Effect of Selection for Increasing Body Weight at Harvest on Immune Response Parameters in Second Generation of Nile Tilapia (Oreochromis Niloticus)

Selection for improved immunity in applied breeding programs may decrease mortality rates in fish farming. The basis for selection is genetic difference in improved quantitative traits such as immunity and disease resistance [1]. Therefore, the value of selective breeding depends on the level of genetic difference to a disease within the population in question, being most advantageous when the variation is relatively large [2]. Selection may be done directly based on survival records under farming conditions, in challenge tests or indirectly using correlated traits as indicators. As the immune system is mostly responsible for cover against disease, immunological markers are being used as indirect measures of disease resistance.


Introduction
Selection for improved immunity in applied breeding programs may decrease mortality rates in fish farming. The basis for selection is genetic difference in improved quantitative traits such as immunity and disease resistance [1]. Therefore, the value of selective breeding depends on the level of genetic difference to a disease within the population in question, being most advantageous when the variation is relatively large [2]. Selection may be done directly based on survival records under farming conditions, in challenge tests or indirectly using correlated traits as indicators. As the immune system is mostly responsible for cover against disease, immunological markers are being used as indirect measures of disease resistance.
The markers showed wide genetic difference, correlate with disease resistance as well as being simple and quick to be quantified from large populations of live fish [3]. Both physiological and biochemical mechanisms conferring resistance to micro parasites can have a strong genetic basis [4]. Previous work showed that potential correlation exists between disease resistance of fish and nonspecific immune parameters viz., serum lysozyme, complement, hemolytic, and bactericidal activities, which affect inherent capacity of fish to resist pathogens prior to generation of a specific immune response [5][6][7][8][9].The objective of the present investigation was to determine the specfic and non specific immune parameters between selected and nonselected breed line of the second generation of full-sib Nile tilapia, Oreochromis niloticus.

Fish Origin and Culture Condition
Nile tilapia used in this work was the second generation of selection program for growth. Sixteen fish were randomly collected from tanks (circular fiber glass tanks with capacity of 500 liter) and each tank represents a full-sib family. Sixteen fullsib families were used. Eight fish were collected from selected line, and the average body weight was 35 ± 1.5. Also, eight fish were collected from control line, and their body weights were 28 ± 1.1 g. These tanks were in laboratory at faculty of Veterinary Medicine, Alexandria University at period from 30 September 2015 to 30 May 2016. At the end of the experiment, blood samples were collected from the fish caudal peduncle of the two groups and the following parameters were measured. the clot is loosened from the wall and the tubes are placed in the incubator for half an hour to enhance retraction of the clot, then put in the refrigerator at 4C overnight, and the sera were separated by centrifugation. The serum samples were kept in deep freezer at 20-30C until being used [10].

Clinico-Biochemical Analysis
At the end of the experiment, blood samples were collected from the fish caudal peduncle of the different groups. Adequate amounts of whole blood in small plastic vials containing heparin were used for the determination of hemoglobin (Hb) by using commercial kits (Diamond Diagnostic, Egypt) and the hematocrit (PCV %) was measured according to Stoskopf [11]. Also, total erythrocytes (RBCs), platelets and total leukocytes (WBCs) were counted according to Dacie & Lewis [12] on an Ao Bright -Line Haemocytometer model (Neubauer improved, Precicolor HBG, Germany). Other blood samples were collected and transferred for centrifugation at 3500 rpm for 15 min to obtain blood plasma for determination of total protein (TP) [13]; albumin (Al) [14]; globulin (Gl) by difference [15] and total cholesterol [16] which were assayed following commercial test kits using a spectrophotometer (model 5010, Germany).

