Aquaculture, 83 (1989) 123-136 123

Elsevier Science Publishers B.V.. Amsterdam - Printed in The Netherlands

Influence of Environmental Variables on

Haematology, and Compendium of Normal
Haematological Ranges of Milkfish, Chanos chanos
(Forskal), in Brackishwater Culture

B. RAM BHASKAR and K. SRINIVASA RAO

Department of Zoology, Andhra University, Waltair, Visakhapatnam 530 003 (India)
(Accepted 26 October 1988)

ABSTRACT

Ram Bhaskar, B. and Srinivasa Rae, K., 1989. Influence of environmental variables on haema-
tology, and compendium of normal haematological ranges of milkfish, Chanos chanos (Forskal ) ,
in brackishwater culture. Aquaculture, 83: 123-136.

The influence of temperature, salinity, dissolved oxygen, and pH of water on nineteen blood
characteristics grouped under erythrocyte parameters, erythrocyte indices, leucocyte parameters,
and plasma constituents of juvenile milkfish, Chanos chanos, from three different brackishwater
fish farms, was evaluated in terms of total (TC) and partial correlation coefficients. There were
no significant differences between fish grown under different farm conditions with regard to most
blood values. The TCs were significant in relationships of salinity of water with total plasma
proteins (negative), plasma sodium (positive), and potassium (positive). Under normal haema-
tological ranges the distributions of most of the blood characteristics were not Gaussian. Two
nonparametric methods, namely tolerance interval and percentile estimation, were used to esti-
mate the normal ranges.

INTRODUCTION

Fish respond readily even to small changes in their ambient environment.

The impact of environmental variables, like temperature, salinity, dissolved
oxygen and pH of water, on fish physiology and metabolism is known to be
reflected in measurable changes of the blood parameters (Blaxhall, 1972).
Healthy growth of fish in any culture system depends on availability of opti-
mum environmental conditions in addition to diet composition, stocking rate,
and pond management. Fluctuations of environmental variables are profound
in a brackishwater culture system, occasionally rendering the water hostile to
its inhabitants. Nevertheless, juveniles of many euryhaline fish congregate in
the shallow brackishwater areas where food is abundant.

0044-8486/89/$03.50 0 1989 Elsevier Science Publishers B.V.

124 B. RAM BHASKAR AND K. SRINIVASA RAO

Several workers have studied the effect of environmental variables on hae-

matological characteristics of fish. The most effective are temperature-related
changes which were observed in fish blood by Allanson et al. (1971), Houston
and Smeda (1979)) Joshi et al. (1980) and Dwyer et al. (1983). The effect of
ambient salinity on blood parameters was studied in brackishwater fish by
Cameron (1970), Farghaly et al. (1973), Courtois (1976) and Usha Rani
(1981)) and results of changes due to reduction in dissolved oxygen content
were described by Soivio et al. (1974), Scott and Rogers (1981) and Swift
( 1981).Limited information concerning responses of blood characteristics to
changes in pH of water is available (Jozuka and Adhachi, 1979). Most of these
studies were experimental and hence related to isolated factors, and usually
fish were acclimated to different environmental conditions.
The present work shows the effects of the changing environmental variables
in a brackishwater culture system on the blood characteristics of milkfish,
Chanos chunos. In their juvenile stages milklish are known to congregate in
brackishwater inlets and hence are amenable for coastal aquaculture. Although
there are descriptions of milkfish ecology (Buri et al., 1981))reproductive cycle
(Kuo and Nash, 1979), and spawning (Marte and Lacanilao, 1986), infor-
mation on its haematology has not been reported so far.
Base-line data for normal haematological ranges are important for the di-
agnosis of fish health (McCarthy et al., 1973) in a culture system. Blood char-
acteristics of fishes, like those of human beings, are often non-Gaussian in
their frequency distributions, i.e., their frequencies do not depict the Gaussian
curve known also as normal distribution (Simpson et al., 1960). The non-
Gaussian distribution results in inaccurate and biassed normal ranges when
parametric methods are applied. Inaccuracies can be avoided by using nonpar-
ametric techniques which can be applied to both Gaussian and non-Gaussian
distributions (Elveback et al., 1970; Reed et al., 1971; Wedemeyer and Nelson,
1975).
The purpose of the present study is to assess, through blood values, the tol-
erance of juvenile milkfish to widely fluctuating environmental conditions in
brackishwater culture, and to present a collation of the results of nonparame-
tric and Gaussian techniques in establishing normal range estimates of the
haematological characteristics.

