afifi2 1. National Institute of Oceanography and Fisheries (NIOF), Fish Nutrition Laboratory, Cairo 24135, Egypt 2. Regional Center for Foods and feeds, Agriculture Research Center, Ministry of Agriculture, Cairo 24135, Egypt Received: August 1, 2011 / Published: January 20, 2012. Abstract: The present study was conducted to evaluate the effect of baker yeast, Saccharomyces cerevisiae (SC) and exogenous digestive enzymes (pepsin, papain and α-amylase, EDE) dietary supplementation on growth performance, feed utilization and hematological indices of Nile tilapia, Oreochromis niloticus fingerlings. A total of 630 Nile tilapia fingerlings with an average body weight of 26.4 ± 0.2 g were divided in the seven experimental net-pen treatments (three replicates each). The experiment was conducted for 119 days. Seven isonitrogenous (26.50%) digestible protein and isocaloric (13.40 MJ kg-1) digestible energy experimental diets were formulated. The control diet had no SC and EDE added. Diets 2-3 each contained SC at levels of 2 and 4 g 100 g diet-1, respectively, while diets 4-5 each contained EDE at levels of (0.64, 1.28, 0.16) and (1.28, 2.56, 0.32) g 100 g diet-1 of pepsin, papain and α-amylase, respectively. Diet 6 contained mixture of SC and EDE at levels of 1 g yeast and 0.32, 0.64, 0.08 g of pepsin, papain and α-amylase, respectively 100 g diet-1 and diet D7 contained 2 g yeast and 0.64, 1.28, 0.16 g of pepsin, papain and α-amylase, respectively 100 g diet-1. Growth performance and feed utilization efficiency of Nile tilapia were significantly (P  0.05) higher in all treatments receiving SC and/or EDE supplemented-diets than the control diet which suggests that the addition of SC and EDE enhanced the growth performance. Red blood cells counts, hematocrit and hemoglobin were significantly (P  0.05) highest in all treatments receiving mixture of SC and EDE supplemented-diets (D6 + D7). The same trend was observed for total plasma protein and total plasma globulin levels. The results of present study suggested that Nile tilapia fingerlings fed diets containing the mixture of 1 g yeast, SC and 0.32, 0.64, 0.08 g of pepsin, papain and α-amylase, respectively 100 g diet-1, for 119 days had enhanced growth performance, diet utilization efficiency and hematological indices. Key words: Baker yeast, digestive enzymes, growth, feed utilization, hematological indices, Nile tilapia.

1. Introduction Nile tilapia, Oreochromis niloticus (L.) is an important species for freshwater aquaculture. Improving fish performance and disease resistance of cultured organisms are major challenges facing fish culturists. Intensive Nile tilapia culture usually Correspondent: Ashraf Mohamed Abdelsamee Goda, Ph.D., research fields: fish nutrition and aquaculture. E-mail: [email protected].

requires balanced diets which are formulated with several ingredients, especially additives that have effectiveness of fish dynamic and physiological functions. The demand for animal protein has gone far beyond supply as a result of the rapid growth of human population in many countries of the world, especially in the developing countries. An urgent need is therefore necessary to increase the production of

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Effect of Using Baker’s Yeast and Exogenous Digestive Enzymes as Growth Promoters on Growth, Feed Utilization and Hematological Indices of Nile tilapia, Oreochromis niloticus Fingerlings

protein sources. Nowadays, aquaculture is one of the fastest growing food production sector and an increasingly important option in animal protein sources in the world. However, high-quality fish diets should contain not only high quality and levels of dietary nutrients but also additives to keep fish healthy and improve growth. Probiotics have received some attention in aquaculture, emphasized a reduction in mortality [1, 2], and improved the ability to antagonize other organisms [3-7], improve fish growth and immune responses [1, 8-13], reduce the number of bacterial cells in kidneys [14], and production of polyamines and digestive enzyme activity [15, 16]. Commercial yeast, Saccharomyces cerevisiae is defined as unicellular eukaryotic micro-organisms [17] and usually used in animal nutrition as an excellent source of protein and vitamins, especially B-complex vitamins, whose functions are related to metabolism. S. cerevisiae contains other minerals and co-factors such as various immunostimulating compounds including β-glucans, nucleic acids, mannan and other oligosaccharides which has the capability to enhance growth as well as immune responses of various fish species [4, 18-24]. Most of the plant origin ingredients have demerits on account of presence of anti-nutritional factors which have an adverse impact on the digestion and nutrient utilization of feed. The presence of anti-nutritional factors within plant feed-stuffs limits their use in aqua-feeds [25]. However, certain enzymes provide an additional powerful tool that can inactivate anti-nutritional factors and enhance the nutritive value of plant based protein in feeds. Enzymes responsible for the digestion of carbohydrates (amylases), proteins (proteases), and fats (lipases) occur in pyloric caeca and intestinal mucosa of fishes. However, the initiation of digestion is in stomach fish species that possess true stomach, where hydrochloric acid secreted leads to the activation of pepsin. The maximum reaction usually

