What future works have the authors mentioned in the paper "A biochemical study of fasting, subfeeding, and recovery processes in yellow-legged gulls" ?

Yellow-legged gulls showed a moderate physiological adaptation to extended fasting but the same model of biochemical changes in plasma that other more adapted species showed. Some differences in the results reported here with respect to a recent study on fasting in herring gulls can be explained by methodological interferences.

(Open Access) A Biochemical Study of Fasting, Subfeeding, and Recovery Processes in Yellow‐Legged Gulls (2001) | Carlos Alonso-Alvarez

A Biochemical Study of Fasting, Subfeeding, and Recovery Processes in Yellow‐Legged Gulls

Author(s): CarlosAlonso‐Alvarez and MiguelFerrer

Reviewed work(s):

Source:

Physiological and Biochemical Zoology,

Vol. 74, No. 5 (September/October 2001), pp.

703-713

Published by: The University of Chicago Press

Stable URL: http://www.jstor.org/stable/10.1086/322932 .

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703

A Biochemical Study of Fasting, Subfeeding, and Recovery Processes

in Yellow-Legged Gulls

Carlos Alonso-Alvarez*

Miguel Ferrer

Department of Applied Biology, Estacio´n Biolo´ gica de

Don˜ana, Pabello´n del Peru´ , Consejo Superior de

Investigaciones Cientiﬁcas, Avenida de Marı´a Luisa s/n 41013

Sevilla, Spain

Accepted 5/22/01

ABSTRACT

An investigation of the effects of fasting, subfeeding, and re-

feeding on plasma biochemistry was carried out on 22 captive

yellow-legged gulls Larus cachinnans Pallas. These birds showed

the same fasting endurance model described in other species,

but with an important decrease in glucose plasma concentration

and very great differences between individuals when reaching

the deterioration limit, suggesting a moderate physiological ad-

aptation to long periods of fasting. A different model was pro-

posed in subfed gulls in relation to fasted gulls, based on lipid

and protein use, which could be reﬂected by changes in nitrogen

wastes and triglyceride levels in this experiment. Thus, the sub-

fed gulls might use protein directly from the diet as an energy

source, thereby reducing the use of fat stores. The gulls quickly

recovered body mass during the refeeding period, but while

some plasma substances quickly reached their initial values,

others showed many changes before the end of the experiment,

which could reﬂect a process of metabolic restabilization. These

results contribute to a better knowledge of fasting, subfeeding,

and refeeding processes in birds and can be added to a recent

study about fasting in gulls.

Introduction

Many studies have been developed on the physiological re-

sponse of birds when enduring food restriction. These works

suggest three different phases based on changes in body weight

and plasma biochemistry (see Fig. 1), which we will name the

“classic model” (e.g., Le Maho et al. 1981; Boismenu et al. 1992;

* Corresponding author; e-mail: alonso@ebd.csic.es.

Physiological and Biochemical Zoology 74(5):703–713. 2001. 䉷 2001 by The

Handrich et al. 1993b). In a ﬁrst phase (phase 1), body weight

shows an important reduction in a short interval of time,

whereas the second phase (phase 2) presents a slow and stable

daily weight descent, during a more or less long period, de-

pending on the species. The ﬁnal phase reveals a quick and

strong increase in body-mass loss to reach a critical level close

to death (phase 3). The fasting-adapted species relies on fat as

the primary energy source during fasting periods, spares pro-

tein, and relies primarily on protein only when fat reserves are

depleted (see, e.g., Cherel et al. 1988a). Protein is spared due

to its key role in body structure and muscle function and as

enzymes (Felig 1979; Castellini and Rea 1992). In this model,

plasma levels of residuals from protein catabolism (urea and

uric acid) decrease during phase 1, maintain a stable low con-

centration or a slow increment in phase 2, and rise suddenly

to reach the highest levels during phase 3 (use of structural

proteins as energy source), which might indicate the bird’s

death. Changes in excretion of these nitrogen residuals are in

fact parallel to those observed in daily body-mass change. How-

ever, in birds, as in mammals, by far the largest reservoir of

body fuel is in the form of fat, stored as triglycerides (Cherel

et al. 1988a; Castellini and Rea 1992). In the classic model, free

fatty acids and ketone bodies increased in blood plasma in phase

1 as a consequence of triglycerides breaking down, which re-

ﬂects their use as an energy source; they maintain high con-

centrations in phase 2 and abruptly decrease in phase 3 (ex-

haustion of fat stores). Plasma triglycerides steadily decrease

during fasting (Fig. 1), though they were not described on the

basis of the classic model (Jenni-Eiermann and Jenni 1994).

