Archives of Animal Nutrition, 2013
Vol. 67, No. 6, 492 502, http://dx.doi.org/10.1080/1745039X.2013.857079
Addition of citrus pulp and apple pomace in diets for dogs: influence
on fermentation kinetics, digestion, faecal characteristics and bacterial
populations
Sebastián Brambillascaa*, Alejandro Britosa, Carolina Delucaa, Martín Fragab and
Cecilia Cajarvillea
a
Departamento de Nutrición Animal, Facultad de Veterinaria, Universidad de la
b
RepÅ›blica, Montevideo, Uruguay; 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]
© 2013 Taylor & Francis
Archives of Animal Nutrition 493
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. Larrańaga 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 źm. 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 + CITż 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
ż ś
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.
494 S. Brambillasca et al.
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 CO2 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 KH2PO4) and 0.5 ml
of a reducing solution (Na2S " 9H2O 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
CO2 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 Ä… 0.4 kg). Animals were housed in 2.0 m × 2.0 m
individual cages and received 27 g DM/kg BW0.75 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 CO2 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 ½mlŠ ź 4:0289 psi þ 0:1687 psi2 n ź 34; R2 ź 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):
hi
G ź A= 1þðB=tÞC
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 (t½, [h]) and t is the time [h]. Maximum fermentation rate (Rmax, [ml/h])
and time at which it occurs (tmax [h]) were also calculated (Bauer et al. 2001):
2
ð B 1Þ B
RmaxźðA CBÞ B htmax = 1 þ CB i tmax Š
tmaxź C ðB 1Þ=ðB þ 1Þð1=BÞ
Archives of Animal Nutrition 495
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 Ä… 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
BW0.75 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%Š
ź ðNutrient intake ½gŠ Faecal nutrient output ½gŠÞ=Nutrient intake ½gŠ 100%:
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
496 S. Brambillasca et al.
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 log10 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 ANKOM220 fibre
analyser (Ankom Technology Corp., Fairport, NY, USA) using nylon bags and heat stable
Ä…-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:
Yi ź źþFiþLjþðF LÞij þ eij;
where Y is the variable to be tested; µ is the mean; Fi is the fixed effect of the fibre
source (i = CIT, APP); Lj 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 eij 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
Yijkź ź þ Diþ Pjþ Tkþ eijk
where Y is the variable to be tested; µ is the mean; Di is the random effect of the dog
(n = 6); Pj is the fixed effect of period (n = 3); Tk is the fixed effect of treatment (k = CON,
CON + CIT, CON + APP) and eij 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 · 102 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 t½ value (p = 0.006) and a higher Rmax (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
Archives of Animal Nutrition 497
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
t1/2ż [h] 8.21 5.46 0.36 0.006
RmaxÅ› [ml/h] 10.84 14.08 1.04 0.048
tmaxĘ% [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
! ż
three replicates per substrate; OMCV, OM cumulative volume; t½, Half-time of the asymptotic gas production;
ś Ę%
Rmax, Maximal rate of gas production; tmax, Time of occurrence of Rmax.
A
B
110 10
100
90
8
80
CIT
70
APP
6
60
Source: p = 0.437
Source: p = 0.144 CIT
Level: p < 0.001
50
Level: p = 0.690
Source × Level: p = 0.637 APP
Source × Level: p = 0.881
40 4
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
C
Fibre source level [g/kg DM] Fibre source level [g/kg DM]
D
12 1.2
1.0
10
0.8
8 0.6
CIT
0.4
APP CIT
6
Source: p= 0.078
Source: p = 0.751
Level: p = 0.004 0.2
APP
Level: p = 0.073
Source × Level: p = 0.552
Source × Level: p = 0.953
4 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Fibre source level [g/kg DM] Fibre source level [g/kg DM]
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 Ä… SEM; measurements were based on three
replicates per fibre source and level).
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
max
R
[ml/h]
OMCV [ml gas/g OM]
1/2
max
t
[h]
t
[h]
498 S. Brambillasca et al.
Table 3. Effect of fibre inclusion on characteristics of faeces and apparent digestibility of nutrients.
Treatment Contrast p-valueż
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-NH3/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,
ż
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).
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 (Rmax), 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 t1/2 and a tendency in the tmax 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
Archives of Animal Nutrition 499
Table 4. Effect of fibre inclusion on faecal bacterial populations [CFU log10/g fresh faeces]ż.
Treatment
CON* CON + CIT# CON + APP p-Values!
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)
EnterococciÅ› 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 perfringensÅ› 7.53 7.00 6.74 0.490
(<2.00 8.23) (<2.00 7.85) (<2.00 8.93)
ż
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;
! Å›
p, probability of diet effect as determined by Kruskal Wallis test; There were four values above the limit of
detection.
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
500 S. Brambillasca et al.
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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