takes place within the pH ranges of 2.0 to 2.2. The ability of fish to convert dietary protein into body flesh is dependent on its ability to hydrolyze dietary protein into digestible peptides and amino acids. Peptides and amino acids are then absorbed from the gut and synthesized into tissue protein. The most important protein digestive enzymes are the endopeptidases, chymotrypsin and trypsin, and the exopeptidases, aminopeptidase and carboxypeptidase. However, pepsin is also an important enzyme in true stomach possessing fishes [26]. Endogenous enzymes found in the digestive tract of fish help to break down large organic molecules like starch, cellulose and protein into the simpler substances. Addition of exogenous enzymes in fish feeds can improve nutrient utilization, thereby reducing nutrient losses. Exogenous enzymes have been proven to improve nutritional value of feed and decrease environmental pollution in terrestrial animals [27]. Nowadays, exogenous enzymes are extensively used all over the world as additives in fish diets to improve the nutritional value of fish feeds, especially with the raise of using plant proteins in aqua feeds and reduce water pollution [28]. Herbal based enzyme “papain” is a cysteine protease hydrolase (EC 3.4.22.2) enzyme present and derived from papaya (Carica papaya) and mountain papaya (Vasconcellea cundinamarcensis). It is a proteolytic enzyme and called as vegetable pepsin [29]. Papaya leaves contain around 9% protein and 5.3% papain and also contain vitamin C (286 mg/100 g) and vitamin E (30 mg/100 mg) [30]. Papain will digest most protein substrates more extensively than the pancreatic proteases. Papain exhibits broad specificity, cleaving peptide bonds of basic amino acids, leucine, or glycine. It also hydrolyzes esters and amides [31]. Papain has also been used in the enzymatic synthesis of amino acids, peptides, and other molecules [32, 33]. An exogenous enzyme such as α-amylase is enzyme that hydrolyses alpha-bonds of large alpha-linked


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polysaccharides such as starch and glycogen, yielding glucose and maltose. The enzyme α-amylase secreted by the pancreas and it is a powerful enzyme and succeeds in digesting most of the carbohydrates in the diet. Fish meal is used globally as dietary protein in formulated fish feeds but the major problems with the use of fish meal as source of protein in the fish diet are its rising cost, uncertain availability, adulteration and variation in quality. The increasing demand, unstable supply and high price of the fish meal with the expansion of aquaculture made it necessary to search for alternative protein sources [26, 29, 34]. Currently, one of the challenges that fish nutritionists face is to partially or totally replace FM with less expensive, untraditional animal and/or plant protein sources. Plant protein sources differ in its carbohydrate content and digestibility which influence the fish growth [35-37]. Most carbohydrate content in plant protein source is starch. Enzymes provide a natural way to transform complex feed components into absorbable nutrients. Endogenous enzymes found in the fishes digestive system help to break down large organic molecules like starch, cellulose and protein into simpler substances. Therefore, the addition of enzymes in feed can improve different nutrients utilization in plant protein sources, reducing feed cost and the excretion of nutrients into the environment [38-40]. The present study was undertaken to determine the effect of supplementation different levels of dietary S. cerevisiae and exogenous enzymes (Pepsin, Papain and α-amylase) on growth performance, feed utilization and hematological indices of Nile tilapia, O. niloticus (L.) fingerlings.

Governorate, Egypt. The fingerlings were stocked into three cement ponds (each with 42 m3) at Fish Research Station, El-Kanater El-Khayria, National Institute of Oceanography and Fisheries (NIOF), Cairo, Egypt. Each cement pond was divided into seven equal compartments (net pens) by netting (each of 6 m3) and each pen was stocked with 30 fish. Three replicate pens were randomly assigned to each treatment. Prior to the start of experiment, fish were acclimated to the experimental conditions for two weeks, during this period fish were fed a control diet at 3% of body weight. The daily ration was divided into three equal amounts and offered three times a day (08:00, 12:00 and 14:00). The fish were fed one of seven experimental diets for 119 days (17 weeks). The cement ponds were supplied with freshwater from the Darawa Irrigation Branch, Kalubiya Governorate where the water turnover rate was 0.3 m3 pond day-1 and fish were held under natural light (12:12 light: dark schedule).

2. Material and Methods

times a day (08:00, 12:00 and 14:00). All fish in each

Water temperature, dissolved oxygen, pH, and ammonia were monitored during the study, to maintain water quality at optimal range for Nile tilapia. Water temperature was recorded daily at 13:00 using a mercuric thermometer suspended at 30 cm depth. Dissolved oxygen (DO) was measured at 05:00 using YSI model 56 oxygen meter (YSI Company, Yellow Springs Instrument, Yellow Springs, Ohio, USA) and pH at 09:00 by using pH meter (Orion pH meter, Abilene, Texas, USA). Ammonia was measured three times a week according to APHA [41]. During the 17-week experimental period, all fish were fed respective diets at a level of 3% of live body weight for 6 days a week. The daily ration was divided into three equal amounts and offered three replicate pen were weighed biweekly and the amount

2.1 Experimental Fish and Culture Technique Nile tilapia (O. niloticus) fingerlings with an initial body weight of 26.4 ± 0.2 g were obtained from Arab Fisheries Hatchery, Abu-Hammad, Sharkia

of daily allowance was adjusted accordingly. 2.2 Experimental Diets Seven isonitrogenous 26.5% digestable protein (DP)

