Ammonotely in a passerine nectarivore: the influence of renal and post-renal modification on nitrogenous waste product excretion
Mitrani Department of Desert Ecology, Jacob Blaustein Institute for Desert Research and Department of Life Sciences, Ben-Gurion University of the Negev, Sede Boqer Campus, 84990 Israel
* e-mail: pinshow{at}bgumail.bgu.ac.il
Accepted 28 March 2002
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Summary |
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Key words: nitrogen, excretion, ammonia, Palestine sunbird, Necatarinea osea, bird, ammonotely
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Introduction |
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Ammonia, urea and uric acid differ in toxicity, solubility and production
costs. Ammonia, as the direct end-product of amino acid metabolism, has no
additional production costs and is highly soluble, but also highly toxic,
requiring 400 ml of water to dilute each gram to a non-toxic concentration
(Wright, 1995). Terrestrial
animals, being water-limited, usually detoxify ammonia and excrete it in the
form of urea or a purine such as uric acid (e.g.
Campbell, 1991
). Urea and uric
acid are less toxic than ammonia and require 10 and 50 times less water,
respectively, for storage and excretion at non-toxic concentrations
(Wright, 1995
). Urea contains
two nitrogen atoms per molecule and uric acid contains four, whereas ammonia
contains just one. Thus, solutions of urea, ammonia or uric acid that contain
the same amount of nitrogen differ in osmotic potential.
The ammonia-detoxifying mechanism in birds results in the production of
uric acid (Campbell, 1994), but
ammonia and urea themselves are also found in avian urine
(Sturkie, 1986
). The
proportions of these three substances in the urine have been shown to vary
according to the protein content of food and the amount of water available to
birds (e.g. Ward et al., 1975
;
McNabb et al., 1980
). Uric
acid requires energy and amino acids for production. The major advantage of
excreting uric acid and its salts (hereafter referred to as urates) rather
than other nitrogenous waste products is in water conservation. Little water
is required to expel them because they contain more nitrogen per molecule and
have a lower toxicity and solubility. Some urate may thus precipitate out of
the urine once it has left the kidneys
(Sturkie, 1986
), although
urate is held in colloidal suspension in urine, in excess of its aqueous
solubility limits, by protein. Potential costs of uricotely include the energy
cost of the waste product, the potential loss of additional proteins used to
package the urates for transport through the renal tubules
(Janes and Braun, 1997
), and
the possibility of excessive ion loss via cations that associate with
urinary urates (McNabb et al.,
1973
; Laverty and Wideman,
1989
; Dawson et al.,
1991
).
The presence of urea in avian urine is indicative of the catabolism of
excess dietary amino acids, citrulline and arginine, and not of a functional
urea cycle (Klasing, 1998).
The urea cycle, which produces urea from ammonia, is incomplete in birds
(Griminger and Scanes, 1986
).
This means that, if birds have flexibility in their pattern of nitrogenous
waste excretion, it is likely to be in varying the proportions of urates and
ammonia rather than in varying urea excretion.
Nitrogenous waste products of nectarivorous birds
A nectar diet is rich in water (60-90 %; e.g.
Baker, 1975;
Calder and Hiebert, 1983
;
Roxburgh, 2000
), but poor in
electrolytes (5-80 mmoll-1;
Roxburgh, 2000
;
Calder and Hiebert, 1983
) and
protein (<0.04 % dry mass; Martinez del
Rio, 1994
). Thus, nectarivorous birds typically have high water
turnover rates (e.g. Williams,
1993
; Powers and Conley,
1994
; McWhorter and Martinez
del Rio, 1999
) and low protein requirements relative to allometric
predictions (e.g. Brice and Grau,
1991
; Roxburgh and Pinshow,
2000
). For such birds, excreting their waste nitrogen as urates
should not have the same advantages as it does for birds that are
water-stressed. Nectarivores may often have little to gain from the low
solubility and possible precipitation of urates, although at high temperature
and/or high sugar concentration they may be water-limited
(Calder, 1979
). When the ratio
of waste nitrogen to urine volume is low, the excretion of an increased
proportion of ammonia is feasible, thereby potentially avoiding the costs
(described above) of urate excretion.
