Hemolytic activity in blood serum of oreochromis niloticcus

Oreochromis Niloticus fish (O. niloticus) were divided into 9 equal experimental groups included: group (1) as a control, group (2) exposed to phenol (150 μg/l water) for 10 days, group (3) exposed to phenol (200 μg/l water) for 10 days, group (4) exposed to hypoxia for 3 days, group (5) exposed to copper sulphate in water (5 ppm) for 10 days, group (6) exposed to acidic water (pH 5) for 3 days, group (7) exposed to ammonia-N (0.2 ppm) in water for 3 days, group (8) exposed to starvation for 14 days, and group (9) exposed to cold condition (14 oC ± 2) for 3 days. Lysozyme activity and serum hemolytic activity were determined in serum of all fish groups. In addition, lysozyme activity was measured in scrapings of skin mucus and scrapping collected from intestinal linings. Hematocrit value, erythrocytic and platelet counts, and concentrations of blood glucose, plasma total protein and hemoglobin were determined.

 The obtained results are summarized in the following points:

1. Intestinal scrapping and serum samples of the control fish had the highest lysozyme activity compared to skin mucus samples which may explain the high inherited disease resistance of O. niloticus.
   
   
2. Phenol (either 150 or 200 μg/l) treatments induced significant decrease in serum and intestinal scrapping lysozyme activity.
   
   
3. Hypoxia, cold water (14oC), acidic water (pH 5) and ammonia (0.2 ppm) significantly reduced lysozyme activity in all samples.
   
   
4. Copper sulphate pollution elevated serum lysozyme activity compared to the control, while the intestinal scrapping and skin mucus samples had low activities.
   
   
5. -Starvation significantly reduced lysozyme activity.
   
   
6. Exposure of fish to phenol, copper sulphate, ammonia, hypoxia, acidic water, starvation and cold water resulted in reduction of serum hemolytic activity as compared to the control.
   
   
7. Concentration of glucose significantly increased and concentration of total protein significantly decreased in all groups (with the exception of copper groups concerning glucose) as compared to the control.
   
   
8. Total leukocytic count (TLC) and differential count were significantly increased in copper and cold treatments. A non significant decrease in TLC was found in fish groups treated with phenol (200 μg/l), acidic water, ammonia and starvation.
   
   
9. Erythrocytic and platelet counts tended to reduce in all groups, except in the hypoxia fish group.
   
   
10. Hematocrit values significantly reduced, except in the hypoxia fish group. Concomitant changes in hemoglobin levels were also found.

This study has shown that both serum lysozyme activity and serum spontaneous activity can be determined easily in blood samples drawn from live fish which make them potentially useful markers traits for indirect monitoring of disease resistance in fish.


INTRODUCTION

Fish diseases or stresses have become an important limiting factor in the fish farming industry. Historically, disease prevention measures have included vaccination, drug therapy and improvement of the environment. The significant genetic variation in disease resistance in different fish species found (Gjedrem et al., 1991) suggesting possibility of selective breeding to increase disease resistance. The genetic control of resistance to disease is highly complex, and involves the interaction of many systems of the body, one of most important of these is the immune system and the possible use of immunological markers as indirect measures for disease resistance is being explored. The defense against pathogenic organisms that infect an animal is first provided by the non-specific factor in fish, the hemolytic activity in fish serum against heterogonous red blood cells is considered to reflect significant components of natural defense mechanism (Ourth and Bachinsk, 1987). Two kinds of hemolytic activity against heterogonous red blood cells are usually present in normal fish sera. These are a specific (antibody-dependent) and a non-specific (natural) hemolytic activity, the latter being characterized by spontaneous hemolysis (Nanaka et al; 1981).

