Microsoft word - artículo 218
Lat. Am. J. Aquat. Res., 41(3): 490-497, The
lactic acid bacteria as growth promoters in fish fed 490
The use of lactic acid bacteria isolated from intestinal tract of Nile tilapia
(Oreochromis niloticus), as growth promoters in fish fed low protein diets
Maurilio Lara-Flores1 & Miguel A. Olvera-Novoa2
1Instituto de Ecología, Pesquerías y Oceanografía del Golfo de México, Universidad Autónoma
de Campeche, Av. Agustín Melgar y Juan de la Barrera S/N, Col. Buenavista
C.P. 24039. San Francisco de Campeche, Campeche, México
2Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional
Unidad Mérida Apdo. Postal 73-Cordemex, C.P. 97310, Mérida, Yucatán, México
In this study, the effect as growth promoter of five lactic acid strains (Enterococcus faecium, E.
sp. II), isolated from intestinal tract of Nile
tilapia (Oreochromis niloticus
), was evaluated. Eight isocaloric diets were formulated: one containing 40% of
protein as positive control, and seven with 27% protein. Five diets with 27% protein were supplemented with
one of the isolated lactic acid bacteria in a concentration of 2.5x106 cfu g-1 of diet. A commercial probiotic
based on S. faecium
and Lactobacillus acidophilus
was added at the same concentration to one 27% protein
diet as a comparative diet, and the last diet was not supplemented with bacteria (negative control). Tilapia fry
(280 mg basal weight) stocked in 15 L aquaria at a density of two per liter were fed for 12 weeks with
experimental diets. Results showed that fry fed with native bacteria supplemented diets presented significantly
higher growth and feeding performance than those fed with control diet. Treatment with Streptococcus
isolated from the intestine of Tilapia produced the best growth and feeding efficiency, suggesting that this
bacteria is an appropriate native growth promoter.
probiotics, Nile tilapia, Oreochromis niloticus
, growth promoter, lactic acid bacteria.
Uso de bacterias ácido lácticas aisladas del tracto intestinal de tilapia
nilótica (Oreochromis niloticus) como promotores de crecimiento en peces
alimentados con dietas bajas en proteína
Se evaluó el efecto como promotores de crecimiento de cinco cepas de bacterias ácido lácticas
(Enterococcus faecium, E. durans, Leuconostoc
II) aisladas del
tracto intestinal de tilapia nilótica (Oreochromis niloticus
). Se formularon ocho dietas isocalóricas: una
conteniendo 40% de proteína como control positivo y siete con 27% de proteína. Cinco dietas con 27% de
proteína fueron suplementadas con cada una de las bacterias aislada a una concentración de 2,5x106 ufc g-1 de
alimento. Un probiótico comercial a base de S. faecium
y Lactobacillus acidophilus
a la misma concentración
de inclusión bacteriana a una dieta con 27% de proteína como dieta comparativa, y la última dieta no fue
suplementada con bacterias (control negativo). Juveniles de tilapia (280 mg de peso basal) fueron distribuidos
en acuarios de 15 L de capacidad, a una densidad de dos juveniles por litro, alimentados durante 12 semanas
con las dietas experimentales. Los resultados mostraron que los organismos alimentados con las dietas
suplementadas con bacterias nativas presentaron crecimiento y asimilación del alimento significativamente
mayor que las dietas control. El tratamiento con Streptococcus
sp. I, aislada del intestino de la tilapia, produjo
el mejor crecimiento y la mejor eficiencia alimenticia, sugiriendo que esta bacteria es apropiada como un
promotor de crecimiento nativo de tilapia. Palabras clave:
probiótico, tilapia nilótica, Oreochromis niloticus
, promotor de crecimiento, bacterias ácido
Maurilio Lara-Flores (firstname.lastname@example.org)
491 Latin American Journal of Aquatic Research
morphology and produce certain enzymes and inhibitory compounds causing improved digestion
Aquaculture is a fast-growing and rapidly expanding
and absorption of nutrients, as well as enhanced
multibillion dollar industry. Marine capture fisheries
immune response (Verschuere et al.
