Effects of Dietary Lipid on the Hematology Channel Catfish

Aquaculture

ELSEVIER Aquaculture 147 (1996) 225-233

Effects of dietary lipid on the hematology of channel catfish, Ictalurus punctatus

RuthEllen C. Klinger * , Vicki S. Blazer ‘, Carlos Echevarria 2

Georgia Cooperative Fish and Wildlife Research Unif, 3 National Biological Service, School of Forest Resources, University of Georgia, Athens, GA 30602, USA

Accepted 19 July 1996

Abstract

Juvenile channel catfish were fed diets containing either soybean oil, menhaden oil, beef tallow, or a combination of these three lipid sources. After 90 days, total erythrocyte, leucocyte, and thrombocyte counts, hematocrit, mean corpuscular volume, plasma recalcification time, erythrocyte osmotic fragility, total serum iron, and iron-binding capacity were measured. Total erythrocyte counts, leucocyte counts, and mean corpuscular volume were not significantly different (P < 0.05) among dietary groups. However, fish fed the menhaden oil diet had significantly lower (P < 0.05) hematocrits, higher thrombocyte counts, and higher serum iron concentrations. They also had the highest concentration of n - 3 fatty acids in the pronephros tissue and their erythrocytes were the least susceptible to osmotic lysis. Catfish fed the beef tallow diet had the lowest level of n - 3 fatty acids in pronephros tissue and their erythrocytes were the most susceptible to osmotic lysis. Results suggest that dietary lipids affect several hematological factors of cultured channel catfish.

Keywords: Channel cattish; Hematology; Dietary lipid; Thrombocytes; Clotting

* Corresponding author at: Department of Fisheries and Aquatic Sciences, University of Florida, 7922 NW 71st Street, Gainesville, FL 32653, USA. Tel.: (352) 392-9617, ext. 230; fax: (352) 8461088. ’ Present address: National Fish Health Research Laboratory, National Biological Service, Box 1700, Keameysville, WV 25430, USA. ’ Present address: Regional Fisheries Center, Fish and Wildlife Service, Route 1, Box 5 15, Warm Springs, GA 31830, USA. 3 The Unit is jointly supported by the National Biological Service, the Georgia Department of Natural Resources, the University of Georgia and the Wildlife Management Institute.

1. Introduction

In fish culture, nutrition obviously plays an important role in the maintenance of a healthy and marketable product. Channel catfish is an economically important cultured species in the United States. Lipids are an important component of the diet, both as an energy source and as the source of essential fatty acids, which fish cannot synthesize but need for basic functions, including growth and maintenance of healthy tissues (Watanabe, 1982). Because most cultured fish are maintained at low temperatures (compared with homeotherms), they require a higher proportion of unsaturated fatty acids to maintain cell permeability (Bell et al., 1986). As a particular fish acclimates to a lower temperature, more highly unsaturated fatty acids are incorporated into the phospholipid portion of the membrane. This is true of blood cells (Bly et al., 1986; Lie et al., 1989) and other cells (Farkas, 1984; Dey et al., 1993). Channel catfish can utilize saturated fats, and several studies have focused on catfish growth rates as a function of dietary lipid (Stickney and Andrews, 1971; Gatlin and Stickney, 1982). In these studies, no difference in growth was observed between fish fed either menhaden oil or beef tallow at temperatures of 20 or 30°C. However, dietary lipid composition was reflected in fatty acid profiles in tissues (Stickney and Andrews, 1971). Fracalossi and Love11 (1995) found that at 28°C young catfish fed a diet with menhaden oil or a combination of menhaden oil, beef tallow, and corn oil had significantly greater weight gains than fish fed only beef tallow, corn oil, or linseed oil. At 17°C fish fed diets with menhaden oil or the mixture gained significantly more weight than those fed beef tallow. Differences in immune response, disease resistance, and toxicological responses as a function of dietary lipid source have also been reported (Ankley et al., 1989; Sheldon and Blazer, 1991; Fracalossi and Lovell, 1994; Fracalossi et al., 1994). Factors involved in clotting, coagulation, and blood cell formation have been studied for fish health assessment (Blaxhall, 1972; Klontz, 1972; Stoskopf, 1993). A number of studies have shown that differences in blood cell formation and function can also be indicative of dietary manipulations (Poston, 1964; Barnhart, 1969; LeRay et al., 1986; Jarboe et al., 1989; Greene and Selivonchick, 1990; Duncan et al., 1993; Wise et al., 1993). We initiated this study to determine if dietary lipids, previously shown to affect disease resistance factors of channel catfish, had any effect on selected hematological parameters. Total erythrocyte and leucocyte counts were used as indicators of hemopoiesis. Measures of mean corpuscular volume and osmotic fragility allowed us to evaluate the physiological state of the erythrocyte. Thrombocyte counts and plasma recalcification time were used to evaluate clotting mechanisms. Serum iron and iron-binding capacity, important in both hemopoiesis and disease resistance, were also measured. The hematological response of cultured catfish to dietary lipid was evaluated by concurrently analyzing these characteristics.