MATERIALS AND METHODS

Juvenile milkfish (around 30 cm total length) were obtained from brackish-

water fish farms at Kakinada ( Andhra Pradesh, India), belonging to Andhra
Pradesh Fisheries Corporation ( APFC ), Andhra Pradesh Agricultural Uni-
versity (APAU), and Central Institute of Fisheries Education (CIFE). Fish
were stocked at the rate of 1500/ha in all the farms, which were situated more
HAEMATOLOGY OF MILKFISH IN BRACKISHWATER CULTURE 125

or less contiguous to one another. The seed stocked in all farms belonged to a
single strain from Kakinada.
Fish were stunned by a blow on the head and blood was drawn into heparin-
ized, plastic disposable syringes by cardiac puncture. Small volumes of whole
blood were used for estimation of erythrocyte and leucocyte parameters. The
remaining blood was centrifuged (before the analysis of the whole blood) at
15 000 rev./min for 10 min and the plasma obtained was stored at - 15’ C for
further analysis.
Erythrocyte and leucocyte numbers, haematocrit, haemoglobin, and eryth-
rocyte indices were estimated as described by Rambhaskar and Srinivasa Rao
(1987). Blood films were prepared manually on microscope slides, fixed in
methanol and stained with Giemsa (Rambhaskar and Srinivasa Rao, 1987).
The diameters of erythrocyte cytosome and nucleus (CD and ND respectively)
were calculated as (a + b) /2; and surface areas (C and N respectively ) as ICab,
where a and b are major and minor semi-axes respectively of cytosome or nu-
cleus (Benfey and Sutterlin, 1984 ). Nucleo/cytoplasmic (N/C) ratio was eval-
uated from N and C values. Differential leucocyte counts were determined as
suggested by McCarthy et al. (1973).
All plasma constituents were estimated by the methods described by Ram
Bhaskar (1985) (total cholesterol by Zak’s 2-stage method; total plasma pro-
tein (TPP) by Biuret method; albumin by dye (bromocresol green) binding
method, and sodium and potassium by flame photometer method). Tempera-
ture, salinity, and pH of water were measured, and dissolved oxygen content
determined following Winkler’s method.
Thirteen fish samples were taken: six in the APFC farm (Station I), one in
the APAU farm (Station II) and six in the CIFE farm (Station III). The sam-
ple size varied for each set of blood characteristics in each sampling.

Statistical analyses
One-way analysis of variance (ANOVA) was performed for each blood char-
acteristic to test for statistically significant (P < 0.01) differences between the
fish samples collected at the different stations. The thirteen mean values of
each blood characteristic were correlated with the corresponding values of each
of the environmental variables by evaluating Pearson’s total correlation coef-
ficient (TC ). From the TC, the partial correlation coefficient (PC) was de-
rived, which is the measurement of relationship between the value of the given
dependent variate (blood characteristic) and that of any one of the indepen-
dent variates (environmental variables ) on which the former variate depends
(Simpson et al., 1960). In evaluation of TC, only two variates would be in-
volved (one blood characteristic and one environmental variable in this study),
whereas in PC more than two would be involved (five in this study: one blood
characteristic and four environmental variables ). Regression equations were
calculated for those relationships having significant (PC 0.01) TCs. The
126 B. RAM BHASKAR AND K. SRINIVASA RAO

