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Biochemical and physiological changes in the blood of dominant and subordinate CF-1 male mice, Mus musculus
Corz~p.Bishrm Phpol.. Vol. 63A. pp. I93 10 197 rg Pqtmon Press Lid 1979. Printed in Great Britain
BIOCHEMICAL AND PHYSIOLOGICAL CHANGES BLOOD OF DOMINANT AND SUBORDINATE MAL”, MICE, MUS MUSCULUS
IN THE CF-1
LESLIE S. CANE,* FREDERICK A. LUCHETTE and FREDERICK K. HILTON Department of Anatomy, University of Louisville, School of Medicine, Health Sciences Center, P.O. Box 35260, Louisville, KY 40232, U.S.A. (Received II Juiy 1978) Abstract-l. When two previously isolated male CF-1 mice are placed together, they will fight to establish a dominant-subordinate relationship. 2. In response to this behaviorly stressful situation, various physiological and biochemical parameters in the mice are altered. 3. When compared to dominants and controls, subordinates showed decreased hemoglobin concentration and bicarbonate levels, with a concomitant increase in hydrogen ion concentration, negative base excess and blood lactate levels. PC02 and PO, remained unchanged. 4. No significant differences were observed in levels of 2,3-diphosphogiyceric acid in dominants, subordinates or controls. 5. Subordinate individuals were anemic with persistent lactic acidemia reflecting the inability of the liver to catabolize excess lactate.
is a major force in the evolution of mammals. ~ammali~ organization may be based partly on hierarchical social rank consisting of dominant and subordinate relationships formed from intraspecific competition. Agonistic behavior is an important factor in establishing and maintaining social rank in rodent populations, such as the house mouse, Mus musculus (Christian, 1970; Leshner, 1975). In malk mice the ~tab~shment of a dominant-subordinate relationship by fighting results in certain anatomical, physiological and biochemical changes. For example, levels of plasma corticosterone, androgen and gonadotropins were altered in male mice {BranSocial behavior
son, 1973; Chapman et al., 1969; McKinney & Desjardins, 1973). Subordinate animals showed increased
adrenal mass and decreased seminal vesicle weights (Davis & Christian, 1957; Bronson & Eleftheriou, 1964). Hematological changes such as splenomegaly with a concomitant decrease in h~mat~rit and an increase in reticulocyte counts occurred in subordinate individuals (Brassard, 1965; Wehle rr al., 1978). To date there is little information regarding the physiological acid-base status, erythrocyte oxygenation or the condition of oxidative metabolism in dominant and subordinate individuals. Since it is proposed (Wehle et al., 1978) that subordinate individuals undergo anemic or isochemic hypoxic states produced by varying degrees of stress and/or wounding, it is the purpose of this investigation to document such physiolo~~al changes as they may occur in dominant-subordinate relationships between male mice. MATERIALSAND METHODS Male CF-I mice were weaned at 21 days post partum. Individuals were isolated in cages and maintained on Pur* Present address: apartment of Anatomy Loyola University Medical Center. Maywood, Illinois 60153. 193
ina lab chow and water ad ~jbjf~rn during a 12 hr light-12 hr dark cycle. After 3 weeks the animals were matched in pairs according to weight ( 5 2.5 g). Non-paired isolated animals were used as controls. Establishment of dominant-subordinate relationships was obtained by exposing the individuals of a “pair” to each other for two 30min encounter periods daily for up to 21 days. Biochemical test procedures were carried out on encounter days 0, 7, 14 and 21. Animals were removed from the fighting cage, anesthetized with sodium nembutai, and placed back into the cage. As soon as the animals showed response to the anesthesia (usually 3-4min), blood samples were obtained by cardiac puncture using a 1cm3 heparinized syringe. A volume of at least 0.5cm3 of mixed venous blood was used and evaluated in an IL Ultramicro Blood Gas Analyzer. The following measurements were obtained or calculated: pH, PCO,, pOZ, oxygen saturation, bicarbonate and base excess. Total body hemoglobin determination using a modified Drabkin’s technique (Drabkin & Austin, 1935) was employed. Blood lactate was measured using the lactic dehydrogenase technique (Kit No. 826 UV, Sigma Chemical Co., St. Louis, MO). Levels of 2,3-diphosphoglyceric acid were measured by a modified Lowry techniquk (Lowry et al., 1964) (Kit -No. 35 UV, Sirrma Chemical Co., St. Louis. Mot. Ail values were reported as means 1S.E. Means were compared statistically using a multivariant analysis. RESULTS
After I and 2 weeks of fighting, subordinates eihibited a considerable and significant decrease in hemoglobin ievels, 8.39 and 10.39 g/lo0 ml of whole blood, whereas values respectively, control were 13.97 g/t00 ml of blood (Fig. 1). After I, 2 and 3 weeks of encounter, subordinates demonstrated an acidotic state with blood pHs of 7.29, 7.19 and 7.28, respectively. Significant decreases in pH are noted among subordinates compared to control value of pH 7.37 (Fig. 2). Subordinates at 1, 2 and 3 weeks showed decreased bicarbonate levels, as did the dominants at 2 weeks of encounter (Fig. 3). Also, subordinates exhibited significant negative base excesses of 10.6.
