Addition of citrus pulp and apple pomace in diets for dogs: influence
on fermentation kinetics, digestion, faecal characteristics and bacterial
populations

Sebastián Brambillasca

a

*

, Alejandro Britos

a

, Carolina Deluca

a

, Martín Fraga

b

and

Cecilia Cajarville

a

a

Departamento de Nutrición Animal, Facultad de Veterinaria, Universidad de la

República, Montevideo, Uruguay;

b

Departamento de Microbiología, Instituto de Investigaciones

Biológicas Clemente Estable, Montevideo, Uruguay

(Received 26 July 2013, accepted 4 October 2013)

Fermentation kinetics, digestibility, faecal characteristics and bacterial populations
(aerobes, anaerobes, lactobacilli, lactic acid bacteria, enterococci, coliforms and clos-
tridia) of dog food mixed with citrus pulp and apple pomace were evaluated. The in
vitro gas production of a pre-digested dog food mixed with 0, 30, 50 and 70 g/kg dry
matter (DM) of citrus pulp or apple pomace was measured, and also an experiment
with dogs fed the same dog food with or without the addition of 70 g/kg of either fresh
citrus pulp or apple pomace was conducted. Gas production increased linearly
(p < 0.001) and quadratically (p < 0.001) as fibre levels augmented. The inclusion
of fibre sources in the diets resulted in higher faecal output (p = 0.005) and defecation
frequency (p < 0.001), and lower faecal pH (p < 0.001) and digestibility values
(p < 0.01). Faecal consistencies and microbial populations did not differ among
treatments. The addition of fresh citrus and apple was effective to stimulate the hindgut
fermentation, but slightly depressed the digestion.

Keywords: apple pomace; bacteria; citrus pulp; digestibility; dogs; feeding;
fermentation; fibre

1.

Introduction

The inclusion of fermentable fibre in the diets of monogastric animals is an interesting
strategy to improve the gastrointestinal health and microbiota ecosystem in the gut. Fibre
sources from dehydrated vegetables and fruits are being used by the pet food industry to
reduce the incidence of constipation and plasma glucose concentrations in diabetic
animals (Biagi et al.

2010

). Citrus pulp and apple pomace contain fermentable fibre that

result in compounds associated with positive impacts on animal gut physiology (Bosch
et al.

2008

), and their beneficial effects on intestinal microbiota and microbial metabolites

have been reported previously through in vitro and in vivo approaches (Sunvold, Fahey,
Merchen, and Reinhart 1995; Sunvold, Fahey, Merchen, Titgemeyer, et al. 1995; Swanson
et al.

2001

; Middelbos et al.

2007

; Biagi et al.

2010

). The aforementioned studies used the

additives in a purified (i.e. pectins) or in a dehydrated form. The dehydration process
involves heating the raw material and the addition of desiccant substances (i.e. calcium
hydroxide), that increase ash and fibre contents of the material (Martínez

–Pascual and

Fernández

–Carmona

1980

) which can affect digestive utilisation. However, little is known

*Corresponding author. Email:

[e-mail]

Archives of Animal Nutrition, 2013
Vol. 67, No. 6, 492

–502, http://dx.doi.org/10.1080/1745039X.2013.857079

© 2013 Taylor & Francis

about animal responses when those fibre sources are used fresh (or just oven dried,
without addition of other substances) as supplements of dog food, which can be useful
in formulation of home-made diets and supplements.

We tested the hypothesis that the inclusion of fresh citrus and apple by-products in the

diet of dogs may improve the hindgut fermentation and may have a positive impact on
microbial ecology as was demonstrated for dehydrated by-products. Therefore, our
purpose was to evaluate fermentation characteristics of two fibre sources (citrus pulp
and apple pomace) added in different levels to a pre-digested dog food, and to determine
whether the supplementation of a dog food with fresh citrus pulp and apple pomace
affects the digestibility of nutrients, stool characteristics and faecal microbial populations.

2.

Materials and methods

Two experiments were conducted: an in vitro gas production trial and an in vivo feeding
trial. All procedures were approved by the Bioethics Committee of the Veterinary Faculty
(Facultad de Veterinaria, UdelaR, Uruguay).

2.1.

