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Dietary probiotic supplementation in juvenile rainbow trout (Oncorhynchus

mykiss) reared under cage culture production: Effects on growth, fish welfare,
flesh quality and intestina...

Article in Aquaculture Research · March 2015

DOI: 10.1111/are.12724

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Aquaculture Research, 2015, 1–16 doi:10.1111/are.12724

Dietary probiotic supplementation in juvenile rainbow

trout (Oncorhynchus mykiss) reared under cage
culture production: effects on growth, fish welfare,
flesh quality and intestinal microbiota

Rodrigo O A Oz
orio1,2, Justyna Kopecka-Pilarczyk1, Maria J Peixoto1,2, Rebecca Lochmann3,

Ricardo J Santos4, Goncßalo Santos5, Barbara Weber5, Joaquim Calheiros6, Lia Ferraz-Arruda7,
Paulo Vaz-Pires1,2 & Jos
e F M Goncßalves1,2
1
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Rua dos Bragas
289, P 4050-123 Porto, Portugal
2
ICBAS – Abel Salazar Institute for the Biomedical Sciences, Universidade de Porto, Rua Jorge Viterbo Ferreira, 228,
4050-313 Porto, Portugal
3
Department of Aquaculture and Fisheries, University of Arkansas at Pine Bluff, 1200 N. University Drive, MailSlot
4912, Pine Bluff, Arkansas 71601, USA
4
LSRE – Laboratory of Separation and Reaction Engineering, Fac Engn, University of Porto, 4200-465 Porto, Portugal
5
BIOMIN Holding GmbH, Austria
6
Quinta do Salm~ao, Pracßa Lu
ıs de Cam~
oes 15, 4490 P
ovoa de Varzim, Portugal
7
Departamento de Agroind
~o, Escola Superior de Agricultura ‘Luiz de Queiroz’ (ESALQ),
ustria, Alimentos e Nutricßa
Universidade de S~ao Paulo, Piracicaba, SP, Brazil

Correspondence: Jos
e F M Goncßalves, Interdisciplinary ICBAS - Abel Salazar Institute for the Biomedical Sciences, Universidade de
Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. E-mail: [email protected]

(GST) (P < 0.001), glutathione peroxidase (GPx)

Abstract
(P < 0.05) increased and lipid peroxidation (LP)
This study was conducted to evaluate the effects of (P < 0.01) decreased with dietary probiotic supple-
dietary probiotic supplementation on growth perfor- mentation. Flesh quality and shelf-life of fish stored
mance, fish welfare, sensory evaluation, freshness on ice was not affected by dietary probiotic supple-
during ice storage and changes in intestinal microbi- mentation. Probiotic supplementation had a limited
ota of rainbow trout (Oncorhynchus mykiss). The 9- effect on the intestinal microbiota. Signal strength of
week feeding trial was conducted in cages under the observed operational taxonomic units (OTUs) dif-
commercial conditions during the winter season, fered between the control and treatment samples and
when water temperature decreased from 14°C to 7°C. could indicate variations in the abundance of certain
Fish were fed either a diet without probiotic supple- OTUs. Microbiota adhering to the intestinal mucus
mentation (control) or with 0.2% (2 9 109 varied between sampling points, indicating that
CFU kg
1 diet) of multi-strain probiotic bacteria changes were associated with water temperature.
(Bacillus sp., Pediococcus sp., Enterococcus sp., Lactoba-
cillus sp.). Fish weight was recorded at 0, 2, 4, 6 and Keywords: rainbow trout, probiotics, growth,
9 weeks. Growth performance was not affected by welfare, sensory evaluation, flesh quality
dietary probiotic supplementation. Alternative comple-
ment activity (ACH50) was significantly higher in fish
Introduction
fed the diet with probiotic (58.4  3.7 units mL
1)
than in the control group (46.9  3.1 units mL
1) Currently, about 1000 tonnes of rainbow trout
(P = 0.03). Several biochemical markers were altered are produced in Portugal in a variety of systems,
by the dietary treatment. 7-ethoxyresorufin-O-deethy- including flow-through systems and cages in lakes
lase (EROD) (P < 0.01), glutathione S-transferase (INE 2011). As with most aquaculture species, the

© 2015 John Wiley & Sons Ltd 1

Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al. Aquaculture Research, 2015, 1–16