, 2000). Several
and aquaculture supplied the world with about 104
studies have demonstrated that use of probiotics
million ton of fish in 2004 (FAO, 2007). Of this total,
improves health of larval and juvenile fish, disease
marine aquaculture accounted for about 18%, where
resistance, growth performance and body compo-
shrimp from aquaculture continues to be the most
sition, however, the mode of action in fish species
important commodity traded in terms of value (2.4
may vary between farmed fish species cultured in
million ton). Worldwide, the aquaculture sector has
been expanding at an average compounded rate of
The use of probiotic in feeds to improve growth of
9.2% per year since 1970, compared with only 1.4%
different fish species including African catfish, (Al-
for capture fisheries and 2.8% for terrestrial-farmed
Dohail et al.
, 2009); Senegalese sole (Sáenz de
meat production systems. During the last decades,
Rodrigáñez et al.
, 2009), Nile tilapia (Lara-Flores et
antibiotics used as traditional strategy for fish
, 2003, 2010; El-Haroun et al.
, 2006), Japanese
diseases management but also for the improvement of
flounder (Taoka et al.
, 2006), gilthead sea bream and
growth and efficiency of feed conversion. However,
sea bass (Carnevali et al.
, 2006) has been inves-
the development and spread of antimicrobial resistant
tigated. The effects of probiotics have been linked to
pathogens were well documented (Kim et al.
modulation of gut microbiota and establish-ment of
the beneficial microorganisms, higher specific and
There is a risk associated with the transmission of
total digestive enzyme activities, in the brush border
resistant bacteria from aquaculture environments to
membrane, which increases the nutrient digestibility
humans, and risk associated with the introduction in
and feed utilization (Verschuere et al.
, 2000; Balcazar
the human environment of nonpathogenic bacteria,
, 2006; Kesarocodi-Watson et al.
, 2008). In
containing antimicrobial resistance genes, and the
addition, the production of vitamins by these gut
subsequent transfer of such genes to human
microbiota could also increase vitamin synthesis and
pathogens (FAO, 2005). Considering these factors, as
improve fish health (Holzapfel et al.
, 1998). This
well as the fatal effect of residual antibiotics of
study was carried out to find the effect of isolated
aquaculture products on human health, the European
acid lactic bacteria from intestinal tract of Nile tilapia
Union and USA implemented bans on, or restricted
), on feed efficiency and
the use of antibiotics (Kesarcodi-Watson et al.
growth of fry Nile tilapia fed with low protein diets.
In connection with the ban of antibiotic growth
MATERIALS AND METHODS
promoters new strategies in feeding and health management in fish aquaculture practice have
received much attention (Balcázar et al.
, 2006). In
Five strains of lactic acid bacteria isolated from Nile
addition, the global demand for safe food has
tilapia intestine were characterized on the basis of
prompted the search for natural alternative growth
morphological, physiological and biochemical test by
promoters to be used in aquatic feeds. There has been
Bergey´s Manual of Systemic Bacteriology (Holt et
heightened research in developing new dietary
, 1993). Axenic cultures of the purified bacteria
supplementation strategies by promoting various
were tentatively identified, using Mini-API System
health and growth compounds as probiotics (Denev,
Bio-Merieux, as Enterococcus faecium, E. durans,
The importance of probiotics in human and
sp. II. Commercial probiotic containing
animal nutrition is widely recognized (Fuller, 1992;
mixture of Lactobacillus acidophilus
and S. faecium
Rinkinen et al.
, 2003), in recent years, the role of
probiotics in nutrition and health of certain
All bacteria were grown aseptically in 10 mL of
aquaculture species have also been investigated
MRS broth for 24 h at 35 ± 2°C. Five mL were
(Gatesoupe, 1999; Verschuere et al.
, 2000; Kesarcodi-
transferred under aseptic conditions into 250 mL of
Watson et al.
, 2008; Ringo et al.
