Science International  Volume 5 Issue 3, 2017

Research Article

Impacts of Pro-Grow® on Growth Performance, Physiological, Immune Responses and Economic Efficiency of Adult Oreochromis niloticus (Linnaeus, 1758) under Stocking Density Stress
Ahmed Ismail Mehrim
Department of Animal Production, Faculty of Agriculture, Mansoura University, Al-Mansoura, PC. 35516, Egypt

Fathy Fatoh Khalil
Department of Animal Production, Faculty of Agriculture, Mansoura University, Al-Mansoura, PC. 35516, Egypt

Mohamed Moaaz Refaey
Department of Animal Production, Faculty of Agriculture, Mansoura University, Al-Mansoura, PC. 35516, Egypt

Aram Muhamad Abdalqadir
Department of Animal Production, Faculty of Agriculture, Mansoura University, Al-Mansoura, PC. 35516, Egypt

ABSTRACT:
Background and Objective: Fish intensification had led to the outbreaks of stress effects and inflicting major problems in the fish farming industry. Hence, the objective of the present study was to evaluate the effects of dietary probiotic Pro-Grow® contrary to the stocking density stress (SD) on adult Oreochromis niloticus (O. niloticus ) for 15 weeks. Methods: The present study was designed to evaluate the effects of graded levels of Pro-Grow® (0, 10 and 20 g kg–1 diet) against SD (40 and 80 fish m–3) stress on water quality parameters, growth performance, physiological, immune responses, besides the economic efficiency parameters of adult O. niloticus. Data was analyzed using SAS. Results: The results revealed that increasing SD (80 fish m–3) significantly (p<0.05) decreased the water dissolved oxygen, while water NH3-N was significantly increased. Also, high SD had drastically effects on all growth and feed efficiency parameters, body composition (ash and crude protein), hematological parameters (hemoglobin, red blood cells and white blood cells) and the economic efficiency compared to O. niloticus reared at low SD (40 fish m–3). No significant (p>0.05) effects of all tested immune responses parameters of fish reared at both SDs rates. However, addition of 10 and 20 g Pro-Grow® kg–1 diet alleviated the severely effects of both SDs stress 40 and 80 fish m–3, respectively on all above tested parameters. Conclusion: Thus, it could be concluded that the Pro-Grow® probiotic was successfully using at these levels into O. niloticus intensive production systems. Regarding its predicted high fish production, improving physiological responses, besides the high economic efficiency or the environmental friendly effects.
 
    How to Cite:
Ahmed Ismail Mehrim, Fathy Fatoh Khalil, Mohamed Moaaz Refaey and Aram Muhamad Abdalqadir , 2017. Impacts of Pro-Grow® on Growth Performance, Physiological, Immune Responses and Economic Efficiency of Adult Oreochromis niloticus (Linnaeus, 1758) under Stocking Density Stress. Science International, 5: 113-126
DOI: 10.17311/sciintl.2017.113.126
 


INTRODUCTION
Tilapias are able to easily bred in captivity, tolerate a wide range of environmental conditions, reproduce rapidly and highly resistant to diseases. Thus, they are the most important fish species for freshwater aquaculture and represent a major protein source in many developing countries1. Aquaculture has been increasing in recent decades as a consequence of the increase of fish consumption, since fisheries have possibly reached their maximum due to overexploitation2. The rapid expansion and intensification of aquaculture production had led to the outbreaks of new pathogens and infectious diseases inflicting major problems in the fish farming industry3.

Attractive food may be looted and consumed quickly, thus reducing losses by leaching of essential water-soluble components. Currently, number of probiotics are commercially available and have been introduced as food additives for fish, shrimp and molluscan4. Probiotics are defined as live microorganisms including many bacteria and yeast, which when administered in sufficient amounts could improve the growth and health of the host5. Immunomodulatory products, including probiotics are increasingly used in aquaculture production. Thus, the research into the use of probiotics for aquaculture is increasing with the demand for environment-friendly sustainable aquaculture6. Many studies have pointed out that probiotics in fish diet improved growth performance and nutrients utilization7, physiological paramaters8 and immune responses9,10. Moreover, probiotic supplementation may provide vitamins, short chain fatty acids and/or digestive enzymes and therefore may also contribute to host nutrition11.

