Metabolism of inorganic cations by quail (Coturnix coturnix) drinking solutions of CaCl2 and MgCl2.

l. In experiments to indicate the abilities of birds to tolerate and excrete Mg and Ca in drinking water, twenty-eight male quail received solutions of CaCl, or MgCl, perenterally for several days or in single doses. The concentrations of Ca, Mg, Na and K were subsequently measured in excretory fluids voided spontaneously or collected by intubating cloaca and rectum, and in plasma. 2. The physiological mechanisms of quail which deal with divalent cations are affected similarly by both Ca and Mg, without distinguishing sharply between the two. 3. Calcium appears to be less readily absorbed in the gut than Mg. 4. Excretion of Na is increased by intake of CaCl, or MgCIP. 5. Water appears to be absorbed from ureteral urine in cloaca or rectum. 6. The effects of CaCl, and MgCl, on salt metabolism of the quail are complex. INTRODUCTION DURING the past two decades physiologists have investigated the abilities of various birds to drink hyperosmotic saline solutions and to obtain physiologically useful water from them. These studies have contributed to an understanding of the adaptive specializations of birds that live in a variety of habitats, including marine situations, salt marshes, and deserts, where the salinity or scarcity of water make it difficult to maintain homeostasis (for reviews see Bartholomew & Cade, 1963: Cade, 1964; Dawson & Bartholomew, 1968). These studies have dealt only with metabolism of NaCl and the tolerance of birds to various concentrations of NaCl or sea water (for example, see Bartholomew & Cade, 1963; Cade, 1964; Dawson et al., 1965; MacMillen & Trost, 1966; Harriman, 1967; Harriman & Nance, 1968) and the results have been interpreted only in terms of the osmotic effects of NaCl. Since sea water contains appreciable concentrations of Ca and Mg-10.4 mM Ca, 54.7 mM Mg (Barnes, 1954)-and since saline desertic waters frequently contain considerable amounts of Ca and Mg (Margat, 1961; Williams & Siebert, 1963), it is desirable to know what physiological effects these biologically * Present address: Division of Science & Mathematics, McKendree College, Lebanon, Illinois 62254. 541 19 542 ERNEST J. WILLOUGHBY active ions may have on birds. Such knowledge would contribute to a better understanding of the physiological adaptations of birds for utilizing such waters. Therefore, I did some experiments with quail (Coturnix coturnix) to learn something about their abilities to tolerate and excrete Mg and Ca in their drinking water, the excretory pathways involved in their salt metabolism, and the possible function of the cloaca and rectum in salt and water balance. I chose the 120-g quail for the experiments because it can be kept in good condition in small individual cages that take up relatively little space, and it is big enough to allow sampling of blood and urine in volumes sufficient for analysis of several cations per sample. The quail employed came from a breeding colony maintained in the Department of Zoology, the University of Rilichigan, and were derived from the Japanese subspecies, C. c. japonica. The species occupies a wide geographic range in Eurasia and Africa. MATERIALS AND METHODS Twenty-eight male quail aged 6-22 months were employed. The mean body weight of these birds during experiments was 1198 g, range 859-1568 g, s.d. 14-l. The birds were housed in individual wire cages 30.4 cm (12 in.) to a side, and were maintained on a commercial diet (Purina Game Bird Breeder Layena) which I found to contain 0.230 m-equiv/g Mg, 1.05 m-equiv/g Ca, 0.141 m-equiv/g Na, and 0.305 m-equiv/g K. Acute and chronic loading regimens To study the effects of the intake of CaCla and MgCla on cation metabolism in the quail, I gave solutions of these salts to the birds either over a period of days, referred to as “chronic loading”, or in a single dose, termed “acute loading”. The regimen for chronic loading consisted of substituting solutions of MgCl, or CaCl, (or distilled water in controls) for their usual drinking water for 4 or more days. In some cases individual quail would not drink the higher concentrations of CaCla or MgCl,, and so these birds were given regular doses of the solution by stomach tube for at least 3 days prior to the next step. Food and fluid were then removed from the cages for 20-24 hr, and at the end of this period of fasting, 3 ml of the solution were injected into the crop or stomach through polyethylene tubing (PE-50/S36, Clay-Adams, Inc., New York) inserted through the esophagus, and the fluids voided by the birds over the subsequent 4 or 5 hr were collected. Acute loading consisted of removing food and water from cages of birds previously maintained on tap water, injecting 3 ml (in a few instances 5 ml, see Table 1) of the designated fluid by stomach tube 20-24 hr later, then collecting fluids voided during the subsequent 4-5 hr. Methods of collecting u~i~ary~u~~ Two methods were employed in collecting the fluids voided by the experimental quail. In one series of experiments, the birds were placed in individual wire cages (their usual living quarters) in a darkened room over pans containing light mineral oil about 1 cm deep so that as the birds voided the fluid spontaneously, it fell into the oil and sank. These fluids were then aspirated from beneath the oil at the end of the collection period in a Pasteur capillary pipette and mixed so that no distinction was made between urinary fluid and any fluid that might have passed unabsorbed through the gut. The fluids thus collected will be referred to as “spontaneously voided urine”. Fasting of the quail before beginning the collection of the fluids minimized fecal contamination. The second method of collecting fluid was designed to obtain urine directly as it passed into the cloaca from the ureters. This would minimize both contamination by unabsorbed fluid from the gut and the