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Comparative Biochemistry and Physiol

The functional morphology of the english sparrow cecum

Levy Reyes, Eldon J. Braun *

Department of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85719, USA

Received 28 January 2005; received in revised form 25 May 2005; accepted 27 May 2005

Available online 29 June 2005

Abstract

As birds do not have a urinary bladder, the kidneys and lower gastrointestinal tract must function in concert to maintain fluid and

electrolyte homeostasis. In birds, urine is conveyed to the cloaca, and moved by reverse peristalsis into the colon and digestive ceca.

Digestive ceca have been well studied for non-passerine birds and have been shown to absorb substrates and water. The ceca of passerine

birds have been suggested to be non-functional because of their small size. The present study was undertaken to examine the morphology and

cytochemistry of the small ceca of the English sparrow (Passer domesticus). Three-dimensional reconstruction of the ceca from serially

sectioned tissue showed these organs to have a central channel with a large number of side channels. Electron micrographs indicated that all

of the channels are lined by epithelial cells with a very dense microvillus brush border as well as a region densely packed with mitochondria

just below the brush border. Specific staining for Na+, K+-ATPase indicated the enzyme to be localized to the brush border. Quantification of

Na+, K+-ATPase activity showed it to be comparable to the coprodeum of domestic fowl. The data suggest that the small ceca of passerine

birds may function in fluid and electrolyte homeostasis.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Birds; Ceca; GI; Kidney; Passerine; Na+; K+-ATPase

1. Introduction

In mammals, kidneys are the organs that function to

regulate the composition of the extracellular fluid. Urine

produced by the mammalian kidney is conveyed to the

urinary bladder where it is stored until it can be conveniently

voided. Birds, unlike mammals, have a much more

integrated system that functions to regulate the composi-

tion of the extracellular fluid. As birds do not have a

urinary bladder, urine produced by the kidneys is conveyed

by the ureters to the terminal portion of the gastrointestinal

(GI) tract, the cloaca. Urine does not remain in the cloaca

to be excreted but rather, is moved by a reverse peristalsis

into the colon and digestive ceca (Fig. 1). In the lower GI

tract, urine produced by the kidneys can be modified

(Anderson and Braun, 1985; Brummermann and Braun,

1995). Thus, the renal and GI systems are anatomically

1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpb.2005.05.053

* Corresponding author. Tel.: +1 520 626 7134; fax: +1 520 626 2382.

E-mail address: ejbraun@u.arizona.edu (E.J. Braun).

and functionally connected in birds, and as such, must

function in concert to maintain fluid and electrolyte

homeostasis.

The digestive ceca of birds are outgrowths that appear at

the junction of the ileum and colon. These organs take a

wide variety of shapes and lengths, and fall under one of

three classifications: long, moderately developed, or small

ceca (McLelland, 1989). The long, saculated ceca have been

well studied and it has been shown that they aid in

maintaining nutritive, fluid, and electrolyte homeostasis

(Moreto and Planas, 1989; Obst and Diamond, 1989). In the

ceca, bacteria ferment small particles that enter from the

colon, the products of which are absorbed by the cecal

epithelial tissue, along with inorganic and organic ions as

well as water (Campbell and Braun, 1986).

The functions of the long, saculated ceca that occur in

most Galleneous birds have been quite clearly defined;

however, there has been little information published on the

function of the very small ceca that are characteristic of

most passerine birds. Moreover, most ornithological liter-

ature relegates the ceca of passerine birds to a non-

ogy, Part A 141 (2005) 292 – 297

The Avian Urinary and

Lower G.I. Tracts

KIDNEYS

URETERS

CLOACACOLON

CECUM

Fig. 1. Diagrammatic illustration of the anatomical relationship between the kidneys and lower gastrointestinal tract of the English sparrow. The small ceca are

shown at the junction between the colon and ileum.

L. Reyes, E.J. Braun / Comparative Biochemistry and Physiology, Part A 141 (2005) 292–297 293

functional role or states that they are rudimentary at best;

however, there are no data to support such conclusions

(McLelland, 1989; Chaplin, 1989; Clench, 1989). The

function of these small ceca may be a unique area of avian

physiology to explore.

Preliminary observations suggest that the small ceca may

function in an absorptive and/or secretive capacity. Unpub-

lished observations by Braun have noted an abundance of

mitochondria near the brush border of these ceca, which

suggests that a significant amount of energy is being

produced in these ‘‘vestigial’’ organs. The lack of parsimony

suggests that the vestigial ceca must serve some function.