We therefore predicted that three characteristics of nectarivore physiology may influence or alter the proportions or concentrations of nitrogenous waste produced in the kidney: (i) high water turnover rates, (ii) a need to conserve electrolytes and (iii) low protein intake rates.
Refluxing and post-renal modification of urine
In birds, unlike in mammals, the ureters open into the cloaca, which serves
as a common receptacle for the urinary, digestive and reproductive systems.
There, the urine may mix with the faeces and be actively refluxed by reverse
peristalsis into the hindgut. This allows the urine contact with populations
of micro-organisms that inhabit the hindgut and with the epithelial tissue of
the colon, both of which can modify the composition of the urine
(Braun, 1999). Refluxing allows
for the uptake of electrolytes and water from urine in the hindgut
(Goldstein and Braun, 1986
).
Protein that is present in ureteral urine may be broken down and reabsorbed,
and recycling of the nitrogen in urates or urea may occur
(Karasawa, 1999
). Thus, the
kidneys and hindgut work in concert to produce the final excreted fluid.
The implications of refluxing for nectar feeders in particular (although applicable to birds in general) are that, if it occurs, the protein present in the urine can be broken down and amino acids reabsorbed. Urates may also be broken down, releasing trapped electrolytes that are then available for reabsorption, together with the products of urate breakdown.
In the light of the above, we tested whether ammonotely would occur in a
small nectarivorous passerine bird, the Palestine sunbird (Nectarinia
osea). The specific questions we addressed in this study were: does
ammonotely occur and, if so, under what conditions; i.e. what influences the
proportions and concentrations of nitrogenous waste products in ureteral urine
and excreted fluid? And, is there a difference between ureteral urine and
excreted fluid; i.e. is there evidence for post-renal modification of urine in
Palestine sunbirds? To answer these questions, we examined the influence of
dietary water, electrolytes and protein on the excretion of the three major
nitrogenous waste products, ammonia, urea and urate. We also compared ureteral
urine with voided cloacal fluid to determine changes that occur by
modification in the hindgut. Finally, we examined the effect of ambient
temperature on the proportions of ammonia found in ureteral and excreted urine
to repeat the experiment of Preest and Beuchat
(1997) on Anna's
hummingbirds.
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Materials and methods |
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Experimental protocol
Three separate experiments were performed. In the first experiment, we
tested the effect of total water, protein and salt intake on excretion of
nitrogenous wastes. In the second, we tested the effect of the salt
concentration of the diet on nitrogenous waste products, and in the third we
tested the effect of ambient temperature (Ta) on excretion
of nitrogenous wastes. All three experiments used the following basic
protocol.
During experiments, birds were individually housed in small cages in a controlled-temperature room (25 °C; 13 h: 11 h L:D, to mimic the natural light cycle at the time of the experiments) and were offered artificial nectar containing sugar, protein and electrolytes, the exact concentrations of which depended on the treatment. The amount of nectar taken in by the birds was determined by weighing the feeders before and after feeding and correcting for evaporative losses by measuring the mass change of feeders that were not available for birds to feed on. Each treatment lasted for 3 days followed by a minimum 1-week recovery period. Birds were allowed to adjust to the cages and diet for 3 days, after which excreted fluid was collected in mineral oil for 2 h, between 07:00 and 09:00 h. Immediately thereafter, ureteral urine and blood samples were collected from each bird, and the birds were returned to the outdoor aviaries.
Protocols specific to each experiment
Experiment 1: the influence of total intake of water, protein and
salt on nitrogenous waste products
Artificial nectars offered to sunbirds in each treatment had one of three
levels each of water, protein and salt. The highest levels corresponded to the
upper extreme of protein, salt and water intake rates that free-living birds
would experience [based on measurements of the sugar and salt concentrations
of nectar (e.g. Baker, 1975;
Calder and Hiebert, 1983
;
Roxburgh, 2000
) and of the
protein requirements of Palestine sunbirds
(Roxburgh and Pinshow, 2000
)].
