Content uploaded by Pedro Gomes Peixoto
Author content
All content in this area was uploaded by Pedro Gomes Peixoto on Apr 12, 2016
Content may be subject to copyright.
304
J Health Sci Inst. 2014;32(3):304-7
Sodium chloride as reducing agent of stress induced in guppy
Poecillia reticulata Peters, 1859
Cloreto de Sódio como agente redutor de estresse induzido em Poecillia reticulata Peters, 1859
Pedro Gomes Peixoto, Renato Ventresqui de Oliveira, Isabela Marcomini de Lima, Afonso Pelli
1Departamento de Ecologia e Evolução da Universidade Federal do Triângulo Mineiro, Uberaba-MG, Brasil.
Abstract
Objective – To evaluate the effects of sodium chloride to control induced stress in juvenile Poecillia reticulata. Methods – The animals
were grown in a closed system, monitored by parameters: temperature, pH, conductivity, turbidity, dissolved oxygen and color. Thermal
stress was induced by temperature variation using ice and hydric stress. Stress was induced by temperature variation, using ice and water,
after standardization of mortality, NaCl was used as a potential agent for prevention of the harmful effects of stress in different concen-
trations of 1, 3 and 5g/L. Results – The results showed a beneficial effect of adding NaCl at concentrations between. Without the addition
of salt, the mortality was 43% and after use of NaCl, there was a decrease in the mortality of 19 % (1g/L), 24 % (3g/L) and 28% (5g/L),
of NaCl. Conclusion – The authors concluded that the addition of sodium chloride, and reduce stress, is a product easily available and
inexpensive, which facilitates its use by developers.
Descriptors: Acclimation
Resumo
Objetivo – Avaliar o efeito do cloreto de sódio (NaCl) para controlar o estresse induzido em juvenis de Poecillia reticulata. Métodos –
Os animais foram cultivados em sistema fechado e monitorados pelos parâmetros: temperatura, pH, condutividade, turbidez, oxigénio
dissolvido e cor. O estresse foi induzido por variação de temperatura, utilizando gelo e estresse hídrico, após a padronização da mor-
talidade, foi utilizado o NaCl como um possível agente de prevenção dos efeitos prejudiciais do estresse em diferentes concentrações
de 1, 3 e 5g/l. Resultados – Os resultados mostraram um efeito benéfico da adição de NaCl a concentrações intermédias. Sem a adição
do sal, a mortalidade foi de 43% e após a utilização de NaCl, houve uma redução da mortalidade para 19, 24 e 28% a respectivas con-
centrações de 1, 3 e 5g/l de NaCl. Conclusão – Os autores concluíram que a adição de cloreto de sódio, além de reduzir o estresse, é
um produto de fácil obtenção e de baixo custo, o que favorece o seu uso por criadores.
Descritores: Aclimatação
Introdução
Epicontinental aquatic environments are characteri-
zed by dynamic processes with variations in the con-
centrations of dissolved oxygen, pH, conductivity and
other physical-chemical parameters1.Such changes are
capable of causing stress and reduce the ability to main-
tain homeostasis of organisms. The stressful factor has
been a major cause of decreased productivity in fish
farms, reflecting in the animal’s metabolism causing
slow the growth and carcass yield2.
The responses of stress are presented in three stages
denominated in together as Adaptation Syndrome Ge-
neral constituting a series of biochemical changes and
physiological. The first is alarm phase, consisting in
physiological reactions that occur in order to compen-
sate the disorder, the second stage is the resistance,
which represents the physiological responses adjustment
or compensatory aiming once again achieve homeos-
tasis, and last one is the exhaustion that occurs when
the duration or severity caused by exposure to agent
promoter of stress exceeds the limits, may cause patho-
logy or death3A-3B-4-5.
Considering that antibiotics are expensive, and that
inappropriate use of these can select pathogen resistant
strains6and be considered a source of environmental
pollution7. In this scenery, the use of prebiotics acquires
great importance, maximizing animal welfare and some
resistance modulation mechanisms of specific and non-
specific defense against opportunistic pathogens by in-
creasing the absorption of food nutrients reducing phy-
siological changes such as hyperglycemia and
hyper cortisolemy, providing alleviation stress2reducing
costs and favoring the profitability, so supplements pro-
mising easy marketing and use8.