Our hypothesis is that an examination of the morphology of

the English sparrow cecum will reveal certain anatomical

and cytochemical features that are consistent with these

organs playing a role in the fluid and electrolyte homeo-

stasis of passerine birds.

2. Methods

2.1. Isolation of ceca

Mature English sparrows (Passer domesticus) were

collected at the University of Arizona Dairy Research

Center by mist net. The birds were euthanized 1–3 h after

capture by the intramuscular injection of 40 AL of sodium

pentobarbital (65 mg/mL). The ceca, located at the junction

of the colon and ileum, were removed and prepared for one

of the following procedures.

2.2. 3-D reconstruction of the ceca

Fresh cecal tissue was fixed in 2.5% glutaraldehyde,

dehydrated in a graded series of alcohols, embedded in

paraffin, and serial cross sections cut at 8 Am yielding 125

sections. The sections were stained with hematoxylin and

eosin and their images captured using an inverted micro-

scope and Simple PCI i v 5.2 software (Compix Inc;

Cranberry Township, PA, USA). Six pictures were taken of

each section; these images were stitched together using the

Simple PCI program to produce a montage of a section. The

resulting montage(s) were uploaded into Adobe Photoshop

to keep the image file size to a minimum without losing

resolution. After the file manipulations were complete, a 3-

D reconstruction of the ceca was created using Amira i v

2.3 3-D modeling software (Indeed-Visual Concepts;

Berlin-Dahlem, Germany).

2.3. Measurement of Na+, K+-ATPase activity

Na+, K+-ATPase was quantified using the method of

Hossler et al. (2002). Following this method, ceca were

homogenized in 250 AL of Tris–HCl buffer (pH 7.5)

containing 0.1 M Tris–HCl, 0.25 M sucrose and 0.1 M of

EDTA. The tissue was homogenized on ice to preserve

enzyme activity and homogenate stored in liquid nitrogen

overnight. The following day, the protein concentration of

the homogenate was determined using a Bio-Rad Assay

with chicken serum albumin as a standard. After the

protein concentration of the homogenate was determined,

the volume necessary to yield 3 Ag of protein in the

experimental incubation medium was calculated. This

medium consisted of 60 mM Tris–HCl buffer (pH 7.5),

10 mM KCl, 6 mM MgCl2 and 20 mM of para-

nitrophenyl phosphate ( p-nPP). Three micrograms of

protein were also added to the control incubation medium

which was identical to the experimental incubation with

the addition of 2 mM of ouabain and no KCl. Once protein

was added to each medium, the samples were incubated for

25 min in a 37 -C water bath with constant agitation. After

25 min the samples were taken out of the bath and placed

TJ

TJ

TJ

1µM

C

A

0.2 mm

BB

0.25 mm

B

Fig. 2. Light micrograph and electron micrographs of the English sparrow cecum. A) A light micrograph of a cecum cut in sagittal section. The image shows

one central channel with a proximal opening to the colon (upper right of section). Side channels (arrows) can be seen branching from the central channel. B)

Higher power light micrograph of a cecum. The image illustrates the columnar cells that line the channels (bracket) with their well developed brush border

(arrow). C) Electron micrograph of an English sparrow cecum. The image shows a portion of the epithelial cells that line channels of the ceca. Evident is a

dense microvillus brush border on the apical surface of the cells and tight junctions (TJ) between the cells. Just below the brush border is very dense layer of

mitochondria (bracket). 7400 �.

1.9 mm

Fig. 3. A 3-D reconstruction of the English sparrow cecum. The image was

created by using the Amira 3-D software. Inside the cecum the channels

(purple color) become highly branched and mimic a bottle brush. (For

interpretation of the references to colour in this figure legend, the reader is

referred to the web version of this article.)

L. Reyes, E.J. Braun / Comparative Biochemistry and Physiology, Part A 141 (2005) 292–297294

in ice. The reaction was stopped with 3 mL of 0.1 N

NaOH. The cooled samples, were centrifuged at 7000 g for

5 min using a Sorvall RC-5B Refrigerated Superspeed

Centrifuge. The supernatants from the samples were

collected and the optical density read using a DU series

600 Spectrophotometer at 420 nm. The amount of p-nPP

activity was determined by subtracting the absorption

found in the control samples from that of the experimental

samples. The activity was measured in AM phosphate/mg

protein/min.

