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Intravenous hypertonic saline injections and drinking in domestic fowls
Physiology &Behavior, Vol. 42, pp. 307-312. Copyright ©Pergamon Press pie, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
Intravenous Hypertonic Saline Injections and Drinking in Domestic Fowls M. R. Y E O M A N S
A N D C. J. S A V O R Y t
*Department o f Psychology, University of Edinburgh, 7 George Square, Edinburgh, EH8 9JZ tA.F.R.C. Institute for Grassland and Animal Production (Poultry Division) Roslin, Midlothian, EH25 9PS, Scotland R e c e i v e d 15 O c t o b e r 1987 YEOMANS, M. R. AND C. J. SAVORY. Intravenous hypertonic saline injections and drinking in domestic fowls. PHYSIOL BEHAV 42(4) 307-312, 1988.--Fowls were given intravenous (IV) injections of hypertonic solutions of NaC1, and subsequent water intakes were recorded. All concentrations of hypertonic NaC1 increased drinking in the 90 min after injection, compared with control treatments. Increments in drinking in this time agreed closely with calculated amounts required to restore normal osmolality. In further experiments, delaying access to water by periods of 60-360 min after injection failed to reduce drinking elicited by hypertonic NaC1. Injections of 2.0 M NaC1 caused increases in plasma osmolality and sodium concentration which were maintained throughout 360 rain water deprivation, and caused prolonged reductions in hematocrit and plasma protein concentrations. These results demonstrate that cellular dehydration is a potent thirst stimulus in fowls, and imply that fowls do not reduce hyperosmolality by excretion of salt when water is unavailable. Cellular-dehydration
Domestic fowl
Drinking
Osmolality
Osmoregulation
Sodium
Thirst
NaC1 in rats, guinea pigs and gerbils further suggested that the amount drunk reflected differences in renal capacity for excretion of salt [1]. Reports of drinking responses of birds to osmotic stimuli have been restricted to pigeons, which drink sufficient water to restore osmolality when injected intraperitoneally (IP; [10]) or IV with hypertonic NaCl [9, 12, 23]. This suggests a relative inability to reduce hyperosmolality by salt excretion like that found in iguanas. However, Hawkins and Corbit [10] found that the volume drunk by pigeons in response to hypertonic NaCI injections was reduced by 57% when access to water was delayed by 4 hours, and Thornton [23] reported that 60% of a similar salt load had been excreted in this time. Thus, pigeons do appear to reduce osmotic thirst by salt excretion if given time enough to do so. The different responses of rats, iguanas and pigeons to hypertonic NaCl injections suggest that there may also be differences in the importance of cellular dehydration in control of normal drinking in these species. With animals that can reduce hyperosmolality by excretion, it is unlikely that cellular dehydration would be as important as in species that rely on drinking to maintain osmotic balance. This suggestion is based on evidence from very few species, however, so the purpose of this study was to obtain further information on osmotic control of drinking in another bird, the domestic fowl.
I N T R A V E N O U S (IV) injections of hypertonic NaC1 solutions have been used widely in investigations of osmotic thirst. Such solutions induce changes in plasma osmolality without causing major changes in ionic balance, since sodium and its associated anions account for some 90% of normal plasma osmolality. Hence, the drinking induced by such injections is likely to be similar to that seen when osmolality is increased during normal water loss, such as during water deprivation, and studies like these therefore provide information on the possible role of osmotic thirst in control o f normal drinking. Amounts of water drunk by animals in response to hypertonic NaC1 injections depend on their ability to reduce the imposed osmotic imbalance by excretion of salt [7]. F o r example, it was found that amounts of water drunk by common iguanas (Iguana iguana) in response to IV hypertonic NaC1 injections matched closely the volumes required to restore normal osmolality [8]. This suggested that iguanas could not reduce the imposed hyperosmolality by excretion of salt, and this was confirmed by analyses of plasma composition after hypertonic NaC1 injections. By contrast, rats drink only about 75% of their osmotic requirement when given IV injections of hypertonic NaCl [4,5], and if access to water is delayed after injection, the drinking response is reduced further [4]. This apparent under-drinking was attributed to excretion of salt by the kidney, since nephrectomised rats were found to drink the full amounts needed to restore osmolality [5], even when access to water was delayed by 240 min. Comparisons of drinking responses to hypertonic
EXPERIMENT 1 The purpose of this experiment was to establish drinking
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YEOMANS AND SAVORY
responses of fowls to IV injections of hypertonic NaCI, and to investigate any effects on feeding.
TABLE 1 MEAN WATER AND FOOD INTAKES FOLLOWING IV INJECTIONS OF DIFFERENT CONCENTRATIONS OF NaCI
Subjects Subjects were 8 hens of a medium-hybrid laying strain (Rhode Island Red x Light Sussex), aged 12-18 weeks and weighing 1.1-1.6 kg at the time of testing. They were housed and tested in single cages (30x45x45 cm), in a room where lights were on for 14 hr each day (0700-2100 hr) and ambient temperature was maintained at 20-25°C. Food (standard layers mash, for composition see [19]) and fresh tap water were available ad lib except where otherwise indicated. Procedure IV injections (2.5 ml/kg by wing vein, at ca. 6 ml/min) of solutions of NaCI were given on consecutive days according to a balanced Latin-square design. All testing was carried out between 1000-1300 hr. Solutions tested were 0.15 (isotonic), 0.5, 1.0 and 2.0 M NaCI, prepared freshly each day from distilled water and analytical grade NaC1. Water and food intakes were recorded to the nearest 0.1 g, at 15, 30, 60, 90 and 120 min after injection. Water loss due to evaporation (which never exceeded 1.5 ml/hour) was estimated by weighing a spare (control) drinker which contained the same amount of water as the other (test) drinkers at the start of testing. Treatment means were compared by analyses of variance, and intakes in hypertonic conditions were compared with 0.15 M NaCI (control) by t-test. Dose relationships were assessed by multiple regression analyses. Observed drinking responses were compared with those calculated to restore normal osmolality (calculated using the formulae of Corbit [4]).