Particular attention has been drawn to stocking density (SD) as one of the key factors influencing the perceived level of stress in fish12. Fish may encounter different types of stress such as, thermal13, SD14, anoxia, hypoxia, chemicals and pesticides15. To avoid these stressful conditions, intervention with immunostimulants, vaccines and probiotic bacteria, either as a feed supplement or in water, could trigger the defense system and thus ameliorate the harmful effects mediated by different stress factors16. Moreover, the intensification systems of fish are more important subject not only in developed countries but also in developing countries for increasing the total fish production and partially for increasing the fish production per capita. Consequently, the present study was aimed to estimate the effects of graded levels of a new commercial probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) against SD (40 and 80 fish m–3) stress on water quality, growth, feed performance, body composition, haematological, biochemical, immune responses and the economic efficiency parameters of adult Oreochromis niloticus for 15 weeks.


MATERIALS AND METHODS
Experimental procedures: This study was conducted during the summer season 2014 in Fish Research Unit, Faculty of Agriculture, Mansoura University, Al-Dakahlia Governorate, Egypt. Adult O. niloticus, with an average initial body weight (55.75±0.6 g) were purchased from Integrated fish farm at Al-Manzala (General Authority for Fish Resources Development-Ministry of Agriculture) Al-Manzala, Al-Dakhalia Governorate, Egypt. Fish were stocked into a rearing glass fiber tanks (1 m3 in volume) for two weeks as an adaptation period, during this period they fed a basal experimental diet. Fish were distributed into six experimental treatments as 3 replicates per each (Table 1). Each tank was supplied with an air stone connected with electric compressor for aeration the water. Fresh underground water was used to change half of the water volume in each tank every day. Water quality parameters were determined as, water temperature was measured two times daily (via a thermometer). While, water pH, dissolved oxygen and NH3-N were measured two times day by day. Water pH was measured using a digital Jenway Ltd., Model 350-pH-meter. Dissolved oxygen was measured using a digital Jenway Ltd., Model 970-dissolved oxygen meter. Water NH3-N was determined by direct Nesslerization method using a CHEMets® test kits (CHEMetrics, Inc, USA) according to APHA17. The fish were weighed every two weeks by a digital scale (accurate to ±0.01 g) to adjust their feed quantity according to the actual body weight changes of the fish present in each tank.

Experimental diet and probiotic: The ingredients of the experimental diet were bought from the local market for fish feed but its proximate chemical analysis was carried out according to AOAC18, as shown in Table 2. The ingredients were ground to add the tested probiotic at levels of 0, 10, 20 g kg–1 diet and then referred to treatments no. T1, T2 and T3, respectively, for 40 fish m–3 and T4, T5 and T6 treatments for 80 fish m–3 (Table 1), then all diets were repelleted by manufacturing machine (pellets size 1 mm).

Table 1: Details of the experimental treatments

Table 2: Ingredients and proximate chemical analysis (% on dry matter basis) of the experimental basal diet

The experimental diets were introduced manually twice daily at 9 am and 15 pm at 3% of the fish biomass in each tank for 9 weeks, then 2% for the rest 6 weeks of the experiment. Pro-Grow® was manufactured by Zagro industry company Ltd., South Korea and distributed by Elyoser Medicine Trading Co., Egypt. One kg contained Saccharomyces cervisae, 4000×1012 CFU (colony forming units), 14×1012 CFU of Bacillus subtilis, 150000 IU protease enzyme, 70000 IU amylase enzyme and up to 1 kg lime stone.

Fish sampling and fish performance parameters: At the start and at the end of the experiment, fish samples were collected and kept frozen (-20°C) till the proximate analysis of the whole body were done according to AOAC18. Their energy content was calculated according to NRC19. Fish growth and feed efficiencey parameters such as average weight gain (AWG, g fish–1), average daily gain (ADG, g fish–1 day–1), specific growth rate (SGR, % day–1), survival rate (SR, %), feed conversion ratio (FCR), protein efficiency ratio (PER) and energy utilization (EU, %) were calculated acording to Halver and Hardy20.