farming of freshwater trout presents some con- performance of ornamental fish fed diets supple-
cerns since intensification of any biological produc- mented with Bacillus subtilis was enhanced. Queiroz
tion system may cause substantial impacts to the and Boyd (1998) reported that a multi-strain probi-
environment and society (Barton & Fløysand otic added to the rearing water increased survival
2010; Burridge, Weis, Cabello, Pizarro & Bostick and production of channel catfish (Ictalurus puncta-
2010). Several countries involved in aquaculture tus). A strain of Carnobacterium sp. previously iso-
activity have developed strategies and standards lated from the intestine of Atlantic salmon was
that may address key impact areas, including effective in controlling infections caused by A. sal-
water and feed quality control, biosecurity and monicida, Vibrio ordalii and Yersinia ruckeri in juvenile
health management (Grøttum & Beveridge 2007). salmonids (Robertson, O0 Dowd, Burrells, Williams &
However, disease outbreaks and declining environ- Austin 2000). Probiotic bacteria are also capable of
mental conditions due to fish crowding and con- enhancing non-specific immune responses.
stant handling have compromised production According to Irianto and Austin (2002), there was
economics (Barton & Fløysand 2010) and raised an increase in lysozyme and phagocytic activity of
public health concerns (Sapkota, Sapkota, Kuchar- head-kidney macrophages in fish exposed to a range
ski, Burke, McKenzie, Walker & Lawrence 2008) of probiotics compared to a control group.
about intensive aquaculture production. Bacterial strains that are commonly used as
Husbandry and management are essential to human probiotics (e.g. Lactobacilli and Enterococci)
maintaining the health and welfare of farmed fish. have been also tested in fish health experiments.
Recently, disease prevention and control measures L. rhamnosus administration to rainbow trout
have substantially increased the use of chemical reduced the mortality caused by A. salmonicida by
additives and veterinary medicines (Barton & Fløy- 53% when compared to control groups (Nikoske-
sand 2010; Burridge et al. 2010). However, the lainen, Salminen, Bylund & Ouwehand 2001). A
use of antimicrobial agents increased drug resis- reduction in V. anguillarium and V. ordalii was also
tance (Collado, Fouz, Sanjuan & Amaro 2000; reported in rainbow trout after oral administration
Nomoto 2005), as well as environmental problems of V. alginolyticus (Sakai, Yoshida, Astuta & Kobay-
associated with the chemical additives (Wang & ashi 1995). In the same study, phagocytic activity
Xu 2004; Burridge et al. 2010). of leucocytes increased in fish after oral treatment
The defence mechanisms of fish may be stimu- with Clostridium butyricum, suggesting an immu-
lated through prior administration of immunostim- nostimulation that might help control disease.
ulants such as probiotics, offering promising This study evaluated the effects of dietary probi-
methods of protecting fish health without using otic supplementation on the growth performance,
antibiotics. Probiotics are defined as ‘live microor- immune and antioxidant responses, sensory evalu-
ganisms, which when consumed in adequate ation and freshness of fish during cold storage,
amounts, confer a health effect on the host’ (FAO/ and changes in the intestinal microbiota of juve-
WHO 2006). The benefits of probiotics in terrestrial nile rainbow trout reared in floating net cages
animals are well documented, but the studies in under natural environmental conditions.
aquatic animals showed benefits at various levels.
The effects of probiotics in farmed fish have
Materials and methods
focused on fish health, farming performance, gut
microbiota changes and environmental impact All procedures were conducted by an expert in
(Garcia de La Banda, Lobo, Le
on-Rubio, Tapia- laboratory animal science and approved by the
Paniagua, Balebona, Morinigo, Moreno-Ventas, Portuguese Veterinary Authority (1005/92,
Lucas, Linares, Arce & Arijo 2010; Ramos, Weber, DGV-Portugal, following FELASA category C rec-
Goncßalves, Santos, Rema & Oz
orio 2013). ommendations), according to the guidelines on the
The potential benefits of probiotics in aquacul- protection of animals used for scientific purposes
ture include improved water quality, enhanced published in the European directive 2010/63/UE.
dietary nutrient utilization through the production
of supplemental digestive enzymes, lower incidence
Fish and husbandry conditions
of diseases and greater survival (Verschuere,
Rombaut, Sorgeloos & Verstraete 2000). Ghosh, The feeding trial was carried out on a commercial
Sinha and Sahu (2008) observed that growth trout farm (Quinta do Salm~ ao, Alto Rabag~
ao dam,

2 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16

Aquaculture Research, 2015, 1–16 Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al.

Nov ´10 Dez ´10 Jan ´11

16

14

12

Temperature (oC)
10

2
Figure 1 Daily variation in water
temperature in a feeding trial with
rainbow trout under natural condi- 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

tions. Days

41°440 23″ N, 7°510 28″ W), using two 500-m3 fish oil was sprayed onto the extruded pellets. The
floating net cages under natural conditions with control diet was coated using the same procedure,
ambient photoperiod and temperature (Fig. 1). but without probiotic supplementation.
Each cage was stocked with 30 thousand rainbow Each diet was distributed to fish twice daily for
trout (90.5  7.5 g initial body weight), in which 9 weeks. The nutrient composition of the diets
a randomly collected sub-population of 5% of fish were as follows: 93% dry matter, 42% crude pro-
in each cage were individually marked with visible tein, 24% crude lipid, 21 kJ g
1 gross energy and
implant tags (Northwest Marine Technology, Shaw 1.6% phosphorus.
Island, WA, USA) for growth measurements.

Sampling
Diet and feeding protocol
All tagged fish were weighted before the start of the
Since the fish trial were carried out under commer- feeding trial (week 0), and then 400 tagged fish
cial conditions, the feeding level used were the were weighted at 2, 4, 6 and 9 weeks after the start
same applied by the commercial fish farm (i.e. of the feeding trial for the calculation of growth per-
50 kg per cage per day). Such fixed feeding level formance parameters. Tissues were collected from
when expressed as % body weight day
1 varied 12 additional fish per treatment at the beginning
along the trial, as determined during the fish sam- and at the end of the feeding trial to assess selected
plings (feed intake: 1.5, 1.1, 0.9 and 0.8% non-specific immune parameters in plasma, selected
BW day
1 for week 2, 4, 6 and 9 respectively. Fish biochemical markers in liver and rheological mea-
were fed with two experimental diets; a basal diet surements in faeces. Prior to tissue collection, fish
without probiotic (control diet) and a basal diet were anaesthetized with 0.4 ml L
1 ethylene-gly-
supplemented with 0.2% (2 9 109 CFU kg
1 diet) col-monophenyl-ether (Merck, Lisboa, Portugal).
of a multi-strain probiotic bacteria (Bacillus sp., Changes in the microbiota attached to the intestinal
Pediococcus sp., Enterococcus sp., Lactobacillus sp.,). mucus were also determined. In addition, whole fish
All dietary ingredients, except the probiotic were kept boxed in ice for sensory evaluation and
product, were grinded below 250 lm and then assessment of fish freshness during cold storage.
mixed accordingly to the target formulation to
attain a basal mixture. After extrusion, the basal
Growth performance
diet was divided into two portions, in which fish
oil was incorporated in both portions by a vacuum The following parameters were used to evaluate
coating process at about 45°C. The probiotic bac- growth performance:
teria were mix to the fish oil before vacuum coat- Weight gain (WG) = BW2
BW1; where BW1
ing process and mix to the basal mixture after and BW2 are initial and final individual body
extrusion. Vacuum was slowly released and the weights (g) respectively.

