Scholarly article on topic 'Physiological and microbiological indices as indicators of evaluating dietary fungi degraded date pits as a probiotic for cultured Nile tilapia Oreochromis niloticus fingerling and its effect on fish welfare'

Physiological and microbiological indices as indicators of evaluating dietary fungi degraded date pits as a probiotic for cultured Nile tilapia Oreochromis niloticus fingerling and its effect on fish welfare Academic research paper on "Animal and dairy science"

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{"Fungi degraded date pits" / " Trichoderma reesei " / "Nile tilapia" / Haemoglobin / Probiotic}

Abstract of research paper on Animal and dairy science, author of scientific article — H. Assem, A. Khalifa, M. ELSalhia

Abstract The main goal of this study is to explore the potentiality of using blood haemoglobin, plasma glucose, plasma triglycerides, plasma cholesterol, growth rate and intestinal bacterial count as biomarkers to evaluate the dietary supplementation of fungi-degraded date pits (FDDP) as growth promoters as well as probiotic for fingerling Nile tilapia Oreochromis niloticus reared in a recirculating system. Five isoenergetics–isonitrogenous diets containing 0, 0.5, 1.0, 1.5 and 2.0g/100g of fungi Trichoderma reesei-degraded date pits (FDDP), as a replacement for dietary α-cellulose, were used to feed triplicate groups of fish fingerlings (2.0g initial weight), for 9weeks. Tilapia weight gain (4.27g/g), was superior only in fish fed on diets containing 0.5g/100g FDDP, when compared with those fish fed on other diets. Blood haemoglobin was found to be unchanged in fish fed on different percentages of FDDP diets. Plasma glucose was high in fish fed on 0.5% FDDP, the accompanied plasma hypertriglyceridemia, could possibly be due to hepatic neo lipogenesis. Cholesterol levels in fish plasma fed on experimental FDDP diets at all concentrations were highly and significantly decreased which may indicate the attenuated ability for transporting lipid out of liver. Although significant correlation among the physiological indices and the dietary FDDP have been observed, blood haemoglobin, plasma triglycerides and cholesterol are shown as more sensitive indicators followed by plasma glucose and then growth rate. The present study showed a possible potential of using FDDP to lower the bacterial population densities in O. niloticus intestine without affecting the fish weight or health welfare condition, that could be an excellent approach in designing subsequent probiotic researches on FDDP. Oestradiol was measured in all tested diets and was found to increase linearly with FDDP content in diets, which indicates that a FDDP may has phytoestrogenic roll.

Academic research paper on topic "Physiological and microbiological indices as indicators of evaluating dietary fungi degraded date pits as a probiotic for cultured Nile tilapia Oreochromis niloticus fingerling and its effect on fish welfare"

Egyptian Journal of Aquatic Research (2014) xxx, xxx-xxx

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National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research

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Egyptian Journal of Aquatic Research

FULL LENGTH ARTICLE

Physiological and microbiological indices as indicators of evaluating dietary fungi degraded date pits as a probiotic for cultured Nile tilapia Oreochromis niloticus fingerling and its effect on fish welfare

H. Assem *, A. Khalifa, M. ELSalhia

Fish Physiology Department, National Institute of Oceanography and Fisheries, Alexandria, Egypt Received 14 May 2014; revised 14 October 2014; accepted 18 October 2014

KEYWORDS

Fungi degraded date pits; Trichoderma reesei; Nile tilapia; Haemoglobin; Probiotic