2.4. Localization of Na+, K+-ATPase

Cytochemical localization of Na+, K+-ATPase in the

cecal tissue was carried out according to the method of

Mayahara et al. (1980), revised by Hossler et al. (2002). In

this method, ceca were removed from birds and fixed for 1

h on ice in 2% p-formaldehyde and 0.25% glutaraldehyde

in 0.1 M cacodylate–HCl buffer, pH 7.2. The tissues were

Table 2

A comparison of English sparrow and domestic fowl Na+/K+-ATPase

activity

Cecaa Coprodeumb Colonb

Male Female High NaCl Low NaCl High NaCl Low NaCl

0.0588 0.0534 0.0300 0.065 0.0767 0.0933

All values are expressed as AMol phosphate/mg protein/min.a Data from present study.b Data from Skadhauge, 1980.

40 µm

Fig. 4. Wire frame model of the internal channels of the cecum obtained

from 3-D reconstructions. The illustration clearly shows the branching

nature of the channels. The tissue has been electronically stripped away to

show only the channels. The proximal end of the cecum is to the lower left.

L. Reyes, E.J. Braun / Comparative Biochemistry and Physiology, Part A 141 (2005) 292–297 295

then washed three times (approximately 1 h each) with ice

cold cacodylate buffer (pH 7.2), containing 25% sucrose

and 10% dimethyl sulfoxide (DMSO). After the third and

final wash, the ceca were separated into control and

experimental samples. The control ceca were washed for

an additional 15 min with a solution identical to the

previous wash with the addition of 5 mM of ouabain. The

experimental tissues were placed directly into an incubation

medium consisting of 250 mM glycine–KOH buffer (pH

8.8), 0.4% Pb–citrate in 0.05 mM KOH, 25% DMSO, 10

mM p-nPP, 20 mM MgCl2, and 2.5 mM of levamisole

(levamisole is a potent inhibitor of non-specific alkaline

phosphatase). The control tissues were incubated in a

similar medium; however, it contained an additional 5 mM

ouabain. The tissues were incubated in their respective

media for 25–30 min at 37 -C under constant agitation.

Upon termination of the incubation cycle, both tissues

(control and experimental) were placed in ice-cold transfer

medium, 0.1 M cacodylate–HCl buffer (pH 7.2) and 25%

sucrose. The tissues were subjected to an additional two

changes of transfer medium, approximately 15 min each

and were then post-fixed in 2% osmium tetraoxide in 0.1

M cacodylate–HCl buffer and processed for electron

microscopy (EM). The critical element of this method is

that the lead–citrate binds to the p-nPP of the tissues to

Table 1

Na+/K+-ATPase activity in cecal tissue as indicated by p-nPPase

Sex Ceca mass (mg) Experimental p-nPP (Ag/mL)

Male 5.33T0.003 2977.9T1912.05

Female 6.98T0.004 2447.6T809.7

Male+Female 6.16T0.003 2712.8T1427.1

There was measurable p-nPP activity found in both the male and female English s

of 12 birds are presented. Values are AMol phosphate/mg protein/min.

form a dark reaction product indicating the presence of an

ATPase.

3. Results

3.1. Ceca morphology

The small ceca (approximately 5 mm in length) of the

English sparrow are outgrowths connected to the gastro-

intestinal tract through one central channel (Fig. 2A). The

epithelium which lines the channels is columnar in nature

and contains a very dense brush border (Fig. 2B). The tight

junctions can be seen joining the columnar cells along with

a row of densely packed mitochondria just below the brush

border (Fig. 2C). The branching of the channels varies in

intricacies and complexity, but they are all connected to the

central channel and terminate in the periphery of the cecum

(Figs. 3 and 4).

3.2. Measurement of Na+, K+-ATPase

The amount of Na+, K+-ATPase present was measured by

quantifying the enzyme para-nitrophenyl phosphatase ( p-

nPP). A large amount of p-nPP was found in the cecal

tissue: the enzyme activity in male English sparrow ceca

was 0.0588 and for females 0.0534 AM p-nPP/Ag protein/

min (Table 1). These values are comparable to those for

lower GI tract and cloaca tissue that have been shown to

function in fluid and electrolyte balance of other birds

(Table 2; Skadhauge, 1980).