LESLIE S. CANE PI u/
194
.
i
2
3
I
123
Fig. 1. Total body hemoglobin in dominant. subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents _C1 S.E.M. and the asterisk denotes significant difference from the control values P 5 0.05.
r
--
ppO1
Fig. 3. Concentration of bicarbonate in the blood of dominant. subordinate and control mice at I, 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P 5 0.05.
L
pr.
WCEW i
__~ Fig. 2. Whole blood pH in dominant, subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
2
3 ___.1_
SLJBORDINATl~ \I EClt.5
f z -.-~---
DNTR
‘--9-.
3
Fig. 4. Base excess of the blood from dominant. subordinate and control mice at 1. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + 1 S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
Changes
*
L-
*
in blood
*
of dominant
1
_!--7Qi
Fig. 5. Concentration of lactic acid in the blood of dominant, subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P 5 0.05. 15.6 and 10.6 mequivjl at weeks I, 2 and 3. respectively. Dominants at 2 weeks also showed a negative base excess of 8.7mequiv/l, compared to a control value of 4.8 mequiv/l (Fig. 4). Subordinate mice showed at least a twofold increase in the blood lactic acid level, compared to.dominant and control values (Fig. 5). Only the dominant animals at 2 weeks of encounter showed a decrease in the PC02 levels; 27.9 mm Hg compared to a control value of 33.6 mm Hg (Fig. 6). There were no significant differences in PO2 (Fig. 7), per cent oxygen saturation (Fig. 8) or 2,3-diphosphoglyceric acid levels (Fig. 9).
and subordinate
mice
195
male house mice continuously exposed to each other in groups of four. In our study, determination of total body hemoglobin revealed that anemia was present in subordinate animals (Fig. I). We believe this anemic condition is associated with repeated wounding of the subordinate and loss of hemoglobin associated with the hemorrhage. An acidotic condition developed in the subordinate mice (Fig. 2) and remained as such for all 3 weeks of encounter. The acidosis occurs when the amount of acid metabolites generated exceeds the body’s buffering capacity. Concomitantly, bicarbonate concentration falls (Fig. 3) since it is consumed in neutralizing the excess acid. With this condition the base excess becomes increasingly negative (Fig. 4). Our biochemical determinations showed that the acidosis in subordinates was caused by significant increases in blood lactate (Fig. 5). Lactic acidosis occurs in a number of conditions such as shock, muscular exercise and severe heart failure, in which tissues fail to receive an adequate oxygen supply. Lactic acid accumulation is formed from the breakdown of carbohydrates in the absence of sufficient oxygen. Compensation for the metabolic acidosis is accomplished both by the lungs and the kidneys. Normally, the acidotic state stimulates the respiratory center in the medulla and causes an increase in both the rate and depth of respiration. Since arterial PC02 would be the best indicator for respiratory compensation, we can only state that our venous PCO, measurements do not reflect hypocapnia (Fig. 6). Further observation of the lactic acidotic state proved that it was not due solely to fighting, with accompanying increase in muscle lactate, since a rest period of 24 hr after fighting failed
r-
;i Q 3b
1
DISCUSSION
Establishment of social rank through intraspecific competition induces a number of biochemical and physiological changes. Hematological changes such as splenomegaly. decreased hematocrit and reticulocytosis. reported by Brassard (1965) and Wehle et al. (1978). were also evident in the subordinate members of the experimental pairs of the present study. The increase in reticulocytes may be due to one or a combination of factors. Both anemic and ischemic hypoxic states produce hemopoietic effects (Kubanek et al., 1968; Fried, 1975). Such effects are reflected by pronounced hematopoiesis in the spleens of the subordinate individuals. We suggest that hypersplenism results primarily from the anemic state, in contrast to Brassard (1965) who proposed that a much greater loss of blood would be required to bring about reticulocytosis and splenomegaly. Further, the mice in his experiments were laboratory reared wild
Fig. 6. PC02 in the blood of dominant, subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents f I S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
LESLIE
196
S.CANE
PC al.