Abstract The main goal of this study is to explore the potentiality of using blood haemoglobin, plasma glucose, plasma triglycerides, plasma cholesterol, growth rate and intestinal bacterial count as biomarkers to evaluate the dietary supplementation of fungi-degraded date pits (FDDP) as growth promoters as well as probiotic for fingerling Nile tilapia Oreochromis niloticus reared in a recirculating system. Five isoenergetics-isonitrogenous diets containing 0, 0.5, 1.0, 1.5 and 2.0 g/100 g of fungi Trichoderma reesei-degraded date pits (FDDP), as a replacement for dietary a-cellulose, were used to feed triplicate groups of fish fingerlings (2.0 g initial weight), for 9 weeks. Tilapia weight gain (4.27 g/g), was superior only in fish fed on diets containing 0.5 g/100 g FDDP, when compared with those fish fed on other diets. Blood haemoglobin was found to be unchanged in fish fed on different percentages of FDDP diets. Plasma glucose was high in fish fed on 0.5% FDDP, the accompanied plasma hypertriglyceridemia, could possibly be due to hepatic neo lipogenesis. Cholesterol levels in fish plasma fed on experimental FDDP diets at all concentrations were highly and significantly decreased which may indicate the attenuated ability for transporting lipid out of liver. Although significant correlation among the physiological indices and the dietary FDDP have been observed, blood haemoglobin, plasma triglycerides and cholesterol are shown as more sensitive indicators followed by plasma glucose and then growth rate. The present study showed a possible potential of using FDDP to lower the bacterial population densities in O. niloticus intestine without affecting the fish weight or health welfare condition, that could be an excellent approach in designing subsequent probiotic researches on FDDP. Oestradiol was measured in all tested diets and was found to increase linearly with FDDP content in diets, which indicates that a FDDP may has phytoestrogenic roll. © 2014 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

* Corresponding author. Peer review under responsibility of National Institute of Oceanography and Fisheries.

http://dx.doi.org/10.1016/j.ejar.2014.10.004

1687-4285 © 2014 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Introduction

The human demand of fish consumption increased all over the world. Fish is about to become the main alternative source of animal protein. Nile tilapia, Oreochromis niloticus, is the predominant cultured species worldwide (Thodesen et al., 2012; Gjerde et al., 2012; El-Sayed et al., 2012). Tilapia aquaculture represents about 42% of world total aquaculture fish production, and about 75% of the total fish production in Egypt (GAFRD, 2012). In addition, tilapia culture has been gradually shifted from traditional semi-intensive systems to more intensive systems, which rely exclusively on artificial feeds. Therefore, the major challenge facing tilapia culture industry is the production of cost effective and environmentally performing feeds for farmed tilapia, using inexpensive, locally available ingredients. Several studies have been conducted to evaluate the incorporation of different unconventional animal and plant proteins and energy sources for farmed tilapia with varying results (El-Sayed, 2006).

Middle East Egypt is one of the largest date-producing countries (1.373.57 MT/year, FAO, 2011). Date pits are by products of the date industry. Depending on the variety and grade quality of the ripe date, date pit comprises from 60 to 120 g/kg of its total weight (Aldhaheri et al., 2004). In Egypt, approximately 120 tons of date pits are available every year as a by-product from date processing plants.

Date pits were tested as a feed ingredient on fish feeds (Belal and Al-Owaifer, 2005; Belal, 2008) but fish production was not significantly affected. High content of indigestible carbohydrates may limit the use of date pits in fish ration (Rahman et al., 2007). In his study, Belal (2008) discussed the use of Xylanase enzymes in feed formulations to liberate nutrients either by hydrolysis of non-degradable cellulose and hemicellu-lose fibres or by liberating nutrients blocked by these fibres.

Because of the high cost of enzyme utilization in animal feed, Belal (2008) tested and recommended the use of the soft-rot fungus, Trichoderma reesei (also known as Hypocrea jecorina), which was able to produce the Xylanases enzymes, as an economical alternative for dietary fibre degradation.

Belal (2008) had investigated the effect of FDDP in the diet of O. niloticus and concluded that FDDP improved the growth performance of the fish. In his study, he tested different concentrations of FDDP in the formulated diet and he depended only on growth parameters as indicators of FDDP effects.

Since fish peripherical blood analysis is used as a diagnosis to assess its physiological state and the effect of hazardous substances, to determine the nonspecific resistance of progenitors and its descendants, to establish the quality of food, the genetic variability and the effect of stress (Assem et al., 2013; Hassan et al., 2013), an objective of the present study was to investigate the potential use of some physiological health parameters of O. niloticus as indices in evaluations of fish diets supplemented with different concentrations of fungi T. reesei degraded date pits (FDDP). The concentrations we used were so far lower than those used by Belal (2008).