2. Materials and methods

Juvenile channel catfish, Ictalurus punctatus, (mean weight, 35.4 g) were maintained in a 90% recirculation system consisting of four 225-gallon circular fiberglass tanks. Fish were fed at 3.0% body weight day-’ for a period of 90 days on a 3-day-on-feed, R.C. Klinger et al./Aquaculture 147 (1996) 225-233 227

1-day-off-feed cycle. The four experimental diets were prepared as described in Sheldon and Blazer (1991). They differed only in the lipid content of 7% beef tallow (BT) (predominantly n - 9 fatty acids), 7% soybean oil (SO) (n - 6 fatty acids), 7% menhaden oil (MO) (n - 3 fatty acids), or 7% combination lipid (CO) (equal portions of the above three lipid sources). Water temperature was maintained at 28 + 1°C. At the end of 90 days, approximately 3 ml of blood was collected from the caudal vein of each fish. A portion of blood was collected into standard heparinized capillary tubes and spun for 5 min at 7000 r.p.m. in a Clay Adams microhematocrit centrifuge. The packed red blood cell volume was measured and recorded as the hematocrit. Total erythrocytes (RBCS), leucocytes (WBCs), and thrombocytes were counted with a Neubauer hemacytometer following the method of Blaxhall and Daisley (1973) with Dacies’ solution as a diluting fluid. Thrombocytes were counted separately from other WBCs because of their role in blood clotting (Stoskopf, 1993). Mean corpuscular volume (MCV; rnp’), which reveals changes in cell size, was determined by the formula, hematocrit X IO/total RBCs. Means of all blood values were calculated for each diet and compared with a one-way analysis of variance (SAS Inc., 1982). Osmotic fragility of red blood cells measures the percent lysis of erythrocytes at various salt concentrations (Miale, 1982). A 0.05-ml sample from each fish was subjected to one of 12 buffered saline solutions, ranging from 0.10 to 0.85%. The 0.85% buffered saline solution was used as a blank. Absorbance of blood cell/saline solutions were measured against this standard at 545 nm and averaged for each diet group. Percent hemolysis at each salt concentration for each diet were compared with a Mann-Whitney non-parametric test (Datamost Corp., 1994). The mean erythrocyte fragility, the salt concentration at which 50% of the erythrocytes are hemolyzed, was calculated (Ezell et al., 1969). Plasma recalcification time is an indicator of the intrinsic blood coagulation system (Fujikata and Ik e d a, 1985a). This test involves mixing equal volumes of titrated plasma and 0.025 M CaCl, in a siliconized glass tube. The tube is mixed gently with a horizontal motion, and clotting time is measured to the nearest second. Mean clotting times were compared with a one-way analysis of variance (SAS Inc., 1982) The remaining blood was centrifuged to separate serum for iron analysis. Serum iron and iron binding capacity from eight fish per diet were measured as described by Caraway (1963). Total serum iron measures both bound and free circulating iron. Iron-binding capacity is measured by first saturating the serum with ferric iron, removing the excess unbound iron, and then following the procedure for total serum iron. Total iron-binding capacity is a measure of iron-binding proteins, primarily the glycoprotein transferrin. Pronephros tissue was removed from five fish in each diet group and pooled for fatty acid analysis. The analysis was performed by the Food Science Department at the University of Georgia using a Varian model 3700 gas chromatograph equipped with a flame ionization detector. Glass columns (180 cm X 0.64 cm X 0.02 cm) were packed with GP 5% DEGS-PS on 100/120 supelcoport (Supelco, Bellefonte, PA). Lipids were extracted using AOAC (1984) methods 43.288-289, and methyl esters were prepared according to AOAC (1984) methods 28.057-059. Fatty acid peaks were identified using an n - 3 fatty acid standard. 228 R.C. Klinger et al./Aquaculture 147 (1996) 225-233

3. Results

RBC and WBC counts and MCV in channel catfish were not significantly different (P < 0.05) among fish fed the test diets (Table 1). Fish fed the MO diet had significantly (P < 0.05) lower hematocrits than fish fed the other three diets. Fish fed the MO diet also had higher numbers of thrombocytes and hence total leucocytes. Although variable, slower recalcification time was observed in plasma from fish fed the CO diet than fish fed the SO diet (P < 0.05). The BT diet group developed Flexibacter columnaris, a common freshwater bacterial infection, and thus was not used for tests of serum iron and total iron-binding capacity. This is the only group that showed any sign of disease. Serum iron was significantly higher (P < 0.05) in the MO group than in other dietary groups, yet this group shared a low total iron-binding capacity with the CO diet group. The SO diet yielded a significantly higher (P < 0.05) total iron-binding capacity than either the MO or CO diets (Table 1). Osmotic fragility curves (Fig. 1) indicate that erythrocytes from fish fed the BT diet were most fragile whereas the MO group cells were most resistant to hemolysis under varying salt concentrations. The mean erythrocyte fragility was significantly higher (P < 0.05) in the BT group (0.550) when compared with CO (0.5081, SO (0.504) or MO (0.478) groups. Fatty acid composition of head kidney tissue from fish fed the MO diet