WEEKS
Fig. 7. PO* in the blood of dominant, subordinate and Each bar control mice at 1. 2 and 3 weeks of encounter. of the histogram represents at least nine animals. The vertical line represents f I S.E.M. and the asterisk denotes significant differences from the control values P < 0.05.
ONTFU3L
‘i,
Fig. 8. Per cent oxyhemoglobin in the blood of dominant, subordinate and control mice at I, 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents +_I S.E.M. and the asterisk denotes significant difference from the control values of P i 0.05.
Fig. 9. Levels of 2,3-diphosphoglyceric acid in the blood of dominant, subordinate and control mice at day 2, I, 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
to cause lactate levels to return to control values. Thus, prolonged hyperlactatemia must reflect an imbalance between lactate production and clearance. (Berry, 1967). This suggests that the liver is unable to metabolize excess lactate in the subordinate individuals. Several circumstances in the latter, i.e. anemia, decreased pH and sympathetic discharge may have affected liver function adversely. For example, pronounced anemia and accompanying restricted oxygen supply reduces hepatic metabolism of lactate in the dog (Cain, 1965). Further, small decreases of blood pH and increases of circulating epinepherine have been shown to reduce significantly canine hepatic blood flow (Gelman & Ernst, 1977; Hirsh et al., 1976), which may exacerbate the problem of sufficient oxygen delivery. The liver has alpha receptor sites in the vascular bed and if stimulated could cause vasoconstriction to that organ with resultant decrease in organ perfusion. Since venous PO, and oxyhemoglobin in subordinates did not differ significantly from levels in dominants and controls (Figs 7 and 8). hypoxemia was not a factor. It was theorized that if the oxygencarrying capacity of the blood was altered as in the case of anemia or ischemia, an erythrocyte oxygenation indicator, 2,3_diphosphoglyceric acid (2,3-DPG), would be increased. Because 2,3-DPG preferentially binds to deoxyhemoglobin in a ratio of 1 mol/ tetramer, it lowers the oxygen affinity of hemoglobin and thus facilitates oxygen unloading to tissues. An increased 2,3-DPG level was not produced in our study (Fig. 9). Since blood samples were first taken
Changes
in blood
of dominant
after 48 hr of encounter, 2,3-DPG could have risen then returned to control levels by the time blood was sampled. Miller et al. (1976) showed increased 2,3-DPG levels in rats bled acutely. Their values began to return to control levels 48 hr after initial bleeding. A decrease in pH will shift the oxygen dissociation curve to the right, allowing for further unloading; however, sustained acidotic states as demonstrated in subordinates, could lead to a decrease in the level of 2,3-DPG (Astrup, 1970). It is interesting to note that mice have one of the highest rates of synthesis of 2,3-DPG (Torrance, 1973) and if 2,3-DPG was synthesized as quickly as it is catabolized, essentially no change would occur. Also, since DPG mutase and phosphatase governing the erythrocyte
play an important role in levels of this organic phos-
phate, some dysfunction in either or both of the enzymes might produce the results obtained in our study. Further investigation of enzyme systems and glycolytic intermediates might reveal why no change in 2,3-DPG occurred in the case of anemic hypoxia produced by bleeding in the subordinate animals. In contrast to anemic hypoxia, hypoxic hypoxia has been shown to produce an increase in 2,3-DPG in rats (Martin et al., 1975). The physiological and biochemical observed in subordinate mice strongly
alterations
support the theory that anemic hypoxia is a major factor in contributing to the changes which include decreased total body hemoglobin and lactic acidemia. At present, we are attempting to quantitate the contribution of ischemic hypoxia arising from the hypersympathetic stimulation as proposed by this laboratory (Wehle et al., 1978). It is evident that social interaction accompanied by intraspecific competition and stress produce important biochemical and physiological changes in individuals.