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and isocaloric 13.40 MJ kg-1 digestible energy (DE) experimental diets were formulated. The control diet had no SC or EDE added. Diets 2 and 3 each contained yeast at levels of 2 and 4 g 100 g diet-1, respectively, while diets 4 and 5 each contained exogenous digestive enzymes at levels of (320, 640 and 80) and (640, 1280, 160) mg 100 g diet-1 of pepsin, papain and α-amylase, respectively. Diet 6 contained mixture of SC and EDE at levels of 1 g yeast and (160, 320, 40) mg of pepsin, papain and α-amylase, respectively 100 g diet-1 and diet D7 contained 2 g yeast and (320, 640, 80) mg of pepsin, papain and α-amylase, respectively 100 g diet-1. The proximate chemical composition of the experimental diets is presented in Table 1. Dietary gross energy (GE) contents were calculated according to gross caloric values of Brett [42] using the values of 23.6, 39.5, and 17.2 kJ g-1 for crude protein, crude fat, and total carbohydrate, respectively. The digestible energy (DE) was estimated by applying the coefficient of 70% from determined GE values according to Hepher et al. [43]. Digestible protein (DP) was calculated using values of 90.2%, 87.2%, 96.5%, 96.2%, 83.6%, and 75.1% for crude protein content of fish meal, meat and bone meal, corn gluten meal, soybean meal, wheat bran, and yellow corn, respectively, according to Sklan et al. [44]. Fish meal, corn gluten meal, soybean meal, yellow corn and wheat bran were purchased from a commercial feed manufacturer (Animal Production Islamic Company (APICO), Dokki-El-Giza, Egypt). The enzymes product used in study Digestin® was obtained from Pharco (El-Horreya Avenue, Boulkly, Alexandria, Egypt). The Digestin® is reported to contain: pepsin (40 mg 5 mL-1), papain (80 mg 5 mL-1) and Sanzyme2000 (10 mg 5 mL-1, Sanzyme2000 is a complex multi-enzymatic extracted from fermented Aspergillus oryzae media, essentially not less than 90% α-amylase and 10% other enzymes (protease, lipase, cellulose, pectinaze, etc.). Dry ingredients were ground by screen diameter (1.0 mm) using a

homogenous mixture grinder (PHILIPS, Mode HL 1616ID, Philips India Limited. 7, Justice Chandra Medhab Road, Calcutta 700020). Dry ingredients were blended for 15 min, then commercial baker’s yeast (Sweet Sham Company for Food Industries, Industrial Zone, 6 October City, Cairo, Egypt) were diluted in water and added; oil and liquid enzymes (in aqueous solution, 100 mL kg-1) were mixed and sprayed onto the mixture during the blending process and homogenized well for 10 minute according to Deguara [45]. 100 mL of water kg-1 diet was added to blended mixture to make a paste of each diet, which were passed through local hand grinder and pelleted to 2 mm diameter size. The same approach has been followed to add enzymes to the diets. All diets were dried (temperature did not exceed 40 ºC) until the moisture was 10%, then packed in cellophane bags and stored at -4 ºC prior to use. 2.3 Blood Samples and Analysis Blood samples were collected at the end of the experiment. Each of the experimental treatment was sampled once, with five fish/pen for hematological indices analysis and five fish/pen bled for plasma content analysis. The fish were anesthetized with t-amyl alcohol and the blood samples were taken by puncturing the caudal vessels. EDTA (Ethylenediaminetetraacetate) was used as anticoagulants. The red blood cell counts (RBCs) were determined by using a Bürker counting chamber and Hayem solution. The findings and instructions published by Blaxhall and Daisley [46] and Hrubec et al. [47] were followed when the RBCs were determined. Hematocrit (Hct) were determined by using microhematocrit-heparinized capillary tubes and a microhematocrite centrifuge (10,000 g for 5 min). The values of Hct were determined within 30 min alter bleeding. Hemoglobin concentrations (Hb) were determined by the cyanhemoglobin method, at 540 nm. RBCs and Hb values were determined within 6 h after blood sampling.

Effect of Using Baker’s Yeast and Exogenous Digestive Enzymes as Growth Promoters on Growth, Feed Utilization and Hematological Indices of Nile tilapia, Oreochromis niloticus Fingerlings Table 1

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Formulation and proximate analysis of the experimental diets (% dry matter).

Ingredients

Control D1 10 10 10 16 29 20 3 2 -

Yeast D3 10 10 10 16 25 20 3 2 4 -

Experimental diets Enzymes D4 D5 10 10 10 10 10 10 16 16 27 25 20 20 3 3 2 2 320 640 640 1,280 80 160