Determination of (S. GOT) and (S. GPT)
The activity of serum aspartate aminotransferase (S. AST), normally known as glutamic oxalacetic transaminase (S. GOT), and serum alanine aminotransferase (S. ALT), usually known as glutamic pyruvic transaminase (S. GPT), were estimated according to Bohnert et al., [17] using commercial kits made by Pasteur Lab. Estimation of serum alkaline phosphatase: Serum alkaline phosphatase was determine according to modified method of Aoki et al., [18] using commercial kits produced by Pasteur Lab. Lysozyme activity and Serum bactericidal activity: Serum lysozyme activity was estimated through the turbidimetry described by Hultmark et al., [19]. Serum bactericidal activity to Aeromonas hydrophila strain (Standard strain ATCC obtained from Dept. Avian and aquatic Animal Med., Fac. Med. Alexandria University) was determined according to Rainger and Rowley [20].

Determination of IgA, IgG and IgM
Immunoglobulin an ELISA Kit (Saliva) -Salimetrics Assays, 1-1602. Chicken IgG ELISA Cat. No. KT-619 PRODUCT the K-ASSAY Ò Chicken IgG ELISA is an enzyme immunoassay for the quantitative determination of IgG in chicken serum plasma. Chicken IgM ELISA Cat. No. KT-621PRODUCT the K-ASSAY Ò Chicken IgM ELISA is an enzyme immunoassay for the quantitative determination of IgM in chicken serum plasma. IgM was measured according to Ohta et al. [21], Nevens et al. [22] & Wendelborn et al. [23].

Total Cholesterol and Triglycerides Determination
Cholesterol was measured enzymatically in serum or plasma in a series of coupled reactions that hydrolyze cholesteryl esters and oxidize the 3-OH group of cholesterol. One of the reaction byproducts, H 2 O 2 was measured quantitatively in a peroxidase catalyzed reaction that produces a color. Absorbance was measured at 500 nm. Triglycerides was measured enzymatically in serum or plasma using a series of coupled reactions in which triglycerides are hydrolyzed to produce glycerol. Glycerol is then oxidized using glycerol oxidase, and H 2 O 2 , one of the reaction products, was measured as described above for cholesterol. Absorbance is measured at 500 nm. High density lipoprotein (HDL) cholesterol Low serum concentrations of HDL-cholesterol was associated with increased risk for CHD. Coronary risk increases markedly as the HDL concentration decreases from 40-to 30 mg/dL.

Statistical Analysis
Data were analyzed by Student's "t" test to compare the different parameters using SPSS (2010) statistical package version. The data values were presented as mean ± standard error of mean (SEM).

Results and Discussion
Mean values of immune parameters were compared between selected bred line and non-selected bred line of the second generation of Nile tilapia, Oreochromis niloticus. The highest means of total protein, albumin, globulin, alpha, beta, gama, ALT, AST, Total lipid, Triglycerol, cholesterol, HDL, LDL, Lysozyme activity, Bactericidal activity, IgA, IgG, IgM, TWBCS, TRBCS, HB, PCV, Lymphocytes, monocytes, Basophil, Eosinophil, Neutrophil, and Heterophil, showed significant superiority for selected bred line compared to those of non-selected bred line of Nile tilapia. While, the means of urea, creatinine, and Alkaline phosphate showed significant decrease for selected bred line compared to non-selected bred line (Table1).
Experimental selection for increasing body weight had a large and significant effect on immune functions. The results of selection for increased immune functions are mixed, however. Significant overall effect was found. Pinard-Van Der Laan [24] found no consistent difference between the arms of the immune system, either when comparing the effects of selection for growth on different immune system components, or when comparing the effects of selection for growth on different components of the immune system. This is not unexpected because the effectiveness of different arms of the immune system need not be genetically correlated. However, the effects of selection for growth were