Medium levels were typical of foods encountered by free-living birds and, in
the case of protein, met the birds' protein requirements. Low levels of
protein were below the protein requirements of sunbirds, and low salt and
water levels were below average values for floral nectars.
Sucrose concentrations were 0.29, 0.58 and 1.46 mol l-1 (10, 20 and 50% sucrose by mass). Birds compensated for low sugar concentrations by drinking more artificial nectar. In this way, energy intake remained constant while intake of water varied across diets. Water intake rates averaged 2, 8 and 17 ml day-1. Salt and protein concentrations were adjusted so that the intake of salt and protein would be similar for each dietary level. These nutrients were added to the diet to produce protein (isolated soy protein) intakes of 9, 26 and 61 mg day-1 and salt (NaCl and KCl) intakes of 3, 15 and 30 mg day-1 for low, medium and high treatments, respectively.
Experiment 2: the influence of salt concentration on nitrogenous
waste products
Birds were offered a 0.58 mol l-1 sucrose solution containing a
2.62 g kg-1 solution of isolated soy protein with three different
concentrations of salts, 0.8, 39.6 and 78.4 mmol l-1 each of NaCl
and KCl. Five birds were used in this experiment. Each bird was tested on
every diet, with at least 1 week between treatments.
Experiment 3: the influence of Ta on nitrogenous waste
products
Birds were placed in a controlled-temperature room at 10°C for a 3-day
experimental period, followed by a 1-week recovery period in the outdoor
aviary, after which they were returned to the controlled temperature room at
30°C for a further 3 days. During the experimental period, birds were
offered artificial nectar containing 0.58 mol l-1 sucrose and 11
mmol l-1 each of sodium chloride and potassium chloride. The nectar
offered to the birds at 10°C contained 0.86 g soy protein kg-1,
whereas the diet at 30°C contained 1.46 g kg-1. To keep the
protein intake of the birds as constant as possible, we increased the protein
content of the 30°C diet, because birds ate less at 30°C than at
10°C.
Fluid sampling and analyses
Blood, ureteral urine and excreted fluid samples were collected during
experiments. Urate, urea and ammonia concentrations of all samples were
determined using Sigma diagnostic kits (no. 685 for uric acid and no. 640 for
urea and ammonia). Sodium and potassium concentrations were measured using a
flame photometer (Corning 435) and chloride concentration with a chloride
titrator (Corning 925). Osmotic potential was measured with a vapour pressure
osmometer (Wescor 5100C). Ureteral urine samples were collected from birds by
briefly inserting a closed-end cannula, made from polyethylene tubing, into
the bird's cloaca. The closed end prevented contamination of ureteral urine by
intestinal fluids. Urine drained into the cannula via a window
positioned under the ureteral papillae
(Thomas et al., 1984). Blood
samples were collected in heparinised capillary tubes from a brachial vein
punctured with a 27-gauge needle.
In addition to analysing whole excreta samples, we also diluted excreta
samples 1:1 in a 0.5 mol l-1 LiOH solution to dissolve all urate
precipitates and solubilise any trapped ions
(Laverty and Wideman, 1989).
We reanalysed these samples for potassium and sodium, and compared them with
samples in which urates had not been dissolved to determine the proportion of
salts bound to uric acid.
Experimental design and statistical analyses
All values are expressed as mean ± standard deviation (S.D.).
Experiment 1: the influence of total intake on nitrogenous waste
products
The first experiment had a fractional factorial design. This design uses
only a fraction of all possible dietary treatment combinations; it was used
because, with three dietary factors, there are too many combinations of water,
protein and salt intake to be tested on every bird
(Table 1). Thus, every possible
dietary combination was tested, but not every bird was tested on every diet.
The data were analysed using a four-way fractional factorial analysis of
variance (ANOVA) (Mead, 1988;
SYSTAT ver. 7.0, SPSS Inc.). Repeated measurements of birds were taken into
account by using bird as a factor in the ANOVA together with water, protein
and salt intake.