The aim of this study it was to evaluate the effects of
NaCl to control induced stress in juvenile
P. reticulata
and standardize mortality (LD50) to a fish widely dis-
tributed and easily accessible in order to minimize or
prevent the deleterious effects induced by stress.
Methods
Adult specimens of
Poecillia reticulata,
Peters, 1859
were grown in a closed system. Juveniles with approxi-
mately 0.8cm (± 0.2cm) of total length were used in
the experiment conducted in aquariums with a capacity
of 1000ml using the volume of 600ml. All specimens
were fed with 55% crude protein in the first month of
life and 36% protein in the diet of juveniles and adults.
To evaluation the total size has been adapted the
anesthesia protocol used by9. The fish were transferred
the aquarium to another with 500ml of water and 3ml
of the stock solution (Eugenol®1ml, 0.5ml of absolute
ethanol and 10mL of distilled water). The period of
anesthesia was approximately 1 minutes and 40 se-
conds, which was already possible to perform the mea-
surement of the animals with the aid of a caliper King
Tools®502,200 BL. Immediately after the evaluation,
the fishes were placed in the home aquarium with ae-
ration until complete recovery, that occurred about five
minutes after anesthesia.
The environment was monitored by measuring the
physical and chemical parameters: temperature, pH,
conductivity, turbidity, color and dissolved oxygen. The
measurements were performed by electrometric met-
hods: HandLab OX1 O2 meter Schott®; meter pH me-
ter-Tec 2®Tecnical; Conductivity of Tecnical 4MP-Tec®;
Turbidity AP 2000 Policontrol®, Colorimeter Aquacolor
of Policontrol®and HandLab OX1 O2 meter Schott®,
respectively.
The aquariums were kept at room temperature (25 ±
2°C). The thermal stress occurred with a significant de-
crease in the temperature with addition of ice (made
with water from the aquarium). Sometime later the
fishes were subjected to water stress, being kept on pa-
per towels for 90 seconds, where a mortality rate was
expected near 50% of the sample LD50 (Lethal Dose
for 50% of subjects tested). Was used as a standard
protocol to induce stress and thus there would be a
treatment aiming at prevention of mortality caused by
stress. Otherwise, there would increase the amount of
ice and / or time of water until it was obtained a value
close to the desired mortality.
The combinations between time out of water and/ or
application of ice both are important factors and recur-
rent stress for fishes2, here we performed13 experiments
types: Control Group; Experiment 1) 60 seconds out of
water; Exp. 2) 90 sec. out of water; Exp. 3) 180 sec. out
of water; Exp. 4) Added 20 ml of ice; Exp. 5) Added 20
ml of ice and ± 30 sec. out of water; Exp. 6) Added 20
ml of ice and ± 60 sec. out of water; Exp. 7) Added 20
ml of ice and ± 90 sec. out of water; Exp. 8) Added 40
ml of ice and ± 90 sec. out of water. Exp. 9) Added 60
ml of ice and ± 90 sec. out of water; Exp. 10) Added 80
ml of ice and ± 90 sec. out of water; Exp. 11) Added 80
ml of ice and ± 90 sec out of water witch 1g NaCl; Exp.
12) Added 80ml of ice and ± 90 sec out of water witch
3g NaCl; Exp. 13) Added 80 ml of ice and ± 90 sec. out
of water witch 5g NaCl.
For the experiment which utilized NaCl as a preven-
tive agent deleterious effect of stress, aquariums were
used at the concentrations of 1 (Exp. 11), 3 (Exp. 12)
and 5 (Exp. 13) grams NaCl per liter. After adding so-
dium chloride to the acclimatization period was set at
three days. We performed a total of 26 repetitions with
each of the methods proposed up to the LD50 was rea-
ched, and 520 used a total of 20 individuals per expe-
riment. On the fourth day the experiment for stress in-
duction was performed.
The morphometric analysis data were done using the
program Minitab®and the analysis of variance were
performed to compare the total length of dead and sur-
vivors individuals. The results were analyzed through
the survival rate obtained for each treatment after the
induction of stress with and without sodium chloride
as an agent for preventing stress.
This study it was submitted and approved by Ethics
Committee local (protocol number 198).
