Comp. Biochem. Physiol.. 1975. |ol. 52A. pp. 431 to 434. Pergamon Press. Printed in Great Britain
METABOLIC AND HEMATOLOGICAL EFFECTS OF STARVATION IN THE EUROPEAN EEL, ANGUILLA ANGUILLA L.--II. HEMATOLOGY MAJ-LIS JOHANSSON-SJ~)BECK,GORAN DAVE, AKE LARSSON, KERSTIN LEWANDER AND ULF LIDMAN Department of Zoophysiology, University of Gtiteborg, Fack, S-400 33 Gtiteborg 33, Sweden
(Received 28 September 1974) A b s t r a c t - - l . The effects of starvation on some hematological parameters were studied in the European eel, Anguilla anguiUa L. after I l, 47, 96 and 164 days. 2. During the first 47 days there was an increase in the hematocrit as well as in the mean corpuscle volume (MCV), probably due to a swelling phenomenon of the erythrocytes. 3. Between 47 and 96 days the red blood cells (RBC) count increased while the hemoglobin content and the MCV decreased. 4. During the last period (96-164 days) the hemoglobin content returned to the starting level, the hematocrit was still slightly elevated, the RBC count increased and the MCV decreased. 5. There was a successive decrease in the white blood cells (WBC) count during the entire starvation period.
INTRODUCTION
MANY fishes are able to survive for long periods without food. These periods are shortened at higher ambient temperatures (Love, 1970). The hematological effects of starvation have been very little studied in fish and the reports of the few investigations made are contradictory. Studies on the carp, Cyprinus carpio, by Murachi (1959) have shown that starvation for 7 weeks caused a decrease in hemoglobin content and hematocrit. Smallwood (1916) observed that the red blood cell count of Amia calva dropped after starvation for 20 months. In Salmo gairdneri Kawatsu (1966) found that the decline in red cell count is preceded by an increase while the white cells decline steadily• However, Larsson & Lewander (1973) found that starvation for about 5 months did not have any significant effects on either the hematocrit value or the hemoglobin content of the European freshwater eel, Anguilla anguilla L. Smirnova (1965), on the other hand, showed that during starvation for 145 days of the burbot, Lota Iota, the hemoglobin content, hematocrit and number of erythrocytes were increased. The present report describes hematological effects on European eels, (Anguilla alu3uilla L.) which are subjected to starvation under semilaboratory conditions during about 5 months. MATERIALS AND METHODS
Animals All fish used in this experiment were caught at a seawater locality, Sm~Sgen,on the Swedish west coast. The animals were transported to Kristineberg Zoological station, Fiskeb~ickskil, where they were kept in a basin with running seawater with a salinity of 32-34%o. The water tern431
perature followed the temperature of the incoming water, i.e. the seasonal temperature variation (0.I-9.5°C). The starvation period lasted from November to April the following year. Sampling was carried out after 11, 47, 96 and 164 days of starvation. At the beginning the average body weight was 133 ___9 g and after 164 days the body weight decreased by 30%. More detailed information about the experimental procedure is reported by Dave et al. (1975).
Blood sampling After stunning the eel by a blow on the head, blood was obtained from caudal vessels with a heparinized syringe. Aliquots of whole blood were used for the preparation of blood smears, for erythrocyte counts and for determination of hematocrit and hemoglobin.