Time After Injection (min)
0.5
1.0
2.0
SED
5.2 "b 1.6a 5.0 ~b 1.8"b 3.2
9.6 be 4.3 a 4.5 ab 5.4 b 3.5
12.0c 12.2b 8.1 b 2.8 ab 3.5
1.5 2.4 2.0 1.5 1.4
1.6 1.3 2.7 3.3 2.4
1.3 1.5 2.9 2.7 3.1
0.9 1.1 3.0 2.1 3.1
0.6 0.7 0.7 1.1 0.9
Food Intake (g/kg) 0-15 15-30 30-60 60-90 90-120
1.7 1.5 2.0 2.9 2.3
SED is the standard error of the difference between means, with 14 degrees of freedom. Within each row, data values with the same superscript do not differ significantly. 40 /
3O
RESULTS
~J
~ z
The observation in Experiment 1 that fowls drank precisely the volume of water required to restore normal osmolality after hypertonic NaCI injections suggests that they did not reduce the imposed osmotic thirst during the 90 min
0.15
Water Intake (ml/kg) 0-15 2.4a 15-30 1.5a 30-60 1.5a 60-90 1.2" 90-120 4.8
Compared with isotonic NaCI, injections of hypertonic NaCI increased water intake at all the concentrations tested (Table 1). Although latencies to drink were not recorded, birds generally appeared to start drinking soon after return to their cages. Most of this increased drinking occurred in the first 15 rain after injection, although there were also significant differences in the periods 15-30 and 30--60 min. Since drinking with 1.0 M NaCI was still raised slightly 60-90 min after injection, cumulative water intakes from 0-90 min were used to assess dose-relationships, and to compare observed increases in drinking with corresponding calculated amounts required to restore normal osmolality (Fig. 1). There was a significant linear relationship between the molarity of injected NaC1 and water intake (p<0.01), so drinking responses were directly proportional to the imposed osmotic imbalance. The difference between observed and expected linear slopes was not significant (by t-test), and fowls drank precisely what was required to restore osmotic balance. Food intakes were unaffected by NaCI injections at any stage (Table 1), and so the drinking elicited by these treatments cannot be attributed to any indirect effect of altered feeding. EXPERIMENT 2
Molarity of Injected Saline
20
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K I~o
2.'o
MOLARITY OF INJECTED NACL FIG. 1. Mean (n=8) water intakes in 90 rain after IV injections of different concentrations of NaC1. The solid line is the linear relationship fitted by multiple regression analysis and the broken line indicates the predicted regulatory requirement for water. SED is the standard error of the difference between means.
response time. One way of testing this suggestion is to increase the time available to excrete salt, and this experiment did this by examining the effect of delaying access to water by one hour on dose-dependent drinking elicited by hypertonic NaC1 injections. Procedure Ten hens, housed and maintained as in Experiment 1,
HYPERTONIC SALINE AND THIRST
309
40 /
~-~ 30
f
~
2.0M NaC1
0.15M NaC1 15
oo
1;o
z J0
MOLA.RITY OF INJECTED NACL FIG. 2. Mean (n= 10) water intake in 120 min after 60 min without water, with different concentrations of NaC1 injected either at the start or end of water deprivation. The solid lines are the linear relationships from multiple regression analyses, and the broken line represents the predicted regulatory requirement for water. SED is the standard error of the difference between means.
TABLE 2 M E A N F O O D I N T A K E S F O L L O W I N G IV I N J E C T I O N S O F D I F F E R E N T C O N C E N T R A T I O N S O F NaCI G I V E N AT T H E START OR
END OF 60 MIN WITHOUTWATER Time Relative to Water Access (rain)
Molarity of Injected Saline 0.15
0.5
1.0
1.5
2.0
SED
5.2 5.1 5.1
5.1 4.3 4.9
0.7 0.9 0.6
Injected at End of Water Deprivation -60-0 0-60 60-120
5.1 5.7 5.1
4.7 7,7 4.1
5.1 5.9 5.2
Injected at Start of Water Deprivation -60-0
0-60 60-120
5.5 ~
4.0 ~
4.5 ~
3.0 b
2.2 b
0.7
5.7 4.6
6.1 5.0
6.0 4.0
6.7 4.1
6.0 3.9
0.9 0.6
SED is the standard error of the difference between means, with 14 degrees of freedom. Within rows, values with the same superscript are not significantly different.
were each injected (2 ml/kg IV) with 0.15, 0.5, 1.0, 1.5 and 2.0 M NaC1, either at the start or end of a 60 min period of water deprivation. All birds received all treatments, according to a balanced design, and treatments were given on consecutive days, excluding weekends. Water intake was measured for the 120 min after its return, since in Experiment 1 all drinking elicited by hypertonic saline injections was completed within this time. Food intake was measured in the 60 rain without water and in the 120 min after its return. RESULTS As in Experiment 1, all concentrations of hypertonic
'o
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zi0
36o
TIME B E T T E E N INJECTION AND WATER ACCESS (MIN)
FIG. 3. Mean (n=8) water imake in the 120 min after IV injections of 2.0 M (broken line) and 0.15 M (solid line) NaC1 with 0--360 rain between injection and access to water. Values given are means_+SEM (vertical bars). NaCI elicited increased drinking (Fig. 2), and significant linear relationships (p<0.001 by t-test) between molarity and water intakes were found both with immediate access to water and when access was delayed by 60 rain. This delay had no effect on subsequent drinking (no significant difference between intercepts), and the interaction of dose with delay was not significant. Moreover, the slopes of the 2 regression lines did not differ from each other, and neither differed from the slope of the drinking response calculated to restore osmolality. These results confirm that fowls compensate precisely to remove the imposed osmotic imbalance, and that they do not appear to reduce osmotic thirst by excretion of salt, at least during the time allowed in this experiment. Food intake was reduced significantly (p <0.01) during the 60 min period of water deprivation following injections of 1.5 and 2.0 M NaC1, compared with the control (0.15 M) treatment and 0.5 and 1.0 M NaCI (Table 2). Presumably this was an adaptive response to prevent further cellular dehydration [15], as seen also in rats [17]. Food intake was not affected by NaCI treatment at any stage when access to water was immediate. EXPERIMENT 3 This experiment examined the effect of longer delays between injection of NaCl and access to water, to test whether the apparent lack of reduction in osmotic thirst found in Experiment 2 with a 60 min delay was due to insufficient time for the excess NaC1 to be excreted. Procedure Eight hens, housed as before, were each injected (2 ml/kg, IV) with 0.15 or 2.0 M NaC1, with either immediate access to water or access delayed by 120, 240 or 360 min, according to a Latin-square design. Water intake was measured in the 120 min after its return, and food was removed 60 rain before injection and returned at the end of testing.