3.3. Localization of Na+/K+-ATPase

The cecal tissue incubated in medium without ouabain

formed a reaction product between the exogenous lead–

Control p-nPP (Ag/mL) AMol p-nPP/Ag protein /min

2365.4T1551.6 0.0588T0.0504

1891.2T526.5 0.0534T0.0346

2128.3T1132.1 0.0561T0.0413

parrow ceca. The mean (TS. D.) values for 6 males and 6 females for a total

C

A B

100 nm

100 nm 100 nm

Fig. 5. A) Cecal tissue incubated in ouabain-free medium. A precipitate of lead–citrate bound to tissue p-nPP is visible at the brush border of the epithelial cell

layer indicating the localization of the enzyme Na+/K+-ATPase. B) Cecal tissue incubated in solutions containing ouabain (control) blocking a possible reaction

with Na+/K+-ATPase. The lack of reaction product indicates that the only ATPase present in Fig. 5A is Na+/K+-ATPase. C) Tissue used as a control for

cytochemical reactions and only prepared for EM. The image resembles that of the control tissue, and is used as a background for the experiment.

L. Reyes, E.J. Braun / Comparative Biochemistry and Physiology, Part A 141 (2005) 292–297296

citrate and tissue p-nPP. The deposits of this reaction

product were localized to the brush border. Staining is

observed around each microvillus (Fig. 5A). The control

tissue was exposed to ouabain which inhibits the activity of

Na+, K+-ATPase and therefore no reaction product was

formed (Fig. 5B).

4. Discussion

In this study, we examine the unsubstantiated claim that

the small ceca of passerine birds are vestigial. Although the

small ceca have been characterized as non-functional, there

are no studies to support this assertion. We conducted a

series of experiments and analyses to better define the role

the small ceca might play in fluid homeostasis of passerine

birds. We first examined the morphology of the cecum

through light and electron microscopy and formed a 3-D

reconstruction using images of serial sections from the

organ (Figs. 3 and 4). The results of the morphological

studies suggested that these small organs might not be

vestigial.

The 3-D reconstruction of the ceca revealed an intricate

system of channels within these small organs. The ceca open

to the gastrointestinal tract through one main channel that

begins to branch immediately into side channels. The main

channel extends the length of the ceca while side channels

deviate and either terminate or continue branching (Figs. 3

and 4). This extensive network of branching has been

likened to that of a bottlebrush.

L. Reyes, E.J. Braun / Comparative Biochemistry and Physiology, Part A 141 (2005) 292–297 297

Because the 3-D model provided insight as to the

intricate nature of the channel system within the ceca, it

suggested that the cells around these channels could

function in transport. Electron micrographs revealed a

dense microvillus brush border on all the columnar

epithelial cells lining the channels (Fig. 5C). Just below

this brush border is a layer of densely packed mitochondria,

suggesting that a significant amount of energy may be

produced close to the lumen. The abundance and location

of mitochondria are often found in epithelial cells involved

in substrate transport.

It is conceivable that the row of mitochondria below the

brush border is present to provide ATP to power substrate

transport. To investigate this hypothesis we assayed

homogenates for ouabain sensitive Na+, K+-ATPase, with

the results showing a large amount of enzyme activity. The

observed activity compared well with that of the coprodeum

of chickens maintained on high and low salt diets (Tables 1

and 2; Skadhauge, 1980). The coprodeal portion of the

chicken cloaca has been shown to be extremely active in

sodium transport (Skadhauge, 1980).

Although the data led us to believe that there is a Na+,

K+-ATPase within the cells of the ceca, they were not

specific as to which portion of the cells the ATPase was

located. To localize the Na+, K+-ATPase, cytochemical

staining using lead–citrate was carried out. A complex

formed between the exogenous lead–citrate and tissue p-

nPP; this reaction product was seen at the apical sides of the

epithelial cells (Fig. 5A). The presence of the Na+, K+-

ATPase at the apical surface raised some speculation as to

whether the staining was indeed that of Na+, K+-ATPase, or

from another ATPase, such as H+-ATPase which is much

more likely to be found at the apical portion of the cell.

However, had this indeed been H+-ATPase, there would

have been reaction product in the control tissues (Araki et

al., 1990). As this was not the case, the localized products

must be Na+, K+-ATPase.

Considering the localization of the Na+/K+ pumps and

the overall morphology of the ceca it is difficult to accept

the current views in literature that classify these organs as

vestigial. While the influence that the small ceca have on

fluid homeostasis remains to be determined, the data

presented suggest they play a role in this regulation.

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