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Nine birds were used, with six treatments on each bird. These numbers were
chosen to keep the experimental design balanced, i.e. all dietary factors were
tested equally. This design allowed us to test second- and third-order
interactions between the three dietary factors
(Mead, 1988), but did not
allow testing of any interactions that included the identity of the birds.
Proportional data were arcsine-square-root-transformed before analysis
(Sokal and Rohlf, 1981
).
Experiments 2 and 3: the influence of salt concentration and
Ta on nitrogenous waste products
Data from these experiments were analysed using a non-parametric, bootstrap
randomisation technique (Manly,
1997; Resampling Stats ver. 5.0.2, Resampling Stats. Inc.). This
technique was chosen because sample sizes were small and birds were used
repeatedly in different treatments. Probability values were calculated by
comparing the observed sum of squared mean differences between treatment
groups with a randomly generated sum of squared mean differences. This was
repeated 1000 times, the final probability value being the proportion of
randomly generated sums of squared mean differences that equalled or exceeded
the observed sum of squared mean differences.
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Results |
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The energy gained on different diets did not differ significantly (ANCOVA using body mass as a covariate; F2,24=0.80, P=0.46 for sugar concentration, F2,24=1.31, P=0.29 for protein concentration; F2,24=0.36, P=0.70 for salt concentration). Thus, sunbirds compensated fully for changes in the sugar concentration of their diet so that mean daily energy intake was 32.5±4.8 kJ (assuming 15.41 kJ g-1 for sucrose), irrespective of artificial nectar intake rate.
Proportions of ammonia in ureteral urine and excreted fluid
The proportion of ammonia in ureteral urine was not significantly different
in any of the treatment groups (three-factor fractional ANOVA;
F2,14=0.69, P=0.52 for water;
F2,14=1.06, P=0.37 for protein;
F2,14=0.33, P=0.73 for salt;
Table 2). The proportion of
ammonia in excreted fluid was significantly influenced by the amount of
protein in the diets (three-factor fractional ANOVA;
F2,17=5.51, P=0.01;
Table 3). Birds eating low and
medium levels of protein had significantly higher proportions of ammonia in
their excreted fluid than did birds on the high-protein diets
(post-hoc pairwise comparison, P=0.02 and P=0.04,
respectively). Water and salt intake rates had no influence on the proportion
of ammonia in excreted fluid (ANOVA; F2,17=0.29,
P=0.75 for water; F2,17=0.48, P=0.63 for
salt).
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Concentrations of nitrogenous waste products in ureteral urine and
excreted fluid
In ureteral urine, the concentrations of ammonia and urea were
significantly different at different water intake rates (ANOVA;
F2,14=10.81, P<0.01 for ammonia;
F2,14=4.62, P=0.03 for urea), but urate
concentration did not differ significantly (ANOVA;
F2,14=1.71, P=0.22;
Table 2). The excreted fluid
concentrations of all three nitrogenous waste products were significantly
different at different water intake rates (ANOVA;
F2,17=19.07, P<0.01 for urate;
F2,17=19.66, P<0.001 for urea;
F2,17=31.19, P<0.001 for ammonia;
Table 3). In addition, the
urate concentration of the excreted fluid was significantly higher on the
high-protein diet than on the other two diets (post-hoc pairwise
comparison, P=0.001 in both cases). Therefore, the change in the
proportion of ammonia in the excreted fluid with increasing protein intake
rate is apparently a result only of the change in the quantity of urate being
excreted.
Incidence of ammonotely
Seven out of 52 excreted fluid samples were indicative of ammonotely (i.e.
the amount of nitrogen excreted as ammonia exceeded the amount excreted as
urate) (Fig. 1). Ammonotely was
significantly more likely to occur in low-protein treatments than in medium-
or high-protein treatments (G-test;
Sokal and Rohlf, 1981;
G=7.07, P<0.05). In all except eight excreted fluid
samples, ammonia concentration exceeded that of urate. However, as uric acid
contains four nitrogen molecules, the amount of nitrogen excreted in the form
of urate usually exceeded that excreted as ammonia. Two out of 29 ureteral
urine samples contained more ammonia-nitrogen than urate-nitrogen, and in both
cases the corresponding excreted fluid sample was also ammonotelic. The
remaining five ammonotelic excreted fluid samples had corresponding ureteral
urine samples that were uricotelic.