Blood samples: At the end of the experiment, five fish from each tank were randomly collected, then fish anaesthetized by transferred in a small plastic tank containing 10 L water supplemented with 3 mL pure clove oil (dissolved in 10 mL absolute ethanol). For the hematological parameters analysis, blood samples, (5 mL of whole blood at each collection), were collected from the fish by puncturing caudal venous with a syringe needle and the samples were kept in small plastic vials containing heparin-anticoagulant. Other blood samples were collected in dried plastic tubes and centrifuged for 20 min at 3500 rpm to obtain the blood serum. Serum samples were kept in deep freezer (-20°C) until the biochemical analysis was carried out.

Hematological parameters: Whole blood samples were used for the determination of hemoglobin (Hb) using commercial colorimetric kits (Diamond Diagnostic, Egypt). Total red blood cells (RBCs×106 mm–3) and total white blood cells (WBCs×103 mm–3) were counted according to Dacie and Lewis21 on an Ao Bright-Line Hämocytometer model (Neubauer improved, Precicolor HBG, Germany). The packed cell volume (PCV, %) was measured according to Stoskopf22.

Biochemical and immune responses parameters: Biochemical constituents of serum were determined calorimetrically using commercial kits produced by Diagnostic System Laboratories, INC, USA. Total protein was determined according to the method described by Tietz23. Serum albumin was determined according to Doumas et al.24. The concentration of serum globulin was obtained by subtracting the albumin from the total serum protein concentration according to Doumas and Biggs25. Competitive ELISA was used to measure the immunoglobulin M (IgM) concentration in serum, which described by Magnadottir and Guomundsdottir26.

Statistical analysis: The obtained data was statistically analyzed using SAS (Version 8.2)27 for users guide, with factorial design (2×3) by the following model:

Yijk = μ+Li+Mj+LMij+eijk

where, Yijk is the data of rearing water quality, growth and feed performance, carcass composition, hematological and biochemical parameters, immune responses and economic efficiency of O. niloticus, μ is the overall mean, Li is the fixed effect of the stocking density (40 and 80 fish m–3), Mj is the fixed effect of the probiotic levels (0, 10 and 20 g kg–1 diet), LMij is the interaction effect between the stocking density and probiotic levels and eijk is the random error. All ratios and percentages were arcsine transformed prior to statistical analyses. The differences between mean of treatments were compared using Tukey’s post hoc significant test and differences were considered statistically significant at p<0.05.


RESULTS
Rearing water quality measurements: Data in Table 3 showed water quality parameters of adult O. niloticus reared under high stocking densities (SDs, 40 and 80 fish m–3) and fed different levels (0, 10 and 20 g kg–1 diet) of tested probiotic Pro-Grow®. The current results indicated that water dissolved oxygen was significantly (p<0.05) decreased, while water NH3-N was significantly (p<0.05) increased by increasing the fish stoking density rate. However, no significant (p>0.05) differences in both water temperature or pH-value were detected by increasing the stoking density rate. On the other hand, no significant differences in all tested water quality parameters were observed of fish groups fed different levels of probiotic or diet free probiotic group. Also, the interaction between SD and Pro-Grow® did not show the significant differences in all water quality parameters, except in case of fish reared at high SD (80 fish m–3) and fed different levels of Pro-Grow® had significantly (p<0.05) decreased the water NH3-N compared with the control group (Fig. 1).

Growth and feed efficiency parameters: Growth and feed efficiency parameters of adult O. niloticus reared under different SDs rates (40 and 80 fish m–3) and fed different levels (0, 10 and 20 g kg–1 diet) of tested probiotic (Pro-Grow®) are illustrated in Table 4. The results revealed that increasing SD (80 fish m–3) caused significantly (p<0.05) decreased all growth and feed efficiency parameters (negatively affected on FCR) compared to the low SD (40 fish m–3), while survival rate (SR, %) not significantly affected by increasing SD rate. From other hand, fish fed 20 g Pro-Grow® kg–1 diet was significantly (p<0.05) increased all growth and feed efficiency parameters (gave the best FCR) compared to fish fed 10 or 0 g Pro-Grow® kg–1 diet, respectively.

No significant differences in all growth and feed efficiency parameters in the interaction between low SD (40 fish m–3) and all dietary probiotic levels. However, fish reared in low SD (40 fish m–3) and fed 10 g Pro-Grow® kg–1 diet had highest (p<0.05) SR among other treatments (Fig. 2a).

