Hematological Parameters of Brackish Water Fish Tilapia zillii in Benghazi, Libya

Hematology can be a useful tool for monitoring health status, detecting illness, and following the progress of disease and response to therapy. Despite advances in fish medicine in recent years, interpretation of fish hematology often is hampered by a lack of meaningful reference values and the bewildering diversity of fish species. Hematology is the science of studying the anatomical, physiological, and pathological aspects of blood. This study gives valuable information on fish Tilapia zillii , Hematological indices (RBC, WBC, Hct, Hb, MCV, MCH, MCHC, GRA, MON, LYM


Introduction
In the broad sense, all the tilapias have in common a mainly herbivorous diet, in distinction to the majority of fishes that feed predominantly on small invertebrates or young or small-sized fishes.They are therefore only one step from the primary producers (plant life) and as they grow to a good size they are a valuable food source for man, the omnivore.The blood transports a variety of materials, including inorganic ions and some organic constituents such as hormones, vitamins, and several plasma proteins.
Cellular constituents of the blood are the red blood cells or erythrocytes, and the white blood cells or leucocytes obtain their characteristic color from hemoglobin.Made up of the colorless protein globin, and the red-yellow pigment heme, which contains iron (Bond, 1979).Recently fish hematological examination is gaining popularity.Several workers have performed hematological studies on fishes; however, data regarding normal health parameters are limited.Few hematologic values of clinically normal tilapia have been reported, but these data may be key for identifying and managing disease issues in recirculating systems (Michael, 2007).
Hematological studies on fishes have assumed greater significance due to the increasing emphasis on pisciculture and greater awareness of the pollution of natural freshwater resources in the tropics.Such studies have generally been used as an effective and sensitive index to monitor physiological and pathological changes in fishes (Chekrabarthy and Benerjee, 1988;and Iwama et al., 1976).
Blood tissue reflects physical and chemical changes occurring in organisms, therefore detailed information can be obtained on the general metabolism and physiological status of fish in different groups of age and habitat (Orun and Erdemli, 2003).In fishery, it is important to find out illness and parasites as the source of these causes may not be generally detectable in the early period of the infection, however, it is also possible early diagnosis of illnesses in case of evaluating hematological data, particularly blood parameters (Rimsh and Adamova, 1973).Although initiated as an adaptive response to destabilizing factors, the physiological stress response can have damaging effects if prolonged.It is well established that continuous stress affects behavior and normal development, with growth reduction (Jobling and Reinsnes, 1986), suppression of reproduction (Gerking, 1980), and increased susceptibility to infections immune-depression, which may cause mortality.Therefore, there have been greater understandings of the need to establish reference hematological and biochemical values in fish to assess health status and the subsequent diagnosis of disease.(Schreck and Bradford, 1990).So, the aims of the present study are; 1) To obtain a basic knowledge of the hematology of Tilapia zillii; and 2) To evaluate the effect of sex, weight, and length on a selection of hematological parameters in T. zillii living in brackish water.

Fish Samples
Thirty Fish of the T. zillii were collected by hand net (30 cm in diameter) in December 2009, from (Ain Elmajdob lake), located north Benghazi city.Fish were collected and transported immediately in pouches filled with lake water to the laboratory of the Zoology Department, University of Benghazi.Fish were divided into two groups each group containing (15) fishes, and kept in a laboratory glass aquarium of 200 L capacity were well aerated and provided with external filtration.They were given a minimum period of 72 hrs. to acclimatize to laboratory conditions.

Water Quality
The water quality parameters such as pH, temperature, dissolved oxygen, and Salinity were measured for two the water systems using a portable meter, (Values are shown in the Results section).The following parameters were measured in the site of collection: pH: the pH of the water was measured employing the Hanna pH meter.Water temperature: the water temperature of two ecosystems was measured using the thermometer attached to the water sampler.The temperature reading was recorded after collecting the water inside the sampler with both lids closed.Five minutes time was allowed to elapse before taking the readings after the water samples were lifted out of the water column, to ensure uniformity in the temperature of the water inside the sampler.Dissolved oxygen of water: was measured using Guard Portable oxygen meter (handy MKI and MKII).Salinity: salinity of the water was measured employing Hanna meter.

Blood Collection and Analysis
Random fish samples of T. zillii were considered normal on the basis of their external appearance and absence of symptoms of diseases.
The fish sample was caught from the containers.After the preliminary investigation of the length, weight, and sex, a damp cloth was used to cover the fish head.They placed the belly upwards and blood samples were obtained from the heart with the aid of a heparinized disposable plastic syringe containing EDTA as an anticoagulant.
Heart puncture was chosen for being easy to perform and excellent for obtaining a large quantity of blood when applied to Tilapia.After obtaining the desired quantity of blood, the use of a plastic syringe is a necessary precaution with fish blood because contact with glass results in decreased coagulation time (Smith et al., 1952).Fish was wiped dry with tissue paper to aid contamination with mucus it was then pushed gently down until blood started to enter in the needle.Thereafter the needle was withdrawn and the blood was gently transferred into heparinized plastic containers, which contained EDTA as an anticoagulant.The samples were then mixed gently.
Then blood samples were transported to Saleem laboratory for analysis using (Diatron instrument) after being adjusted to animal analysis and measurement of Hematocrit (Hct) or Alrwab (2021) Faculty of Marine Resources, Alasmarya Islamic University, Libya.

