Accepted Manuscript

Title: Effect of fasting in the digestive system: Histologicalstudy of the small intestine in house sparrows

Author: Samanta Celeste Funes Veronica Palmira FilippaFabricio Damian Cid Fabian Mohamed EnriqueCaviedes-Vidal Juan Gabriel Chediack

PII: S0040-8166(14)00056-1DOI: http://dx.doi.org/doi:10.1016/j.tice.2014.06.007Reference: YTICE 875

To appear in: Tissue and Cell

Received date: 31-8-2013Revised date: 27-5-2014Accepted date: 20-6-2014

Please cite this article as: Funes, S.C., Filippa, V.P., Cid, F.D., Mohamed, F.,Caviedes-Vidal, E., Chediack, J.G.,Effect of fasting in the digestive system:Histological study of the small intestine in house sparrows, Tissue and Cell (2014),http://dx.doi.org/10.1016/j.tice.2014.06.007

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Effect of fasting in the digestive system: Histological study of the small intestine in house

sparrows.

Samanta Celeste Funes1, Verónica Palmira Filippa3, Fabricio Damián Cid1,2, Fabián

Mohamed3, Enrique Caviedes-Vidal1,2 and Juan Gabriel Chediack1,2*

1Laboratorio de Biología Integrativa. Instituto Multidisciplinario de Investigaciones

Biológicas de San Luis (IMIBIO-SL). CCT-San Luis. CONICET. San Luis, Argentina; 2Área

de Biología; 3Área de Morfología. Departamento de Bioquímica y Ciencias Biológicas.

Facultad de Química, Bioquímica y Farmacia. Universidad Nacional de San Luis. San Luis,

Argentina.

Figures: 6, tables: 2

Running title: Histological changes of intestine during fasting

*Correspondence to: Juan Gabriel Chediack. Postal address: Chacabuco 917, post code:

D5700HHW, San Luis, Argentina. Telephone number: 54-0266 4423789 ext. 6259. Fax

number: 54-0266 4430224 E-mail: jg.chediack@gmail.com.

Grant sponsor. Consejo Nacional de Investigaciones Científicas y Técnicas; Grant number

998 PIP2010-2012 CONICET and Grant number 0110, Ciencia y Técnica – Universidad

Nacional de San Luis to Juan Gabriel Chediack. Grant number 9502, Ciencia y Técnica –

Universidad Nacional de San Luis to Enrique Caviedes-Vidal.

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ABSTRACT

In birds and mammals the metabolic response to fasting has been studied and can be

characterized by three consecutive phases reflecting metabolic and physiological adjustments.

An effective way to minimize energy expenditure during food scarcity is to decrease the mass

of the organs. As the digestive system is metabolically expensive to maintain, the small

intestine and the liver are the most affected organs. We evaluated the effects of phase III

starvation on the mass of the different organs and histological parameters on house sparrows,

a small non-migrant bird. In a short period of time (34 hours) we observed a larger reduction

in the digestive organ mass when compared to the mass of the body and non-alimentary

tissues. Furthermore, the intestinal mass was proportionally more reduced than its length and

nominal surface area. A reduction on the intestinal mucosal layer also resulted in a shortening

of villus (length and thickness) and crypt depth. Moreover, the morphology of the enterocytes

changed from cylindrical to cubical, suggesting that the surface exposed to the lumen was

conserved. This may indicate an adaptive response to the moment of refeeding. The nominal

surface area/body mass remained constant in both groups and several histological parameters

were reduced, suggesting that starving induces the atrophy of the small intestine. However,

the goblet cells were conserved after fasting indicating a protective tendency.

Keywords: Starvation, gastrointestinal tract, histological parameters, non-migrant birds.

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INTRODUCTION

In birds and mammals the effects of fasting can be described by three consecutive phases.

