summary of phd thesis - usamv clujaurelian cosmin barbu summary of phd. thesis 12 0.2 % + the...

Aurelian Cosmin BARBU SUMMARY of PhD. THESIS

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UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICICNE

CLUJ-NAPOCA

DOCTORAL SCHOOL

FACULTY OF ANIMAL SCIENCES AND BIOTECHNOLOGY

Eng. BARBU AURELIAN COSMIN

THE USE OF FODDER ADDITIVES IN SOME

FISH SPECIES ALIMENTATION

SUMMARY OF Ph.D. THESIS

Scientific coordinator:

Prof. univ. dr. eng. AUREL ŞARA

CLUJ-NAPOCA

2010


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Part I. BIBLIOGRAPHIC DOCUMENTATION The first part of this thesis has five chapters as follows: CHAPTER I. SALMONIDS AND WELS CATFISH ALIMENATION

SPECIFICITY. This chapter present in short terms the nutritional substances digestion and

valorification particularities of salmonids and wels catfish, the digestive apparatus morphology, the phisiology of digestion and salmonids and wels catfish energy and nutritional substances demands (one of the three factors that determine the succes of a fish holding is represented by the choice of the best dietary regime for the cultured fish, diet whch must be corelated with the ecophysiological demands of the cultured speceies; regardless of the growth system and food supply, natural or artificial, the aquatic organisms nutritional demands don’t change; fish need proteins, lipids, glucids, vitamins and minerals for the normal deployment of somatic and gonadal development processes; to obtain production, meaning profit, regardless of the growth system and the cutlured species, the fish farmer must assure an optimum balance between the nutritional components according to the cultured species particularities).

CHAPTER II. THE FODDER ADDITIVES WITH STIMULATING ACTION ON THE PRODUCTIONS AND HEALTH STATUS OF FISH.

This chapter presents on short terms the fodder additives used in fish farming. To satisfy the physiological demands of cultured species, the fodders must contain a series of ingredients. The fodder additives represent a category of substances or products which are introduced in small quantities in animals food to assure some specific demands and/or to influence in a positive way (direct or undirect) the zootechnical performances and the health status (POP, 2006).

CHAPTER III. PREBIOTICS AND THEIR ROL IN FISH ALIMENTATION. Prebiotics are natural organic substances which favourises the development of useful micro organisms in the digestive tube. Prebiotics are represented by oligosaccharides, fruit and mannan oligosaccharides. Mannan oligosaccharides are yeast derivates, obtained through extraction from the cell wall of Saccharomyces cerevisiae, and it contains mannans, glucans and glicoproteins. Bio-Mos® is a mannan oligosaccharide derived from the outer cell wall of a specific strain of Saccharomyces cerevisiae which blocks the developement of pathogens acting like a sponge on whose surface pathogens can bond themselves preventing thus the pathogens development. Recently, its effectiveness in aquaculture has been established. Improvements in the growth performances and health status of several species of fish are being seen as a result of supplementing the fish feeds with mannan oligosaccharide.Bio-Mos® has also imunostimulating properties determining the improvement of organism resistence to sickness by boosting the white blood cells phagocitosis capacity. Bio-Mos® acts from the intestine, increasing the absorbtion area through the levelling of the microvilli layer, improving the nutrient usage and the health status, all those leading to a better somatic development (TORECILLAS et all., 2006; DIMITROGLOU, 2005).


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CHAPTER IV. PROTEIC PRODUCT NUPRO® AND ITS ROL IN FISH ALIMENTATION. A new trend in fish nutrition is to replace the animal protein from fish feeds. One of the alternatives is NuPro®. NuPro® has been shown to have significant benefits as a functional nutrient. NuPro® represents a rich source of nucleotides and amino acids with attractant and feeding stimulation roles in fish.. Some researches made showed the efficiency of some yeast extracts, extracts that can be a viable alternative protein source, an ecological source that can successfully replace the animal and vegetal protein (fish and soy meal) from fish fodder structures (LI and GATLIN, 2004).

CHAPTER V. ORGANIC MINERALS AND THEIR ROL IN FISH ALIMENTATION.

The researches made worldwide in the nutrition area showed the bioavalibility and the increased biological activity of organic minerals comparative with the anorganic forms (MCDOWELL, 2003; SARKAR et all., 2005). Among organic minerals of highly importance in fish nutrition is selenium. In the biological system of fish Selenium fulfills several functions, but its main role consists in the fact that it is a component of the glutathione peroxidase, an enzyme that detoxifies the lipid peroxides and protects the cell membranes and also it can act like an antioxidant if it has a supply of vitamin E. In fish the organic Selenium maintains fish health, assures the improvement of survival percentage and the hatching percent (Wang et al, 1997).

PART II.

PERSONAL RESEARCHES CHAPTER VI. GENERAL EXPERIMENTS ORGANIZATION

6.1.RESEARCHES GOAL AND THE OBJECTIVES PROPOSED The goal of these researches was to pinpoint the effects of some fodder additives

(prebiotic Bio-Mos® 0.2 %, organic mineral SelPlex® 0.03 % and proteic extract NuPro® 2 %) on the productive performances and health of salmonids (Salvelinus fontinalis M. and Onchorhynchus mykiss W.) raised in a classic system in production conditions and the evaluation of the effects of prebiotic Bio-Mos® 0.2 % and proteic extract NuPro® 2 % on the productive performances and health of wells catfish (Silurus glanis L.) raised in a recirculating system in experimental conditions.

In the first experiment “RESEARCHES REGARDING THE EFFECTS OF SOME FODDER ADDITIVES (BIO-MOS®, SELPLEX® AND NUPRO®) ON THE PRODUCTIVE PEFORMANCES AND HEALTH OF BROOK TROUT (Salvelinus fontinalis M.)” it was followed the effect of the prebiotic Bio-Mos® 0.2 %, organic mineral SelPlex® 0.03 % and proteic extract NuPro® 2 % on the brook trout (Salvelins fontinalis M.) growth and consumption indices, hematological and immunological indices, meat quality and to pinpoint the effect of Bio-Mos® at intestinal level.

In the second experiment “RESEARCHES REGARDING THE COMBINED FODDER ADITIVES (BIO-MOS®+SELPLEX®+NUPRO®) EFFECTS ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF RAINBOW TROUT (Oncorhynchus mykiss W.)” it was followed the combined effect of the prebiotic Bio-Mos®


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0.2 % + the organic mineral SelPlex® 0.03 % + the proteic extract NuPro® 2 % on the rainbow trout (Oncorhynchus mykiss W.) growth and consumption indices, hematological indices and on the meat quality as well as to pinpoint the effect of Bio-Mos® at intestinal level. The third experiment “RESEARCHES REGARDING THE EFFECTS OF SOME FODDE ADDITIVES (BIO-MOS® AND NUPRO®) ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF WELS CATFIFH (Silurus glanis L.)” it was followed the effect of the prebiotic Bio-Mos® 0.2 %, and proteic extract NuPro® 2 % on the wells catfish (Silurus glanis L.) growth and consumption indices, hematological and immunological indices.

Table 1 EXPERIENCES ORGANIZATION SCHEME

Issue Experimental variants

Species Number of

fish/group

The variable

Control group

Brook trout (Salvelinus

fontinalis M.)

250

-

Experimental group I (Bio-

Mos)


fontinalis M.)

250

Prebiotic Bio-Mos

0.2 % Experimental

group II (NuPro)


fontinalis M.)

250

Proteic extract

NuPro 2 %

Experiment I - RESEARCHES

REGARDING THE EFFECTS OF SOME

FODDER ADDITIVES (BIO-MOS, SELPLEX AND

NUPRO) ON THE PRODUCTIVE

PEFORMANCES AND HEALTH OF BROOK

TROUT (Salvelinus fontinalis M.)

Experimental group III (Sel-

Plex)


fontinalis M.)

250

Organic Selenium SelPlex 0.03%

Control group

Rainbow trout (Oncorhynchus

mykiss W.)

250

-

Experiment II - RESEARCHES

REGARDING THE COMBINED FODDER

ADITIVES (BIO-MOS+SELPLEX+NUPRO)

EFFECTS ON THE PRODUCTIVE

PERFORMANCES AND HEALTH OF RAINBOW TROUT (Oncorhynchus

mykiss W.)

Experimental group (Bio-

Mos + SelPlex + NuPro)

Rainbow trout (Oncorhynchus

mykiss W.)

250

Bio-Mos 0.2 % + SelPlex 0.03% + NuPro 2

%


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Table 1 continuing Issue Experimental

variants Species Number

of fish/group

The variable

Control group

Wels catfish (Silurus

glanis L.)

50

-

Experimental group I (Bio-

Mos)


glanis L.)

50

Prebiotic Bio-Mos

0.2 %

Experiment III - RESEARCHES

REGARDING THE EFFECTS OF SOME FODDE ADDITIVES

(BIO-MOS AND NUPRO) ON THE

PRODUCTIVE PERFORMANCES AND HEALTH OF WELS CATFIFH (Silurus glanis L.)