Statistical Analysis
Results were analyzed with SPSS for windows by using (T-test, Regression).

Result
In the study some parameters of water quality were measured (Table 1); the result shows the water quality parameters.In the present study, the white blood cells of T. zillii were found depending on the length and weight of T. zillii studied by regression of white blood cells on the length and weight of Tilapia zillii, as well as Lymphocytes and Monocytes, increased with the increase of weight.In this study, the result value of Red blood cells counts of fish was very closely significant RBC is (0.91±0.33) as shown in Figure (3).

Discussion
In this study, the result, such as water temperature was (21.4°C).In shallow water, there may be little or no differences between the temperature at the surface and the bottom.In deep water, temperatures vary with the coolest water at the bottom.Sun et al. (1995) concluded that low temperatures have the significant effects of either increasing or decreasing direction on the changes of various blood parameters in tilapias depending on the environmental salinity.An increase in water temperature increased most of the parameters (Ezzat et al., 1973).
In this study, the water pH result was (7.7).The pH value vary depending on a number of factors such as pH levels of the pond water change depending on the aquatic life within the pond, Carbon dioxide produced by aquatic organisms respire has an acidic reaction in the water, the pH in ponds rise during the day as phytoplankton and other aquatic plants remove CO2 from the water during photosynthesis.The pH decreases at night because of respiration and the production of CO2 by all organisms.The fluctuation of pH levels will depend on algae levels within the pond.(Aquaculture, 2003).
Also, the Salinity was (6.1%), Salinity is a measure of the amount of salt in water, brackish water is a mixture of fresh and marine water.Salinity significantly caused the decrease of hemoglobin concentrations at 24°C (Sun et al., 1995).High salinity caused an increase in the concentration of most of the blood characteristics, Temperature and salinity changes produce several variations in the blood characteristics of T. zillii (Ezzat et al., 1973).Although the dissolved oxygen was not high (3.16mg/L) because warm water holds less oxygen than cold water does.Dissolved oxygen in the water is obtained through diffusion from air into water, mechanical aeration by wind or aeration systems, and via photosynthesis by aquatic plants (Aquaculture, 2003).
The weight and length of T.zillii in Brackish water between males and females were 16 males weight between (9.4 to 41.9 g) and total length between (7.4 to13.2 cm); 14 females weight between (9.2 to 28.2) g, and total length between (8 to12 cm).The effect of length-weight variables on blood parameters such as the enhanced formation of lymphocytes was an essential component of the immune system in the early stages of the growth period (Aldrin et al., 1982).In research for Nile tilapia Oreochromis niloticus, there was an effect of fish size (between small and big fishes) on all the parameters except for MCV (Tran-Duy et al., 2008).In a study on Mugil it was verified that mean values for HCT, Hb, MCV, MCH, and MCHC showed a slightly increasing tendency as individuals got larger (Ranzani-Paiva, 1995).Also, larger individuals present higher mean values for MCV, MCH, and MCHC for Dourado Salminus maxillosus (Ranzani-Paiva et al., 2001).Al-Hassan et al. (1993) observed the hemoglobin and hematocrit values of Acanthopagrus latus were having increased with both body weight and length, but to a certain weight and length group after which a slight fall in their value was observed .
In this work the values of RBC of T. zillii in brackish water, in this study is similar to Tilapia zillii (0.98±0.32) by Ghazaly et al. (1992).But lower than control values those reported by Gbore et al. (2006) in T. zillii from state Government Hatchery (1.10±0.04)when studied the effect of transportation and handling stress on hematology and plasma biochemistry in T. zillii, and normal result of T. zillii was (3.4±0.23) by Zaki et al. (2010a).
The Red Blood Cells have the important function of hemoglobin transport, which carries oxygen to all tissue in the body (Hibiya, 1982).Red Blood Cells count significant decrease may be attributed to the decrease in the erythropoietin activity of the kidney (Santhakumar et al., 1999), decreased Red Blood Cells count indicates that the erythrocytes are being affected or destroyed with the infection of fish (Martins, 2008), the reduction in the erythrocyte value may be due to destruction of the Red Blood Cells which might have resulted in the liberation of the hemoglobin content of the cells (Gbore, 2006).On the other hand, the increased Red Blood Cells count may be due to stimulation of erythropoietin by elevated demands for oxygen or carbon dioxide transport as a result of increased metabolic activity or destruction of gill membranes causing faulty gaseous exchange (Cyria et al., 1989).High Red Blood Cells could be explained by the increase in reproductive activity (Collasozn et al., 1998).Changes in metabolism and hormonal activity, triggered by cooler water temperatures and declining photoperiod during fall, may result in anemia (Lane, 1979) and reduced erythropoietin production (Zanuy and Carrillo 1985;and Lane 1979).
The hemoglobin within those erythrocytes is the main transport mechanism for oxygen and carbon dioxide in the blood.Alterations in blood oxygen capacity reflect a seasonal adjustment in oxygen transport (Anderson et al., 1985;and Cameron, 1970).The result of hemoglobin concentration in brackish water T. zillii is similar to the result of T. zillii by Gbore et al. (2006), and lower than those described by Zaki et al. (2010b), and T. zillii by Badawi and Said (1971) when compared the blood of four Tilapia species.Hemoglobin is the oxygen-carrying component in the blood of fish and its concentration can be used as a good indicator of anemia (Blaxhall and Daisley, 1973).The hemoglobin can vary with season, temperature and nutritional state, and health of the fish (Bond, 1979).The increased hemoglobin content could be explained as a process where the body tries to replace the oxidized denatured hemoglobin (Cyria et al., 1989).Changes in the hemoglobin content of Alrwab (2021) Faculty of Marine Resources, Alasmarya Islamic University, Libya.