Yeast + Enzymes D7 10 10 10 16 25 20 3 2 2 320 640 80

D2 D6 Fish meal 10 10 Meat and bone meal 10 10 Corn gluten meal 10 10 Soybean meal 16 16 Yellow corn 27 27 Wheat bran 20 20 Corn oil 3 3 Vitamin and mineral premix1 2 2 Yeast 2 1 Enzymes Pepsin (mg) 160 Papain (mg) 320 α-amylase (Sanzyme 2000, mg)* 40 Proximate composition Dry matter (%) 89.99 89.86 89.68 89.86 89.68 89.86 89.68 Crude protein (%) 30.68 30.52 31.70 30.51 30.34 30.51 30.34 Lipid (%) 6.18 6.11 6.04 6.11 6.04 6.11 6.04 Total carbohydrate (%) 55.92 56.19 56.46 56.19 56.46 56.19 56.46 Ash (%) 7.22 7.19 7.16 7.19 7.16 7.19 7.16 2 -1 Gross energy , MJ 100g 19.28 19.26 19.24 19.26 19.24 19.26 19.23 Digestible protein (DP) 3 26.57 26.54 27.60 26.55 26.90 26.54 26.42 Digestible energy (DE) 4 , MJ 100g-1 13.496 13.482 13.468 13.482 13.468 13.482 13.461 DP: DE ratio, mg CP KJ-1 19.69 19.69 20.49 19.69 19.97 19.69 19.63 D1: control; D2: content 2 g S. cerevisiae yeast 100 g diet-1; D3: content 4 g S. cerevisiae yeast 100 g diet-1; D4: content 320, 640 and 80 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D5: content 640, 1280, 160 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D6: content mixture of 1 g S. cerevisiae yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively 100 g diet-1 and D7: content 2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, respectively100 g diet-1. 1 Vitamin and mineral mixture kg-1 of mixture contains: 4800 I.U. Vit A, 2400 IU cholecalciferol (Vit. D), 40 g Vit E, 8 g Vit K, 4.0 g Vit B12, 4.0 g Vit B2, 6 g Vit B6, 4.0 g Pantothenic acid, 8.0 g Nicotinic acid, 400 mg Folic acid, 20 mg Biotin, 200 gm Choline, 4 g Copper, 0.4 g Iodine, 12 g Iron, 22 g Manganese, 22 g Zinc, 0.04 g Selenium. Folic acid, 1.2 mg; niacin, 12 mg; d-calcium pantothenate, 26 mg; pyridoxine. HCl, 6 mg; riboflavin, 7.2 mg; thiamin. HCl, 1.2 mg; sodium chloride (NaCl, 39% Na, 61% Cl), 3077 mg; ferrous sulfate (FeSO4·7H2O, 20% Fe), 65 mg; manganese sulfate (MnSO4, 36% Mn), 89 mg; zinc sulfate (ZnSO4·7H2O, 40% Zn), 150 mg; copper sulfate (CuSO4·5H2O, 25% Cu), 28 mg; potassium iodide (KI, 24% K, 76% I), 11 mg; Celite AW521 (acid-washed diatomaceous earth silica), 1000 mg. 2 Calculated using gross calorific values of 5.65, 9.45 and 4.11 Kcal g-1 for protein, fat and carbohydrate, respectively according to Brett [42]. 3 Digestible protein was calculated using values of 90.2%, 87.2%, 96.5%, 96.2%, 83.6%, and 75.1 % for crude protein content of fish meal, poultry by-product meal, corn gluten meal, soybean meal, wheat bran, and yellow corn, respectively, according to Sklan et al. [44]. 4 Digestible energy was estimated as 70% of Gross energy values according to Hepher et al. [43]. * Sanzyme2000 a complex multi-enzymatic extracted from fermented Aspergillus oryzae media, essentially not less than 90% α-amylase and 10% other enzymes (protease, lipase, cellulose, pectinaze, etc.).

The derived mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using standard formulae. The total WBC

counts and differential cell counts were determined according to the method of Stoskopf [48] and Terry et al. [49]. Blood plasma was obtained by centrifugation of the blood samples at 3,000 g for 5 min and stored at

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-20 ºC for further analysis. Total plasma protein (TPP) and albumin (TPA) were determined according to Doumas et al. [50] and Reinhold [51], respectively using commercial kits produced by Pasteur labs (Egyptian American Co. for Laboratory Services, Egypt). However, the total plasma globulin (TPG) was calculated by subtracting the total plasma protein from total plasma albumin according to Coles [52]. The haematological parameters are expressed in international units (SI). 2.4 Analytical Methods At the beginning of the trial, a random pooled sample of 25 fish was collected, anesthetized with t-amyl alcohol and sacrificed for determination of initial whole-body proximate composition. At the termination of the feeding trial, six fish were randomly selected from each pen and anesthetized with t-amyl alcohol, sacrificed, and homogenized in a blender for final whole-body proximate composition. The fish were pooled for each pen, oven-dried, ground, and stored at -20 ºC for subsequent analysis. The chemical composition of fish and diet samples were determined according to procedures of AOAC [53]. Dry matter was determined after drying the samples in an oven (105 ºC) for 24 h. Ash by incineration at 550 ºC for 12 h. Crude protein was determined by micro-Kjeldhal method, N% × 6.25 (using Kjeltech autoanalyzer, Model 1030, Tecator, Höganäs, Sweden) and crude fat by Soxhlet extraction with diethyl ether (40-60 ºC). 2.5 Growth Indices Mean final body weight (FBW) of each experimental treatment was determined by dividing total fish weight in each pen by number of fish. Weight gain (WG), specific growth rate (SGR%), Average daily gain (ADG), feed conversion ratio (FCR), protein efficiency ratio (PER), protein productive value (PPV), fat retention (FR) and energy retention (ER) were calculated according to Goda [54]

using the following equations: WG = Final body weight (g) － Initial body weight (g); Average daily gain (ADG) (g fish-1 day-1) = Total gain/Duration period; SGR% = (ln FBW － ln IBW)/t × 100; where: FBW is final body weight (g); IBW is initial body weight (g); ln = natural logarithmic; t = time in days; FCR = feed intake (g)/weight gain (g); PER = weight gain (g)/protein intake (g); PPV% = (protein gain (g)/protein intake (g)) × 100; FR = (fat gain (g)/fat intake (g)) × 100 and ER% = (energy gain (kJ)/energy intake (kJ)) × 100. Economical conversion rate (ECR) was calculated according to Abdel Rahman et al. [55] using the following equation: ECR = Cost of diet ($ kg-1)  Feed conversion ratio (FCR). 2.6 Statistical Analysis Data were statistically analyzed by ANOVA using MSTAT-C version 4 software (MSTAT-C, 1987) [56]. Duncan’s multiple range test was used to compare differences between treatment means when significant F values were observed [57], at P  0.05 level. All percentage data were arc-sin transformed prior to analysis [58], however data are presented untransformed to facilitate comparisons. The relationship between hematological indices was tested using simple correlation analysis.