, 2010; Merrifield et
MRS broth and held on a shaker at 150 rpm for 24-48
, 2010). It appears that probiotics provide benefits
h at 35 ± 2°C. The cells of each isolate were
by establishing favorable microbial communities,
harvested by centrifugation at 10,000 rpm for 15 min
such as lactic acid bacteria and Bacillus
and washed twice with phosphate buffer (PB) having
gastrointestinal track, which may alter gut
The use of lactic acid bacteria as growth promoters in fish fed 492
after which the different treatments were randomly
Eight isocaloric diets were formulated: one
assigned to the aquaria, with four replicates per
containing 40% protein, and the other seven with
27% of protein level. The lower protein inclusion in
Feed was manually administered ad libitum
the latter diets was used as a stress factor since that
times a day, for 12 weeks. A daily record was kept of
the optimum protein level for fry tilapia is 40%
feed offered. Bulk weight was measured weekly to
(Tacon, 1984). Each one of the lactic acid bacteria
follow growth in weight and calculate survival and
isolates was added to lower protein diets in a
feeding ration. Briefly, the fish were taken from each
concentration of 2.5x106 cfu g-1 of diet. The
tank using a net previously disinfected with a 1%
commercial probiotic was added to one diet with 27%
benzalkonium chloride solution. Initial mean weight
protein in a concentration of 2.5x106 cfu g-1 of diet
(IMW), final mean weight (FMW), specific growth
for comparison. Finally, positive and negative control
rate (SGR), Feed conversion ratio (FCR), survival,
diets were formulated with 40 and 27% of protein
protein efficiency ratio (PER), apparent nitrogen
level, respectively, both diets without bacterial
utilization (ANU), apparent organic matter diges-
supplements. To all diets, 0.5% chromic oxide was
tibility (AOMD) and apparent protein digestibility
added for determining digestibility. Tables 1 and 2
(APD) were measured using the following equations:
shows diet formulation and proximate composition
SGR = 100[(log. final body weight-log initial body
FCR = individual food intake/individual weight gain
PER = individual protein intake/individual weight
Population density was also used as a stress factor,
under the assumption that overpopulation is one of the main growth-inhibiting factors in intensive
ANU = 100(carcass nitrogen deposition/N intake)
aquaculture systems. To this end, 32 glass aquaria of
Beginning in the third week of the experiment,
15 L capacity were stocked at a 30 organisms per
feces were collected by siphoning the aquaria 30 min
aquaria (2 fry per liter). All fry had similar average
after the second daily feeding, to minimize leaching.
initial weights (280 ± 10 mg). The different diet
Scales were removed from the collected feces, the
formulations were assigned within the aquaria. The
feces were oven dried at 105°C for 24 h, and then
animals were allowed to adapt to the experimental
stored in hermetic containers under refrigeration to
system for a week, and fed with a conventional diet,
Formulation of experimental diets.
CON 40: Positive control, CON 27: Negative control, ED: Diet supplemented with E. faecium
, B2: Diet supplemented with E. durans
, B3: Diet supplemented with Leuconostoc
sp., A1: Diet supplemented with Streptococcus
sp. I, A2: Diet supplemented with Streptococcus
sp. II; ALL 27: Diet supplemented with commercial probiotic. 1Jauncey & Ross, (1982). 2Tacon, (1984).
493 Latin American Journal of Aquatic Research
Proximate composition of experimental diets (% dry matter).
Gross energy (MJ kg-1) 19.95 19.75 20.09
For water quality control, temperature and
dissolved oxygen were measured daily, and weekly analyses were done of total ammonium, nitrite, nitrate
The growth performance including IMW, FMW,
and pH levels, using standard methods (APHA,
SGR, FCR, PER, ANU, AOMD, APD and survival
1989). The following values (±SD), appropriate for
rate of Nile tilapia are shown in Table 3. No
tilapia cultivation, were used: temperature, 28.83 ±
significant differences were observed in IMW among
0.45°C; dissolved oxygen, 5.71 ± 1.16 mg L-1; pH
treatments. Fish fed with CON 27 diet showed significantly lower survival (66.7%) than those fed
7.98 ± 0.45; ammonia, 0.09 ± 0.04 mg L-1; nitrite,
with bacteria-supplemented and positive control diets
0.08 ± 0.02 mg L-1 and nitrate, 5.93 ± 0.61 mg L-1.