REFERENCES ASTRUPP. (1970) Red cell pH and oxygen affinity of hemoglobin. New Engl. J. Med. 281, 202-203. BERRY M. N. (1967) The liver and lactic acidosis. Proc. R. Sot.
Med.
60, 126&1262.
BRA~SARD A. (1965) Splenic hematopoiesis, morphological, and hematological relationships in grouped male mice (Mus musculus). Rev. can. Biol. 24, 83-100. BRONSN F. H. (1973) Establishment of social rank among grouped male mice: relative effects on circulating FSH. LH and corticosterone. Physiol. Behav. 10, 947-951.
and subordinate
mice
197
BRONSON F. H. & ELEFTH~RIOU B. E. (1964) Chronic physiological effects of fighting mice. (;CU c’ornp. Endocr. 4, 9-14. CAIN S. M.
(1965) Appearance of excess lactate in anesthetized dogs during anemic and hypoxic hypoxia. Am. 1. Physiol. 209, 604610. CHAPMAN V. M.. DESJARDINS C. & BRONSON F. H. (1969) Social rank in male mice and adrenocortical response to open field exposure. Proc. Sot. c\p. RIO/. Mrd. 130, 624-627. CHRISTIAN J. J. (1970) Social subordination. population density and mammalian evolution Scirvw. N. Y. 168. 8490. DAVIS D. E. & CHRISTIAN J. J. (1957) Relation of adrenal weight to social rank of mice. Pro,. So<,. (‘\-p. Biol. Med. 94, 728-73
I.
DRABKIN D. L. & AUSTIN J. H. (1935) Spectrophotometric studies. II Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoplobin. J. hio[. Chrm. 112, 51.-55. FRIED W. (1975) Erythropoietin and the kidney. Nephron 15, 327-349. GELMAN S. & ERNST E. A. (1977) Role of pH, PCO, and O2 content of portal blood in hepatic circulatory autoregulation. Am. J. Physiol. 233, E255-E262. HIRSCH L. J.. AYABE T. & GLICK G. (1976) Direct effects of various cotecholamines on liver circulation in dogs. Am. J. Physiol. 230. 1394-1399. KUBANEK B.. FERRARI L., TYLFR W. S.. HOWARI~ D.. JAY S. & STOHLMAN F. (1968) Regulation of erythropoiesis. XXIII Dissociation between stem cell and erythroid response to hypoxia. Blood 32, 586-596. LE~HNER A. I. (1975) A model of hormones and agonistic behavior. Physiol. Behao. 15, 225. 235. LOWRY 0. H.. PA~~ONNEAU J. V.. HAIISRFRGER F. X. & SCHULZ D. W. (1964) Effects of ischemia on known substrates and cof&ors of the glycolytic pathway in brain. J. biol. Chem. 239. 18-22. MARTIN L. G., CONNORS J. M., MCGRATH J. J. & FREEMAN J. (1975) Altitude induced erythrocytic 2,3-DPG and hemoglobin changes m rats of various ages. J. uppl. Physiol. 39, 258-261. MCKINNEY T. D. & DESJARDINS C. (1973) Postnatal development of testis. fighting behavior, and fertility in house mice. Biol. Reprod. 9, 279-294. MILLER M. E., RORTH M., S~OHLMAN F.. VALERI C. R., LOWRIE G., HOWARD D. & MCGILVRAY N. (1976) The effects of acute bleeding on acid-base balance, erythropoietin (Ep) production and in uiuo P,, in the rat. Br. J. Haemat.
33, 379-385.
TORRANCE J. D. (1973) Erythrocytes, Thromhocytrs. Leukocytes, pp. 161-163. George Thieme, Stuttgart. WEHLE M., HARRIS B. W. & HILTON F. K. (1978) The influence of aggression on erythropoiesis -the hypersympathetic syndrome. Physiol. Behag. in press.