Experiment 1: in vitro gas production

In this trial mixtures of a pre-digested dog food (PRED) with either apple pomace (APP) or
citrus pulp (CIT) were prepared in order to test four different inclusion levels: 0, 30, 50, and
70 g/kg of each fibre source on a dry matter (DM) basis. Mixtures and the PRED were used as
substrates in an in vitro gas production experiment (a total of seven substrates). Citrus pulp
and apple pomace were obtained from a juice processing industry (Frigorífico Uruguayo S.A.,
Damaso A. Larrañaga 3551, Montevideo, Uruguay). The material collected was the pulp and
peel remaining after the mechanical fruit squeezing. In order to assure the freshness and
hygienic quality of the material, it was collected immediately after the extraction of juice,
chopped with a blender in the laboratory and stored at

–20°C throughout the study. PRED was

obtained by a pepsin

–pancreatin hydrolysis of a commercial dog food (Purina Excellent

®

based on chicken by-product meal, cereals, meat and bone meal, vegetable protein meal,
animal fat, minerals and vitamins; Nestlé Argentina, Buenos Aires, Argentina) as described by
Cone et al. (

2005

) and filtered with nylon gauze with pores of 43

μm. Prior to the mixing,

PRED, APP and CIT were oven-dried at 55°C for 48 h and ground to pass a 1-mm screen.
Chemical composition of the substrates is presented in

Table 1

.

Table 1.

Chemical composition of the substrates used in Experiment 1 and experimental diets in

Experiment 2 [g/kg, DM basis].

Experiment 1

Experiment 2

PRED*

CIT

#

APP

†

CON

‡

CON + CIT

§

CON + APP

Dry matter

929.7

145.0

210.0

928.5

679.7

746.2

Organic matter

945.8

964.8

982.5

929.7

930.3

931.3

Ash

54.2

35.2

17.5

70.3

69.7

68.7

Crude protein

140.3

61.0

26.5

243.6

229.0

223.9

Neutral detergent fibre

318.3

220.0

340.2

105.7

113.7

122.2

Acid detergent fibre

80.5

162.7

237.7

47.5

56.3

61.5

Note: *PRED, Pre-digested control dry dog food;

#

CIT, Citrus pulp;

†

APP, Apple pomace;

‡

CON, Commercial

dog food;

§

CON + CIT, Commercial dog food supplemented with 70 g/kg DM of citrus pulp;

¶

CON + APP,

Commercial dog food supplemented with 70 g/kg DM of apple pomace.

Archives of Animal Nutrition

493

The in vitro gas production procedure was performed using 125 ml fermentation bottles.

Each substrate was incubated in triplicate, and three bottles with no substrate were also
included as inoculum blank (a total of 24 bottles). Substrates were weighed (0.5 g DM) and
placed in the bottles. Then, three solutions as described by Williams et al. (

2005

) were added

separately to each fermentation bottle under continuous CO

2

stream. Briefly, these solutions

were 38 ml of a basal solution (macro- and micro-minerals, short-chain fatty acids and
haemin), 0.5 ml of a vitamins/buffer phosphate solution (vitamins and KH

2

PO

4

) and 0.5 ml

of a reducing solution (Na

2

S

∙ 9H

2

O and cysteine HCl). Afterward, bottles were sealed with

butyl rubber stoppers and stored at 4°C for 8 h to hydrate substrates. Prior to inoculation,
bottles were pre-warmed in a water bath at 39°C for 2 h, and 2 ml of a bicarbonate buffer
was added. Then, each bottle was inoculated with 10 ml of diluted dog faeces (1:5 w/v), and
butyl stoppers were fastened with aluminium crimp seals and remained in the water bath
throughout the measurement period. All manipulations were performed under continuous
CO

2

stream. The whole procedure was conducted in one run.

Faecal inoculum was prepared using faeces obtained from three adult Cocker Spaniel

dogs (two females, one male; BW: 12.6 ± 0.4 kg). Animals were housed in 2.0 m × 2.0 m
individual cages and received 27 g DM/kg BW

0.75

of Purina Excellent

®

for 12 days prior

faecal collection. Faeces were collected immediately after defecation, sealed in plastic
bags under anaerobic conditions, transported in a pre-warmed container to the laboratory
(<1 h between collection and inoculation) and pooled quantitatively to provide a unique
representative inoculum. Faeces were diluted with a saline sterile solution (9 g NaCl/l) in
a 1:5 ratio (w/v), homogenised using a hand-mixer and strained through four layers of
cheesecloth. The fluid obtained was then continually flushed with CO

2

and stirred until

inoculation.