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ISSN (Print): 2413-5267 ISSN (Online): 2706-9966 the blood in response to the environment might come about either by a change in the number of erythrocytes or by a change in the hemoglobin concentration of the individual cells (Anthony, 1961).
Hematocrit provides a measurement of red blood cells (erythrocytes) in whole blood (Anderson et al., 1985;and Cameron, 1970).The result of hematocrit value in brackish water Tilapia zillii in this study is lower than values obtained from T. zillii; 20.07±0.07(Gbore et al., 2006), the result of T. zillii 19.80±1.20 ( Zaki et al., 2010a), and result of T.zillii 25.0 (Badawi et al., 1971).The increase of hematocrit value may be attributed to swelling of red blood cells due to increased carbon dioxide in blood, hypoxia, or stress of fish (Ellis,1981), and high hematocrit levels may be caused by metabolic activities (Collasozn et al., 1998).These differences may be due to diverse strains used in previous studies and different environmental and cultured conditions.Low temperature caused the decrease of hematocrit (Sun et al.,1995).This work shows a decrease in lymphocytes in T. zillii Brackish water contrarily to that observed in T. zillii (74.4±2.13) by Ghazaly (1992).However, these values were higher than the values obtained for Nile tilapia (20.5±9.7) by Martins et al. (2008), and values of Tilapia guineensis (35.46±4.11)by Akinrotimi et al. (2010).
The result of Monocytes value in brackish water T. zillii in this research was lower than those studied by Martins et al. (2008) in Nile tilapia (14.3±7.7) and Tilapia guineensis (3.12±1.01)by Akinrotimi et al. (2010).High Monocytes could be explained by the increase in reproductive activity and defense mechanism of fish (Orun and Erdemli, 2003).
The result of Granulocytes of T. zillii in this research is lower than the value referred by Akinrotimi et al. (2010) in Tilapia guineensis (20.95±2.21),but higher than the value obtained for Nile tilapia (6.5±4.6) by Martins et al. (2008), and T. zillii (4.8±1.18) by Ghazaly (1992).Granulocytes are phagocytic, involved in combating disease, and may increase in number when the fish is infected by bacteria (Bond, 1979).
The lower value of Mean Corpuscular volume was found in the present study in T. zillii than those referred by Bittencouret et al. (2003) in Nile tilapia (148.80±153.19)and the result of T. zillii (181.78±0.06)by Gbore et al. (2006).Nevertheless, the value of Mean Corpuscular volume in the present work T. zillii was higher than the value obtained in Tilapia guineensis; 80.76±2.11by Akinrotimi et al. (2010), and T. zillii 104.29±13.7 (Ghazaly, 1992).Changes in the hemoglobin content of the blood in response to the environment might come about either by a change in the number of erythrocytes or by a change in the hemoglobin concentration of the individual cells (Anthony, 1961).
In this study, the value of Granulocytes (9.52±7.58),and the value of Mean Corpuscular volume (143.96±29.51) as shown in Figures (8 and 9) respectively.

Table 1 .
The water quality parameters The summary of the mean values of weight, length and hematological parameters for T. zillii are represented in Table (2), together with the standard deviation, and Standard Error.

Table 2 .
Hematological parameters for T. zillii in fresh water

Table 3 .
Regression of blood parameters on length and weight of T.zillii in fresh water (Red=sig at 0.05)