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Effect of temperature and salinity changes on the blood characteristics of Tilapia zilli G. In egyptian littoral lakes
Camp. Biochem. Physiol.,1973, Vol. 46A, pp. 183
to 193. Pergamon Press. Printed in Great Britain
EFFECT OF TEMPERATURE AND SALINITY CHANGES ON THE BLOOD CHARACTERISTICS OF TILAPIA ZILLI G. IN EGYPTIAN LITTORAL LAKES A. M. FARGHALY,
A. A. EZZAT
and M. B. SHABANA
Faculty of Science, Alexandria University, Egypt (Received 10 October 1972) Abstract-l. Temperature and salinity changes produce several variations in the blood characteristics of Tilapia zilli G. 2. An increase in water temperature increased most of the parameters except the agranular leucocyte count. 3. A decrease in water temperature caused the disappearance of gamma globulins, and a depression in the other characteristics. 4. Creatinine was not affected. 5. High salinity caused an increase in the concentration of most of the blood characteristics, although the alpha and beta globulins, and the blood coagulation time decreased. 6. A depression in salinity has a slight effect on the haemoglobin content, the erythrocyte count and the total leucocyte count. Nevertheless the granulocytes showed a remarkable increase. INTRODUCTION SALINITY
and temperature are two important factors affecting the survival, metabolism and distribution of many fishes, especially those that live in waters exposed to variations in temperature and salinity, such as estuarine waters. Fishes of the species Tilapia zilli G. live in estuarine waters where fluctuations of the above two important factors frequently occur. Stress caused by low temperatures may account for winter mortalities in various fish species in temperate countries. Failure to adapt to temperature fluctuations (Graham, 1966) and an upset of osmoregulation (Hoar, 1952) h ave been suggested as possible causes of mortalities in many fishes. The variations taking place inside the fish body as a function of temperature and salinity changes in the outside medium can be clarified by studying the changes taking place in the blood characteristics of the fish. This paper is one of a series of studies which were done in order to clarify the internal physiological changes taking place inside the fish body, as a function of the changes taking place in the outside medium. MATERIALS
AND
METHODS
Fishes used in this work are adult healthy fishes which were maintained in suitable continually aerated aquaria. The average temperature of water in the aquaria was about 16.5%‘. 183
184
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
To study the effect of temperature, fishes were put in tanks of 60 1. capacity with temperatures of 30, 32, 34, 36, 37 and 38°C respectively. The fishes were kept for 2 days at the required temperature, before taking the blood sample. To study the effect of cold, the aquarium containing the fishes was put inside an incubator adjusted at 4°C. For the effect of salinity changes, four groups of fishes from 6 to 9 cm in total length were used. Each group was placed in an aquarium of 60 1. capacity. The salinities were as follows: 1, sea water 39-S g salt per 1. ; 2, diluted sea water S%, = 19.5x,; 3, diluted sea water S%, = 9~87%~; 4, fresh water of salinity 0~03%~. Techniques followed in the blood analysis Blood samples were collected by severing of the caudal peduncle. An aliquot of 6.5% potassium citrate was used as an anticoagulant. For erythrocyte counts fish saline (7% NaCl) and Hendrick’s (1952) diluting fluid were used. Shaw’s (1930) solution was used for white cell count as recommended by Hesser (1960). For differential leucocyte counts Giemsa stain was found suitable. The microhaematocrit method was used to measure the packed cell volume. Blood mixed with anticoagulant was collected in duplicate standard capillary tubes and centrifuged for 15 min at 500 rev/min. Haemoglobin levels were measured by the alkali hematin method (Oser 1965). Blood coagulation time was determined according to M&night (1966). Total serum proteins, albumin and globulin were measured using the method adopted by Gornal et al. (1949). Specific electrophoretic procedures were adopted according to Briere & Mull (1964). Separated fractions were scanned using the Carl Zeiss densitometer. Blood sugar was determined according to the Roe (1955) procedure. Blood creatinine content was measured by the Folin and Wu method, based on the Jaffe reaction.
RESULTS EY$zrocyte
count
The acclimatization of Tilapia zilli to high temperatures increased the red cell count to a considerably high value. This increase in the red blood cells was accompanied by a decrease in their mean cell volume. On the other hand, acclimatization to low temperatures of (4°C) leads to a diminution of the erythrocyte count, as shown in Table 1, a slight increase in the mean cell volume also took place. Haematocrit value High temperature increases the haematocrit value of the blood of T. xilli. A noticeable decrease in the value of this blood property accompanies acclimatization of the fish to a low temperature of 4°C. Haemoglobin content Haemoglobin content of Tilapia fish increases at temperatures around 30°C. It is also reduced during acclimatization to low temperatures. The mean erythrocytic haemoglobin content shows only very slight variations with changes in the environmental temperatures.
30
(“C)
1,530,OOO 2,060,OOO 7.65 5.45 34.8 44.3 227.45 215.04 37.13 35.49 7570 15,843 70.5 43.5 5.5 12.0 13.0 10.5 52.0 21.0 29.5 56.5 25.5 39.0 16.0 4.0 0 I.5 3.42 4.38 8.75 17.23 0.20 0.09 95.7 70.5 2.82 1.61
16
Temperature
970,000 3.75 22.4 230.92 36.07 4680 93.5 2.5 6.5 84.5 6.5 6.5 0 0 3.17 9.34 0.10 53.2 1.97
4 1,680,000 5.80 38.2 227.38 34.52 8270 71.5 8.5 Il.5 51.5 28.5 23.0 5.0 0.5 3.20 IO.60 0.12 73.2 I.34
0.03 1,730,000 6.15 38.7 223.69 35.54 6402 65.0 6.0 4.0 45.0 35.0 25.5 9.0 0.5 3.55 2.81 0.02 68.8 1.55
9.87
G.