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YEOMANS AND SAVORY
A
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314
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306
298
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130
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30 10 25
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0 Time
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120
240
360
Post-ln|ectlon (rain)
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0
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120
240
360
Time Pollt-lnlectlon (rain)
FIG. 4. Mean (n=8) plasma osmolality (A), plasma sodium concentration (B), hematocrit (C) and plasma protein concentration (D) immediately and during 360 min without water, after injections of 2.0 M NaCI (broken lines) ~and 0.15 M NaCI (solid lines). All values are means_+SEM (vertical bars). See text for statistical analyses.
RESULTS
The difference between amounts drunk in the 120 min after injections of 0.15 and 2.0 M NaCI was not affected by delaying access to water by up to 360 min (Fig. 3). Both the dose of NaCI and time of access had significant effects on the response (p<0.001 by analysis of variance), but the interaction between them was not significant. These results indicate that even delays as long as 360 min do not reduce osmotic thirst produced by hypertonic NaCI injections. EXPERIMENT 4
This experiment examined changes in blood parameters immediately after injections of hypertonic NaCI and during 360 min subsequent water deprivation. If the inability to excrete salt implied in Experiments 1-3 is correct, then increases in plasma osmolality and plasma sodium concentration produced by such injections should remain unaltered in the absence of drinking. Procedure
Two groups of 8 birds, housed as before, were injected (2 ml/kg IV) with either 0.15 or 2.0 M NaCI. Water was removed immediately after the injection, and blood samples (1 ml) were withdrawn into heparinised syringes from alternate wing veins, directly before and 0, 60, 120, 240 and 360 min after injection. Duplicate readings of hematocrit were made on a micro-hematocrit centrifuge (Hawkesley), and the remaining blood was centrifuged (5 min at ca. 2000×g) and plasma collected. Duplicate readings of plasma osmolality were made using a freezing-point osmometer (Advanced Digimatic, Advanced Instruments). Plasma protein levels were
assayed using a commercial assay kit (Bio-Rad) and plasma sodium levels read on an atomic absorption spectrophotometer (Varian Model AA-875) using plasma diluted 1:5000 with deionised water. RESULTS
Plasma osmolality and sodium concentration both increased significantly (p<0.001) immediately after injection of 2.0 ml/kg 2.0 M NaCI, compared with pre-injection values, and both remained raised throughout the succeeding 360 min without water (Fig. 4). Injection of 0.15 M NaC1 did not alter either plasma osmolality or sodium concentration. Plasma osmolality increased significantly further during the 360 min post-injection with both 2.0 M and 0.15 M NaC1. This may have been due to dehydration, since water was unavailable during this period. The fact that the increases in both measures induced by injections of 2.0 M NaCI were not reduced during the 360 min water deprivation supports the earlier suggestions that fowls do not reduce hyperosmolality by excretion of NaC1. Significant reductions in both hematocrit (p<0.001) and plasma protein concentration (p<0.01) occurred immediately after injections of 2.0 M NaCI. Hematocrit then rose to an intermediate level in the following 60 min and remained there for the remaining 300 min (Fig. 4). Plasma protein rose throughout the post-injection period, and did not differ significantly from the control (0.15 M NaC1) level after 360 min. No significant changes in either measure were seen after injections of 0.15 M NaCI. DISCUSSION
Fowls responded to IV hypertonic NaCI injections by
HYPERTONIC S A L I N E AND THIRST
311
drinking precisely the volume of water required for cellular rehydration. The accuracy with which this rehydration was achieved at least equals that previously reported in iguanas [8] and pigeons [10,12]. However, whereas delaying access to water after injecting hypertonic NaCI reduced subsequent drinking in pigeons [10], no such reduction was found here with fowls even when access was delayed by 360 min. This implies that fowls had either not reduced the imposed hyperosmolality by excretion of NaC1, or that any such excretion failed to alter the subsequent drinking response. The observation that the increases in plasma osmolality and sodium concentration produced by hypertonic NaCI injections were maintained during 360 min water deprivation supports the former suggestion. This result is perhaps surprising since fowls can produce a slightly hypertonic urine [14], and since IV injections of hypertonic NaCI have been shown to induce natriuresis in fowls [2,18]. Although excretion of NaC1 was not measured here, it is possible that fowls did increase such excretion by the kidney after hypertonic NaCI injections, but that their ability to reabsorb NaCI in the cloaca [20,21] prevented any change in plasma osmolality or sodium concentration. Indeed, when fowls were transferred between low and high salt diets in a previous study, changes in salt excretion in faeces lagged 1.5 days behind changes in salt intake, and this was attributed mainly to changes in cloacal salt resorption [22]. Although pigeons responded to IV infusions of NaCI by excreting 60% of this load within 4 hours, no further excretion could be detected and the remaining (40%) sodium could not be accounted for [23]. This led to the suggestion that pigeons, and possibly other birds, may have some form of sodium store [24,25]. The apparent inability of fowls to excrete NaCI here tends to support this suggestion. Furthermore, the fact that plasma osmolality rose during the period without water after hypertonic NaCI injection, while plasma sodium concentration did not (Fig. 4), suggests that sodium ions could have been removed from the extraceUular fluid and been replaced by another osmotically active ion. The increases in hematocrit and plasma protein concentrations in the hour after hypertonic NaCI injections imply a decrease in plasma volume during this time, compared with immediately post-injection (Fig. 4), presumably through redistribution of extracellular fluid. How such redistribution might be controlled is unclear, although fowls do have a mammal-like juxtaglomerular complex [3] and so should have vascular baroreceptors which may detect the large, initial increase in plasma volume resulting from hypertonic NaC1 injections. It has been suggested that pigeons also possess extra-vascular baroreceptors [11], that may be involved in control of distribution of extracellular fluid. The consistent drinking responses elicited by hypertonic NaC1 in fowls suggest that any increase in osmolality caused by normal water loss, such as during water deprivation [13],
should also be a potent stimulus to drink. Indeed, the apparent inability to reduce hyperosmolality by salt excretion implies that fowls must rely on drinking to maintain osmotic balance, at least in the short term. This contrasts with rats, which moderate the amount of water required to restore osmotic balance by excretion of salt [4], and therefore rely less on drinking to osmoregulate. Pigeons appear to be between fowls and rats, since they drink sufficient to rehydrate when access to water is immediate after hypertonic NaCI administration [10, 12, 23], but drink less when access to water is delayed [10]. In general, responses of fowls to hypertonic NaCI injections are most like those of iguanas, which also rely entirely on drinking to osmoregulate [12]. However, whereas fowls complete most of the drinking elicited by hypertonic NaCI injections within 15 min of the injection, iguanas take up to 360 rain to complete the response. The linear relationship between the molarity of injected NaCI and resulting drinking (Figs. 1 and 2) implies that even slight increases in osmolality above isotonicity should result in drinking. The smallest hypertonic dose used here (2 ml/kg of 0.5 M NaC1, Experiment 2) would have increased plasma osmolality by less than 1% (calculated according to the formula of Fitzsimons [6]). Studies on osmotic thirst thresholds in pigeons found that increases of 2.3% were necessary to elicit drinking [23]. However, in such studies animals are usually infused slowly IV, with no food during testing [6,28], and so spontaneous drinking is minimal. Here, however, food was usually available during testing, and the drinking elicited by small doses of NaCl could have resulted from birds taking earlier or longer spontaneous bouts of drinking, even though the osmotic stimulus may not have been sufficient to initiate drinking in a threshold-test situation. Even so, the fact that small hypertonic NaCl doses did increase drinking reliably indicates that small changes in osmolality are detected, probably by central osmoreceptors like those described in pigeons [25,26] and mammals [16,27]. To conclude, these experiments demonstrate that the cellular dehydration produced by IV hypertonic NaCl injections is a potent dipsogenic stimulus in fowls, and that the resulting drinking response is precisely that required to restore isotonicity. Fowls appear to be unable to reduce the need to drink by reducing hyperosmolality when access to water is delayed. The results suggest that increased osmolality from normal water loss should also be a potent dipsogenic stimulus, and hence that osmotic thirst may have an important role in control of normal drinking. ACKNOWLEDGEMENTS I would like to thank the Director of the A.F.R.C. Edinburgh Research Centre for providing the facilities for these experiments, Miss Elaine Seawright for technical assistance and the BEMB for financial support through a research studentship.