Figure 1: Effect of the interaction of high stoking density (80 fish m–3) and dietary probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) levels on rearing water NH3-N of adult Oreochromis niloticus

Table 3: Effect of stoking density (fish m–3) and dietary probiotic Pro-Grow® (g kg–1 diet) levels on rearing water quality parameters of adult Oreochromis niloticus

Table 4: Effect of stoking density (fish m–3) and dietary probiotic Pro-Grow® (g kg–1 diet) levels on growth and feed efficiency parameters of adult Oreochromis niloticus

Figure 2(a-h): Effect of the interaction of stoking density (40 and 80 fish m–3) and dietary probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) levels on (a) Survival rate (%), (b) Final weight (g), (c) Average weight gain (g fish–1), (d) Average daily gain (g fish–1 day–1), (e) Specific growth rate (% day–1), (f) Feed conversion ratio, (g) Protein efficiency ratio and (h) Energy utilization (%) of adult Oreochromis niloticus

Meanwhile, all growth and feed efficiency parameters significantly increased in case of fish reared in high SD (80 fish m–3) and fed 20 g Pro-Grow® kg–1 diet (Fig. 2b-e, g and h) and gave the best FCR among other treatments (Fig. 2f). From the other hand, no significant differences were detected in SR of the interaction between high SD (80 fish m–3) and all probiotic levels.

Figure 3: (a-c): Effect of the interaction of high stoking density (80 fish m–3) and dietary probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) levels on some body composition parameters, (a) Ash (%), (b) Ether extract (%) and (c) Energy content Kj 100 g–1 DM of adult Oreochromis niloticus

Table 5: Effect of stoking density (fish m–3) and dietary probiotic Pro-Grow® (g kg–1 diet) levels on body composition parameters of adult Oreochromis niloticus

Fish body composition: Proximate chemical analysis of the whole body of adult O. niloticus at the start or at the end of the experiment was summarized in Table 5. Fish reared at high SD (80 fish m–3) had significantly (p<0.05) increased DM, EE and EC, while ash and CP were significantly decreased compared to the low SD rate (40 fish m–3). While, fish fed Pro-Grow® probiotic in both levels (10 or 20 g kg–1 diet) showed significant (p<0.05) decrease of ash and CP, while significantly increased EE and EC contents of fish body compared to the free probiotic diet (control group). However, no significant differences in DM among all probiotic levels.

From the other hand, no significant differences in all body composition parameters in case of fish reared at low SD (40 fish m–3) and fed different levels of Pro-Grow® probiotic. However, fish reared at high SD (80 fish m–3) and fed different levels of Pro-Grow® probiotic showed significant (p<0.05) increase of EE (Fig. 3b), EC (Fig. 3c), while ash (Fig. 3a) was significantly decreased compared to the control group (free diet from the tested probiotic). However, no significant differences in DM or CP were detected among all treatments.

Table 6: Effect of stoking density (fish m–3) and dietary probiotic Pro-Grow® (g kg–1 diet) levels on hematological parameters of adult Oreochromis niloticus

Table 7: Effect of stoking density (fish m–3), dietary probiotic Pro-Grow® (g kg–1 diet) levels and their interaction on biochemical and immune responses parameters of adult Oreochromis niloticus

Haematological parameters: In the present study, data of haematological parameters of O. niloticus reared in high SD (80 fish m–3) showed significant decrease (p<0.05) of Hb, RBCs, WBCs, while MCH and MCHC significantly increased compared to the fish reared in low SD (40 fish m–3). No significant differences of PCV and MCV were detected among fish groups at two SDs. On the other hand, fish fed 20 g Pro-Grow® kg–1 diet led to significant increase of RBCs, PCV and WBCs, but significantly (p<0.05) decreased all the blood indices (MCV, MCH and MCHC) compared to fish fed other probiotic levels. No significant effects of Hb content were detected among all dietary probiotic levels (Table 6).