1,950,000 6.5 43.0 220.51 33.33 5980 49.5 4.5 20.0 25.0 so*5 35.5 13.5 I.5 3.74 18-11 0.22 67.3 1.41
19.75
Salinity (%,)
OF TEMPERATURE AND SALINITY CHANGESON BLOOD CHARACTERISTICS OF T. zilli
Erythrocyte count (per mm3) Haemoglobin content (g %) Haematocrit value (%) Mean erythrocytic volume ($) Mean erythrocytic haemoglobin (r) Total leucocytes (mm”) Agranulocyte (%) Monocytes (%) Large lymphocytes (%) Small lymphocytes (%) Granulocytes (%) Neutrophils (%) Eosinophils (%) Basophils (%) Total serum proteins (g %) Albumin (%) Albumin globulin ratio Blood sugar level (mg %) Blood creatinine level (mg ?A)
Blood characteristic
TABLE I-EFFECT
2,210,000 7.35 46.4 209.95 33.25 5647 31.0 3.0 17.5 10.5 69.0 49.5 18.0 I.5 4.67 6.62 0.07 61.7 2.92
39.50
186
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
---- Ii ----
i3
!=I u
q
0
Pi
_ ----
El E3
u B-4
EFFJXT OF TEMPERATURE
AND SALINITY
ON TILAPIA
BLOOD
187
White blood cells At a temperature of 3O”C, the Tilupia blood is characterized by a high total leucocyte count, while those acclimated to a lower temperature of 4°C have a lower count. At 3O”C, the leucocyte count is distinguished by a drop in the number of agranular white cells. From these the small lymphocytes markedly decreased in number. All types of granulocytes showed a remarkable elevation. On the other hand at 4°C monocytes and large lymphocytes decreased markedly while small lymphocytes increased in abundance. From the granulocytes the neutrophils only were represented but in low numbers, i.e. 6-S per cent. Blood coagulation time High temperature accelerated the blood coagulation time while low temperature decreased the blood clotting activity. The coagulation time recorded at a high temperature was 37 set, while at a low temperature it was 176 sec. Total serum proteins A high temperature of 30°C caused a considerable increase in the total serum protein level. This increase is also accompanied by an increase in the albumin fraction of about twice its value at the normal temperature of 16°C. Both alpha and beta globulins slightly decreased while the gamma globulin disappeared. Accordingly, the increase in the total serum protein may be attributed to the increase in the albumin fraction. That is why the A/G ratio at 30°C increased to more than twice the normal value. On the other hand, a low temperature of 4°C caused a marked decrease in the total serum proteins while the albumin remains more or less within the normal range. Alpha globulins greatly increased, beta globulin decreased, while the gamma globulin disappeared as in the case of high temperature. Thus the decrease in the total serum proteins observed at a low temperature may be explained as being due to the decrease in beta globulin and the disappearance of gamma globulin. At a low temperature the A/G ratio diminished to half its value of the high temperature. This variation in the A/G ratio was reported previously by Meisher & Hickman (1962) w h o were able to show alterations in the A/G ratio of the rainbow trout, Salmo gairdneri, at two different temperatures. The electrophoretic pattern of Tilapia fishes acclimatized to both high and low temperatures are visually similar and are characterized by the presence of only three bands, albumin, alpha and beta globulins respectively. These three fractions possess more or less very similar electrophoretic mobilities for both acclimatization temperatures. Thus the present findings agree with those of Falkner & Houston (1966) who showed that the number of serum protein fractions typically seen in the goldfish, Carassius auratus, varied directly with acclimation temperature. Blood sugar level High temperatures ones showed a drop.
caused an elevation in the level of blood sugar whereas low
188
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
Blood creatinine level Acclimatization to either high or low temperatures caused an elevation of the blood creatinine level of Tilapia fishes. This is contrary to what was noted with respect of all other blood characteristics. Effect of salinity changes Erythrocyte count. The erythrocyte count was slightly changed by lower salinities. Acclimatization of fishes to higher salinities caused a considerable increase in the red blood count. The increase in the red blood cells was accompanied by a marked decrease in their mean cell volume, especially in fishes acclimatized to sea water. Haematocrit value. Haematocrit values were not greatly affected by lower salinities, although high salinities provoked an elevation in the haematocrit value. Haemoglobin content. Higher salinities of 20-25x, caused an increase in the blood haemoglobin content of Tilapia fish. In these salinities, the mean corpuscular haemoglobin remained more or less without any variation. Leucocyte count. The total leucocyte count as well as the agranulocytes decreased at high salinities. The percentage of granulocytes bear a direct proportionality to salinity. Low salinities (9~8%~)did not modify the leucocyte pattern of Tilapia to any extent. However, a salinity of 19*75x, caused a slight decrease in monocytes and an increase in the large lymphocytes. In addition a marked decrease in small lymphocytes occurred. Meanwhile, the three granulocytes, namely neutro-, eosino- and basophils show a considerable increase. At higher salinities (39*50%,) th e p ercentage of both monocytes and large lymphocytes are more or less similar to those at salinities of 17*75x,. Small lymphocytes, on the other hand, dropped clearly. Neutrophils as well as eosinophils show a considerable increase and the percentage of basophils remained the same as the value recorded at a salinity of 19*75x,. Blood coagulation. A high salinity of 39*5%,, caused a decrease in the blood coagulation time to 40 sec. Blood serum protein. Serum protein appears to take part in the maintenance of a normal blood volume and in the maintenance of normal water content in the tissue fluids. By virtue of their colloidal dimensions the serum proteins cannot normally diffuse through the blood capillary membrane into the relatively proteinfree tissue fluids. They exert an osmotic pressure which acts as a force to hold a certain volume of water within the blood. Although they constitute less than 1 per cent of the total osmotic pressure of the plasma, nevertheless it becomes the dominant osmotic force in the blood capillaries, since the other plasma constituents are freely diffusible across the capillary membranes. An increase in the salinity of the medium was found to be accompanied by a gradual increase in the total serum protein of the fish. Each group of our Tilapia fishes, which were acclimatized to a certain water salinity, has its own characteristic electrophoretic pattern. For Tilapia fishes acclimated to a salinity of about lo%,, the amount of albumin fractions showed a marked decrease accompanied by an increase in the
EFFECT OF TEMPERATURE AND SALINITY
ON TILAPIA
BLOOD
189
alpha globulins. At 20x,, the albumin fraction greatly increased, while the amount of alpha-globulin was more or less the same as that of the control specimens. An electropherogram of fishes acclimatized to sea water showed a slight decrease in the albumin fraction which was accompanied by a considerable increase in the alpha globulin. From this we can see that alpha I and II globulins undergo considerable variations with salinity changes. At low salinities the albumin decreased markedly. This diminution is substituted by an increase in both alpha I and alpha II globulins, the increase of which is more or less equivalent to the loss in albumin. In the case of the 20x0 salinity, when the albumin fraction greatly increases, the alpha Alternatively, in the case of sea globulin levels remain more or less constant. water, when albumin decreases, a considerable decrease takes place in alpha globulin. Further, it should be noted that during acclimation to various salinities the beta-globulin fraction changes very slightly. These observations apparently mean that beta-globulin does not take an effective part in osmoregulation of Tilapia blood. This also applies to the gamma globulin. Blood glucose level. The blood glucose level remains more or less within the normal especially at lo%,. At 39x,, the blood glucose level showed a slight decrease. Blood creatinine level. Low salinities caused a small change in the level of blood creatinine level. On the other hand, higher salinities such as sea water showed a marked increase in this constituent.