In aquaculture, infectious diseases are the major cause of economic losses. Probiotic supplementation may change the microbiota of the digestive tract and modulate the immune defences and nutritional performance (Ramos et al., 2013). The use of bacterial count in the intestine of the fish as indicator in evaluating the effect of fungi degraded date pits as feed

ingredient in diminution of bacterial infection and hence leading to health benefits to O. niloticus, was another goal of our present study. The possibility of using FDDP as probiotic was also discussed.

Aldhaheri et al. (2004), proposed that date pits have an oestrogen-like substance, which acts as a phytoestrogen in the body of animal fed date pits, also El-Sayed et al. (2012) discussed the possible effect of phytoestrogen on sex reversal on Nile tilapia O. niloticus larvae fed diets treated with 17a ethyl-testosterone. In order to investigate, if any, the possible role of hormone in FDDP, we measured the concentration of testosterone and oestradiol in the diet supplemented with increasing percentage of FDDP (0.5-2%).

Materials and methods

Preparation of the fish for experiments

Nile tilapia O. niloticus fingerlings (1.95 g average initial weight) were collected from local hatcheries. The fish were stocked into 20 L fibreglass tanks in a closed, recirculating indoor system. The tanks were provided with a continuous flow of aerated dechlorinated tap water at 25 0C (hardness 5.2 mg/L as CaCO3; Ca2+ 0.045-0.069 mM/L; Na+ 0.024 mM/L; dissolved oxygen 80% saturation; pH 7.2-7.5).

Experimental design

Groups of ten fish were randomly stocked into each aquarium, with four replicates per each treatment. In order to avoid the influence of any systematic stress factors, the fish groups were randomly redistributed half way through the experiment. In each trial, one aquarium was allocated to fish fed on the diet without FDDP to serve as control. Each aquarium was considered as an experimental unit. The fish was fed ad lib at 09.00, 12.00, and 15.00 h. Approximately 15 to 20 min after all feeding activity had subsided, the uneaten feed was removed and weighed to determine the amount of eaten feed. Faecal waste was siphoned from each aquarium every day. Group weight measurements were done at weekly intervals. Experimental periods lasted for 9 weeks for all tested diets.

Culture system

The feeding trials were conducted in 3 L plastic aquaria. Water was recirculated through biological and mechanical filters. The recirculating system used a 200 L vertical screen filter system utilizing high-density polyester screens (FIAB fish technic, GmbH, Germany) to remove particulate matter and provide substrate for Nitrosomonas and Nitrobacter bacteria. A blower and air stone provided continuous aeration. Water exchange rate for the system was approximately 5% of the total volume per day. Each aquarium was supplied with water at a rate of 200 mL/h and cleaned daily. A PVC screen covered the aquarium bottom to prevent the experimental fish from eating their faeces. A black plastic screen covered the back and sides of all aquaria to minimize disturbances caused by personnel present in the laboratory. Illumination was supplied by fluorescent ceiling lighting, controlled by an automatic electric timer to maintain a photoperiod of 18:6 h light:dark per day cycle.

Table 1 Composition of the test diets (g/100 g dry matter).

Degraded date pits (g/100 g)

Feed ingredients 0 0.5 1.0 1.5 2.0

Fish meal 46.15 46.15 46.15 46.15 46.15

Yellow corn 34 34 34 34 34

FDDP 0 0.5 1.0 1.5 2.0

a-Cellulose 15.85 15.35 14.85 14.35 13.85

Vitamins premix 1 1 1 1 1

Minerals premix 3 3 3 3 3

Total 100 100 100 100 100

Table 2 Changes of blood haemoglobin, plasma glucose, plasma triglycerides, plasma cholesterol, weight gain, water content and

survival percentage for Oreochromis niloticus fed test diets with graded levels (0-2 g/100 g) of FDDP.