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16 3

Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al. Aquaculture Research, 2015, 1–16

Daily growth index (DGI) chrom Ltd., Cambridge, UK), and the analysis of
= 100 9 [(BW2)1/3
(BW1)1/3]/feeding days. EROD activity was conducted using a microplate
Thermal growth coefficient (TGC) fluorometer (Fluoroskan Ascent) (Kopecka-Pila-
= [(BW2 1/3
BW1 1/3)/(∑°C)] 9 1000, rczyk & Coimbra 2010). Briefly, total protein con-
where ∑°C is the sum of daily temperatures (°C) tent was determined in the hepatic S15 fraction
for the whole experimental period. according to the method of Lowry, Rosebrough,
Farr and Randall (1951) using bovine serum albu-
min as a reference standard, adapted for micro-
Non-specific immune responses
plate reader. Hepatic biomarkers were analysed in
Blood from 12 individual fish per treatment was the post-mitochondrial fraction (S15-PMS) of fish
collected by caudal puncture with lithium-heparin liver using the following protocols adapted for mi-
coated syringes. Plasma was immediately separated croplates: EROD, Galgani and Payne (1991) and
by centrifugation and stored at
80°C until subse- Kopecka and Pempkowiak (2008); GST, Habig,
quent analysis. Lysozyme activity was determined Pabst and Jakoby (1974); SOD, McCord and Frido-
by measuring the decrease in the turbidity of a vich (1969); CAT, Claiborne (1985); LP, Niki
Micrococcus lysodeikticus suspension, as described (2000); LPO, Sol
e, Baena, Arnau, Carrasson, May-
by Ellis (2001) and Hutchinson and Manning nou and Cartes (2010); POx, Levine, Williams,
(1996). Lysozyme concentration (lg mL
1 or Stadman and Shacther (1994) and GPx, L
opez-
U mL
1) was estimated using lyophilized hen egg- Galindo, Vargas-Chacoff, Nebot, Casanueva, Rubio,
white lysozyme (Sigmaâ; Sigma-Aldrich, Sintra, Mancera and Sol
e (2010).
Portugal) serially diluted in sodium phosphate buf-
fer (0.05 M; pH 6.2), enabling the establishment of
Rheological analysis of the faeces
a standard curve. A unit of lysozyme activity was
defined as the amount of plasma causing a The rheology of the trout faeces was determined in
decrease in the absorbance of 0.001 min
1. a Paar Physica UDS 200 rotational rheometer
Alternative haemolytic complement pathway with the following configuration: UM 200 measur-
(ACH50) activity was determined following the ing drive, MC 200 electronics control unit and
method described by Sunyer and Tort (1995), VT2 thermostatic bath. The UM 200 measuring
using rabbit red blood cells (RaRBC) as target cells drive was equipped with an air-bearing mecha-
to haemolysis as described previously (Chen, Loch- nism to minimize friction, and a permanent mag-
mann, Goodwin, Praveen, Dabrowski & Lee 2003). netic synchronous drive motor with adjustable
The reciprocal of the serum dilution causing 50% rotational drive speeds used to measure torque. A
lysis of RaRBC was designated as the ACH50 cone and plate (CAP) measuring system was used
(Tort, Gomez, Montero & Sunyer 1996). Results (Fig. 2; MK22, Paar Physica; Paar GmbH, Graz,
are presented as ACH50 units mL
1. Austria). The cone tips were truncated at a dis-
tance f = 50 lm from the vertices, so the cones
did not contact the plate. A sample of 700 lL of
Biochemical markers
faeces solution was placed on the plate (TEK 180
The following biomarkers were measured in liver from Paar Physica) and analysed at 8°C and 16°C,
samples: EROD, glutathione S-transferase (GST) to simulate the range of gut temperature fish were
and three enzymatic antioxidants: superoxide exposed during the feeding trial. The cone was
dismutase (SOD), catalase (CAT) and glutathione lowered over the sample until its tip was at a dis-
peroxidase (GPx). In addition, lipid peroxidation tance f + ξ from the plate, where ξ is an addi-
(measured in two ways: using the thiobarbituric tional clearance set to the CAP when solids of the
acid, and using 1-methyl-2-phenylindole, further sample can adhere to the cone and slow its rota-
referred to as, respectively, LP and LPO), and pro- tion. Since the samples of fish faeces had some lar-
tein oxidase (POx) were determined. The activities ger suspended solid materials that could affect the
of GST, SOD, LP, LPO, POx and protein content rheogram, the rheological measurements were
were measured with a microplate reader (BIO-TEK made with oscillatory tests where the material was
PowerWave 340; BIO-TEK Instruments Inc., not flowing inside the CAP measuring system. The
Winooski, VT, USA), CAT activity – cuvette spec- oscillation amplitude was 10 mrad and frequency
trophotometer (LKB BIOCHROM, Ultrospec II; Bio- values between 6 and 10 Hz were reported. After

4 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16

Aquaculture Research, 2015, 1–16 Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al.