Finally, glucose maintains its concentration, only decreasing in

phase 3 (Cherel et al. 1988b; Boismenu et al. 1992), reﬂecting

its importance in birds’ metabolism. In fact, this carbohydrate

is a critical fuel for the central nervous system, and its circu-

lating concentration is tightly regulated (Castellini and Rea

1992).

On the other hand, the physiological means for supporting

absolute fasting could be different from a limited food restric-

tion, a phenomenon probably more extended in species with

very diverse food resources, such as gulls (Cramp and Simmons

1983; Munilla 1997). Comparison between the effects of a re-

duced diet in relation to the effects of fasting in wild species

of birds has not been documented in bibliographies, as far as

we know. This kind of study might help develop understanding

of the usual physiological state of many individuals during

subfeeding periods.

In contrast with the studies on fasting, the refeeding period

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704 C. Alonso-Alvarez and M. Ferrer

Figure 1. Ideal pattern of changes in plasma composition and body

mass during fasting in bird species (classic model). Daily body-mass

loss is the body-weight proportion with respect to the previous fasting

day (the scale on Y-axis is only illustrative).

in wild bird species has been poorly studied. Handrich et al.

(1993a) proposed a model consisting of two phases. The ﬁrst

one is the recovery of initial body weight and the restoration

of prefasting metabolic rates. The second phase is a period of

steady body mass and metabolism. The Garcı´a-Rodrı´guez et al.

(1987) study showed a slow recovery of body weight in buzzards

(Buteo buteo Linnaeus) throughout the refeeding period, while

plasma uric acid levels declined abruptly at the beginning but

slowly reached the original concentrations of fed birds, in agree-

ment with the second phase proposed by Handrich et al.

(1993a). More research on this process is needed.

In summary, this study has three objectives: (1) to know

whether a seabird, the yellow-legged gull Larus cachinnans Pal-

las, uses the above-cited classic model of resource allocation

during starvation, as shown in other bird species (thus, we will

analyze differences with respect to the described pattern; Fig.

1). Moreover, (2) this species was chosen in order to compare

changes during periods of moderate food restriction with pe-

riods of absolute fasting. Therefore, we might obtain a more

realistic approach to some bird life histories. Finally, (3) we

also studied the recovery process in the yellow-legged gull. With

these three objectives, we analyzed 12 plasma parameters, rep-

resentatives of proteins and their catabolic residuals (total pro-

tein, urea, and uric acid), fats such as triglycerides and cho-

lesterol, carbohydrates such as glucose, and some enzymes and

ions.

Material and Methods

Experimental Procedure

On February 28, 1999 (2 mo before the laying date in the

colony), we captured 22 adult yellow-legged gulls on a refuse

dump close to the city of Porrin˜o (Pontevedra, Spain). These

birds were transported to the wildlife recovery center La Can˜ada

de los Pa´jaros (Huelva, Spain) and were housed in individual

cages ( m). The study was performed with the per-4 # 4 # 4

mission of the appropriate authorities, avoiding any damage to

the birds. The gulls were divided into three groups, and the

sex ratio was balanced. Sex was determined through the PCR

ampliﬁcation of CHD gene fragment sequences following Grif-

ﬁths et al. (1998). In this way, nine individuals formed the

fasting group (four males, ﬁve females), nine formed the re-

stricted group (four males, ﬁve females), and the last four birds

constituted the control group (two males, two females). For 2

wk, sardines (Sardina pilchardus Walbaum) were provided ad

lib. Fish is present in 32% of the pellets in the original pop-

ulation of the experimental birds (sardines included; Munilla

1997), and its biomass proportion might be even higher. After

this, from day 0 of the experiment the fasting group remained

without food, the restricted group was fed one-third of the

mean daily intake (calculated individually for each gull during

the previous 2 wk), and the control group remained with ad

lib. food. This interval was called the deterioration period. All

birds had water ad lib. during the experiment. Variable total

body-mass loss was deﬁned as the proportion of body-mass

loss regarding weight at the beginning of the experiment. The

return to feeding (recovery period) was planned when birds

would reach phase 3 of the classic model described in other

species (i.e., Boismenu et al. 1992), but since there was no

previous information about critical levels of body-mass change

or biochemical parameters in this species, an a priori limit of

total body-mass loss was ﬁxed at 25% to start refeeding the

birds. This limit was estimated conservatively from the pro-

portion of total body-mass loss of three ill individuals (captured

in previous years), which was calculated from the expected body

weights regarding their body size in the original population (C.