Evidence has been obtained that the antibody dependent and the non-specific hemolytic activities in fish sera arise via the classical and the alternative pathways, respectively of a complement system similar to that present in mammals (Roed et al., 1992).The complement system is known to play a prominent role both in the inflammatory process and in the humoral and cellular immunity against pathogens (Ingram, 1990). Lysozyme, an enzyme which splits peptidoglycan in the bacterial cell wall, is another important part of the natural defense mechanisms (Chipman and Sharon, 1969). In higher vertebrates, this enzyme also appears to be involved in opsonization, immune response potentiation, and as anti- viral activity (Jolles and Jolles, 1984). The presence of both lysozyme activity (Mayner et al., 1993), and antibody- dependent and spontaneous hemolytic activity (Roed et al., 1992).have been detected in the Atlantic Salmon Salmo solar. Previous reports has been indicated that a positive correlation exists between a high level of lysozyme activity and serum hemolytic activity (SH) in fish and the survivability potentials of fish flocks which points to un urgent further study of these two properties. So, the aim of the present investigation was to study the effects of some fish disease predisposing factors (including toxicity of organic and inorganic agents, hypoxia, acidic water, starvation, ammonia and cold) on the lysozyme and SH activity in Oreochromis. Niloticus. Total and differential leukocytic count, RBCs count, hemoglobin level, total plasma protein and glucose levels were also determined.


MATERIALS AND METHODS

1-Experimental fish

Males and females of the Nile tilapia, Oreochromis niloticus, and (70-80 g body weight) were purchased from a local private fish farm. Fish were allocated similarly into 18 aquaria, each of 10 fish, two aquaria per treatment (group). The glass aquaria (100 x 40 x 30 cm) were filled to their 2/3 volumes with dechlorinated tap water which stocked in large tanks 24 hours before use for evaporation of chlorine. One third of the water in each aquarium was daily exchanged with paying attention for removing excreta and food residuals from the bottom of the aquaria. Fish were acclimatized for laboratory conditions for 7 days before the beginning of the experiment as where fish were fed during this adaptation period. Also each aquarium was supplied with an air pump all over the period of adaptation and experimentation, except in the experiment of hypoxia, where aeration was removed. This supplementary aeration provided oxygen levels not less than 4 mg/lit which substantially determined twice daily using oxy-90 apparatus to keep this optimum dissolved oxygen for Oreochromis Niloticous (Magid and Babiker, 1975). Water temperature was kept stable at 27±3 CO as optimum temperature for Oreochromis Niloticous (Beamish, 1970) during the whole period of study except in experiment of cold groups. The pH was detected twice daily using pH-papers, where the water pH was kept at 7-8 as optimum level for Oreochromis Niloticous (Huet, 1972) except in experiment of acidic water and ammonia groups. Fish were fed on a balanced formulated tilapia feed (28% protein) at a rate of 5% of fish body weight once daily from the day of arrival until the end of the experiment, except fasting aquaria. Daily change of water was around 10% that in the aquaria. The light provided for all aquaria during adaptation and experimental period was the natural photoperiod without interference, which was about 12 hours of light: 12 hours of dark/day during the days of study, where the present study was carried out during winter season on February 2003.


2-Treatments

Group 1 (Untreated): As a control group, and was kept under optimal environmental conditions.

Groups 2 & 3 (Low and high concentrations of phenol): In these groups, phenol was added to the aquarium's water at concentrations of 150 and 200 μg/L water, respectively. Water was renewed every 2 days with the same concentrations of phenol for 10 days. At day 11, blood was collected from caudal vessels.

Group 4 (Hypoxia): In this group (fish were exposed to hypoxic conditions by removing aeration from their aquaria (Graham and Baird, 1984); the dissolved oxygen level was 1.5+_0.5 mg/l during the whole experimental period. Blood samples were collected after 3 days from the onset of hypoxic stress, and all other environmental conditions were kept at optimum levels as in the control group.

Group 5 (Copper sulphate): Copper sulphate at a concentration of 5 ppm was added to aquaria water. Water was renewed every 2 days with the same concentration of CuSO₄ for 10 days after which blood samples were collected.

Group 6 (Acidic water): Water was made acidic by adding drops of concentrated HCl till the pH 5. Fish were kept in aquarium water and pH was kept adjusted to 5 ± 0.5 for 3 days after which blood samples were collected.

Group 7 (Ammonia): Ammonia level in water was raised by addition of ammonium chloride (0.2 ppm) with frequent renewal of water containing the same concentration of ammonia. The experiment continued for 3 days after which blood samples were collected.

Group 8 (Starvation): these groups of fish were subjected to complete feed deprivation as fasting stress and sampling was carried out after 14 days. The other environmental conditions were kept at optimum levels as in the control group.

Group 9 (Cold): This experiment was carried out during winter season in February. The water temperature was around 14±2CO. Fish were killed after 3 days for collection of blood samples.