Amplification of the 16S rDNA variable region V3

The universal primer pairs 518r and 341f-GC
(Muyzer, de Waal & Uitterlinden 1993) and the
KAPA 2G Robust PCR kit from PEQlab (Germany)
were used to amplify a 217 bp from the region V3
of the 16S rDNA. A KAPA 2G Robust PCR kit was
used according to the manufacturer’s instructions
with 100 ng genomic DNA as a template. The
amplification steps on an Eppendorf thermocycler
included an initial denaturation at 95°C for
5 min, followed by 30 cycles of 95°C for 30 s,
56°C for 20 s and 72°C for 40 s, with final elon-
gation at 72°C for 7 min.

Denaturing gradient gel electrophoresis and

cluster analysis
Figure 2 Schematic of the cone and plate measuring
systems. The INGENYphorU system (Ingeny International
BV, Goes, the Netherlands) was employed for
the cone was lowered over the sample, the denaturing gradient gel electrophoresis (DGGE). To
cone was rotated to see whether solids had counteract individual variations, PCR products
adhered to it. from 2 to 3 fish per group and time point were
pooled. The PCR products, as well as reference
Microbiota in the intestinal mucus samples consisting of a mix of 16S rDNA V3 frag-
ments from various bacteria with different G/C
DNA extraction contents, were separated in a 30–60% gradient,
After faeces were removed for rheological analysis, 8% (w/v) polyacrylamide gel containing 32%
a method adapted from Griffiths, Whiteley, O0 Don- formamide and 5.6 M urea heated to 60°C for
nell and Bailey (2000) was used to extract geno- 16 h. After electrophoresis, the DGGE profiles were
mic DNA from 300 mg mucus and hind gut tissue visualized with the Sybr Green I Nucleic Acid Gel
from 12 fish per treatment at the beginning and Stain (Sigma-Aldrich) according to the manufac-
end of the trial. In brief, samples were transferred turer’s instructions and documented with the Bio-
into bead-beating tubes [150 mg glass beads Vision system and Vision-cap software (PeqLab,
(0.5 mm) and 150 mg ceramic beads (1.4 mm)] Germany). The DGGE profiles were analysed with
from PEQlab (Erlangen, Germany) and incubated the Gel compare II software version 6.0 (Applied
for 2 h at 37°C with 0.5 mL hexadecyltrimethy- Maths NV, Sint-Martens-Latem, Belgium) using
lammonium bromide buffer and 0.1 mL lysozyme the reference samples for normalization. Dendro-
(100 mg mL
1). Cells were lysed with 1 vol phe- grams were created based on Pearson’s correlation
nol-chloroform-isoamylalcohol (25:24:1; pH 8.0; and unweighted pair group method with arithme-
Sigma-Aldrich) for 30 s
1 in a bead beater (Precel- tic mean. The reliability of the dendrogram was
lys-PEQlabs, Germany) set to 5.5 m s
1. The fol- determined with the cophenetic correlation. Inten-
lowing centrifugation steps and treatments with sity and number of the operational taxonomic
RNAaseA and ProteinaseK were conducted accord- units (OTU) of each profile were determined with
ing to protocols described by Sambrook (1989). the software Gel compare II version 6.0 (Applied
DNA was precipitated with 0.6 vol isopropanol Maths NV).
and 0.1 vol 3 M sodium acetate (pH 5.2) for 2 h
at room temperature. After two washing steps
Sequence analysis
with 70% ethanol, the DNA pellet was dried and
taken up in 60 lL double-distilled water. DNA DNA bands representing OTUs were aseptically
quality and quantity were assessed with gel elec- excised from the gel and eluted in 50 lL sterile
trophoresis and the Nanodrop spectrophotometer. water. The eluate (8–10 lL) was used as a template

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16 5

Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al. Aquaculture Research, 2015, 1–16

in a PCR reaction with the oligos 518r and 341f, Table 1 Spreadsheet evaluation and score for quality
as described above. The resulting PCR products index method in rainbow trout (Wuennenberg 2008)
were gel purified with the Illustra GFX PCR
DNA and gel band purification kit (GE health- Quality parameters Description Score