3. Results All conditions of the experimental evaluation in the present study were apparently satisfactory and fell under the optimal standards defined for nutritional evaluations in Nile tilapia. The survival rate of Nile tilapia after 17 weeks of feeding either yeast and/or enzymes supplemented diets was 100%. Water temperature ranged from 27.1 to 28.3 °C, DO from 5.6 to 6.5 mg/L, pH from 6.7 to 8.1, and ammonia (NH3) from 0.22 to 0.31. Results in Table 2 revealed that with the higher dietary levels of S. cerevisiae or EDE, the FBW, SGR, ADG and SGR% improve significantly (P ≤ 0.05), while

Effect of Using Baker’s Yeast and Exogenous Digestive Enzymes as Growth Promoters on Growth, Feed Utilization and Hematological Indices of Nile tilapia, Oreochromis niloticus Fingerlings Table 2

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Growth performance and feed nutrient efficiency of Nile tilapia fed the different experimental diets. Experimental treatments

Items

Control

Yeast

D1

D2

Enzymes

D3

D4

Yeast + Enzymes

D5

D6

D7

Growth performance Initial body weight (g fish-1)

26.11 ± 0.99 a

-1

Final body weight (g fish )

170.89 ±1.55

Gain (g 119 days -1 fish -1)

e

26.36 ± 1.13 a 26.60 ± 1.65 a 197.64 ±1.06

c

213.40 ±1.58

b

26.73 ± 0.86 a

26.99 ± 1.12 a 27.13 ± 1.59 a

d

184.70 ± 1.05 213.70 ± 2.04

b

226.67 ±2.92

a

26.53 ± 0.85 a 207.57±1.43 bc

144.78 ± 1.55 e 171.28 ± 1.06 c 186.80 ± 1.58 b 157.98 ± 1.05 d 186.71± 2.04 b 199.54 ± 2.92 a 181.04 ± 1.43 b -1

-1

Average daily gain (g fish day ) -1

Specific growth rate (% day )

1.22 ± 0.07e

1.44 ± 0.11c

1.57 ± 0.18b

1.33 ± 0.08d

1.57 ± 0.18b

1.68 ± 0.23a

1.52 ± 0.15b

e

c

b

d

b

a

2.58 ± 0.17 b

2.40 ± 0.13

2.54 ± 0.39

2.61 ± 0.17

2.46 ± 0.17

2.60 ± 0.30

2.65 ± 0.21

Feed nutrient efficiency Feed intake ( g fish period-1 ) Feed conversion ratio Protein efficiency ratio Protein productive value Fat retention Energy retention

231.67 ± 1.52e 263.43 ± 1.66 c 282.24 ± 1.14 a 248.64 ± 1.98d 288.01± 1.44 a 279.47± 2.00 b 275.30 ± 1.95b 1.60 ± 0.22 e

1.54 ± 0.16 c

1.51 ± 0.15 c

1.57 ± 0.22 d

1.56 ± 0.09 b

1.40 ± 0.17 a

1.52 ± 0.15 c

e

c

bc

2.09 ± 0.08

d

ab

2.34 ± 0.09

a

2.17 ± 0.10 bc

29.71 ± 2.12

c

36.12± 1.38

a

35.31 ± 2.92 b

67.08 ± 2.80

a

60.96 ± 2.17

b

56.83 ± 2.44 c

19.54 ± 0.79

c

21.65 ± 1.60

a

21.32 ± 1.37 ab

2.03 ± 0.08 35.95 ± 1.36

b

54.68 ± 2.04

d

20.52 ± 0.77

b

2.13 ± 0.07 32.60 ± 1.15

bc

55.03 ± 1.95

c

19.10 ± 0.67

c

2.18 ± 0.08

33.96 ± 0.80 59.24 ± 2.06

b

bc

20.01 ± 0.81

b

2.25 ± 0.10

34.86 ± 1.50

b

64.71 ± 1.81

b

18.26 ± 0.83

d

Values are means ± SD of triplicate analyses. Means in the same row bearing different superscript differ significantly (P ≤ 0.05). D1: Control; D2: content 2 g S. cerevisiae yeast 100 g diet-1; D3: content 4 g S. cerevisiae yeast 100 g diet-1; D4: content 320, 640 and 80 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D5: ontent 640, 1280, 160 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D6: content mixture of 1 g S. cerevisiae yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively 100 g diet-1 and D7: content 2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, respectively100 g diet-1.

the opposite trend was recorded with the higher dietary mixture of S. cerevisiae or EDE (D7). Overall, the highest significant (P ≤ 0.05) values for FBW, SGR, ADG and SGR% was recorded for Nile tilapia fed the diet D6 (1 g yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively). In the present study, feed conversion ratio (FCR) values decreased significantly (P ≤ 0.05) with increasing S. cerevisiae and/or EDE supplemented level compared to fish fed the control diet with the optimum for fish fed the diet D6 (Table 2). The same trend was observed for highest significant (P ≤ 0.05) values for PER, PPV% and ER, while the highest value of FR was recorded for tilapia fed the diet D4. No clear trend was showed for the lowest PER, PPV, FR and ER values. Whole-body composition data are presented in Table 3. No significant (P ≥ 0.05) differences were detected among treatments for body moisture content (%) as the influence of dietary S. cerevisiae and/or EDE levels. The protein content (%) and gross energy (kJ 100g-1/ body weight) were significant (P ≤ 0.05)

higher for fish fed the control diet (D1), while the lowest body protein content (%) and the highest (P ≤ 0.05) fat content (%) were recorded for the fish fed diet D4 (320, 640 and 80 of pepsin, papain and α-amylase, respectively 100g diet-1). However, the lowest (P ≤ 0.05) values for fat and ash contents (%) were recorded for fish fed diets D5 (640, 1280, 160 mg of pepsin, papain and α-amylase, 100 g diet-1) and D7 (2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, 100 g diet-1). Hematological investigated parameters are presented in Table 4. Mean RBCs (106/μL), Hct (%), and Hb (g/dl) of Nile tilapia were significantly (P ≤ 0.05) higher with fish fed diet either D6 (1 g yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, 100g diet-1) or D7 (2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, 100 g diet-1) compared to the fish fed other experimental diets. No significant differences (P ≥ 0.05) were observed in total WBC for Nile tilapia fed BY and/or EE supplemented diets compared with fish fed the control diet, and the same observation was also recorded for WBC differential

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Table 3

Effect of Using Baker’s Yeast and Exogenous Digestive Enzymes as Growth Promoters on Growth, Feed Utilization and Hematological Indices of Nile tilapia, Oreochromis niloticus Fingerlings Chemical composition of the whole body of Nile tilapia fed the experimental diets.

