Hematology and Plasma Chemistry Reference Intervals for Cultured Tilapia (Oreochromis Hybrid

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Terry C. Hrubec, DVM, PhD; Jenifer L. Cardinale, DVM; Stephen A. Smith, DVM, PhD
Abstract: Tilapia are a commonly aquacultured fish yet little is known about their normal physiology and response to disease. In this study we determined the results of complete hematologic (n = 40) and plasma biochemical profiles (n = 63) in production tilapia (Oreochromis hybrids). The fish were raised in recirculating systems with a high stocking density (120 g/L), and were in the middle of a 15-month production cycle. Blood was analyzed using standard techniques, and reference intervals were determined using nonparametric methods. Non-production tilapia (n = 15) from low-density tanks (4 g/L) also were sampled; the clinical chemistry results were compared to reference intervals from the fish raised in high-density tanks. Differences were noted in plasma protein, calcium and phosphorus concentrations, such that reference intervals for high-density production tilapia were not applicable to fish raised under different environmental and management conditions. (Vet Clin Pathol 2000;29:7-12) Key Words: Clinical chemistry, fish, hematology, reference intervals, tilapia

N
Tilapia are the second most commonly cultured fish in the world,1,2 and are a food staple in many parts of Africa, Asia and South America. Tilapia consumption has increased in the United States, where it is the fourth most commonly cultured food fish. Aquaculture of tilapia, as with other species of finfish, is adversely affected by production related disorders and infectious diseases.1 Unfortunately, there are few diagnostic tools available to veterinarians and fish health professionals to evaluate disease in fish. Many of the clinical tools used to evaluate mammalian health are not developed for use in fishes. As the aquaculture industry expands, there is an increasing need for improved diagnostic methods. Hematology and clinical chemistry analysis, although not used regularly in fish medicine, can provide substantial diagnostic information once reference values are established. In this study, we determined reference intervals for hematologic and plasma chemistry analytes in cultured tilapia. We also evaluated clinical chemistry results from a small group of tilapia raised under different culture conditions. To our knowledge, this is the first study to determine complete hematologic and clinical chemistry results for tilapia, and to report the values as reference intervals suitable for diagnostic use. Aquaculture Center.The fish, in the middle of their production cycle (mean weight 240 g, mean length 22 cm), were fed a commercial tilapia feed (Southern States, Richmond, VA, USA) at 4% of body weight per day. Tanks received a 12% fresh water exchange per day. Water quality was monitored daily and was considered acceptable for high-density production systems (Table 1). Each of the 8 tanks was sampled once, with 10 fish per tank bled for biochemical analysis and 10 fish bled for hematologic analysis. Fish with any gross abnormality were not included in the study. Careful netting and handling was implemented to minimize stress. Fish were rapidly netted, anesthetized in aerated buffered tricaine methanesulfonate (MS-222, Sigma Chemical Co. St. Louis, MO, USA) and bled with a needle and syringe from the caudal vessels. The blood was placed in tubes containing either lithium heparin for chemistry analysis, or EDTA for hematologic analysis. Any hemolyzed, clotted or insufficient volume samples were discarded. After sampling, fish were placed in a separate tank of fresh water for recovery. Blood in heparinized tubes was centrifuged immediately at 14,000ϫg for 5 minutes. The plasma was collected and frozen at –10°C until all fish were sampled. Plasma samples were analyzed using an automated dry chemistry system (Kodak Ektachem 700, Eastman Kodak Co., Rochester, NY, USA) for total protein, albumin, creatinine, ammonia, total bilirubin, cholesterol, sodium, chloride, potassium, calcium, magnesium and phosphorus concentrations, and alkaline phosphatase

Materials and Methods
Production tilapia (Oreochromis nilotica ϫ O. mossambicusϫO. aureus hybrids) were maintained indoors in 8 high-density (120 g/L) tanks (10,220 L) at Virginia Tech’s

From the Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. Address correspondence to Dr. Hrubec (thrubec@vt.edu).