care, Madrid, Spain) according to the manufac- Skin

turer’s instructions. The purified fragments were Colour/appearance Bright, iridescent 0
sent for sequencing with the 518r oligo to LGC pigmentation, 1
clear red stripe
genomics (Germany). Resulting DNA nucleotide
Rather dull, becoming
sequences were compared to the BLAST database discoloured
nucleotide collection (blastn) using Megablast Gloss of skin Bright 0
to search for highly similar sequences (http:// Dull 1
blast.ncbi.nlm.nih.gov). Mucus Clear 0
Milky 1
Viscosity of mucus Thin, low viscosity* 0
Sensory evaluation and assessment of fish Thick, high viscosity* 1
Texture In rigour, pre-rigour 0
freshness during storage on ice
Finger mark disappears 1
To evaluate sensory quality of the fish stored on rapidly 2
Finger leaves mark over
ice, the quality index method (QIM) and two instru-
3 seconds
mental methods (Torrymeter and Freshmeter) were Eyes
used. The analyses were performed in fish collected Corona Clear 0
at the start and at the end (9 weeks) of the feeding Milky 1
trial. A randomly sampled group (n = 13 fish per Form Convex 0
Flat 1
treatment) was packed in cooling boxes containing
Sunken 2
ice. Evaluations were performed on days 1, 3, 5, 7
Gills†
and 10 of ice storage. Day 10 of ice storage was Odour Fresh, metallic 0
considered the point of rejection, since the fish Neutral 1
become unacceptable for consumption and/or com- Sweet, slightly rancid 2
Metal, rotten 3
mercialization (Soccol & Oetterer 2003).
Abdomen
For the QIM, fish were individually subjected to
Odour Fresh pond water 0
sensory analysis according to several evaluation Neutral 1
criteria (Table 1) developed for rainbow trout Rotten 2
(Wuennenberg 2008). The QIM is based on objec- Quality Index (0–14)
tive evaluation of certain attributes of raw fish *Looking at the ropy from the fish when lifting it from the
(e.g. skin, eyes, gills, etc.) using a point scoring table.
system (from 0 to 3). The QIM was calculated as †The gills were removed during the slaughter process. The
the sum total of the demerit points for every indi- odour analyses were carried out for the gills cavity.
vidual attribute.
To evaluate physical freshness, both sides of
the fish were measured in triplicate (six values
Statistical analyses
per fish) with the RT Freshmeter type RT-2E (Ra-
fagnataekni Eletronics, Reykjavik, Iceland) and All parameters were analysed by one-way ANOVA.
GR Torry Fish Freshness Meter (Torrymeter) type Treatment means were considered significantly dif-
295 (Distell Industries, Scotland, UK). The Fresh- ferent at P ≤ 0.05. Immune parameters were anal-
meter is fitted with electrodes that take a series ysed with the STATVIEW program (SAS Institute,
of measurements based on electrical impedance version 5.0.1, Cary, NC, USA). Biochemical mark-
of the fish skin. The measurements were taken ers were analysed using the STATSOFT STATISTICAâ
with the electrodes continuously touching the 10.0 software package. The sensory evaluation
whole body of the fish (Vaz-Pires & Barbosa data were analysed using SAS version 9.1 (SAS
2004). The Torrymeter was positioned so that Institute). Growth performance and rheological
the electrodes remained in the fish longitudinally, parameters were analysed using IBM SPSS Statistics
and the measurements were taken at the head, for Windows, Version 21.0 (Armonk, NY, IBM
mid-body and tail. Corp).

6 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16

Aquaculture Research, 2015, 1–16 Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al.

such as temperature and photoperiod (Handeland,

Results and discussion
Imsland & Stefansson 2008; T€ urker & Yıldırım
2011). Although salmonids are eurythermal,
Growth performance
extreme environmental conditions negatively influ-
Fish grew 2½ fold throughout the feeding trial ence their farming production efficiency (Hande-
from 87  5 g to 214  15 g (Fig. 3), with no land et al. 2008). The selection of bacterial strains
significant effect of dietary treatment. The biomass as probiotic candidates for aquaculture applica-
increase observed in this study is similar to those tions should consider not only their antagonism
achieved under similar rearing conditions in other towards pathogens and growth improvement qual-
studies (Kaushik 1998; Dumas, France & Bureau ities but also how the probiotic agent responds
2007; Davidson, Good, Welsh & Summerfelt under a wide range of rearing/environmental con-
2011). In Table 2, body weight, weight gain, DGI ditions. Panigrahi, Kiron, Satoh, Hirono, Kobay-
and TGC are presented for each sampling period ashi, Sugita, Puangkaew and Aoki (2007)
(0–2, 2–4, 4–6 and 6–9 weeks). The DGI ranged demonstrated that probiotic feeding of Lactobacillus
from 1.5 to 4.0% BW day
1 and TGC ranged from rhamnosus, Enterococcus faecium or Bacillus subtilis
1.3% to 3.7% BW day
1. The DGI and TGC to rainbow trout had a positive effect on the
showed a reduction during sampling 3 (4– immune response of the fish. In particular, the fish
6 weeks) and sampling 4 (6–9 weeks trial). fed the E. faecium strain showed better perfor-
Growth rates decrease as temperature decreased mance, probably because the bacteria’s tempera-
(Fig. 1), but no dietary treatment effect was ture optima was compatible with that of the
observed (P > 0.05). The growth rate of fish is culture environment (Panigrahi et al. 2007). Tem-
size-dependent and strongly influenced by factors perature is a major environmental factor affecting

250
Control
Probiotic
200
Live weight (g)

150

100

50 Ycontrol = 1,0308x 2 + 25,929x + 66,394 Yprobiotic = –1,1322x 2 + 37,292x + 45,887

R² = 0,99 R² = 0,99

Figure 3 Temporal variation in

live weight of rainbow trout fed 0 2 4 6 9
the experimental diets for 9 weeks. Trial duration (weeks)

Table 2 Temporal variation in growth performance of rainbow trout (90.5  7.5 g initial body weight) fed the control
and probiotic diets

0–2 weeks 2–4 weeks 4–6 weeks 6–9 weeks

Control Probiotic Control Probiotic Control Probiotic Control Probiotic

BW 128.1  9.9 110  2.9 158.1  9.1 150.1  3.9 179.8  6.8 175.6  6.7 225.5  4.1 204.8  4.4
WG 35.5  17.0 29.5  11.7 31.9  17.1 40.3  10.9 28.5  15.1 24.8  11.9 43.7  14 29.9  12.5
TGC 2.60  1.19 2.34  0.93 2.81  1.54 3.70  1.02 2.38  1.29 2.02  1.06 1.75  0.57 1.30  0.48
DGI 3.86  1.57 3.33  1.32 3.06  1.67 4.02  1.11 2.24  1.22 1.90  1.0 2.02  0.66 1.44  0.59

BW, body weight; WG, weight gain; TGC, thermal growth coefficient; DGI, daily growth index.

