Can birds be ammonotelic? Nitrogen balance and excretion in two frugivores
1 Department of Biology, Technion - Israel Institute of Technology, Haifa
32000, Israel
2 Department of Zoology and Physiology, University of Wyoming, Laramie WY,
82071, USA
3 Department of Biology, University of Haifa at Oranim, K. Tivon 36006,
Israel
* Author for correspondence (e-mail: elat{at}techunix.technion.ac.il)
Accepted 12 January 2005
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Summary |
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Key words: yellow-vented bulbul, Pycnonotus xanthopygos, Tristram's grackle, Onychognathus tristram, nitrogen balance, nitrogen requirements, uric acid, ammonia, frugivores
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Introduction |
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Preest and Beuchat (1997)
suggested that it might be advantageous for birds that ingest large amounts of
dilute, protein-poor nectar to shift from uricotely to ammonotely. Thus,
ammonia can be voided rapidly, and the costs of synthesizing urates can be
reduced. Preest and Beuchat
(1997
) called the shift from
uricotely to ammonotely in hummingbirds `facultative ammonotely' In their
study on Anna's hummingbird Calypte anna, Preest and Beuchat
concluded that these birds were facultatively ammonotelic. At low ambient
temperatures and high water intakes, Anna's hummingbirds excreted more than
50% of their total nitrogen excretion as ammonia (i.e. they became
ammonotelic), whereas at higher ambient temperatures and lower food intake
they were uricotelic (Preest and Beuchat,
1997
). The hypothesis of Preest and Beuchat
(1997
) proposes that high
energy demands and high water fluxes favor ammonotely.
McWhorter et al. (2003)
challenged the generality of Preest and Beuchat's results
(Preest and Beuchat, 1997
).
They fed black-chinned hummingbirds Archilochus alexandri,
magnificent hummingbirds Eugenes flugens and blue-throated
hummingbirds Lampornis clemenciae dilute, protein-poor diets, but
found that neither of these species became ammonotelic. The hummingbirds
remained uricotelic even when they fed on the most dilute food and when they
ingested and excreted prodigious amounts of water (>3 times their body mass
per day). Urate constituted over 60% of all nitrogenous waste in all species
and water intake had no effect on the proportion of nitrogen excreted as
ammonia.
To further cast doubt on the generality of facultative ammonotely in
nectarivorous birds, Roxburgh and Pinshow
(2002) found that Palestine
sunbirds Nectarinia osea were generally uricotelic. However, in seven
out of 52 tests, N. osea excreted more nitrogen as ammonia than as
uric acid. Because the fraction of nitrogen excreted as ammonia never exceeded
50%, these birds were not ammonotelic. However, they were clearly more
ammonotelic than uricotelic. Roxburgh and Pinshow
(2002
) noted that in birds
with high water intake, the concentration of urate was higher in ureteral
urine than in excreta. They argued that ammonotely in Palestine sunbirds was
only `apparent' as it was not a result of excessive excretion of ammonia, but
rather the result of a reduction in excreted urate resulting from post-renal
modification of urine.
Although apparent ammonotely has not yet been observed in non-nectarivorous
species it might not be a unique feature of nectarivorous birds
(Roxburgh and Pinshow, 2002).
We hypothesized that apparent ammonotely is related to low nitrogen intake and
high water fluxes. We only tested the second component of Preest and
Bucheaut's hypothesis (Preest and Bucheaut, 1997), but added nitrogen intake
as an interacting factor. Our experiments did not address the effect of
increased energy demands on the composition of excreted nitrogenous products.