Table 1 Effects of dietary lipid source on selected hematological parameters in channel cattish Indices Diet group a

nb MO SO BT co

Hematocrit 18 21.4a 33.0b 32.9b 31.6b (5.1) (2.4) (2.5) (2.7) Corpuscular volume (m #> 11 118.6a 115.0a 113.4a 117.9a (12.2) (18.4) (9.7) (19.3) Recalcification time (s) 18 580.6a,b 37 1.8a 844.la,b 979.41, (310.1) (187.8) (881.8) (914.1) RBC count (X 106) 11 2.55a 2.898 2.97a 2.63a (0.36) (0.49) (0.28) (0.52) WBC count (X lo31 18 58.0a 56.9a 51.6a 57.la (10.60) (13.7) (6.3) (8.1) Thrombocyte count (X 103) 11 96.8a 67.5b 62.713 76.lb (13.0) (12.5) (13.9) (11.7) Total leucocyte count (X lo? 11 154.7a 124.4b 118.8b 132.2b (19.8) (18.2) (11.11 (15.6) Serum iron ( pg per 100 ml) 8 133.la 120.3b NT 82.9~ (6.2) ( 14.0) (8.1) Iron-binding capacity ( pg per 100 ml) 8 119.la 196.4b NT 129.8a a Fish were fed purified diets containing menhaden oil (MO), soybean oil (SO), beef tallow (BT), or a combination of all three lipid sources (CO), for 90 days at 28°C. Values are means with SD in parentheses. Means, in the same row, followed by the same letter are not significantly different (P < 0.05). NT, not tested. b n, total fish sampled. R.C. Klinger et al./Aquaculture 147 (1996) 225-233 229

+ Beef Tallow

*Combination

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Salt Concentration (%)

Fig. I. Effect of dietary lipid source on erythrocyte osmotic fragility in channel catfish. had the highest total n - 3 fatty acids and the highest n - 3/n - 6 ratio (Table 2). Head kidneys from the BT group were substantially higher in monounsaturated and saturated fatty acids.

Table 2 Effect of diet on fattv acid comoosition of channel catfish rwoneohros tissue Fatty acid a Diet MO SO BT co 14:o 4. I 1.1 1.6 2.3 16:0 19.1 17.0 21.0 19.3 l&O 6.8 5.2 6.3 6.2 24:0 0.7 0.0 0.0 0.0 Total SFA ’ 30.7 23.3 28.9 27.8 16:ln-9 9.3 4.3 5.5 6.5 18:ln-9 34.9 40.4 52.6 41.2 Total n-9 44.2 44.7 58.1 47.7 18:2n-6 6.1 23.1 10.7 13.3 20:4n-6 2.4 3.2 1.4 1.8 Total n-6 8.5 26.3 12.1 15.1 18:3n -3 1.4 3.7 0.9 3.4 20:5n-3 6.5 0.6 0.0 2.1 22:6n - 3 8.7 1.4 0.0 2.1 Total n-3 16.6 5.7 0.9 9.4 n-3/n-6 2.0 0.2 0.1 0.6 a Fatty acids presented as percent of total lipid. ’ SFA refers to saturated fatty acids. 230 R.C. Klinger et ul./Aquaculture 147 (19961225-233