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16 7

Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al. Aquaculture Research, 2015, 1–16

microbial growth, and optimal temperatures for key component of the non-specific defence mecha-
growth differ among microorganisms. E. faecium nism, that is, broadly protective against potential
was found to be more psychrotolerant than L. pathogens, as well as parasites. Merrifield, Dimi-
rhamnosus or B. subtilis, with E. faecium growing troglou, Foey, Davis, Baker, Bøgwald, Castex and
well at temperatures ranging from 12 to 30°C. In Ringo (2010) reviewed the effects of probiotics on
this study, water temperature decreased signifi- the immune responses of salmonids. Kim and Aus-
cantly from 12.5°C (0–2 weeks) to 10.1°C (2– tin (2006) observed significant increases in lyso-
4 weeks) and from 8.8°C (4–6 weeks) to 7.8°C zyme activity in rainbow trout fed diets with
(6–9 weeks). Since fish are poikilotherms, their Carnobacterium sp. Panigrahi, Kiron, Kobayashi,
intestinal temperature is similar to the ambient Puangkaew, Satoh and Sugita (2004) observed
temperature. It is likely that the very low tempera- significant increases in both lysozyme and alterna-
ture that occurred after week 2 of the feeding trial tive complement activity in response to the probi-
repressed proliferation of the probiotic bacteria, otic L. rhamnosus. However, the increases were
inhibiting their positive actions. Efficacy of the pro- only seen in the latter study in fish fed the highest
biotics in terms of improved growth performance dose of probiotic tested. Batista, Ramos, Cunha,
might be more obvious at higher temperatures. Barros, Crist
ov~
ao, Rema, Pires, Valente and Oz
orio
(2014) showed in sole (Solea senegalensis) that die-
tary probiotic supplementation did not affect the
Non-specific immune responses
non-specific immune responses, although lysozyme
Alternative complement activity (ACH50) and and ACH50 activities tended to increase with pro-
Lysozyme are depicted in Figs 4a and b respec- biotic treatment. This trend was also observed by
tively. Lysozyme did not vary significantly between D
ıaz-Rosales, Arijo, Chabrill

on, Alarc
on, Tapia-Pa-
treatment (555.5  18.9 to 575.2  15.9 niagua, Mart
ınez-Manzanares, Balebona and Mor-
units mL
1 for the control and probiotic treated i~
nigo (2009) working with sole, where probiotic
fish, respectively). The ACH50, however, was sig- treatment induced a slightly increase in the respi-
nificantly higher in the probiotic treated fish ratory burst activity. The effects of probiotics on
(58.4  3.7 units 25 lL
1 plasma) than in the the immune response of fish are often contradic-
control (46.9  3.1 units 25 lL
1 plasma) tory, allegedly as a consequence of differences in
(P = 0.03). Alternative complement activity is a type and concentration of probiotic used, duration
of treatment (Merrifield et al. 2010), among other
factors. In this study, only one level of dietary pro-
70 (a) biotic was tested, and it is possible that other doses
a
60 and/or different probiotic bacteria species would
ACH50 (units/25 µL)

b
50 lead to differences in non-specific immune
40 responses. Nevertheless, the multi-strain probiotic
30
was effective in enhancing the alternative comple-
ment activity, which confirms the immunostimula-
20
tory potential, when supplemented at
10
1 9 1010 CFU kg
1 diet.
0
Control Probiotic

600 Biochemical markers

Lysozyme (units mL–1)

(b)
590
580 The results of biomarker measurements are shown
570 in Fig. 5. Several biochemical markers were
560 altered by the dietary treatment. EROD (Fig. 5a,
550
540
P < 0.01), GST (Fig. 5c, P < 0.001), GPx (Fig. 5f,
530 P < 0.05) increased and LP (Fig. 5h, P < 0.01)
Control Probiotic
decreased with dietary probiotic supplementation.
Treatment
CAT, POx and LPO values (Figs. 5d, 5g and 5i)
Figure 4 Non-specific immune responses. Alternative were lower in the probiotic group than in the con-
complement activity (ACH50, (a, units 25 lL
1, trol group, but changes were not statistically
P < 0.05.) and lysozyme (b, units mL
1). different.

8 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16

Aquaculture Research, 2015, 1–16 Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al.

(a) (b) (c)

Control Probioc Control Probioc Control Probioc

(d) (e) (f)

Control Probioc Control Probioc Control Probioc

(g) (h) (i)

Control Probioc Control Probioc Control Probioc

Figure 5 Results of biomarker measurements (* indicates P < 0.05). Full caption: Hepatic biomarkers (mean  SD)
in rainbow trout fed the experimental diet for 9 weeks. –control (day 0 and week 9) and –probiotic treatment
group (week 9).