Vol. 29 / No. 1 / 2000

Veterinary Clinical Pathology

Page 7

29 / No. to give a range of diameters for each cell type.1 0.6-4.9 7. Percentages of RBC and WBC + thrombocytes were determined by counting 1. If thrombocyte clumping was more severe. and cell size estimates. Globulins were calculated from the difference between total protein and albumin values. The RBC.3 136 31. † Hardness is the sum of the calcium and magnesium ions in the water.0 281.8 2.000ϫg. and the percentages of each WBC type and of thrombocytes were multiplied by the total WBC + thrombocyte count to obtain absolute differential cell counts.3-2. The total RBC count was determined manually with a Neubauer hemacytometer using Natt-Herrick’s solution as a diluent stain.4 128-142 13.3 Slight thrombocyte clumping prevented accurate enumeration of a combined WBC + thrombocyte count on the hemacytometer. the samples were discarded.7 7.1-0.5 0.6 1.4 9 Low Density 26. MCH. 1 / 2000 . Thrombocyte numbers were subtracted from the WBC + thrombocyte count to obtain a total WBC count. Non-production fish conditions were characterized by lower stocking density (4.3 ND ND Reference Interval 2.9-3. Parameter Temperature (°C) pH NH3 non-ionized (mg/L) NO2-N (mg/L) NO3-N (mg/L) Alkalinity (mg/L)* Hardness (mg/L) † Table 2. AST = aspartate aminotransferase thrombocyte clumping (< 4 cells clumped) were used for differential counts.2 22 26 151 4.5 5.5-22.This method of manually determining total WBC and differential counts has been recommended for avian4 and fish5 blood because nucleated RBC prevent accurate enumeration using automated analysis.7 As suggested in these guidelines.6 1.0 9. and used to determine the WBC + thrombocyte count. Leukocyte size was measured in 10 WBC of each type.5 111-414 0. and MCHC were calculated by standard formulas. using the cyanomethemoglobin method (Sigma).0 2.5% were discarded. For the differential count.Tilapia Hematology and Plasma Chemistry Values Table 1. MCV.4 1. Hemoglobin was determined. in 10 different fish.3 g/L) tilapia production systems.9-6.500 cells.The range of the remaining values provided the reference interval.6 Only smears with mild ALP = alkaline phosphatase.2 0.5 9. Mean water quality values for high-density (120 g/L) and low-density (4. differential WBC counts.5-5.3 15-39 9-102 139-160 3. Plasma protein was determined with a clinical refractometer using plasma from the microhematocrit tube. Plasma chemistry values from 15 non-production tilapia (mean weight 350 g) were compared to reference intervals determined for the production fish. outliers were determined using the 1/ ratio difference/range ratio. The WBC + thrombocyte percentage was multiplied by the RBC count from the hemacytometer to determine the WBC + thrombocyte absolute count.020 0. Analyte Total Protein (g/dL) Albumin (g/dL) Globulins (g/dL) Creatinine (mg/dL) Ammonia (µg/dL) Total bilirubin (mg/dL) ALP (U/L) AST (U/L) Sodium (mEq/L) Potassium (mEq/L) Chloride (mEq/L) Calcium (mg/dL) Magnesium (mg/dL) Phosphorus (mg/dL) Glucose (mg/dL) Cholesterol (mg/dL) High Density 29. Two hematologic values 3 and one biochemical value were identified as outliers and were deleted.9 1. Reference intervals were determined following the guidelines proposed by the National Committee for Clinical Laboratory Standards (NCCLS). ND = not determined (ALP) and aspartate aminotransferase (AST) activities. Prior to reading the absorbance.01 3 34. Plasma chemistry reference intervals for hybrid tilapia (n = 63) raised in high-density production systems. and the high and low 2. WBC and thrombocytes were counted until 200 WBC were enumerated on blood smears.1 46 189 Dissolved oxygen (mg/L) Turbidity (NTU) *Alkalinity is a measure of the buffering capacity of the water.3 g/L).0-0.2 249 0. Remaining values were then ranked.1 30-69 110-318 Median 3. lower feeding rate of the Page 8 Veterinary Clinical Pathology Vol.2 51.36 70 105. Blood smears were made within 45 minutes of sample collection.004 0. stained with Wright-Giemsa. hemoglobin test samples were centrifuged to remove dispersed nuclear material. Blood from the EDTA tubes was drawn into microhematocrit tubes and the PCV was determined after centrifugation for 5 minutes at 10.4 0.6-69.