REFERENCES
1. Almli, C. R. and C. S. Weiss. Behavioural and physiological responses to dipsogens: A comparative analysis. Physiol Behav 14: 633-641, 1975. 2, Bailey, J. R. and H. Nishimura. Renal responses of fowls to hypertonic saline infusion into the renal portal system. Am J Physiol 246: R624-R632, 1984. 3. Christensen, J. A., I. Morild, E. Mikeler and A. Bohle. Juxtaglomerular apparatus in the domestic fowl (Gallus domesticus). Kidney Int 22: Suppl 12, 24-29, 1982.
4. Corbit, J. D. Osmotic thirst: theoretical and experimental analysis. J Comp Physiol Psychol 67: 3-14, 1969. 5. Fitzsimons, J. T. Drinking by nephrectomised rats injected with various substances. J Physiol 155: 565-579, 1961. 6. Fitzsimons, J. T. The effects of slow infusions of hypertonic solutions on drinking and drinking thresholds in rats. J Physiol 167: 344-354, 1963. 7. Fitzsimons, J. T. The physiology of thirst and sodium appetite. Monogr Physiol Soc 35: 96-127, 1979.
312 8. Fitzsimons, J. T. and S. Kaufman. Cellular and extracellular dehydration, and angiotensin as stimuli to drinking in the common iguana, Iguana iguana. J Physiol 265: 443-463, 1977. 9. Fitzsimons, J. T., M. Massi and S. N. Thornton. The effects of changes in osmolality and sodium concentration on angiotensin-induced drinking and excretion in the pigeon. J Physiol 330: 1-15, 1982. 10. Hawkins, R. C. and J. D. Corbit. Drinking in response to cellular dehydration in the pigeon. J Comp Physiol Psychol 89: 265267, 1973. 11. Kaufman, S., H-P. Kaesermann and G. Peters. The mechanism of drinking induced by parentera] hyperoncotic solutions in the pigeon and the rat. J Physiol 301: 91-99, 1980. 12. Kaufman, S. and G. Peters. Regulatory drinking in the pigeon Columba livia. Am J Physiol 239: R219-R225, 1980. 13. Koike, T. I., L. R. Pryor and H. L. Neldon. Plasma volume and electrolytes during progressive water deprivation in chickens (Gallus domesticus). Comp Biochem Physiol [A] 74: 83-87, 1983. 14. Korr, I. M. The osmotic function of the chicken kidney. J Cell Cornp Physiol 13: 175-193, 1939. 15. McFarland, D. J. and P. W. Wright. Water conservation by inhibition of food intake. Physiol Behav 4: 95-99, 1969. 16. McKinley, M. J., D. A. Denton and R. S. Weisiager. Sensors for antidiuresis and thirst: osmoreceptors or C.S.F. sodium detectors? Brain Res 141: 89-103, 1978. 17. Oatley, K. and F. M. Toates. Osmotic inhibition of eating as a subtractive process. J Comp Physiol Psycho182: 268-277, 1973. 18. Ruth, F. R. and M. R. Hughes, The effects of hypertonic sodium chloride injection on body water distribution in ducks (Anas platyrhychos), gulls (Larus glaucescens) and roosters (Gallus domesticus). Comp Biochem Physiol [A] 52: 21-28, 1975.
YEOMANS AND SAVORY 19. Savory, C. J. and J. P. Hodgkiss. Influence of vagotomy in domestic fowls on feeding activity, food passage, digestibility, and satiety effects of two peptides. Physiol Behav 33: 937-944, 1984. 20. Skadhauge, E. In vivo perfusion studies of the cloaca] water and electrolyte reabsorption in the fowl (Gallus gallus domesticus). Comp Biochem Physiol 23: 483-501, 1967. 21. Skadhauge, E. Osmoregulation in Birds. Berlin: SpringerVedag, 1981. 22. Skadhauge, E., D. H. Thomas, A. Chadwick and M. Jallageas. Time course of adaptation to low and high NaC1 diets in the domestic fowl: effects on electrolyte excretion and on plasma hormone levels. Pflugers Arch 396: 302-307, 1983. 23. Thornton, S. N. Drinking and renal responses to peripherally administered osmotic stimuli in the pigeon (Columba livia). J Physiol 351: 501-515, 1984. 24. Thornton, S. N. A central Na ÷ receptor and its influence on osmotic and Angiotensin II induced drinking in the pigeon. Columba livia. J Physiol (Paris) 79: 505-510, 1984. 25. Thornton, S. N. The influence of central infusions on drinking due to peripheral osmotic stimuli in the pigeon (Columba livia). Physiol Behav 36: 229-233, 1986. 26. Thornton, S. N. Drinking in the pigeon (Columba livia) in response to water deprivation and the influence of intracerebroventricular infusions of NaC1 or sucrose solutions. Physiol Behav 38: 719-724, 1986. 27. Thrasher, T. N., C. J. Brown, L. C. Keil and D. J. Ramsay. Thirst and vasopressin release in the dog: an osmoreceptor or sodium receptor mechanism. Am J Physiol 238: R333-R339, 1980. 28. Wolf, A. V. Osmometric analysis of thirst in man and dog. Am J Physiol 161: 75-86, 1950.