Results and discussion
The creation of anesthesia protocol was efficient for
the species and from that measurement of all individuals
used in the experiments. Statistic tests showed that the
variation the size does not represented an survival factor
for individuals, but random events, linked to intra-popu-
lation variation and plasticity of the population, 10 show
significant differences in the different wild populations
of
Poecillia reticulata
, when phenotypic and genotypic
variables are in question. For individuals who survived,
the average total length was 7,988 ± 1,361mm, 2,556
mm versus 8,304 ± of those who died, with p-value
equal to 0.475. Within the range of variation studied,
the size did not appear as a factor of influence on the
survival of
P. reticulata
when subjected to stressors.
When analyzing the values of water temperature in
the control group and the experiments in which there
was no addition of ice, it was observed average tempe-
rature of 25.04 ± 0.61°C. All experiments until the tenth
no significant changes in the values of electrical con-
ductivity, turbidity, color and dissolved oxygen percen-
tage of aquariums before and after the experiment. The
same way to mortality rate has not reached proposed
by this work.
In all experiments performed the pH change was not
statistically significant with mean values of 6.1 (± 0.5)
before the experiment and 5.9 (± 0.4) after the experi-
ment. Even though this parameter scale is logarithmic
and that the pH 5 is 10 times more acid that your pH 6,
the variation of 0.2 units has little biological signifi-
cance, and the animals are capable of acclimation, that
is to shape the environment showing variation, this ca-
pability being genetically determined11-13 found that du-
ring transport of tambaqui (
Colossoma macropomum
)
the physico-chemical parameters measured also remai-
ned with low variation before and after treatment.
The experiment 10 neared the LD50 being used as
standard with 43% mortality in an “n” of 37 individuals,
the temperature variation was approximately 9 + 1ºC
the other variables showed no significant change during
this experiment.
It sets out the 11, 12 and 13 using NaCl, had initial
average temperature of 25.1°C (± 0.4) and the final
mean temperatures respectively of 17.1°C (± 1) 18.1°C
(± 1) and 18.5°C (± 1).
Higher the variation in temperature was greater mor-
tality rate within the population, the fish has an optimal
temperature range and tolerance is variable between
species, stage of development and acclimation period
in which they were submitted14. These attributes are
Peixoto PG, Oliveira RV, Lima IM, Pelli A. J Health Sci Inst. 2014;32(3):304-7
305
themselves known phenotypic plasticity may be adap-
tive adjustment which increases the half of the indivi-
duals15. Various environments subject to changes like
time and space, such as aquarium fish that were tested
represent a micro-environment that was subject to va-
riations in physic-chemical parameters constantly whet-
her by experiments or by the normal fluctuation of these
parameters by seasonality, the theory of “habitat tem-
plate”17 proposes that temporal and spatial variability
of habitat influences the evolution of behavior, physio-
logy and life history characteristics of the species18. As
the test subjects are all the same species, were at the
same stage of development (juvenile) and were pre-
viously acclimated to laboratory conditions. Thus, one
cannot attribute mortality to these external factors.
In nature animals are free to escape these adverse
conditions that represent stress, like in fish farm animals
are confined, the EMBRAPA19 recommends that the ideal
temperature for a good production quality is maintained
between 26 and 28ºC, rapid changes in temperature by
water dynamics and temporal factors are increased stress
to the fish, which decrease productivity. The temperature
variation caused exceeded the limits of optimal tempe-
rature, thus verifying high mortality rates. Evolutionary
pressures shape the ecological strategies of a population
where the middle is the model of those changes. In con-
trast, the phylogenetic characteristics limit some specific
characteristics, regardless of habitat20.
Fish subjected to low temperatures led certain period
of time so that there was a compensatory response to
heat stress which were undergoing. According to 21
fish thermoregulation is activated second or minutes
after the heat stress, but adaptation can take hours, days
or weeks. This Fact that observed during the experiments
in which a portion of 60% of the surviving population
continued to be tested, resisting better to the induction
of stress than fish that replaced the dead.
It was observed that there was a decrease in the morta-
lity rate and reducing the deleterious effects induced by
stress, by adding sodium chloride to the corresponding
low concentrations of 1 and 3 g/l NaCl (Figure 1). All ex-
periments were performed out in triplicates, and tested a
total of 60 individuals of
P. reticulata
for the control group
and 60 for the other test groups of each experiment.
