mammals, have hardly been considered in birds. The only information found in the literature has been that of the chicken (Hogan & Parrot, 1961; Hunter, 1961;.
48
Laboratory Animals (I982) 16, 48-50
Method for determining the osmotic fragility curves of erythrocytes in birds G. VISCOR
& J. P ALOMEQUE
Departamento de Fisiolog{a Animal. Facultad de Biolog{a, Universidad de Barcelona (Central). Barcelona-?-, Spain
Summary The osmotic fragfllty of erythrocytes in 3 species of birds (Gallus gallus domesticus, Cotumix cotumix Japonica, Columba IMa) and the rat was determined. The results of this study point to a smaller osmotic fragility. Birds, with eIllptical erythrocytes, have a lower osmotic fragility than species with round erythrocytes, like most of the mammals. The osmotic fragility values that are so well known and useful as diagnostic and research aids in man and mammals, have hardly been considered in birds. The only information found in the literature has been that of the chicken (Hogan & Parrot, 1961; Hunter, 1961; March, Coates & Biely, 1966; Sturkie, 1965) and ostrich (Sturkie, 1965). This scarcity of data could be a consequence of inaccuracy in the application of the usual saline-solution technique used in man (Parpart, Lorenz, Parpart, Gregg & Chase, 1947) to the measurement of osmotic fragility of the erythrocytes of birds. With this technique it was found that haemolysis was not complete and that part of the haemoglobin remained attached to the broken erythrocytes in the most hypotonic solutions. When the freezing-defreezing and the saline methods using distilled water were compared, the saline method showed only a partial haemolysis of 50-60% as opposed to 100% haemolysis when the former method was used. Such a difference of results couid explain why some authors (March et al., 1966) apparently calculate the percentage of haemolysis in relation to the highest value obtained with the spectrophotometer. However, in the bird blood this value does not always represent total haemolysis, which could be a source of error in the determination of osmotic fragility. We have therefore developed a small modification of Parpart et al. (1947) technique for determining osmotic fragility curves, which we believe makes the technique applicable to the blood of birds. Materials and methods In this experiment 3 species of birds, the hen Gallus gallus domesticus, the pigeon Columba livia, and the Received1 June 1981. Accepted27 July 1981.
quail Coturnix coturnix japonica, as well as the Wistar rat Rattus norvegicus, were analyzed. The bird blood wa~ withdrawn from the basilic vein of the wing with a heparinized syringe, as it has been shown that heparin is the anticoagulant which least affects blood osmolarity (Fourie, 1977). The rat blood was obtained by cardiac puncture_
Analyses were carried out immediately. In birds
50.ul blood was added to each of a series of tubes with a saline concentration that ranged from nil (distilled water) to 1·0 g sodium chloride/100 ml. The tubes were agitated continuously in an incubator at 41°C for 1 h in order to minimize the retention of haemoglobin by the broken, haemolyzed cells. In order to help free haemoglobin still retained when the incubation period was completed, 3 ml saline solution was added and the tube shaken again. Although the addition of saline decreased the hypotonicity of the solution it did not change the haemolysis produced during incubation, but it helped to complete freeing the haemoglobin retained in the haemolyzed erythrocytes, as was later confirmed by means of spectrophotometric analysis of the stroma. The tubes were then centrifuged for 15 min at 4000 rev/min and the supernatant was analyzed in a spectrophotometer at a wavelength of 540 nm. The highest value of optical density, which always corresponded to an incubation concentration of 0·0 or 0·1 g sodium chloride/100 ml, was taken as 100% haemolysis. The same technique was followed for the rat blood, except that 25 ,Ill of blood was used and the 3 ml saline solution was not added after incubation because no haemoglobin was retained by the haemolyzed red blood cells. Incubation was at 37°C. Results and discussion Curves obtained by plotting percentage haemolysis against saline concentration (Fig. 1) represent the osmotic fragility at these concentrations. There are various ways of expressing these curves quantitatively to compare species or individuals. Traditionally the initial and final haemolysis concentrations have been used (Hogan & Parrot, 1961; Sturkie, 1965). The exact determination of these is difficult, however, because it is necessary to use many saline solutions at
49
Osmotic fragility in birds
100
80
~ III
'iii
>- 50
0 E Q)
'"
I
20
o
---, ---, ---, --,---,---,-- ...•,--...•,---,-- ..•, o·g
0·8
O·7
0·6
0·5
0·4
0·3
0·2
0·1
0