0031-9384/88 $3.00 + .00
Intravenous Hypertonic Saline Injections and Drinking in Domestic Fowls M. R. Y E O M A N S
A N D C. J. S A V O R Y t
*Department o f Psychology, University of Edinburgh, 7 George Square, Edinburgh, EH8 9JZ tA.F.R.C. Institute for Grassland and Animal Production (Poultry Division) Roslin, Midlothian, EH25 9PS, Scotland R e c e i v e d 15 O c t o b e r 1987 YEOMANS, M. R. AND C. J. SAVORY. Intravenous hypertonic saline injections and drinking in domestic fowls. PHYSIOL BEHAV 42(4) 307-312, 1988.--Fowls were given intravenous (IV) injections of hypertonic solutions of NaC1, and subsequent water intakes were recorded. All concentrations of hypertonic NaC1 increased drinking in the 90 min after injection, compared with control treatments. Increments in drinking in this time agreed closely with calculated amounts required to restore normal osmolality. In further experiments, delaying access to water by periods of 60-360 min after injection failed to reduce drinking elicited by hypertonic NaC1. Injections of 2.0 M NaC1 caused increases in plasma osmolality and sodium concentration which were maintained throughout 360 rain water deprivation, and caused prolonged reductions in hematocrit and plasma protein concentrations. These results demonstrate that cellular dehydration is a potent thirst stimulus in fowls, and imply that fowls do not reduce hyperosmolality by excretion of salt when water is unavailable. Cellular-dehydration
Domestic fowl
Drinking
Osmolality
Osmoregulation
Sodium
Thirst
NaC1 in rats, guinea pigs and gerbils further suggested that the amount drunk reflected differences in renal capacity for excretion of salt [1]. Reports of drinking responses of birds to osmotic stimuli have been restricted to pigeons, which drink sufficient water to restore osmolality when injected intraperitoneally (IP; [10]) or IV with hypertonic NaCl [9, 12, 23]. This suggests a relative inability to reduce hyperosmolality by salt excretion like that found in iguanas. However, Hawkins and Corbit [10] found that the volume drunk by pigeons in response to hypertonic NaCI injections was reduced by 57% when access to water was delayed by 4 hours, and Thornton [23] reported that 60% of a similar salt load had been excreted in this time. Thus, pigeons do appear to reduce osmotic thirst by salt excretion if given time enough to do so. The different responses of rats, iguanas and pigeons to hypertonic NaCl injections suggest that there may also be differences in the importance of cellular dehydration in control of normal drinking in these species. With animals that can reduce hyperosmolality by excretion, it is unlikely that cellular dehydration would be as important as in species that rely on drinking to maintain osmotic balance. This suggestion is based on evidence from very few species, however, so the purpose of this study was to obtain further information on osmotic control of drinking in another bird, the domestic fowl.
I N T R A V E N O U S (IV) injections of hypertonic NaC1 solutions have been used widely in investigations of osmotic thirst. Such solutions induce changes in plasma osmolality without causing major changes in ionic balance, since sodium and its associated anions account for some 90% of normal plasma osmolality. Hence, the drinking induced by such injections is likely to be similar to that seen when osmolality is increased during normal water loss, such as during water deprivation, and studies like these therefore provide information on the possible role of osmotic thirst in control o f normal drinking. Amounts of water drunk by animals in response to hypertonic NaC1 injections depend on their ability to reduce the imposed osmotic imbalance by excretion of salt [7]. F o r example, it was found that amounts of water drunk by common iguanas (Iguana iguana) in response to IV hypertonic NaC1 injections matched closely the volumes required to restore normal osmolality [8]. This suggested that iguanas could not reduce the imposed hyperosmolality by excretion of salt, and this was confirmed by analyses of plasma composition after hypertonic NaC1 injections. By contrast, rats drink only about 75% of their osmotic requirement when given IV injections of hypertonic NaCl [4,5], and if access to water is delayed after injection, the drinking response is reduced further [4]. This apparent under-drinking was attributed to excretion of salt by the kidney, since nephrectomised rats were found to drink the full amounts needed to restore osmolality [5], even when access to water was delayed by 240 min. Comparisons of drinking responses to hypertonic
EXPERIMENT 1 The purpose of this experiment was to establish drinking
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responses of fowls to IV injections of hypertonic NaCI, and to investigate any effects on feeding.
TABLE 1 MEAN WATER AND FOOD INTAKES FOLLOWING IV INJECTIONS OF DIFFERENT CONCENTRATIONS OF NaCI
Subjects Subjects were 8 hens of a medium-hybrid laying strain (Rhode Island Red x Light Sussex), aged 12-18 weeks and weighing 1.1-1.6 kg at the time of testing. They were housed and tested in single cages (30x45x45 cm), in a room where lights were on for 14 hr each day (0700-2100 hr) and ambient temperature was maintained at 20-25°C. Food (standard layers mash, for composition see [19]) and fresh tap water were available ad lib except where otherwise indicated. Procedure IV injections (2.5 ml/kg by wing vein, at ca. 6 ml/min) of solutions of NaCI were given on consecutive days according to a balanced Latin-square design. All testing was carried out between 1000-1300 hr. Solutions tested were 0.15 (isotonic), 0.5, 1.0 and 2.0 M NaCI, prepared freshly each day from distilled water and analytical grade NaC1. Water and food intakes were recorded to the nearest 0.1 g, at 15, 30, 60, 90 and 120 min after injection. Water loss due to evaporation (which never exceeded 1.5 ml/hour) was estimated by weighing a spare (control) drinker which contained the same amount of water as the other (test) drinkers at the start of testing. Treatment means were compared by analyses of variance, and intakes in hypertonic conditions were compared with 0.15 M NaCI (control) by t-test. Dose relationships were assessed by multiple regression analyses. Observed drinking responses were compared with those calculated to restore normal osmolality (calculated using the formulae of Corbit [4]).