Methods of analyses The hematocrit was determined in heparinized capillary tubes, which were centrifuged at 15,000 rev/min for 3 rain (MSE microcentrifuge). The hemoglobin content was determined spectrophotometrically as cyanmethemoglobin (Hainline, 1958). The erythrocyte counts (RBC) were estimated by drawing blood into a standard RBC diluting pipette, diluting 1:200 with Hendrick's solution (Hesser, 1960) and counting by the aid of a microscope and a Bilrker hemocytometer. The obtained hematocrit, hemoglobin and RBC values were used for calculating th~ following parameters (cf. Seiverd, 1964). Mean corpuscle hemoglobin Hemoglobin(g/100 ml) x 100 concentration (%) = (MCHC) Hematocrit (%) Mean corpuscle hemoglobin Hemoglobin (g/100 ml) x 10 (pg) = (MCH) RBC in millions per mm 3 Mean corpuscle volume (/~m3) (MCV)
Hematocrit (%) x 10 RBC in millions per mm 3
432
MAJ-LIs JOHANSSON-SJOBECKet al. Table 1. Hematological effects of starvation in yellow eels
Hemetocrit (%) Hemoglobin (g/100 ml)
ii days
47 days
96 days
164 days
28.9~0.9 (12) 8.5~0.3 (12)
33.2±1.5 x (12) S.8t0.4 (12)
32.7~1.3 x (12) 7.6~0.3 x (12)
30.6±1.3 (8) 8.8±0.6 (8)
1.3±0.i (12)
1.3±0.i (12)
1.5±0.1 (12)
RBC count (number of erythrocytes/~l} x i06
WBC count (number of white blood cells/1000 erythrocytes) 47.9±3.0 (12) MCHC (%) 29.5±0.3 (12) MCH (~g) 66.2±4.0 (12) MCV (cu.mlcrons) 223.7~iI.8 (12)
37.7±3.5 (12) 26.4±2.4 (12) 67.9±3.5 (12) 261.1±20.3 (12)
29.7±i.8 x (12) 23.4~0.5 x (12) 53.5±2.3 x (12) 228.3~7.4 (12)
1.6~0.I x (8) 19.4±2.1 x (8) 27.9±0.8 (8) 54.5±2.6 x (8) 192.6±10.6 (8)
x p ~ 0 . 0 5 is considered to be significant. Results are expressed as mean ~ S.E. Number of fishes in parentheses. The data after 47, 96 and 164 days are tested against those after Ii days (Student's t-test).
Table 2. Percentage of white blood cells
Lymphocytes Spindle cells ("Thrombocytes") Neutrophilic granulocytea
ii days
47 days
96 days
164 days
54.8~6.6
50.4~3.4
46.5~6.3
56.5~7.3
34.1~5.8 ii.i±1.6
38.1~3.7 11.5±2.1
37.8~5.1 15.7±2.8
27.7~6.9 15.8±2.0
X P < 0.05 iS considered to be significant. Results are expressed as mean ~ S.E. Number of fishes in parentheses. The data after 47, 96 and 164 days are tested against those after ii days (Student*a t-test~.
The blood smears were prepared and stained using the methods of Giemsa & Pappenheim (cf. Wintrobe, 1967). These preparations were used for the microscopic observations, which included studies of cell differentiation and cell numbers. The white blood cells (WBC) count were made by calculating the number of white blood cells per 1000 erythrocytes. The cells were identified and the percentages of lymphocytes, neutrophilic granulocytes and spindleshaped cells Cthrombocytes") were calculated. About four slides were counted per fish.
Hematocrit and hemoglobin Starvation caused a significant increase in the hematocrit after 47 and 96 days (Table 1 and Fig. I). After 164 days the hematocrit value had returned nearly to the starting level. The hemoglobin content was significantly reduced after 96 days but this decrease was transient and did not persist after 164 days (Table I and Fig. 2). Red blood cells count
RESULTS The effects of starvation on hematological parameters are seen in Tables 1 and 2 and Fig. 1-5. The results after 47, 96 and 164 days were tested against those after 11 days by Student's t-test Significant differences were established at the 0.05 level. Ht (%)
During the first 96 days the red blood cells (RBC) c o u n t remained unchanged but after 164 days there was a significant increase (Table 1 and Fig. 2). White blood cells count There was a successive decrease in the white blood ceils (WBC) count during the entire starvation period (Table 1 and Fig. 5). Between 96 and 164 days the Hb (g/lOOml)
]
3O 8.0 11
47
96
1~i4
days /
Fig. 1. Effect of starvation on hematocrit values in eels. Each point represents the mean value and vertical bars indicate the standard error of the mean.
I 11
I 47
I 96
L da 8 164 Y
Fig. 2. Effect of starvation on hemoglobin content in the blood of eels. Symbols as in Fig. 1.
Effects of starvation in the European eel--II
433
WBC--count
MCV (cu.microns)
20O
91
47
96
164 days
Fig, 3. Effect of starvation on mean corpuscle volume in
eels. Symbols as in Fig. 1. WBC count was reduced to only 40~ of the initial level.
2O
¢1
417
916
164 days
Fig. 5. Effect of starvation on white blood cells count (number of white blood cells/1000 erythrocytes) in eels. Symbols as in Fig. I.