The interaction effects between low SD and dietary Pro-Grow® levels did not show any significant differences in all haematological parameters. Also, no significant differences of Hb and PCV were detected among all treatments. However, fish reared in high SD and fed high Pro-Grow® level (20 g kg–1 diet) showed significant increase of RBCs (Fig. 4a), WBCs (Fig. 4e) but all blood indices (MCV, MCH and MCHC) were significantly decreased compared to other treatments (Fig. 4b, c and d, respectively).

Biochemical and immune responses measurements: Serum biochemical and immune responses parameters of adult O. niloticus reared in high SD and fed dietary Pro-Grow® probiotic are presented in Table 7. Data revealed that both of SDs, Pro-Grow® probiotic, as well as their interaction had no significant effects of all tested immune responses parameters of O. niloticus, except the case of fish fed 20 g Pro-Grow® kg–1 diet, which significantly (p<0.05) increased the globulin concentration compared to other levels of tested probiotic.

Economic efficiency: Fish reared at high SD (80 fish m–3) led to significantly (p<0.05) increase the total feed costs (LE kg–1 diet) and total outputs (LE kg–1 fish), consequently led to significantly decrease of the net return (LE) and economic efficiency (%) compared to the low SD. Fish fed 10 and 20 g kg–1 diet of Pro-Grow®, respectively have positively effects on the economic efficiency parameters compared to the control group (Table 8).

In case of the interaction effects between SDs rates and dietary Pro-Grow® levels, fish reared at low SD (40 fish m–3) and fed 10 g Pro-Grow® kg–1 diet or fish reared at high SD (80 fish m–3) and fed 20 g Pro-Grow® kg–1 diet had gave the highest significant (p<0.05) increased of the economic efficiency parameters compared to the control group in each case (Fig. 5 and 6a-d, respectively).


DISCUSSION
All water quality parameters measured herein were within the acceptable ranges as recommended for rearing O. niloticus28. Where, increasing SD rate of adult O. niloticus did not affected on water quality parameters, except significantly decreased water dissolved oxygen and increased water NH3-N. Increasing of SD may cause deterioration in water quality, resulting in stressful conditions.

Figure 4: (a-e):Effect of the interaction of high stoking density (80 fish m–3) and dietary probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) levels on some haematological parameters, (a) Red blood cells (×106 mm–3), (b) Mean corpuscular volume (μ3), (c) Mean corpuscular hemoglobin (Pg), (d) Mean corpuscular hemoglobin concentration (%) and (e) White blood cells (×103 mm–3) of adult Oreochromis niloticus

Figure 5: (a-d):Effect of the interaction of low stoking density (40 fish m–3) and dietary probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) levels on the economic efficiency parameters of adult Oreochromis niloticus

Figure 6: (a-d):Effect of the interaction of high stoking density (80 fish m–3) and dietary probiotic Pro-Grow® (0, 10 and 20 g kg–1 diet) levels on the economic efficiency parameters of adult Oreochromis niloticus

Table 8: Effect of stoking density (fish m–3) and dietary probiotic Pro-Grow® (g kg–1 diet) levels on the economic efficiency parameters of adult Oreochromis niloticus

Water quality plays a significant role in the biology and physiology of fish and may impact on the health and productivity of the culture system29. Thus, negative correlationship between high SD and fish rearing water quality parameters was previously detected by M’balaka et al.30. Meanwhile, the tested probiotic enhanced the water quality parameters of O. niloticus under high SD rate. This improvement of rearing water quality parameters may be due to the tested probiotic formula, which it contains S. cervisae, 4000×1012 CFU and 14×1012 CFU of B. subtilis. These effective microorgansims played a potential role for improvement the rearing water quality parameters, which confirmed also by Mehrim8 on O. niloticus reared under different SD conditions. Also, Dalmin et al.31, stated that improved water quality parameters, SR, growth rates and increased the health status of juvenile Penaeus monodon has especially been associated with dietary Bacillus sp. This improvement of rearing water quality herein not only refluxing on fish growth performance or survival rate (Table 4), but also may be on the total fish production, fish health and final product quality, besides the economic efficiency (Table 8) or the expected friendly environmental effects.