Experimental group II (NuPro)


glanis L.)

50

Proteic extract

NuPro 2 %

6.2. MATERIALS AND METHODS

6.2.1. The biological material. The biological material was represented by three fish species, two salmonids species

(brook trout, Salvelinus fontinalis M. and rainbow trout, Oncorhynchus mykiss W.) and wels catfish (Silurus glanis L.). The researches from the first experiment had been made on 1000 fish (Salvelinus fontinalis M.) from ICAS Gilău, Cluj county, divided into four groups 250 fish/group. At the beggining of the trial brook trout juveniles had 20 g body weight and 12 cm body lenght. The experimental period was 250 days. The researches from the second experiment had been made on 500 fish (Oncorhynchus mykiss W.) from ICAS Gilău, Cluj county, divided into two groups 250 fish/group. At the beggining of the trial rainbow trout juveniles had 10 g body weight. The experimental period was 161 days. The researches from the third experiment had been made on 150 fish (Silurus glanis L .) from Budurleni Fish Farm, Bistrita-Nasaud county, divided into three groups, 50 fish/group. At the beggining of the trial wels catfish juveniles had an age of 8 months, 110 g body weight and 20 cm body lenght. The experimental period was 150 days

6.2.2. The growth system Salmoinds were raised in a classic system in concrete tanks (4 x 1 x 1.5 m) in

identical enviromental conditions. The experiments took place in concrete tanks which offered the same rearing conditions (the same water quality, rearing density, feeding hours ad food quantity). The water inflow was 25 l/min/tank. Wels catfish juveniles were raised in a recirculating aquacultural system in controled enviromental conditions.

6.2.3. The water physico-chemical characteristics The characteristics of water have been monitored on a daily basis during the entire

period with a pH tester (model HI 8424, range -2.00 – 16.00 upH, accuracy ±0.01pH, Hanna Instruments) and a portable oxymeter (model HI 9146-04 range 0.00-45.00 mgO2/l). The characteristics of water have been supervised on a daily basis during the entire period and


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water analyses were made according to the known methods (ŞTEłCA şi HEGHEDUŞ, 2008; LUJERDEAN şi BUNEA, 2005). The supervised and analysed water physico-chemical characteristics were represented by dissolved oxygen, water pH, water temperature, nitrates and nitrites concentrations, chloride concentration and organic substances concentration.

6.2.4. The administered fodder. The administerd fodder was represented by the combined granulated fodder

Skretting® Classic Extra for salmonids and a romanian combined granulated fodder for wels catfish. The salmonids fodder had the following nutritional characteristics: 41 % CP, 12 % Cfat, 7.8 % Ash and 2.5 % Cellulose. The wels catfish fodder had the following nutritional characteristics: 48.90 % CP, 1.67 % Cellulose, 11.13 % Ash and 14.95 % CFat with a diammeter of 2 and 4 mm. In the first trial, every group received the same basal diet, 41 % CP, 12 % Fat, 2,5 % Cellulose. Every experimental group received one additive:

▪ Bio-Mos® 0,2 % (bacteriostatic and stimulator of phagocitosis and immunity, extracted from Saccharomices cerevisiae)at experimental group 1.

▪ NuPro® 2 % (stimulator of the fodder consumption, made from free aminoacids, glutamic acid, nucleotids and tripeptids) at experimental group 2. ▪ SelPlex® 0,03 % (stimulator of several functions of the biological system of fish, component of the glutathione peroxidase, an enzyme that detoxifies the lipid peroxides and protects the cell membranes) at experimental group 3. All the batches received two meals per day, at 8.30 am and 5 pm. In the second trial the experimental group recieved the combined fodder additives in its granulated foder (Bio-Mos® 0.2 % + SelPlex® 0.03 % + NuPro® 2 %).

Table 2 The structure and nutritional value of the salmonids granulated combined fodder.

Specification U.M. Value Fish meal % 31

Soybean meal NGMO % 39 Wheat meal % 20

Fish oil % 4,5 Soybean oil % 4

Vitamino-mineral premix % 1,5 Nutritional characteristics

Crude Protein % 41 Crude Fat % 12

Ash % 7,8 Cellulose % 2,5 Phosphor % 1,1 Copper mg 6 Vit. A U.I. 10 000 Vit. D3 U.I. 1 250 Vit. E mg 150


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Classic Extra fodder ingredients: fish meal, NGMO soybean meal, wheat meal, fish oil, soybean oil, vitamins and minerals.

In the third trial, the first experimental group received in its fodder Bio-Mos® 0.2 % and the second experimental group received in its fodder NuPro® 2 %. The wels catfish fodder had the following nutritional characteristics: 48.90 % CP, 1.67 % Cellulose, 11.13 % Ash and 14.95 % CFat with a diammeter of 2 and 4 mm. Proteic content of the fodder eas established through the known methods (ŞARA and ODAGIU, 2005).

Table 3 Wels catfish combined granulated fodder structure.

Control group Group E1 (Bio-Mos 0,2 %)

Group E2 (NuPro 2 %)

Issue

% % % Fish meal I class (FF

Skagen Austral) 40 39,80 38

Bio-Mos 0,2 NuPro 2

Fish meal II class II (Peru)

15 15 15

Soybean meal 19 19 19 Premix 3 3 3 Total 100 100 100

Nutritional characteristics Pb % 48,90 48,90 48,90 Gb % 14,95 14,95 14,95

Cenuşă % 11,13 11,13 11,13 Celuloză % 1,67 1,67 1,67

6.2.5. The working methods

During the trials the evolution of the main growth indices was followed through periodical measurements as well as the survival rate and the fodder consumption.

Blood samples were collected from the caudal vein using 2 ml desposable plastic syringe. The blood samples were placed into vacutaineers tubes with EDTA and Li-Heparine (to determine the glutathion-peroxidase concentration) . The haematological investigations, based on known methods (OGNEAN et al., 2007), included: microhaemotocrit (Ht), haemoglobin (Hb), the total count of erythrocytes (E), among the blood morphological exam on MGG blood smears. Glutathion-peroxidase concentration had been determined with Screen master Touch spectrometre through kinetic method in UV domain at 340 nanometres lambda wavelenght using GPx reactive from Randox Laboratories UK.

The chemical composition of meat, based on known methods (ŞARA and ODAGIU, 2005) followed CP content, Humidity, Fat and Dry Matter content.

The Selenium content of brook trout meat was determined through atomic absorbtion spectrophotometry (SAA-HVG) with the Shimadzu AAS-6300 spectrophotometer.


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For the histological exam two intestine samples were took from 2 and 8 cm from the stomach. The tissue samples (intestinal mucosa) were kept in 10 % formol, pH 7 for 24 hours, than the samples had been proccesed with Leica RM 2125 RT microtome and HE coloured and than the samples were examed with Olympus BX 51 microscope. The images were taken with Olympus SP 350 digital camera and analysed with Olympus DP soft.

Lysozime concentration was determined through lamella diffusion method. Total imunoglobulines were determined through Serb reactive precipitation test. The circulant imune complexes were determined through Haskova method.

The experimental data had been analyzed with GraphPad Instat 3 and the meaning of the differences between groups was established using Tukey-Kramer multiple comparissons test, Dunn test and Kruskall-Wallis test

CHAPTER VII. RESEARCHES REGARDING THE EFFECTS OF SO ME FODDER

ADDITIVES (BIO-MOS ®, SELPLEX® AND NUPRO®) ON THE PRODUCTIVE PEFORMANCES AND HEALTH OF BROOK TROUT ( Salvelinus fontinalis M.)

The goal of this research was to pinpoint the effects of some fodder additives (prebiotic Bio-Mos® 0.2 %, organic mineral SelPlex® 0.03 % and proteic extract NuPro® 2 %) on the productive performances and health of brook trout (Salvelinus fontinalis M.). It was followed the effect of the prebiotic Bio-Mos® 0.2 %, organic mineral SelPlex® 0.03 % and proteic extract NuPro® 2 % on the brook trout (Salvelinus fontinalis M.) growth and consumption indices, hematological and immunological indices, meat quality and the effect of Bio-Mos® at intestinal level.

The researches from the first experiment had been made on 1000 fish (Salvelinus fontinalis M.) from ICAS Gilău, Cluj county, divided into four groups 250 fish/group. The first experimental group recived Bio-Mos® 0,2 % (bacteriostatic and stimulator of phagocitosis and immunity, extracted from Saccharomices cerevisiae) . The second experimental group received NuPro® 2 % (stimulator of the fodder consumption, made from free aminoacids, glutamic acid, nucleotids and tripeptids) and the third experimental group received SelPlex® 0,03 % (stimulator of several functions of the biological system of fish, component of the glutathione peroxidase, an enzyme that detoxifies the lipid peroxides and protects the cell membranes). All the batches received two meals per day, at 8.30 am and 5 pm. At the beggining of the trial brook trout juveniles had 20 g body weight and 12 cm body lenght. The experimental period was 250 days.