Mean corpuscle hemoglobin concentration (MCHC) The mean corpuscle hemoglobin concentration (MCHC) showed a significant decrease after 96 days (Table 1). However, after 164 days this parameter had returned to the starting level.
Mean corpuscle hemoglobin (MCH) After 47 days no change was observed in the mean corpuscle hemoglobin (MCH), but after 96 and 164 days there was a significant decrease (Table 1).
Mean corpuscle volume (MCV) During the first 47 days the mean corpuscle volume (MCV) increased slightly (Table I and Fig. 3) followed by a successive decrease (26~).
Lymphocytes, spindle cells ("thrombocytes") and neutrophilic 9ranulocytes There were no significant changes in the % of lymphocytes, spindle cells or neutrophilic granulocytes (Table 2). DISCUSSION
Conflicting results exist in the literature concerning the effects of starvation in fish on blood parameters such as hemoglobin content and hematocrit value. In the present study there was an increase in the hematocrit and the MCV between 11 and 47 days, while the hemoglobin content and RBC count were almost constant. These effects indicate an increased water content in the erythrocytes. Such a swelling phenomenon might be a result of a chloride shift (Gunn et al., 1973). This assumption is supported by the observation, in a study parallel to this, that starvation in eels causes a decrease in plasma chloride at the same stage of the starvation period (Dave et al., 1975), The hematocrit change is in good agreement with that reported by Smirnova (1965), who found a hematocrit increase in the burbot starved for 145 days. Kamra (1966) found a slightly increased hematocrit value in cod after 30 days of starvation. Sano (1962) showed
RBC- count (x106) 1'6t 1.4
1
1
1
Fig. 4. Effect of starvation on red blood cells count (number of erythrocytes//~l) in eels. Symbols as in Fig. 1.
that starvation caused a marked fluctuation of the hematocrit values of the Japanese eel with the highest hematocrit value found at the end of the experiment (90 days). The swelling phenomenon observed in this study seems to have disappeared after 96 and 164 days. This is indicated by a return of MCV to the starting level, which occurred contemporaneously with an increase in RBC count and a persisting high hematocrit value. The increase in RBC count and the high hematocrit after 96 days may be explained by evacuation of erythrocytes from the blood depots. However, the increased number of red blood cells was not accompanied by a corresponding increase in hemoglobin biosynthesis. This is indicated by a transient decrease in hemoglobin content observed after 96 days of starvation. Consequently also the MCHC and MCH values were reduced at the same time. This slight anaemic response might be an effect of iron deficiency. Seiverd (1964) claimed that a decrease in MCV, MCH and MCHC indicates an iron deficiency anaemia in mammals. Some studies have yielded results which disagree with the present results. In an earlier study on the European eel, Anguilla anguilla, (Larsson & Lewander, 1973) it was shown that starvation did not affect the values of hematocrit and hemoglobin. Furthermore, Murachi (1959) and Kawatsu (1966) have shown that starvation causes a decrease in hematocrit value and hemoglobin content. A possible explanation to these contradictory results may be differences in experimental conditions. In the present study, the number of white blood cells declined steadily. After 164 days their number was diminished by about 60~o. This is in good agreement with Smirnova (1965), who found that the number of white blood cells was reduced during starvation of the burbot, Lota Iota. Such a reduction in number of white blood cells might indicate an impaired capacity of the immunological defence during prolonged starvation. It may be concluded from this study that long starvation has a limited effect on the red blood cell picture of the eel. The transient swelling of the erythrocytes occurring initially and probably caused by a chloride shift, is followed by a slight anaemic response during the later phase of starvation. This anaemic effect may be a result of an iron deficiency. In contrast to the slight effect on red blood cells the starvation markedly affected the number of white blood cells. The successive decrease in the number of these cells suggests a reduced immunological defence in the eels during long-term starvation.
MAJ-LIS JOHANSSON-SJOBECKet al.