Experimental treatments Control Yeast Enzymes Yeast + Enzymes D1 D2 D3 D4 D5 D6 D7 Moisture (%) 71.77 ± 0.04 73.89 ± 0.81 73.34 ± 0.32 74.04 ± 1.00 75.23 ± 0.90 73.79 ± 0.97 73.66 ± 0.25 Crude protein (%) 17.64 ± 0.47 a 15.30 ± 0.96 c 15.57 ± 0.85c 14.27 ± 0.12 d 15.48 ± 0.41 c 14.93 ± 0.07 d 17.16 ± 0.71 b Lipid (%) 5.41 ± 0.65 b 5.17 ± 0.78 b 5.41 ± 0.18 b 6.45 ± 0.01 a 3.75 ± 0.08 d 5.16 ± 0.41 b 4.40 ± 0.09 c Ash (%) 5.18 ± 0.47 b 5.64 ± 0.98 b 5.68 ± 0.06 b 5.24 ± 0.94 b 5.54 ± 0.47 b 6.12 ± 0.42 a 4.78 ± 0.78 c Gross energy 630.61 ± 1.35 565.83 ± 1.89 581.72 ± 1.95 592.08 ± 1.37 513.97 ± 1.79 556.71 ± 2.60 579.33± 1.69 (kj 100g-1) Values are means ± SD of triplicate analyses. Means in the same row bearing different superscript differ significantly (P ≤ 0.05). D1: control; D2: content 2 g S. cerevisiae yeast 100 g diet-1; D3: content 4 g S. cerevisiae yeast 100 g diet-1; D4: content 320, 640 and 80 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D5: ontent 640, 1280, 160 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D6: content mixture of 1 g S. cerevisiae yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively 100 g diet-1 and D7: content 2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, respectively100 g diet-1. Items

Table 4

Hematological characteristics of Nile tilapia after 17 weeks of feeding experimental supplemented-diets.

Experimental treatments Control Yeast Enzymes Yeast + Enzymes D1 D2 D3 D4 D5 D6 D7 RBC (106/μL) 1.91 ± 0.12 b 1.59 ± 0.14 d 1.87 ± 0.08 c 1.85 ± 0.35 c 1.83 ± 0.22 c 1.98 ± 0.11 a 1.99 ± 0.23 a a d b c b a Hct (%) 30.60 ± 1.12 25.90 ± 1.23 29.00 ± 1.55 26.30 ± 1.12 28.30 ± 1.34 30.70 ± 1.85 30.70 ± 2.10 a b a c e c c MCV (fl) 160.00 ± 0.10 163.00± 0.30 155.11 ± 0.61 142.00 ± 0. 41 153.00 ± 1.11 155 .00 ± 0.52 144.00 ± 0.22 d Hb (g/dl) 11.70 ± 0.75 b 10.61 ± 0.65 c 11.42 ± 0.90 b 11.10 ± 0.81 b 10.10 ± 0.53 d 12.21 ± 1.01 a 12.20 ± 0.15 a MCH (pg) 61.3 ± 0.6 67.4 ± 1.0 61.4 ± 0.9 60.4 ± 0.1 55.3 ± 0.6 62.4 ± 1.6 55.2 ± 1.4 MCHC (g/dl) 30.2 ± 0.2 40.9 ± 0.9 39.3 ± 0.1 32.2 ± 0.7 36.1 ± 0.2 39.7 ± 0.8 38.3 ± 0.1 WBC (105/μL) 4.90 ± 0.23 4.71 ± 0.45 5.21 ± 0.43 4.72 ± 0.18 4.51 ± .55 4.72 ± 0.13 4.51 ± 0.22 -LYM (%) 92.90 ± 0.99 87 0.1 ± 1.22 89.00 ± 1.45 86.50 ± 0.52 90.00 ± 0.71 86.20 ± 1.62 86.40 ± 1.42 -MON (%) 6 .00 ± 0.12 11.90 ± 0.22 10.00 ± 0.24 10.80 ± 0.32 6.00 ± 0.52 11.70 ± 0.09 10.74 ± 0.17 -GRA (%) 1.10 ± 0.07 1.00 ± 0.03 1.00 ± 0.08 2.70 ± 0.08 4.00 ± 0.36 2.10 ± 0.11 2.86 ± 0.09 TPP (g/dl) 2.70 ± 0.12 d 2.81 ± 0.08 c 2.91 ± 0.07 c 3.50 ± 0.10 b 3.10 ± 0.09 b 3.20 ± 0.11 b 3.61± 0.13 a c c c a d c TPA (g/dl) 1.30 ± 0.11 1.31 ± 0.07 1.30 ± 0.10 1.50 ± 0.09 1.20 ± 0.11 1.30 ± 0.09 1.41± 0.11 b d cd c b b b TPG (g/dl) 1.40 ± 0.09 1.50 ± 0.10 1.61 ± 0.07 2.00 ± 0.05 1.90 ± 0.06 1.90 ± 0.05 2.20 ± 0.10 a a b b c d c TPA/ TPG ratio 0.93 ± 0.10 0.87 ±0.18 0.81 ±0.21 0.75 ± 0.14 0.63 ±0.24 0.68 ±0.09 0.64 ±0.11 d Values are means ± SD of triplicate analyses. Means in the same row bearing different superscript differ significantly (P ≤ 0.05). D1: control; D2: content 2 g S. cerevisiae yeast 100 g diet-1; D3: content 4 g S. cerevisiae yeast 100 g diet-1; D4: content 320, 640 and 80 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D5: ontent 640, 1280, 160 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D6: content mixture of 1g S. cerevisiae yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively 100 g diet-1 and D7: content 2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, respectively100 g diet-1. RBC= Red blood cell counts; Hct = Hematocrit; Hb = Hemoglobin concentrations; MCV = Mean corpuscular volume; MCH = Mean corpuscular hemoglobin; MCHC = Mean corpuscular hemoglobin concentration; WBC = The total white blood cell; LYM = lymphocytes; MON = monocytes; GRA = granulocytes; TPP = Total plasma protein; TPA = Total plasma albumin; TPG = Total plasma globulin. Items