Time After Injection (min)
0.5
1.0
2.0
SED
5.2 "b 1.6a 5.0 ~b 1.8"b 3.2
9.6 be 4.3 a 4.5 ab 5.4 b 3.5
12.0c 12.2b 8.1 b 2.8 ab 3.5
1.5 2.4 2.0 1.5 1.4
1.6 1.3 2.7 3.3 2.4
1.3 1.5 2.9 2.7 3.1
0.9 1.1 3.0 2.1 3.1
0.6 0.7 0.7 1.1 0.9
Food Intake (g/kg) 0-15 15-30 30-60 60-90 90-120
1.7 1.5 2.0 2.9 2.3
SED is the standard error of the difference between means, with 14 degrees of freedom. Within each row, data values with the same superscript do not differ significantly. 40 /
3O
RESULTS
~J
~ z
The observation in Experiment 1 that fowls drank precisely the volume of water required to restore normal osmolality after hypertonic NaCI injections suggests that they did not reduce the imposed osmotic thirst during the 90 min
0.15
Water Intake (ml/kg) 0-15 2.4a 15-30 1.5a 30-60 1.5a 60-90 1.2" 90-120 4.8
Compared with isotonic NaCI, injections of hypertonic NaCI increased water intake at all the concentrations tested (Table 1). Although latencies to drink were not recorded, birds generally appeared to start drinking soon after return to their cages. Most of this increased drinking occurred in the first 15 rain after injection, although there were also significant differences in the periods 15-30 and 30--60 min. Since drinking with 1.0 M NaCI was still raised slightly 60-90 min after injection, cumulative water intakes from 0-90 min were used to assess dose-relationships, and to compare observed increases in drinking with corresponding calculated amounts required to restore normal osmolality (Fig. 1). There was a significant linear relationship between the molarity of injected NaC1 and water intake (p<0.01), so drinking responses were directly proportional to the imposed osmotic imbalance. The difference between observed and expected linear slopes was not significant (by t-test), and fowls drank precisely what was required to restore osmotic balance. Food intakes were unaffected by NaCI injections at any stage (Table 1), and so the drinking elicited by these treatments cannot be attributed to any indirect effect of altered feeding. EXPERIMENT 2
Molarity of Injected Saline
20
o.o
K I~o
2.'o
MOLARITY OF INJECTED NACL FIG. 1. Mean (n=8) water intakes in 90 rain after IV injections of different concentrations of NaC1. The solid line is the linear relationship fitted by multiple regression analysis and the broken line indicates the predicted regulatory requirement for water. SED is the standard error of the difference between means.
response time. One way of testing this suggestion is to increase the time available to excrete salt, and this experiment did this by examining the effect of delaying access to water by one hour on dose-dependent drinking elicited by hypertonic NaC1 injections. Procedure Ten hens, housed and maintained as in Experiment 1,
HYPERTONIC SALINE AND THIRST
309
40 /
~-~ 30
f
~
2.0M NaC1
0.15M NaC1 15
oo
1;o
z J0
MOLA.RITY OF INJECTED NACL FIG. 2. Mean (n= 10) water intake in 120 min after 60 min without water, with different concentrations of NaC1 injected either at the start or end of water deprivation. The solid lines are the linear relationships from multiple regression analyses, and the broken line represents the predicted regulatory requirement for water. SED is the standard error of the difference between means.
TABLE 2 M E A N F O O D I N T A K E S F O L L O W I N G IV I N J E C T I O N S O F D I F F E R E N T C O N C E N T R A T I O N S O F NaCI G I V E N AT T H E START OR
END OF 60 MIN WITHOUTWATER Time Relative to Water Access (rain)
Molarity of Injected Saline 0.15
0.5
1.0
1.5
2.0
SED
5.2 5.1 5.1
5.1 4.3 4.9
0.7 0.9 0.6
Injected at End of Water Deprivation -60-0 0-60 60-120
5.1 5.7 5.1
4.7 7,7 4.1
5.1 5.9 5.2
Injected at Start of Water Deprivation -60-0
0-60 60-120
5.5 ~
4.0 ~
4.5 ~
3.0 b
2.2 b
0.7
5.7 4.6
6.1 5.0
6.0 4.0
6.7 4.1
6.0 3.9
0.9 0.6
SED is the standard error of the difference between means, with 14 degrees of freedom. Within rows, values with the same superscript are not significantly different.
were each injected (2 ml/kg IV) with 0.15, 0.5, 1.0, 1.5 and 2.0 M NaC1, either at the start or end of a 60 min period of water deprivation. All birds received all treatments, according to a balanced design, and treatments were given on consecutive days, excluding weekends. Water intake was measured for the 120 min after its return, since in Experiment 1 all drinking elicited by hypertonic saline injections was completed within this time. Food intake was measured in the 60 rain without water and in the 120 min after its return. RESULTS As in Experiment 1, all concentrations of hypertonic
'o
lzo
zi0
36o
TIME B E T T E E N INJECTION AND WATER ACCESS (MIN)
FIG. 3. Mean (n=8) water imake in the 120 min after IV injections of 2.0 M (broken line) and 0.15 M (solid line) NaC1 with 0--360 rain between injection and access to water. Values given are means_+SEM (vertical bars). NaCI elicited increased drinking (Fig. 2), and significant linear relationships (p<0.001 by t-test) between molarity and water intakes were found both with immediate access to water and when access was delayed by 60 rain. This delay had no effect on subsequent drinking (no significant difference between intercepts), and the interaction of dose with delay was not significant. Moreover, the slopes of the 2 regression lines did not differ from each other, and neither differed from the slope of the drinking response calculated to restore osmolality. These results confirm that fowls compensate precisely to remove the imposed osmotic imbalance, and that they do not appear to reduce osmotic thirst by excretion of salt, at least during the time allowed in this experiment. Food intake was reduced significantly (p <0.01) during the 60 min period of water deprivation following injections of 1.5 and 2.0 M NaC1, compared with the control (0.15 M) treatment and 0.5 and 1.0 M NaCI (Table 2). Presumably this was an adaptive response to prevent further cellular dehydration [15], as seen also in rats [17]. Food intake was not affected by NaCI treatment at any stage when access to water was immediate. EXPERIMENT 3 This experiment examined the effect of longer delays between injection of NaCl and access to water, to test whether the apparent lack of reduction in osmotic thirst found in Experiment 2 with a 60 min delay was due to insufficient time for the excess NaC1 to be excreted. Procedure Eight hens, housed as before, were each injected (2 ml/kg, IV) with 0.15 or 2.0 M NaC1, with either immediate access to water or access delayed by 120, 240 or 360 min, according to a Latin-square design. Water intake was measured in the 120 min after its return, and food was removed 60 rain before injection and returned at the end of testing.