< 0.05). The highest survival was recorded for
Every third day, each aquaria was partially cleaned
CON 40 and diet supplemented with E. durans
and the water partially changed (1:l). Once a week,
(100%). The treatment CON 27 presents the lower
the same day bulk weight measurement was done, the
FMW (5.95 g). Fish fed with diets supplemented with
aquaria were completely cleaned and a total change
native bacteria exhibited higher FMW compared to
The ALL 27 treatment resulted with the signifi-
cantly higher FCR (2.02) among the bacteria-
Proximate chemical analyses of diet ingredients were
supplemented diets, thought all the other bacteria-
made and a sample of fish, at the beginning and end
containing diets showed FCR significantly lower than
of the experiment, according to standard methods
those for the controls diets (P
< 0.05). The best FCR
(AOAC, 1995). Gross energy in the feed was
determined by combustion in a Parr adiabatic
The PER was significantly higher in treatment A1
calorimeter. To evaluate digestibility, the chromic
(2.53) than in the others treatments. The lower PER
oxide content of each diet and the collected feces
was recorded for the CON 40 treatment (1.36). Fish
were analyzed using the acid digestion method
from A1 treatment presented ANU significantly
(Furukawa & Tsukahara, 1966). Protein content was
greater (48.4%), in comparison with the other
also determined for the feces, to assess protein
treatments. The lowest biological value was observed
In general, AOMD and APD were variable among
treatments. The maximum value were obtained in the
Growth performance and feed utilization efficiency
A2 treatment (AOMD = 95.08%; APD = 94.28%),
parameters were statically compared using one-way
which was statically different from the rest of the
< 0.05), and differences among means
were identified using Duncan Multiple Range Test.
Whole body composition data are presented in
Analyses were carried out with the StatGraphics Plus
Table 4. The moisture content showed no significant
Version Centurion XV computer software. Arcsin
difference among fish fed with the experimental
transformation of raw data were made when
diets, and it ranged from 72.9 to 76.4%. The
uppermost two values (18.7 and 18.4%) of crude
The use of lactic acid bacteria as growth promoters in fish fed 494
Growth and feeding performance of fish feed with diets supplemented with bacteria.
1Values with the same superscript in the same row are not statistically different (P
> 0.05), 2Standar error, calculated from mean-square error of the ANOVA, 3Specific Growth Rate, 4Food Conversion Ratio, 5Protein Efficiency Ratio, 6Apparent Nitrogen Utilization, 7Apparent Organic Mater Digestibility, 8Apparent Protein Digestibility.
Body composition of fish fed diets supplemented with bacteria.
1Values with the same superscript in the same row are not statically different (P
protein were achieved for fish fed diets A1 and
promotion (Gatesoupe, 2002; Lara-Flores et al.
ALL27, with no significant difference. Fish from B2
treatment showed lower lipid content (4.7%) in
In this study, groups administered diets with lactic
comparison with the other treatments. Statistical
acid bacteria showed similar and superior survival
differences were observed also in the body ash
results when compared with positive and negative
content among fish fed with the different diets, with
control groups. Similar results were observed by
significantly lower content in fish from ALL 27
Suyanandana et al.
(2002) when administered Lacto-
sp. isolated from the intestine of Nile tilapia.
Probiotics are biopreparations containing living
microbial cells that optimize the colonization and composition of the growth and gut micro flora in
Many studies on probiotics in aquaculture have used
animals, and stimulate digestive processes and
models of specific bacteria as antagonists of
immunity (Bomba et al.
, 2002). The results of the
pathogens (Vine et al.
, 2004, 2006), measured the
present study confirm the results from other studies
survival of probiotic in fish gut (Andlid et al.
that the incorporation of probiotic in the diets can
or evaluated the beneficial effect of probiotic on
improve growth performance in terms of SGR, FCR
health management, disease resistance and immune
and PER. Gatesoupe (1991) reported increased
response of fish (Li & Gatlin III, 2004; Shelby et al.
weight gain in Scophital mus
larvae fed a diet
2006). Other important effect of the use of probiotic,
incorporating lactic acid bacteria and Bacillus toyoi
that it is not extensively study, but demonstrated an
In the present study, fish fed lactic acid bacteria grew
important effect, is the feed efficiency and the growth
faster than those fed a control. It has been reported
495 Latin American Journal of Aquatic Research
that the improvement of growth by using probiotics is
The present investigation showed that the addition
related to an enhancement of nutrition (El-Haroun et
of native bacteria in Nile tilapia fry diets improved
, 2006), as some probiotic strains may serve as a
animal growth and mitigated the effect of stress
supplementary source of food and their activity in the
factors, such as the low protein level in diets. All
digestive tract may be a source of essential nutrients
native bacterial strains used in the present study were
(Balcazar et al.