counts, including lymphocytes (LYM, 86.20%-90.0% vs. 92.90%), monocytes (MON, 6.00%-11.90% vs. 6.0%), and granulocytes (GRA, 1.00%-4.00% vs. 1.10%). All fish fed yeast and/or digestive enzymes had significantly (P ≤ 0.05) higher levels of TPP (g/dl) and

TPG (g/dl) compared to fish fed control diet. The TPP ranged from 2.81 to 3.61 versus 2.70 (g/ dl), and TPG 1.5-2.20 versus 1.4 (g/dl) in fish fed different experimental diets compared with those fed the control diet, respectively. The highest significant (P ≤ 0.05) values of TPP (g/dl) and TPG (g/dl) were


recorded for fish fed D6 and D7 diets. A highly positive correlation was observed between TPP and TPG (N = 5, r2 = 0.97), while a moderate and lower positive correlation was found between TPP and TPA (N = 5, r2 = 0.65) and between TPA and TPg (N = 5, r2 = 0.44). The data of the present study observed that addition of yeast and/or digestive enzyme to the experimental Nile tilapia diets improves the FCR compared to control. The same tendency was observed for economical conversion rate (ECR) values (Table 5).

4. Discussion The survival rate of Nile tilapia after 17 weeks of feeding either yeast and/or enzymes supplemented diets was 100%, this may be due to the fulfillment of dietary requirement. All the water quality parameters were within the acceptable range for Nile tilapia [59]. Data in Table 2 show that all fish fed yeast and/or digestive enzymes recorded significantly (P ≤ 0.05) higher growth than fish fed the control diet, suggesting that the addition of S. cerevisiae or EDE enhanced the growth performance. The effects of different dietary supplementation of yeast and exogenous enzyme on several fish species growth have been demonstrated previously for Channel catfish [60], Pangasius pangasius [61], Clarias batrachus x Clarias gariepinus, [62], Nile tilapia [1, 10, 13, 63-65]; rainbow trout, Oncorhynchus mykiss Table 5

23

[8] and common carp, Cyprinus carpio [29]. Tewary and Patra [66] found a superior growth performance in terms of weight gain percentage and SGR in Labeo rohita fed dietary supplemented with 5% S. cerevisiae feed compared to 7.5% and 10% levels. Tolan [67] reported that increasing total gain of Nile tilapia by increasing level of dry yeast supplementation from 1 up to 3g/kg diet. Moreover, Diab et al. [68] stated that dietary dried yeast fed for Nile tilapia from 1% up to 5% recorded high average body weight compared to fish fed control group (without yeast). The same trend was reported by Tolan and Sherif [69] for Nile tilapia. These observations determine the optimum doses of yeast, S. cerevisiae supplementation in feed, which might be helpful for optimum dietary utilization. The same trend was recorded in the present study with the dietary levels of 4%. In this connection, Singh et al. [29] reported that papain did not affect the water quality parameters and had no adverse effect on feeding response of common carp, Cyprinus carpio. The results indicate that supplementing diets with either S. cerevisiae and/or EDE significantly (P ≤ 0.05) improves feed conversion ratio (FCR), dietary protein and energy utilization compared to control diet. Similar results have been reported for S. cerevisiae used in diets for carp [70], and Nile tilapia [22]. In this connection Pooramini et al. [71] reported that the use of probiotics can decrease the amount of food necessary for animal growth, resulting in production

Economical analysis for Nile tilapia, Oreochromis niloticus fed the experimental diets.

Experimental treatments Items Control Yeast Enzymes Yeast + Enzymes D1 D2 D3 D4 D5 D6 D7 Cost of diet (kg/$)* 0.44 0.44 0.45 0.43 0.44 0.43 0.44 Feed conversion ratio 1.60 ± 0.22 e 1.54 ± 0.16 c 1.51 ± 0.15 c 1.57 ± 0.22 d 1.56 ± 0.09 b 1.40 ± 0.17 a 1.52 ± 0.15 c ECR 0.71 0.67 0.68 0.68 0.69 0.61 0.67 Values are means ± SD of triplicate analyses. Means in the same row bearing different superscript differ significantly (P ≤ 0.05). D1: control; D2: content 2 g S. cerevisiae yeast 100 g diet-1; D3: content 4 g S. cerevisiae yeast 100 g diet-1; D4: content 320, 640 and 80 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D5: ontent 640, 1,280, 160 mg 100 g diet-1 of pepsin, papain and α-amylase, respectively; D6: content mixture of 1 g S. cerevisiae yeast and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively 100 g diet-1 and D7: content 2 g yeast and 320, 640, 80 mg of pepsin, papain and α-amylase, respectively100 g diet-1. ECR = Economical conversion rate; FCR = Feed conversion ratio. ECR = Cost of diet ($ kg-1) × Feed conversion ratio (FCR) * 1$. = 5.93 LE