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YEOMANS AND SAVORY
A
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314
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306
298
0
130
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I
I
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I
I
I
I
C
30 10 25
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-
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20 ----.I
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0 Time
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I
120
240
360
Post-ln|ectlon (rain)
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0
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I
I
|
120
240
360
Time Pollt-lnlectlon (rain)
FIG. 4. Mean (n=8) plasma osmolality (A), plasma sodium concentration (B), hematocrit (C) and plasma protein concentration (D) immediately and during 360 min without water, after injections of 2.0 M NaCI (broken lines) ~and 0.15 M NaCI (solid lines). All values are means_+SEM (vertical bars). See text for statistical analyses.
RESULTS
The difference between amounts drunk in the 120 min after injections of 0.15 and 2.0 M NaCI was not affected by delaying access to water by up to 360 min (Fig. 3). Both the dose of NaCI and time of access had significant effects on the response (p<0.001 by analysis of variance), but the interaction between them was not significant. These results indicate that even delays as long as 360 min do not reduce osmotic thirst produced by hypertonic NaCI injections. EXPERIMENT 4
This experiment examined changes in blood parameters immediately after injections of hypertonic NaCI and during 360 min subsequent water deprivation. If the inability to excrete salt implied in Experiments 1-3 is correct, then increases in plasma osmolality and plasma sodium concentration produced by such injections should remain unaltered in the absence of drinking. Procedure
Two groups of 8 birds, housed as before, were injected (2 ml/kg IV) with either 0.15 or 2.0 M NaCI. Water was removed immediately after the injection, and blood samples (1 ml) were withdrawn into heparinised syringes from alternate wing veins, directly before and 0, 60, 120, 240 and 360 min after injection. Duplicate readings of hematocrit were made on a micro-hematocrit centrifuge (Hawkesley), and the remaining blood was centrifuged (5 min at ca. 2000×g) and plasma collected. Duplicate readings of plasma osmolality were made using a freezing-point osmometer (Advanced Digimatic, Advanced Instruments). Plasma protein levels were
assayed using a commercial assay kit (Bio-Rad) and plasma sodium levels read on an atomic absorption spectrophotometer (Varian Model AA-875) using plasma diluted 1:5000 with deionised water. RESULTS
Plasma osmolality and sodium concentration both increased significantly (p<0.001) immediately after injection of 2.0 ml/kg 2.0 M NaCI, compared with pre-injection values, and both remained raised throughout the succeeding 360 min without water (Fig. 4). Injection of 0.15 M NaC1 did not alter either plasma osmolality or sodium concentration. Plasma osmolality increased significantly further during the 360 min post-injection with both 2.0 M and 0.15 M NaC1. This may have been due to dehydration, since water was unavailable during this period. The fact that the increases in both measures induced by injections of 2.0 M NaCI were not reduced during the 360 min water deprivation supports the earlier suggestions that fowls do not reduce hyperosmolality by excretion of NaC1. Significant reductions in both hematocrit (p<0.001) and plasma protein concentration (p<0.01) occurred immediately after injections of 2.0 M NaCI. Hematocrit then rose to an intermediate level in the following 60 min and remained there for the remaining 300 min (Fig. 4). Plasma protein rose throughout the post-injection period, and did not differ significantly from the control (0.15 M NaC1) level after 360 min. No significant changes in either measure were seen after injections of 0.15 M NaCI. DISCUSSION
Fowls responded to IV hypertonic NaCI injections by
HYPERTONIC S A L I N E AND THIRST
311
drinking precisely the volume of water required for cellular rehydration. The accuracy with which this rehydration was achieved at least equals that previously reported in iguanas [8] and pigeons [10,12]. However, whereas delaying access to water after injecting hypertonic NaCI reduced subsequent drinking in pigeons [10], no such reduction was found here with fowls even when access was delayed by 360 min. This implies that fowls had either not reduced the imposed hyperosmolality by excretion of NaC1, or that any such excretion failed to alter the subsequent drinking response. The observation that the increases in plasma osmolality and sodium concentration produced by hypertonic NaCI injections were maintained during 360 min water deprivation supports the former suggestion. This result is perhaps surprising since fowls can produce a slightly hypertonic urine [14], and since IV injections of hypertonic NaCI have been shown to induce natriuresis in fowls [2,18]. Although excretion of NaC1 was not measured here, it is possible that fowls did increase such excretion by the kidney after hypertonic NaCI injections, but that their ability to reabsorb NaCI in the cloaca [20,21] prevented any change in plasma osmolality or sodium concentration. Indeed, when fowls were transferred between low and high salt diets in a previous study, changes in salt excretion in faeces lagged 1.5 days behind changes in salt intake, and this was attributed mainly to changes in cloacal salt resorption [22]. Although pigeons responded to IV infusions of NaCI by excreting 60% of this load within 4 hours, no further excretion could be detected and the remaining (40%) sodium could not be accounted for [23]. This led to the suggestion that pigeons, and possibly other birds, may have some form of sodium store [24,25]. The apparent inability of fowls to excrete NaCI here tends to support this suggestion. Furthermore, the fact that plasma osmolality rose during the period without water after hypertonic NaCI injection, while plasma sodium concentration did not (Fig. 4), suggests that sodium ions could have been removed from the extraceUular fluid and been replaced by another osmotically active ion. The increases in hematocrit and plasma protein concentrations in the hour after hypertonic NaCI injections imply a decrease in plasma volume during this time, compared with immediately post-injection (Fig. 4), presumably through redistribution of extracellular fluid. How such redistribution might be controlled is unclear, although fowls do have a mammal-like juxtaglomerular complex [3] and so should have vascular baroreceptors which may detect the large, initial increase in plasma volume resulting from hypertonic NaC1 injections. It has been suggested that pigeons also possess extra-vascular baroreceptors [11], that may be involved in control of distribution of extracellular fluid. The consistent drinking responses elicited by hypertonic NaC1 in fowls suggest that any increase in osmolality caused by normal water loss, such as during water deprivation [13],
should also be a potent stimulus to drink. Indeed, the apparent inability to reduce hyperosmolality by salt excretion implies that fowls must rely on drinking to maintain osmotic balance, at least in the short term. This contrasts with rats, which moderate the amount of water required to restore osmotic balance by excretion of salt [4], and therefore rely less on drinking to osmoregulate. Pigeons appear to be between fowls and rats, since they drink sufficient to rehydrate when access to water is immediate after hypertonic NaCI administration [10, 12, 23], but drink less when access to water is delayed [10]. In general, responses of fowls to hypertonic NaCI injections are most like those of iguanas, which also rely entirely on drinking to osmoregulate [12]. However, whereas fowls complete most of the drinking elicited by hypertonic NaCI injections within 15 min of the injection, iguanas take up to 360 rain to complete the response. The linear relationship between the molarity of injected NaCI and resulting drinking (Figs. 1 and 2) implies that even slight increases in osmolality above isotonicity should result in drinking. The smallest hypertonic dose used here (2 ml/kg of 0.5 M NaC1, Experiment 2) would have increased plasma osmolality by less than 1% (calculated according to the formula of Fitzsimons [6]). Studies on osmotic thirst thresholds in pigeons found that increases of 2.3% were necessary to elicit drinking [23]. However, in such studies animals are usually infused slowly IV, with no food during testing [6,28], and so spontaneous drinking is minimal. Here, however, food was usually available during testing, and the drinking elicited by small doses of NaCl could have resulted from birds taking earlier or longer spontaneous bouts of drinking, even though the osmotic stimulus may not have been sufficient to initiate drinking in a threshold-test situation. Even so, the fact that small hypertonic NaCl doses did increase drinking reliably indicates that small changes in osmolality are detected, probably by central osmoreceptors like those described in pigeons [25,26] and mammals [16,27]. To conclude, these experiments demonstrate that the cellular dehydration produced by IV hypertonic NaCl injections is a potent dipsogenic stimulus in fowls, and that the resulting drinking response is precisely that required to restore isotonicity. Fowls appear to be unable to reduce the need to drink by reducing hyperosmolality when access to water is delayed. The results suggest that increased osmolality from normal water loss should also be a potent dipsogenic stimulus, and hence that osmotic thirst may have an important role in control of normal drinking. ACKNOWLEDGEMENTS I would like to thank the Director of the A.F.R.C. Edinburgh Research Centre for providing the facilities for these experiments, Miss Elaine Seawright for technical assistance and the BEMB for financial support through a research studentship.