These phases can be defined by progressive metabolic and physiological changes (Chediack

et al., 2012; McCue, 2010; Wang et al., 2006). Phase I occurs immediately after the last food

has been absorbed by the small intestine. It is characterized by the use of the liver glycogen

stores and a significant reduction in body mass in a short period of time. Phase II is

characterized by energy preservation and lipid oxidation. Since lipids have a high amount of

energy, weight loss is slow during this stage. Finally, during phase III, when lipid deposits are

almost depleted, muscle protein catabolism begins combined with a decrease in protein

synthesis. During this phase, there is a remarkable reduction in body and organ mass (Wang

et al., 2006) resulting from starvation combined with the energy demands required to maintain

the body homeostasis (Bauchinger et al., 2005). Thus, an effective way to minimize energy

expenditure during food scarcity is to decrease the mass of certain organs.

The digestive system is metabolically expensive to maintain, the regulation of its

physiological functions requires about 17-25% of the whole body oxygen consumption (Cant

et al., 1996 ). It has been found that the small intestine and the liver are the most affected

organs during fasting (Chediack et al., 2012; Ferraris and Carey, 2000; Starck, 2003).

Throughout starvation animals display morphological changes in the gastrointestinal tract.

These include a decrease in the length and mass of the small intestine, a decrease in mucosal

weight, changes in villus length and thickness, and phenotypic changes in the enterocytes

(Dunel-Erb et al., 2001; Karasov et al., 2004; Zeng et al., 2012). In rats and birds the

generative components (crypts) of the mucosa are preferentially preserved in comparison with

the absorptive part (villi) during fasting (Dunel-Erb et al., 2001; Karasov et al., 2004). The

effects of fasting on the muscular layer are important in rats (Dou et al., 2002), while absent

in fish and passerine wild birds (Karasov et al., 2004; Zeng et al., 2012). Several factors

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including body mass affect the rate at which starving animals (birds and mammals) lose body

mass, making it highly variable. On a comparative level, mammals and birds with similar

body mass, such as sparrows and mice (~ 25 g each), have a notable difference in their fasting

tolerance times, 3-4 days for the mouse (Sokolovic et al., 2007; Wang et al., 2006) and 31-36

hours for the sparrow (Chediack et al., 2012; Khalilieh et al., 2012). This disparity between

mammals and birds may be explained by the higher basal metabolic rate of the latter, between

30 to 40% higher than that of mammals (McNab, 2009). In mice there are controversial

observations about the significance of the structural changes observed during starvation,

mainly in the third phase, where some researchers found varied histological changes while

others did not (Chappell et al., 2003; Nian et al., 2002; Shin et al., 2005; Sokolovic et al.,

2007). In birds, observations in a migrant passerine wild bird (Sylvia atricapilla) showed a

significant effect of fasting in the reduction of the structure of the gastrointestinal tract

(Karasov et al., 2004). Despite, previous studies in house sparrows reports changes in organ

morphology during fasting (Chediack et al, 2012), this is the first exhaustive analysis of

intestinal histology changes on a non-migrant adult passerine bird undergoing fasting.

Our objectives were to evaluate the effects of long-term starvation on organ mass loss,

and show how starvation affects the histology of the small intestine (perimeter, muscular

layer, mucosal layer, villus length, crypt size, enterocyte morphology and goblet cell).

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MATERIALS AND METHODS

1.1. Animal care and housing

Adult house sparrows (Passer domesticus) were captured with a live trap near the

Universidad Nacional de San Luis campus (San Luis, Argentina). The birds were housed in

cages (40 x 25 x 25 cm) indoors under relatively constant environmental conditions (23 ± 1

°C and 40 ± 10% of relative humidity) on a photoperiod of 14:10 hours (Light:Dark) with

food and water ad libitum (seeds supplied with vitamins and minerals). Animals were

acclimatized to laboratory conditions for at least two weeks prior experimentation. Animal

care and trial protocols (protocol number N° B69/09) were approved by the committee of

Universidad Nacional de San Luis (CICUA).

1.2. Experimental design

1.2.1. Experiment: Effect of long-term fasting on organ mass and intestinal

morphometric parameters.