Specifically, we predicted that birds with low nitrogen intake recover some of
the uric acid in the lower gastrointestinal tract. This reduction leads to an
apparent increase in the fraction of nitrogen excreted as ammonia. Because
fruit-eating birds appear to have unusually low nitrogen intake
(Witmer, 1998
;
Pryor et al., 2001
), we
hypothesized that we would find apparent ammonotely in two frugivorous birds:
the yellow-vented bulbul Pycnonotus xanthopygos and the Tristram's
grackle Onychognathus tristrami. To test this idea we manipulated
protein and water intake by simultaneously varying the protein and water
contents of the diet and by measuring their effects on the composition of
nitrogenous waste in excreta and in ureteral urine. Our experimental protocols
allowed us to also measure minimal nitrogen requirements (MNR) and total
endogenous nitrogen loss (TENL) in two desert frugivorous birds.
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Materials and methods |
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Care and maintenance
Tristram's grackle
Seven birds were mist-netted under license from the Israel Nature and
National Parks Authority in the Dead Sea region one month before the
experiments. Two other birds were long-term captives on the campus of Haifa
University at `Oranim', Kyriat Tivon, where all birds were kept and where all
experiments were conducted. Birds were held in a large open outdoor cage and
maintained on fruits (apples, grapes, melons) and chopped boiled eggs.
Yellow-vented bulbuls
Ten birds were mist-netted on the `Oranim' campus, Kyriat Tivon, and at
Sde-Yaakov, northern Israel (under license from the Israel Nature and National
Parks Authority). Birds were maintained in separate cages (60 cmx30
cmx60 cm) in an outdoor shed. Maintenance food contained fruits (apples,
grapes, melons), vegetables (tomatoes, cucumbers) and chopped boiled eggs. In
all experiments birds were housed in individual cages (40 cmx30
cmx30 cm) in a room maintained at 20±2°C with a photoperiod
of 12 h:12 h light:dark.
Intake response experiment
On the day before experiments, birds were transferred to the experimental
cages (bulbuls N=10, grackles N=9) and offered a fluid diet
in plastic feeders containing different concentrations of casein acid
hydrolysate (Sigma Chemical, St Louis, MO, USA), and a 1:1 mixture of glucose
and fructose. Each bird was fed a solution containing one of the following
sugar concentrations: 5, 10, 15, 20 or 25% (g sugar per 100 g of solution).
The sugar solutions also contained one of two possible casein hydrolysate
concentrations. Bulbuls were fed 2, and 6 g l-1, and grackles were
fed 1.2 and 6 g l-1. After 8 h, the amount of solution consumed was
measured. The variation in protein intake that we observed in these
experiments allowed us to control the protein intake in subsequent
experiments.
Nitrogen requirements and composition of urine and excreta
Experiments lasted 3 days. During experiments birds were offered synthetic
fluid diets, modified from those of Brice and Grau
(1989). These diets varied in
sugar and protein concentrations (Table
1). NaCl concentration was constant in all diets (9.07 mmol
l-1). Birds were transferred to individual experimental cages 3
days before experiments commenced. During this period, the birds received the
maintenance diet. On the day prior to an experiment food was removed from the
cages 2 h before dark. We measured body mass on the first morning of the
experiment and every 24 h thereafter. Plastic pans containing 200 ml of
mineral oil were placed at 08:00 h under the cages for collection of excreta.
Pans were removed after 24 h and the excreta and mineral oil were collected
into plastic bottles and frozen at -20°C for later analysis. New pans were
placed under the cages for another 24 h collection period. Only the 24 and 72
h (first and third day, respectively) collections were analyzed. Two birds
were randomly assigned to each of the five diets. Food was offered ad
libitum in plastic feeders. Fluid consumption was measured every 24
h.
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Collection of ureteral urine and plasma
Ureteral urine samples were collected at 08:00 h after the birds had
consumed the experimental diet for 24 h and 72 h. Samples were obtained by
briefly inserting a closed-ended perforated cannula
(Goldstein and Braun, 1986),
custom-made of polyethylene tubing (Tristram's grackle PE300, yellow-vented
bulbuls PE200; Intramedic, MD, USA) into the bird's cloaca. The samples were
then kept at -20°C for later analysis. We collected blood (
100 µl)
in heparinized microhematocrit tubes by puncturing the brachial vein with a
28-gauge needle. Plasma was separated from cells after centrifugation
(micro-hematocrit centrifuge, International Equipment CL A4922X-1, Needham,
MA, USA) for 3 min.