Alonso-Alvarez and M. Ferrer, unpublished observations).Nev-

ertheless, after 8 d, three gulls from the fasting group died on

the same day, without symptoms, and their lesser total body-

mass loss the day before (15%) was ﬁnally established as the

ﬁnal limit before placing the gulls in the recovery period, re-

feeding them with food ad lib. Another two birds, one from

the fasting group and the other from the restricted group, were

retired after phase 3 because of a clear risk of death (inability

to walk), and they were treated with glucosaline serum and

vitamins so that they could recover their initial healthy state.

The experiment ﬁnished when all the birds reached the con-

ﬁdence limits of total body-mass loss in relation to capture.

After this, the gulls were set free in the original capture location.

Blood Extraction and Weighing Procedures

Blood samples were taken from the humeral vein (2.5 mL)

every 2 d throughout the experiment, always before feeding, at

the middle of the day (1100–1500 hours) to avoid any variation

in blood chemicals caused by the circadian rhythm (Ferrer

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Fasting, Subfeeding, and Recovery in Yellow-Legged Gulls 705

1993). Winged infusion sets (Valu-Set, Becton Dickinson,

Sandy, Utah) were used to prevent damage to the veins, ap-

plying them on alternate wings each time. Blood sampling was

done immediately after capture. Lithium-heparin was employed

as anticoagulant, and the samples were stored between 0⬚ and

4⬚C until they were carried to the laboratory a few hours after

collection. Plasma was separated by centrifugation (550 g for

10 min) and was stored in a freezer for 1 d until the analysis.

The gulls were weighed after blood collection with a dyna-

mometer (Pesola; accuracy Ⳳ5 g). Daily body-mass loss rep-

resented the change in body weight with respect to the day

before sampling, allowing us to see small changes from day to

day.

Tested Parameters

Twelve biochemical components of blood were measured using

a spectrophotometer (Hitachi 747, Tokyo, Japan) and com-

mercial kits (Boehringer-Mannheim Biochemica, Mannheim,

Germany). The analyzed biochemical parameters were (abbre-

viations and methods indicated in parentheses): urea (UREA;

urease method), uric acid (URIC; uricase method), triglycerides

(TRIG; enzymatic method that includes amounts of free glyc-

erol), total protein (TP; biuret reaction), creatinine (CREA;

kinetic Jaffe´ reaction), inorganic phosphorus (iP; molybdenum

blue reaction), calcium (Ca; cresolphtalein complexone reac-

tion), magnesium (Mg; blue xilidil reaction), glucose (GLUC;

hexocinase method), cholesterol (CHOL; cholesterol esterase),

amylase (AMY; maltoheptaose reaction), and alkaline phos-

phatase (AP; paranitrophenyl-phosphate method).

Data Analysis

Mean values of parameters were tested for differences between

groups on the same day or in the same mass-loss rank by the

Mann-Whitney U-test for independent samples. Within-group

variations were tested with Wilcoxon matched pairs signed-

ranks test. These nonparametric tests were used as a precaution

since, as a result of small sample sizes in some analyses, normal

distribution could not be ascertained for all parameters. The

experiment effects were examined with repeated-measures

ANOVA, where the treatment (fasting or subfeeding) was used

as a factor (between-subject effect) and the samples obtained

from the same bird throughout the experiment were used as

repeated measures (within-subject effect). Moreover, repeated-

measures ANOVA was used to analyze changes in mass or

biochemical parameters in each group separately. A general

linear model of variance analysis was developed in order to

determine the inﬂuence of the treatment (group as ﬁxed factor)

and the inﬂuence of proportion of body-mass loss (total body-

mass loss as covariable) in each biochemical parameter (de-

pendent variable) throughout the experiment, using the indi-

vidual as a random factor in order to avoid pseudoreplication.

All tests were performed with SPSS software (Norusis 1993).