Adults house sparrows (n=22) were randomly assigned to either the feeding or long-term

fasting group with water ad libitum (n=11 for each experimental group). The mean body

mass of both groups was similar at the beginning of the experiment (25.66 ± 0.88; 25.56 ±

0.40, p>0.92). On the fasted group, food was removed 2 hours after lights turned on (8:00

h). 24 hours later the bird’s body mass was measured every 2–3 hours until phase III of

fasting, when animals lost more than 15% of their body mass (~31–34 hours). At the end

of the experiment, birds were anesthetized using ketamine and xilacyne (Paul-Murphy and

Fialkowski, 2001), the abdominal cavity opened and the entire gastrointestinal tract

removed and chilled in ice-cold avian saline buffer. The small intestine, stomach,

pancreas, heart and liver were removed, cleaned of extraneous tissue and weighed. The

small intestine lumen was washed with ice-cold avian saline buffer to remove digesta and

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measured. For histological procedures four animals were used in each group, the

remaining animals were used for another experiment.

1.2.2. Enterocytes isolation

Enterocytes were isolated using a modification of a classical chemical method

adjusted for birds (MacDonal et al., 2008). Briefly, small intestinal segments were washed

with ice-cold Hanks' balanced salt solution supplemented with mannitol (HBSS–mannitol, pH

7.2). Intestinal segments were submerged in 1 mL of ice-cold chelation buffer containing: 30

mM EDTA, 1.94 mM DL-dithiothreitol, 52 mM NaCl, 4.39 mM KCl, 10 mM L-1N-2-

hydroxyethylpiperazine-N′-2- ethanosulfonic acid (HEPES), 60 mL/L HCl 1 N (pH 7.1) for

75 min at 4 °C without shaking. After that the chelation buffer was gently discarded and 1 mL

of HBSS–mannitol added and cells vigorously shaken for 30 s. Cells were collected and

centrifuged at 100g during 5 min at 4 °C. The pellet was washed three times by successive

resuspension in 1 mL of HBSS–mannitol. The cells were dispersed by passing them several

times through a hypodermic needle and centrifugation at 100g for 5 min at 4 °C. The protein

concentrations of the enterocytes samples in the three intestinal portions were estimated using

the Bradford protocol.

1.3. Analytical procedures

1.3.1. Histological procedure

Proximal, medial and distal intestinal portions were placed in individual vials

containing fresh Bouin solution (saturated solution of picric acid, with glacial acetic acid and

formol 40%) and allowed to fix for 6 to 12 hours at room temperature, for posterior

processing. The intestinal pieces were dehydrated in increasing ethanol

concentrations, cleared in xylene, embedded in paraffin and five-µm thick sections were

obtained with a microtome (Microm HM 325). Sections were mounted on slides, stained with

hematoxylin-eosin or Periodic acid-Schiff (PAS) methods and covered with cover glasses.

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A computer-assisted image analysis system was used to measure the percentage of

area of PAS-positive vesicles and the number of cells per reference area, circumference of the

serosal surface, length and width of villi, width of the crypts, length and width of enterocytes.

The system consisted of an Olympus BX-40 binocular microscope interfaced with a host

computer, image processing and recording system. The images were captured by a Sony SSC-

DC5OA camera (Sony Corp., Tokyo, Japan) and processed with Image-Pro Plus 5.0 software

(Media Cybernetics Inc., Bethesda, MD, USA) under control of a Pentium IV computer. The

software allowed the following processes: image acquisition, automatic analogous

adjustment, thresholding, background subtraction, distance calibration, area and diameter

measuring, and disk data logging. The image was displayed on a color monitor, and the

parameters were measured with the image analysis system. Before counting, a standard area

of 18,200 µm2 (reference area, RA) was defined on the screen, and distance calibration was

done using a slide with a micrometric scale for microscopy (Reichert, Austria).

• Percentage of area of PAS-positive vesicles (% AV) was calculated using the

formula % AV=∑Av/∑RA×100, where ∑Av is the sum of the area of PAS-positive vesicles

and ∑RA is the sum of the small intestine area of every microscopic field.

• The number of goblet cells (No. cell/RA) with a visible nucleus was counted in 10

microscopic fields per section. The result was expressed as number of cells per RA.

• In each section the circumference of the serosal surface, length and width of villi,

width of the crypts and length and width of enterocytes was measured. At least 30

measurements per section were taken, resulting in more than 90 measurements per individual.