Sample analysis
Excreta samples were thawed and separated from the mineral oil by
centrifugation (5000 r.p.m. for 3 min, Sorvall RC 5B Plus, Asheville, NC,
USA). Feather parts were removed with the oil and an aliquot sample of each
sample was taken for ammonia, urea and soluble protein analysis. The samples
were dissolved in 0.5 mol l-1 LiOH. LiOH dissolves the urate
precipitates and any trapped ions
(Roxburgh and Pinshow, 2002).
Clinical diagnostic kits (Sigma Chemical, St Louis, MO, USA) were used to
analyze uric acid (procedure no. 685) and urea (procedure no. 535). Ammonia
was assayed using the Roche ammonia UV-test (catalog no. 1-112-732, Roche,
Indianapolis, IN, USA). Total soluble protein was assayed with the Bio-Rad
Protein Assay Kit II (catalog no. 500-0002, Bio-Rad Laboratories, Hercules,
CA, USA). Because the uric acid in excreta samples was dissolved in lithium,
we constructed standard curves using both lithium and de-ionized water. Adding
lithium to our samples had no significant effect on the standard curves of
uric acid (ANCOVA on intercepts and slopes, P>0.1).
Nitrogen requirements
Nitrogen requirements and endogenous losses were determined by the
regression of the apparent nitrogen balance (nitrogen intake minus nitrogen
excretion) on nitrogen intake (Brice and
Grau, 1990; Korine et al.,
1996
; Witmer,
1998
; Roxburgh and Pinshow,
2000
; Pryor et al.,
2001
), using the total nitrogen recovered from the different
assays.
Statistical analysis
We used linear models to determine the effect of protein and sugar
concentrations on the intake response and to compare the nitrogen requirements
between the experimental days. ANCOVA was also used to compare plasma urate
concentrations between the species. Least-squares linear regression was used
to test for correlations between excretions of all forms of nitrogenous waste
and the water or nitrogen intake. Paired t-tests were used to
determine the significance of changes in Mb over the
experimental period and to compare the concentrations of nitrogenous waste
forms in ureteral urine and in excreta. A bird was considered ammonotelic if
more than 50% of its total nitrogen excretion was excreted as ammonia. Thus,
we used a one sample t- test to test for ammonotely in both species.
The exponent of the power relationship between volumetric intake and sugar
concentration in food should equal -1 if birds compensate perfectly for energy
dilution by increasing intake
(Martínez del Rio et al.,
2001). Thus, we used one sample t-test to test whether
the exponent of the power relationship differed from -1. Multiple regressions
were used to determine the effect of water and protein intake and of
experimental day on the percentage of nitrogen excreted as ammonia and as uric
acid. Values are reported as means ±
S.E.M.
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Results |
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Nitrogen requirements
Both species maintained constant Mb during experiments
(paired t-test before and after experiments: bulbuls,
t9=-1.7, P>0.1; grackles,
t8=1.1, P>0.1). During experiments, nitrogen
intake ranged from 0 to 1.1 g per day in grackles and from 0 to 0.58 g per day
in bulbuls (assuming 1 g protein, 0.16 g nitrogen). MNR and TENL were
calculated based on the nitrogen recovered from chemical assays. In a previous
study (Tsahar et al., 2005
),
we recovered over 80% of the elemental nitrogen in chemical assays. Thus, we
assumed that using the assayed nitrogen underestimates nitrogen requirements
only slightly.
In both species, a significant positive correlation was found between
nitrogen balance and nitrogen intake (Fig.