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A comparison of the nitrogenous waste products of excreted fluid and
ureteral urine
The ammonia, urate and urea concentrations of excreted fluid samples were
compared with those of ureteral urine samples. A complicating factor when
comparing the concentrations of nitrogenous waste products is that the volume
of ureteral urine and excreted fluid may not be the same. T. J. McWhorter
(unpublished data) found that in Palestine sunbirds not all water in the diet
was taken up across the gut wall and processed by the kidneys and, thus, that
the greater the water intake rate, the lower the volume of ureteral urine
would be relative to the volume of excreted fluid. We therefore assumed that,
at low water intake rates (i.e. a diet with a high sugar concentration),
ureteral urine and excreted fluid volume would be almost identical, or even
that water is reabsorbed from the hindgut, making the volume of excreted fluid
less than that of ureteral urine. At medium and high water intake rates,
excreted fluid volume would be increasingly greater than ureteral urine
volume. We analysed the data and interpreted our results in the light of these
assumptions.
We compared ureteral urine and excreted fluid using repeated-measures ANOVA, with water intake rate (low, medium or high) as an additional factor. The urate concentration of excreted fluid was significantly lower than that of ureteral urine (F1,27=10.58, P<0.01), regardless of water intake rate (interaction F2,27=0.94, P=0.40) (Fig. 2A). There were significant differences in the urea concentration of ureteral urine and excreted fluid (F1,27=11.41, P=0.01), and these differences were dependent on water intake rate (interaction F2,27=14.91, P<0.01). In general, the ammonia concentration did not differ between ureteral urine and excreted fluid (F1,27=1.16, P=0.29). However, there were significant differences between diets with different water intake rates (interaction F2,27=5.22, P=0.01).
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Both urea and ammonia concentrations were higher in excreted fluid than in ureteral urine at low water intake rates (Fig. 2B,C). At medium water intake rates, urea and ammonia concentrations were similar in ureteral urine and excreted fluid, while at high water intake rates, urea and ammonia concentrations were lower in excreted fluid than in ureteral urine.
The proportions of ammonia were significantly higher in excreted fluid than in ureteral urine (repeated-measured ANOVA; F1,27=4.31, P=0.05). The percentage of ammonia in ureteral urine was 20.7±16.0% (N=30), whereas it was 27.4±12.1% in matching excreted fluid samples.
Osmotic potential and cation concentrations of excreted fluid and
ureteral urine
The changes in osmotic potential between ureteral urine and excreted fluid
samples followed a similar pattern to that found for concentrations of ammonia
and urea. That is, whereas there were no overall significant differences
between ureteral urine and excreted fluid (repeated-measures ANOVA;
F1,22=2.83, P=0.11), there were significant
differences between diets with different water intake rates (interaction
F2,22=20.85, P<0.001;
Table 4). When water intake
rate was low, excreted fluid osmotic potential was higher than that of
ureteral urine, while at high water intake rates, excreted fluid osmotic
potential was less than that of ureteral urine.
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The osmotic potential of both ureteral urine and excreted fluid was strongly correlated with salt and water intake rates, with the lowest osmolalities occurring on the low-salt high-water diets (47±20 mosmol kg-1; N=6; for excreted fluid) and highest on the high-salt low-water diets (754±233 mosmol kg-1; N=5; for excreted fluid). Neither the sodium nor the potassium concentration of ureteral urine was significantly different from that of excreted fluid (repeated-measures ANOVA, F1,15=0.03, P=0.87 for sodium; F1,15=0.99, P=0.34 for potassium; Table 4).