Kolmogorov-Smirnov D statistic and x2 statistic (Reed et al., 1971) were per-

formed to test the normality of the distribution in the case of every blood pa-
rameter, based on the null hypothesis that data values were a random sample
from a normal distribution. The null hypothesis was rejected wherep< 0.05
for both D and x2. Nonparametric and parametric range estimates were deter-
mined for each characteristic following Reed et al. (1971). The tables of
Somerville (1958) were used to compute the 90% (95-90) and 50% (95-50)
probability ranges of nonparametric tolerance interval (TI). The 2.5-97.5 per-
centile estimate (PE) and median were evaluated in nonparametric methods
and 95% estimate (mean ? 1.96 SD) was performed to obtain parametric
Gaussian range.
Observations of changes in environmental variables under field conditions,

TABLE 1

Blood characteristics of milkfish, C. chanos, at different stations

Blood characteristics Station I Station II Station III

Mean* SD (n) Mean? SD (n) Mean? SD (n)

Erythrocyte parameters
Erythrocytes ( x 106/mm3) 2.41? 0.55 (138) 2.17? 0.77 (25) 2.53f 0.70 (120)
Haematocrit ( % )* 44.21? 9.27 (138) 33.90f 6.61 (25) 43.50 + 6.53 (120)
Haemoglobin (g/100 ml) 7.96+ 2.78 (138) 7.63f 1.93 (25) 8.09? 2.88 (120)
Cytosome diameter (pm) 7.46? 1.76 (90) 7.05t 1.50 (15) 7.40? 1.80 (90)
Nucleus diameter (pm ) 3.58? 0.36 (90) 3.30t 0.20 (15) 3.50* 0.40 (90)
N/C ratio 0.26+ 0.07 (90) 0.27+ 0.08 (15) 0.28* 0.03 (90)
Erythrocyte indices
MCV (pm3) 192.67 k67.67 (138) 158.2Ok35.9 (25) 216.30 + 46.8 (120)
MCH (pg) 31.62& 5.70 (138) 35.50 + 5.60 (25) 32.59? 9.60 (120)
MCHC (%,) 18.182 7.40 (138) 23.40f 5.70 (25) 19.16 & 7.90 (120)
Leucocyte parameters
Leucocytes ( X 10’/mm3)* 4.50t 2.10 (138) 5.90f 2.80 (25) 6.20f 2.50 (120)
Differential
Lymphocytes (% ) 63.03+ 4.20 (90) 63.13f 3.90 (15) 62.51* 4.30 (90)
Thrombocytes (% ) 18.22* 3.10 (90) 17.18? 3.00 (15) 16.61% 4.30 (90)
Monocytes (% ) 13.83t 1.90 (90) 13.09& 1.80 (15) 13.36 f 2.00 (90)
Granulocytes (% ) 5.66+ 1.60 (90) 5.00* 1.40 (15) 5.48+ 1.30 (90)
Plasma constituents
Cholesterol (mg/lOO ml)* 205.40+84.10 (60) 139.61?31.30 (IO) 255.23 i 84.20 (59)
TPP (g/100 ml)* 3.26? 1.80 (60) 3.62* 0.70 (10) 4.30f 1.10 (59)
Albumin (g/100 ml)* 2.50? 1.60 (60) 3.62? 0.70 (10) 3.67+_ 0.90 (59)
Sodium (mEq/l ) * 200.10 f 23.90 (60) 159.80f 7.10 (10) 164.58k 24.50 (59)
Potassium (mEq/l)* 6.8Of 1.60 (60) 4.69? 0.72 (10) 4.53f 0.79 (59)
Results of one way ANOVA: *P< 0.01.
n is the number of fish examined.
HAEMATOLOGY OF MILKFISH IN BRACKISHWATER CULTURE 127

and their influence on blood of fish, eliminate certain disadvantages of assum-

ing some arbitrary acclimation period (Cameron, 1970). Constant environ-
mental conditions are not realistic for most fishes, especially those like milk-
fish in culture. In this investigation the fish were assumed to be acclimated to
the ambient conditions, which were subjected to natural variations.