GENERAL
DISCUSSION
Acclimatization of Tilupiu to a high temperature of 30°C leads to higher values of haematological parameters, while low temperatures cause a depression in these values. The effect of temperature variations on the different blood characteristics of fresh-water teleosts and amphibians has been the subject of several papers published during the past 20 years. A well-defined increase in the erythrocytic count was observed during acclimatization of goldfish, C. auratus (Spoor, 1951), the river shiner, Notropis blennius (Bonder, 1957) and in the amphibian Ranu esculenta (Straub, 1957) and Bufo melunosticus (Deb & Boral, 1964). Recently De Wilds & Houston (1967) investigated the blood oxygen capacity of the rainbow trout, S. gairdneri, as a function of thermal acclimation in terms of erythrocyte abundance, packed cell volume, haemoglobin concentration, mean erythrocytic volume and mean erythrocytic haemoglobin. These authors reported very similar results to those obtained with TiZupia fish. Trout acclimatized to lower temperatures (3 and 7°C) are characterized by relatively low cell counts and a decrease in haematocrit and haemoglobin levels. The mean erythrocytic volumes tend to be relatively high, while the erythrocytic haemoglobin is somewhat below that at the higher temperature. Finally, fishes adapted to 21°C typically have larger numbers of somewhat smaller red cells, more haemoglobin and higher levels of haemoglobin per erythrocyte than the low temperature adapted fishes.
190
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
Anthony (1961) found an inverse relationship between red cell count and temperature. Haws & Goodnight (1962), comparing the haematological characteristics of the channel catfish, Ictalurus punctutus, with those of the brown bullhead, I. nebulosus, found that the former species (which is a cold-water fish), typically possesses higher counts of smaller erythrocytes. Moreover marked differences in the haematological responses of closely related species have been observed (Straub, 1957; Cline & Waldman, 1962). From this we note a marked contradiction as regards the response of fishes in the variations of the ambient temperature, the results reported for one and the same species was found to vary from one author to another (e.g. Spoor, 1951; Anthony, 1961; Falkner, 1964). Falkner & Houston (1966) explained the variations of responses and the contradiction of the results by various species and related species (even the same species) may represent the reliance of the animals upon adaptive responses other than presumably more metabolically expensive processes of erythrocyte elaboration and haemoglobin synthesis. It was noted in the present study that the increase or decrease in the noncellular blood constituents run hand in hand with the changes in the cellular elements. An increase in the various blood constituents at higher temperatures may be explained on the assumption that dehydration of blood takes place as a result of the osmotic transference of water from blood to muscles. This same phenomenon was found to occur in T. xilli G. during its suffering from asphyxia (Shabana, 1972). This viewpoint finds support in the results given for both the brown trout, Salmo trutta (Swift, 1962) and the brook trout, Salvelinus fontinalis @lack et al., 1966) acclimatized to higher temperatures. The decrease in blood constituents occurring at lower temperature may be attributed to a certain mechanism leading to the dilution of these constituents. This proposal can possibly be accepted if we assume that acclimatization of Tilapia fish to a lower temperature is associated with induced kidney dysfunction. This is because even at a lower temperature the blood creatinine level increases, as it does at higher temperatures. Pora & Pekup (1960a) showed that the excretory functions of either gills or kidneys of a fish are greatly diminished under the effect This fact may account for the elevation of blood creatinine of a low temperature. level of Tilapia fish adapted to 4°C. The specific electrophoretic serum protein pattern at different temperatures is characterized by the presence of only three bands corresponding to albumin, alpha and beta globulins respectively. This is completely different from the normal pattern of five bands corresponding to albumin, alpha 1, alpha 2, beta and gamma These results are concordant with those of Falkner & globulins respectively. Houston (1966) who found different patterns of the electropherograms at various temperatures. Salinity changes clearly modify the blood picture of Tilapia fish. The most striking modifications are those observed in the serum protein pattern. The total serum protein level was found to increase in salinity, in order to maintain the
EFFECTOF TEMPERATURE ANDSALINITYON TILAPIA
BLOOD
191
osmoregulatory function of the blood. These modifications seem to be adaptive features to the new higher saline media. Although T. nilli is a fresh-water fish, yet, in most cases, it inhabits brackish waters prevailing in the Egyptian lakes. Like the majority of fresh-water bony fishes, the serum albumin content is decidedly small (not more than 11 per cent of the total serum proteins in contrast to the sea-water teleosts in which albumin exceeds more than 50 per cent in many cases). In order to overcome this deficiency, when placed in higher salinities, variations in both albumin and globulin fractions take place in addition to the general increase in the serum total protein. The results derived from the present investigation show that changes in the serum globulin fraction (especially alpha-globulin) are due to the shorter half life time of the globulin molecule (Miller et al., 1949). This suggests that the serum globulin fraction is more important than the albumin, in osmoregulation of the fish, because of the rapid variability with respect to salinity tolerance. Cordier et al. (1959) studied the effect of osmotic pressure of the outside medium on the serum proteins of the euryhaline fish, AnguiZZa vulgaris. They reported that as the osmotic pressure increases hyperproteinemia occurs. Lecal (1958) obtained the same result when he placed a number of fishes from the species Blennius pavo in different concentrations of salt water for various periods of time. Robertson et al. (1961) reported some variations in the total serum proteins, albumin and globulins of the Pacific salmon, Onchorhyncus spp., moving from sea to their spawning sites. A considerable variation in the average total serum protein took place in Mugil cephalus L. migrating from fresh water to the sea (Ezzat, 1965). Keys (1933) f ound that the total serum proteins of eels dropped from 8.4 g “/o in sea water to 68 g y0 in fresh water. Cordier & Barnoud (1958, 1959) g ave results which are contradictory to those of our results. According to these authors the two stenohaline fishes, Time tinca and Scorpaena porcus, show hypoproteinemia with increasing salinity. They attributed the diminution in the total proteins to an increase in the protein catabolic rate, which occurs at higher saline environments. From the preceding discussion we conclude that the most important response of euryhaline fishes to higher salinities is an increase in their total serum proteins. On the other hand, stenohaline fishes respond to those media by a decrease in these blood constituents. REFERENCES ANTHONY E. H. (1961) The oxygen capacity of goldfish (Curassi~s auratus L.) blood in relation to thermal environment. J. exp. Biol. 38, 39-107. BONDERM. J. (1957) A haematological study of the genus Notropis. M.Sc. thesis, University of Manitoba, Winnipeg. BLACK E. C., DONALDK. & HAROLDH. (1966) Oxygen dessication curves of the blood of brook trout (Salvelinus fontinalis) acclimated to summer and winter temperatures. J. Fish Res. Bd Can. 23, 1-13. BRIERE R. 0. & MULL J. D. (1964) Electrophoresis of proteins with cellulose acetate. Am. r. clin. Path. 34, 547-551.