FDDP Haemoglobin Glucose Triglycerides Cholesterol Body water Weight gain Survival%

(g/dL) (mg/dL) (mg/dL) (mg/dL) (%) (g/g)

0.0 8.47 ± 1.0 59.0 ± 3.0 51.5 ± 2.0 228 ± 20.4 75.4 ± 2.0 3.20 ± 0.4 94.2

0.5% 7.96 ± 0.9 65.5 ± 2.0* 64.0 ± 3.1** 145 ± 11.1** 75.9 ± 3.2 4.27 ± 0.2** 100.0

1.0% 8.37 ± 1.2 42.9 ± 3.0** 69.0 ± 4.0** 152 ± 14.2** 75.8 ± 2.5 3.43 ± 0.6 100.0

1.5% 7.98 ± 0.9 46.5 ± 2.0** 73.0 ± 2.6** 151 ± 12.5** 76.5 ± 1.9 3.16 ± 0.5 99.0

2.0% 8.11 ± 0.7 55.0 ± 4.0 84.7 ± 1.8** 122 ± 10.7** 76.1 ± 2.6 3.56 ± 0.3 96.0

Values are means of 10 replicates for each treatment ± standard error of the mean. Mean values are significant at the level of p < 0.05. Mean values are significant at the level of p < 0.01.

Water temperature and dissolved oxygen levels were measured daily. Total ammonia and nitrite levels were measured twice weekly (spectrophotometer, Hach Company, Loveland, CO). Total alkalinity and hardness were monitored once a week (colorimeter, Orion Company); pH was monitored daily using an electronic pH metre. Salinity was checked every other day with a salinity refractometer (Erma, Tokyo, Japan). Over the duration of the study, the average values of water-quality parameters (average ± S.D.) were as follows: water temperature 26.0 ± 1.0 0C; dissolved oxygen 7.0 ± 0.5mg/L; total ammonia 0.4 ± 0.03 mg/L; nitrite 0.05 ± 0.01 mg/L; total alkalinity 107.0 ± 25.2 mg/L; hardness 5.2 ± l .0mg/L; and pH 7.7 ± 0.3.

Sample collection: blood and tissue samples

At the end of the experiment (9 weeks), three groups of 10 fish were killed for each of the experimental diet. Handled controls were subjected to the same amount of disturbance as the experimental fish but fed FDDP free diet. Fish were caught by hand net quickly to minimize the disturbance. Then they were placed upside down and the blood was obtained by incision directly into the heart using heparinized glass pipette. Plasma was separated directly by centrifugation trying to avoid haemolysis and stored at —20 0C until analysis. After blood sampling the fish was decapitated, intestine was dissected and prepared for bacterial analysis.

Analytical techniques and feed efficiency

Glucose concentration was determined in 10 iL plasma using glucose-liquizyme GOD-PAP kit (SPECTRUM, MDSS GmbH Schiffgraben 41 30175 Hannover, Germany, Catalog

# 250 001). Haemoglobin concentration in blood was determined by the cyanmethaemoglobin method. Plasma triglyceride and cholesterol concentrations were determined using commercial kit. Muscle water contents were analysed by taking a piece of a specific weight of white epaxial muscle and drying it to constant weight at 100 0C for 24 h. It was then reweighed after drying, and then the water content was measured as a percentage to the muscle weight. Weight gain (g/ g) was calculated as the final weight minus the initial weight divided by the initial weight.

Hormonal assay

The steroid hormones oestradiol and testosterone in the diets were directly analysed by using Enzyme Linked Immuno Sor-bent Assay (ELISA) techniques.

Preparation of fungus culture and process of degradation

These parameters were described in detail by Belal (2008). In short, T. reesei is grown on potato dextrose agar (PDA) at 25 0C for 7 days in the dark. To confirm that the fungi could degrade the date pits a ground date pit inoculum was prepared by adding half a kilogram of ground date pits and 150 mL of distilled water into 1 L flasks. The flasks were autoclaved at 121 0C for 30 min on 3 consecutive days. Under aseptic conditions the ground date pits were then inoculated with 8 agar plugs (6 mm diameter) from actively growing margins of the T. reesie colony. The flasks were incubated at 25 0C in the dark for 3 weeks. The flasks were shaken occasionally to ensure uniform colonization of the date pits by the fungi. Colonized date pits which had been autoclaved twice served as a control. Small amounts of the colonized and control ground date pits

Table 3 Microbial population densities in log10 colony-forming units (cfu) per g dry fish intestine tissue for total bacterial counts, Salmonella spp., Shigella spp. and Escherichia coli.