Activity of the enzyme cytochrome P4501A, Some statistical differences in biomarkers were
measured as EROD activity, is an indicator of also found between the control at day 0 and the
membrane activity, because this enzyme is control at week 9 (in EROD, SOD, GST, POx and
involved in Phase I of metabolism and detoxifica- LPO), as well as between the control at day 0 and
tion of drugs and xenobiotics (Ankley, Blazer, Pla- the treatment group at week 9 (EROD, SOD, GST,
kas & Reinert 1989), and it is tethered to the GPx, LP and LPO). The differences in EROD, SOD
microsomal membrane via a single transmem- and LPO are most likely due to the different tem-
brane region (Bus & Gibson 1979). GST is an peratures at different sampling points, and not to
enzyme of Phase II metabolism that plays an fish growth.
important role in maintaining cell homoeostasis.
Dietary probiotics apparently increased synthesis
Rheological analysis
of both enzymes, thereby accounting for the
increased activity. The activities of SOD, CAT and The results of the rheological analyses are shown
GPx are important markers of oxidative stress, in Table 3. The values of complex viscosity and
since these enzymes tend to inhibit the formation the loss modulus were obtained at a strain of 60%
of reactive oxygen species (ROS) (Van der Oost, Be- by averaging the measurements in the frequency
yer & Vermeulen 2003). ROS can cause formation range 6–10 Hz. The group fed the diet with probi-
of fatty acid hydroperoxides (measured as LP) otic showed a decrease in the complex viscosity
(Stegeman, Brouwer, Di Giulio, F€ orlin, Fowler, and the loss modulus of the faeces. The faeces are
Sanders & Van Veld 1992) and induction of car- pseudoplastic and dietary probiotic supplementa-
bonyl content (oxidised protein – POx) (Parvez & tion had very little effect on the rheological behav-
Raisuddin 2005). The lower level of LP observed iour of the faeces (Fig. 6). Furthermore, the effect
in the probiotic group, together with the slight of probiotic supplementation on the stability of
changes in SOD (Fig. 5b) and CAT, suggest that faecal matter is not clear (Brinker, Koppe & R€ osch
the probiotic mediated a beneficial decrease in 2005; Brinker & Friedrich 2012).
oxidative stress. This occurred although the level The values for viscosity and loss modulus of the
of GPx was higher in fish fed probiotic diet. faeces decreased with temperature for both fish

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16 9

Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al. Aquaculture Research, 2015, 1–16

00
Table 3 Average value and standard deviation of the complex viscosity, r(g∗), and of the loss modulus, r(g ), in faeces
of trout fed control and probiotic diets for 9 weeks

8 °C 16 °C
00 00
r(g∗), Pa.s r(g ), Pa r(g∗), Pa.s r(g ), Pa

Control 2.50  1.40 92.0  20.0 1.00  0.46 57.0  0.8

Probiotic 0.91  0.59 33.0  23.0 0.58  0.28 26.0  13.0

In the resulting dendrogram, control samples from

both time points and treatment samples from the
initial time point built a cluster, whereas treat-
ment samples taken at 60 days built another clus-
ter. Although the number of OTUs was limited,
treatment samples at 60 days were separated from
the others, with the exception of one 60-day con-
trol sample.
Overall, there was a limited effect of the probiot-
ics on the number of OTUs that were detected in
the DGGE analysis. Signal strength of the observed
OTUs differed between control and treatment sam-
ples and could indicate variations in the abun-
dance of certain OTUs. However, DGGE is a PCR-
Figure 6 Average flow curve of faeces of rainbow
based method and thus differences in signal
trout fed the dietary treatments.
strength could be due to different levels of amplifi-
cation. Ramos et al. (2013) working with rainbow
trout reared at constant water temperature (17°C)
groups. Brinker et al. (2005) reported storage and fed the same probiotic (Bacillus sp., Pediococcus
modulus values around 100 Pa to 160 Pa for fae- sp., Enterococcus sp., Lactobacillus sp.) as in this
ces of rainbow trout, measured with a strain of study showed changes in gut microbial composi-
60% at 1 Hz frequency and 4°C. The values in tion. Number of OTUs and Shannon index were
Table 3 are similar to those findings, considering higher in fish fed probiotic diet than in untreated
the standard deviation of the results and also the group. It is plausible to infer that a sharp decrease
fact that the elastic modulus decreased with tem- in water temperature were the major factor affect-
perature. ing the changes in the microbiota in the intestinal
mucus.
To determine the identity of several OTUs and to
Microbiota of the intestinal mucus
ensure that DNA fragments with the same migra-
DGGE is a culture-independent technique com- tion from different profiles corresponded to the
monly used to analyse the bacterial composition of same OTU, bands were excised, reamplified and
various environmental and animal samples (Muy- sent to a commercial lab for sequencing. The
zer et al. 1993). Because DNA was isolated after resulting nucleotide sequences were compared to
samples had been withdrawn for rheology, only the blastn using Megablast to search for highly
one to three dominant OTUs were detected in each similar sequences. Results of the BLAST search are
DGGE profile (Fig. 7). Species reflected by these listed in Table 4. Of 20 total bands, the resolution
OTUs likely adhere to the intestinal mucus. The of only one band (#19) was too low to permit a
most obvious changes were observed when the blastn search. Unfortunately, almost all bands pos-
two time points were compared. Few OTUs were sessed high similarities to uncultured bacteria that
present initially (0 days), but at 60 days additional were previously detected in DGGE analyses.
bands appeared. These OTUs were present in the However, most of the isolates are associated with
control and treatment samples. Based on the DGGE fish, indicating that these are species found in
profile, a cluster analysis was performed (Fig. 7). aquatic animals. Compared to mammals, fish have

10 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16

Aquaculture Research, 2015, 1–16 Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al.

Figure 7 Cluster analysis and 16S rDNA PCR-DGGE profiles from intestinal mucus samples. Dendrogram (left) and
DGGE profiles (right). Samples from fish fed with control diet at time point 0 days (0 day ctrl) and 60 days (60 day
ctrl) were compared to fish fed a diet supplemented with probiotics at time point zero days (0 day pro) and 60 days
(60 days pro). Numbers in the DGGE profile indicate the DNA fragments that were excised and sent for sequence
analysis.