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Table 4 The general organization scheme of the first trial RESEARCHES REGARDING THE EFFECTS OF SOME FODDER ADDITIVES (BIO-MOS ®, SELPLEX® AND NUPRO®) ON THE PRODUCTIVE PEFORMANCES AND HEALTH

OF BROOK TROUT ( Salvelinus fontinalis M.) Experimental

period Experiment

location Groups

(fish/group) Species Experimental

factors

Followed indicators

21.08.2008 –28.04.2009 (250 days)

ICAS trout farm

Gilău, Cluj county

Control (250)

1E (Bio-Mos 0.2%)

(250)

2E (NuPro 2 %)

(250)

3E (SelPlex 0.03 %)

(250)


fontinalis M.)

-

Prebiotic Bio-Mos

0.2%

Proteic extract NuPro 2%

Organic Selenium SelPlex 0.03%

-biometrical and growth indices evolution - feed conversion rate - survival rate - hematological indices immunological indices - histological indices - meat chemical composition and meat Selenium content.


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7.1. The effects of the fodder additives (Bio-Mos®, SelPlex® and NuPro®) on the growth and consumption indices of brook trout (Salvelinus fontinalis M.).

The trout weighing was made individualy at the beggining of the trial, at the end of the trial and during the trial to observe the growth dynamic of the trouts. The average values of trout body weight at the end of the trial are listed in the table below:

Table 5 The initial and final body weight of brook trout

Group

n

Initial body weight (g/fish)

Final body weight (g/fish) _ _ X ± Sx

v %

Minimum body

weight (g)

Maximum body

weight (g)

Control

30

20

204.65±5.90

12.90

153

250

1 E (Bio-Mos

0.2%)

30

20

xx

238.55±7.97

14.94

170

314

2 E (NuPro

2%)

30

20

225.25±6.21

12.33

179

287

3 E (SelPlex 0.03%)

30

20

212.2±7.14

15.04

153

279

xx - P < 0,01

Analyzing the body weight of brook trout it can be seen that the experimental group 1 (Bio-Mos 0.2%) riched an average body weight of 238.55 g/specimen with 16.56 % higher than the average body weight of the Controls (204.65 g/specimen). This result is similar with the one reported by Staykov et al., 2005 in rainbow trout (13.7 % higher average body weight). At the other experimental groups (NuPro and SelPlex) were also registered superior values comparative with the Control group regarding the final body weight (225.25 g/specimen and 212.20 g/specimen vs 204.65 g/specimen in Control group). All the groups, Control and experimental groups showed a middle variability (10% < v% < 20%). The body weight evolution during the trial showed that the introduction of Bio-Mos® and NuPro® in the combined granulated fodder structure had a positive influence on the growth dynamic of brook trout, fact reported by other reserchers too (CRAIG and MCLEAN, 2005; HOSSU et all., 2005; DIMITROGLOU et all., 2005; TORRECILLAS et all., 2006). To have a realistic apraisal of the growth dynamic of brook trout, at the end of the trial was determined the growth rhytm (final body weight – initial body weight) and the specific growth rate (g/day) this one bing the main indicator of the biomass acumulation rhythm assessment.


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Table 6 The growth and consumption indices values at the end of the trial at brook trout.

Issue U.M. Control group

Group 1 E (Bio-

Mos 0.2%)

Group 2 E

(NuPro 2%)

Group 3 E (Sel-

Plex 0.03%)

g 184.45 218.35 205.05 192 Growth rate % 100 118.37 111.16 104.09

g/day 0.73 0.87 0.82 0.76 Specific growth

rate % 100 119.17 112.32 104.01

FCR (feed conversion

rate)

kg fodder/kg

body weight

1.57:1

1.48:1

1.65:1

1.59:1

Analyzing the main production indices recorded during the trial it can be seen the favourable influence of the fodder additives administered in brook trout feed, Bio-Mos® 0.2 %, SelPlex® 0.03 % and NuPro® 2 % on the growth rate. The growth rate incresed by 18.37 % at the experimental group 1 comparative with the Control group (218.35 g versus 184.45 g value recorded at the Control group). Also in groups 2E and 3E there was noticed an increasement of the growth rate by 11.16 % and 4.09 %. Analyzing the specific growth rate it can be noticed that the highest value was recorded at group 1E, 0.87 g/day, with 19.17 % higher than the specific growth rate of the Controls. Also in groups 2E and 3E there was noticed a slightly increasement of the specific growth rate, 0.82 g/day at group 2E and 0.76 g/day at group 3E, values higher than the Control (0.73 g/day). Analyzing the feed conversion rate one can see that its values are characteristic to the values recorded at trout. The addition of Bio-Mos® 0.2 % in group 1E feed led to an abatement of the feed conversion rate at group 1E (1.48:1) by 5.74 % comparative with the Control group (1.65:1). This abatement of the feed conversion rate as a response to Bio-Mos® feed supplementation was reported to other fish species in whose diet Bio-Mos® was introduced in proprotion of 0.2 % (STAYKOV et all., 2005; CULJAK et all., 2006; BOGUT et all., 2006)

7.2. The effects of the fodder additives (Bio-Mos®, SelPlex® and NuPro®) on the histological indices of brook trout (Salvelinus fontinalis M.).

The researches made on Diplodus sargus (DIMITROGLOU et all., 2005), and Salmo salar revealed the influence of Bio-Mos® at intestinal level. The addition of Bio-Mos® in this species feed led to an uniformization of the villi structures, to an increasment of their lenght and to the increasement of the microvilli lenght too. To reveal the effects of Bio-Mos® at intestinal level, there have been made a few histological investigations: the measurement of the villi and microvilli lenght. The data regarding the brook trout villi lenght are listed in the table below:


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Table 7 The brook trout intestinal villi lenght values (µm).

Issue Control group

Group 1E (Bio-Mos 0.2 %)

Group 2E (NuPro 2 %)

Group 3E (SelPlex 0.03 %)

n 68 69 24 31

_ _ X±Sx

361.95±23.07

402.78±18.38

391.78±21.71

368.15±19.33

v % 13.44 17.90 17.15 19.23 P>0,05

From the values presented in the table above it can be seen an increasement of the villi lenght at group 1E (Bio-Mos 0.2 %) with 11.28 % higher comparative with the Control group. Thi sdevelopment of the villi structures reported also by Sweetman et all., 2008 at rainbow trout, determined the enlargement of the nutrient absorbtion area, fact that explains the superior growth rhytm of the Bio-Mos® treated group. Also it can be observed a slightly increasement of the villi lenght at the other experimental groups, by 8.24 % at group 2E (NuPro 2%) and by 1.71 % at group 3E (SelPlex 0.03%). Regarding the brook trout microvilli lenght it has increased from 2.06 µm at 3.16 µm at group 1E, as a result of entherocytes stimulation with mannanoligosaccharids. This stimulation determined a uniformization of the microvilli brush border and an increased lenght of them.

Table 8 The brook trout microvilli length (µm) values.

Issue Control

group Group 1E (Bio-Mos 0.2 %)



n 50 50 38 46 _ _

X±Sx 2.06±0.11

xxx

3.16±0.06 xxx

2.82±0.13 xx

2.56±0.09

v % 17.86 13.92 19.78 14.60 xxx - P<0.001; xx - P<0.01

7.3. The effects of the fodder additives (Bio-Mos®, SelPlex® and NuPro®) on the survival rate of brook trout (Salvelinus fontinalis M.).

One of the most important indices followed in a fish farm is the survival rate of the biological material. During the trial this aspect was followed and at the end of the trial the final brook trout survival rate was established.


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Table 10 The brook trout survival percent and the losses recorded during the trial.

Issue Control group

Group 1 E (Bio-Mos

0.2%)

Group 2 E (NuPro

2%)

Group 3 E (Sel-Plex 0.03%)

Initial number of fish

250

250

250

250

Final number of fish

193

207

200

236

Survival (%) 77.2 82.8 80.0 94.4

Losses (%) 22.8 17.2 20.0 5.6

Analyzing tha data from the table above it can be seen an improvement of the survival rate at the experimental groups, +5.6 % at group 1E ( Bio-Mos 0.2%), +2.8 % at group 2E (NuPro 2%) and +17.6 % at group 3E (SelPlex 0.03%), results similar with the ones reportde by YAMBO et all., 2006.

7.4. The effects of the fodder additives (Bio-Mos®, SelPlex® and NuPro®) on the hematological indices of brook trout (Salvelinus fontinalis M.). The corelation and the analysis of indices regarding the quantification of the

health status (hematological indices) and the survival and losses, shows the benefical biological effects of the additives that had been incorporated in the basal diet of brook trout. (SAKAI et all., 1996; VERBURG et all., 1994).

Table 11 The hematological indices values of

brook trout recorded at the end of the trial (n=20)(X±Sx).