RESULTS

Mean and standard deviation (SD) of all blood characteristics in the fish
from Stations I, II and III along with results of one-way ANOVA are given in
Table 1. Only seven of the nineteen blood characteristics showed significant
differences (P < 0.01) between stations and this would account for the homo-
geneous nature of all the samples. Apart from all the plasma constituents, the
haematocrit and leucocyte numbers also showed significant differences be-
tween stations and this shows the flexibility of these parameters to changing
environmental variables in different stations.
The mean values of blood characteristics in each of the thirteen samples and
the corresponding environmental variables at the three stations are given in
Table 2. Apart from high salinity values in some of the samples of Station I
(located nearer to the bay), all the environmental variables are devoid of wide
fluctuations. In each of the Stations I and III the mean values given in Table
1 are overall mean values of six samples shown in Table 2.
The total and partial correlation coefficients (TC and PC respectively) be-
tween blood characteristics and environmental variables are given in Table 3.
Most of the blood characteristics were robust, with only total plasma proteins
(TPP) showing significant negative TC with salinity. Significant positive TCs
of plasma sodium and potassium with salinity of water were also observed.
Certain blood characteristics which did not show significant TCs with envi-
ronmental variables were significant with regard to their PCs. The relation-
ships of plasma sodium with temperature; monocytes with dissolved oxygen
and pH; TPP with dissolved oxygen; and plasma potassium with dissolved
oxygen did not have significant TCs, but had significant PCs. In these cases
where there was drastic difference between the values of TC and PC, only the
latter was taken into consideration as it would give the correct picture of the
effect of one environmental variable on each blood value, suppressing the ef-
fect of other environmental variables.
Regression lines were fitted (Fig. 1) for the relationships of TPP, plasma
sodium and potassium with salinity of water, in the case of TCs which were
statistically significant (P < 0.01) .
In Table 4, nonparametric and parametric normal range estimates, median
and mean values of the blood characteristics are given. Among the nonpara-
metric ranges 95% (2.5%-97.5%) PE estimate was narrower compared with
95-90 TI estimate (having 90% probability of covering 95% of the target pop-
TABLE 2

Blood characteristics of milkfish, C. chunos. in each sample and the corresponding environmental variables

Blood Station I Station II Station III

characteristics/
Environmental
variables

Erythrocyte parameters
Erythrocytes 2.03 3.21 2.33 2.83 2.49 1.88 2.17 3.75 1.87 2.31 2.88 2.11 2.23
( X 106/mm3) (23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20)
Haematocrit 47.30 42.30 40.50 45.30 44.30 48.50 33.90 45.80 38.60 40.00 45.30 47.20 44.00
(%I (23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20)
Haemoglobin 9.30 12.40 8.10 9.00 6.50 5.70 7.60 13.10 8.60 7.20 8.50 5.40 5.70
(g/l00 ml) (23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20) m
Cytosome 6.50 8.50 7.20 7.80 7.30 7.30 6.10 7.50 7.80 7.20 7.90 7.30 6.80 g
diameter (pm) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Nucleus 3.40 3.80 3.40 2.80 4.20 3.90 3.30 3.70 3.80 2.90 3.80 3.50 3.20 !!
diameter (pm) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) %
N/C ratio 0.25 0.18 0.22 0.11 0.33 0.29 0.37 0.23 0.21 0.14 0.22 0.23 0.18 F
(15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) Fz
z
Erythrocyte indices 7:
MCV (pm3) 247.50 134.90 174.90 165.80 180.20 269.50 158.20 124.40 208.20 175.00 159.30 226.30 196.50 g
(23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20) z
MCH (pg) 47.30 39.60 34.30 32.50 26.10 31.50 35.50 35.40 46.70 31.70 30.10 25.90 25.70
(23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20) k
MCHC (%) 19.20 30.20 21.00 20.00 14.70 11.70 23.40 29.10 24.20 18.10 19.10 11.60 12.90 $
(23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20) 0
Leucocyte parameters
Leucoc~s 5.10 4.10 2.80 2.60 4.70 2.50 6.00 6.20 2.60 2.40 6.50 2.60 2.30 F
( X 10’/mm3) (23) (15) (25) (25) (25) (25) (25) (20) (20) (20) (20) (20) (20) F
Differential r;
Lymphocytes 72.60 50.60 62.70 64.30 63.60 64.40 63.60 51.70 61.60 67.70 65.10 63.50 65.30
(%) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) 2
Thrombocytes 10.20 37.30 19.20 15.40 14.20 13.20 15.20 20.20 25.10 14.20 14.10 13.30
(%) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Monocytes 9.00 7.00 12.50 17.20 19.30 18.30 11.10 19.10 10.20 15.10 16.60 18.10 19.20 F:
(%) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Granulocytes 8.50 5.20 5.50 3.00 3.90 10.10 9.10 3.00 4.20 5.10
(%) (15) (15) (151 (15) (15) (15) (15) (15) (15) (15) (15) 2