192
A. M. FARGHALY,A. A. EZZATANDM. B.
SHABANA
BROU~NM. E. (1957) The Physiology of Fishes, Vol. I-Metabolism. Academic Press, New York. CLINE M. J. & WALDMANT. A. (1962) Effect of temperature on erythrophoresis and red cell survival in the frog. An.J. Physiol. 3,401-403. CORDIER D. R. & BRANDONA. M. (19.58) Influence du passage de l’eau deuce a salee sur la proteinernie de la Tanche (Time vulgaris L.). J. Physiol., Lond. 150, 235-237. CORDIERD. R. & BARNOUDJ. R. (1959a) Effet de l’agression osmotique sur la proteinernie de la Rascasse (Scorpaena porcus L.). C. Y. Seanc. Sot. Biol. 153, 368. CORDIERD. R. & BARNOUDJ. R. (1959b) Variations de la protein&nie chez l’anguille de mer (Anguilla vulgaris L.) sous l’influence de l’agression osmotique. C. Y. Sbunc. Sot. Biol. 11, 1802-1805. DEB C. & BORALM. C. (1964) Body fluids and haematological changes in a poikilothermic animal on cold exposure. Am.J. Physiol. 207, 865-867. EZZAT A. A. (1965) Contribution P l’etude de la biologie de quelques Mugilidae de la region de l’etang de Berre et de port de Bout. These de Doctorat es Sciences Naturelles, FacultC des Sciences de l’universite d’Aix Marseille, France. FALKNERN. W. (1964) A study of some haematological changes in the goldfish (Carussius aurutus) following thermal acclimation and nonlethal heat shock. M.Sc. thesis, University of Manitoba, Winnipeg. FALKNER N. W. & HOUSTONA. H. (1966) Some haematological responses to sublethal thermal shock in the goldfish, Carassius auratus L. ‘j. Fish Res. Bd Can. 23, 1109-l 120. GORNALA. G., BARDAWILLC. L. & DAVIDM. M. (1949) Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751. HAWS T. G. & GOODNIGHTC. J. (1962) Some aspects of haematology of two species of caffish in relation to their habitat. Physiol. Zoiil. 35, 8-17. HEUSERE. F. (1960) Methods for routine fish haematology. Progr. Fish. Cult. 22, 164-171. HENDRICKSL. J. (1952) Erythrocyte counts and haemoglobin determination for two species of suckers, genus Lutostomus, from Colorado. Copeia 4, 265-266. LE CAL (1958) Influence du facteur salinite sur les protides seriques chez Blennius pavo. C. Y. Seam. Sot. Biol. 152, 1708-1711. M&NIGHT I. M. (1966) A haematological study on the mountain whitefish Prospoium williamsoni. J. Fish Res. Bd Can. 23,45-46. MILLER L. L., BALE W. F., YUILE C. L., MASTERYR. E., TISHKOFF G. H. & WIPPLE G. H. (1949) The use of radioactive lysine in studies of protein metabolism. Synthesis and utilization of plasma proteins. r. exp. Med. 90, 297-313. OSER B. L. (1965) Hawks Physiological Chemistry, 14th Edn. McGraw-Hill, New York. PORAA. C. & PEKUP 0. U. (1960a) Ob izoocheni vudelutelnukh protsesov o precnovodnukh ru soobchenu, 11. vuluyanu temperatouru sredunna vudelnee protsesu o karpa u karasya bopr. UkhtuZ bep. 15,138-147, str. (With an English summary.) ROBERTSON 0. H., KRUPP M. B., FAVOURC., HANE S. & THOMASS. F. (1961) Physiological changes occurring in the blood of the Pacific salmon (Oncorhyncus tshuwytschu) accompanying sexual maturation and spawning. Endocrinology 68, 733-746. ROE J. H. (1955) The determination of sugar in blood and spinal fluid with aromatic reagents. r. biol. Chem. 212, 335. SHABANA M. B. (1972) Effect of asphyxia on blood characteristic of Tihzpiu zilli. (In press.) SHAW A. F. B. (1930) A direct method for counting the leucocyte and thrombocytes of bird’s blood. J. Path. Bact. 33, 833-835. SPOORW. A. (1951) Temperature and the erythrocyte count of goldfish. Fedn PYOC. Fedn Am. Sots exp. Biol. 10, 131. STRAUBM. (1957) Weitere Untersuchungen zur Temperatur-Adaptation der SauerstoffBindung des Blutes von Rana esculentes L. Z. vergl. Physiol. 39, 507-523.
EFFECTOF TBMPEBATUBE ANDSALINITYON TILAPIA BLOOD SWIFT D. R. naturally. DE WILDE M. process in
193
(1962) Activity cycles in brown trout (Sulmo trutta L.)-I. Fish feeding Hydrobiology 20, 241-247. A. & HOUSTONH. H. (1967) Haematological aspects of the thermoacclimatory the rainbow trout Salmo gairdnui. J. Fish Res. Bd Can. 24, 2267-2281.