3-Blood parameters

Blood samples collected from caudal blood vessels in heparinized centrifuge tubes were used to estimate serum hemolytic activity and serum lysozyme activity as well as blood parameters (Smith and Hattingh, 1980).

This collected blood was used for blood picture determination, plasma separation and hemolysate preparation. On the other hand, another blood samples were collected in centrifuge tubes without anticoagulant for serum separation. Blood samples were directly placed on ice as quickly as collected, where the whole blood analysis was completed within 2-3 hours immediately after collection. After blood picture was determined in the whole blood. Blood was centrifugation at 3000 rpm for 15 min in cooling centrifuge for separation of plasma which was stored at –18 CO till used for biochemical analysis. Hematocrit measurements were made by drawing well mixed sample of blood into heparinzed capillary tubes and centrifuging at 10000 rpm for 15 min (Siwicki and Anderson, 1993). Counts of erythrocytes and thrombocytes were calculated using a Newbauer hemocytometer and Natt-Herrick solution as a diluent and stain (Stoskopf, 1993). Natt and Herrick solution (Natt and Herrick, 1952) is used for counting red and white cells. Hemoglobin concentration was determined using the cyanomethemoglobin method (Blaxhall and Daisley, 1973). Plasma glucose concentration was determined according to Trinder (1969) using an enzymatic kit (Bio-Merieux-France). Total protein was determined by the method of Garnall et al. (1949).


4-Mucus scrapping

Intestinal scrapping were collected using a clean scalpel and mucus was flushed out with the aid of 0.5 ml sterile saline skin and pharyngeal mucus were collected in the same manner and all samples were frozen until analysis within 7 days.


5-Serum hemolytic activity

The used method (Alton, et al., 1988) depends on using sheep red cells sensitized with Guinea pig serum as a source of complement suspended in Alsever’s solution. Hemolytic activity was measured as the amount of hemoglobin released. Sheep blood was aseptically withdrawn and thoroughly mixed with an equal volume of sterile Alsever’s solution. Sheep blood was then transferred aseptically to screw-capped bottles and stored in the refrigerator. It was not used until 5 days after collection; thereafter, it was used for up to 6 weeks if it is not contaminated).On the day of use, sheep RBCs were washed 3times with Veronal buffered saline by repeated centrifugation and resuspension. A 3% suspension was obtained by centrifuging the RBCs at 1500 g. for 10 min and suspending the deposit in 32.3 times its volume of Veronal buffered saline. One volume of hemolysin was added to 1 volume of the 3% RBCs suspension. The mixture was gently stirred on a magnetic stirrer at room temperature for 15min. various amounts of hemoglobin solution and sheep erythrocyte suspension were mixed to obtain various degrees of hemolysis ranging from 0.0 to 100.0%. A number of 20 guinea pigs were aseptically bled and serum was collected within not more than one hour.

Clear sera were pooled and used as complement. The solution mentioned in Richardson (1941) was used for preserving complement in the liquid state at 4oC. Five C’H50 units (the quantity of complement required to lyses 50% of optimally sensitized RBCs called C`H50) were used for the test. The optical density (OD) of the supernatants was determined spectrophotometrically. The C’H50 of complement was determined graphically by plotting the degrees of hemolysis given by the doses of complement on log/log paper. Percent hemolysis = obtained OD/OD of 100% hemolysis.


6-Serum lysozyme activity

The method of Litwach (1955) modified by Sankaran and Gurnani (1972) was used. The substrate was 0.025% (w/v) suspension of Micrococcus lysodcikticus cells (High Institute of Health, Alex. Univ.)

made up in acetate buffer (0.02 M, pH 5.5). Lyophilized hen egg white (Sigma L-6876) was used as a standard. A new standard curve was made for each assay. Test serum (0.2 ml) was added to the substrate (2.5 ml) solution at 25oC. The decrease in optical density at 450nm was measured after 30-min incubation. The unit of enzyme activity (U) was defined as the amount of enzyme that caused a decrease in absorbance of 0.001 per min.


7-Statistical analysis

One-way ANOVA was performed and the differences were evaluated at the 0.05 probability. When F-test was significant, the comparisons among treatments were done according to Steel and Torrie (1980).