Plasma constituents E
Cholesterol 321.00 220.60 105.00 185.80 199.40 200.60 139.60 378.28 222.80 284.30 256.60 199.00
(mg/lOO ml) (10) (10) (10) (10) (10) (10) (10) (10) (10) (9) (10) (10)
TPP 5.14 5.61 1.45 2.08 1.95 2.08 3.62 4.41 4.93 4.79 4.50 3.48
(g/100 ml) (10) (10) (10) (10) (10) (10) (10) (10) (10) (9) (10) (10)
Albumin 4.37 5.04 1.04 1.46 1.48 1.60 3.13 3.92 3.79 4.02 4.11 3.00
(g/100 ml) (10) (10) (10) (10) (10) (10) (10) (10) (10) (9) (10) (10)
Sodium 164.50 173.00 213.60 212.60 211.50 224.90 159.80 153.80 182.90 142.40 153.90 147.20
(n-&/l) (10) (10) (10) (10) (10) (10) (10) (10) (10) (9) (10) (10)
Potassium 5.87 5.43 5.64 7.85 9.32 6.93 4.69 3.88 4.94 4.24 4.01 4.27
(m&/l) (10) (10) (10) (10) (10) (10) (10) (10) (10) (9) (10) (10)

Environmental variables
Temperature
(OC) 24.50 25.00 25.50 23.00 27.00 28.00 26.00 30.00 26.00 25.00 21.50 29.50 29.00
Salinity (%o) 17.30 26.60 32.60 32.80 34.80 35.10 12.00 13.60 14.40 18.30 18.70 18.00 19.00
Dissolved
oxygen (ml/l) 5.30 8.00 7.20 4.60 8.30 4.00 5.00 4.50 5.40 4.20 2.80 8.60 3.60
PH 7.50 7.00 7.50 9.00 8.00 8.50 7.50 7.50 7.00 7.50 8.50 7.00 8.50

Values given as means (number of fish examined ) .

130 B. RAM BHASKAR AND K. SRINIVASA RAO

TABLE 3

Total and partial correlation coefficients (TC and PC respectively) of the blood characteristics
of milkfish, C. chanos, in relation to environmental variables