Key Word Index-Temperature; salinity; T&pita zilli; blood cells; leucocytes; agranular leucocytes; gamma globulin; erythrocyte count.
to 193. Pergamon Press. Printed in Great Britain
EFFECT OF TEMPERATURE AND SALINITY CHANGES ON THE BLOOD CHARACTERISTICS OF TILAPIA ZILLI G. IN EGYPTIAN LITTORAL LAKES A. M. FARGHALY,
A. A. EZZAT
and M. B. SHABANA
Faculty of Science, Alexandria University, Egypt (Received 10 October 1972) Abstract-l. Temperature and salinity changes produce several variations in the blood characteristics of Tilapia zilli G. 2. An increase in water temperature increased most of the parameters except the agranular leucocyte count. 3. A decrease in water temperature caused the disappearance of gamma globulins, and a depression in the other characteristics. 4. Creatinine was not affected. 5. High salinity caused an increase in the concentration of most of the blood characteristics, although the alpha and beta globulins, and the blood coagulation time decreased. 6. A depression in salinity has a slight effect on the haemoglobin content, the erythrocyte count and the total leucocyte count. Nevertheless the granulocytes showed a remarkable increase. INTRODUCTION SALINITY
and temperature are two important factors affecting the survival, metabolism and distribution of many fishes, especially those that live in waters exposed to variations in temperature and salinity, such as estuarine waters. Fishes of the species Tilapia zilli G. live in estuarine waters where fluctuations of the above two important factors frequently occur. Stress caused by low temperatures may account for winter mortalities in various fish species in temperate countries. Failure to adapt to temperature fluctuations (Graham, 1966) and an upset of osmoregulation (Hoar, 1952) h ave been suggested as possible causes of mortalities in many fishes. The variations taking place inside the fish body as a function of temperature and salinity changes in the outside medium can be clarified by studying the changes taking place in the blood characteristics of the fish. This paper is one of a series of studies which were done in order to clarify the internal physiological changes taking place inside the fish body, as a function of the changes taking place in the outside medium. MATERIALS
AND
METHODS
Fishes used in this work are adult healthy fishes which were maintained in suitable continually aerated aquaria. The average temperature of water in the aquaria was about 16.5%‘. 183
184
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
To study the effect of temperature, fishes were put in tanks of 60 1. capacity with temperatures of 30, 32, 34, 36, 37 and 38°C respectively. The fishes were kept for 2 days at the required temperature, before taking the blood sample. To study the effect of cold, the aquarium containing the fishes was put inside an incubator adjusted at 4°C. For the effect of salinity changes, four groups of fishes from 6 to 9 cm in total length were used. Each group was placed in an aquarium of 60 1. capacity. The salinities were as follows: 1, sea water 39-S g salt per 1. ; 2, diluted sea water S%, = 19.5x,; 3, diluted sea water S%, = 9~87%~; 4, fresh water of salinity 0~03%~. Techniques followed in the blood analysis Blood samples were collected by severing of the caudal peduncle. An aliquot of 6.5% potassium citrate was used as an anticoagulant. For erythrocyte counts fish saline (7% NaCl) and Hendrick’s (1952) diluting fluid were used. Shaw’s (1930) solution was used for white cell count as recommended by Hesser (1960). For differential leucocyte counts Giemsa stain was found suitable. The microhaematocrit method was used to measure the packed cell volume. Blood mixed with anticoagulant was collected in duplicate standard capillary tubes and centrifuged for 15 min at 500 rev/min. Haemoglobin levels were measured by the alkali hematin method (Oser 1965). Blood coagulation time was determined according to M&night (1966). Total serum proteins, albumin and globulin were measured using the method adopted by Gornal et al. (1949). Specific electrophoretic procedures were adopted according to Briere & Mull (1964). Separated fractions were scanned using the Carl Zeiss densitometer. Blood sugar was determined according to the Roe (1955) procedure. Blood creatinine content was measured by the Folin and Wu method, based on the Jaffe reaction.
RESULTS EY$zrocyte
count
The acclimatization of Tilapia zilli to high temperatures increased the red cell count to a considerably high value. This increase in the red blood cells was accompanied by a decrease in their mean cell volume. On the other hand, acclimatization to low temperatures of (4°C) leads to a diminution of the erythrocyte count, as shown in Table 1, a slight increase in the mean cell volume also took place. Haematocrit value High temperature increases the haematocrit value of the blood of T. xilli. A noticeable decrease in the value of this blood property accompanies acclimatization of the fish to a low temperature of 4°C. Haemoglobin content Haemoglobin content of Tilapia fish increases at temperatures around 30°C. It is also reduced during acclimatization to low temperatures. The mean erythrocytic haemoglobin content shows only very slight variations with changes in the environmental temperatures.
30
(“C)
1,530,OOO 2,060,OOO 7.65 5.45 34.8 44.3 227.45 215.04 37.13 35.49 7570 15,843 70.5 43.5 5.5 12.0 13.0 10.5 52.0 21.0 29.5 56.5 25.5 39.0 16.0 4.0 0 I.5 3.42 4.38 8.75 17.23 0.20 0.09 95.7 70.5 2.82 1.61
16
Temperature
970,000 3.75 22.4 230.92 36.07 4680 93.5 2.5 6.5 84.5 6.5 6.5 0 0 3.17 9.34 0.10 53.2 1.97
4 1,680,000 5.80 38.2 227.38 34.52 8270 71.5 8.5 Il.5 51.5 28.5 23.0 5.0 0.5 3.20 IO.60 0.12 73.2 I.34
0.03 1,730,000 6.15 38.7 223.69 35.54 6402 65.0 6.0 4.0 45.0 35.0 25.5 9.0 0.5 3.55 2.81 0.02 68.8 1.55
9.87
G.