REFERENCES
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4. Corbit, J. D. Osmotic thirst: theoretical and experimental analysis. J Comp Physiol Psychol 67: 3-14, 1969. 5. Fitzsimons, J. T. Drinking by nephrectomised rats injected with various substances. J Physiol 155: 565-579, 1961. 6. Fitzsimons, J. T. The effects of slow infusions of hypertonic solutions on drinking and drinking thresholds in rats. J Physiol 167: 344-354, 1963. 7. Fitzsimons, J. T. The physiology of thirst and sodium appetite. Monogr Physiol Soc 35: 96-127, 1979.
312 8. Fitzsimons, J. T. and S. Kaufman. Cellular and extracellular dehydration, and angiotensin as stimuli to drinking in the common iguana, Iguana iguana. J Physiol 265: 443-463, 1977. 9. Fitzsimons, J. T., M. Massi and S. N. Thornton. The effects of changes in osmolality and sodium concentration on angiotensin-induced drinking and excretion in the pigeon. J Physiol 330: 1-15, 1982. 10. Hawkins, R. C. and J. D. Corbit. Drinking in response to cellular dehydration in the pigeon. J Comp Physiol Psychol 89: 265267, 1973. 11. Kaufman, S., H-P. Kaesermann and G. Peters. The mechanism of drinking induced by parentera] hyperoncotic solutions in the pigeon and the rat. J Physiol 301: 91-99, 1980. 12. Kaufman, S. and G. Peters. Regulatory drinking in the pigeon Columba livia. Am J Physiol 239: R219-R225, 1980. 13. Koike, T. I., L. R. Pryor and H. L. Neldon. Plasma volume and electrolytes during progressive water deprivation in chickens (Gallus domesticus). Comp Biochem Physiol [A] 74: 83-87, 1983. 14. Korr, I. M. The osmotic function of the chicken kidney. J Cell Cornp Physiol 13: 175-193, 1939. 15. McFarland, D. J. and P. W. Wright. Water conservation by inhibition of food intake. Physiol Behav 4: 95-99, 1969. 16. McKinley, M. J., D. A. Denton and R. S. Weisiager. Sensors for antidiuresis and thirst: osmoreceptors or C.S.F. sodium detectors? Brain Res 141: 89-103, 1978. 17. Oatley, K. and F. M. Toates. Osmotic inhibition of eating as a subtractive process. J Comp Physiol Psycho182: 268-277, 1973. 18. Ruth, F. R. and M. R. Hughes, The effects of hypertonic sodium chloride injection on body water distribution in ducks (Anas platyrhychos), gulls (Larus glaucescens) and roosters (Gallus domesticus). Comp Biochem Physiol [A] 52: 21-28, 1975.
YEOMANS AND SAVORY 19. Savory, C. J. and J. P. Hodgkiss. Influence of vagotomy in domestic fowls on feeding activity, food passage, digestibility, and satiety effects of two peptides. Physiol Behav 33: 937-944, 1984. 20. Skadhauge, E. In vivo perfusion studies of the cloaca] water and electrolyte reabsorption in the fowl (Gallus gallus domesticus). Comp Biochem Physiol 23: 483-501, 1967. 21. Skadhauge, E. Osmoregulation in Birds. Berlin: SpringerVedag, 1981. 22. Skadhauge, E., D. H. Thomas, A. Chadwick and M. Jallageas. Time course of adaptation to low and high NaC1 diets in the domestic fowl: effects on electrolyte excretion and on plasma hormone levels. Pflugers Arch 396: 302-307, 1983. 23. Thornton, S. N. Drinking and renal responses to peripherally administered osmotic stimuli in the pigeon (Columba livia). J Physiol 351: 501-515, 1984. 24. Thornton, S. N. A central Na ÷ receptor and its influence on osmotic and Angiotensin II induced drinking in the pigeon. Columba livia. J Physiol (Paris) 79: 505-510, 1984. 25. Thornton, S. N. The influence of central infusions on drinking due to peripheral osmotic stimuli in the pigeon (Columba livia). Physiol Behav 36: 229-233, 1986. 26. Thornton, S. N. Drinking in the pigeon (Columba livia) in response to water deprivation and the influence of intracerebroventricular infusions of NaC1 or sucrose solutions. Physiol Behav 38: 719-724, 1986. 27. Thrasher, T. N., C. J. Brown, L. C. Keil and D. J. Ramsay. Thirst and vasopressin release in the dog: an osmoreceptor or sodium receptor mechanism. Am J Physiol 238: R333-R339, 1980. 28. Wolf, A. V. Osmometric analysis of thirst in man and dog. Am J Physiol 161: 75-86, 1950.