434
Acknowledgements---We are indebted to Mrs. S. C)stling for her excellent technical assistance during the course of this study. Professor R. F~inge is also greatly acknowledged for his kind help and valuable advice. The financial support of the National Board of Fisheries is also gratefully acknowledged. REFERENCES DAVE G., JOHANSSON-SJI)BECK M.-L., LARSSON A., LEWANDER K. t~ LIDr~,N U. (1975). Metabolic and hematological effects of starvation in the European eel, Anguilla anguilla L.--I. Carbohydrate, lipid protein and inorganic ion metabolism. Comp. Biochem. Physiol. 52A, 423-430. GUNN R. B., DALMARK M., TOSTESON D. C. & WIETH J. O. (1973) Characteristics of chloride transport in human red blood cells. J..qen. Physiol. 61, 185-206. HAINLINE A., JR. (1958) Standard Methods of Clinical Chemistry, Vol. 2, p. 49. Academic Press, New York. HESSER E. F. (1960) Methods for routine fish hematology. Prog. Fish-cult. 22, 164-17 I. KAMRA S. K. (1966) Effect of starvation and refeeding on some liver and blood constituents of Atlantic cod (Gadus morhua L.).J. Fish. Res. Bd Can. 27, (7), 975-982. KAWATSU H. (1966) Studies on the anaemia of fish--l. Anaemia of rainbow trout caused by starvation. Bull. Freshwater Fish. Res. Lab., Tokyo 15, 167-173.
LARSSON A. & LEWANDER K. (1973) Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A, 367-374. Love R. M. (1970) The Chemical Biology of Fishes, pp. 222-257. Academic Press, New York. MURACHI S. (1959) Haemoglobin content, erythrocyte sedimentation rate and haematocrit of the blood in the young of the carp (Cyprinus carpio). J. Fac. Fish. Anita. Hush. Hiroshima Univ. 2, 241-247. SANO T. (1962) Haematological studies of the culture fishes in Japan. J. Tokyo Univ. Fish. 48, 105-109. SUVERO C. E. (1964) Haematology Jbr Medical Technologists, p. 334. Lea & Febiger, Philadelphia. SMALLWOOO W. M. (1916) Twenty months of starvation in Amia Calva. Biol. Bull. mar. biol. Lab., Woods Hole 31,453-464. SMIRNOVA L. J. (1965) Blood indices of the burbot during prolonged total fasting and subsequent feeding. Dokl. Acad. Sci. U.S.S.R. Biol. Sci. Sect. (English translation) 160, 107 109. WINTROaE M. M. (1967) Clinical Haematology, p. 448. Henry Kimpton, London.
Key Word Index--Starvation; Anguilla anguilla L.; eel; fish; blood cells; hemoglobin; hematocrit.
METABOLIC AND HEMATOLOGICAL EFFECTS OF STARVATION IN THE EUROPEAN EEL, ANGUILLA ANGUILLA L.--II. HEMATOLOGY MAJ-LIS JOHANSSON-SJ~)BECK,GORAN DAVE, AKE LARSSON, KERSTIN LEWANDER AND ULF LIDMAN Department of Zoophysiology, University of Gtiteborg, Fack, S-400 33 Gtiteborg 33, Sweden
(Received 28 September 1974) A b s t r a c t - - l . The effects of starvation on some hematological parameters were studied in the European eel, Anguilla anguiUa L. after I l, 47, 96 and 164 days. 2. During the first 47 days there was an increase in the hematocrit as well as in the mean corpuscle volume (MCV), probably due to a swelling phenomenon of the erythrocytes. 3. Between 47 and 96 days the red blood cells (RBC) count increased while the hemoglobin content and the MCV decreased. 4. During the last period (96-164 days) the hemoglobin content returned to the starting level, the hematocrit was still slightly elevated, the RBC count increased and the MCV decreased. 5. There was a successive decrease in the white blood cells (WBC) count during the entire starvation period.
INTRODUCTION
MANY fishes are able to survive for long periods without food. These periods are shortened at higher ambient temperatures (Love, 1970). The hematological effects of starvation have been very little studied in fish and the reports of the few investigations made are contradictory. Studies on the carp, Cyprinus carpio, by Murachi (1959) have shown that starvation for 7 weeks caused a decrease in hemoglobin content and hematocrit. Smallwood (1916) observed that the red blood cell count of Amia calva dropped after starvation for 20 months. In Salmo gairdneri Kawatsu (1966) found that the decline in red cell count is preceded by an increase while the white cells decline steadily• However, Larsson & Lewander (1973) found that starvation for about 5 months did not have any significant effects on either the hematocrit value or the hemoglobin content of the European freshwater eel, Anguilla anguilla L. Smirnova (1965), on the other hand, showed that during starvation for 145 days of the burbot, Lota Iota, the hemoglobin content, hematocrit and number of erythrocytes were increased. The present report describes hematological effects on European eels, (Anguilla alu3uilla L.) which are subjected to starvation under semilaboratory conditions during about 5 months. MATERIALS AND METHODS
Animals All fish used in this experiment were caught at a seawater locality, Sm~Sgen,on the Swedish west coast. The animals were transported to Kristineberg Zoological station, Fiskeb~ickskil, where they were kept in a basin with running seawater with a salinity of 32-34%o. The water tern431
perature followed the temperature of the incoming water, i.e. the seasonal temperature variation (0.I-9.5°C). The starvation period lasted from November to April the following year. Sampling was carried out after 11, 47, 96 and 164 days of starvation. At the beginning the average body weight was 133 ___9 g and after 164 days the body weight decreased by 30%. More detailed information about the experimental procedure is reported by Dave et al. (1975).
