Comp. Biochem.Physiol.Vol. 103B,No. 4, pp. 909-911, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
STUDIES ON THE BLOOD OF FALLOW DEER (DAMA DAMA) AND RED DEER (CERVUS ELAPHUS)" HAEMATOLOGY, RED CELL ENZYMES, METABOLIC INTERMEDIATES AND GLYCOLYTIC RATES N. S. AGAt and I. R. GODWIN Department of Physiology, University of New England, Annidale, N.S.W. 2351, Australia (Tel. 067-73-2485; Fax 067-73-3234)
(Received 14 May 1992; accepted 17 June 1992) AImract--l. Blood samples were obtained from fallow deer (Dama dama) and red deer (Cerv~ elaphu~). Basic haematology, red cell enzymes, and metabofic intermediates and the glycolytic rate of the red cells incubated with different substrates were measured. 2. The major findings were (i) the activity of glucose phosphate isomerase was notably high in the red blood cells of the red deer; (ii) red deer cells also utilized adenosine more efficiently than those of fallow deer and (iii) red cells of both species utilized galactose more efficiently than other species of ruminants.
INTRODUCTION The red blood cells of many species of deer exhibit sickiing (Chapman, 1977). Unlike humans where sickling is associated with an abnormal haemoglobin ( H b ) - - H b S, some normal H b types cause sickling in deer while others do not (Kitchen et al., 1964). Because of this and the fact that deer red cells may serve as a natural reservoir for the haemoparasite Anaplasma marginale, transmissible between deer and cattle (Jain, 1986), extensive studies have been carried out on the haematology of deer (see review by Chapman, 1977; Jain, 1986). Although studies on the haemoglobin polymorphism and its structure (Kitchen et al., 1964, 1967, 1968), and the oxygen transport (Ochiai and Enoki, 1975), have been undertaken in deer, reports on red cell metabofism are lacking. Because of this together with a continuous growing interest in the farming and management of deer, we have now examined several aspects of red cell metabolism in two species of deer; the fallow deer (Dama dama) and the red deer (Cervus elaphus) including the activities of 10 enzymes, concentrations of adenosine triphosphate (ATP), 2,3-diphosphoglycerate (DPG) and reduced glutathione (GSH) and the glycolytic rate of the red cells incubated with 8 different substrates. The results of these studies are reported in the present communication.
mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). GSH levels in whole blood were measured by the method of lkutler (1984). Red cell GSH values were then calculated using the haematocrit. ATP and DPG concentrations were analysed in TCA extracts by the methods of Godwin et al. (1983). The activities of red cell hexokinase (HK), glucose phosphate isomerase (GPI), glyceraldehyde phosphate dehydrogenase (GAPD), phosphoglycerate kinase (PGK), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), NADH-methaemoglobin reductase (NADH-MR) and NADPH-MR were measured. The methods used for the preparation of haemolysates and assay systems were those described by Beutler (1984). Assays were carried out at 37°C in cuvettes with a I em fight path and a final volume of I ml. Changes in absorbance were recorded mdng a Beckman Du-64 spectrophotometer. The method used to assess the glycolytic rate was essentially that of Bethlenfalvay et al. (1984), with slight modification. Briefly, the red cells were washed three times in cold 145 mM NaCI and suspensions with haematocrits of approximately 15% were prepared in the following buffer; NaCI 120 mM, Na2HPO4 30raM, KCI 5mM, MgCI2 1.2mM, Tris, 50mM, pH 7.4. To the red cell suspensions were added one of the following sutntrates: glucose, galactme, fructose, mannose, dihydroxyacetone (DHA), glyceraldehyde (GAD) (each to give a final concentration of 10/~mol/l), adenosine and inosine (in a final concentration of 5/tmol/l). The red cell susp~siom were incuhatcd in a shaking water bath at 37°C. Aliquots were taken at 0, 1 and 2 hr for the measurement of lactate (Godwin eta/., 1983).
MATERIALS AND METHODS Four fallow deer (maintained at the University farm) and four red deer (kept at a local commercial farm) were used. All animalo were adult female. About 10 ml of blood was obtained by jugular venepunctme and collected in hepexinised tubes. Immediately after collection, 0.5 mi blood was added to 0.5 ml of ico-cold 8% triohloroacetic acid (TCA), mixed and kept on ice, along with the remaining blood. Haematologleal parameten, determined on a Coulter model T-890 haematology analyser (Coulter Eleetron~ Ltd., Hem., Enlllaad) inO_w4A~lpeeked cell volmne (PCV), haemoglobin concentration (Hb conc), red cell number (RBC no), ~m
io31s,-x
RESULTS AND DISCUSSION The most comprehensive study of the haematology of the fallow deer appears to have been undertaken by Chapman et aL (1982) who examined 42 fallow deer with respect to age, season and sex. Our values given in Table 1 for fallow deer are in good agreement with those reported by Chapman et al. (1982). Although PCV, Hb, RBC and MCV were similar in the two types of deer, the values for MCH and MCHC were significantly (P < 0.05) greater in the
910
N . S . AOAg and I. R. C.¢ODW1N
Table 1, Haematologicai parameters in fallow and red deer (mean ± SEM, N = number of animals) Parameters Packed cell volume (%) Haemoglobin (g/dL) Red blood cell (number × 106//tl) Mean cell volume (fL) Mean cell haemoglobin (pg) Mean cell haemoslobin concentration (g/dL)
Fallow deer N ffi 4
Red deer N =4
47.1±0.9 17.2 ± 0.3 9.91 ± 0.10 47.5 ± 0.63 17.4 ± 0.2
45.6±2.81 18.9 ± 0.97 9.67 ± 0.51 47.0 ± 0.46 19.6 ± 0.41 *
36.6 ± 0.8
41.8 + 1.12"
*P < 0,05.