, 2006). According with Ghosh et al.
effective in stimulating fish performance. Strepto-
(2007), most of this enhancement is reflected in the
sp. I produced the best results, and it could be
whole body proximal composition of fish. In the
a good candidate for optimizing growth and feed
present experiment, and regardless of the treatments
utilization in intensive tilapia culture.
with lactic acid bacteria, the whole body composition of O. niloticus
showed a trend of higher values of
protein, which might indicates a better utilization of diet nutrient provided by the probiotic cells.
Al-Dohail, M.A., R. Hashim & M. Aliyu-Paiko. 2009.
The mechanisms by which probiotic bacteria
Effects of the probiotic, Lactobacillus acidophilus
stimulate growth rate are not yet clearly. The
on the growth performance, haematology parameters
improvement of feed utilization for fish fed diet,
and immunoglobulin concentration in African
supplemented with probiotics, could be due to
Catfish (Clarias gariepinus
, Burchell, 1822)
improvement in the intestinal microbial flora balance
fingerling. Aquacult. Res., 40: 1642-1652.
which, in turn, will lead to better absorption quality,
Andlid, T., R. Vazquez & L. Gustafsoon. 1998. Yeast
increased enzyme activities (Tovar-Ramírez et al.
isolated from the intestine of rainbow trout adhere to
2002; Balcazar et al.
, 2006; Waché et al.
, 2006; Al-
and grow intestinal mucus. Mol. Mar. Biol. Biotech.,
Dohail et al.
, 2009; Lara-Flores et al.
, 2010), and
more degradation of higher molecular weight protein
Association of Official Analytical Chemist. 1995.
to lower molecular weight peptides and amino acids
Official Methods of Analysis of the Association of
(De Schrijever & Ollevier, 2000). Especially, the
Official Analytical Chemist. AOAC, Washington,
stimulating growth by probiotics containing LAB
strains has been associated with improved feed
American Public Health Association (APHA), 1989.
conversion ratio and protein efficiency ratio
Standard methods for the examination of water and
attributed to an increase in lactic acid and cellulolytic
and amylolytic enzyme production (Kesarcodi-
Watson et al.
, 2008). These contribute towards
Balcazar, J.L., D. Vendrell, I. De Blas, D. Cunninghem,
optimizing the digestion and use of protein for
D. Vandrell & J.L. Muzquiz. 2006. The role of
growth, that will result in more efficient protein in
probiotic in aquaculture. Vet. Microbiol., 114: 173-
fish diets. The probiotic, after transit thought the
stomach, they attach in the intestine and use a large
Bomba, A., R. Nemcoa, S. Gancarefkova, R. Herich, P.
number of carbohydrates for their growth and
Guba & D. Mundronova. 2002. Improvement of
produce a range of relevant digestive enzymes
probiotic effects in microorganisms by their
(amylase, protease and lipase), that increase the
combination with maltodextrins, fructo-oligosac-
digestibility of organic matter and protein, produce a
charides and poly unsaturated fatty acids. British J.
higher growth, prevent intestinal disorders and
produce or/and stimulate a pre-digestion of secondary
Cabello, F.C. 2006. Heavy use of prophylactic anti-
compounds present principal in plant sources (El-
biotics in aquaculture: a growing problem for human
Haroun et al.
, 2006; Lara-Flores et al.
and animal health and for the environment. Environ.
Moreover, the nutritional benefits of probiotic
bacteria have been attributed to the synthesis of B
Carnevali, O., L. De Vivo, R. Sulpizio, G. Gioacchini, I.
vitamins and short chain fatty acids in the intestine,
Olivotto, S. Silvi & A. Cresci. 2006. Growth
and the higher availability of trace elements
improvement by probiotic in European sea bass
(Holzapfel et al.
, 1998; Lara-Flores & Aguirre-
juveniles (Dicentrarchus labrax
L.), with particular
Guzmán, 2009). Our observation shows that a
attention to IGF-1, myostatin and cortisol gene
significant increase in body weight, and better
efficiency, occur in fish fed with native bacteria
De Schrijever, R. & F. Ollevier. 2000. Protein digestion
supplemented specifically with the Streptococcus
in juvenile turbot (Scophtalmus maximus
The use of lactic acid bacteria as growth promoters in fish fed 496
effects of dietary administration of Vibrio proteo-
Kesarocodi-Watson, A., H. Kaspar, M.J. Lategan & L.