oblong cells measuring 7.0-9.2 39-96 64-299 2. there were three types of circulating blood cells: RBC. or polychromatophilic RBC.8-7.1 0. Immature RBC.3-2.5-19.0-0. Cell morphology Six types of WBC were distinguished and counted: small lymphocytes. condensed nuclei with clumped chromatin. monocytes. these cells were included in RBC counts. Analyte PCV (%) Hemoglobin (g/dL) MCV (fL) MCH (pg) MCHC (g/dL) Plasma protein (g/dL) RBC (X 10 /µL) WBC (/µL) Small lymphocytes (/µL) Large lymphocytes (/µL) Neutrophils (/µL) Monocytes (/µL) Eosinophils (/µL) TLC (/µL) Thrombocytes (/µL) TLC = thrombocyte-like cell 6 Reference Interval 27-37 7. Protein concentrations in tilapia raised in high-density systems were slightly higher.5-7.068-85.0ϫ12. and had dark purple. while calcium and phosphorus levels were much higher compared to fish in low-density systems. Fish were sampled and the blood was processed as described for fish raised in high-density tanks.0 2. 1 / 2000 Veterinary Clinical Pathology Page 9 . and thrombocytes.6 0 0 26 18 150 3. As in other species of fish.645 35-4.3 22-29 4. Plasma chemistry results for hybrid tilapia (n = 15) raised in low-density systems compared to reference intervals from tilapia raised in high-density systems (see Table 2). Small lymphocytes ranged from 4. Cardinale.2-4.0-1. Nuclei stained purple and cytoplasm stained reddish-gray. large lymphocytes.7 34. Analyte Range* Median Compared to Ref. Reference intervals for hematology analytes were summarized (Table 4). had a blue-grey tinge to the usual eosinophilic cytoplasm.91-2.8 2.2 135.31 75.6 1.3-3.873 400-4. Results Analyses Reference intervals for plasma chemistry analytes were summarized (Table 2). Small lymphocyte cytoplasm was deep blue and often consisted of only a thin rim encircling the nucleus.6 1.83 21.9 µm in size. Both small and large lymphocytes had high N:C ratios.0 µm in diameter.3-2. WBC Vol. lymphocytes had cytoplasmic pseudopods and azurophilic cytoplasmic granules. Large lymphocyte cytoplasm was more deeply basophilic and abundant than that of small lymphocytes.7 to 6. consequently.4 µm in diameter.6 to 5.805 1.559-154. Intervals (n) Low Total protein (g/dL) Albumin (g/dL) Globulins (g/dL) Creatinine (mg/dL) Total bilirubin (mg/dL) ALP (U/L) AST (U/L) Sodium (mEq/L) Potassium (mEq/L) Chloride (mEq/L) Calcium (mg/dL) Magnesium (mg/dL) Phosphorus (mg/dL) Glucose (mg/dL) Cholesterol (mg/dL) 2.7 6.8 115-183 28.2-1.390 2.720 1. eosinophils and thrombocyte-like cells (TLC) (Figures 1-6). Large lymphocytes measured 5. Smith Table 3. Frequently.659 61.1 16-38 5-124 140-156 3.762 *Minimum-maximum values ALP = alkaline phosphatase. The RBC were nucleated.3-42. neutrophils. AST = aspartate aminotransferase same diet (1% of body weight per day).8 3.164 10. and had round nuclei that were larger and had a more open chromatin pattern than small lymphocytes.9 1. more optimal water quality (Table 1).833 557-9.852-30.9 25. Plasma chemistry values from non-production tilapia were summarized and compared to reference intervals from production tilapia (Table 3).1 2.Hrubec. 29 / No.520 334 972 52. Hematology reference intervals for hybrid tilapia (n = 40) raised in high-density production systems.8 1.286 35-1.776-136.9 141 11. round to oval.336 25.2 1.5 4. greater fresh water exchange rate (35% per day) and.690 6.3 136-147 10.216 Median 33 8.6 52 156 7 9 6 0 0 0 1 0 2 0 14 0 13 0 2 Within 8 6 9 14 15 15 13 15 13 10 1 15 2 13 13 High 0 0 0 1 0 0 1 0 0 5 0 0 0 2 0 Table 4.1 0.