Degraded date pits (g/kg) Total bacterial counts Salmonella spp. Shigella spp. Escherichia coli

0.0 7.14 ± 0.10 5.0 6.77 ± 0.21* 10.0 6.47 ± 0.03* 15.0 5.89 ± 0.02** 20.0 5.23 ± 0.04** 0.97 ± 0.03 0.71 ± 0.01* 0.61 ± 0.04** 0.55 ± 0.05** 0.34 ± 0.02** 0.72 ± 0.10 0.33 ± 0.06** 0.31 ± 0.04** 0.25 ± 0.02** 0.19 ± 0.03** 1.11 ± 0.06 0.82 ± 0.04* 0.71 ± 0.02** 0.43 ± 0.05** 0.32 ± 0.07**

Values are means of 10 replicates for each treatment ± standard error Mean values are significant at the level of p < 0.05. Mean values are significant at the level of p < 0.01. of the mean.

were plated onto PDA to confirm that T. reesei was present or absent respectively. The process of fungi degraded date pits began with the addition of a starter culture of the fungi to the glassware containing some sterilized medium and substrate. The rest of the medium and date pits were then added. In the feed batch process, the feeding continued until a certain volume of fungi was reached, after which the process is stopped and the DDP with the fungi mass being collected and dried. During degradation, continuous aeration is needed because the process is primarily aerobic. Cooling is also necessary because large amounts of heat are evolved during micro-bial growth. Stirring is needed to intensify cooling and to provide air transport to the organisms.

Experimental diets (Table 1)

Five isocaloric-isonitrogenous diets (300 g/kg crude protein and 107 kJ gross energy/100 g) and test diets were prepared as described by Belal (2008) Fungi degraded date pits (FDDP) was incorporated into the test diets, as a replacement of a-cellulose at 0, 0.5, 1.0, 1.5 and 2.0 g/100 g. The diets were fed to triplicate groups of 15 fish (2.0 g average initial weight) at 4% body weight/day, twice a day (09:00-16:00 h) for 63 days. Fish weighed collectively at 7 day intervals, their average weights recorded and the daily rations was readjusted accordingly.

Enumeration of microbial populations

The microbial populations of the intestine samples were estimated using dilution plate method (Johnson and Curl, 1972). Intestines were macerated in 100 mL of distilled water using a sterile mortar and pestle under aseptic conditions, and then shaken for 20 min on a shaker at 250 rpm at 25 0C. Aliquots (0.2 mL) were spread with a sterile glass rod over the surface of different general purposes and selective agar media in sterile plastic Petri dishes (90 mm diameter). Plates were dried in a laminar flow-cabinet for 20 min before incubation at 37 0C in the dark for 4 days and colony counts were carried out from day 2 onwards. Five plates per dilution were made for each sample. Population densities were expressed as log10 colony forming units (CFU)/g dry intestine weight (Hallmann et al., 1997). The groups of organisms selected for enumeration and the media used were as follows: (i) total aerobic bacteria on 1/5 M32 medium (Sivasithamparam et al., 1979), incubated for 2-4 days; (ii) Escherichia coli on Eosin methylene blue (EMB) (M317) (HiMedia Laboratories Limited, Mumbai, India) incubated for 2-4 days; (iii) Salmonella spp. on

(Bismuth sulphite agar (modified Wilson & Blair medium, CM0201) (Oxoid Limited, Basingstoke, Hampshire, UK), incubated for 2-4 days; (iv) Shigella spp. on xylose lysine deoxycholate agar (XLD agar M031) (HiMedia Laboratories Limited, Mumbai, India), incubated for 2-4 days.

Statistical analysis

Statistical analysis was performed using SPSS (SPSS 20.0 for Windows, SPSS Inc., USA). Normal distributions were checked by Shapiro-Wilk test and homogeneity of variances by Levene test. One-way analysis of variance (ANOVA) was carried out to evaluate the effect of fungi-degraded and unde-graded date pit amendment on blood haemoglobin, plasma biochemical variables and intestinal total and specific micro-bial population. A value of p < 0.05 was considered to be statistically significant.