Figure 1. Mortality rate in the control group and the various con-
centrations of sodium chloride during the experiment.
The experiment was based on NaCl table proposed
by22, where 0.5 to 0.8% salt concentrations already
have beneficial effects for the maintenance of osmore-
gulation in freshwater fish and stress relief resulting
from handling and transportation of these animals. The
presence of sodium ions (Na+) in water favors the me-
chanism of active transport of ammonium in the blood
of fish to water. In active transport occurs at the entrance
of a sodium ion and an ammonium ion output, favoring
the elimination of ammonia even negative gradient of
ammonia concentration. It promotes good results will
aquaculture minimizing stress-related problems and
health issues of the activity.
The number of experiments 11, 12 and 13 correspon-
ding concentrations of 1, 3 and 5g/l NaCl showed positive
results in reducing mortality stress, accordingly, there
was a further fall to a lower concentration (19 ± 2%)
and intermediate (24 ± 4%) 23obtained similar results for
matrinxã –
Brycon cephalus
. After four hours of transport
density of 150kg/m3, using 6g of salt / L, with a suppres-
sion of stress responses. Sodium chloride at the highest
concentration tested (8g/L water) was efficient to remove
most of the physiological responses to stress tambaqui –
Colossoma macropomum
24. The use of sodium chloride
as a reducing agent of stress is widely spread in aqua-
culture to approximate the osmotic pressure between
the middle and the plasma inside the freshwater fish are
hypertonic in relation to the external environment (con-
centration of dissolved salts in freshwater is about 5 g/L)
so that there is a reduction in diffusion of ions into water24.
The salt also stimulates the secretion of mucus on the
gills epithelium, hindering the passage of ions through
the cell membranes25. Besides reducing stress, salt also
has prophylactic effect easy to obtain and inexpensive,
with proven efficacy in several species26-28.
Conclusion
With the obtained results we can conclude that the
anesthesia protocol was efficient for
P. reticulata
.
The initial temperature does not interfere in the mor-
tality rate, but this variation by external factors, that the
temperature variation is a factor highly stressful and let-
hal for
P. reticulata
, but should take into account other
physical and chemical parameters in the context in
which the fish live, because despite low significance
and/or little variation of this parameters in this study
are also stress factors and lethality.
And that the reduction of deleterious effects caused
by stress can be reduced preferentially with the addition
of NaCl in the concentration of 1 and 3g/L.
References
1. Moura RS, Pelli A, Sarreta AP, Okura MH. Qualidade da água
de minas em área urbana na cidade de Uberaba-MG. Rev Baiana
Saúde Pública. 2009;33(2):85-96.
2. Oba ET, Mariano WS, Santos RLB. Estresse em peixes cultiva-
dos: agravantes e atenuantes para o manejo rentável. Em: Tava-
res-Dias M. (org.) Manejo e sanidade de peixes em cultivo.
Amapá: Embrapa. 2009.
306
Sodium chloride as reducing agent in guppyJ Health Sci Inst. 2014;32(3):304-7
Peixoto PG, Oliveira RV, Lima IM, Pelli A. J Health Sci Inst. 2014;32(3):304-7
307
3A. Selye H. Stress and the general adaptation syndrome. Br Med
J. 1950;1 (4667): 1383-92.
3B. Selye H. The evolution of the stress concept. Am Scientist.
1973;61:699-9.
4. Pickering AD. Stress responses in farmed fish. In: Black K, Pic-
kering AD, ed. London: Academic Press. 1998. p. 222-43.
5. Roberts RJ, Agius C, Saliba C, Bossier P, Sung YY. Heat shock
proteins (chaperones) in fish and shellfish and their potential role
in relation to fish health: a review. J Fish Dis. 2010; 33:789-801.
6. Vázquez JA, González MP, Murado MA. Effects of lactic acid
bacteria cultures on pathogenic macrobiotic from fish. Aquacul-
ture. 2005. 245:149-61.
7. Boyd CE, Massaut L. Risks associated with the use of chemicals
in pond aquaculture. Aquaculture Engineering. 1999;20:113-32.