Blood sampling After stunning the eel by a blow on the head, blood was obtained from caudal vessels with a heparinized syringe. Aliquots of whole blood were used for the preparation of blood smears, for erythrocyte counts and for determination of hematocrit and hemoglobin.
Methods of analyses The hematocrit was determined in heparinized capillary tubes, which were centrifuged at 15,000 rev/min for 3 rain (MSE microcentrifuge). The hemoglobin content was determined spectrophotometrically as cyanmethemoglobin (Hainline, 1958). The erythrocyte counts (RBC) were estimated by drawing blood into a standard RBC diluting pipette, diluting 1:200 with Hendrick's solution (Hesser, 1960) and counting by the aid of a microscope and a Bilrker hemocytometer. The obtained hematocrit, hemoglobin and RBC values were used for calculating th~ following parameters (cf. Seiverd, 1964). Mean corpuscle hemoglobin Hemoglobin(g/100 ml) x 100 concentration (%) = (MCHC) Hematocrit (%) Mean corpuscle hemoglobin Hemoglobin (g/100 ml) x 10 (pg) = (MCH) RBC in millions per mm 3 Mean corpuscle volume (/~m3) (MCV)
Hematocrit (%) x 10 RBC in millions per mm 3
432
MAJ-LIs JOHANSSON-SJOBECKet al. Table 1. Hematological effects of starvation in yellow eels
Hemetocrit (%) Hemoglobin (g/100 ml)
ii days
47 days
96 days
164 days
28.9~0.9 (12) 8.5~0.3 (12)
33.2±1.5 x (12) S.8t0.4 (12)
32.7~1.3 x (12) 7.6~0.3 x (12)
30.6±1.3 (8) 8.8±0.6 (8)
1.3±0.i (12)
1.3±0.i (12)
1.5±0.1 (12)
RBC count (number of erythrocytes/~l} x i06
WBC count (number of white blood cells/1000 erythrocytes) 47.9±3.0 (12) MCHC (%) 29.5±0.3 (12) MCH (~g) 66.2±4.0 (12) MCV (cu.mlcrons) 223.7~iI.8 (12)
37.7±3.5 (12) 26.4±2.4 (12) 67.9±3.5 (12) 261.1±20.3 (12)
29.7±i.8 x (12) 23.4~0.5 x (12) 53.5±2.3 x (12) 228.3~7.4 (12)
1.6~0.I x (8) 19.4±2.1 x (8) 27.9±0.8 (8) 54.5±2.6 x (8) 192.6±10.6 (8)
x p ~ 0 . 0 5 is considered to be significant. Results are expressed as mean ~ S.E. Number of fishes in parentheses. The data after 47, 96 and 164 days are tested against those after Ii days (Student's t-test).
Table 2. Percentage of white blood cells
Lymphocytes Spindle cells ("Thrombocytes") Neutrophilic granulocytea
ii days
47 days
96 days
164 days
54.8~6.6
50.4~3.4
46.5~6.3
56.5~7.3
34.1~5.8 ii.i±1.6
38.1~3.7 11.5±2.1
37.8~5.1 15.7±2.8
27.7~6.9 15.8±2.0
X P < 0.05 iS considered to be significant. Results are expressed as mean ~ S.E. Number of fishes in parentheses. The data after 47, 96 and 164 days are tested against those after ii days (Student*a t-test~.