1,950,000 6.5 43.0 220.51 33.33 5980 49.5 4.5 20.0 25.0 so*5 35.5 13.5 I.5 3.74 18-11 0.22 67.3 1.41
19.75
Salinity (%,)
OF TEMPERATURE AND SALINITY CHANGESON BLOOD CHARACTERISTICS OF T. zilli
Erythrocyte count (per mm3) Haemoglobin content (g %) Haematocrit value (%) Mean erythrocytic volume ($) Mean erythrocytic haemoglobin (r) Total leucocytes (mm”) Agranulocyte (%) Monocytes (%) Large lymphocytes (%) Small lymphocytes (%) Granulocytes (%) Neutrophils (%) Eosinophils (%) Basophils (%) Total serum proteins (g %) Albumin (%) Albumin globulin ratio Blood sugar level (mg %) Blood creatinine level (mg ?A)
Blood characteristic
TABLE I-EFFECT
2,210,000 7.35 46.4 209.95 33.25 5647 31.0 3.0 17.5 10.5 69.0 49.5 18.0 I.5 4.67 6.62 0.07 61.7 2.92
39.50
186
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
---- Ii ----
i3
!=I u
q
0
Pi
_ ----
El E3
u B-4
EFFJXT OF TEMPERATURE
AND SALINITY
ON TILAPIA
BLOOD
187
White blood cells At a temperature of 3O”C, the Tilupia blood is characterized by a high total leucocyte count, while those acclimated to a lower temperature of 4°C have a lower count. At 3O”C, the leucocyte count is distinguished by a drop in the number of agranular white cells. From these the small lymphocytes markedly decreased in number. All types of granulocytes showed a remarkable elevation. On the other hand at 4°C monocytes and large lymphocytes decreased markedly while small lymphocytes increased in abundance. From the granulocytes the neutrophils only were represented but in low numbers, i.e. 6-S per cent. Blood coagulation time High temperature accelerated the blood coagulation time while low temperature decreased the blood clotting activity. The coagulation time recorded at a high temperature was 37 set, while at a low temperature it was 176 sec. Total serum proteins A high temperature of 30°C caused a considerable increase in the total serum protein level. This increase is also accompanied by an increase in the albumin fraction of about twice its value at the normal temperature of 16°C. Both alpha and beta globulins slightly decreased while the gamma globulin disappeared. Accordingly, the increase in the total serum protein may be attributed to the increase in the albumin fraction. That is why the A/G ratio at 30°C increased to more than twice the normal value. On the other hand, a low temperature of 4°C caused a marked decrease in the total serum proteins while the albumin remains more or less within the normal range. Alpha globulins greatly increased, beta globulin decreased, while the gamma globulin disappeared as in the case of high temperature. Thus the decrease in the total serum proteins observed at a low temperature may be explained as being due to the decrease in beta globulin and the disappearance of gamma globulin. At a low temperature the A/G ratio diminished to half its value of the high temperature. This variation in the A/G ratio was reported previously by Meisher & Hickman (1962) w h o were able to show alterations in the A/G ratio of the rainbow trout, Salmo gairdneri, at two different temperatures. The electrophoretic pattern of Tilapia fishes acclimatized to both high and low temperatures are visually similar and are characterized by the presence of only three bands, albumin, alpha and beta globulins respectively. These three fractions possess more or less very similar electrophoretic mobilities for both acclimatization temperatures. Thus the present findings agree with those of Falkner & Houston (1966) who showed that the number of serum protein fractions typically seen in the goldfish, Carassius auratus, varied directly with acclimation temperature. Blood sugar level High temperatures ones showed a drop.
caused an elevation in the level of blood sugar whereas low
188
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
Blood creatinine level Acclimatization to either high or low temperatures caused an elevation of the blood creatinine level of Tilapia fishes. This is contrary to what was noted with respect of all other blood characteristics. Effect of salinity changes Erythrocyte count. The erythrocyte count was slightly changed by lower salinities. Acclimatization of fishes to higher salinities caused a considerable increase in the red blood count. The increase in the red blood cells was accompanied by a marked decrease in their mean cell volume, especially in fishes acclimatized to sea water. Haematocrit value. Haematocrit values were not greatly affected by lower salinities, although high salinities provoked an elevation in the haematocrit value. Haemoglobin content. Higher salinities of 20-25x, caused an increase in the blood haemoglobin content of Tilapia fish. In these salinities, the mean corpuscular haemoglobin remained more or less without any variation. Leucocyte count. The total leucocyte count as well as the agranulocytes decreased at high salinities. The percentage of granulocytes bear a direct proportionality to salinity. Low salinities (9~8%~)did not modify the leucocyte pattern of Tilapia to any extent. However, a salinity of 19*75x, caused a slight decrease in monocytes and an increase in the large lymphocytes. In addition a marked decrease in small lymphocytes occurred. Meanwhile, the three granulocytes, namely neutro-, eosino- and basophils show a considerable increase. At higher salinities (39*50%,) th e p ercentage of both monocytes and large lymphocytes are more or less similar to those at salinities of 17*75x,. Small lymphocytes, on the other hand, dropped clearly. Neutrophils as well as eosinophils show a considerable increase and the percentage of basophils remained the same as the value recorded at a salinity of 19*75x,. Blood coagulation. A high salinity of 39*5%,, caused a decrease in the blood coagulation time to 40 sec. Blood serum protein. Serum protein appears to take part in the maintenance of a normal blood volume and in the maintenance of normal water content in the tissue fluids. By virtue of their colloidal dimensions the serum proteins cannot normally diffuse through the blood capillary membrane into the relatively proteinfree tissue fluids. They exert an osmotic pressure which acts as a force to hold a certain volume of water within the blood. Although they constitute less than 1 per cent of the total osmotic pressure of the plasma, nevertheless it becomes the dominant osmotic force in the blood capillaries, since the other plasma constituents are freely diffusible across the capillary membranes. An increase in the salinity of the medium was found to be accompanied by a gradual increase in the total serum protein of the fish. Each group of our Tilapia fishes, which were acclimatized to a certain water salinity, has its own characteristic electrophoretic pattern. For Tilapia fishes acclimated to a salinity of about lo%,, the amount of albumin fractions showed a marked decrease accompanied by an increase in the
EFFECT OF TEMPERATURE AND SALINITY
ON TILAPIA
BLOOD
189
alpha globulins. At 20x,, the albumin fraction greatly increased, while the amount of alpha-globulin was more or less the same as that of the control specimens. An electropherogram of fishes acclimatized to sea water showed a slight decrease in the albumin fraction which was accompanied by a considerable increase in the alpha globulin. From this we can see that alpha I and II globulins undergo considerable variations with salinity changes. At low salinities the albumin decreased markedly. This diminution is substituted by an increase in both alpha I and alpha II globulins, the increase of which is more or less equivalent to the loss in albumin. In the case of the 20x0 salinity, when the albumin fraction greatly increases, the alpha Alternatively, in the case of sea globulin levels remain more or less constant. water, when albumin decreases, a considerable decrease takes place in alpha globulin. Further, it should be noted that during acclimation to various salinities the beta-globulin fraction changes very slightly. These observations apparently mean that beta-globulin does not take an effective part in osmoregulation of Tilapia blood. This also applies to the gamma globulin. Blood glucose level. The blood glucose level remains more or less within the normal especially at lo%,. At 39x,, the blood glucose level showed a slight decrease. Blood creatinine level. Low salinities caused a small change in the level of blood creatinine level. On the other hand, higher salinities such as sea water showed a marked increase in this constituent.