8. Oliveira MC, Cancherini LC, Gravena RA, Rizzo PVR, Moraes
VMB. Utilização de nutrientes de dietas contendo monondigos-
sacarídeo e/ou complexo enzimático para frangos de corte. Rev
Bras Zootec. 2007;36(4):825-31.
9. Okamoto MH, Tesser MB, Louzada LR, Santos RA, Sampaio,
LA. Benzocaína e eugenol como anestésicos para juvenis do
pampo Trachinotus marginatus. Ciênc Rural. 2009;39(3)866-70.
10. Harris S, Ramnarine WI, Smith HG, Pettersson LB. Picking
personalities apart: estimating the influence of predation, sex and
body size on boldness in the guppy Poecillia reticulata. Oikos.
2010;119:1711-18.
11. Belck MC, & Tuckfield RC. Changing costs of reproduction:
age-based differences in reproductive allocation and escape per-
formance in a live bearing fish. Oikos. 2010;119:163-9.
12. Rochet MJ, Cornillion PA, Sabatier R, Pontier D. Comparative
analysis of phylogenetic and fishing effects in life history patterns
of teleost fishes. OIKOS. 2000;91:255-70.
13. Carvalho ES, Gomes LC, Brandão FR, Crescêncio R, Chagas,
EC, Anselmo AAS. Uso do probiótico Efinol®durante o transporte
de tambaqui (Colossoma macropomum). Arq Bras Med Vet Zoo-
tec. 2009:61(6).
14. Companhia de Tecnologia e Saneamento Ambiental. Altera-
ções Físico Químicas. São Paulo: Cetesb. 2006.
15. Ricklefs RE. Economia da natureza. Rio de Janeiro: Guana-
bara Koogan. 2010.
16. Poff NL, Allan D, Bain MB, Karr JR, Prestegaard KL, Richter
BD. The natural flow regime: A paradigm for river conservation
and restoration. BioScience. 1999. 47:769-84.
18. Townsend CR, Begon M, Harper JL. Essentials of ecology. 2ª
ed. Oxford UK: Blackwell Science. 2003.
19. Rotta MA, Queiroz JF. Boas práticas de manejo (BPMs) para
produção de peixes em Tanques-redes. Corumbá-MS: Embrapa.
2003.
20. Gould SJ, Lewontin RC. The spandrels of San Marco and the
Panglossian paradigm: a critique of the adaptationist programme.
Proc R Soc London B. 1979;205:581-98.
21. Crawshaw LI. Temperature regulation in vertebrate. Annu Rev
Physiol. 1980;42:473-91.
22. Kubitza FA. Versatilidade do sal na piscicultura. Panorama
Aquic. 2007.
23. Urbinati EC, Carneiro PCF. Metabolic and hormonal respon-
ses of matrinxã, Brycon cephalus (Teleost: Characidae), to trans-
port stress under influence of benzocaine. J Aquac Trop. 2001;
16(1):75-85.
24. Gomes LC, Araujo-Lima CARM, Roubach R, Urbinati EC. Ava-
liação dos efeitos da adição de sal e da densidade no transporte
de tambaqui. Pesq Agropec Bras. 2003;38(2).
25. Wurts WA. Using salt to reduce handling stress in channel
catfish. World Aquac. 1995;26(3):80-1.
26. Barton BA, Zitzow RE. Physiological responses of juvenile
walleyes to handling stress with recovery in saline water. Progres
Fish-Culturist. 1995;57:267-76.
27. Marchioro MI, Baldisserotto B. Sobrevivência de alevinos de
Jundiá (Rhamdia quelen Quoy & Gaimard, 1824) à variação de
salinidade da água. Ciênc Rural, Santa Maria. 1999;29(2):315-8.
28. Allyn ML, Sheehan RJ, Kohler CC. The effects of capture and
transportation stress on white bass semen osmolarity and their al-
leviation via sodium chloride. Trans Am Fish Soc. 2001;130(4):
706-11.
Endereço para correspondência:
Afonso Pelli
Universidade Federal do Triângulo Mineiro
Instituto de Ciências Biológicas e Naturais
Av. Frei Paulino, 30 – Bairro Abadia
Uberaba-MG, CEP 38025-010
Brasil
E-mail: apelli@terra.com.br
Recebido em 16 de maio de 2014
Aceito em 30 de agosto de 2014