and a thrombocyte (T). Thrombocyte-like-cell (TLC). Bar = 10 µm. Bar = 10 µm. Eosinophil (E) with an eccentric nucleus and moderate number of eosinophilic granules. Bar = 10 µm. Page 10 Veterinary Clinical Pathology Vol. Neutrophil (N) with an irregular cytoplasmic border. The insert depicts a neutrophil with a bean shaped nucleus. Photograph taken with a didymium filter. 29 / No. A small lymphocyte (SL) and a thrombocyte (T) are also present. large lymphocyte (LL). Wright-Giemsa. A small lymphocyte (SL) is also present. Figure 2. Small lymphocyte (SL). Wright-Giemsa. Thrombocytes can be distinguished from lymphocytes by the highly condensed nucleus and grey cytoplasm. Bar = 10 µm. 1 / 2000 . Eosinophil (E) with a large number of granules. Wright-Giemsa.Tilapia Hematology and Plasma Chemistry Values Figure 1. Wright-Giemsa. Bar = 10 µm. Photograph taken with a didymium filter. Monocyte (M) with an indented nucleus and vacuoles. Figure 6. Figure 3. Figure 4. A thrombocyte (T) with a slightly indented nucleus and one with a segmented nucleus (insert) are present. thrombocyte (T) and a small lymphocyte (SL). Bar = 10 µm. Figure 5. Wright-Giemsa. Wright-Giemsa. The TLC can be distinguished by a more open nucleus and more abundant grey cytoplasm than is seen in either the thrombocyte or lymphocyte.