The benefical biological effects of the fodder additives that have been

incorporated in the basal diet of brook trout are exprimated through the annealing of leucogramme and the abatement of losses recorded, noticing the high value of lymphocites (79-92 %) at the groups that were fed with additives comparative with the Control group where neutrophillia (30 %) associated with a higher percent of losses was recorded. The hematological indices values recorded at the end of the trial can be

Issue Control group

Group 1E (Bio-Mos)

Group 2E (NuPro)

Group 3E (Sel-Plex)

Neutrophils (%) 28.60±8.15 5.94±1.68 5.40±1.03 3.60±0.67 Eosinophils (%) 0.50±0.22 0.20±0.15 0 0 Basophils (%) 0.60±0.40 0.20±0.20 0 0.20±0.20 Lymphocytes (%)

53±12.07 82±2.09 93.40±0.67 90.6±1.36

Monocytes (%) 12±1.54 11.40±1.74 3.40±1.16 6.80±0.80 Hematocrit (%) 40.80±1.77 39.60±3.02 41±1.44 41.60±2.42 Hemoglobin (g/dl)

7.30±0.45 7.08±0.90 6.66±0.36 6.12±0.30

Erythrocytes (x 103)

2.12±0.06 2.04±0.07 2.09±0.08 2.07±0.07


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placed in the normal physiological range of the salmonids (SVOBODOVA, 1991; MODRÁ et all., 1998).

Glutathion-peroxidase (GPx) is an enzyme involved in the body defense against free radicals action. The main role of GPx is to stop the propagation of radicals reactions. In the active situs of GPx was identified Se, which means that Se has a major role in the prevention of free radicals action.

Table 12 Gluthation-peroxidase values of brook trout.

_________________GPx (UGPx/ml PCV)___________________________________ Control group Group 3E (SelPlex 0.03%)_______ _ _ X ± Sx 4.34 ± 0.57 5.63 ± 0.28_______________ PCV = hematocrit. P>0.05

The high blood levels of glutathion-peroxidase recorded at fish fed with SelPlex 0.03 % (5.63 ± 0.28), comparative with those registered at the Control group (4.34 ± 0.57 ), is an extra evidence of the biological benefical effects of Selenium in fish. The values obtained show an improvement of GPx level with 29.7 % at group 3E as a response to the supplementation of their feed with organic Selenium in form of SelPlex®.

7.5. The effects of the fodder additives (Bio-Mos®, SelPlex® and NuPro®) on the imunological indices of brook trout (Salvelinus fontinalis M.). To explain the superior survival rates recorded at the experimental groups and

to show the imunostimulator role of the fodder additives that have benn used in brook trout diet, there have been made some imunological investigations (the establishment of the lysozime concentration and the total imunoglobulins concentration).

Table 13 Brook trout lysozime concentration (µg/ml)

P>0.05

The increasement of lysozime concentration at the experimental groups (+7.52 % at group 1E, 19.57 % at group 2E and +11.06 % at group 3E) shows the positive influence of the used additives on the humoral body defense factors (substances with antiviral action, bactericid and fungic action present in tissues and in internal liquids, lysozime, alexin, interferon and properdin), from which one of the most importance is the lysozime.

Issue Control group




n 5 5 5 5 _ _

X±Sx 9.04±0.17

9.72±0.51

10.81±0.17

10.04±0.71

v % 4.42 11.83 3.51 15.93


23

Table 14 Total immunoglobulines concentration (Vernes degrees) of brook trouts Issue Control

group Group 1E (Bio-Mos 0.2 %)



n 6 7 7 7 _ _

X±.Sx 4.31±0.70

3.59±0.30

3.97±0.20

4.04±0.50

v % 19.44 12.28 17.63 17.12 Total immunoglobulines concentration represents an indicator of the humoral

reactivity which characterizes the specific imune system. The values recorded show a normal status of the specific imune system, the differences recorded being unsignificant statisticaly.

7.6. The effects of the fodder additives (Bio-Mos®, SelPlex® and NuPro®) on the meat quality of brook trout (Salvelinus fontinalis M.).

Table 15 The chemical composition of brook trout meat (n=20).

Issue D.M. % Humidity % CP % CFat % Control 24.74±0.4 75.25±0.4 17.59±0.33 6.30±0.36


26.05±0.44

73.94±0.44

18.04±0.37

6.80±0.15


25.92±0.39

74.07±0.39

17.57±0.28

6.72±0.29


25.76±0.55

74.22±0.56

18.09±0.40

6.40±0.47

P>0.05

From the data presented in the table above it can be seen that the addition of Bio-Mos®, NuPro® and SelPlex® in brook trout feed did not have a major impact on the chemical composition of brook trout meat, the differences recorded being insignificant. The values obtained are similar with the ones given in the literature (Bud I. and Vioara Miresan, 2008; Stephen J. et al., 2005).

Analyzing the values from the table above it can be seen a slight increasement of the protein content of meat at groups 1E and 3E, 18.04 % CP at group 1E (Bio-Mos 0.2%) and 18.09% CP at group 3E (SelPlex 0.03%) and an abatement of the water content, 73.94 % at group 1E (Bio-Mos 0.2%) respectiv 74.07 % at group 2E (NuPro 2%) and 74.22 % at group 3E (SelPlex 0.03%) comparative with 75.25 % at Control group.

From the data presented in the table below it can be seen a very significant

improvement of selenium content of brook trout meat, with 287.15 % higher than the Control group (238.99 µg/kg comparative with 61.73 µg/kg) as a response to brook trout diet supplimentation with organic selenium in form of SelPlex®.


24

Table 16

The brook trout meat Selenium content (n=10). ____________________________Se (µg/kg)__________________________ Control group Group 3E (SelPlex 0.03%)____ _ _ xxx X ± Sx 61.73 ± 2.67 238.99 ±7.95____________ xxx – P < 0.001

This high content of brook trout meat in Selenium can be a first step in the making of a functional food, brook trout meat enriched with Selenium.

7.7. Economical efficiency considerations In fish farming industry, the fodder costs have the highest proportion from the

total production cost. To achieve positive production performances is highly necessary to assure a quantitative and qualitative feed level that coresponds with the ecophisyological requirements of the farmed species. A major role in assuring this feed level can be played by the fodder additives. Improving the production performances of the cultured species can also improve the economical indicators. The economical effect of Bio-Mos addition in brook trout feed was a plus of 2,64 RON/5 fish. The economical effect of NuPro addition in brook trout feed was a plus of 1,35 RON/5 fish and the economical effect of SelPlex addition in brook trout feed was a plus of 0,61 RON/5 fish.

7.8. PARTIAL CONCLUSIONS • Through the use of prebiotic Bio-Mos® 0.2 %, proteic extract NuPro® 2 % and organic mineral SelPlex® 0.03 % in brook trout feed had been showed their biostimulating effects exprimated through the improvement of brook trout main production and health indices.

▪ Regarding the brook trout trial, the best results regarding the body weight evolution were recorded at group 1E as a result of supplementing its feed with mannanoligosaccharids in form of Bio-Mos® 0.2 %.

▪ The growth rate showed an improvement by 18.37 % at group 1E (Bio-Mos 0.2%) respectiv 11.16 % at group 2E (NuPro 2%) comparative with the Control group.

▪ Regarding the survival rate it has benn noticed the positive effect of organic Selenium. At group 3E (SelPlex 0.03 %) was recorded the highest survival rate, 94.4 %, with 17.6 % higher than the Control group survival rate.

▪ The benefical biological effects of the fodder additives that have been incorporated in the basal diet of brook trout are exprimated through the annealing of leucogramme and the abatement of losses recorded, noticing the high value of lymphocites (79-92 %) at the groups that were fed with additives comparative with the Control group where neutrophillia (30 %) associated with a higher percent of losses was recorded

▪ The high blood levels of glutathion-peroxidase recorded at fish fed with SelPlex 0.03 % (5.63 ± 0.28), comparative with those registered at the Control group (4.34 ± 0.57 ), is an extra evidence of the biological benefical effects of Selenium in fish


25

▪ The addition of Bio-Mos®, NuPro® and SelPlex® in brook trout feed did not have a major impact on the chemical composition of brook trout meat. It was observed a slight increasement of the protein content of meat at groups 1E and 3E, 18.04 % CP at group 1E (Bio-Mos 0.2%) and 18.09% CP at group 3E (SelPlex 0.03%) and an abatement of the water content, 73.94 % at group 1E (Bio-Mos 0.2%) respectiv 74.07 % at group 2E (NuPro 2%) and 74.22 % at group 3E (SelPlex 0.03%) comparative with 75.25 % at Control group.

▪ Regarding the meat Selenium content, it was noticed a very significant improvement of the selenium content of brook trout meat, with 287.15 % higher at group 3E than the Control group (238.99 µg/kg comparative with 61.73 µg/kg) as a response to brook trout diet supplimentation with organic selenium in form of SelPlex®

▪ Regarding the histological indices, it was noticed an increasement of the villi lenght at group 1E (Bio-Mos 0.2 %) with 11.28 % higher comparative with the Control group (402.78 µm at group 1E vs 361.95 µm at Control group). This development of the villi structures reported also by Sweetman et all., 2008 at rainbow trout, determined the enlargement of the nutrient absorbtion area, fact that explains the superior growth rhytm of the Bio-Mos® treated group.