Plasma urate concentrations
Plasma urate concentrations did not vary with salt or water intake rates,
but were significantly higher on the high-protein diet (ANOVA;
F2,17=20.2, P<0.01). Plasma urate
concentrations were 1.1±0.6 mmol Nl-1 (N=17) on the
high-protein diet and 0.4±0.2 mmol Nl-1 (N=17) and
0.5±0.3 mmol Nl-1 (N=18) on the low- and
medium-protein diets, respectively. Urea and ammonia concentrations were too
low and the sample volumes were too small to allow reliable measurement using
the Sigma diagnostic kits.
Experiment 2: the influence of dietary salt concentration on
nitrogenous waste products
The electrolyte concentration of the diet did not affect the proportion of
ammonia in excreted fluid (P=0.27), although the proportion of
ammonia in low-salt treatments was less than in the medium- or high-salt
treatments (Table 5). However,
the proportion of ammonia in ureteral urine was significantly lower in the
low-salt treatment than in the other two treatments (P=0.008).
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The ammonia and urea concentrations in ureteral urine did not differ among treatments (P=0.49 and P=0.63, respectively). However, the urate concentration of ureteral urine was significantly higher in the low-salt treatment than in the medium- and high-salt treatments (P=0.05). Plasma urate concentrations were also significantly different among treatments (P=0.03), with birds on the high-salt diet having lower plasma urate concentrations than birds on the low-salt diet. Feeding rates of sunbirds were not significantly different among the three treatment groups (ANOVA; F2,11=2.9, P=0.1).
Experiment 3: the influence of Ta on nitrogenous waste
products
Sunbirds drank significantly more artificial nectar at 10 than at 30 °C
(P<0.001): 11.5±0.8 ml day-1 compared with
8.24±1.1 ml day-1, respectively. Temperature had no
significant influence on the proportion of ammonia in excreted fluid
(P=0.19) or ureteral urine (P=0.68)
(Fig. 3A). While the average
concentration of all three nitrogenous waste products was greater at 30 than
at 10 °C, only the ammonia concentration of excreted fluid was
significantly greater (P<0.001)
(Fig. 3B).
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Sodium and potassium trapping with urates
Neither the sodium nor the potassium concentrations of excreted fluid and
ureteral urine were different when samples were solubilised with LiOH compared
with when they were not, suggesting that insignificant amounts of these ions
were trapped with urate (paired t-test for excreted fluid,
t8=2.1, P=0.07 for sodium;
t8=1.7, P=0.13 for potassium; for ureteral urine,
t5=1.9, P=0.13 for sodium;
t5=0.6, P=0.7 for potassium).
Samples collected from wild sunbirds
Eight free-ranging sunbirds were caught with mist-nets or drop nets on the
Sede Boqer campus in August and September, and blood and ureteral urine
samples were collected from these birds. Birds were released immediately
thereafter. It was possible to collect ureteral urine samples from four of
these birds. The mean proportion of ammonia in ureteral urine was
8.7±5.3 % (N=4) and mean ammonia, urate and urea
concentrations were 4.7, 48.4 and 2.2 mmol Nl-1 respectively.
Plasma urate concentration was 1.6±0.5 mmol Nl-1
(N=8).
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Discussion |
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Table 6 summarises data on the concentrations and proportions of urate, urea and ammonia in the ureteral urine of five bird species. Palestine sunbirds have concentrations of nitrogenous waste products that are considerably lower (ranging from only 0.5 to 15 %) than those for the other species. However, the proportions of nitrogenous waste products in ureteral urine are comparable with those of the other species.
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Post-renal modification of ureteral urine
A comparison of excreted fluid and ureteral urine in Palestine sunbirds
shows that post-renal modification of urine occurred in the hindgut. Excreted
fluid contained on average 64 % of the urate present in ureteral urine. In
comparison, in Gambel's quail (Callipepla gambelii), the voided
excreta contained 68 % of the urate present in the ureteral urine
(Anderson and Braun, 1985). As
mentioned above, excreted fluid volume was probably greater than ureteral
urine volume. It could be argued that the decline in urate concentration of
excreted fluid relative to ureteral urine is simply an effect of dilution by
addition of liquid excreta from the gut. However, the difference in the
dilution patterns of urate versus urea and ammonia suggests that
urate breakdown occurred.