The blood smears were prepared and stained using the methods of Giemsa & Pappenheim (cf. Wintrobe, 1967). These preparations were used for the microscopic observations, which included studies of cell differentiation and cell numbers. The white blood cells (WBC) count were made by calculating the number of white blood cells per 1000 erythrocytes. The cells were identified and the percentages of lymphocytes, neutrophilic granulocytes and spindleshaped cells Cthrombocytes") were calculated. About four slides were counted per fish.
Hematocrit and hemoglobin Starvation caused a significant increase in the hematocrit after 47 and 96 days (Table 1 and Fig. I). After 164 days the hematocrit value had returned nearly to the starting level. The hemoglobin content was significantly reduced after 96 days but this decrease was transient and did not persist after 164 days (Table I and Fig. 2). Red blood cells count
RESULTS The effects of starvation on hematological parameters are seen in Tables 1 and 2 and Fig. 1-5. The results after 47, 96 and 164 days were tested against those after 11 days by Student's t-test Significant differences were established at the 0.05 level. Ht (%)
During the first 96 days the red blood cells (RBC) c o u n t remained unchanged but after 164 days there was a significant increase (Table 1 and Fig. 2). White blood cells count There was a successive decrease in the white blood ceils (WBC) count during the entire starvation period (Table 1 and Fig. 5). Between 96 and 164 days the Hb (g/lOOml)
]
3O 8.0 11
47
96
1~i4
days /
Fig. 1. Effect of starvation on hematocrit values in eels. Each point represents the mean value and vertical bars indicate the standard error of the mean.
I 11
I 47
I 96
L da 8 164 Y
Fig. 2. Effect of starvation on hemoglobin content in the blood of eels. Symbols as in Fig. 1.
Effects of starvation in the European eel--II
433
WBC--count
MCV (cu.microns)
20O
91
47
96
164 days
Fig, 3. Effect of starvation on mean corpuscle volume in
eels. Symbols as in Fig. 1. WBC count was reduced to only 40~ of the initial level.
2O
¢1
417
916
164 days
Fig. 5. Effect of starvation on white blood cells count (number of white blood cells/1000 erythrocytes) in eels. Symbols as in Fig. I.
Mean corpuscle hemoglobin concentration (MCHC) The mean corpuscle hemoglobin concentration (MCHC) showed a significant decrease after 96 days (Table 1). However, after 164 days this parameter had returned to the starting level.
Mean corpuscle hemoglobin (MCH) After 47 days no change was observed in the mean corpuscle hemoglobin (MCH), but after 96 and 164 days there was a significant decrease (Table 1).
Mean corpuscle volume (MCV) During the first 47 days the mean corpuscle volume (MCV) increased slightly (Table I and Fig. 3) followed by a successive decrease (26~).
Lymphocytes, spindle cells ("thrombocytes") and neutrophilic 9ranulocytes There were no significant changes in the % of lymphocytes, spindle cells or neutrophilic granulocytes (Table 2). DISCUSSION
Conflicting results exist in the literature concerning the effects of starvation in fish on blood parameters such as hemoglobin content and hematocrit value. In the present study there was an increase in the hematocrit and the MCV between 11 and 47 days, while the hemoglobin content and RBC count were almost constant. These effects indicate an increased water content in the erythrocytes. Such a swelling phenomenon might be a result of a chloride shift (Gunn et al., 1973). This assumption is supported by the observation, in a study parallel to this, that starvation in eels causes a decrease in plasma chloride at the same stage of the starvation period (Dave et al., 1975), The hematocrit change is in good agreement with that reported by Smirnova (1965), who found a hematocrit increase in the burbot starved for 145 days. Kamra (1966) found a slightly increased hematocrit value in cod after 30 days of starvation. Sano (1962) showed
RBC- count (x106) 1'6t 1.4
1
1
1
Fig. 4. Effect of starvation on red blood cells count (number of erythrocytes//~l) in eels. Symbols as in Fig. 1.