▪ Regarding the brook trout microvilli lenght it has increased from 2.06 µm at 3.16 µm at group 1E, as a result of enetrocytes stimulation with mannanoligosaccharids. This stimulation determined a uniformization of the microvilli brush border and an increased lenght of them.

• The economical effect of Bio-Mos, NuPro and SelPlex addition in brook trout feed represented a plus of 2,64 RON/5 fish at Bio-Mos treated group, 1,35 RON/5 fish at NuPro treated group and 0,61 RON/5 fish at organic selenium treated group.

CHAPTER VIII. RESEARCHES REGARDING THE COMBINED FOD DER

ADITIVES (BIO-MOS ®+SELPLEX®+NUPRO®) EFFECTS ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF RAINBOW TROUT

(Oncorhynchus mykiss W.). In the second experiment “RESEARCHES REGARDING THE COMBINED

FODDER ADITIVES (BIO-MOS®+SELPLEX®+NUPRO®) EFFECTS ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF RAINBOW TROUT (Oncorhynchus mykiss W.)” it was followed the combined effect of the prebiotic Bio-Mos® 0.2 % + the organic mineral SelPlex® 0.03 % + the proteic extract NuPro® 2 % on the rainbow trout (Oncorhynchus mykiss W.) growth and consumption indices, hematological indices and on the meat quality as well as to pinpoint the effect of Bio-Mos® at intestinal level. The researches from the second experiment had been made on 500 fish (Oncorhynchus mykiss W.) from ICAS Gilău, Cluj county, divided into two groups 250 fish/group. The experimental group received Bio-Mos® 0.2 % (bacteriostatic and stimulator of phagocitosis and immunity, extracted from Saccharomices cerevisiae) + NuPro® 2 % (stimulator of the fodder consumption, made from free aminoacids, glutamic acid, nucleotids and tripeptids) +SelPlex® 0.03 % (stimulator of several functions of the biological system of fish, component of the glutathione peroxidase, an enzyme that detoxifies the lipid peroxides and protects the


26

cell membranes). The both groups (Control and Experimental) received two meals per day, at 8.30 am and 5 pm. At the beggining of the trial rainbow trout juveniles had 10 g body weight.. The experimental period was 161 days.

Table 17 General organisation of the second experiment Researches regarding the combined

effect of some fodder additives (Bio-Mos+SelPlex+NuPro) on the productive performances and the health status of rainbow trout (Oncorhynchus mykiss W.) Experi- mental period

Experiment location

Groups (fish/group and species)

Experimental factors

The followed indicators

05.05.2009-30.09.2009

ICAS Gilău, trout farm, Cluj county

Control (250 fish, rainbow

trout)

E (250 fish, rainbow trout)

-

Addition of 0.2 %Bio-Mos + 2 % NuPro

+ 0.03 % SelPlex in the

granulated combined

fodder

-biometrical and growth indices evolution - feed conversion rate - survival rate - hematological indices - histological indices - meat chemical composition

8.1. The combined fodder additives (Bio-Mos®+SelPlex®+NuPro®) effects on the

growth and consumption indices of rainbow trout(Oncorhynchus mykiss W.). The aim of the addition of the fodder addititves in the combined granulated

fodder it was to obtain superior bioproductive indices at the experimental group.

0

50

100

150

200

250

300

m aig/exem plar

iunieg/exem plar

iulieg/exem plar

augustg/exem plar

septem brieg/exem plar

M

E

Fig. 2. The rainbow trout body weight evolution during the trial Analyzing the body weight evolution chart during the trial it can be seen that

the introduction of Bio-Mos®, NuPro® and SelPlex® in the combined granulated fodder structure had a positive influence on the growth dynamic of rainbow trout, fact reported by other reserchers too (CRAIG and MCLEAN, 2005; HOSSU et all., 2005; DIMITROGLOU et all., 2005; TORRECILLAS et all., 2006). At the end of the trial, the experimental group had an average body weight of 253.20 g with 4.81 % higher than the final average body weught of the Control group (241.10 g).


27

To have a realistic apraisal of the growth dynamic of rainbow trout, at the end of the trial was determined the growth rhytm (final body weight – initial body weight) and the specific growth rate (g/day) this one bing the main indicator of the biomass acumulation rhythm assessment.

Table 18 The growth and consumption indices values at the end of the trial at rainbow trout

Issue U.M. Control group

Experimental group (Bio-Mos 0.2%+ NuPro 2%+ SelPlex 0.03%)

g 231.1 243.2 Growth rate % 100 105.23

g/day 1.43 1.51 Specific growth rate % 100 105.59

FCR (feed conversion

rate)

kg fodder/kg

body weight

1.42:1

1.39:1

Analyzing the main production indices recorded during the trial it can be seen the favourable influence of the fodder additives administered in rainbow trout feed, Bio-Mos® 0.2 %, SelPlex® 0.03 % and NuPro® 2 % on the growth rate. The growth rate incresed by 5.23 % at the experimental group comparative with the Control group (243.2o g at group E vs 231.10 g value recorded at the Control group).

Analyzing the specific growth rate it can be noticed that the highest value was recorded at group E, 1.51 g/day, with 5.59 % higher than the specific growth rate of the Controls. Analyzing the feed conversion rate one can see that its values are characteristic to the values recorded at trout. The addition of fodder additives in group E feed led to an abatement of the feed conversion rate (1.39:1) by 2.12 % smaller comparative with the Control group (1.42:1). This abatement of the feed conversion rate as a response to fodder additives incorporation in the basl diet of fish was reported also by STAYKOV et all., 2005; CULJAK et all., 2006; BOGUT et all., 2006.


histological indices of rainbow trout(Oncorhynchus mykiss W.). Due to the fact that Bio-Mos® fulfills different roles in fish oragnism

(eliminates the pathogens from the intestinal level and through its constitution of oligosaccharid it offers energetical support for enterhocyres development) and to explain the superior growth rhytm of the experimental group, there were made some histological investigations at intestinal level. The investigations consisted in the measurement of the villi and microvilli lenght, both of them being responsible with nutrient absorbtion.


28

Table 19 The villi and microvilli lenght values of rainbow trout.

Issue Control group Experimental group Intestinal villi lenght (µm)

n 57 46 _ _ X±Sx

422.69±12.68

x

468.02±19.48

v % 12.65 18.24 Microvilli lenght( µm)

n 69 38 _ _ X±Sx

3.20±0.10

xx

3.83±0.44

v % 15.93 12.71 x - P<0.05 xx - P<0.01

From the values presented in the table above it can be seen the favourable influence of Bio-Mos® at intestinal level, noticing an increasement of the villi lenght at group E with 10.72 % higher comparative with the Control group. This development of the villi structures reported also by Sweetman et all., 2008 at rainbow trout, determined the enlargement of the nutrient absorbtion area, fact that explains the superior growth rhytm of the group. Also it can be observed a slightly increasement of the microvilli lenght with 19.68 % higher than the Controls as a result of enetrocytes stimulation with mannanoligosaccharids. This stimulation determined a uniformization of the microvilli brush border and an increased lenght of them which explains the superior growth rhytm of the experimental rainbow trouts. 8.3. The combined fodder additives (Bio-Mos®+SelPlex®+NuPro®) effects on the

meat quality of rainbow trout(Oncorhynchus mykiss W.). Among the body indices and the growth rhytm dynamic it was also followed

the chemical composition of rainbow trout meat. 78.46 77.88

21.54 22.1217.02 18.14

6.45 5.61

0

20

40

60

80

Apa % S.U. % Pb % Gb %

M

E

Fig. 3. The chemical composition of rainbow trout meat

From the data presented in the chart above it can be seen that the addition of Bio-Mos®, NuPro® and SelPlex® in rainbow trout feed did not have a major impact on the chemical composition of its meat, the differences recorded being insignificant. The values obtained are similar with the ones given in the literature (Bud I. and Vioara Miresan, 2008; Stephen J. et al., 2005).

It can be seen a slight increasement of the protein content of meat at group E 18.14 % CP with 2.69 % higher than the one recorded at Control group.


29

From the data presented in the table below it can be seen a very significant improvement of selenium content of rainbow trout meat as a response to rainbow trout diet supplimentation with organic selenium in form of SelPlex®.

Table 20

The rainbow trout meat Selenium content (n=10). ____________________________Se (µg/kg)__________________________ Control group Group 3E (SelPlex 0.03%)____ _ _ xxx X ± Sx 51.63 ± 2.46 219.78 ±7.35____________ xxx – P < 0.001


survival rate of rainbow trout( Oncorhynchus mykiss W.). Table 21

The survival percent and the losses recorded during the trial. Issue Control

group Experimental group (Bio-Mos 0.2% + SelPlex

0.03% + NuPro 2%) Initial fish number

250

250

Final fish number

209

217

Survival (%) 83.6 86.8

Losses (%) 16.4 13.2

Analyzing the values from the table above it can be seen an improvement of the survival rate at the experimental group, an improvement of 3.2 %. This improvement of the survival rate is due to the presence of mannanoligosaccharids that stop the colonization of the intestine with pathogens and to the presence of Selenium which stops the radicalic reactions. This improvement of the survival rate shows the benefical effect of the fodder additives that have been incorporated in rainbow trout diet.


hematological indices of rainbow trout(Oncorhynchus mykiss W.). The hematological investigations followed the corelation of some erythrocitary and leucocitary indices needed to elaborate a hematological pattern to evaluate the health of the fish through the cuantification of the toatl erythrocytes and leucocytes nomber and erythrocytes/leucocytes and lymphocytes/granulocytes ratio.