RESULTS AND DISCUSSION

1-Lysozyme activity

Table (1) shows the effect of different treatments on lysozyme activity (unit/ml) of samples assayed. In the control fish, the intestinal scrapping and serum samples had the highest lysozyme activity, while skin mucus had the lowest activity. The effect of treatment on serum lysozyme, intestinal scrapping and skin mucus was significant. Exposure of fish to phenol (150 or 200 μg/l) induced significantly decreased lysozyme activity in serum and intestinal scrapping while it was undetectable in samples collected from skin mucus. However, increasing level of phenol significantly increased lysozyme activity in serum and decreased it in intestinal scrapping. Serum, intestinal scrapping and skin mucus samples collected from fish exposed to hypoxia for 3 days had significantly lower lysozyme activity than the control fish, being more pronounced in serum than intestinal scrapping. Lysozyme activity of fish exposed to Cu sulphate showed significantly increase in serum samples and significantly decrease in the intestinal scrapping samples, while undetectable levels in skin mucus samples were observed. Exposed fish to acidic water had adverse effect on lysozyme activity of serum, intestinal scrapping and skin mucus samples being significantly lower in treated than in the control fish.

Increased ammonia level significantly reduced lysozyme activity in serum and intestinal scrapping samples. Levels fell to undetectable values in skin mucous samples collected from this group. Lysozyme activity in serum and tissue scrapings collected from fish starved for 14 days was significantly comparable to the control fish, showing the lowest effect of all treatment in serum samples, but not to level observed in intestinal scrapping for fish treated with phenol. Cold conditions significantly reduced lysozyme activity in all samples collected from exposed fish while, levels fell to undetectable values in skin mucus samples. It can be concluded that exposed fish to phenol pollution had the most drastic effect on lysozyme activity in serum and tissue scrapings. Also, it can be observed that levels of lysozyme activity fell to undetectable values in skin mucus samples collected from fish exposed to phenol, copper sulphate, ammonia and cold.

The immune system regulates foreign molecules entering the body and foreign molecules generated within the body. Commonly encountered antigenic molecules are associated with bacteria, viruses, foreign proteins and chemicals. In the normal course of events these antigens are neutralized by the immune system and eliminated without causing any adverse effect on the host. Under other circumstances, the immune response is excessive and detrimental to the host.These detrimental immune reaction is an important mechanism in chemical toxicity. Chemicals may evoke these allergic detrimental reactions causing immune injury in form of anaphylaxis, phagocytosis, cytolysis, and/or inflammation (Glaister, 1986). However, natural decay of cells releases enzymes into the plasma, where activities are usually low. Raised levels are due to cell damage. Patterns of enzyme changes together with clinical findings are used for interpretation (Zilva and Pannall, 1983). However, some chemicals inhibit the enzmatic activity of brain and gills of tilapia, particularly in acute exposure (Alkhail et al., 2004). Also, bronchial enzymes activity is altered when assayed at varying temperatures of rearing water of tilapia fish (Sardella et al., 2004).

The present work also shows that Oreochromis Niloticous have high lysozyme activity in serum and tissue scrapings studied which is comparable to the high activity reported previously in rainbow trout and catfish, which possess high lysozyme activity as compared to low lysozyme activity in species such as cod (Godus morhua) and Atlantic salmon (Salmo salar) (Grinde et al., 1988). The high-inherited lysozyme activity in Oreochromis Niloticous species may explain its wide spread distribution and disease tolerance as farm fish. Stress induced variations in lysozyme activity were previously investigated by Muona and Soivio (1992), who reported that transportation stress and different environmental stressors during stocking may increase the disease susceptibility of fish. Cheng et al. (2003) reported significant decrease in phagocytic activity of giant fresh water prawn exposed to temperature stress. In the present work, the drastic reduction of lysozyme activity in Oreochromis Niloticous fish exposed to cold stress (14OC) may explain the high mortality rate in this species during severe winter months. The relative tolerance of Oreochromis aurea (Saad et al., 1999) to lowered temperature may attract the attention to genetic variation in lysozyme activity between different tilapia species and necessitate further studies on hybrids between Oreochromis niloticus and Oreochromis aurea as cold resistant hybrid. Studies of Chiayvareesajja et al. (1999) on lysozyme activity in Nile tilapia exposed to 15OC are helpful in this respect.