Gas production was measured in the bottles at 2, 4, 6, 8, 10, 12, 18, 24, 48 and 67 h

after inoculation with a transducer fixed to a pressure meter (840065, Sper Cientific,
Scottsdale, AZ, USA) and registered in psi units. Gas volume in millilitre was predicted
from psi values. For this purpose in a parallel trial, gas volume was measured with a
syringe and simultaneously gas pressure was recorded in psi from each bottle following
the procedure described by Theodorou et al. (

1994

). Then, gas volume in millilitres was

calculated from pressure using the equation

Volume ml

½ ¼ 4:0289 psi þ 0:1687 psi

2

n

¼ 34; R

2

¼ 0:9822

:

Cumulative gas volume recorded during the fermentation was related to the incubated OM
(organic matter cumulative volume, OMCV). The data for cumulative gas production
were fitted to the model (Groot et al.

1996

):

G

¼ A= 1þ B=t

ð

Þ

C

h

i

where G is the total gas produced [ml/g]; A is the asymptotic gas production [ml/g]; B is
the switching characteristic of the curve; C is the time at which one-half of the asymptote
has been reached (t

½

, [h]) and t is the time [h]. Maximum fermentation rate (R

max

, [ml/h])

and time at which it occurs (t

max

[h]) were also calculated (Bauer et al.

2001

):

R

max

¼ ðA C

B

Þ B t

max

ðB1Þ

= 1 þ C

B

t

B

max

2

t

max

¼ C B 1

ð

Þ= B þ 1

ð

Þ

ð1=BÞ

h

i

494

S. Brambillasca et al.

2.2.

Experiment 2: in vivo feeding trial

This trial was performed to test the influence of fresh citrus pulp and apple pomace included
in the diet of dogs on digestion of nutrients, faecal characteristics and bacterial populations.
Six healthy adult Cocker Spaniel dogs (three males, three females; 12.7 ± 0.7 kg BW) were
randomly assigned to three diets according to a 3 × 3 Latin square design. The feedstuffs
used in this experiment were the same as used in Experiment 1, but the dry food was used as
is (without pre-digestion) and the fibrous sources were used fresh. Diets consisted in Purina
Excellent

®

(CON), 930 g/kg CON plus 70 g/kg citrus pulp on a DM basis (CON + CIT) or

930 g/kg CON plus 70 g/kg apple pomace on a DM basis (CON + APP). Portions of citrus
and apple were daily thawed and mixed with CON immediately prior to feeding. Chemical
composition of the diets is presented in

Table 1

. Each dog was fed daily 27 g DM/kg

BW

0.75

of each diet offered in equal meals at 09:00 h and 16:00 h. Citrus and apple were

included at the highest level without compromising the fixed intake. For this purpose, a pre-
experimental period was performed beginning with 120 g/kg of fibre sources on DM basis
(following Fahey, Merchen, Corbin, Hamilton, Serbe, Lewis, et al. 1990) and controlling
intake. The level of citrus an apple was reduced until no refusals were observed. Therefore,
the level of inclusion of both fibre sources was equaled at 70 g/kg in a DM basis. On as fed
basis it represented on average 208, 277 and 254 g/d, for CON, CON + CIT and
CON + APP respectively. Animals had free access to fresh water throughout the experiment.
Each experimental period of the Latin square consisted of a 5-day diet adaptation phase,
followed by 3 day for collection of faeces, and 1 day of faecal sampling for bacterial
enumeration.

During the collection phase dogs were checked hourly for defecation from 08:00 to

18:00 h. After defecation, faecal consistency and pH were determined individually and
immediately. Faecal consistency was scored using a scale of 1 (indicating liquid consis-
tency) to 5 (indicating firm consistency) as described by Strickling et al. (

2000

). Faecal pH

was measured with a digital pH-meter (eChem Instruments Pte. Ltd., Oakton, Singapore)
diluting 1 g of faeces in 10 ml of distilled water as described by Hesta, Janssens, et al.
(