Only villi that were cut in their midline, from tip to base, were measured, as verified by

observations of similarly sized and shaped enterocytes. These data were used to estimate the

surface area enlargement factor (SEF) by a simple method (Kisielinski et al., 2002). To avoid

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inflation of degrees of freedom by repeated measurements within individuals, means and

standard deviation were calculated for individual birds. These means were used in statistical

analyses.

1.4. DATA ANALYSIS

Results are given as means ± 1 SE, the number of individuals (n) was 11 for the

morphological and 4 for the histological study. Morphological (body mass, intestine,

pancreas, liver, stomach and heart mass) and histological (perimeter, muscular layer, mucosal

width, villus, crypts, enterocytes, goblet cells and PAS-positive vesicles) parameters were

analyzed by T-tests for independent samples. Repeated measures analysis of variance (RM-

ANOVA) followed by Tukey post-hoc test was used to examine the effect of fasting on

histological parameters at different intestinal positions. If required, data were first

transformed to achieve homogeneity of variances and normality of distribution, using natural

logarithm or square root transformations. Normality of data was checked by Shapiro-Wilk

test, and Homoscedasticity by Levene´s test and homocedasticity of the covariance tested

with the Box M test. The significance level selected to accept a difference for all statistical

analyses performed was α<0.05. Association between histological parameters (perimeter,

muscular layer, mucosal width, villus, crypts and enterocytes) was analyzed by Pearson

product-moment correlation coefficient.

2. RESULTS

2.1. Variation in organ masses

The mass of all the measured organs (g) was reduced after prolonged fasting (liver

0.82 ± 0.066 vs. 0.45 ± 0.023; pancreas 0.13 ± 0.011 vs. 0.10 ± 0.005; stomach 0.78 ± 0.060

vs. 0.59 ± 0.025; heart 0.37 ± 0.015 vs. 0.28 ± 0.016). The length (cm) and mass (g) of the

small intestine was significantly reduced (~14% and ~40% respectively) compared to non-

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fasted animals (length 17.23 ± 0.742 vs. 14.83 ± 0.505; mass 1.08 ± 0.150 vs. 0.65 ± 0.048),

in all cases p<0.05, see Table 1A).

When normalized to body mass, fasted animals showed no difference in intestine

length, heart, pancreas, and stomach mass compared to controls (small intestine cm/g 0.66 ±

0.069 vs. 0.70 ± 0.022; heart g/g 0.0153 ± 0.0006 vs. 0.0136 ± 0.0007; pancreas g/g: 0.0051 ±

0.0004 vs. 0.0048 ± 0.0003; stomach g/g: 0.0305 ± 0.0019 vs. 0.0282 ± 0.001). However, the

differences in liver and intestine mass were still significant after normalization to body mass

(liver g/g: 0.0301 ± 0.0035 vs. 0.021 ± 0.001; small intestine g/g: 0.0413 ± 0.0044 vs. 0.030 ±

0.022). Similarly, the ratio between intestinal mass and length was decreased in fasting

animals (0.062 ± 0.006 vs. 0.043 ± 0.003) (see Table 1B for p-values).

Moreover, the nominal surface area (cm2) shrank (12.82 ± 0.773 vs. 9.65 ± 0.859;

unpaired t-test p<0.018) and the protein concentration (µg per mg of enterocytes) decreased

35% between the control and fasted groups when looking at the whole intestine (0.114 ±

0.0149 vs. 0.073 ± 0.0104, unpaired t-test p<0.028).

2.2. Intestinal morphologic observations

Fasting reduced the mucosal layer thickness in the whole intestine, and in the proximal

portion this reduction was more noticeable (Figure 1). The connective tissue (lamina propria)

decreased in the center of the villi in fasting birds, concomitantly with decreased villi

thickness (Figure 2b and c). The intestinal epithelium conserved its characteristic

morphology; however, less developed lacteal ducts and enterocyte height reduction were

observed after fasting (Figure 3). Also, Lieberükhn crypts and the number of mitotic cells

were reduced in fasting birds compared to controls (Figure 2).