2). The estimated MNR and TENL for both species on the first and
third days and their expected values (based on
Robbins, 1993) are summarized
in Table 2. In both species MNR
and TENL after 72 h were lower than after 24 h of feeding on the experimental
diets. MNR decreased by 44% in grackles and by 47% in bulbuls, and TENL
decreased by 45% in grackles and by 40% in bulbuls. In grackles, only the
intercept changed significantly between days while the slope did not
(F(slope)1,14= 0.004, P>0.9;
F(intercept)1,14=-3.81, P<0.002), whereas in
bulbuls both differed between days (F(slopes)1,16=4.65,
P<0.05; F(intercepts)1,16=-6.04,
P<0.0001).
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Factors affecting the chemical composition of the nitrogenous waste
We used multiple regressions to assess the effect of water and protein
intake and experimental day on the percentage of nitrogen excreted as either
ammonia or urate. We included two data points for each of the nine grackles or
ten bulbuls in these regressions and hence our analysis can be considered
pseudoreplicated. When we included bird identity as a factor in the linear
model, its contribution was not significant. Hence we dropped individual
identity from the model. In bulbuls, the percentage of total nitrogen excreted
as ammonia did not depend on day (F1,16=0.03,
P=0.85), was negatively correlated with protein intake
(F1,16=5.9, P=0.03), and was positively
correlated with water intake (F1,16=18.7,
P=0.0005; Fig. 3,
Table 3). The percentage of
nitrogen excreted as uric acid was tightly and negatively related to the
percentage of nitrogen excreted as ammonia (%uric acid 100-%ammonia).
Thus, in bulbuls the proportion of nitrogen excreted as uric acid did not
depend on experimental day (F1,16=0.13, P=0.73),
but was positively related to protein intake (F1,16=8.68,
P=0.009) and negatively related to water intake
(F1,16=15.69, P=0.001). In grackles, the
percentage of nitrogen excreted as either ammonia or urate did not depend on
day (F1,14=4.29, P=0.06;
F1,14=1.72, P=0.21, respectively), protein intake
(F1,14=1.55, P=0.23;
F1,14=3.17, P=0.097, respectively), or water
intake (F1,14=1.21, P=0.29;
F1,14=1.07, P=0.32, respectively). Protein and
urea represented a very small percentage of all nitrogen excreted in both
species (Table 4). In both
species water intake decreased significantly between the first and third
experimental days (Table 4). In
bulbuls the percentage of nitrogen excreted as urate increased significantly
between the first and third day and the percentage of nitrogen excreted as
ammonia decreased significantly. In Tristram's grackles, the percentage of
nitrogen excreted as urate and ammonia did not change significantly between
experimental days.
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Taken together, we can conclude that bulbuls were ammono-uricotelic (Fig. 4). The percentage of ammonia in their excreta did not differ significantly from 50% (Table 4, one sample t19=0.13, P>0.5). In contrast, grackles were significantly uricotelic (t19=7.3, P<0.0001; Table 4, Fig. 4). However, pooling all individuals may not be appropriate for bulbuls. The results presented in Fig. 4A suggest that the percentage of nitrogen excreted as ammonia increased significantly with water intake. About 80% of all bulbuls that ingested more than 60 ml of water per day were ammonotelic. In contrast, all but one of the grackles were uricotelic under all conditions.
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Comparison between excreta and ureteral urine
In bulbuls, the concentrations of urate and soluble proteins were higher in
ureteral urine than in excreta (Table
5). However, the concentration of ammonia and urea did not differ
between ureteral urine and excreta. In grackles, the concentrations of urea,
soluble protein and ammonia were significantly higher in ureteral urine than
in excreta, but the concentration of uric acid did not differ between ureteral
urine and excreta (Table
5).
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Plasma uric acid
The plasma concentration of uric acid was positively correlated with
protein intake in grackles (r2=0.65,
F1,6=11.22, P=0.015;
Fig. 5). However, protein
intake did not affect the plasma uric acid concentration in the bulbuls
(F1,6=0.07, P=0.8;
Fig. 5). The average plasma
concentration of uric acid was lower in grackles (2.1±0.3 mg
dl-1) than in bulbuls (3.3±0.7 mg dl-1). Thus,
per unit of protein intake standardized by
mb0.75, bulbuls had a higher plasma uric acid
concentration than grackles (ANCOVAspecies:
F1,12=7.86, P<0.02).
