0300-9629 79 0301-0555602.00~0

EFFECTS OF DENSITY ON GOLDFISH BLOOD-I HEMATOLOGY

COLETTE B. BURTON and SYLVIA A. MURRAY

Water Quality and Ecology Branch, Division of Environmental Planning. Tennessee Valley Authority, Muscle Shoals. AL 35660, U.S.A.

(Rrceiwd 5 May 1978)

Abstract-l. Adult goldfish were held at two loading densities (0.80 and 4.19 fish cm/l water) to deter- mine the effects of density on hematology.

2. Consistent decreases of hematocrit and hemoglobin concentrations, with an increase in erythrocyte sedimentation rate, were found to be related to density.

3. A highly significant reduction in hemoglobin was associated with a high density of males. 4. Density effects are apparently masked by fish size and sex. 5. Density was found to have no effect on other hematological indices and plasma osmolality. These

are discussed in the text.

INTRODUCTION

Many studies have been done to determine the effects of environmental stress on fish hematology. No stan- dard stress response has been identified, however, since many factors, such as length, sex, diet, photo- period and temperature are known to alter hemat- ology (Haws & Goodnight, 1962; Falkner & Hous- ton, 1966; Kamra, 1966; Kaplan, 1971; Summerfelt, 1967). Moreover, there is little mention in the litera- ture of the fish loading densities used to determine hematological responses to environmental stresses, despite the basic tenet that growth rate is a function of population density (Lagler et al., 1962; Silliman & Gutsell, 1958). Since fish blood is sensitive to a variety of environmental changes, it is possible that certain characteristics of fish blood may register den- sity effects. The purpose of this study is to determine whether hematological characteristics are influenced by density under experimental conditions.

MATERIALS AND METHODS

Adult goldfish, Carassius auratus L., were raised, held and analyzed at Carbon Hill National Fish Hatchery in Carbon Hill, AL. Two holding densities were used in this study. Group 1 consisted of 60 fish held in 400 gallons (151.4 1.) of water in a concrete tank, thus giving a holding densitv of 1.2 fish in./sallon (0.80cmil.) of water. This is considered a desirable density (Lewis. 1963). Group 2 con- sisted of 240 fish held in a 400-gallon (151.4 1.) tank, giving a holding density of 6.24 fish in./gallon (4.19 cm/l.) of water. The fish in group I were held from June to August 1976 before sampling, and those in group 2 were held from December 1975 to September 1976. The holding tanks were aerated continuously, and fresh well water at 17’C was continuously circulated once through the tanks at a rate of 4 gallons (15.14 I.) per minute. Although the light was somewhat subdued in the tank house, the fish were exposed to a natural photoperiod. Fish were fed ad libitum daily a diet of Purina Trout Chow.

Approximately 60 healthy fish, free of epizootics, from each density group, were analyzed. Fish from group 1 were analyzed between August 19 and September 16, 1976, and

fish from group 2 were analyzed between June 22 and August 5, 1976. Three to 10 fish were analyzed during each of the 10 analysis sessions for each group. To facilitate blood extraction, fish were placed in a special holder and pithed. Blood was collected from the exposed caudal vein according to the blood extraction method detailed by McKnight (1966). A heparinized syringe and needle were used to extract the blood. Heparinized microhematocrit tubes were used to collect blood to determine hematocrit (Hc) concentration and erythrocyte sedimentation rate (ESR). A drop of whole blood was used for hemoglobin (Hb) determinations.

Hc was determined according to Hesser (1960) (i.e. cen- trifuging the sample for 5 min in a hematocrit centrifuge and measuring the percentage of packed-cell volume in the capillary tube). The micro-Wintrobe method, described by Blaxhall & Daisley (1973). was used to determine the percentage of ESR. Hb. expressed in g/lOOml, was measured calorimetrically by an A & 0 Hemoglobin- ometer with the Spencer modification of Kennedy’s method (Jones, 1967). Whole blood centrifuged at medium speed for 5-15 min in a fixed-angle clinical centrifuge. Plasma was drawn off the separated blood and frozen for later determinations of plasma osmolality (PO). PO, expressed in mOsm/l, was measured with 5 ~1 of plasma in a Wescor dewpoint microosmometer (Kopp. 1975). Counts of red blood cells, determined by methods de- scribed by Murray 81 Burton (1979) were used to compute three hematological indices (Faulkner & King, 1970; DeWilde & Houston, 1967): (1) mean erythrocytic volume (MEV) in pm3; (2) mean erythrocytic hemoglobin (MEH) in ng; and (3) mean erythrocytic hemoglobin concentration (MEHC) in per cent (g/l00 ml).