4. Discussion

The utility of hematology in assessing fish health and as an aid to fish disease diagnosis has been questioned. The lack of standardized methods and nomenclature, species differences, and effects of age, sex, water quality, water temperature, and capture methods, all contribute to variability which is difficult to interpret. For this reason it is difficult to compare results from different studies or set ‘normal ranges’ and it has been suggested that reference intervals for each set of culture conditions may need to be determined. In this study, all conditions, other than diet, were held constant. Red blood cells and thrombocytes were the cells most affected by dietary manipulations. Previous work with blackfish, Tautoga on&s, (Doolittle and Surgenor, 1962) and common carp (Fujikata and Ikeda, 1985b) suggests that thrombocytes are important in intrinsic blood coagulation (conversion of prothrombin to thrombin) and are responsible for clot retraction. Srivastava (1969) studied several freshwater fish and suggested that clotting rate is a function of the number of thrombocytes; blood clotting time shortens with increasing number of thrombocytes. Aggregation of warmblooded animal platelets is induced by thromboxane A, that is metabolized from arachidonic acid, a long chained n - 6 fatty acid (Hamberg et al., 1975). Diets high in fish meal and/or fish oil are believed to be responsible for decreased clot formation in other animals because high levels of certain n - 3 fatty acids interfere with the production of thromboxanes (Sanders and Roshanai, 1983). Fish thrombocytes were also shown to produce a variety of prostaglandins although fish species seem to differ in the types of produced prostaglandins and therefore the mechanism of thrombocyte aggregation (Kayama et al., 1985, 1986, 1987; Matsumoto et al., 1989). If the high levels of long chained n - 3 fatty acids in the MO diet inhibited or reduced production of the prostaglandins responsible for thrombocyte aggregation, perhaps catfish on this diet compensated by increasing the number of thrombocytes available to induce clotting. To our knowledge, information is not available for dietary effects on thrombocytes and clotting in fish. When studying the effects of stress on blood coagulation in common carp, Fujikata and Ikeda (1985b) showed that plasma recalcification time decreased with an increase in thrombocyte number. In our study recalcification times were difficult to interpret because of the variability in all diet groups. However, when comparing the two extremes in regards to n - 3 fatty acids, fish fed the MO diet (highest in n - 3 fatty acids, Table 2) had the highest number of thrombocytes and a shorter recalcification time than fish on the BT diet (lowest in n - 3 fatty acids) which exhibited a long recalcification time and lower thrombocyte numbers. Conversely, the SO diet had a low thrombocyte count and low recalcification time. The present study suggests that diet affects not only the number but perhaps also the function of the thrombocytes. Osmotic fragility curves of channel catfish erythrocytes followed the typical sigmoid nature observed in other fish species (Lewis and Ferguson, 1966; Ezell et al., 1969). However, as shown in rainbow trout by LeRay et al. (19861, the rate of osmotic hemolysis increased as the percentage of red cell membrane unsaturated fatty acids increased. In this study the red cell membrane fatty acids were not determined; however, head kidney tissue from fish fed the BT diet had the highest monounsaturated and R.C. Klinger et al./Aquaculture 147 (1996) 225-233 231 saturated fatty acid content, followed by the SO, CO, and MO groups. Red blood cells from fish fed the BT diet were also most susceptible to lysis. The dietary group with the highest II - 3/n - 6 ratio (MO) was the least susceptible to lysis, suggesting a more permeable cell membrane. The ratio of n - 3 to II - 6 in each diet also coincides with the position of each diet’s osmotic fragility curve (Fig. I, Table 2) at the low salt concentrations ( < 0.45%). Serum iron and iron-binding capacity were also significantly affected by dietary lipid, however, these data are difficult to interpret. The only group of fish to develop any signs of disease were those fed the BT diet and they developed a Flexz’bacter columnar& infection. For this reason they were not used for the iron and iron-binding assays. Iron and iron-binding proteins have long been known to affect both fish (Suzumoto et al., 1977; Hershberger and Pratschner, 1981) and homeotherm (Weinberg, 1974) disease resistance. More recently, both iron and iron-binding proteins in homeotherms have been shown to act as regulators of immune function. Lymphocytes, macrophages, and natural killer cells respond (differently) to changes in iron concentration (DeSousa et al., 1988). Because related studies have not been done with fish, we have no comparison for our findings. However, this is another mechanism by which dietary lipid could affect disease resistance in channel catfish. As immunological studies continue to include dietary manipulation, hematology becomes a necessary research tool for further interpretation of diet effects. Sheldon and Blazer (1991) showed that increasing levels of n - 3 fatty acids enhanced intracellular killing of the bacteria Edwardsiellu ictaluri by pronephros macrophages, particularly at the optimum channel catfish culture temperature of 28°C. Fracalossi and Love11 (1994) demonstrated significantly higher antibody titers to E. ictuluri 2 weeks after immuniza- tion, in catfish fed menhaden oil versus corn oii, linseed oil or mixed oils. However, fish fed menhaden oil or linseed oil had lower survival rates when challenged with E. ictuluri. The present study showed osmotic fragility and clotting factors (thrombocytes) and serum iron and iron-binding capacity are also influenced by levels of n - 3 in the diet. Results from this study support findings by others (Fracalossi and Lovell, 1994, 1995) that the immune response in fish can be affected by dietary lipid. Therefore, at least some portion, but probably not all, of the dietary lipid in channel catfish feeds should be fish oil.

Acknowledgements

The authors thank Wade Sheldon for providing the fish for this research and Dr. Philip Koehler and Ms. Anne Morrison for the lipid analysis. The menhaden oil for this study was contributed by Dr. Anthony Bimbo, Zapata Haynie Corp. The project was jointly funded by Cooperative States Research Service USDA agreement #87-CRSR-2- 3040 and Federal Hatch Forestry Research project #25-26-CC295001.

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