effects of cloacal or rectal reabsorption of water or solutes in the METABOLISM OF SALTS BY QUAIL 543 urine. A collecting device of polyethylene tubing (Clay-Adams, Inc.) and rubber balloons was fashioned (Fig. 1) so that when it was inserted into the cloaca and rectum of the quail and held in place by stitches between the polyethylene sheet collar and the skin surrounding the cloaca, one tube opened near the ureter on one side of the cloaca, and the other tube extended into the large intestine and opened near the level of the paired ceca. During the insertion of this device, birds were anaesthetized with 040 ml of Equi-Thesin (JensenSalisbery Laboratories, Kansas City, MO.) injected into the pectoral muscles. The quail remained under the effects of the drug for varying periods during the subsequent injection of the salt solution by stomach tube and collection of fluids. The quail then were held in individual cages in a darkened room while the fluids accumulated in the balloons attached to the tubes. This method of fluid collection is subsequently referred to as “intubation”. In an effort to check against contamination of urine by intestinal fluids, the solutions injected into the crops of the intubated quail were colored by dye (Fast Green FCF), which passed unabsorbed through the gut. Rarely was fluid in the urinary collecting bag colored by dye, whereas about one-third of the collections in the intestinal bag had traces of green dye. Urine that was colored by green dye was discarded. Polyethylene XLarge intestine (rectum) FIG. 1. Diagram of the device employed to collect ureteral urine from the cloaca, and intestinal fluids from the anterior end of the rectum, showing the manner of insertion. The diagram shows the quail on its back, as it was held during insertion of the tubes. Blood samples Blood was obtained from unanaesthetized quail by heart puncture with a 26 ga., 1 in. long hypodermic needle on a 1 cm3 heparinized glass tuberculin syringe. The needle was inserted between the furculum along the dorsal side of the sternum. Sodium-heparin was used as the anticoagulant, and distilled water drawn into heparinized syringes and treated in the same manner as the blood had 1.5-3.0 m-equiv/l. Na. Therefore, the plasma Na values presented here must be considered to be elevated by 2-3 per cent owing to the presence of 544 ERNEST J. WILLOUGHBY the heparin. Four-tenths to 0.8 ml of blood was drawn into the syringe, and the syringe was rocked gently to mix the blood with the heparin. The blood was then placed in capillary tubes (Kimax No. 34500, 100 mm long) which were closed and centrifuged for 20 min at 3000 rev/mm. The hematocrit was determined, the tube broken just above the packed cells, and the plasma sealed in the remainder of the capillary tube and stored in a freezer for subsequent analysis. Treatment of urinaryfluids The fluid voided by the birds was centrifuged in 15 ml graduated tubes and the volume of solids (including uric acid and occasional small amounts of fecal material) and of clear fluid estimated. Concentrated nitric acid was then added to the fluid in the proportion of 0.1 ml acid to 1 ml of clear liquid, and the mixture was shaken and allowed to stand at room temperature overnight. The purpose of this acidification was to put into solution Mg and Ca that may have been in the form of insoluble carbonates or urates, and to release Na or K which might be bound as insoluble urates (the urates thus being converted to the less soluble uric acid with release of the metal ions). The next day the acidified fluids were centrifuged again and the clear supemate sealed in capillary tubes and frozen for future analysis. In calculating the ion concentrations of the urinary fluids as they came from the quail, all subsequently determined values were multiplied by the appropriate factor owing to the dilution of the fluid with the acid, so that the concentrations given in the results reflect total ions excreted per volume of water, although some of these ions may have initially been in insoluble compounds. Ion analysis Sodium and potassium were determined by flame photometry (Coleman Model 21 Flame Photometer). Magnesium was determined with the dye Clayton Yellow as described by Natelson (1961, in conjunction with a Coleman Junior Spectrophotometer Model 6C. Calcium was determined by two methods. For plasma, total Ca and Mg were determined by a modification of the method described by Grette (1953), and the concentration of Ca was taken as the difference between this value and the value for Mg alone as determined by the use of Clayton Yellow. For urinary fluids, Ca was determined by flame photometry using standards that contained the same ranges of Na, K and Mg as the samples. Precision of these methods was within 6 per cent or better. Plasma ions and hematocrit RESULTS Table 1 lists plasma values of birds subjected to both chronic and acute loading. Birds given chronic loads of MgCI, had significantly elevated hematocrits (PC O-05, two-tailed t-test) compared to the control group given distilled water, whereas none of the other experimental groups had hematocrits significantly different from the controls. Quail receiving chronic loading of MgCl, showed reduced levels of Ca in the plasma. In birds receiving O-084 M MgCl, this resulted in a level of plasma Ca significantly different from the controls (P<O*O2, two-tailed t-test). Quail receiving loads of MgCl, showed a tendency to have elevated Mg concentrations in the plasma (0.099 M MgCI, acute, P-C 0.05 ; 0.084 M MgCl, chronic, P<O*Ol). Sodium concentrations in the plasma varied considerably, but showed no significant change relative to experimental regimens. Potassium showed no significant changes in relation to regimen. T A B L E ~ P L A S M A I O N C O N C E N T R A T I O N S A N D N E M A T O C R I T O F Q U A I L D R I N K I N G O R R E C E I V I N G B Y S T O M A C H T U B E S O L U T I O N S O F M gC l,