The effect of water pollution with phenol on lysozyme activity observed in the present work may be attributed to the toxic and hepatic degenerative changes induced by phenol. It was reported previously (Naylor, 1992) that phenol may be found in the aquatic environment in a wide range of concentrations, from low micromolar range in river to 1 mg/l in the effluents from pulp mills. The decrease in lysozyme activity in stressed fish in the present work coincedes with the previous reports which confirm that lysozymes are produced by leukocytes and that distribution of lysozymes in fish tissues is closely related to the occurrence of leukocytes in these tissues (Lindsay, 1986). The decrease of immunocompetent cells, which is connected with increased cortisol response during stress and probably the lysing effect of corticosteroid stress hormone (Clarean et al., 1971) leads to impair specific immune response and reduce disease resistance.

Table (1):Lysozyme activity (unit/ml) in serum intestinal scrapping and skin of control and different group treated, (¯X ± SE).

a-i: Means in the same column having different superscripts are significantly different (P<0.05).

2-Total leukocytic count and differential percentages

The total leukocytic count (Table 2) was significantly increased in blood of fish exposed to copper and cold treatment. A non significant decrease in total leukocytes (TLC) was found in fish groups treated with phenol (200 μ g/l), acidic water, ammonia and starvation. Lysozymes are produced by leukocytes and the distribution of lysozyme in rainbow trout was in tissues rich in leukocytes (Lindsay, 1986). The results showed a decrease in circulating lymphocytes of Oreochromis Niloticous exposed to copper and cold. This is in agreement with previous results of Maule et al. (1987). A significant decrease in neutrophil percentage was evident in fish groups exposed to phenol (200 μ g/l) and starvation. Muona and Soivio (1992) found a correlation between Atlantic salmon plasma lysozyme activity and the decrease in number of adult neutrophils during starvation period during per-smolt transformation.


3-Erythrocytic and platelet counts

The effect of treatments on count of erythrocytes (RBCs) and thrombocytes in blood of fish groups was not significant. All counts of erythrocytes and thrombocytes were lower in all treatment groups, than the control one (Table 3). The decrease in erythrocytic and platelet count observed in the present study was more pronounced in phenol and starvation treated fish which may be attributed to instability of red cell membrane as a sequence to liver damage (Harding et al., 1978), necrosis of hematopoietic elements of spleen and anterior kidney caused by phenol intoxication (Minami et al., 1979), and increased formation of immature red cells (erythroblests), since maturation need as long as 35 days (Lane et al., 1981). Low hemoglobin level is correlated by low RBCs count resulted in anemia (Merck, 1974). Absolute lower erythrocytes count is found by anemia and after hypervolemia resulted from speedy-rich fluid taking up (Merck, 1976). Chemicals induced chromosomal aberrations in the kidney, and increased micronuclei erythrocytes and the concentration of DNA in liver tissues in Nile tilapia fish (Mahmoud et al., 2004).

Additionally, platelets count is lower by thrombopenia, i.e. acute leucosis and allergic and toxic thrombopenia as a result of treating by benzol and cytostatiks. Yet, increasing platelets count may accur by essential thrombocythemia, polycythemia and sometimes in chronic myeloic leukemia and fibrosis or osteomyelosclerosis (Merck, 1974). Some chemicals affect the function of circulating platelets, where chemicals may affect the ability of platelets to adhere and aggregate or to release their granular constituents. Some chemicals act on the surface membrane of the platelet, other chemicals act intracellularly (Glaister, 1986).

Table (2):Total leukocytic count (TLC) and differential leukocytic percentages of O.Niloticus (¯X ± SE)

a-i: Means in the same column having different superscripts are significantly different (P<0.05).

Table (3):Average count of erythrocytes (X106 ul-1) and thrombocyte (X 103) in blood of O. niloticus (¯X ± SE).

*All differences among treatment are not significant at (P ≥ 0.05).

4-Serum hemolytic activity

The effect of treatments on hemolytic activity of the serum is shown in Table 4. Exposure of fish to phenol caused significantly severe reduction of hemolytic activity, being significantly lower with the high level of phenol as compared to the low level. The reduction in hemolytic activity was also observed clearly in fish exposed to copper sulphate or ammonia pollution (44.9 and 33.3%, respectively). The reduction in hemolytic activity in fish exposed to hypoxia, acidic water, starvation or cold was much less evident (56.5, 68.8, 79.9 and 80.9%, respectively). The result of this experiment demonstrates clearly that hemolytic activity in fish serum can be modulated by pollution and other environmental conditions such as elevated ammonia levels. Ingram (1987) reported that a single injection of trout, Salmo trutte, with sheep erythrocytes stimulated the production of antibody-secreting cells in lymphoid organs and increased the levels of natural hemolysins. A second injection of sheep erythrocytes further raised the hemolysin values and antibody secreting cell counts. Concerning the effect of phenol, Mehrim (2001) found that the maximum tolerable level (30 ppm in water) of phenol at the chronic exposure to Nile tilapia led to significant decreases in both of phagocytic activity and phagocytic index as well as to hypoproteinemia, increased activity of serum transaminases (AST, ALT, ALP) and indicating severe tissue damage and phenol accumulation in the muscles.