BIOCHEMICAL AND PHYSIOLOGICAL CHANGES BLOOD OF DOMINANT AND SUBORDINATE MAL”, MICE, MUS MUSCULUS
IN THE CF-1
LESLIE S. CANE,* FREDERICK A. LUCHETTE and FREDERICK K. HILTON Department of Anatomy, University of Louisville, School of Medicine, Health Sciences Center, P.O. Box 35260, Louisville, KY 40232, U.S.A. (Received II Juiy 1978) Abstract-l. When two previously isolated male CF-1 mice are placed together, they will fight to establish a dominant-subordinate relationship. 2. In response to this behaviorly stressful situation, various physiological and biochemical parameters in the mice are altered. 3. When compared to dominants and controls, subordinates showed decreased hemoglobin concentration and bicarbonate levels, with a concomitant increase in hydrogen ion concentration, negative base excess and blood lactate levels. PC02 and PO, remained unchanged. 4. No significant differences were observed in levels of 2,3-diphosphogiyceric acid in dominants, subordinates or controls. 5. Subordinate individuals were anemic with persistent lactic acidemia reflecting the inability of the liver to catabolize excess lactate.
is a major force in the evolution of mammals. ~ammali~ organization may be based partly on hierarchical social rank consisting of dominant and subordinate relationships formed from intraspecific competition. Agonistic behavior is an important factor in establishing and maintaining social rank in rodent populations, such as the house mouse, Mus musculus (Christian, 1970; Leshner, 1975). In malk mice the ~tab~shment of a dominant-subordinate relationship by fighting results in certain anatomical, physiological and biochemical changes. For example, levels of plasma corticosterone, androgen and gonadotropins were altered in male mice {BranSocial behavior
son, 1973; Chapman et al., 1969; McKinney & Desjardins, 1973). Subordinate animals showed increased
adrenal mass and decreased seminal vesicle weights (Davis & Christian, 1957; Bronson & Eleftheriou, 1964). Hematological changes such as splenomegaly with a concomitant decrease in h~mat~rit and an increase in reticulocyte counts occurred in subordinate individuals (Brassard, 1965; Wehle rr al., 1978). To date there is little information regarding the physiological acid-base status, erythrocyte oxygenation or the condition of oxidative metabolism in dominant and subordinate individuals. Since it is proposed (Wehle et al., 1978) that subordinate individuals undergo anemic or isochemic hypoxic states produced by varying degrees of stress and/or wounding, it is the purpose of this investigation to document such physiolo~~al changes as they may occur in dominant-subordinate relationships between male mice. MATERIALSAND METHODS Male CF-I mice were weaned at 21 days post partum. Individuals were isolated in cages and maintained on Pur* Present address: apartment of Anatomy Loyola University Medical Center. Maywood, Illinois 60153. 193
ina lab chow and water ad ~jbjf~rn during a 12 hr light-12 hr dark cycle. After 3 weeks the animals were matched in pairs according to weight ( 5 2.5 g). Non-paired isolated animals were used as controls. Establishment of dominant-subordinate relationships was obtained by exposing the individuals of a “pair” to each other for two 30min encounter periods daily for up to 21 days. Biochemical test procedures were carried out on encounter days 0, 7, 14 and 21. Animals were removed from the fighting cage, anesthetized with sodium nembutai, and placed back into the cage. As soon as the animals showed response to the anesthesia (usually 3-4min), blood samples were obtained by cardiac puncture using a 1cm3 heparinized syringe. A volume of at least 0.5cm3 of mixed venous blood was used and evaluated in an IL Ultramicro Blood Gas Analyzer. The following measurements were obtained or calculated: pH, PCO,, pOZ, oxygen saturation, bicarbonate and base excess. Total body hemoglobin determination using a modified Drabkin’s technique (Drabkin & Austin, 1935) was employed. Blood lactate was measured using the lactic dehydrogenase technique (Kit No. 826 UV, Sigma Chemical Co., St. Louis, MO). Levels of 2,3-diphosphoglyceric acid were measured by a modified Lowry techniquk (Lowry et al., 1964) (Kit -No. 35 UV, Sirrma Chemical Co., St. Louis. Mot. Ail values were reported as means 1S.E. Means were compared statistically using a multivariant analysis. RESULTS
After I and 2 weeks of fighting, subordinates eihibited a considerable and significant decrease in hemoglobin ievels, 8.39 and 10.39 g/lo0 ml of whole blood, whereas values respectively, control were 13.97 g/t00 ml of blood (Fig. 1). After I, 2 and 3 weeks of encounter, subordinates demonstrated an acidotic state with blood pHs of 7.29, 7.19 and 7.28, respectively. Significant decreases in pH are noted among subordinates compared to control value of pH 7.37 (Fig. 2). Subordinates at 1, 2 and 3 weeks showed decreased bicarbonate levels, as did the dominants at 2 weeks of encounter (Fig. 3). Also, subordinates exhibited significant negative base excesses of 10.6.