30

Table 22

Comparative average values between the two groups

Issue Average values of Control group

Average values of Experimental group

Nr. % Nr. % Erythrocytes (total) 541.20 96.94 528.30 96.94 Leucocytes (total) 17.40 3.02 16.60 3.02 Neutrophils 1.80 0.12 0.60 0.12 Lymphocytes 15 2.79 15.16 2.79 Monocytes 3.00 0.23 0.50 0.23 Total Erythrocytes+Leucocytes

558.60 99.98 544.60 99.98

Erythrocytes/leucocytes 31.1/1 96.72/3.12 31.8/ 1 96.95/3.04 Lymphocytes/granulocytes 8.3/1 89.27/10.75 22.75/1 95.05/4.9

The values obtained are in the normal range revealing a good level of welfare and health of the rainbow trouts. The corelation of those indices with the improvement of the growth rhytm indices and the survival rate show the positive biological effects of the used fodder additives.

8.6. PARTIAL CONCLUSIONS • Through the combined use of prebiotic Bio-Mos® 0.2 %, proetic extract

NuPro® 2 % and organic mineral SelPlex® 0.03 % in rainbow trout feed had been showed their biostimulating effects exprimated through the improvement of rainbow trout main bioproductive performances and health indices.

▪ Regarding the rainbow trout trial, the results obtained regarding the body weight evolution showed the positive effect of the fodder additives that have been incroporated in rainbow trout feed, the Experimental group reaching a higher body weight.

▪ The growth rate and the specific growth rate showed an improvement by 5.23 % respectiv 5.59 % at group E comparative with the Control group

▪ Regarding the survival rate it has been noticed the positive effect of the fodder additives that have been incroporated in rainbow trout feed. At the Experimental group it was recorded the highest survival rate, 86.8 %, with 3.2 % higher than the Control group survival rate.

▪ The benefical biological effects of the fodder additives that have been incorporated in the basal diet of brook trout are exprimated through the annealing of leucogramme and the abatement of losses recorded

▪ The addition of Bio-Mos®, NuPro® and SelPlex® in rainbow trout feed did not have a major impact on the chemical composition of its meat. It was observed a slight increasement of the protein content of meat at the Experimental group 18.14 % CP comparative with 17.02 % at Control group.

▪ Regarding the meat Selenium content, it was noticed a very significant improvement of the selenium content of rainbow trout meat (219.78 Se µg/kg at the Experimental group comparative with 51.63 Se µg/kg value recorded at the Control


31

group) as a response to rainbow trout diet supplimentation with organic selenium in form of SelPlex®.

▪ Regarding the histological indices, it was noticed an increasement of the villi lenght at group E with 10.72 % higher comparative with the Control group (468.02 µm at group E vs 422.69 µm at Control group). Regarding the brook trout microvilli lenght it has increased from 3.20 at 3.83 µm at group E, as a result of enetrocytes stimulation with mannanoligosaccharids. This development of the villi and microvilli structures reported also by Sweetman et all., 2008 at rainbow trout, determined the enlargement of the nutrient absorbtion area, fact that explains the superior growth rhytm of the treated group.

CHAPTER IX. RESEARCHES REGARDING THE EFFECTS OF SOM E FODDER ADDITIVES (BIO-MOS ® AND NUPRO®) ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF WELS CATFIFH ( Silurus glanis L.).

Through this experiment it was aimed to establish the influence of prebiotic Bio-Mos® 0.2 % and proteic extract NuPro® 2 % on the main growth and consumption indices of wels catfish (Silurus glanis L.) raised inside a mini recirculating aquacultural system. The goal of this research was to pinpoint the posibility to adapt and raise wels catfish juveniles (Silurus glanis L.) to a recirculating aquacultural system and to evaluate the effects of some fodder additives (prebiotic Bio-Mos® 0.2 % and proteic extract NuPro® 2 %) on the growth performances and the health status of wels catfish expresed through some hematological and immunological indices.

Table 23 General organisation of the second experiment Researches regarding the effect of

some fodder additives (Bio-Mos and NuPro) on the productive performances and the health status of wels catfish (Silurus glanis L.)

Experimental

period Experiment

location Groups

(fish/group and species)

Experimental factors

The followed indicators

01.11.2008-01.04.2009

Own biobase, Cluj-Napoca,

Cluj county

M (50 fish, wels catfish)

1E (Bio-Mos 0.2 %) (50 fish, wels catfish)

2E (NuPro

2%)(50 fish, wels catfish)

-

Addition of Bio-Mos 0.2

%

Addition of NuPro 2 %

-biometrical indices evolution - feed conversion rate - survival rate - hematological indices -imunological indices


32

RESULTS AND DISCUSSIONS 9.1. The effects of prebiotic Bio-Mos® and proteic extract NuPro® on the growth

and consumption indices of wels catfish juveniles (Silurus glanis L.). Table 24

The wels catfish juveniles body weght evolution during the trial (01.11. 2008-01.04.2009).

Group Date n Control (g) n 1E (Bio-Mos

0.2%)(g) n 2E (NuPro

2%)(g)

01.11.08

50

110±0.009

50

110±0.009

50

110±0.009

20.11.08

20

88.80±2.17

20

90.15±2.15

20

90.75±2.09

20.12.08

20

96.65±1.82

20

96.60±2.32

20 98.01±2.26

20.01.09

20

101.40±1.48

20

110.35±2.19

20

109.65±2.03

20.02.09

20

112.20±2.05

20

128.65±2.09

20

125.90±2.24

15.03.09

20

126.55±2.29

20

139.90±2.15

20

136.15±2.06

01.04.09

20

139.40±2.85

20

149.95±3.09

20

142.45±3.48

P>0.05

During the trial it can be noticed the positive effect of the prebiotic Bio-Mos® 0.2 % on the growth rhytm of wels catfish juveniles. It can be observed that group 1E had a higher growth rhytm comparative with the other two groups. At the end of the trial, the fish from group 1E reached an average body weight of 149.95±2.81g, comparative with the Control group 139.40±2.85g respective 142.45± 2.99g value recorded at group 2E. The results obtained are similar with the results reported by Bogut et all., 2006 who studied the effects of Bio-Mos® on wels catfish (Silurus glanis L.).

The effects of Bio-Mos® 0.2 % and NuPro® 2 % on the wels catfish juveniles growth and consumption indices are listed the in table below:

Table 25 The growth and consumption indices values at the end of the trial at wels catfish

juveniles Issue U.M. Control

group Group

1E Group

2E g 29.40 39.95 32.45 Growth rate

% 100 135.88 110.37 Specific growth rate g/day 0.19 0.26 0.21

Feed conversion rate

kg fodder/kg body weight

1.36:1

1.28:1

1.30:1


33

Analyzing the main production indices recorded during the trial it can be seen the favourable influence of the fodder additives administered in wels catfish juveniles feed, Bio-Mos® 0.2 % and NuPro® 2 % on the growth rate. The growth rate incresed by 35.88 % at the experimental group 1 comparative with the Control group. (39.95 g at group 1E vs 29.40 g value recorded at the Control group). Analyzing the specific growth rate it can be noticed that the highest value was recorded at group 1E, 0.26 g/day, with 36.84 % higher than the specific growth rate of the Controls. Also the specific growth rate of the second experimental group increased by 10.52 % comparative with the Control group. The addition of fodder additives in the experimentals groups feed led to an abatement of the feed conversion rate (1.28:1 and 1.30:1). Those coeficients were a little bit higher than the ones reported by Mioara Costache et all., 2008 (1,12-1,17:1) in a similar study. This abatement of the feed conversion rate as a response to fodder additives incorporation in the basal diet of fish was reported also by BOGUT et all., 2006.

9.2. The effects of prebiotic Bio-Mos® and proteic extract NuPro® on the

survival rate of wels catfish juveniles (Silurus glanis L.). The survival rate and the losses redocrded during the trial are listed in table 26:

Table 26

The survival rate and the losses recorded during the trial. Issue Control group Group 1 E (Bio-Mos

0.2%) Group 2 E (NuPro

2%) Initial fsih number 50 50 50 Final fish number 41 46 47

Survival (%) 82 92 94

Losses (%) 18 8 6

Analyzing the values from the table above it can be seen an improvement of the survival rate at the experimental groups, an improvement of 10 % at group 1E and 12 % at group 2E (NuPro 2 %), improvement also reported by BOGUT et all., 2005; STAYKOV et all., 2005; BRIAN et all., 2008. This improvement of the survival rate shows the benefical effect of the fodder additives that have been incorporated in wels catfish juveniles diet.