Results

Initial and Final Values in the Deterioration Period

Body mass ( ; males: ; females: 733.1 Ⳳmean Ⳳ SE 898.9 Ⳳ 24.2 g

7.9 g) and plasma biochemical values were measured the ﬁrst

day of the experiment in all birds. That day, there were no

differences among the three groups or between sexes (Mann-

Whitney: in all parameters). The four birds from theP

1 0.05

control group did not show signiﬁcant variations in total body-

mass loss (repeated-measures ANOVA: , )F p 1.07 P p 0.41

10, 30

and plasma biochemical traits (always ) throughout theP 1 0.05

experiment and are not used in the rest of the statistical anal-

yses. There were many signiﬁcant differences (Wilcoxon: P

) in plasma concentrations between the ﬁrst day and the0.05

last day of the deterioration period in gulls suffering fasting or

food restriction (see Table 1). Urea, uric acid, cholesterol, glu-

cose, and alkaline phosphatase changed in both groups. There

were no signiﬁcant differences between these two groups the

last day of the deterioration period regarding all the parameters,

but inorganic phosphorus, calcium, and magnesium showed a

tendency toward higher values in the fasting group (Mann-

Whitney: ).P

! 0.12

Weight and Biochemical Changes with Respect to the Classic

Model

In order to explain the changes in body mass throughout the

deterioration period, we analyzed daily body-mass loss and total

body-mass loss during the fasting phases according to the classic

model (Fig. 2). For both variables, data of the ﬁrst four sam-

pling days from the beginning and, separately, data of the last

four sampling days to reach the ﬁnal limit of the deterioration

period were analyzed ( in each group) in order to equil-n p 9

ibrate the sample size between the groups. The sample size on

some days was not equilibrated because of the highly variable

number of days to reach the ﬁxed deterioration limit among

individuals (fasting group: 8–12 d; restricted group: 10–18 d).

Daily body-mass loss did not show signiﬁcant within-subject

differences in the ﬁrst four sampling days (repeated-measures

ANOVA: , ), but it did show such a dif-F p 1.41 P p 0.25

3, 48

ference in the last 4 d ( , ). The differencesF p 15.77 P ! 0.001

3, 48

between groups in the ﬁrst four measurements and in the last

4 d (Fig. 2) showed a tendency to statistical signiﬁcance

( , ; and , , respec-F p 3.07 P p 0.09 F p 3.47 P p 0.08

1, 16 1, 16

tively), showing lower values in the restricted group (Fig. 2).

A descent in daily body-mass loss between the second and the

fourth day (proposed phase 1) were not signiﬁcant in either

group (Wilcoxon: , in both groups). DailyZ p 0.77 P p 0.44

body-mass loss measurements in the last day of the deterio-

ration period (in proposed phase 3) were higher in the fasted

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706 C. Alonso-Alvarez and M. Ferrer

Table 1: Mean values (ⳲSE) of plasma parameters the ﬁrst and the last day of the

deterioration period, differentiated by groups

Biochemicals

Fasting Group

Restricted Group

First Day Last Day First Day Last Day

Urea (mg/dL) 5.78 (.22) 13.22 (1.63)* 5.33 (.33) 11.67 (1.54)**

Uric acid (mg/dL) 9.97 (1.05) 20.32 (2.93)* 10.51 (1.98) 18.17 (2.49)*

Total protein (g/dL) 3.35 (.36) 3.05 (.46) 3.17 (.19) 2.22 (.28)**

Triglycerides (mg/dL) 75.56 (10.34) 53.56 (9.90) 73.67 (4.92) 32.22 (4.42)**

Cholesterol (mg/dL) 324.6 (35.4) 201.9 (24.9)* 375.6 (19.7) 181.6 (15.5)**

Glucose (mg/dL) 365.8 (15.9) 267.8 (11.9)** 325.8 (9.49) 282.4 (9.0)*

Amylase (U/L) 815.4 (62.7) 653.9 (22.4) 927.6 (88.3) 890.1 (115.3)

Creatinine (mg/dL) .26 (.003) .22 (.003) .26 (.002) .17 (.002)**

AP (U/L) 230 (52.2) 99.56 (34.1)* 223.2 (57) 172.9 (44.1)*

Pi (mg/dL) 3.28 (.25) 4.75 (.63)* 3.18 (.16) 3.57 (.30)

Ca (mg/dL) 8.73 (.50) 8.25 (.54) 8.69 (.23) 7.17 (24)**

Mg (mg/dL) 2.23 (.006) 2.26 (.13) 2.22 (.008) 2.01 (.005)