GENERAL
DISCUSSION
Acclimatization of Tilupiu to a high temperature of 30°C leads to higher values of haematological parameters, while low temperatures cause a depression in these values. The effect of temperature variations on the different blood characteristics of fresh-water teleosts and amphibians has been the subject of several papers published during the past 20 years. A well-defined increase in the erythrocytic count was observed during acclimatization of goldfish, C. auratus (Spoor, 1951), the river shiner, Notropis blennius (Bonder, 1957) and in the amphibian Ranu esculenta (Straub, 1957) and Bufo melunosticus (Deb & Boral, 1964). Recently De Wilds & Houston (1967) investigated the blood oxygen capacity of the rainbow trout, S. gairdneri, as a function of thermal acclimation in terms of erythrocyte abundance, packed cell volume, haemoglobin concentration, mean erythrocytic volume and mean erythrocytic haemoglobin. These authors reported very similar results to those obtained with TiZupia fish. Trout acclimatized to lower temperatures (3 and 7°C) are characterized by relatively low cell counts and a decrease in haematocrit and haemoglobin levels. The mean erythrocytic volumes tend to be relatively high, while the erythrocytic haemoglobin is somewhat below that at the higher temperature. Finally, fishes adapted to 21°C typically have larger numbers of somewhat smaller red cells, more haemoglobin and higher levels of haemoglobin per erythrocyte than the low temperature adapted fishes.
190
A. M. FARGHALY, A. A. EZZAT AND M. B. SHABANA
Anthony (1961) found an inverse relationship between red cell count and temperature. Haws & Goodnight (1962), comparing the haematological characteristics of the channel catfish, Ictalurus punctutus, with those of the brown bullhead, I. nebulosus, found that the former species (which is a cold-water fish), typically possesses higher counts of smaller erythrocytes. Moreover marked differences in the haematological responses of closely related species have been observed (Straub, 1957; Cline & Waldman, 1962). From this we note a marked contradiction as regards the response of fishes in the variations of the ambient temperature, the results reported for one and the same species was found to vary from one author to another (e.g. Spoor, 1951; Anthony, 1961; Falkner, 1964). Falkner & Houston (1966) explained the variations of responses and the contradiction of the results by various species and related species (even the same species) may represent the reliance of the animals upon adaptive responses other than presumably more metabolically expensive processes of erythrocyte elaboration and haemoglobin synthesis. It was noted in the present study that the increase or decrease in the noncellular blood constituents run hand in hand with the changes in the cellular elements. An increase in the various blood constituents at higher temperatures may be explained on the assumption that dehydration of blood takes place as a result of the osmotic transference of water from blood to muscles. This same phenomenon was found to occur in T. xilli G. during its suffering from asphyxia (Shabana, 1972). This viewpoint finds support in the results given for both the brown trout, Salmo trutta (Swift, 1962) and the brook trout, Salvelinus fontinalis @lack et al., 1966) acclimatized to higher temperatures. The decrease in blood constituents occurring at lower temperature may be attributed to a certain mechanism leading to the dilution of these constituents. This proposal can possibly be accepted if we assume that acclimatization of Tilapia fish to a lower temperature is associated with induced kidney dysfunction. This is because even at a lower temperature the blood creatinine level increases, as it does at higher temperatures. Pora & Pekup (1960a) showed that the excretory functions of either gills or kidneys of a fish are greatly diminished under the effect This fact may account for the elevation of blood creatinine of a low temperature. level of Tilapia fish adapted to 4°C. The specific electrophoretic serum protein pattern at different temperatures is characterized by the presence of only three bands corresponding to albumin, alpha and beta globulins respectively. This is completely different from the normal pattern of five bands corresponding to albumin, alpha 1, alpha 2, beta and gamma These results are concordant with those of Falkner & globulins respectively. Houston (1966) who found different patterns of the electropherograms at various temperatures. Salinity changes clearly modify the blood picture of Tilapia fish. The most striking modifications are those observed in the serum protein pattern. The total serum protein level was found to increase in salinity, in order to maintain the
EFFECTOF TEMPERATURE ANDSALINITYON TILAPIA
BLOOD
191
osmoregulatory function of the blood. These modifications seem to be adaptive features to the new higher saline media. Although T. nilli is a fresh-water fish, yet, in most cases, it inhabits brackish waters prevailing in the Egyptian lakes. Like the majority of fresh-water bony fishes, the serum albumin content is decidedly small (not more than 11 per cent of the total serum proteins in contrast to the sea-water teleosts in which albumin exceeds more than 50 per cent in many cases). In order to overcome this deficiency, when placed in higher salinities, variations in both albumin and globulin fractions take place in addition to the general increase in the serum total protein. The results derived from the present investigation show that changes in the serum globulin fraction (especially alpha-globulin) are due to the shorter half life time of the globulin molecule (Miller et al., 1949). This suggests that the serum globulin fraction is more important than the albumin, in osmoregulation of the fish, because of the rapid variability with respect to salinity tolerance. Cordier et al. (1959) studied the effect of osmotic pressure of the outside medium on the serum proteins of the euryhaline fish, AnguiZZa vulgaris. They reported that as the osmotic pressure increases hyperproteinemia occurs. Lecal (1958) obtained the same result when he placed a number of fishes from the species Blennius pavo in different concentrations of salt water for various periods of time. Robertson et al. (1961) reported some variations in the total serum proteins, albumin and globulins of the Pacific salmon, Onchorhyncus spp., moving from sea to their spawning sites. A considerable variation in the average total serum protein took place in Mugil cephalus L. migrating from fresh water to the sea (Ezzat, 1965). Keys (1933) f ound that the total serum proteins of eels dropped from 8.4 g “/o in sea water to 68 g y0 in fresh water. Cordier & Barnoud (1958, 1959) g ave results which are contradictory to those of our results. According to these authors the two stenohaline fishes, Time tinca and Scorpaena porcus, show hypoproteinemia with increasing salinity. They attributed the diminution in the total proteins to an increase in the protein catabolic rate, which occurs at higher saline environments. From the preceding discussion we conclude that the most important response of euryhaline fishes to higher salinities is an increase in their total serum proteins. On the other hand, stenohaline fishes respond to those media by a decrease in these blood constituents. REFERENCES ANTHONY E. H. (1961) The oxygen capacity of goldfish (Curassi~s auratus L.) blood in relation to thermal environment. J. exp. Biol. 38, 39-107. BONDERM. J. (1957) A haematological study of the genus Notropis. M.Sc. thesis, University of Manitoba, Winnipeg. BLACK E. C., DONALDK. & HAROLDH. (1966) Oxygen dessication curves of the blood of brook trout (Salvelinus fontinalis) acclimated to summer and winter temperatures. J. Fish Res. Bd Can. 23, 1-13. BRIERE R. 0. & MULL J. D. (1964) Electrophoresis of proteins with cellulose acetate. Am. r. clin. Path. 34, 547-551.