On a high-sugar/low-water diet, urate concentrations were lower in excreted
fluid than in ureteral urine, whereas urea and ammonia concentrations were
greater (Fig. 2). On this diet,
Palestine sunbirds probably absorbed almost all their water from the food in
the gut (T. J. McWhorter, unpublished data). Volumes of ureteral urine and
excreted fluid were thus unlikely to differ significantly unless reabsorption
of water from ureteral urine had occurred in the hindgut. Reabsorption of
water from ureteral urine has been demonstrated in, for example, house
sparrows Passer domesticus
(Goldstein and Braun, 1986)
and Gambel's quail (Anderson and Braun,
1985
). Whether water was reabsorbed or not, our suggestion that
urate was broken down is supported. If water had been reabsorbed in the
hindgut, one would expect that ammonia, urate and urea concentrations would
all increase. But this was not the case. Urate concentration dropped by an
average of 9 %, while ammonia and urea concentrations increased by 104 and 97
%, respectively.
On the low-sugar/high-water diet, we expected the volume of ureteral urine to be significantly less than that of excreted fluid because not all water in the food is taken up across the gut wall (T. J. McWhorter, unpublished data). Thus, dilution of all three nitrogenous waste products should have occurred (Fig. 2). However, urate concentration fell to a much greater extent than ammonia or urea concentration (an average decrease of 62 % versus 34 % and 25 % for ammonia and urea, respectively), again providing evidence indicating the breakdown of urate.
The breakdown and recycling of urate is known to occur in the caeca of
chickens, turkeys and other galliforms
(Clench, 1999). The caeca
contain large microbial populations that are capable of breaking down urates
(Laverty and Skadhauge, 1999
).
The products of this breakdown (ammonia) are either incorporated into
microbial protein or may be absorbed by the caecal epithelium. Mortensen and
Tindall (1981
) have shown that
caecal ammonia can be used in the enzymatic synthesis of glutamic acid and
that this amino acid can then be absorbed.
However, many (if not most) bird species, such as the Palestine sunbird, do not possess caeca or have vestigial caeca. How do these birds recycle the nitrogen in urates? It is possible that uricolytic microbial populations occur in the colon of these birds and that urate is broken down and recycled in the same way as in birds with caeca. C. A. Beuchat (personal communication) found uricolytic bacteria in the hindgut of hummingbirds, and such bacteria are probably responsible for urate breakdown in sunbirds as well. The mechanisms of nitrogen recycling, particularly in birds that do not possess caeca, are unknown.
Osmotic potential and post-renal modification of ureteral urine
The osmotic potential of the excreted fluid of Palestine sunbirds in the
lowest salt and sugar treatments were 47±20 mosmol kg-1.
This matches the lowest values found in free-living hummingbirds
(Calder and Hiebert, 1983) and
in freshwater fish (e.g. Talbot et al.,
1992
) and amphibians (e.g.
Shpun et al., 1992
). In the
highest sugar and salt treatments, excreted fluid osmotic potential rose to
754±233 mosmol kg-1, which is approximately double the
plasma osmotic potential. The osmotic potential of ureteral urine ranged from
a mean value of 61 mosmol kg-1 to a mean value of 585 mosmol
kg-1. The ability to produce urine that is twice as concentrated as
plasma is within the range typically found for birds (e.g.
Skadhauge, 1974
).
In Palestine sunbirds, we found no significant trapping of electrolytes in
urates. In contrast, in Gambel's quail, 16% of the sodium and 36% of the
potassium found in ureteral urine were trapped in urates
(Anderson and Braun, 1985),
while in domestic hens (Gallus domesticus) 9% of sodium and 23% of
potassium were trapped (Long and
Skadhauge, 1983
). In comparison with other birds, electrolyte
concentrations in nectarivore urine are typically very low (e.g.