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Discussion |
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Low nitrogen requirements in yellow-vented bulbuls and Tristram's grackles
Available evidence indicates that nectarivorous and frugivorous bird
species have unusually low nitrogen requirements and our results were
consistent with this observation (Brice and
Grau, 1990; Witmer,
1998
; van Tets and Nicolson,
2000
; Pryor et al.,
2001
; Roxburgh and Pinshow,
2000
; McWhorter et al.,
2003
). The nitrogen requirements of both bulbuls and grackles were
extremely low. Because we did not recover all the nitrogen in excreta, the
nitrogen requirements reported here are underestimated. However, in other
experiments in which birds have been fed nectar-like diets, we have found only
traces of other nitrogen-containing compounds such as creatine, creatinine,
free amino acids and bile salts in excreta
(McWhorter et al., 2003
). We
can conclude that our method underestimates nitrogen requirements only
slightly (Tsahar et al.,
2005
).
How birds can have such low nitrogen requirements remains enigmatic. They
may have lower rates of endogenous protein turnover and/or high nitrogen
recycling capacity (Witmer,
1998; Pryor et al.,
2001
). Birds excrete urate in their urine in the form of small
spherical concretions (Goldstein and
Skadhauge, 2000
). These concretions contain protein and inorganic
ions in addition to urate (Casotti and
Braun, 1997
). We found that protein concentration was lower in
excreta than in ureteral urine (Table
5). We hypothesize that some of the protein associated with urate
spheres is digested in the lower intestine and recovered. This hypothesis is
consistent with the findings of high activities of membrane-bound digestive
peptidases in the distal small intestine of the cedar waxwing Bombycilla
cedrorum (Witmer and Martínez
del Rio, 2001
). As protein represents only a small fraction of all
nitrogen excreted, this mechanism probably contributes to the low nitrogen
requirements of frugivores, but it does not explain it fully.
Are yellow-vented bulbuls facultatively ammonotelic, apparently ammonotelic, or both?
Preest and Beuchat (1997)
were the first to challenge the long-held notion of avian uricotely. They
showed that under certain conditions Anna's hummingbirds could excrete more
nitrogen as ammonia than as uric acid. They termed the ability to switch from
uric acid to ammonia as the primary waste product `facultative ammonotely'.
Roxburgh and Pinshow (2002
)
found that the Palestine sunbirds also switched from uric acid to ammonia
excretion under some conditions. In sunbirds, ammonotely was correlated with
low protein intake, whereas in hummingbirds it was associated with low ambient
temperature. Roxburgh and Pinshow
(2002
) found that ammonia
excretion remained relatively constant, whereas uric acid excretion increased
with increased protein intake. They claimed that the ammonotely observed in
sunbirds was only `apparent'. Since uric acid was the major nitrogen waste
form in the ureteral urine, no modification took place in the metabolic
pathway of nitrogen waste production in the birds; hence the birds were not
`truly' ammonotelic. In sunbirds, as in bulbuls, the concentration of urate
was higher in ureteral urine than in excreta. Hence, the apparent ammonotely
observed by Roxburgh and Pinshow
(2002
) and found in this study
in bulbuls is probably the result of post-renal urine modification. The term
`apparent ammonotely' complements the hypothesis of `facultative ammonotely'
by offering it a potential mechanism: post-renal urine modification.
The excretion of ammonia in the yellow-vented bulbul was positively
correlated with water intake. These results can explain the difference between
the results of the present study and those of van Tets et al.