gray to lightly basophilic. While these measures do not always ensure the cells are accurately identified.8 µm in diameter. there are surprisingly few reports of normal blood values. 5).Hrubec. Because TLC were identified in all individual tilapia.The reason for higher calcium and phosphorus values is unknown. cytoplasmic vacuoles. The blast cells were not included in cell counts. however.8-10 Terao and Ogawa8 reported much higher mean concentrations of cholesterol (567 mg/dL).7 µm in diameter and had round or indented.12 Leukocyte counts in these same species maintained under low production densities are lower by 40% to 50%. hemoglobin concentration (6. and had round. slightly stippled and more abundant than that of thrombocytes. O.12 The cells were first described in hybrid striped bass. calcium and phosphorus concentrations. often eccentric nuclei with open chromatin (Figures 4. Cardinale. the cell probably represents a maturational stage of one of the WBC. our results were similar for most analytes. as well as in other fish species. Cytoplasmic borders were irregular. and had very dark. Eosinophils were 5. Thrombocyte cytoplasm was clear to slightly basophilic. 6).6 to 10. purple nuclei that were round.9 were similar to those for Oreochromis hybrids reported herein.5 µm in diameter. they were termed TLC to distinguish them from other unidentified cells. eccentric nuclei with an open chromatin pattern (Figure 2).7 g/dL) and RBC (0. due to their immaturity. dense. In tilapia. 1 / 2000 Veterinary Clinical Pathology Page 11 . three granulocyte types were distinguished by ultrastructural observation. including routine repetitions of differential counts. Neutrophil cytoplasm was stippled. light purple. 5. Thrombocytes (counted separately from WBC) were 4. factors which may influence WBC counts. mossambicus. but is Vol. while the third granulocyte was unidentified by light microscopy. The function and origin of TLC were not determined.90 x106/µL) values. mossambicus.31 x106/µL) than those in our study. Distinguishing lymphocytes from thrombocytes can be difficult. Eosinophil cytoplasm was lightly basophilic and contained eosinophilic granules ranging from small and few to large and numerous.7 µm in diameter.4 to 10. although the cell’s lineage and function remain unknown.7 to 5.The earlier studies are of limited use for comparison and diagnostic purposes because too few fish were sampled and the fish populations were not well defined. we were unable to morphologically determine the cell line. Similar differences in blood values were observed in hybrid striped bass raised in high-density production systems.12 Fish in high-density production systems are exposed constantly to high bacterial loads in the water and less than optimal water quality. Thrombocyte-like cells were clearly identified in WBC differentials as a distinct cell type (Figure 6).7 to 6. Leukocyte numbers reported for one parent species.6 to 7. Haniffa and Vijayarani9 reported even lower PCV (10%). TLC appeared similar to a small neutrophil with lighter cytoplasm. Hussein et al10 obtained slightly lower values for PCV (20%).8-10 Compared with previously reported values. and had round. oval or reniform nuclei with an open chromatin pattern (Figure 3). Two granulocytes represented the neutrophils and eosinophils seen by light microscopy.11 In O. glucose (408 mg/dL) and creatinine (4. In a few fish. The cytoplasm of TLC was pale gray.3 mg/dL). Additionally. In addition. published values are severely limited by low fish numbers or by the few analytes measured. When reference intervals for production tilapia were compared to clinical chemistry values obtained from tilapia raised in low-density tanks. large cells with extremely blue cytoplasm were rarely noted. The TLC is a distinct cell type that has been observed and described in other fish species.13 although the differences in calcium and phosphorus values were greater in these tilapia. including hybrid striped bass. there were notable differences in protein. in our laboratory we do a number of procedures to help ensure cells are identified correctly. Monocyte cytoplasm was deeply basophilic and often contained clear.These differences may be due to the fact that the production fish in our study were hybrids of parental species used in previous studies. they do provide consistency. The TLC were larger (5. Discussion Although tilapia are the second most frequently cultured fish in the world. Smith Monocytes measured 9. intracytoplasmic inclusion bodies. Protein concentrations in tilapia raised in high-density systems were slightly higher.0 g/dL) and RBC count (1. This third cell type may be the TLC described here. These cells were considered undifferentiated blast cells. and had occasional vacuoles and basophilic. WBC types and morphology in this study were similar to those described for O. 29 / No. punctate. polygonal or segmented (Figures 1.2 µm diameter) and had a less condensed nuclear chromatin pattern than thrombocytes. while calcium and phosphorus levels were much higher compared to fish in low-density systems. and occasionally vacuolated.The eosinophil granules occasionally obscured the nucleus. yellow perch and tilapia (unpublished observations). hemoglobin (3. 3. and having more than one person repeat the differential counts.The high WBC and lymphocyte counts were consistent with values obtained in our laboratory for other fish species raised in high-density recirculation systems. Neutrophils were 9. mossambicus. in which they superficially resembled thrombocytes.