Goblet cells were studied by the technique of PAS. The abundance of goblet cells

increased from the proximal to the distal portions of the small intestine in both groups (Table

2, Figure 4). There was no quantitative difference between the abundance of goblet cells on

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control and fasted groups (Table 2). Big PAS-positive vesicles were observed “moving” from

crypts to intestinal lumen in both groups in different intestinal portions (Figure 5). The

abundance (%) of PAS-positive vesicles was higher in fasted than in the control group (Table

2).

2.3. Intestinal morphometric parameters

The perimeter (mm) of the intestine of fasted animals was significantly reduced in all

portions (Proximal: 9.25 ± 0.289 vs. 7.61 ± 0.369; Medial 7.20 ± 0.100 vs. 6.55 ± 0.256;

Distal: 6.33 ± 0.219 vs. 5.76 ± 0.087). However, the muscular layer thickness (µm) did not

show significant differences after fasting (Proximal: 40.93 ± 2.628 vs. 36.813 ± 1.999; Medial

32.80 ± 1.163 vs. 29.06 ± 1.636; Distal: 24.86 ± 1.811 vs. 24.64 ± 1.122) in contrast to

observations in mucosal layer thickness (mm) where a significant reduction was observed

(Proximal: 0.865 ± 0.02 vs. 0.638 ± 0.018; Medial 0.586 ± 0.026 vs. 0.404 ± 0.014; Distal:

0.402 ± 0.019 vs. 0.273 ± 0.010). In fasting animals, villus size (µm) showed a significant

reduction in length (Proximal: 572.48 ± 23.805 vs. 413.66 ± 30.084; Medial 366.05 ± 14.859

vs. 296.49 ± 19.929; Distal: 291.76 ± 18.146 vs. 249.33 ± 18.091) and width (expressed in

µm Proximal: 78.18 ± 3.653 vs. 57.84 ± 1.410; Medial 73.09 ± 5.444 vs. 52.51 ± 1.781;

Distal: 68.03 ± 2.083 vs. 56.17 ± 1.732). Crypth width (µm) was reduced in all intestinal

portions compared to fed controls (Proximal: 60.71 ± 1.718 vs. 45.81 ± 1.443; Medial 52.94 ±

2.196 vs. 40.91 ± 1.893; Distal: 46.38 ± 1.581 vs. 38.33 ± 2.037). Also the enterocytes

parameters changed after fasting, length (Proximal: 25.86 ± 1.420 vs. 19.39 ± 0.519; Medial

23.45 ± 1.519 vs. 16.43 ± 0.558; Distal: 21.17 ± 0.265 vs. 16.47 ± 0.331) and width

(Proximal: 8.49 ± 0.401 vs. 7.49 ± 0.206; Medial 8.16 ± 0.345 vs. 7.019 ± 0.151; Distal: 7.28

± 0.195 vs. 6.04 ± 0.075) were significantly reduced (Figure 6 to see p-values). The surface

area enlargement factor or SEF did not show differences on fasted animals compared to

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controls (Proximal: 9.97 ± 0.357 vs. 9.65 ± 0.762; Medial 7.44 ± 0.309 vs. 7.90 ± 0.664;

Distal: 6.73 ± 0.289 vs. 6.90 ± 0.273), p > 0.4 in all cases.

2.4. Correlations among parameters

We performed correlations among histological parameters (perimeter, nominal surface

area, muscular layer, mucosal width, villus, crypt width, length and width of enterocytes and

enlargement mucosal factor). We found some interesting correlations between perimeter with

muscular layer, mucosal width, villus length and width of enterocytes (r > 0.85; p < 0.05);

villus length is more correlated with width of enterocytes (r=0.86; p < 0.05) than length of

enterocytes (r=0.7; p < 0.05).

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DISCUSSION

The digestive system is energetically extremely expensive. The regulation of its

physiological functions requires about 17-25% of the total oxygen consumption of the body

(Cant et al., 1996) and a high cellular turnover rate of the intestinal epithelium (Secor, 2005).