(2001), who also studied the
effect of nitrogen intake on the composition of nitrogenous waste products in
the ureteral urine of yellow-vented bulbuls. The concentrations of urate and
urea that they measured in the ureteral urine were similar to those we found,
but those of ammonia were 4-30% lower. This discrepancy can be explained by
differences in the sugar concentration of the food that the birds ingested.
van Tets et al. (2001
) fed
their birds on a diet that contained 20% sugar, whereas we fed most of our
birds a diet that contained only 10% sugar. The water intake of birds on a 20%
sugar diet is 50% lower than that of birds fed on a 10% sugar
(Fig. 1). Although such a
correlation is expected from the observation that ammonia is highly toxic, and
its disposal requires large amounts of water, the mechanisms that lead to it
are unclear.
The percentage of nitrogen excreted as urate and ammonia was not affected by water intake in Tristram's grackles. However, we only tested this species with 20% sugar. Hence, grackles had relatively lower and less variable water intakes than bulbuls. It is possible that with a higher water intake the grackles would also have demonstrated ammonotely and that with a larger range of intakes, ammonotely and water intake would have been positively correlated.
How do bulbuls and sunbirds reduce the urate content in excreta: the mechanisms behind apparent ammonotely
The modification of urine in the lower gut of Palestine sunbirds and
yellow-vented bulbuls may be the result of two non-exclusive mechanisms:
bacterial breakdown of uric acid or reabsorption of uric acid by the birds. We
currently have no strong evidence for or against either of these mechanisms.
Hence this section must be conceived more as a description of hypotheses in
need of testing than an account of established mechanisms. Preest et al.
(2003) documented microbial
degradation of potassium urate (but curiously not of sodium urate) by microbes
extracted from the distal intestine of Anna's hummingbirds. Bulbuls also have
microbes with uricolytic activity in their lower gastrointestinal tract (E.
Tsahar, unpublished data). Microbial activity in the lower gut can have two
effects: (1) it can lower the concentration of urate, and (2) it can increase
the concentration of ammonia, which is a by-product of the microbial
degradation of urate (Karasawa et al.,
1988
). If birds recover the ammonia resulting from microbial urate
degradation, then this mechanism can improve the nitrogen economy of birds.
Some bird species have large ceca that contain sizeable populations of
microorganisms (Clench, 1999
).
In these species, nitrogen conservation through the breakdown of uric acid is
of significant physiological importance (Mortensen and Tindel, 1981;
Campbell and Braun, 1986
;
Karasawa et al., 1988
;
Karasawa et al., 1993
;
Son and Karasawa, 2000
).
Bulbuls and sunbirds have tiny vestigial ceca and hummingbirds have no cecum
at all. The physiological importance of the microbial degradation of urate, if
any, in these species remains unknown.
An alternative mechanism that can reduce the concentration of uric acid in
excreta is the potential presence of a specific urate transporter in the lower
guts of birds. Although we lack the evidence, we speculate that the bulbuls'
lower intestine might have the capacity to reabsorb urate. This speculation
prompts the question, why would it be advantageous for birds to recover a
nitrogenous metabolic waste? Although uric acid is considered primarily as a
nitrogenous waste, it also has a major function as a powerful antioxidant in
both mammals (Davies et al.,
1986; Becker, 1993
;
Schlotte et al., 1998
;
Spitsin et al., 2000
) and
birds (Simoyi et al., 2002
;
Somoyi et al., 2003). Uric acid may be an especially important antioxidant in
mammalian species, such as humans, that do not express the enzyme
L-gulonolactone oxidase (GLO) and hence lack the ability to
synthesize ascorbic acid (Chatterjee,
1973
), another potent antioxidant. Although GLO expression has not
been measured in yellow-vented bulbuls, these birds are members of a bird
lineage that lacks it. Tristram's grackles belong to a lineage that expresses
the enzyme (Martínez del Rio,
1997
). We suggest that uric acid may shift its functional role
from a waste product to a useful antioxidant in birds species that lack the
ability to synthesize ascorbic acid.
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Acknowledgments |
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