that starvation caused a marked fluctuation of the hematocrit values of the Japanese eel with the highest hematocrit value found at the end of the experiment (90 days). The swelling phenomenon observed in this study seems to have disappeared after 96 and 164 days. This is indicated by a return of MCV to the starting level, which occurred contemporaneously with an increase in RBC count and a persisting high hematocrit value. The increase in RBC count and the high hematocrit after 96 days may be explained by evacuation of erythrocytes from the blood depots. However, the increased number of red blood cells was not accompanied by a corresponding increase in hemoglobin biosynthesis. This is indicated by a transient decrease in hemoglobin content observed after 96 days of starvation. Consequently also the MCHC and MCH values were reduced at the same time. This slight anaemic response might be an effect of iron deficiency. Seiverd (1964) claimed that a decrease in MCV, MCH and MCHC indicates an iron deficiency anaemia in mammals. Some studies have yielded results which disagree with the present results. In an earlier study on the European eel, Anguilla anguilla, (Larsson & Lewander, 1973) it was shown that starvation did not affect the values of hematocrit and hemoglobin. Furthermore, Murachi (1959) and Kawatsu (1966) have shown that starvation causes a decrease in hematocrit value and hemoglobin content. A possible explanation to these contradictory results may be differences in experimental conditions. In the present study, the number of white blood cells declined steadily. After 164 days their number was diminished by about 60~o. This is in good agreement with Smirnova (1965), who found that the number of white blood cells was reduced during starvation of the burbot, Lota Iota. Such a reduction in number of white blood cells might indicate an impaired capacity of the immunological defence during prolonged starvation. It may be concluded from this study that long starvation has a limited effect on the red blood cell picture of the eel. The transient swelling of the erythrocytes occurring initially and probably caused by a chloride shift, is followed by a slight anaemic response during the later phase of starvation. This anaemic effect may be a result of an iron deficiency. In contrast to the slight effect on red blood cells the starvation markedly affected the number of white blood cells. The successive decrease in the number of these cells suggests a reduced immunological defence in the eels during long-term starvation.
MAJ-LIS JOHANSSON-SJOBECKet al.
434
Acknowledgements---We are indebted to Mrs. S. C)stling for her excellent technical assistance during the course of this study. Professor R. F~inge is also greatly acknowledged for his kind help and valuable advice. The financial support of the National Board of Fisheries is also gratefully acknowledged. REFERENCES DAVE G., JOHANSSON-SJI)BECK M.-L., LARSSON A., LEWANDER K. t~ LIDr~,N U. (1975). Metabolic and hematological effects of starvation in the European eel, Anguilla anguilla L.--I. Carbohydrate, lipid protein and inorganic ion metabolism. Comp. Biochem. Physiol. 52A, 423-430. GUNN R. B., DALMARK M., TOSTESON D. C. & WIETH J. O. (1973) Characteristics of chloride transport in human red blood cells. J..qen. Physiol. 61, 185-206. HAINLINE A., JR. (1958) Standard Methods of Clinical Chemistry, Vol. 2, p. 49. Academic Press, New York. HESSER E. F. (1960) Methods for routine fish hematology. Prog. Fish-cult. 22, 164-17 I. KAMRA S. K. (1966) Effect of starvation and refeeding on some liver and blood constituents of Atlantic cod (Gadus morhua L.).J. Fish. Res. Bd Can. 27, (7), 975-982. KAWATSU H. (1966) Studies on the anaemia of fish--l. Anaemia of rainbow trout caused by starvation. Bull. Freshwater Fish. Res. Lab., Tokyo 15, 167-173.
LARSSON A. & LEWANDER K. (1973) Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A, 367-374. Love R. M. (1970) The Chemical Biology of Fishes, pp. 222-257. Academic Press, New York. MURACHI S. (1959) Haemoglobin content, erythrocyte sedimentation rate and haematocrit of the blood in the young of the carp (Cyprinus carpio). J. Fac. Fish. Anita. Hush. Hiroshima Univ. 2, 241-247. SANO T. (1962) Haematological studies of the culture fishes in Japan. J. Tokyo Univ. Fish. 48, 105-109. SUVERO C. E. (1964) Haematology Jbr Medical Technologists, p. 334. Lea & Febiger, Philadelphia. SMALLWOOO W. M. (1916) Twenty months of starvation in Amia Calva. Biol. Bull. mar. biol. Lab., Woods Hole 31,453-464. SMIRNOVA L. J. (1965) Blood indices of the burbot during prolonged total fasting and subsequent feeding. Dokl. Acad. Sci. U.S.S.R. Biol. Sci. Sect. (English translation) 160, 107 109. WINTROaE M. M. (1967) Clinical Haematology, p. 448. Henry Kimpton, London.
Key Word Index--Starvation; Anguilla anguilla L.; eel; fish; blood cells; hemoglobin; hematocrit.