. Aquaculture, 186: 107-116.
Gibson. 2008. Probiotics in aquaculture: The need,
Denev, S.A. 2008. Ecological alternatives of antibiotic
principles and mechanisms of action and screening
growth promoters in the animal husbandry and
aquaculture. DSc. Thesis, Deparment of Bioche-
Kim, S., L. Nonaka & S. Suzuki. 2004. Occurrence of
mestry Microbiology, Trakia University, Stara
tetracycline resistance genes tet(M) and tet(S) in
bacteria from marine aquaculture sites. FEMS
El-Haroun, E.R., A.M.A.-S. Goda & M.A. Kabir
Chowdury. 2006. Effect of dietary probiotic Biogen
Lara-Flores, M., M.A. Olvera-Novoa, B.E. Guzmán-
supplementation as a growth promoter on growth
Méndez & W. López-Madrid. 2003. Use of the
performance and feed utilization of Nile tilapia
bacteria Streptococcus faecium
(L.). Aquacult. Res., 37:
, and the yeast Saccharomyces cerevisiae
as growth promoters in Nile tilapia (Oreochromis
Food and Agriculture Organization (FAO). 2005.
). Aquaculture, 216: 193-201.
Responsible use of antibiotics in aquaculture. In:
Lara-Flores, M. & G. Aguirre-Guzmán. 2009. The use
P.H. Serrano (ed.). FAO Fish. Tech. Pap. 469,
of probiotic in fish and shrimp aquaculture. A
review. In: N. Pérez-Guerra & L. Pastrana-Castro
Food and Agriculture Organization (FAO). 2007. The
(eds.). Probiotics: production, evaluation and uses in
state of world fisheries and aquaculture 2006. Food
animal feed. Research Signpost, Kerala, pp. 75-89.
and Agriculture Organization of United Nations,
Lara-Flores, M., L.C. Olivera-Castillo & M.A. Olvera-
Novoa. 2010. Effect of the inclusion of a bacterial
Fuller, R. 1992. History and development of probiotics.
mix (Streptococcus faecium
In: R. Fuller (ed.). Probiotics: the scientific basis.
), and the yeast (Saccharomyces
) on growth, feed utilization and intestinal enzymatic activity of Nile tilapia (Oreochromis
Furukawa, H. & H. Tsukahara. 1966. On the acid
). Int. J. Fish. Aquacult., 2: 93-101.
digestion method for determination of chromic oxide
Li, P. & D.W. Gatlin III. 2004. Dietary brewers yeast
as an index substance in the study of digestibility of
and the probiotic GrobioticTM AE influence growth
fish feed. Bull. Jpn. Soc. Sci. Fish., 32: 207-217.
performance, immune response and resistance of
Gatesoupe, F.J. 1991. The effect of three strains of lactic
hybrid striped bass (Morone chrypsops
bacteria on the production rate of rotifers Brachionus
) to Streptococcus iniae
and their dietary value for larval turbot
. Aquaculture, 96: 335-342.
Merrifield, D.L., A. Dimitroglou, A. Foey, S.J. Davies,
Gatesoupe, F.J. 1999. The use of probiotics in
R.T.M. Baker, J. Bøgwald, M. Castex & E. Ringo.
2010. The current status and future focus of
Gatesoupe, F.J. 2002. Probiotic and formaldehyde
probiotic and prebiotic application for salmonids.
treatments of Artemia nauplii
as food for larval
pollack Pollachius pollachius
. Aquaculture, 212:
Ringo, E., R.E. Olsen, T.Ø. Gifstad, R. A. Dalmo, H.
Amlund, G.I. Hemre & A.M. Bakke. 2010.
Ghosh, S., A. Sinha & C. Sahu. 2007. Effect of
Prebiotics in aquaculture: a review. Aquacult. Nutr.,
probiotic on reproductive performance in female
livebearing ornamental fish. Aquacult. Res., 38: 518-
Rinkinen, M., E. Westermarck, S. Salminen & A.C.
Ouwehand. 2003. Absence of host specificity for in
Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Stayley &
adhesion of probiotic lactic acid bacteria to
S.T. Williams. 1993. Bergey´s manual of determina-
intestinal mucus. Vet. Microbiol., 97: 55-61.
tive bacteriology. Williams & Wilkins, Baltimore,
Sáenz de Rodrigáñez, M.A., P. Díaz-Rosales, M.