Note. Wilcoxon matched pairs signed-ranks test; in each group. There are no differences among groupsn p 9

on the ﬁrst ( ) or the last day ( ).P

1 0.1 P 1 0.05

*.P

! 0.05

** .P

! 0.01

gulls than in the restricted gulls (Mann-Whitney: ,Z p 1.99

; see Fig. 2).P p 0.047

Concerning the biochemical parameters, we focused on uric

acid and triglycerides as representatives of nitrogen residuals

and fat use, respectively, synchronizing newly recorded data

with respect to the ﬁrst and the last day of the deterioration

period (in Fig. 3, backward from last day). In the fasting gulls,

uric acid increased in the ﬁrst 4 d of the sampling and in the

last four (repeated-measures ANOVA: , ;F p 7. 2 1 P p 0.001

3, 24

and , , respectively). In the same group,F p 4.48 P p 0.012

3, 24

triglycerides increased in the ﬁrst four measures ( ,F p 6.66

3, 24

) and decreased in the last four ( , ).P ! 0.01 F p 4.03 P ! 0.05

3, 24

Differences between groups were detected in the last 4 d of the

deterioration period (uric acid: , ; triglyc-F p 8.24 P p 0.01

1, 16

erides: , ), but they were not signiﬁcant inF p 11.23 P ! 0.01

1, 16

the ﬁrst 4 d (uric acid: , ; triglycerides:F p 4.29 P p 0.06

1, 16

, ). When we observed the deteriorationF p 2.25 P p 0.16

1, 16

period as a whole (see Table 2, “Group” column), we observed

signiﬁcant differences in triglycerides but not in uric acid, al-

though it was close to statistical signiﬁcance ( ; see alsoP p 0.07

Fig. 5).

Weight Changes in the Recovery Period

Changes in total body-mass loss during the recovery period

were used to explain the return of our gulls to the initial body

weight (Fig. 4). The values at last day of the deterioration period

were signiﬁcantly higher than the values at the ﬁrst sampling

day of the recovery period (Wilcoxon; fasting group: Z p

, ; restricted group: , ). Thus,2.02 P p 0.043 Z p 2.52 P p 0.012

afteronly2dofrefeeding, the birds recovered a great part of

mass they had lost, without differences between both groups

( ; fasting group: ; restricted group:mean Ⳳ SE 49.3% Ⳳ 10.5%

; Mann-Whitney: , ). There69.9% Ⳳ 6.17% Z p 1.71 P p 0.24

were not signiﬁcant differences between groups throughout the

recovery period (repeated-measures ANOVA: ,F p 0.04

1, 11

). However, there was a signiﬁcant decrease in totalP p 0.86

body-mass loss in the fasting group (within-subject effect:

, ) but not in the restricted groupF p 7. 9 0 P p 0.001

4, 24

( , ). Nevertheless, only a nonsigniﬁcantF p 1.77 P p 0.16

4, 39

tendency to higher values of mass loss in the restricted group

in the last day was detected (Mann-Whitney: ,Z p 1.70 P p

; Fig. 4).0.09

Changes in Biochemical Variables throughout the Experiment

In this study we confronted two problems in the interpretation

of data. The ﬁrst one was the individual differences in the

number of days to attain the deterioration limit (commented

on above), which prevents changes from being analyzed with

respect to a chronological order. The second problem was the

obvious differences in total body-mass loss level between the

groups in the same day, promoted by the effect of the treatment.

This issue prevents between-group comparisons. With the aim

of avoiding these problems, the changes were analyzed using a

general linear model (Table 2), which allowed testing of the

linear relationship of each biochemical trait with the proportion

of total body-mass loss, but not with time.

In addition, we analyzed data by means of total body-mass

loss ranks (Fig. 5), which allowed comparison of the plasma

levels among the groups when the birds were in a similar body-

mass proportion. Figure 5 included in the deterioration period

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A Biochemical Study of Fasting, Subfeeding, and Recovery Processes in Yellow‐Legged Gulls

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References

Handbook of the birds of Europe, the Middle East and North Africa

A DNA test to sex most birds

A DNA test to sex most birds.

Physiology and biochemistry of long-term fasting in birds

Gastrointestinal Anatomy and Physiology

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Frequently Asked Questions (2)

Q1. What are the contributions mentioned in the paper "A biochemical study of fasting, subfeeding, and recovery processes in yellow-legged gulls" ?

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