Sodium chloride (g/100 mil
Fig. 1. OsmoticrragOltycurvesIn rat (A), pigeon(B),quaD(C) and hen (0). Bars representthe confidenceintervals(x ± /. s/ VIi), wherex = mean value, t = value of Student's t for probabilitylevelP = 0·05 and degreesof freedom= n - 1, s = standard deviation,n = numberof animalsanalyzed. the concentration at which haemolysis is supposed to begin and end. Other authors (Fourie, 1977) used the mean corpuscular fragility (MCF), which represents the saline concentration at which 50% haemolysis is produced. Neither the MCF nor the initial and final haemolysis can be used to determine the shape of the curve and therefore the partial haemolysis. This may be seen (Fig. 1) with the curves of the pigeon and hen, which both have approximately the same values of salt concentration for initial and final haemolysis, but whose curves differ. The rat and pigeon likewise showed a similar MCF but different curves. Detraglia, Cook, Stasiw & Cerny (1974) and Try (1980), using normal humans and a hospital population, tried to solve the problem by lineation of the osmotic fragility curve of erythrocytes in such a way that this would be expressed by 2 values: the saline concentration that produces 50% haemolysis (Cso), and the slope of the linear curve (P). The application of this lineation method to the experimental data in this study was not adequate in 2 of the species analyzed (pigeon and quail). This is because their curves are asymmetrical as the final part of the haemolysis process is more gradual than the initial, showing a partial deviation which makes the application of the equation as described by Detraglia et al. (1974) for man unsuitable. The variability of the osmotic fragility curves among different species makes it difficult to establish a universal mathematical model. The curve could be partially defined with a few values of haemolysis. The
Table 1. Saline concentrations for 3 values of haemolysls C20, CIO and C80 were obtained by extrapolation of the curves of Fig. I. n = number of animals analyzed.
n Rat Quail Pigeon Hen
19 25
17 17
C20
CIO
C80
0·44
0·39
0·43 0·27
0·32 0·18
(g salt/JOO ml)
0·48
0·43
0·46 0·35
0·39
0·33
utilization of 3 values-C2o, Cso and Cgo (saline concentrations at 20%, 50% and 80% of haemolysis)would be advantageous as representative of 3 important parts of the curve, the lower, middle and upper segments (Table 1). The Cso and the global osmotic fragility values were greater in rats than in pigeons, quails and hens, in that order. The data presented for hens and rats are in agreement with some of the values found in the literature (Hogan & Parrot, 1961 ; Hunter, 1961; Sturkie, 1965). They suggest that osmotic fragility is higher in mammals than in birds. An exception among mammals is the Camelidae; in llamas initial haemolysis is at O· 17 g salt/100 ml (Sturkie, 1965). The fact that the erythrocytes pf both Camelidae and birds are elliptical suggests that the structural characteristics and the elastic properties of the membrane of these erythrocytes may play an important role in osmotic resistance.
50
Viscor & Palomeque
References Detraglia, M., Cook, F. B., Stasiw, D. M. & Cerny, L. C. (1974). Erythrocyte fragility in aging. Biochimica et biophysica acta 345,213-219. Fourie, F. R. (1977). Effects of anticoagulants on the hematocrit, osmolarity and pH of avian blood. Poultry Science 56, 1842-1846. Hogan, A. G. & Parrot, E. M. (1961). In Blood and other body fluids (ed D. S. Dittmer), p. 124. Washington: Federation of American Societies for Experimental Biology. Hunter, F. T. (1961). In Blood and other body fluids (ed D. S. Dittmer), p. 124. Washington: Federation of American Societies for Experimental Biology. March, B. E., Coates, V. & Biely, J. (1966). The effects of oestrogen and androgen on osmotic fragility and fatty
acid composItion of erythrocytes in the chicken. Canadian Journal of Physiology and Pharmacology 44, 379-387. Parpart, A. K., Lorenz, P. B., Parpart, E. R., Gregg, J. P. & Chase, A. M. (1947). The osmotic resistance (fragility) of human red cells. Journal of Clinical Investigation 26, 636-640. Sturkie, P. D. (1965). Avian physiology, pp. 6-7. Ithaca, New York: Cornell University Press. Try, K. (1980). Lineation of the osmotic fragility curve of erythrocytes. Scandinavian Journal of Haematology 24, 157-161.
Eine Methode zur Bestimmung der osmotischen Resistenzkurven bei Vogelerythrozyten G. VISCOR & J. PALOMEQUE Zusammenfassung Die osmotische Erythrozytenresistenz wurde bei 3 Vogelarten (Gallus gallus domesticus, Coturnix coturnixjaponica, Columba /ivia) und der Ratte bestimmt. Die Ergebnisse dieser Arbeit weisen auf eine gro13ereosmotische Resistenz
hin. Vogel, mit elliptischen Erythrozyten, haben eine gro13ere osmotische Resistenz, als Arten mit runden Erythrozyten, wie sie die meisten Sanger aufweisen. (G)