Blood characteristics Environmental variables

Temperature Salinity Dissolved pH

oxygen

Erythrocyte parameters
Erythrocytes TC - 0.0472 -0.0241 0.0341 0.0543
PC - 0.4836 - 0.0793 0.1175 0.0812
Haematocrit TC 0.1756 0.3329 0.0269 0.3728
PC 0.4082 0.2596 0.0775 0.4372
Haemoglobin TC -0.1811 -0.2031 - 0.0055 -0.2085
PC - 0.3005 -0.1695 -0.1470 -0.2681
Cytosome diameter TC -0.2104 0.0099 0.1876 - 0.0923
PC - 0.3095 0.0414 0.261 I 0.1123
Nucleus diameter TC 0.2152 0.1863 0.3250 -0.2029
PC 0.1278 0.2834 0.1479 -0.0713
N/C ratio TC 0.2628 - 0.0864 0.1972 -0.1807
PC 0.1750 - 0.0353 0.0795 -0.0714
Erythrocyte indices
MCV TC 0.1889 0.1653 -0.0181 0.0818
PC 0.2616 0.1966 -0.0952 0.0767
MCH TC - 0.2907 -0.3141 -0.0273 - 0.4622
PC - 0.5870 -0.1967 -0.2134 - 0.7309
MCHC TC -0.2142 - 0.3058 0.0348 - 0.4283
PC -0.4816 - 0.2084 0.1183 - 0.5633
Leucocyte parameters
Leucocytes TC -0.2014 -0.3702 -0.1450 -0.0516
PC - 0.2724 -0.5788 0.0676 -0.0377
Differential
Lymphocytes TC - 0.0603 0.0039 -0.2874 0.3277
PC -0.1700 -0.1421 - 0.0400 0.1421
Thrombocytes TC -0.0711 0.0300 0.3706 - 0.4936
PC - 0.3976 0.2056 0.1498 - 0.5385
Monocytes TC 0.4250 0.2213 -0.4412 -0.1484
PC 0.6628 0.3351 - 0.9294* - 0.8265*
Granulocytes TC 0.0586 -0.4113 -0.0126 - 0.3673
PC 0.1492 -0.4165 -0.1212 - 0.2250
Plasma constituents
Cholesterol TC 0.0840 -0.4451 -0.3136 0.0586
PC 0.1491 - 0.4683 - 0.2630 0.1931
TPP TC -0.1345 -0.7241* -0.1714 - 0.4960
PC -0.7813 - 0.6692 - 0.8380* - 0.7345
Albumin TC -0.1245 - 0.6620 -0.1668 - 0.4754
PC - 0.5855 - 0.5846 - 0.5288 0.6784
Sodium TC 0.0596 0.7922* -0.1644 0.5387
PC 0.9653* 0.8854* - 0.5091 0.1936
Potassium TC - 0.0603 0.7a99* 0.2623 0.4879
PC - 0.2630 0.7537 0.8542* 0.5414

*P<o.ol.
Degrees of freedom for: TC = 11; PC = 8.
HAEMATOLOGY OF MILKFISH IN BRACKISHWATER CULTURE 131

Y- 6.3I-O.Il69X
Y--O.7241

n= I3

PC 0.01

0
y = 118.89 +2.7345 X

I I I I I I I .I

14 I8 22 26 30 34

SALINITY OF WATER (%o)

Fig. 1. Regression lines of TPP, plasma sodium and plasma potassium of milkfish, C. chanos, on
salinity of water.

ulation) and 95-50 TI estimates (having 50% probability of covering 95% of

target population). The Gaussian range in the parametric estimates, on the
other hand, had varying widths. In the cases where the distributions are not
Gaussian (all parameters except CD, ND, N/C ratio and lymphocytes in this
TABLE 4

Normal range estimates, median, and mean for the blood chnracteristics of C. chanos

Blood characteristics N Nonparametric Parametric

Median 95-90 TI 95-50 TI PE Mean Gaussian

Range Width Range Width Range Width Range Width

Erythrocyte parameters
Erythrocytes (x 10s/mm3) 283 2.35 1.60- 4.25 2.65 1.65- 4.15 2.50 1.70- 4.00 2.30 2.44 1.26- 3.62 2.36
Haematocrit (%) 283 43.00 20.00- 49.00 29.00 21.00- 48.00 27.00 22.00- 48.00 26.00 42.81 28.74- 56.90 28.16
Haemoglobin (g/100 ml) 283 7.50 4.00- 16.00 12.00 4.50- 15.50 11.00 5.00- 15.00 10.00 8.29 3.43- 13.15 9.72
Cytosome diameter (pm )” 195 6.45 5.10- 9.00 3.90 5.25- 8.90 3.65 5.35- 8.85 3.50 7.40 4.09- 10.71 6.62
Nucleus diameter (pm)” 195 2.95 1.75- 4.45 2.70 1.80- 4.35 2.55 2.00- 4.30 2.30 3.52 2.05- 4.99 2.94
N/C ratio” 195 0.21 0.09- 0.43 0.34 O.lO- 0.40 0.30 O.lO- 0.39 0.29 0.27 0.07- 0.47 0.40