Results

Changes of blood haemoglobin, plasma glucose, plasma triglyceride, plasma cholesterol, weight gain, water content and survival% (Table 2)

Mortalities were low, no infection symptoms recorded during the complete experimental time. Haemoglobin content remained more or less unchanged in fish fed the different FDDP diets. Plasma glucose increased slightly but significantly (p < 0.05, 10%) in fish fed the 0.5% FDDP diet, a strong and significant decrease in glucose level was recorded in fish plasma fed the 1% (p < 0.01, 27%) and 1.5% (p < 0.02, 20%) FDDP

Table 4 Concentrations of oestradiol (pg/g) and testosterone

(ng/g) of the experimental diets.

FDDP Oestradiol (pg/g) Testosterone (ng/g)

0.0 15.16 ± 1.04 27.36 ± 4.2

0.5% 20.60 ± 2.0* 28.10 ± 3.2

1.0% 25.50 ± 5.0** 26.40 ± 2.8

1.5% 33.80 ± 4.2** 27.50 ± 6.2

2.0% 45.74 ± 2.0** 26.10 ± 5.0

Values are means of 10 replicates for each treatment ± standard

error of the mean.

* Mean values are significant at the level of p < 0.05.

Mean values are significant at the level of p < 0.01.

diets, the level of plasma glucose in fish fed the 2% FDDP diet was not significantly different from the control. As compared to controls plasma triglycerides increased in fish fed all FDDP diets and this increase in FDDP concentration dependent being higher in fish fed the 2% diet. The levels of plasma cholesterol were strongly and highly significantly decreased in fish fed all FDDP diets and reached a minimum in fish fed the 2% diet. A negative linear correlation expresses the relation between cholesterol and FDDP percentage in diet (r = —0.8119, r2 = 0.6592). Fish fed diets containing 0.5% FDDP grew better than those fed the diets containing 0%, 1.0%, 1.5% and 2.0% FDDP (p < 0.02). Water content remained unchanged in fish fed the different levels of FDDP diets as compared to controls.

Microbial population densities for total bacterial counts, Salmonella spp, Shigella spp. and E. coli (Table 3)

Samples of intestine dissected from control fish fed the diet without FDDP have a significantly (p < 0.01) higher total bacterial count than the sample of intestine from fish fed the different percentages of FDDP diets as determined by the hydrolysis of the FDA technique. There was a significant (p < 0.01) decrease in the bacterial populations (colony forming units/g dry sample) in the sample of experimental fish fed diet with increasing percentage of FDDP compared to the sample of control fed diets without FDDP. The estimated total populations of Salmonella spp. Shigella spp. and E. coli were significantly (p < 0.01) lower in the sample from fish fed diet with FDDP compared to those fed diets without FDDP. The magnitudes of reductions were FDDP concentrations dependent in the diet being higher with the highest FDDP level (2.0%).

Sex hormones testosterone and oestradiol in diets (Table 4)

The levels of testosterone (ng/g dry diet) were higher than that of oestradiol (pg/g dry diet) in all experimental diets. While the level of testosterone remained unchanged in FDDP diets when compared to control diet without FDDP, oestradiol level had increased with increasing FDDP percentage in diets. A positive linear regression expresses the relation between oestradiol concentration and the FDDP percentage in the diet (r = 0.9797, r2 = 0.9598, sample covariance = 9.125).

Discussion

Belal (2008) was the first who introduced FDDP into tilapia diet, his aim was to improve the fish growth rate. During his experiment fish weight increased than controls when fed diets with 150 and 300 g/kg FDDP. In his study, he related these high levels of growth rates to a combination of effects among which are the increase of digestible carbohydrates, the liberation of nutrients and to a high level of the free sugar mannose. In the present work, and although we used the same FDDP as his but at lower percentages, we also recorded a highly significant growth increase only in fish fed the lowest 0.5% FDDP, and fish growth remained unchanged when FDDP percentage increased in diet. We may agree with Belal (2008) last conclusion only when the fish grew better than the control, the increase of plasma glucose in our experimental fish fed the

0.5% FDDP supports this assumption. However, we agreed not fully with his explanation of the negatively affected growth when FDDP increased in tilapia diets above 300 g/kg in his experiments or above 0.5% FDDP in ours.