Table 4 BLAST search results for 16SV3 DNA fragments

Band no. Closest GenBank entry % Identity Comments

1, 3, 6, 10, 12 and 15 Uncultured bacterium (HM216398) 98, 100, 100, 97, DGGE isolate from the gut of grouper
100 and 93
2 Enterococcus sp. (JQ595484) 92
4, 5, 11 and 18 Uncultured bacterium (AM179931) 98, 98, 99 and 99 DGGE isolate from rainbow trout gut
7 Acinetobacter sp. 100 Isolated as part of the fish microflora
(Kapetanovi
c 2005)
8 Uncultured bacterium (JN032764) 100 Isolated from grass carp
Maybe related to Clostridium
9 and 16 Uncultured bacterium (GU939591) 98 and 97 Related to Ruminococcus?
13 Uncultured bacterium (EU764095) 95
14 and 20 Uncultured bacterium (AB649436) 100 and 100 Environmental DGGE isolate
17 Uncultured Weissella sp. (HM359077) 98 DGGE isolate

No sequence data for sample #19 could be obtained, due to incomplete PCR reactions. That might indicate that this band is either
an artefact or could not be eluted efficiently enough from the gel.

higher variability in the species composition of contact of the intestine with the surrounding envi-
their intestinal microbiota that strongly depends ronment. Many of the dominant species in fish gut
on environmental conditions and the constant are opportunists that are not permanent constitu-

© 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16 11

Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al. Aquaculture Research, 2015, 1–16

13 (a) Ycontrol = 1.92X + 1.35 Yprobiotic = 1.85X + 1.44 biota in this study included bacterial species that
R2 = 0.97 R2 = 0.91 cannot be cultured.
11

Sensory evaluation of freshness

Quality index

9
Immediately after fish samples were collected, the
Control eyes were clear and there was little mucus on the
7
Probiotic skin. The skin colour was bright, the gills were red
and the scales strongly adhered to the skin. The
5
skin was firm and elastic and the odour was
appropriate for fresh fish. All individuals presented
3
0 1 2 3 4 5 6 7 8 9 10
no injuries, stains or indications of disease.
The earliest modifications observed were in the
14 (b) Ycontrol = –1.10X + 13.95 eyes and the skin. By day 1 of ice storage, the eyes
Yprobiotic = –0.95x + 13.65 were milky and skin mucus increased. Between
R2 = 0.96
R2 = 0.94 day 3 and day 5, the intensity of skin brightness
decreased gradually. The skin became opaque and
Freshmeter value

12 the muscle texture had reduced firmness and elas-

ticity. By day 5 of ice storage, the gills started to
lose their red colour. At 7 days, there was a
change in the odour of the gills and loss of abdom-
10
inal elasticity. The final sign of deterioration was a
putrefaction odour at day 9 of ice storage.
The sensory attributes are presented in Figs 8
8 and 9. The QIM of the fish sampled initially and at
0 1 2 3 4 5 6 7 8 9 10 the end of the feeding trial (Figs 8a and 9a)
showed no significant effect of the dietary treat-
12 (c)
Ycontrol = –2.08X + 13.85 ment (P > 0.05). According to the QIM method,
R2 = 0.96 Y = –1.93X + 13.2
probiotic the shelf-life of farmed rainbow trout reared under
R2 = 0.98 the current conditions was not affected by the use
of probiotics.
The Freshmeter values for the initial samples
Torrymeter value

9
decreased from 13 to 8 (from day 1 to day 10 of
ice storage respectively) (Fig. 8b). By the end of
the feeding trial (week 9), the values had
6
decreased from 13 to 9 (Fig. 9b). The Torrymeter
values followed the same pattern: during the
10 days of ice storage they decreased from 12 to
3 and from 12 to 6 for fish sampled initially
3
0 1 2 3 4 5 6 7 8 9 10 and at the end of the trial respectively (Figs 8c
Time in ice (days) and 9c).
The pattern of degradation occurred similarly in
Figure 8 Regression analyses (mean  SD) of quality
the two dietary treatments, so the probiotic did
index method (a), Freshmeter (b) and Torrymeter (c) of
not affect product quality or shelf-life of the fish.
rainbow trout before the start of the feeding trial. Each
point represents the mean of 13 animals.
Loss of freshness in refrigerated fish is normally
attained at day 8–10 of ice storage (Soccol & Oet-
terer 2003), similar to the storage period in this
ents of the normal fish microflora (Gatesoupe study. Therefore, it appears that the probiotic had
2005). Aquatic environments are highly variable no effect on the natural bacteria associated with
habitats, and only a small proportion of the bacte- decomposition during storage. However, the good
ria they contain is culturable and has been correlation of results obtained using the three
described. Thus, it is not surprising that the micro- methods (QIM, Freshmeter and Torrymeter) indi-

12 © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–16

Aquaculture Research, 2015, 1–16 Probiotic supplementation in juvenile rainbow trout R O A Ozorio et al.

(a) Y product. Additional studies are needed to deter-

12 = 1.26X + 0.40
control mine how probiotic-induced changes in the gut
R2 = 0.92
10 microflora mediate improvements in fish perfor-
mance.
8
Quality index

Acknowledgments
6
Control
Probiotic This work was carried out as part of the ECOPI-
4
SCIS project with the financial support of Quadro
Yprobiotic = 1.20X + 0.53 de Refer^encia Estrat
egico Nacional – QREN and
2 R2 = 0.91
Programa Operacional Regional do Norte – ON2
0 (Ref. no. 3442), supported by the European Regio-
0 1 2 3 4 5 6 7 8 9 10 nal Development Fund (ERDF). This research was
partially supported by the European Regional
14 (b) Ycontrol = –0.4577X + 12.939 Development Fund (ERDF) through the COMPETE
R2 = 0.79
– Operational Competitiveness Programme and
13 Yprobiotic = –0.4237X + 12.822
national funds through FCT – Foundation for Sci-
R2 = 0.78
Freshmeter value

ence and Technology, under the project ‘PEst-C/

12
MAR/LA0015/2013’. The oxidative stress analysis
were funded by FCT-MCTES (SFRH/BPD/32125/
11
2006).

10
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