Erythrocyte indices
MCV (pm3) 283 180.05 110.23-315.24 205.01 125.40-305.48 180.08 132.83-302.36 169.53 187.38 81.95-292.81 210.86
MCH (pg) 283 31.23 18.24- 49.23 30.99 19.14- 48.14 29.00 20.92- 47.23 26.31 33.08 13.60- 52.56 38.96
MCHC (%) 283 17.23 7.24- 41.15 33.91 9.83- 40.26 30.43 10.93- 38.14 27.21 19.23 5.02- 33.44 28.42

Leucocyte parameters
Leucocytes (X lO’/mms) 283 2.90 1.65 9.35 7.70 1.70- 9.30 7.60 1.75- 9.25 7.50 3.99 1.93- 6.05 4.12
Differential
Lymphocytes ( %)” 195 63.00 47.00- 71.00 24.00 50.00- 72.00 22.00 51.00- 68.00 3.00 62.80 52.80- 72.80 20.00
Thrombocytes ( %) 195 19.00 lO.OO- 33.00 23.00 1 l.OO- 32.00 21.00 12.00- 32.00 20.00 17.40 4.07- 30.73 26.66
Monocytes ( % ) 195 11.20 3.00- 20.00 17.00 4.00- 19.00 15.00 4.00- 19.00 15.00 13.60 6.35 20.85 14.50
Granulocytes ( %) 195 5.00 2.00- 8.00 6.00 2.oc- 7.00 5.00 3.00- 7.00 4.00 5.20 1.67- 8.73 7.06

Plasma constituents
Cholesterol (mg/lOO ml) 129 204.00 107.00-510.00 403.00 109.00-509.00 400.00 110.00~475.00 365.00 222.40 127.99-316.81 188.82
TPP (g/100 ml ) 129 3.75 l.OOm 6.45 5.45 1.25- 6.35 5.10 1.59- 6.10 4.51 3.67 1.55- 5.79 4.24
Albumin (g/100 ml) 129 3.18 1.00. 5.51 4.51 1.15- 5.23 4.08 1.48- 5.10 3.62 3.08 1.12- 5.04 3.92
Sodium (mEq/l) 129 170.00 133.00-234.00 101.00 134.00-227.00 93.00 135.00-225.00 90.00 180.36 122.03-238.69 46.66
Potassium (mEq/l) 129 5.30 3.60- 11.30 7.70 3.70- 9.90 6.20 3.70- 8.20 4.50 5.60 2.35- 8.85 6.50

“Characteristics exhibiting normal Gaussian distribution (Prob D and Prob x2> 0.05).
N is the number of fish examined.
HAEMATOLOGY OF MILKFISH IN,BRACKISHWATER CULTURE 133

study), the PE method should be preferred because it has fixed and definitive
end points for the normal ranges.

DISCUSSION

Different blood characteristics of milkfish in the present study showed pos-

itive or negative relationships with environmental variables, few of which were
significant. The technique of PC used in this study offers a method to deter-
mine the influence of environmental variables on blood components. A review
of the literature (Cameron, 1970; Soivio et al., 1974; Courtois, 1976; Houston
and Smeda, 1979; Smit et al., 1981; Usha Rani, 1981) shows that trends in
relationships of blood characteristics of fishes with environmental variables
are more or less species specific. The effects of environmental variables are
observed to be profound on the survival and oxidative metabolism of fishes,
affecting the blood characteristics (Farghaly et al., 1973 ) .
An increase in salinity causes an increase in plasma volume, resulting in a
decrease in haematocrit (Courtois, 1976). Although the dilution of plasma is
indicated by a strong negative relationship of TPP with salinity, in the present
study there was a weak TC between haematocrit and salinity. The increase of
plasma sodium with salinity of water showed an obviously strong positive cor-
relation. This was also observed by Courtois (1976) in Morone saxatilis accli-
mated to salt water, but he noted a fall in plasma potassium in the same inves-
tigation, contrary to what was observed in C. chunos.
A direct relationship of plasma sodium and water temperature was reported
by Allanson et al. (1971) in Z’iZupiamossambica, and by Smit et al. (1981) in
Sarotherodon mossambicus, Cyprinus carpio and Salmo gairdneri. In the pres-
ent study there was a positive relationship between plasma sodium and water
temperature wherein the PC was significant but the TC was not. This indicates
that the effect of temperature on plasma sodium was significant only when the
effects of salinity, dissolved oxygen, and pH were removed or suppressed. Al-
lanson et al. (1971) found an increase in serum sodium and osmolarity of T.
mossambica when the temperature was reduced to 11 ‘C in 5%0 sea water, while
in freshwater they noted a steady decrease of these serum values with reduction
in temperature. The authors interpreted this to mean that euryhaline tropical
fishes should move into waters of higher salinity to prevent osmoregulatory
failure brought about by decrease in temperature. This problem was not ex-
perienced by kill&h, Fundulus heteroclitus, a euryhaline temperate fish which
has mechanisms to prevent complete osmoregulatory failure even at low tem-
peratures (Umminger, 1970). In the present study the minimum water tem-
perature recorded in winter was 21.5”C; this allows no possibility for distur-
bance in the plasma sodium concentration and osmoregulation of euryhaline
tropical milkfish.
A negative relationship of haemoglobin and haematocrit with dissolved ox-
134 B. RAM BHASKAR AND K. SRINIVASA RAO