2003

). Total individual faeces were weighed, placed in plastic bags and immediately

frozen at

−20°C. Faecal samples were later thawed and mixed so that pooled samples were

representative of each dog and period. Apparent digestibility of nutrients [DM, OM, crude
protein (CP), neutral detergent fibre (NDF), acid detergent fibre (ADF)] were calculated as:

Apparent digestibility

%

½

¼ Nutrient intake g

½ Faecal nutrient output g

½ Þ=Nutrient intake g

½ 100%:

ð

For bacterial culture a unique faecal sample per dog was collected the last day of each
experimental period. One g of fresh faeces was mixed with 10 ml of sterile phosphate
saline solution containing 0.5 g/l of cysteine and processed immediately after excretion in
a Stomacher blender (Seward, UK). Serial dilutions were made (10

−2

to 10

−9

) and

triplicates were spread onto different culture media for enumeration of colony forming
units (CFU). Total aerobes were grown in Trypticase Soy Agar (TSA; Difco, Inc., Detroit,
USA), and total anaerobes were cultured in TSA with 0.5 g cysteine per litre. Rogosa agar
was used for lactobacilli counts (Merck, Darmstadt, Germany) following Swanson et al.
(

2002

), deMan

– Rogosa – Sharpe (MRS) agar (Merck, Darmstadt, Germany) for lactic

acid bacteria, mEnterococcus agar (Becton Dickinson, Massachusetts, USA) for entero-
cocci, McConkey agar (Merck, Darmstadt, Germany) for coliforms and Sulfite Polymyxin
Suphadiazine (SPS) agar (Difco, Inc., Detroit, MI, USA) for Clostridium perfringens. All

Archives of Animal Nutrition

495

culture media were incubated 37°C for 48 h, TSA/cysteine and SPS plates were incubated
in anaerobic jars (Oxoid, UK), MRS and Rogosa plates under microaerophilic atmosphere
in a candle jar, whereas the rest of the plates were incubated under aerobic conditions.
Bacterial counts were expressed as log

10

CFU per gram of fresh faeces.

2.3.

Chemical analysis

Feeds, substrates and faeces were analysed using AOAC (

1990

) methods for DM (ID 967.03),

ash (ID 942.05) and CP (ID 984.13). OM was calculated by difference (OM = 1000

− Ash).

NDF and ADF fractions were determined sequentially using an ANKOM

220

fibre

analyser (Ankom Technology Corp., Fairport, NY, USA) using nylon bags and heat stable
α-amylase and expressed inclusive of residual ash. Individual faecal samples were thawed
and analysed for ammonia content by steam distillation (Hesta, Roosen, et al.

2003

).

2.4.

Statistical analysis

Data were analysed using the MIXED procedure of SAS software (version 8.2; SAS
Institute, Cary, NC, USA). In Experiment 1, the effects of the fibre source included and
the inclusion level were tested using the model:

Y

i

¼ μþF

i

þL

j

þ ðF LÞ

ij

þ e

ij

;

where Y is the variable to be tested; µ is the mean; F

i

is the fixed effect of the fibre

source (i = CIT, APP); L

j

is the fixed effect of the inclusion level (j = 0, 30, 50 and

70 g/kg); (F

∙ L)

ij

is the interaction between fibre source and inclusion level and e

ij

the

error term. Linear and quadratic effects for increasing inclusion levels of fibre sources
were also tested. Additionally, the fermentation parameters of citrus pulp and apple
pomace incubated solely were compared. For Experiment 2, the model used was

Y

ijk

¼ μ þ D

i

þ P

j

þ T

k

þ e

ijk

where Y is the variable to be tested; µ is the mean; D

i

is the random effect of the dog

(n = 6); P

j

is the fixed effect of period (n = 3); T

k

is the fixed effect of treatment (k = CON,

CON + CIT, CON + APP) and e

ij

is the error term. Means were separated by pre-planned

orthogonal contrasts (independent linear comparisons between groups, Doncaster and
Davey

2007

) in order to study the effects of fibre inclusion in the diet (CON vs.

CON + CIT + CON + APP) and the fibre source used (CON + CIT vs. CON + APP).
Faecal microbial populations were compared among treatments after logarithmic trans-
formation of microbial counts, by the NPAR1WAY procedure of SAS. For values below
the detection limit, 1 · 10

2

was used as count value. Significance was declared at p < 0.05,

and tendencies at p < 0.10.

3.