Regarding the effect of NH3 on hemolytic activity of fish, El-Shafai et al. (2004) concluded that the toxic level of un-ionised ammonia nitrogen (UIA-N) and its negative effect on the Nile tilapia lies between 0.07 and 0.14 mg/l. They recommended that UIA-N concentration be maintained below 0.1 mg/l.

Table (4):Serum hemolytic activity (%) of O. niloticus

a-i: Means in the same column having different superscripts are significantly different at (P ≤ 0.05).

5-Plasma glucose and total protein

The effect of treatments on concentration of glucose and total protein in plasma of fish was significant. Results shown in Table (5) clear that concentration of glucose in blood plasma significantly increased in all treatments as composed to the control, expect for copper sulphate group, it significantly decreased. On the other hand, all treatment groups showed significant reduction in concentration of total protein as compared to the control. The observed hyperglycemia in treatment groups may be due to increased glucocorticoid secretion associated with stressful conditions. This suggestion agrees with previous result of Pickering et al. (1988), who cited that plasma glucose concentration was significantly greater in stressed fish as compared to the controls. Moreover, Pasanen et al. (1979), used blood glucose level as a stress indicator in Coregonus albula. Elevated blood glucose level is recorded in cases of diabetes mellitus, morbus cushing, acromegalia, pheochromocytomas, acute pancreatitis, pancreas carcinoma, myocardinfarct, hemorrhage, toxicity, brain disease and tumor (Marck, 1974 and 1976). Hyperglycemia may be caused by hyperactivity of the thyroid, pituitary and adrenal glands, states of emotional stress, pancreatitis, carcinoma, hemorrhage, and asphyxia (Varley, 1978). During fasting, glycogen breakdown in the liver (and kidney) releases the glucose into the blood. Diabetes mellitus is the result of relative or absolute insulin deficiency. It is characterized by hyperglycemia, lipolysis, ketosis, and acidosis. Coma may be due to hyperosmalality associated with severe water and electrolyte depletion (Zilva and Pannall, 1983).

Hypoproteinemia is a result of protein loss via kidneys (massive proteinuria, hyperlipidemia and edema caused by intoxication), intestine (idiopathic exsudative enteropathy, enteritis and metastasis), and skin (inflammation). It is recorded during tumor, collagenosis, sarcoidosa, chronic inflammation and plasmocytoma (Merck, 1974). A reduction in the total protein is one of the causes of edema (nephritic edema). Sever hemorrhage causes hypoproteinemia. A similar effect is observed in shock, increased protein breakdown, diabetes mellitus, hyperthyroidism, severe liver disease, starvation, defective absorption due to malignant disease of stomach, intestine, and pancreas, and in peptic ulcer (Varley, 1978). Hypoproteinemia is almost due to hypoalbuminemia. Causes of low total protein concentration include over hydration, excessive loss of protein (nephritic syndrome, severe burns, and enteropathy) and/or decreased synthesis of protein (dietary deficiency, liver disease, malabsorption). Changes in plasma volume will cause a change in total protein concentration and in hematocrit value (Zilvu and Pannall, 1983).

Table (5): Concentration of plasma glucose and total protein in the experimented tilapia (¯X ± SE)

a-i: Means in the same column having different superscripts are significantly different (P < 0.05).