Statistical methods were used to determine differences between the densities. The Student’s t-test was used to determine statistically significant differences between group means, whereas correlation coefficients were computed to determine whether significant associations existed between fish size and blood characteristics (Snedecor & Cochran. 1968).

RESULTS

Comparative results between the two different den-

sity groups (Table 1) show significant differences in

Hc, Hb and ESR values. Higher values for Hc (5%)

555

556

.’ 60 fish per 4Wgallon tank.

“240 fish per 400-gallon tank. *x P > O.@l ; NS not significant

-tN’ I::.,R

* P 2 0.05; ** P 2 0.01 ; NSSnot significant

* P > 005: ** P 2 0.01: NS- not signiticant _

Effects of density on goldfish blood-1

Table 4. Comparisons of selected blood characteristics between two loading densities for each sex

*P 2 0.05; **P > 0.01; NS-

and Hb (87,) were associated with lower density (group l), whereas a higher value for ESR (71%) was associated with higher density (group 2). MEV, MEH, MEHC and PO values were virtually the same for both groups.

Because (1) the fish in group 2 were, on the average, significantly larger (by 26%) than those in group 1 and (2) fish length is known to influence hematologi- cal characteristics in some fish (Kaplan, 1971; McCarthy et al., 1973), the data were further analyzed to identify effects influenced by length (Table 2). Sig- nificant inverse correlations were found for associ- ations between length and Hc (L-HC) and between length and Hb (L-Hb), and a significant positive cor- relation was found for the association between length and ESR (L-ESR). Further analysis was therefore required to factor out influences of length in Table 1. The data show that length had no influence, except on ESR, when fish were grouped according to sex. Significant sex-related differences existed for Hc and Hb (Table 3). The significant differences in ESR could not be separated from influences of the length in this table. When the data were further stratified by density and sex within each density group, influences of both length and sex became extinct for these responses, except for the inverse L-Hb relationship in group 1, particularly for the females.

Hematofogicdl differences related to sex are shown in Table 3. Table 3 indicates that Hc values (40.67%) and Hb values (10.47 g/100 ml) in males were signifi- cantly higher (by about 10%) than those found in females. When the data were stratified by density between the sexes, these differences persisted for fish in group 1, but became insignificant for fish in group 2. Although length may have influenced Hb values for the fish in group 1, length probably did not in- fluence Hc values (Table 2). Similarly, ESR values were apparently not related to size within either group. Differences in ESR values in Table 3 were apparently sex-rebated. since the ESR values were sig- nificantly higher (36-55’~) for females than for males in both density groups.

To identify density-related hematological responses, sexual differences were isolated by grouping the data according to sex and comparing the hematological values at each density within each sex. Results of these comparisons are shown in Table 4. When the

not significant.

data were grouped in this way,. neither Hc nor Hb values were seen to be influenced by fish length (Table 2). The densities in this study apparently have no effect on Hc and Hb values in the females. Because the apparent decrease (about 7%) in Hc for males in group 2 was significant only at the 900/A probability level, a possible trend is indicated. but not resolved, for effects of density on Hc in males. The test was sensitive enough to resolve a trend for effects of den- sity on Hb in males, since the lo”/, decrease in Hb for males in group 2 was highly significant. Although significant increases in ESR values are indicated for the fish in group 2, these differences are linked to size-related influences (Table 2). To clarify the effect of density on ESR values, data were analyzed from 12 male fish of similar size from each group. The lengths were 20.49 k 0.32 cm for group 1 and 22.41 k 0.47 cm for group 2. There were no signifi- cant correlations between length and ESR for these 24 fish. ESR values obtained from group 1 were 1.92 f 9.28% and 3.37 + 1.25”/, for group 2. Because no significant difference exists between these values, there is apparently no effect of density on ESR values in males. There were not enough females of similar size to test for effects of density.

DISCUSSION

Many of the hematological values given in Table 1 compare favorably with those reported by others. For example, PO values of 263 mOsmj1 fall within the 95% confidence intervals of values reported by Catlett & Millich (1976). Hc values (38 and 4O”J are almost the same as other values reported for goldfish (Catlett & Millich, 1976; Falkner & Houston, 1966; Houston & Cyr, 1974). Hb values in our study (9.70 and 10.6Og/1OOml) agree with values for goldfish reported by Falkner & Houston (1966). but are higher than those reported by Houston & Cyr (1974). The MEV value for group 1 (Table l), 241 c/c, falls within the range of values in goldfish given by Falkner & Houston (1966) although slightly lower MEV values have been reported (Catlett & Millich. 1976). In addi- tion, MEH, MEHC and ESR values fall within the ranges of values reported for other teleosts (McCarthy et al., 1973; DeWilde & Houston, 1967; Houston & DeWilde. 1968, 1969).