Results

The fermentation characteristics of citrus pulp and apple pomace incubated solely are
shown in

Table 2

. Both fibre sources presented similar values for gas production, never-

theless APP presented a lower t

½

value (p = 0.006) and a higher R

max

(p = 0.048).

The effects of each fibre source added to PRED and the inclusion levels used (0, 30, 50
and 70 g/kg DM) on the in vitro fermentation parameters are presented in

Figure 1

. No

significant interactions between fibre source and inclusion level were detected for any of

496

S. Brambillasca et al.

the parameters studied, so the responses to the different inclusion levels were analysed for
CIT and APP altogether. Increasing the inclusion level produced similar responses for
both fibre sources. Gas production measured (OMCV) increased similarly for both fibre
sources at an increasing rate of inclusion (linear p < 0.001, quadratic p < 0.001). Half-time

Table 2.

In vitro fermentation parameters of fibre sources incubated solely.

Fibre source

CIT*

APP

#

SEM

†

p-values

Fermentation parameter
OMCV

‡

[ml/g OM]

120.95

129.75

6.09

0.365

t

1/2

§

[h]

8.21

5.46

0.36

0.006

R

max

¶

[ml/h]

10.84

14.08

1.04

0.048

t

max

◊

[h]

0.78

1.33

0.31

0.276

Notes: *CIT, Citrus pulp;

#

APP, Apple pomace;

†

SEM, Standard error of means; measurements were based on

three replicates per substrate;

‡

OMCV, OM cumulative volume;

§

t

½

, Half-time of the asymptotic gas production;

¶

R

max

, Maximal rate of gas production;

◊

t

max

, Time of occurrence of R

max

.

40

50

60

70

80

90

100

110

0

10

20

30

40

50

60

70

OMCV [ml gas/g OM]

Fibre source level [g/kg DM]

CIT

APP

Source: p = 0.437
Level: p < 0.001
Source × Level: p = 0.637

A

4

6

8

10

0

10

20

30

40

50

60

70

R

max

[ml/h]

Fibre source level [g/kg DM]

CIT
APP

Source: p = 0.144

Level: p = 0.690

Source × Level: p = 0.881

B

4

6

8

10

12

0

10

20

30

40

50

60

70

Fibre source level [g/kg DM]

CIT

APP

Source: p= 0.078
Level: p = 0.004
Source × Level: p = 0.552

C

0

0.2

0.4

0.6

0.8

1.0

1.2

0

10

20

30

40

50

60

70

Fibre source level [g/kg DM]

CIT

APP

Source: p = 0.751

Level: p = 0.073

Source × Level: p = 0.953

D

t

1/2

[h]

t

max

[h]

Figure 1.

In vitro fermentation parameters of fibre sources added to a pre-digested dog food at 0,

30, 50 and 70 g/kg DM.
Note: CIT, citrus pulp; APP, apple pomace; Panel A, organic matter cumulative volume; Panel B,
maximal rate of gas production; Panel C, half-time of the asymptotic gas production; Panel D, time
of occurrence of maximal rate of gas production; (means ± SEM; measurements were based on three
replicates per fibre source and level).

Archives of Animal Nutrition

497

of the asymptotic gas production was higher (p = 0.004) and the time needed to reach the
maximal tended to be higher (p = 0.073) with the inclusion of both fibre sources.

In the second experiment, all dogs remained healthy and diets were totally consumed

throughout the study. Faecal parameters and apparent nutrients digestibility for the
different treatments are presented in

Table 3

. The addition of fibre sources in the diets

led to a higher wet faecal output (p = 0.005) and defecation frequency (p < 0.001) with a
lower DM content of faeces (p = 0.004), without varying faecal consistency. A lower
faecal pH (p < 0.001) was observed with the addition of fibre, but ammonia concentration
in faeces was similar among treatments. Apparent digestibility of nutrients decreased with
the addition of both fibre sources, and microbial groups quantified were neither influenced
by the addition of citrus pulp nor apple pomace (

Table 4

).

4.

Discussion

According to other studies, and considering the rates of gas production obtained in our
experiment (R

max

), citrus pulp and apple pomace can be characterised as slowly fermen-

table fibre sources (Bosch et al.