Herrick CA. 13. As for mammals.17:275-284.58:126-130.14 Techniques to properly determine reference intervals have been established by the NCCLS. Clin Chem 1971.Total protein. Robertson JL. 8. Schalm's Veterinary Hematology. 9. Fish Medicine. A new blood diluent for counting erythrocytes and leukocytes of the chicken. Pa: Lea and Febiger. 1992. which could have induced a generalized immune response. The majority of blood values determined for fishes have been reported as mean ± SD. therefore nonparametric methods are more accurate for determining reference intervals. Ind J Exp Biol 1989. Effects of temperature on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops X Morone saxatilis). particularly Brian Brazil. Comparison of hematologic reference intervals between culture system and type of hybrid striped bass. In: Stoskopf MK. Smith SA. J Aquatic Animal Health 1997.Tilapia Hematology and Plasma Chemistry Values probably an effect of water quality or stocking density. 1995. Robertson JL.31:735738. Proposed Guidelines. Influence of statistical method used on the resulting estimate of normal range. Ultrastructure of the peripheral leucocytes of Oreochromis mossambicus. and may have influenced blood calcium levels. Smith SA. Aquaculture Magazine Buyer’s Guide ’96. 2nd ed.39:83-88. Bull Environ Contam Toxicol 1996. Zinkl JG. Water hardness was much higher in production fish tanks compared to the low-density tanks. tools to monitor the health status of fish using standardized nonlethal and inexpensive methods will be needed. will contribute to more specific. 1993:113-131.9:239-248. Lumsden JH. Effects of ammonia and nitrate concentration on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops X Morone saxatilis). Smith SA.13. Ogawa T.57:503-510. et al. 1986:256-260. 5. Pa: WB Saunders. Page 12 Veterinary Clinical Pathology Vol. Robertson JL. Evaluation of hematologic and blood chemistry analytes will enhance the culture of fish by facilitating early detection of infectious disease and identification of sublethal conditions affecting production performance. Document C28-P. Determine. et al. Am J Vet Res 1997. Stoskopf MK. Vet Clin Pathol 1998. Mason WB. Calculation of reference intervals as ± 2 SD from the mean is valid only when blood values follow a normal distribution. ◊ Acknowledgements The authors thank Butch Kukanich for technical assistance. 57:624-627. Pa: NCCLS. albumin. Harris JE. References 1.57:618-623. Avian hematology. Am J Vet Res 1996. 1996:6-27. 29 / No. Centers for Epidemiology and Animal Health. timely and effective disease treatments in the future. and the staff and students of the Virginia Tech Aquaculture Center. Casillas E. It is incorrect to assume that biological parameters are distributed normally. J Fish Biol 1989. 2. since culture conditions and environmental variables can markedly affect blood values. ed. Status of the world aquaculture 1995. In: Jain NC.7 and suggestions for their use and interpretation have been discussed. for the culture and maintenance of the tilapia used in this study. Hrubec TC. 3. Doggett TA. 15. 17.33:747-756. 12. Poult Sci 1952. This. Haniffa MA. 6. How to Define. This project was funded in part by the Office of Research and Graduate Studies at the Virginia-Maryland Regional College of Veterinary Medicine. Clinical pathology. Am J Vet Res 1996. Blood biochemical reference intervals for sunshine bass (Morone chrysopsϫMorone saxatilis) in three culture systems. Sci Rep Hokkaido Fish Hatchery 1984.27:102-106. 14. 4.12. ed. Vijayarani SM. Reed AH. recirculating systems may be influenced by the characteristic high organic load and bacterial count. Ahmed SM. reference values are not used on a routine basis in fish medicine. 10. El-Nasser MA. 11. Natt MP. Comparative studies on the effect of the herbicide atrazine on freshwater fish Oreochromis niloticus and Chrysichthyes auratus at Assiut. reference intervals should be determined for different pop- ulations of fish within a single species. since the two groups of tilapia were from the same stock source and were fed the same diets. National Committee for Clinical Laboratory Standards. Arkoosh MR. Characteristics of peripheral blood cells from rainbow trout evaluated by particle counter image analysis and hemocytometric techniques. “Normal” or reference values: questions and comments.15 Unfortunately. Egypt. Huffman PA. Terao T. On the biochemical components in the blood of the cultured cichlid fish Tilapia nilotica. Henry RJ. APHIS. Overview of Aquaculture in the United States. 1 / 2000 .27:476-478. 16. As the aquaculture industry expands. Philadelphia. Hrubec TC.16. and the number of studies in which reference intervals have been determined for fish species is limited. in turn. Hematological effects of textile mill effluent on freshwater fish Oreochromis mossambicus (Trewaves). Colo: US Dept Agriculture. and globulin concentrations in fish from high-density. reference intervals developed in highdensity production tilapia are not applicable to fish raised under different environmental and management conditions. Smith SA. and Utilize Reference Intervals in the Clinical Laboratory. Fort Collins. Philadelphia. Hrubec TC. 7.58:131-136. Villanova. Hrubec TC.17 As demonstrated in our comparison of blood values from tilapia in high and lowdensity systems. Robertson JL. Hussein SY. Am J Vet Res 1997.