9.3. The effects of prebiotic Bio-Mos® and proteic extract NuPro® on the hematological indices of wels catfish juveniles (Silurus glanis L.).

To establish the health status of wells catfish juveniles there have been made some hematological investigations. The results are listed in the tables below:


34

Table 27 The hemoleucogramme values of wells catfish juveniles recorded at the end of the trial (n=15). Issue Leucocytes

Erythrocytes Hemoglobin

Hematocrit

Average erythrocitary hemoglobin

U.M. *10³/µL 10³*10³/µL g/dl % pg/cel Control group

133.48±0.6 1.72±0.009 8.20±0.14 28.32±0.59 47.21±0.38

Group E1 (Bio-Mos

0.2 %)

125.60±1.72

1.60±0.07

7.73±0.02

26.24±0.24

48.82±0.27

Group E2 (NuPro 2

%)

126.46±0.89

1.58±0.005

7.44±0.03

24.69±0.21

47.17±0.15

Analyzing the values presented in the table above it can be seen the relative similarity of them with the values given in the speciality literature (CARUSO et all., 2002; SOBECKA, 2001; TAVARES M., 2007; MODRÁ, 1998). The lack of neutrophily and the normal values of erythrocytes show that the used fodder additives do not have negative effects on the health status of fish. To evaluate the hepatic and pancreatic function of the fish, there were made some investigations regarding the proteins and enzymes levels that characterizes the liver and pancreas status: Table 28 The enzymogramme results at the end of the trial (n=15)

Issue ALAT ASAT GGT PAL Αlfa-amilase

Lipase

U.M. U/L U/L U/L U/L U/L U/L Control group

16.60±0.81 1112±47.15 1.00 185.20±5.91 4.80±0.80 35.26±1.59

Group E1

16.40±1.28 924±60.46 2.00 198.80±4.70 2.00±0.31 55.04±2.32

Group E2

20.20±1.02 1212±19.59 1.00 203.40±4.61 2.00±0.31 27.74±1.12

The values of ALAT, ASAT, GGT recorded show a physiological normal status of the liver and with amylase and lipase values which are in the normal range (SOBECKA., 2001; EKREM, 2004) pinpoint a normal deployment of the wells catfish juveniles digestive activity. To evaluate the nutritional status of wells catfish juveniles there were determined the values of the seric proteins.


35

0

2

4

Prot.tot.g/dl G-globulineg/dl

M1E

2E

M

1E

2E

Fig. 4. The proteinogramme values recorded at the end of the trial at wels catfish

juveniles The total proteins determination allows to evaluate the nutritional status and the

imunitary status of fish through gamma-globulines. Lower values of those gamma-globulines show a secondary immune deficiency. The values recorded correspond with the values reported by other researchers (TAVARES, 2007). The differences between the three groups regarding the proteinogramme values are unsignificant statistically (P>0.05).

9.4. The effects of prebiotic Bio-Mos® and proteic extract NuPro® on the

immunological indices of wels catfish juveniles (Silurus glanis L.). The initial lysozime concentrations increasement in different affections show the envolvment of the unspecific immune system and its state of alarm. Controlling lysozime values and maintaining them in the normal range stabilizes the first line of antiinffections body defense, stoping the infection evolution. For this reason fish diet supplementation with imunostimulating substances becames more and more important. Lysozime is an enzyme with antimicrobial properties and it was detected in fish serum and in different tissues rich in leucocytes (GRINDE et all., 1988; LIE et all., 1989). Lysozime has the property to break down the mucopolisaccharids from the bacterian cell wall causing their lizis (ELLIS, 1990).

Table 29 Wels catfish juveniles lysozime concentration at the end of the trial.

Issue Control group (µg/ml)


(µg/ml)

Group 2E (NuPro 2

%)(µg/ml) n 7 7 7

_ _ X±Sx

10.73±0.60

11.34±0.56

10.80±0.31

v % 14.91 13.13 7.59

P>0.05

Allthough the differences recorded are unsignificant statistically, it can be seen an increasement of lysozime concentration at group 1E, an increase of 5.68 % comparative with the Control group. This increasement of lysozime concentration at Bio-Mos treated group shows an immunostimulating action of Bio-Mos® on the unspecific immunitary system, fact reported by other authors too (STAYKOV, 2005 cited by SWEETMAN and DAVIES, 2006; DIMITROGLOU, 2006; BOGUT, 2006).


36

The immunoglobulines levels represent an indicator of the humoral reactivity. The imunoglobulines levels modifications (upwards or downwards) signalises the initiation of an imune response or the presence of some pathological states accompanied by imunological areactivity or a very weak reactivity under the influence of some intrenal or external factors.

Table 30 Wels catfish juveniles total immunoglobulines concentration at the end of the trial.

Issue Control group ( Vernes degrees)

Group 1E (Bio-Mos 0.2 %) (

Vernes degrees)

Group 2E (NuPro 2 %)(

Vernes degrees) n 12 12 12

_ _ X±Sx

3.57±0.20

3.70±0.40

3.68±0.24

v % 10.16 17.83 12.82

P > 0.05

Analyzing the values presented in the table above, it can be observed a slight increasement of the total imunoglobulines concentrations at the experimental groups (+ 3.78 % at group 1E and + 3.13 % at group 2E) which shows a slightly immunostimulator effect of the two fodder additives. The circulant immune complexes are formed by imunoglobulines binding to the detected antigenes forming thus a circulant complex antibody-antigene. Those complexes are responsable with the complement cascade triggering.

Table 31

Wels catfish juveniles circualnt immune complexes concentration at the end of the trial.

Issue Control group (conventional

units)


(conventional units)

Group 2E (NuPro 2 %) (conventional

units) n 7 5 7

_ _ X±Sx

27±3.30

32.40±3.70

40.20±6.00

v % 12.96 15.61 19.80

P>0.05.

Analyzing the values presented in the table above, it can be observed a slight increasement of the circualnt immune complexes concentrations at the experimental groups which shows a slightly immunostimulator effect of the two fodder additives that have been used (prebiotic Bio-Mos® 0.2 % and proteic extract NuPro® 2 %).


37

9.5. PARTIAL CONCLUSIONS • Through the use of prebiotic Bio-Mos® 0.2 % and proetic extract NuPro® 2

% in wels catfish juveniles feed had been showed their biostimulating effects exprimated through the improvement of the main bioproductive performances .

▪ Regarding the wels catfish trial, the results obtained regarding the body weight evolution showed the positive effect of the fodder additives that have been incroporated in wels catfish juveniles feed, the Experimental group 1 reaching a higher body weight (+ 7.56 % higher than the Control group).

▪ The growth rate and the specific growth rate showed an improvement at the experimental groups comparative with the Control group. The growth rate had been improved by 35.88 % at group 1E and by 10.37 % at group 2E. Also the specific growth rate of the experimental groups was improved by 36.84 % at group 1E and 10.52 % at group 2E as a result of diet supplimentation with Bio-Mos® 0.2 % and NuPro® 2 %.

▪ At the end of the trial the feed conversion rate had lower values at the experimental groups (1.28:1 at group 1E and 1.30:1 at group 2E) comparative with the Control group (1.36:1).

▪ Regarding the survival rate it has been noticed the positive effect of the fodder additives that have been incroporated in wels catfish juveniles feed. At the experimental group 2 it was recorded the highest survival rate with 12 % higher than the Control group survival rate. Also group 1E had a superior survival rate, with 10 % higher than the Control group survival rate.

▪ The benefical biostimulator effects of the fodder additives that have been incorporated in the basal diet of wels catfish juveniles had been assured through the maintenence in the normal range of the leucocitary indices, the lack of neutrophily and the abatement of the losses.

▪ Lysozime concentration increased at group 1E with 5.68 % comparative with the Control group, thus showing the immunostimulator role of Bio-Mos. This increasement of lysozime concentration among the increasement of the total immunoglobulines concentrations (+ 3.78 % at group 1E and +3.13 % at group 2E) pinpoint a slightly immunostimulating role of the foder additives that have been incorporated in wels catfish juveniles feed.


38

CHAPTER X. GENERAL CONCLUSIONS AND RECOMMENDATIONS

The results obtained during the first experiment “RESEARCHES REGARDING THE EFFECTS OF SOME FODDER ADDITIVES (BIO -MOS®, SELPLEX® AND NUPRO®) ON THE PRODUCTIVE PEFORMANCES AND HEALTH OF BROOK TROUT ( Salvelinus fontinalis M.)” allow the formulation of several conclusions:

• The use of Bio-Mos® prebiotic, organic selenium – Selplex® and proteic extract NuPro® in the brook trout feed determined the improvement of the growth performances of brook trout through the multiple effects of the used additives.

• Due to its action at intestinal level, Bio-Mos® prebiotic positively influenced the development of villi and microvilli structures determining a better nutrient utilization which led to superior growth indices compared with the Control group.