2008

). This implies that these sources of fibre are

expected to be fermented in the hindgut, and to produce moderate amounts of organic
acids through the GIT. Both fibrous added to the PRED enhanced the extent of fermenta-
tion by the microbiota, evidenced by the increase in the gas produced and consistent with
the increase in fermentable substrates. By contrast, the inclusion of citrus and apple
slowed down the fermentation (higher t

1/2

and a tendency in the t

max

to be greater in

the fibre containing substrates) compared to PRED incubated solely. This can be related to
the origin of the inoculum, as it was provided by dogs fed without fibre added. This fact
suggests that the number of microbes capable of degrading the substrates was not high
enough, and/or the microbiota needed to adapt to the fibrous substrates. The fermentative
profiles of the mixtures were similar for both fibre sources at the levels tested, although

Table 3.

Effect of fibre inclusion on characteristics of faeces and apparent digestibility of nutrients.

Treatment

Contrast p-value

§

CON* CON + CIT

#

CON + APP

†

SEM

‡

Fibre

Source

Faecal parameters

Wet faeces [g/d]

95.6

148.0

146.0

20.92

0.005

0.912

Dry faeces [g/d]

35.7

46.3

45.7

7.22

0.035

0.909

Faecal DM [%]

38.1

31.2

31.7

0.16

0.004

0.814

Defecation frequency [d

−1

]

1.17

1.83

1.78

0.11

<0.001

0.735

Consistency [1: liquid; 5: firm]

4.41

4.32

4.24

0.22

0.147

0.777

Faecal pH

6.99

6.58

6.51

0.05

<0.001

0.245

Ammonia [mg N-NH

3

/g DM]

2.23

1.92

2.14

0.18

0.375

0.391

Apparent digestibility [%]

Dry matter

84.90

81.07

80.42

1.171

0.004

0.645

Organic matter

88.67

84.53

84.12

0.937

<0.001

0.713

Crude protein

86.15

83.41

81.68

1.243

0.007

0.202

Neutral detergent fibre

50.72

36.05

32.85

3.282

<0.001

0.466

Acid detergent fibre

45.10

26.37

23.25

3.583

<0.001

0.548

Notes: *CON, commercial dog food;

#

CON + CIT, commercial dog food supplemented with 70 g/kg DM of

citrus pulp;

†

CON + APP: commercial dog food supplemented with 70 g/kg DM of apple pomace;

‡

SEM,

standard

error

of

means;

§

Contrast

probability,

Fibre,

effect

of

fibre

inclusion

(CON

vs.

CON + CIT + CON + APP); Source, effect of fibre source (CON + CIT vs. CON + APP).

498

S. Brambillasca et al.

apple incubated solely was fermented faster than citrus. Altogether, we found an improve-
ment in the fermentation activity which can be of interest for intestinal health according to
other studies (Bosch et al.

2008

; Biagi et al.

2010

).

In the in vivo trial, feeding regime was adjusted for suitable maintenance of the dogs.

Despite the fibre sources were used fresh and this caused an enlargement of the volume of
the diets, the levels of fibre in the diets (NDF and ADF) were not larger than 12% and 6%,
respectively, for the fibre supplemented groups. These levels of fibre were similar or
above levels used in other experiments (Fahey, Merchen, Corbin, Hamilton, Serbe, Lewis,
et al. 1990; de-Oliveira et al.

2012

). Interestingly, diets were completely eaten, even

though the volume of the diets was enlarged with fibre addition. As suggested by others
(Diez et al.

1998

), adding fibre may have a dilution effect on energy density but this effect

may not be important when animals receive the amount of feed based on individual
energy requirements, at least at the fibre levels used in our study.

The inclusion of both citrus and apple in the diet led to higher faecal outputs and

defecation frequencies, in agreement with previous reports that communicated the
decrease in the transit time through the GIT (Fahey, Merchen, Corbin, Hamilton, Serbe,
Lewis, et al. 1990), higher output and moisture content of stools, and higher defecation
frequency (Fahey, Merchen, Corbin, Hamilton, Serbe, and Hirakawa 1990; Wakshlag
et al.

2011

) as a consequence of the increase in the fibre levels of the diet. The higher

amounts of faeces excreted in the fibrous diets were as a consequence of an increase on
DM and water output, and can be related to less digestible components and the high
water-binding capacity of fibre sources (Sunvold, Fahey, Merchen, Titgemeyer, et al.

1995

; Swanson et al.