Chabrillón, H. Smidt, S. Arijo, J.M. León-Rubio,
Holzapfel, W.H., P. Harberer, J. Snel, U. Schillinger &
F.J. Alarcón, M.C. Balebona, M.A. Moriñigo, J.B.
J. Huis in´t Vel. 1998. Overview of gut flora and
Cara & F.J. Moyano. 2009. Effect of dietary
probiotics. Int. J. Food Microbiol., 41: 85-101.
administration of probiotics on growth and intestine
Jauncey, K. & B. Ross. 1982. A guide to tilapia feeds
functionally of juvenile Senegalese sole (Solea
and feeding. Institute of Aquaculture, University of
, Kaup 1858). Aquacult. Nutr., 15: 177-
497 Latin American Journal of Aquatic Research
Shelby, R., R. Lim, M. Aksoy & M.A. Delane. 2006.
Tovar-Ramírez, D., J. Zambonino-Infante, C. Cahu, F.J.
Effects of probiotic feed supplements on disease
Gatesoupe, R. Vázquez-Juárez & R. Lésel. 2002.
resistance and immune response of young Nile
Effect of live yeast incorporation in compound diet
tilapia (Oreochromis niloticus
). J. Appl. Aquacult.,
on digestive enzyme activity in sea bass
) larvae. Aquaculture, 204: 113-123.
Sørum, H. 2006. Antimicrobial drug resistance in fish
pathogens. In: F.M. Aerestrup (ed.). Antimicrobial
Verschuere, L., G. Rombaut, P. Sorgeloos & W.
resistance in bacteria of animal origin. ASM Press,
Verstraete. 2000. Probiotic bacteria as biological
control agents in aquaculture. Microbiol. Mol. Biol. Rev., 64: 655-671.
Suyanandana, P., P. Budhaka, P. Sassanarakkit, P.
Saman, P. Disayaboot, Y. Cai & Y. Benno. 2002.
Vine, N.G., W.D. Leukes, H. Kaiser, S. Daya, J. Baxter
New probiotic lactobacilli and enterococci from fish
& T. Hecht. 2004. Competition for attachment of
intestine and their effect of fish production.
aquaculture candidate probiotic and pathogenic
Proceedings of the International Conference of
bacteria on fish intestinal mucus. J. Fish Dis., 27:
Asian network on microbial researches, Yogyakarta,
Vine, N.G., W.D. Leukes & H. Kaiser. 2006. Probiotic
Tacon, A.G.J. 1984. Use of solvent extracted sunflower
in marine larviculture. FEMS Microbiol. Rev., 30:
seed in complete diets for rainbow trout fingerling
). Aquaculture, 43: 381-389.
Waché, Y., F. Auffray, F.J. Gatesoupe, J. Zambonino,
Taoka, Y., H. Maeda, J.Y. Jo, M.J. Jeon, S.C. Bai, W.J.
V. Gayet, L. Labbé & C. Quentel. 2006. Cross
Lee, K. Yuge & S. Koshio. 2006. Growth, stress
effects of the strain of dietary Saccharomyces
tolerance and non-specific immune response of
and rearing conditions on the onset of
Japanese flounder Paralichthys olivaceus
intestinal microbiota and digestive enzymes in
probiotics in a closed recirculating system. Fish.
rainbow trout Onchorhynchus mykiss,
Received: 13 November 2012; Accepted: 9 June 2013
* Vitaleño: Persona proactiva, entusiasta y vitalista, de costumbres productivo-creativas y comunitario-solidarias en atención y asistencia del prójimo, para su propio bienestar y salud. (Emilio Franchi Roussel (22, junio, 2008) “Tenga menos miedo y espere más; coma menos y mastique mejor; quéjese menos y respire más; hable menos y diga más; odie menos y ame más; y todas las cosas
Cryptographic Methods for Storing Ballots on a Voting Machine Abstract The subliminal-free property ensures that malicious soft- ware loaded on the terminal cannot leak covert information A direct recording electronic (DRE) voting machine must exposing how voters voted. Note that we must assume the satisfy several requirements to ensure voter privacy and the user of the voting machine