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SHABANA
BROU~NM. E. (1957) The Physiology of Fishes, Vol. I-Metabolism. Academic Press, New York. CLINE M. J. & WALDMANT. A. (1962) Effect of temperature on erythrophoresis and red cell survival in the frog. An.J. Physiol. 3,401-403. CORDIER D. R. & BRANDONA. M. (19.58) Influence du passage de l’eau deuce a salee sur la proteinernie de la Tanche (Time vulgaris L.). J. Physiol., Lond. 150, 235-237. CORDIERD. R. & BARNOUDJ. R. (1959a) Effet de l’agression osmotique sur la proteinernie de la Rascasse (Scorpaena porcus L.). C. Y. Seanc. Sot. Biol. 153, 368. CORDIERD. R. & BARNOUDJ. R. (1959b) Variations de la protein&nie chez l’anguille de mer (Anguilla vulgaris L.) sous l’influence de l’agression osmotique. C. Y. Sbunc. Sot. Biol. 11, 1802-1805. DEB C. & BORALM. C. (1964) Body fluids and haematological changes in a poikilothermic animal on cold exposure. Am.J. Physiol. 207, 865-867. EZZAT A. A. (1965) Contribution P l’etude de la biologie de quelques Mugilidae de la region de l’etang de Berre et de port de Bout. These de Doctorat es Sciences Naturelles, FacultC des Sciences de l’universite d’Aix Marseille, France. FALKNERN. W. (1964) A study of some haematological changes in the goldfish (Carussius aurutus) following thermal acclimation and nonlethal heat shock. M.Sc. thesis, University of Manitoba, Winnipeg. FALKNER N. W. & HOUSTONA. H. (1966) Some haematological responses to sublethal thermal shock in the goldfish, Carassius auratus L. ‘j. Fish Res. Bd Can. 23, 1109-l 120. GORNALA. G., BARDAWILLC. L. & DAVIDM. M. (1949) Determination of serum proteins by means of the biuret reaction. J. biol. Chem. 177, 751. HAWS T. G. & GOODNIGHTC. J. (1962) Some aspects of haematology of two species of caffish in relation to their habitat. Physiol. Zoiil. 35, 8-17. HEUSERE. F. (1960) Methods for routine fish haematology. Progr. Fish. Cult. 22, 164-171. HENDRICKSL. J. (1952) Erythrocyte counts and haemoglobin determination for two species of suckers, genus Lutostomus, from Colorado. Copeia 4, 265-266. LE CAL (1958) Influence du facteur salinite sur les protides seriques chez Blennius pavo. C. Y. Seam. Sot. Biol. 152, 1708-1711. M&NIGHT I. M. (1966) A haematological study on the mountain whitefish Prospoium williamsoni. J. Fish Res. Bd Can. 23,45-46. MILLER L. L., BALE W. F., YUILE C. L., MASTERYR. E., TISHKOFF G. H. & WIPPLE G. H. (1949) The use of radioactive lysine in studies of protein metabolism. Synthesis and utilization of plasma proteins. r. exp. Med. 90, 297-313. OSER B. L. (1965) Hawks Physiological Chemistry, 14th Edn. McGraw-Hill, New York. PORAA. C. & PEKUP 0. U. (1960a) Ob izoocheni vudelutelnukh protsesov o precnovodnukh ru soobchenu, 11. vuluyanu temperatouru sredunna vudelnee protsesu o karpa u karasya bopr. UkhtuZ bep. 15,138-147, str. (With an English summary.) ROBERTSON 0. H., KRUPP M. B., FAVOURC., HANE S. & THOMASS. F. (1961) Physiological changes occurring in the blood of the Pacific salmon (Oncorhyncus tshuwytschu) accompanying sexual maturation and spawning. Endocrinology 68, 733-746. ROE J. H. (1955) The determination of sugar in blood and spinal fluid with aromatic reagents. r. biol. Chem. 212, 335. SHABANA M. B. (1972) Effect of asphyxia on blood characteristic of Tihzpiu zilli. (In press.) SHAW A. F. B. (1930) A direct method for counting the leucocyte and thrombocytes of bird’s blood. J. Path. Bact. 33, 833-835. SPOORW. A. (1951) Temperature and the erythrocyte count of goldfish. Fedn PYOC. Fedn Am. Sots exp. Biol. 10, 131. STRAUBM. (1957) Weitere Untersuchungen zur Temperatur-Adaptation der SauerstoffBindung des Blutes von Rana esculentes L. Z. vergl. Physiol. 39, 507-523.
EFFECTOF TBMPEBATUBE ANDSALINITYON TILAPIA BLOOD SWIFT D. R. naturally. DE WILDE M. process in
193
(1962) Activity cycles in brown trout (Sulmo trutta L.)-I. Fish feeding Hydrobiology 20, 241-247. A. & HOUSTONH. H. (1967) Haematological aspects of the thermoacclimatory the rainbow trout Salmo gairdnui. J. Fish Res. Bd Can. 24, 2267-2281.
Key Word Index-Temperature; salinity; T&pita zilli; blood cells; leucocytes; agranular leucocytes; gamma globulin; erythrocyte count.