The intestinal structure and function have the ability to adaptively respond to changes in food

intake and energy requirement. Here we show that when adult house sparrows undergo long

term fasting their intestinal mass is drastically reduced, -44% after 34 hours of fasting

(corresponding with phase III). These results are consistent with what has been previously

described in the migrating passerine bird Sylvia atricapilla, -45% after 48 hours of fasting

(Karasov et al., 2004), and mice, -50% after 72 hours of fasting (corresponding with phase

III) (Sokolovic et al., 2007). The reduction in the mass of the digestive organs (-40% small

intestine and -46% liver) was larger compared to the decline in the body mass and non-

alimentary tissues such as the heart (around 24%), in agreement with the high-energy

requirements necessary for the maintenance of the digestive organs (Table 2). The ratio of

organ to body mass remained unchanged when comparing intestine length, heart mass,

pancreas mass and stomach mass with controls. Interestingly, the intestinal mass was reduced

~40% but its length declined by only ~14% compared to control animals. Consistent with this,

we observed a ~30% reduction of the intestinal mucosal layer, resulting in a shortening of

about 20% in villus length (28% in proximal portion to 15% in distal portion), a similar value

(19%) was obtained by Karasov (Karasov et al., 2004). The results obtained so far in mice are

controversial; some researchers found that villus height and crypt depth remained unchanged

during fasting (Chappell et al., 2003; Sokolovic et al., 2007), while others found a marked

effect (Nian et al., 2002; Shin et al., 2005). However, the muscle layer thickness remained

unchanged in birds and mammals, indicating no effect of fasting (Dou et al., 2002; Karasov et

al., 2004; Sokolovic et al., 2007)

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In starved animals the intestinal enterocyte width and length was reduced in different

proportions (~14% and ~26% respectively) resulting in a phenotypic change. Interestingly,

the cell morphology changed from cylindrical to cubic, suggesting that the surface exposed to

the lumen space is conserved. Accordingly, we found no changes in the surface area

enlargement factor (SEF) following long term fasting. The relation SEF/intestinal mass was

significantly increased (~69%), indicating that the food contact surface remained constant

despite the changes in the intestinal weight and length. This is supported by a 19% increase in

the nominal surface area/intestine mass ratio, suggesting an adaptive response in preparation

to refeeding. A reduction in the mucosal layer could affect the biochemical digestive capacity.

However, we found that the sparrows increased their digestive function and intestinal

enzymatic activities (sucrase-isomaltase and aminopeptidase-N) during fasting (Chediack et

al., 2012). Yet, the decrease in several parameters (e.g., lacteal ducts, Lieberükhn crypts)

suggests that the small intestine atrophied in response to starving, but the goblet cells

preservation may indicate a protective mechanism (Specian and Oliver, 1991) of the intestine.

ACKNOWLEDGEMENTS

Samanta Funes is a doctoral fellowship from CONICET. The authors thank to Dr. Jöel

Walicki and Dr. Yanina Ditamo for their thorough and critical reading of the manuscript.

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Figure legends

Figure 1. Proximal portion of small intestine of control (a-b) and long-term fasting (c)

groups. a Section showing mucosal (m), submucosal (sm), and muscular layers (mu); and

serosal surface (se). Scale bar 500 µm. b and c Images that show a reduction of the mucosal

layer thickness (m) and villi length (v). Scale bar 250 µm. Hematoxylin-eosin staining.

Figure 2. Medial portion of small intestine of control (a, b, d, e) and fasting birds (c, f, g). a

Lower magnification of medial portion. Scale bar 500 µm. b and c Photographs of the villi

center showing a reduction of the lamina propia thickness (lm) and the enterocyte height (e).

Scale bar 25 µm. d-g Lieberükhn crypts (L) and the number of mitotic cells (arrows) were

reduced in fasting birds compared with controls. d and f Scale bar 100 µm. e and g Scale bar

25 µm. Hematoxylin-eosin staining.

Figure 3. Distal portion of small intestine of feeding (a-c) and starving (d) birds. a Lower

magnification of distal portion. Scale bar 500 µm. b Parts of the mucosal epithelium (ep) and

the connective tissue core (lm) of villus. Inset Columnar epithelium with a prominent brush

border (*) and a globet cell (arrow). Scale bar 25 µm. c Developed lacteal ducts (ld) in the

connective tissue core of villi; d the length of the lacteal ducts (ld) declined in fasted birds.