We rose the question what might explain these paradoxes? Our first approach to answer that question we measured the changes of haematological and plasma chemistry for our cultured tilapia. Fish peripherical blood analysis is used as a diagnosis to assess its physiological state , effect of hazardous substances and to establish the quality of food (Assem et al., 2013; Belal and Assem, 2011). When we compare O. niloticus normal haemoglobin values with those obtained previously, we find that Hassan et al., 2013 reported similar concentration (8.2g/dL). In the present study haemoglobin in fish blood remained more or less unchanged in fish fed all diets, which may indicate that FDDP neither had anaemic nor healthy effects and consequently we cannot rely on blood haemoglobin solely as an indicator in evaluating FDDP.

The lipid metabolism of the liver includes the secretion, transport, and uptake of lipid. A balance between the secretion and uptake of lipid occurred under normal physiological conditions. Karavia et al., 2013 using rats as experimental animal, attributed the hepatic lipid accumulation to the blocking of lipid secretion. In our study, the plasma TG was significantly higher in fish fed FDDP diet than that of fish fed control diet. These results might indicate that the ability for transporting lipid out of the liver increased after the intake of high FDDP diet. In addition, the plasma TG concentrations may indicate a more active endogenous lipid transport (Du et al., 2005; Gatesoupe et al., 2014). The possible increase of lipid absorption from the gut to the liver may explain the increase of TG in plasma concentrations after feeding the fish with increasing FDDP supplement (Lu et al., 2013). Studies on the distribution and characterization of plasma lipoproteins in teleost fish have shown that they generally are similar to those observed in mammals (Lu et al., 2013).

In his investigation, Belal (2008) explained the negative effects of FDDP in diet higher than 300 g/kg as results of significant reduction of feed intake as indicated by an increase of body water content and the reduction of body fat. This was in odd with our results where body water content remained unchanged while plasma triglycerides increased with increasing FDDP in diet. Lower plasma cholesterol concentrations in fish fed increased percentage of FDDP diets were recorded in our study, which might indicate an increased cholesterol release from the liver. Although significant correlation among the physiological indices and the dietary FDDP in our experimental fish was observed, blood haemoglobin, plasma triglycerides and cholesterol are shown as more sensitive indicators followed by plasma glucose and then growth rate.

The general concept that the use of probiotics in aquaculture may produce various beneficial effects has been proven beyond doubt (Gaggia et al., 2010; Oliva-Teles, 2012). The scientific application of this concept to fish health and disease, although still in its infancy, has already produced some positive results (Merrifield et al., 2010;Dimitroglou et al., 2011; Ramos et al., 2013). Research is now being directed towards the vast unexplored source of plant-based antimicrobials and immunostimulants for disease management, many of which are without the negative side effects associated with synthetic chemotherapy (Hutson et al., 2012; Militz et al., 2013).

Should FDDP considered as a plant-based probiotic? To the author's best knowledge there are no studies examining the immunoregulatory response or pathogen resistance associated with FDDP supplementation in diet of cultured O. niloti-cus. In the current study, only samples from fish fed with FDDP diets had decreased number of total bacterial counts, Salmonella spp., Campylobacter spp., Shigella spp. and E. coli compared to controls. These data suggest that the FDDP might have a probiotic action and significantly affect the microbiota in O. niloticus intestine, and the response was FDDP concentration dependent. These changes were not translated into increased weight gain. Thus, changes in the diversity or richness of the gut microbiota may not per se reflect improvements in fish performance, but the remaining of haemoglobin content unchanged in the blood of fish fed all FDDP diets indicate that FDDP helps fish maintain their health status. The results of this study reinforce our view that dietary supplementation of the FDDP extract was beneficial to fish health by conferring protection against pathogens. However, the premise of FDDP's mode of action in controlling pathogen bacteria in our experimental tilapia is not well understood.

The oestrogenic content of FDDP, which increased linearly with FDDP percentage in diets, may help in explaining its mode of action whether it is direct or indirect, a phytoestrogenic roll of FDDP may be possible.

Concluding remarks and future perspectives

The present study showed a possible potential of using FDDP to lower the bacterial population densities in O. niloticus intestine without affecting fish weight or health welfare condition, that could be an excellent approach in designing subsequent probiotic researches on FDDP. Untargeted metabolite analysis is a new approach that may contribute to the understanding of the function of the intestinal microbiota and the interactions with the host (Matsumoto et al., 2012; Zheng et al., 2011; Lazado and Caipang, 2014).

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