LESLIE S. CANE PI u/
194
.
i
2
3
I
123
Fig. 1. Total body hemoglobin in dominant. subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents _C1 S.E.M. and the asterisk denotes significant difference from the control values P 5 0.05.
r
--
ppO1
Fig. 3. Concentration of bicarbonate in the blood of dominant. subordinate and control mice at I, 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P 5 0.05.
L
pr.
WCEW i
__~ Fig. 2. Whole blood pH in dominant, subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
2
3 ___.1_
SLJBORDINATl~ \I EClt.5
f z -.-~---
DNTR
‘--9-.
3
Fig. 4. Base excess of the blood from dominant. subordinate and control mice at 1. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + 1 S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
Changes
*
L-
*
in blood
*
of dominant
1
_!--7Qi
Fig. 5. Concentration of lactic acid in the blood of dominant, subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P 5 0.05. 15.6 and 10.6 mequivjl at weeks I, 2 and 3. respectively. Dominants at 2 weeks also showed a negative base excess of 8.7mequiv/l, compared to a control value of 4.8 mequiv/l (Fig. 4). Subordinate mice showed at least a twofold increase in the blood lactic acid level, compared to.dominant and control values (Fig. 5). Only the dominant animals at 2 weeks of encounter showed a decrease in the PC02 levels; 27.9 mm Hg compared to a control value of 33.6 mm Hg (Fig. 6). There were no significant differences in PO2 (Fig. 7), per cent oxygen saturation (Fig. 8) or 2,3-diphosphoglyceric acid levels (Fig. 9).
and subordinate
mice
195
male house mice continuously exposed to each other in groups of four. In our study, determination of total body hemoglobin revealed that anemia was present in subordinate animals (Fig. I). We believe this anemic condition is associated with repeated wounding of the subordinate and loss of hemoglobin associated with the hemorrhage. An acidotic condition developed in the subordinate mice (Fig. 2) and remained as such for all 3 weeks of encounter. The acidosis occurs when the amount of acid metabolites generated exceeds the body’s buffering capacity. Concomitantly, bicarbonate concentration falls (Fig. 3) since it is consumed in neutralizing the excess acid. With this condition the base excess becomes increasingly negative (Fig. 4). Our biochemical determinations showed that the acidosis in subordinates was caused by significant increases in blood lactate (Fig. 5). Lactic acidosis occurs in a number of conditions such as shock, muscular exercise and severe heart failure, in which tissues fail to receive an adequate oxygen supply. Lactic acid accumulation is formed from the breakdown of carbohydrates in the absence of sufficient oxygen. Compensation for the metabolic acidosis is accomplished both by the lungs and the kidneys. Normally, the acidotic state stimulates the respiratory center in the medulla and causes an increase in both the rate and depth of respiration. Since arterial PC02 would be the best indicator for respiratory compensation, we can only state that our venous PCO, measurements do not reflect hypocapnia (Fig. 6). Further observation of the lactic acidotic state proved that it was not due solely to fighting, with accompanying increase in muscle lactate, since a rest period of 24 hr after fighting failed
r-
;i Q 3b
1
DISCUSSION
Establishment of social rank through intraspecific competition induces a number of biochemical and physiological changes. Hematological changes such as splenomegaly. decreased hematocrit and reticulocytosis. reported by Brassard (1965) and Wehle et al. (1978). were also evident in the subordinate members of the experimental pairs of the present study. The increase in reticulocytes may be due to one or a combination of factors. Both anemic and ischemic hypoxic states produce hemopoietic effects (Kubanek et al., 1968; Fried, 1975). Such effects are reflected by pronounced hematopoiesis in the spleens of the subordinate individuals. We suggest that hypersplenism results primarily from the anemic state, in contrast to Brassard (1965) who proposed that a much greater loss of blood would be required to bring about reticulocytosis and splenomegaly. Further, the mice in his experiments were laboratory reared wild
Fig. 6. PC02 in the blood of dominant, subordinate and control mice at I. 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents f I S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
LESLIE
196
S.CANE
PC al.