In the present study, high SD rate had negatively effects on all growth and feed efficiency parameters. However, addition of high level of tested probiotic (20 g kg–1 diet) enhancement this picture. This improvement may be due to the probiotic itself, its formula components with beneficial microorganisms. Also, the positive effects of probiotic on the rearing water quality parameters (Table 3), may be led to decrease the stress effects of high SD rate on fish, enrichment the growth and feed efficiency parameters of fish (Table 4). Regarding the depressing effects of high SD on fish growth and feed efficiency parameters in the present study are in agreement with those obtained by Bakeer et al.32, for O. niloticus. Also, Sorphea et al.33, reported that growth performance and SR of tilapia are adversely affected by high SDs. Although, in some cases this effect is either temporary34 or absent35. Some fish species like tilapias can tolerate extreme crowding, nevertheless tilapias are territorial and aggressive fish so that the density effect on growth might be explainable by their competition for territories, as well as the permanent stress caused by crowding36.

Functional additive, like probiotics, is a new concept on aquaculture37, where the additions of microorganisms on diets show a positive effect on growth and feed efficiency caused by the best use of carbohydrates, protein and energy5. All the probiotic-supplemented diets resulted in fish growth higher than that of the control diets, suggesting that the addition of probiotics mitigated the effects of the stress factors38. Affirmative results of O. niloticus growth and feed efficiency parameters related with dietary supplemented probiotics were reported39. Also, the same positive effects on growth performance, feed conversion and protein efficiency ratio were detected for O. niloticus fed Biobuds® and yeast40, some biological additives41, or native bacteria supplemented diets42. Hence, Mehrim8 suggested that Biogen®, can enhance the metabolism and energy of body cells, raise the efficiency of feed utilization, increase the vitality of cells by supplying oxygen to whole body and improve the immune responses of O. niloticus. Conversely, He et al.43, found that supplementation of dietary DVAQUA® showed no effects on growth performance, feed conversion and SR of the hybrid tilapia (O. niloticus ♀×O. aureus ♂). The reasons for the differences between fish species have not been elucidated, but might be due to the differences in aquaculture and physiological conditions, besides the type of basal ingredients in diets. Accordingly, to the positive effects of the tested probiotic on growth performance in the present study and those obtained by other attempts, probiotics may stimulate appetite and improve nutrition of fish by the production of vitamins, detoxification of compounds in the diet and by breakdown of indigestible components5.

As the current findings in fish carcass composition affected by SD or tested probiotic levels, the yeast supplementation significantly affected O. niloticus body composition10, where yeast supplementation plays a role in enhancing feed intake with a subsequent enhancement of fish body composition. Also, results in the present study are in close agreement with those reported by Mehrim8 and Abdelhamid et al.41. Reversely, no significant differences were detected on performance and proximal composition of O. niloticus fed probiotic B. amyloliquefaciens44. Additionally, Khalil et al.38, suggested that body composition of adult O. niloticus males and females fed Hydroyeast Aquaculture® probiotic were took unclear trends, which may be due to the differ in sexes, metabolism, physiological responses and sexual behaviors of fish during this stage of life.

Haematological parameters of fish are useful tools that aid in studying the immunopotentiators45. In the present study, high SD had an adversely effects on haematological parameters of O. niloticus. In this respect, Mehrim8 found the deleterious effects on haematological parameters of O. niloticus reared under different SDs. Moreover, Kpundeh et al.46, reported that haematological indices, RBCs, WBCs, Hb, PCV and blood platelets significantly (p<0.05) decreased by increasing SD rate of O. niloticus. Meanwhile, current positive effects of tested probiotic Pro-Grow® on fish haematological parameters are in agreement with the results obtained by Mehrim8. Furthermore, Abdel-Tawwab et al.10, reported that O. niloticus fed 1.0-5.0 g yeast kg–1 diet exhibited higher RBCs, Hb and PCV values. In a recent srudy, the clearly positive effects on the homeostatic state were observed of O. niloticus fed probiotic B. amyloliquefaciens44. Inversely, Rawling et al.47, found that PCV, Hb and RBCs levels were not affected of red tilapia (O. niloticus) by dietary inclusion of Sangrovit®. Also, Zhu et al.48, reported that Ictalurus punctatus fed yeast polysaccharides had no effects on total number of WBCs, granulocytes and lymphocytes.