Oxygen deprivation is a very common cause of cell and tissue injury. Adequate oxygen supply to the cell depends on adequate ventilatory function, adequate diffusion from alveoli into blood, adequate numbers of functional erythrocytes and an adequate cardiovascular system to transport oxygenated erythrocytes to the cell. Any one or more of these sites may be the target site for chemical attack, e.g. severe irritants and chemicals. A reduction in the number of functional erythrocytes may result from lack of erythrocyte production in bone marrow damaged by chemicals. A primary oxygen deficiency in the circulating blood is known as hypoxia. All cells in the body are at risk to this type of oxygen deprivation, but the cells most at risk are those with high oxygen requirements such as the brain, heart, liver and kidney. Cardiac arrest from toxic substances is an obvious cause of inadequate generalized blood flow (Glaister, 1986). Delaney and Klesius (2004) found that hypoxia led to a highly significant increase in serum glucose levels of juvenile Nile tilapia.


6-Hematocrit value and hemoglobin concentration

Hematocrit (PCV) values and hemoglobin (Hb) concentration were significantly reduced in phenol, sulphate, ammonia and starvation fish groups (Table 6). Concomitant changes in hemoglobin levels were also observed. However, only hypoxia resulted in significant increase in PCV values. Hematocrit levels are routinely used in fish health survey and differences may appear when fish are under stress (Anderson, 1990).

The decrease in hemoglobin concentration may be due to decreased number of erythrocytes and increased formation of immature red cells which were small in size with less of hemoglobin (Bruna and Mumro, 1986). Concentration of hemoglobin being lower during anemia (Merck, 1974 and 1976). Among the blood chemistry parameters which were used as good indicators of deteriorating health in tilapia are hematocrit value and concentration of iron, and glucose and activity of enzymes (Chen et al., 2004). In addition, Garcia-Abiado et al. (2004), reported significantly lower hemoglobin and hematocrit values as well as significantly greater occurrence of immature and abnormal erythrocytes besides polychromatocytes in tilapia fish exposed to a toxic chemical (gossypol). Toxic chemicals such as mycotoxins affect blood picture, hematologically and biochemically. They reduced hemoglobin, PCV, RBC`s count, enzymes activity, total protein concentration and increased glucose, DNA, RNA and cholesterol levels (Abdelhamid, 1990;Abdelhamid and Dorra, 1993; Abdelhamid and Saleh, 2000 and Abdelhamid et al., 2002a & b and 2004a & b).

Urea, ammonia and ammonium compounds are used extensively. In addition, copper salts are widely employed. Successive non-toxic doses of copper have a cumulative effect. Copper is slowly eliminated from the body and is stored in the liver, if liver content reaches a dangerous level, copper is released into the blood stream, excessive hemolysis may occur, and symptoms of poisoning manifest themselves. Phenol (carbolic acid) and the related compounds, the cresols, are extremely poisonous and are contained in many disinfectant preparations, of which lysol is probably the most familiar. Poisoning from preparations containing phenol is occasionally seen in animals. The toxicity symptoms of phenol are anemia, anorexia, in coordination, weakness and dyspepsia. Centrilobular necrosis and hemorrhage of the liver were found at post mortem (Clarke and Clarke, 1978). Phenol is excreted to some extent unchanged, in part conjugated with glucuronic and sulphuric acids, but the greater part is found as hydroquinone and pyrocatechol and their oxidation products (Varley, 1978). Generally urea and ammonia decrease blood hemoglobin and total protein concentrations (El-Ayoty and Abdelhamid, 1989).

Table (6): Hematocrit values and hemoglobin concentration of O.Niloticus (¯X ± SE)

a-i: Average in the same column having different superscripts are significantly different (P<0.05).

Generally, heavy metals may increase blood glucose and serum enzymes activity, but decreases serum total protein as well as affect hemoglobin concentration (Abdelhamid, 1988 a&b). Heavy metals caused pathological findings, e.g. hemorrhages and congestion of the gastrointestinal tract and kidneys. They decrease protein and affect electrolytes (Abdelhamid and El-Ayouty, 1991 and Abdelhamid et al., 2000). They are responsible for physiological dysfunction, mainly in the form of anemia (low hematocrit, hemoglobin, and total protein) and hepatic (low enzymatic activity) lesions (Abdelhamid and Dorra, 1992). Biochemical analysis of tilapia muscles revealed its toxic contents of copper (Abdelhamid and El-Zareef, 1996). Copper is a causative of low muscular protein and different types of chromosomal aberrations in tilapia (Magouz et al., 1996). However, heavy metal concentrations in different fish and crustaceans species from the local water bodies are over the permissible levels which recommended by the national (and international) authorities (Abdelhamid et al., 1997 & 2000 and Abdelhamid and Gawish, 1998).


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