• The addition of the proteic extract NuPro® in the feed administered to brook trout positively influenced the weight gain rhythm, NuPro® being a feed stimulator.

• In the case of survival rate, SelPlex® had a positively influence. The highest survival rate (94.4%) was recorded at the experimental group 3E, fed on SelPlex® containing diet, compared to the Control group which showed a 17.6% lower survival rate. This increase of the survival rate certifies the biostimulating effect of organic selenium.

• The blood indices values recorded in fish from the experimental groups, the increase in the glutathione peroxidase concentration in 3E group and the normal physiologic values of the leukocitary parameters mark the positive influence of the additives used, on the fish health.

• The supplementation of brook trout feeds with the tested additives, determined a slight increase of the whole body protein content in experimental groups 1E and 3E, 18.04% CP in group 1E (Bio-Mos 0.2%), 18.09% CP in group 3E (SelPlex 0.03%) respectively; the selenium content of meat had superiors values in group 3E, who was fed on a diet containing organic selenium (238.99 Se µg/kg) compared to the Control group (61.73 Se µg/kg).

• Bio-Mos® is a mannan-oligosaccharide and so it offers energetic support for the entherocytes; this led to a better entherocytes development which in turn, positively influenced the development of villi and microvilli structures in group 1E; the microvilli length increased from 2.06 to 3.16µ in experimental group 1E, as a response to the entherocyte stimulation with mannan-oligosaccharide, which explains the superior growth performances recorded in group 1E.

The results obtained during the second experiment “RESEARCHES REGARDING THE COMBINED FODDER ADITIVES (BIO-MOS®+SELPLEX®+NUPRO®) EFFECTS ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF RAINBOW TROUT ( Oncorhynchus mykiss W.)” allow the formulation of several conclusions:

• During the experiment carried out on rainbow trout, the results obtained certify the favorable effect of the additives used; the fish in the experimental group showed a superior body weight compared to the control group and also an improved


39

weight gain and growth rate (improved with 5.23% and 5.59% in the experimental group, compared to the Control group). These superior growth performances can be explained by the positive effect that the Bio-Mos® supplementation had on the villi and microvilli morphology at intestinal level. A major contribution to this performances can be attributed to NuPro® proteic extract, which provides all the essentials amino-acids needed for proper somatic development.

• In the case of survival rate, all the additives had a positive influence, especially the organic selenium. The survival rate of the experimental group (86.8%) was enhanced by 3.2% compared to the Control group due to the selenium action on a cellular level, where it assures protection against the action of free radicals, knowing that water is the richest environment in free radicals.

• Leukocytary subpopulation configuration, in the fish from the experimental group is dominated by lymphocytes while in the fish from the Control group neutropihls had the higher proportion; this configuration along with the physiologically normal leukocytary parameters reflect the biostimulating effects of the tested substances.

• Following the meat quality, has been observed a slight increase in protein in experimental group (18.14% CP) compared to the Control group (17.02%) and also a high level of selenium assimilation in the rainbow trout meat (219.78 Se µg/kg in the Experimental group compared to the Control group – 51.63 µg/kg).

• The presence of mannan-oligosaccharides and their mode of action at the intestinal level determined an increase in the villi height in the experimental group, being 10.72% bigger compared to the control group (468.02µm in the experimental group vs. 422.69µm in the control group) and an increase in the villi length (3.20µm in the control group compared 3.83µm in the experimental group 1E) as a response of the entherocytes stimulation with mannan-oligosaccharides

The results obtained during the third experiment “RESEARCHES REGARDING THE EFFECTS OF SOME FODDER ADDITIVES (BIO -MOS® AND NUPRO®) ON THE PRODUCTIVE PERFORMANCES AND HEALTH OF WELS CATFISH (Silurus glanis L)” allow the formulation of several conclusions:

• The addition of Bio-Mos® prebiotic and NuPro® proteic extract in the wells catfish juveniles had a positive influence on the bioproductive indices of fish from the experimental groups. Regarding the body weight evolution during the trial, the best results were recorded at group 1E as a response to diet supplementation with mannan-oligosaccharides in form of Bio-Mos® 0.2 %. The body weight of wels catfish juveniles from group 1E at the end of the trial registered an increase of 7.56 % comparative with the Control group.

▪ The use of Bio-Mos® prebiotic and NuPro® proteic extract in the wells catfish juveniles had a positive influence on the growth rate of fish from the experimental groups, the growth rate recording higher values at the experimental groups, 39.95 g at group 1E, with 35.88 % higher than the value recorded at the Control group, respective 32.45 g at group 2E, with 10.37 % higher than the value recorded at the Control group (29.40 g). Also the specific growth rate (g/day) was positively influenced by the addition of Bio-Mos® and NuPro® (0.26 g/day at group


40

1E, with 36.84 % higher than the value recorded at the Control group and 0.21 g/day at group 2E, with 10.52 % higher than the value recorded at the Control group).

▪ By increasing nutrient use efficiency as a result of wels catfish juveniles diet supplementation with Bio-Mos® and proteic extract NuPro® which assures the nutritional requierments of fish, at the end of the trial the feed conversion rate had lower values at the experimental groups (1.28:1 at group 1E and 1.30:1 at group 2E comparative with 1.36:1 at the Control group).

▪ Maintaining the hematological parameters in the normal range, the abatement of the losses recorded and the increasement of the lysozime concentrations, immunoglobulines and imune circulant complexes shows the favourable influence of the tested additives on the health status of the bilogical material. The survival rate increased by 10 % at group 1E (Bio-Mos 0.2 %) respective 12 % at group 2E (NuPro 2 %), fact recorded by other researchers too; lysozime concentration also increased by 5.68 % at group 1E; immunoglobulines concentration increased by 3.78 % at group 1E and 3.13 % at group 2E. The immune circulant complexes concentration responsable by complement activation also increased at the experimental groups, especially at group 2E from 27 conventional units (value recorded at the Control group) to 32.40 at group 1E and 40.20 at group 2E.

▪ The enzymes values recorded at wels catfish juveniles show a normal physiological status and together with amilase and lipase values pinpoint a normal digetive processes. The proteins values which allow the characterization of the nutritional status and imunitary status through gamma-globulines show a good nutritional status of wels catfish juveniles and the lack of a secondary imune deficit

Based on these conclusions formulated at the end of each trial, there are some genral conclusions that can be made: 1. The use of prebiotic Bio-Mos® 0.2 %, proteic extract NuPro® 2 % and organic selenium SelPlex® 0.03 % in salmonids feed (brook trout and rainbow trout) and the use of prebiotic Bio-Mos® 0.2 % and proteic extract NuPro® 2 % in wels catfish juveniles feed determined the improvement of the main bioproductive indices, the survival rate and the health status of fish. 2. The addition of these fodder additives on salmonids and wels catfish diet led to a superior growth rhythm and to a faster biomass accumulation. 3. At the end of the trials the feed conversion rate registered lower values at the experimental groups comparative with the Control groups. 4. The hematological indices values showed the positive effects of the tested additives on the fish physiological status. 5. The imunological indices values pinpoint the imunostimulating effect of the tested substances. 6. At intestinal level it was noticed the positive influence of prebiotic Bio-Mos® on the histological intestine morphology. 7. The superior survival rates and the gluthathion-peroxidase values of SelPlex® treated groups show the beneficial effect of organic selenium on fish organism and ite efficiency to stop the radicalic reactions within fish organism. 8. The results regarding the selenium meat content of fish (both brook trout and rainbow trout) show an increased selenium assimilation and the improvement of selenium deposit in the tissues that provide the selenium reserves needed by the


41

organism. The values obtained show the cumulative effect of organic selenium in trout meat, meat that could be considered as a functional food.

Recommedations Based on the results obtained and the conclusions that have been formulated we

recommend the use of prebiotic Bio-Mos® 0.2 %, proteic extract NuPro® 2 % and organic mineral SelPlex® 0.03 % in the feed of salmonids and wels catfish juveniles because at fish organism level these substances fulfill different roles with positive effects on the growth rhythm and health status of fish.

The prebiotic Bio-Mos® acts at intestinal level by eliminating the present pathogens and by offering energetic support for entheroctyes development which determined the increasement of the nutrient absorbtion area and a better nutrient usage thus favourising a higher growth rhythm, reason for which we recommend the use of Bio-Mos® 0.2 % in brook trout, rainbow trout and wels catfish juveniles feed.

Due to the high grade of organic selenium assimilation and the improvement of selenium storage in the tissues that provide the selenium reserves needed by the organism and due to its positive effect on the health status of fish, effect showed through the substantial abatement of losses through the reduction of intracellular oxidation and cell membranes protection against free radicals action, we recommend the use of organic selenium SelPlex® 0.03 % in brook trout, rainbow trout and wels catfish juveniles feed.

The proteic extract NuPro® through its components (aminoacids, vitamins and minerals) assures the fish physiological requirements satisfaction preventing thus the aparission of nutritional disorders and anemia, reason for which we recommend the use of NuPro® in fish alimentation.

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