The effects of stress on the immune system of fish showed how complex these interactions could become49. In the present study, the adversely effects of high SD on immune responses of O. niloticus were not cleared, but dietary Pro-Grow® probiotic had slightly improved of immune responses parameters of the experimental fish. This improvement may be due to the seriously experimental management and/or functional microorganisms formula of tested probiotic, namely S. cervisae, 4000×1012 CFU, 14×1012 CFU of B. subtilis. Hence, probiotics supplementation improves the systemic immune response in fish50. Additionally, some factors can regulate their activities such as adhesion properties, attachment site, stress factors, diet, environmental conditions determine the colonization of probiotics in the gut of host, probiotics often exert host specific51, strain specific differences in their modes of action52, the source of probiotics53, achievability54, dose55 and duration of supplementation56.

Modulation of host immunity is one of the most purported benefits of probiotics consumption and fish is no exception51. In this context, enhanced the innate immune responses and disease resistance of O. niloticus fed 1.0-5.0 g yeast kg–1 diet10, dietary Lactobacillus sp.57, or L. plantarum58 compared to the control group. Recently, Guzman-Villanuev et al.59, confirmed strong synergy in stimulation of immune system between glucan and probiotic strain Shewanella putrefaciens. However, not all studies result in enhanced immune function by dietary probiotic supplementation. Thus, Shelby et al.9, did not find any affect on lysozyme activity, alternative complement or total serum immunoglobulin of O. niloticus fed probiotic containing B. subtilis, B. licheniformis, P. acidilactici and S. cerevisiae. Generally, there is far less evidence available suggesting that dietary probiotics influence the humoral immune response in tilapia.

SD and SR are important indicators that determine the economic viability of a production system60. In the present study, fish reared at high SD led to significantly decrease of economic efficiency compared to the low SD. Fish fed 10 and 20 g kg–1 diet of Pro-Grow® respectively have positively effects on the economic efficiency parameters compared to the control group. From the economic point of view, these positive effects may be due to the role of probiotic to alleviate the stress of SDs and significantly improved the water quality parameters, growth performance, feed utilization, carcass composition, physiological and immune responses parameters of fish reared at high SD, which consequently led to highly economic efficiency. Similarly with our findings regarding the economic efficiency, O. niloticus fed Biogen® probiotic8 or Biobuds® and yeast40 significantly improved the economic efficiency parameters compared to the fish fed the diet free probiotics. Furthermore, probiotics can reduce the use of antibiotics and synthetic chemicals in the fish food5, consequently the addition of probiotics to fish diets has become widespread on aquaculture farms. The application of probiotics results in reduced food costs, which plays an important role in determining the practices of aquaculture. Interestingly, previous findings have shown that the beneficial effects of probiotics can manifest as enhanced feed utilization of cultured aquatic animals through the supplementation of digestive enzymes, improved feed efficiency and higher growth, the prevention of intestinal disorders and the pre-digestion of anti-nutritional factors present in mixed feed61.


CONCLUSION AND FUTURE RECOMMENDATIONS
Finally, the obtained results revealed the adversely effects of high SD (80 fish m–3) stress on the tested parameters of the adult O. niloticus compared to the fish reared at SD (40 fish m–3). Also, the potential positive effects of Pro-Grow® at levels 10 and 20 g kg–1 diet against the different SDs 40 and 80 fish m–3, respectively were detected too. Consequently, it could be concluded that the using of dietry Pro-Grow® with these levels at the different intensive fish culture systems may be useful and more applicable in the large scale fish farms. Regarding its predicted high fish production, improving physiological responses, besides the high economic efficiency or the environmental friendly effects. Thus, advanced studies are needed not only of this probiotic, but also for other different levels or types of probiotics, prebiotics, synbiotics or other function food additives of different fish species reared under intensive production systems.


SIGNIFICANCE STATEMENTS
This study discover the effects of graded levels of dietary Pro-Grow® probiotic against the stocking density stress on adult Oreochromis niloticus that can be beneficial for avoiding the drastic effects of stocking density stress on fish and increasing fish growth and production, improving the physiological responses. So, this study will help the researcher to uncover the critical areas of intensification of Aquaculture that many researchers were not able to explore. Thus a new theory on this useful probiotic only or possibly with other combinations by prebiotic may be arrived at.


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