2001

). An interesting observation in the present study is that faecal

scores were not affected by treatments, and that the inclusion of fresh citrus and apple at
this level led to well-formed faeces. This was not expected as DM content of faeces and
digestibility of nutrients were depressed with the inclusion of fibre, and according to other

Table 4.

Effect of fibre inclusion on faecal bacterial populations [CFU log

10

/g fresh faeces]

§

.

Treatment

p-Values

‡

CON*

CON + CIT#

CON + APP

†

Total aerobes

8.31

7.95

7.92

0.834

(6.48

–8.93)

(6.66

–9.48)

(6.89

–8.88)

Total anaerobes

8.60

8.50

7.71

0.623

(6.00

–9.51)

(6.95

–9.89)

(6.38

–8.93)

Lactic acid bacteria

8.25

8.24

7.65

0.823

(5.45

–9.41)

(6.45

–9.28)

(6.08

–9.11)

Lactobacilli

6.18

7.59

6.23

0.692

(5.30

–8.98)

(4.00

–9.00)

(4.48

–8.63)

Enterococci

¶

6.10

5.39

6.81

0.672

(<2.00

–7.48)

(5.00

–8.30)

(4.77

–7.62)

Coliforms

8.41

8.15

7.91

0.895

(6.46

–9.34)

(6.639.10)

(6.70

–9.15)

Clostridium perfringens

¶

7.53

7.00

6.74

0.490

(<2.00

–8.23)

(<2.00

–7.85)

(<2.00

–8.93)

Notes:

§

Median values for each treatment are reported; the minimum and maximum values are shown

in parenthesis; *CON, commercial dog food;

#

CON + CIT, commercial dog food supplemented with 70 g/kg

DM of citrus pulp;

†

CON + APP, commercial dog food supplemented with 70 g/kg DM of apple pomace;

‡

p, probability of diet effect as determined by Kruskal

–Wallis test;

¶

There were four values above the limit of

detection.

Archives of Animal Nutrition

499

authors, low faecal DM content is associated with soft faeces and low digestibility values
(Twomey et al.

2003

; Carciofi et al.

2006

).

A positive response derived from the addition of fibre was the lower faecal pH values

observed, consistent with the higher gas volume obtained in the in vitro experiment,
suggesting a higher fermentation activity and organic acid production in the hindgut
(Flickinger et al.

2003

; Twomey et al.

2003

). Indeed, the low pH values in the intestine

can be protective against pathogenic bacteria in the gut (Seifert and Waltz

2007

).

However, faecal ammonia was not reduced with the addition of fibre, and bacterial
populations were similar among treatments. Therefore we could not confirm that this
addition increased bacterial mass and/or ammonia incorporation into bacterial mass, as
reported by others (Flickinger et al.

2003

; Biagi et al.

2010

).

Apparent digestibilities were diminished by the inclusion of both fibre sources, as

reported by others (Fahey, Merchen, Corbin, Hamilton, Serbe, and Hirakawa 1990;
Lewis et al.

1994

; Burkhalter et al.

2001

). The decrease in apparent CP digestibility

could be due to a lower intestinal digestion of proteins itself, but not as a conse-
quence of higher faecal CP from bacterial mass, as no differences were noticed in
aerobes and anaerobes total counts among treatments. This fact may be considered
specially when using these by-products in diets for growing dogs. The reduction in
apparent digestibility was more evident in the fibre fractions analysed, NDF and
ADF, which suffered a reduction of about 30 and 40% respectively. Despite both
citrus and apple caused a higher fermentation activity in the hindgut, this was not
reflected in higher digestibility values for the fibre fractions.

To conclude, citrus pulp and apple pomace included in a dog diet enhanced the

fermentation activity in the hindgut, and led to well-formed faeces with small reductions
in nutrient digestion. These by-products can be considered for being included fresh in
home-made diets, but it would be necessary increasing the concentration of nutrients to
compensate the reductions on digestibility. Furthermore, additional studies concerning the
most suitable levels of addition of these feedstuffs are needed.

Acknowledgements

The authors thank CIDEC

– Facultad de Veterinaria, Lucía Reyes for her contribution in the gas in

vitro trial and Nestlé, Uruguay for kindly providing the pet food.

Funding
The authors thank CIDEC

– Facultad de Veterinaria for the financial support of this project.

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