Scale bar 25 µm. Hematoxylin-eosin staining.

Figure 4. The number of globet cells (arrows) in the mucosal epithelium increase from

proximal to distal portions of small intestine in feeding (a-c) and long-term fasting (d-e)

groups. a and d Proximal portions; b and e Medial portions; c and f Distal portions. Scale bar

25 µm. Periodic acid-Schiff (PAS) staining.

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Figure 5. Vesicles PAS-positive are moving from crypts to intestinal lumen in the control (a-

c) and fasted (d-f) groups. a and d Vesicles at the proximal portion (arrows). Scale bar 250

µm. b, c, e and f Images of medial portions, the vesicles PAS-positive are numerous in the

fasted birds. Scale bar 25 µm. Periodic acid-Schiff (PAS) staining.

Figure 6. Mean histological parameters (± SE) in all intestinal regions and treatments (Fed

group: filled circle, solid line; Fasting group: open circle, dotted line). Asterisk represent

significantly different between treatments in each intestinal portion (p < 0.05 by ANOVA). *

(p < 0.05) ** (p < 0.01).

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Table I: Effect of fasting on morphological parameters (table A) and morphological parameters relativized by body mass (table B) of sparrows

(n=11 en both experimental groups).

Control Fasted p-value

Small intestine wet mass (g) 1.08 ± 0.15 0.65 ± 0.05 0.017

Small intestine length (cm) 17.23 ± 0.74 14.83 ± 0.51 0.02

Liver wet mass (g) 0.86 ± 0.067 0.45 ± 0.02 0.0001

Pancreas wet mass (g) 0.13 ± 0.01 0.10 ± 0.005 0.016

Stomach wet mass (g) 0.78 ± 0.06 0.59 ± 0.02 0.0018

Heart wet mass (g) 0.37 ± 0.015 0.28 ± 0.016 0.001

Nominal surface area (cm2) 12.82 ± 0.77 9.65 ± 0.86 0.018

Control Fasted p-value

Small intestine wet mass/body mass 0.0413 ± 0.0044 0.030 ± 0.022 0.041

Small intestine length/body mass 0.70 ± 0.02 0.66 ± 0.07 0.319

Liver wet mass/body mass 0.0301 ± 0.0035 0.021 ± 0.001 0.001

Pancreas wet mass/body mass 0.0051 ± 0.0004 0.0048 ± 0.0003 0.52

Stomach wet mass/body mass 0.0305 ± 0.0019 0.0282 ± 0.001 0.31

Heart wet mass/body mass 0.0153 ± 0.0006 0.0136 ± 0.0007 0.76

Nominal surface area (cm2) /body mass 0.49 ± 0.17 0.45 ± 0.38 0.79

Table A

Table B

Table I

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Table II: Abundance of goblet cells and vesicles PAS-positive on small intestine in control and fasted groups.

The values are expressed as mean ± SEM (n= 4). * represent significant differences between control and fasted groups in each intestinal region p

< 0.05. RM-ANOVA followed by Tukey test was used to comparisons between intestinal regions in each group; a p < 0.05;

b p < 0.001.

Control Group Fasted Group

Small intestine position Proximal Medial Distal Proximal Medial Distal

Nº goblet cells/AR 1.72 ± 0.46 7.62 ± 1.24a 22.19 ± 1.36b 2.27 ± 0.53 7.85 ± 0.54a 21.59 ± 1.89b

Area of vesicles PAS-positive (% AV)

0.71 ± 0.12 1.23 ± 0.25 0.24 ± 0.11a 2.36 ± 0.64* 2.09 ± 0.21* 0.49 ± 0.23a

Table II

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Figure

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Research Highlights

We evaluated intestinal histology changes on a non-migrant bird undergoing fasting.

Starvation induces small bowel atrophy.

Starvation induced a decrease in the mass of the digestive organs around 40%.

Histological parameters of small intestine decrease after starvation.

Goblet cells were conserved after fasting indicating a protective tendency.