24


cost reductions. The addition of probiotics improved the digestibility of the diet and protein, which may in turn explain the better growth and feed efficiency seen in using supplemented diets [22]. Singh et al. [29] found that yeast increased the rate of feed intake and conversion efficiency of Labeo rohita. Swain et al. [72] and Tewary and Patra [66] found that S. cerevisiae stimulate the digestion through the supply of digestive enzymes and certain essential nutrients to the fish, where yeast it produces several enzymes, which is not, produces by the host and dietary yeast in the diet improves feed efficiency, organic phosphorus (phytic acid) utilization and fiber digestion. Exogenous application of enzyme in fish feed resulted in improvement in FCR may be attributed to fast metabolism which in turn resulted in better FCR. Singh et al. [29] reported that papain is a protease enzyme that hydrolyzes proteins to short peptides in diet, which is the key factor to increase protein digestibility and fast absorption, and helps to increase growth factors [73]. Majed and Hammad [74] reported that enzymes in yeast consist of invertase, maltase, zymase, protease and others. The protease enzyme in yeast is only active if the liberating after digestion of yeast cell wall. Recently, Singh et al. [29] reported that supplementation of papain to feed of common carp resulted in better growth rate, high protein digestibility, higher protein efficiency ratio, good gross energy retention, better apparent net protein utilization, energy conversion efficiency (%) and nitrogen retention efficiency compared to fish fed control diet (zero enzyme supplemented). Blood is a pathophysiological reflector of the whole body and therefore, blood parameters are important in diagnosing the status of fish health [75], particularly when some additives used in the feed. A highly positive correlation was observed between Hct and RBCs (N = 5, r2 = 0.91), while a moderate positive correlation was found between RBC and Hb (N = 5, r2 = 0.78) and between WBCs and MCH (N = 5, r2 = 0.69). Moreover, a moderate negative correlation was

observed between RBCs and MCH (N = 5, r2 = 0.79) and RBCs and MCV (N = 5, r2 = 0.71). A lower negative correlation was shown between Hct and MCV (N = 5, r2 = 0.29) while a lower positive correlation was observed between Hb and MCV (N = 5, r2 = 0.19). The higher hemo-concentration observed in present study for Nile tilapia fed D6 and D7 diets suggests that the optimum mixture level of S. cerevisiae and exogenous digestive enzymes (pepsin, papain and α-amylase) enhances hematological function of fish. These results could be attributed to optimal health conditions in all fish. Moreover, LYM were presented at higher proportion in the WBC of fish. The values of WBC and differential cell counts in the present study are also similar to the values obtained by Dobšíková et al. [76] for normal healthy common carp, Cyprinus carpio (L.) (5.7 105/μL, 89.5%, 1.8%, and 0.5% for WBC, LYM, MON, and GRA, respectively). The results of present study revealed that all fish fed yeast and/or digestive enzymes had significantly (P ≤ 0.05) higher levels of TPP (g/dl) and TPG (g/dl) compared to fish fed control diet. Helmy et al. [77] reported that the increase in serum protein would result when anabolic processes exceeded catabolic ones, and reserved protein is being produced in greater quantity to meet increased metabolic requirements of fish. The authors added that an increased catabolic rate would explain the decreases in serum protein level and the cyclic nature of the total serum protein is an indicator of the changes taking place in the serum globulin fraction. However, Tizard [78] reported that most techniques employed to investigate the immune state of an animal are those that depend on detection and measurement of antibody in blood serum and other body fluids. Because the source of antibody in blood serum is globulin, the total serum globulin level probably reflects the level of specific immunoglobulin (antibody) [79]. The ratio of TPA:TPG decreased when fish fed the diet supplemented with S. cerevisiae and exogenous digestive enzymes (pepsin, papain and


α-amylase) compared to control diet. Similar observation was reported for rainbow trout [18]; Gilthread Sea bream [20, 80, 81]. Tewary and Patra [66] reported that the possible role of yeast as an imunostimulant may be attributed to its cell wall which composed of lipopolysaccharide such as glucan, which enhanced phagocytic activity of macrophages and globulin level as observed in the present experiment. In this connection, Sahoo and Mukherjee [82] reported that the reduction of fish serum TPA:TPG ratios might be due to the increase of total serum globulin level which increasing significance protective mechanisms for fish. The increasing price of feed is considered one of the most important factors limiting profitability in fish culture. The economical evaluation of the present study showed that the diet D6 (1 g S. cerevisiae and 160, 320, 40 mg of pepsin, papain and α-amylase, 100 g diet-1) was the cheapest and are recommended for culturing Nile tilapia fingerlings.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

5. Conclusion The present results revealed that use of S. cerevisiae yeast and/or exogenous digestive enzymes (pepsin, papain and α-amylase) yeast as a feed additive for Nile tilapia is recommended to stimulate growth performance, nutrient utilization and some hematological variables. Improved diet utilization efficiency was observed in Nile tilapia fingerlings fed diets containing at least 1 g S. cerevisiae and 160, 320, 40 mg of pepsin, papain and α-amylase, respectively 100 g diet-1 for 17 weeks.

[10]

[11]

[12]

[13]

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