WEEKS
Fig. 7. PO* in the blood of dominant, subordinate and Each bar control mice at 1. 2 and 3 weeks of encounter. of the histogram represents at least nine animals. The vertical line represents f I S.E.M. and the asterisk denotes significant differences from the control values P < 0.05.
ONTFU3L
‘i,
Fig. 8. Per cent oxyhemoglobin in the blood of dominant, subordinate and control mice at I, 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents +_I S.E.M. and the asterisk denotes significant difference from the control values of P i 0.05.
Fig. 9. Levels of 2,3-diphosphoglyceric acid in the blood of dominant, subordinate and control mice at day 2, I, 2 and 3 weeks of encounter. Each bar of the histogram represents at least nine animals. The vertical line represents + I S.E.M. and the asterisk denotes significant difference from the control values P I 0.05.
to cause lactate levels to return to control values. Thus, prolonged hyperlactatemia must reflect an imbalance between lactate production and clearance. (Berry, 1967). This suggests that the liver is unable to metabolize excess lactate in the subordinate individuals. Several circumstances in the latter, i.e. anemia, decreased pH and sympathetic discharge may have affected liver function adversely. For example, pronounced anemia and accompanying restricted oxygen supply reduces hepatic metabolism of lactate in the dog (Cain, 1965). Further, small decreases of blood pH and increases of circulating epinepherine have been shown to reduce significantly canine hepatic blood flow (Gelman & Ernst, 1977; Hirsh et al., 1976), which may exacerbate the problem of sufficient oxygen delivery. The liver has alpha receptor sites in the vascular bed and if stimulated could cause vasoconstriction to that organ with resultant decrease in organ perfusion. Since venous PO, and oxyhemoglobin in subordinates did not differ significantly from levels in dominants and controls (Figs 7 and 8). hypoxemia was not a factor. It was theorized that if the oxygencarrying capacity of the blood was altered as in the case of anemia or ischemia, an erythrocyte oxygenation indicator, 2,3_diphosphoglyceric acid (2,3-DPG), would be increased. Because 2,3-DPG preferentially binds to deoxyhemoglobin in a ratio of 1 mol/ tetramer, it lowers the oxygen affinity of hemoglobin and thus facilitates oxygen unloading to tissues. An increased 2,3-DPG level was not produced in our study (Fig. 9). Since blood samples were first taken
Changes
in blood
of dominant
after 48 hr of encounter, 2,3-DPG could have risen then returned to control levels by the time blood was sampled. Miller et al. (1976) showed increased 2,3-DPG levels in rats bled acutely. Their values began to return to control levels 48 hr after initial bleeding. A decrease in pH will shift the oxygen dissociation curve to the right, allowing for further unloading; however, sustained acidotic states as demonstrated in subordinates, could lead to a decrease in the level of 2,3-DPG (Astrup, 1970). It is interesting to note that mice have one of the highest rates of synthesis of 2,3-DPG (Torrance, 1973) and if 2,3-DPG was synthesized as quickly as it is catabolized, essentially no change would occur. Also, since DPG mutase and phosphatase governing the erythrocyte
play an important role in levels of this organic phos-
phate, some dysfunction in either or both of the enzymes might produce the results obtained in our study. Further investigation of enzyme systems and glycolytic intermediates might reveal why no change in 2,3-DPG occurred in the case of anemic hypoxia produced by bleeding in the subordinate animals. In contrast to anemic hypoxia, hypoxic hypoxia has been shown to produce an increase in 2,3-DPG in rats (Martin et al., 1975). The physiological and biochemical observed in subordinate mice strongly
alterations
support the theory that anemic hypoxia is a major factor in contributing to the changes which include decreased total body hemoglobin and lactic acidemia. At present, we are attempting to quantitate the contribution of ischemic hypoxia arising from the hypersympathetic stimulation as proposed by this laboratory (Wehle et al., 1978). It is evident that social interaction accompanied by intraspecific competition and stress produce important biochemical and physiological changes in individuals.
REFERENCES ASTRUPP. (1970) Red cell pH and oxygen affinity of hemoglobin. New Engl. J. Med. 281, 202-203. BERRY M. N. (1967) The liver and lactic acidosis. Proc. R. Sot.
Med.
60, 126&1262.
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