red deer compared to the fallow deer. All animals studied were female adult, although the exact age of the animals could not be ascertained. However age does not appear to affect these parameters at least in the fallow deer (Chapman et al., 1982). Overall our results for the two species of deer are similar to those reported in the literature (Chapman, 1977; Jain, 1986). Values for red cell GSH and ATP (Table 2) are also within the range of the majority of mammalian species (Agar and Board, 1983; Harvey, 1989). The concentration of red cell DPG was very low in both species (Table 2). This agrees with other reports in the literature (Bunn et al., 1974; Ochiai and Enoki, 1975; Kay, 1977). It is relevant to mention here that Bunn et al. (1974) classified mammals into two groups; the first group, which contains the majority of mammalian species tested, have abundant red cell DPG and have a haemoglobin of high oxygen atfmity that is markedly lowered in the presence of DPG. The second group which includes sheep, goats, cattle, deer and cats is characterised by low levels of red cell DPG and haemoglobins of low oxygen affinity which do not react strongly with DPG. The lack of interaction of these haemoglobins with DPG has been explained on the basis of the primary structure of the haemoglobin molecule. Kay (1977) extended these observations and reported that the low DPG group tend to have moderate Bohr effects which are probably the result of structural differences in haemoglobin rather than differences in arterial Pcov pH or body size. The values for the enzyme activities in the red blood cells (Table 3) indicate that of the 10 enzymes under investigation, the activities of most were similar in the two species. The differences in HK and NADHMR were significant (P < 0.05). However, the red blood cells of red deer had GPI activity (72.6 + 5.87 IU/gHb) which was more than five times higher than that in the red cells of fallow deer (14.16 + 0.38 IU/ glib) (P
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
STUDIES ON THE BLOOD OF FALLOW DEER (DAMA DAMA) AND RED DEER (CERVUS ELAPHUS)" HAEMATOLOGY, RED CELL ENZYMES, METABOLIC INTERMEDIATES AND GLYCOLYTIC RATES N. S. AGAt and I. R. GODWIN Department of Physiology, University of New England, Annidale, N.S.W. 2351, Australia (Tel. 067-73-2485; Fax 067-73-3234)
(Received 14 May 1992; accepted 17 June 1992) AImract--l. Blood samples were obtained from fallow deer (Dama dama) and red deer (Cerv~ elaphu~). Basic haematology, red cell enzymes, and metabofic intermediates and the glycolytic rate of the red cells incubated with different substrates were measured. 2. The major findings were (i) the activity of glucose phosphate isomerase was notably high in the red blood cells of the red deer; (ii) red deer cells also utilized adenosine more efficiently than those of fallow deer and (iii) red cells of both species utilized galactose more efficiently than other species of ruminants.
INTRODUCTION The red blood cells of many species of deer exhibit sickiing (Chapman, 1977). Unlike humans where sickling is associated with an abnormal haemoglobin ( H b ) - - H b S, some normal H b types cause sickling in deer while others do not (Kitchen et al., 1964). Because of this and the fact that deer red cells may serve as a natural reservoir for the haemoparasite Anaplasma marginale, transmissible between deer and cattle (Jain, 1986), extensive studies have been carried out on the haematology of deer (see review by Chapman, 1977; Jain, 1986). Although studies on the haemoglobin polymorphism and its structure (Kitchen et al., 1964, 1967, 1968), and the oxygen transport (Ochiai and Enoki, 1975), have been undertaken in deer, reports on red cell metabofism are lacking. Because of this together with a continuous growing interest in the farming and management of deer, we have now examined several aspects of red cell metabolism in two species of deer; the fallow deer (Dama dama) and the red deer (Cervus elaphus) including the activities of 10 enzymes, concentrations of adenosine triphosphate (ATP), 2,3-diphosphoglycerate (DPG) and reduced glutathione (GSH) and the glycolytic rate of the red cells incubated with 8 different substrates. The results of these studies are reported in the present communication.
mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). GSH levels in whole blood were measured by the method of lkutler (1984). Red cell GSH values were then calculated using the haematocrit. ATP and DPG concentrations were analysed in TCA extracts by the methods of Godwin et al. (1983). The activities of red cell hexokinase (HK), glucose phosphate isomerase (GPI), glyceraldehyde phosphate dehydrogenase (GAPD), phosphoglycerate kinase (PGK), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), NADH-methaemoglobin reductase (NADH-MR) and NADPH-MR were measured. The methods used for the preparation of haemolysates and assay systems were those described by Beutler (1984). Assays were carried out at 37°C in cuvettes with a I em fight path and a final volume of I ml. Changes in absorbance were recorded mdng a Beckman Du-64 spectrophotometer. The method used to assess the glycolytic rate was essentially that of Bethlenfalvay et al. (1984), with slight modification. Briefly, the red cells were washed three times in cold 145 mM NaCI and suspensions with haematocrits of approximately 15% were prepared in the following buffer; NaCI 120 mM, Na2HPO4 30raM, KCI 5mM, MgCI2 1.2mM, Tris, 50mM, pH 7.4. To the red cell suspensions were added one of the following sutntrates: glucose, galactme, fructose, mannose, dihydroxyacetone (DHA), glyceraldehyde (GAD) (each to give a final concentration of 10/~mol/l), adenosine and inosine (in a final concentration of 5/tmol/l). The red cell susp~siom were incuhatcd in a shaking water bath at 37°C. Aliquots were taken at 0, 1 and 2 hr for the measurement of lactate (Godwin eta/., 1983).
MATERIALS AND METHODS Four fallow deer (maintained at the University farm) and four red deer (kept at a local commercial farm) were used. All animalo were adult female. About 10 ml of blood was obtained by jugular venepunctme and collected in hepexinised tubes. Immediately after collection, 0.5 mi blood was added to 0.5 ml of ico-cold 8% triohloroacetic acid (TCA), mixed and kept on ice, along with the remaining blood. Haematologleal parameten, determined on a Coulter model T-890 haematology analyser (Coulter Eleetron~ Ltd., Hem., Enlllaad) inO_w4A~lpeeked cell volmne (PCV), haemoglobin concentration (Hb conc), red cell number (RBC no), ~m
io31s,-x
RESULTS AND DISCUSSION The most comprehensive study of the haematology of the fallow deer appears to have been undertaken by Chapman et aL (1982) who examined 42 fallow deer with respect to age, season and sex. Our values given in Table 1 for fallow deer are in good agreement with those reported by Chapman et al. (1982). Although PCV, Hb, RBC and MCV were similar in the two types of deer, the values for MCH and MCHC were significantly (P < 0.05) greater in the
910
N . S . AOAg and I. R. C.¢ODW1N
Table 1, Haematologicai parameters in fallow and red deer (mean ± SEM, N = number of animals) Parameters Packed cell volume (%) Haemoglobin (g/dL) Red blood cell (number × 106//tl) Mean cell volume (fL) Mean cell haemoglobin (pg) Mean cell haemoslobin concentration (g/dL)
Fallow deer N ffi 4
Red deer N =4
47.1±0.9 17.2 ± 0.3 9.91 ± 0.10 47.5 ± 0.63 17.4 ± 0.2
45.6±2.81 18.9 ± 0.97 9.67 ± 0.51 47.0 ± 0.46 19.6 ± 0.41 *
36.6 ± 0.8
41.8 + 1.12"
*P < 0,05.
red deer compared to the fallow deer. All animals studied were female adult, although the exact age of the animals could not be ascertained. However age does not appear to affect these parameters at least in the fallow deer (Chapman et al., 1982). Overall our results for the two species of deer are similar to those reported in the literature (Chapman, 1977; Jain, 1986). Values for red cell GSH and ATP (Table 2) are also within the range of the majority of mammalian species (Agar and Board, 1983; Harvey, 1989). The concentration of red cell DPG was very low in both species (Table 2). This agrees with other reports in the literature (Bunn et al., 1974; Ochiai and Enoki, 1975; Kay, 1977). It is relevant to mention here that Bunn et al. (1974) classified mammals into two groups; the first group, which contains the majority of mammalian species tested, have abundant red cell DPG and have a haemoglobin of high oxygen atfmity that is markedly lowered in the presence of DPG. The second group which includes sheep, goats, cattle, deer and cats is characterised by low levels of red cell DPG and haemoglobins of low oxygen affinity which do not react strongly with DPG. The lack of interaction of these haemoglobins with DPG has been explained on the basis of the primary structure of the haemoglobin molecule. Kay (1977) extended these observations and reported that the low DPG group tend to have moderate Bohr effects which are probably the result of structural differences in haemoglobin rather than differences in arterial Pcov pH or body size. The values for the enzyme activities in the red blood cells (Table 3) indicate that of the 10 enzymes under investigation, the activities of most were similar in the two species. The differences in HK and NADHMR were significant (P < 0.05). However, the red blood cells of red deer had GPI activity (72.6 + 5.87 IU/gHb) which was more than five times higher than that in the red cells of fallow deer (14.16 + 0.38 IU/ glib) (P