ygen, as observed by Cameron (1970) in Lagodon rhomboides, and by Usha

Rani (1981) in Nematoha nasus, was not found in the present study. The
effect of a rise in haematocrit due to oxygen deficiency was described by Swift
( 1981) in rainbow trout as a result of swelling of erythrocytes and/or fluid loss
to the tissue with a subsequent decrease in plasma volume. Though the de-
crease in plasma volume was indicated in C. churu~sby significant negative PC
of TPP with dissolved oxygen, there were weak TCs and PCs of haematocrit,
MCV, and CD of erythrocytes with dissolved oxygen. This questions whether
negative PC of TPP with dissolved oxygen is due to a decrease in plasma vol-
ume or not. Bouk (1972) attributed the increase in TPP of rock bass, Amblo-
plites rupestris, undergoing sublethal hypoxia, to cellular enzymes entering the
blood. It should be noted that the lowest dissolved oxygen value recorded in
the present investigation (2.8 ml/l or 4.3 mg/l) was beyond the threshold and
within the limits of tolerance for C. chunos in brackishwater culture.
Oryzias k&es (Jozuka and Adhachi, 1979) showed a decrease in plasma
sodium with decrease in water pH. In C. chanos there were no significant TCs
of blood characteristics with water pH, but the PCs of monocytes with dis-
solved oxygen and pH were found to be significant.
Wedemeyer and Nelson (1975)) reporting on the normal range estimates for
blood chemistry parameters of 200 rainbow trout, suggested that the PE method
should be preferred to the TI method because there may be a degree of uncer-
tainty in the selection of endpoints in the latter. In C. chunos also the PE
method, which gives narrower width, is preferable for establishing normal hae-
matoiogical range. In view of the physiological capability of milkfish to with-
stand environmental variations and maintain homeostasis, deviations of blood
parameters from normograms are of high diagnostic value. Milkfish affected
by malnutrition or disease can be readily detected in terms of deviations of
haematological values from normal PE ranges established in this study.

ACKNOWLEDGEMENTS

We wish to thank the authorities of the brackishwater fish farms of Kaki-

nada, belonging to APFC, APAU and CIFE for providing the fish samples. We
are also grateful to the Officer-in-Charge, Central Marine Fisheries Research
Institute, Kakinada; the Principal, Andhra Medical College; and the Head of
the Department of Zoology, Andhra University, for providing facilities for the
analyses of whole blood and plasma. One of us (B. Ram Bhaskar) thanks the
Indian Council of Agricultural Research, New Delhi, for the award of Senior
Research Fellowship. We acknowledge the technical assistance rendered by
Mr. G. Maheswarudu, and statistical help extended by Mr. K. Srinivasa I&IO.
HAEMATOLOGY OF MILKFISH IN BRACKISHWATER CULTURE 135

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