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consistent with respect to the aspect of immune function that was measured, while the variation in growth response to immune function selection was independent of the type of immune response that was selected. Variation in innate immune response was examined for fish collected randomly from tanks. The selected fish were obtained from the second generation of selection program of Nile tilapia for growth showed significant superiority compared to non-selected bred line. El-Zaeem [25], reported that serum total protein and globulin of genetically modified Tilapia zillii were significantly enhanced compared to non-genetically modified fish. Moreover El-Zaeem & Assem [26] and Assem & El-Zaeem [27], stated that serum total protein, globulin and IgM of both genetically modified Oreochromis niloticus and Tilapia zillii were improved significantly compared to non-genetically modified fish. The results of the present work are consistent with these findings. RBC and WBC levels were enhanced, and there was also variation in Hb and PCV levels between selected breed line and control breed line. The amounts of hemoglobin and hematocrit are a function of RBC changes [28]. Previous studies have also reported that fish selected for increased body weight show increased WBC and RBC levels and the cellular innate immune response is enhanced [29]. Selected breed line fish show a significant increase in cholesterol and triglyceride levels compared to non-selected breed line. In the contrary Wang et al., [30] reported declining pattern of triglycerides and total serum cholesterol levels in tilapia fed with feed containing Bacillus cereus. However, levels of total protein were significantly higher in selected breed line fish compared to the control breed line fish.

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In general, an increase in serum protein level is an indicator of innate immunity, which is considered important in invertebrates and a fundamental defense mechanism of fish [31]. Such mechanisms form a series of essential functions that keep host cells alive, healthy, and protected from pathogens [32]. The two liver enzymes of ALT and AST increased in selected breed line fish group compared to the control line. This is in accordance with the results of Lin & Luo [33]. Among the innate immune parameters, lysozyme activity was found to rise significantly in the selected fish compared to the control. Similar results were observed in the study of Mohapatra et al., Bandyopadhyay et al., [34,35]. Lysozyme is found in fish mucus, serum and ova, which helps in degrading peptidoglycan layer of the cell wall of bacteria. It also promotes phagocytosis by activitating polymorphonuclear leucocytes and macrophages. The relatively large variation observed in the levels of lysozyme in selected and control bred line reflects the variation in their ability to destory bacteria. Such variation in lysozyme levels was also observed in Atlantic salmon and rainbow trout by Roed et al., [5,7] selected for disease resistance.
The enhancement of immune response parameters with selection breeding program decreased mortality rate in Nile tilapia there by protecting the fish against different diseases by increasing the immunity of fish. For our analysis we found usable data for fish selection lines, but data from other taxa do indicate that selection for production traits had adverse effects on metabolic, reproduction and health traits [36]. Furthermore, productivity and immune function were negatively associated in sheep and pigs [37,38]. Studies involving commercial poultry lines were excluded from our analyses because the selection criteria are unknown and control lines are absent, but their results support our findings. Modern commercial broilers had a decreased humoral response compared with older, lighter lines [39]. Furthermore, differences reported between broiler and layer chicken lines are consistent with our results. A comparative study between a broiler and a layer line suggested that broilers are more specialized in mounting strong short-term humoral immune responses, while layers employ in a long-term humoral immune and cellular response.
The effect of selection for immune function on growth differed between selection lines, while there was no significant overall effect. This contrasts with the effects of selection for growth on immune function, which were uniform across selection lines and immune traits. There are several possible explanations for the difference in results between selection for growth or immune function, which are not mutually exclusive. First, the costs of growth are likely to be high in comparison to immune function [40]. Therefore, when growth is selected for, there is likely to be less leeway when compared with selection for a less resource demanding process such as immune function, and this asymmetry may cause the different selection effect. The potential effect of an increase in immune function on growth is substantially smaller than the potential effect of an increase in growth on immune function, which may explain why selection for growth affected immune function while selection for immune function did not affect growth. Secondly, because selection was for single immune traits rather than for a more comprehensive measure of immune function. It is possible that the selection success was achieved at the expense of other arms of the immune system, or through the selection of a more specific response. In this way it is possible that selecting for an increase in components of immune function did not result in an increase in the resource allocation to all the immune function, and hence no effect on growth [41][42][43][44].

Conclusion
In selection for increased body weight of Nile tilapia at harvest increased immune response parameters in selected breed line than non-selected breed line, and this play important role in disease resistance and improve immunity against diseases.