Calder and Hiebert, 1983
;
Beuchat et al., 1990
), as are
concentrations of urate (e.g. Table
6; Preest and Beuchat,
1997
). Some cations must be bound to uric acid because uric acid
has a pK1 of 5.4 (Goldstein and
Skadhauge, 2000
) and thus, under most physiological conditions,
must occur as a monobasic urate salt. However, the concentration of cations
bound to uric acid is unlikely to be more than a few mmol l-1
because of the low concentrations of uric acid/urate (3.5±3.2 mmol
l-1 in excreted fluid). We rarely observed precipitated urate in
sunbird excreta, and thus co-precipitation is also unlikely to be important in
these birds.
Although we predicted that a reduction in the salt concentration of the diet would lead to an increase in the proportion of ammonia excreted, this was not the case. As little or no precipitation of urates occurred and thus no trapping of electrolytes, our original prediction that a decline in urate concentration or conversely an increase in ammonia concentration would occur was not valid.
However, we did find that, when the salt concentration of the diet was low, the proportion of ammonia in the ureteral urine was significantly lower than in birds offered diets with higher salt concentrations (Table 5). Urate concentration in ureteral urine increased significantly at low salt concentrations. The plasma urate concentration of Palestine sunbirds was higher on the low-salt diet and decreased as salt intake increased; it was unusually low in birds on the high-salt diet. The reason for this fall in plasma urate concentration is not clear.
Comparison with ammonotely in hummingbirds
In contrast to the results of the study on hummingbirds by Preest and
Beuchat (1997), we found that
Ta had no effect on the proportions of ammonia found in
either excreted fluid or ureteral urine. Preest and Beuchat
(1997
) found that 50% of
hummingbirds tested at 10°C were ammonotelic, while the birds were all
uricotelic at 20 and 40°C. The experimental protocols of Preest and
Beuchat's study were different from ours. Anna's hummingbirds were offered a
0.7 mol l-1 sucrose solution, which contained neither protein nor
electrolytes. Birds were not acclimated to the experimental
Ta (other than 20°C) for more than a few hours (M. R.
Preest, personal communication). In our study, birds were offered a 0.58 mol
l-1 sucrose solution containing some salts and protein and were
acclimated to the experimental Ta for 3 days. In addition,
hummingbirds appear to absorb essentially all water from their food and
process it in the kidney (McWhorter and
Martinez del Rio, 1999
), whereas there is evidence that Palestine
sunbirds modulate water uptake from their food (T. J. McWhorter, C. Martinez
del Rio and B. Pinshow, unpublished data). Thus, water loads processed by the
kidney may be much larger at lower Ta for hummingbirds
than for sunbirds, especially considering the hummingbirds' lack of
acclimation to the experimental Ta. This may account for
differences in ammonia and urate excretion.
Other studies report that birds excreted large quantities of ammonia. Moss
and Parkinson (1972) found
that red grouse Lagopus lagopus scoticus eating heather excreted most
of their digested nitrogen as ammonia and ornithurates, while Moss and
Parkinson (1975
) found that
rock ptarmigan Lagopus mutus eating berries excreted approximately
half their metabolised nitrogen as urates and half as ammonia. They did not
collect ureteral urine from these birds, but is it likely that these birds
harbour large populations of uricolytic micro-organisms in the caeca and that
urate is extensively decomposed in the hindgut
(Mortensen and Tindall,
1981
).
Concluding remarks
The proportion of ammonia in the ureteral urine of Palestine sunbirds was
not influenced by total water, salt or protein intake. In excreted fluid,
however, the proportion of ammonia increased significantly with decreasing
protein intake. This change was not due to a change in ammonia concentration
but rather to a change in urate concentration. Urate appeared to be broken
down in the hindgut, leading to a fall in urate concentration in excreted
fluid. The reduction in the urate concentration of excreted fluid led to
`apparent' ammonotely. We suggest that ammonotely may not be a unique feature
of nectarivores. It could occur in any species in which the breakdown of urate
in the hindgut allows the uric acid nitrogen concentrations in the excreta to
fall below that of ammonia nitrogen concentrations.
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