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Hydrogen peroxide, iodine solution and methylene solution highly enhance the hatching rate of freshwater ornamental fish species

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The aim of this study was to evaluate the effect of hydrogen peroxide, iodine solution (PVP) and methylene blue on eggs disinfection of three ornamental fish species, Danio rerio, Pterophyllum scalare and Gymnocorymbus ternetzi. The main idea was to create conditions to enhance the hatching rates. Eggs of each species were exposed to different concentrations of hydrogen peroxide (5, 10, 15 and 25 mg/L), PVP (0.25, 0.5, 0.75 and 1 mg/L) and methylene blue (0.5 1, 2 and 3 mg/L). The optimal doses ranged between species and chemicals: for G. ternetzi, the concentrations that high enhanced the hatching rate were 1 mg/L for the PVP treatment, 25 mg/L for the hydrogen peroxide treatment and 3 mg/L for methylene blue treatment; for P. scalare, the best results were achieved with 25 mg/L for hydrogen peroxide treatment and 3 mg/L for methylene blue treatment. By contrast, for all the different chemical did not increased the D. rerio hatching rate. Results showed that hydrogen peroxide and methylene blue are the most versatile, effective and safe to use in these species. On the other hand, PVP can be used but with many precautions due to very low safety margin. Results clearly show that the optimal concentration of chemicals for eggs disinfection is fish species dependent and it is completely wrong to extrapolate concentrations between different chemicals and fish species. Our study suggests that P. scalare can be used as a model in study of effectiveness of new chemicals with potential to disinfect water and increase hatching rates.
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Hydrogen peroxide, iodine solution and methylene
solution highly enhance the hatching rate of freshwater
ornamental fish species
Joa
˜o Chambel Ricardo Costa Mo
´nica Gomes Susana Mendes
Teresa Baptista Rui Pedrosa
Received: 6 January 2014 / Accepted: 7 April 2014
ÓSpringer International Publishing Switzerland 2014
Abstract The aim of this study was to evaluate the effect of hydrogen peroxide, iodine
solution (PVP) and methylene blue on eggs disinfection of three ornamental fish species,
Danio rerio,Pterophyllum scalare and Gymnocorymbus ternetzi. The main idea was to
create conditions to enhance the hatching rates. Eggs of each species were exposed to
different concentrations of hydrogen peroxide (5, 10, 15 and 25 mg/L), PVP (0.25, 0.5,
0.75 and 1 mg/L) and methylene blue (0.5 1, 2 and 3 mg/L). The optimal doses ranged
between species and chemicals: for G. ternetzi, the concentrations that high enhanced the
hatching rate were 1 mg/L for the PVP treatment, 25 mg/L for the hydrogen peroxide
treatment and 3 mg/L for methylene blue treatment; for P. scalare, the best results were
achieved with 25 mg/L for hydrogen peroxide treatment and 3 mg/L for methylene blue
treatment. By contrast, for all the different chemical did not increased the D. rerio hatching
rate. Results showed that hydrogen peroxide and methylene blue are the most versatile,
effective and safe to use in these species. On the other hand, PVP can be used but with
many precautions due to very low safety margin. Results clearly show that the optimal
concentration of chemicals for eggs disinfection is fish species dependent and it is com-
pletely wrong to extrapolate concentrations between different chemicals and fish species.
Our study suggests that P. scalare can be used as a model in study of effectiveness of new
chemicals with potential to disinfect water and increase hatching rates.
Keywords Ornamental fish Disinfection fish eggs Hydrogen peroxide Iodine
solution (PVP) Methylene blue
J. Chambel (&)R. Costa M. Gomes S. Mendes T. Baptista R. Pedrosa
School of Tourism and Maritime Technology, Marine Resources Research Group, Polytechnic Institute
of Leiria, 2520-641 Peniche, Portugal
e-mail: joao.chambel@ipleiria.pt
123
Aquacult Int
DOI 10.1007/s10499-014-9779-1
Introduction
The ornamental fish sector is a widespread and global component of international trade,
with annual value of the world’s wholesale of one billion dollars, most of the aquaculture
production of ornamental fish focuses on freshwater species (Chambel et al. in press).
Currently, the development of an ornamental aquaculture protocol faces several critical
bottlenecks related to production processes and the competition of less expensive speci-
mens collected from the wild (Calado 2006).
The main constraint in the reliable production of most fish species is mortality during
the early developmental stages. The quality of eggs and larvae produced in hatcheries is
considered an important limiting factor in the larvae production and, consequently, in the
development of the aquaculture industry (Kjørsvik et al. 1990; Peck et al. 2004).
The damage by fungi in fish eggs results in an average annual lack of production of
about 20 %, with peaks higher than 40 % (Forneris et al. 2003). Due to heavily coloni-
zation by pathogens on the external surface of fish eggs, disinfection of eggs has been
widely used to reduce egg mortality and to improve rearing success during the yolk sac and
first feeding stages and also to reduce mortality of fish eggs incubated in hatchery tanks
(Morehead and Hart 2003; Madsen et al. 2005; Stuart et al. 2010).
There are some protocols for fish eggs disinfection, which include the use of malachite
green solutions, formalin, hydrogen peroxide, iodine solution (PVP), methylene blue,
ozone or sodium hypochlorite (Gaikowski et al. 1999; Arndt et al. 2001; Small and Wolters
2003; Rasowo et al. 2007). However, most published egg disinfection drug efficacy studies
were conducted on salmonids or on cool and warm water fish species and concentration of
the chemical to be used depends on fish species, the contact time of the chemical and the
water temperature (Rach et al. 1998,2004; Rasowo et al. 2007; Hirazawa et al. 1999; Eissa
et al. 2013), To the best of our knowledge, no studies have reported for the chemicals
antifungal efficacy on the ornamental fish eggs disinfection.
The goal of this study was to evaluate the effect of hydrogen peroxide, PVP and
methylene blue on eggs disinfection of three ornamental fish species, Danio rerio, verte-
brate fish model in research, Pterophyllum scalare and Gymnocorymbus ternetzi, two of
the most popular ornamental fish species, in order to promote high hatching rates and larval
survival to the first feeding.
Materials and methods
Eggs and experimental facilities
The study was conducted at the Ornamental Aquaculture Laboratory of Polytechnic
Institute of Leiria. All eggs were hatched in the laboratory. Males and females of D. rerio
and G. ternetzi were maintained separately in glass aquaria (60L) and P. scalare brood-
stocks were maintained in glass aquaria (100 l), both with external filtration, constant
aeration, photoperiod 14:10 light–dark cycle, temperature of 27 ±1°C, pH 6.5–7.5 and
fishes were fed ad libitum three times per day with commercial granulate food. Tanks were
cleaned daily, and water quality parameters were measured twice a week.
One day before reproduction males and females of each species D. rerio and G. ternetzi
were joined and in next morning fish spawned. After spawning, eggs were gently siphoned
and equally divided among experimental design. In the case of P. scalare 2–3 days before
spawning, the pair selects and begins cleaning the spawning site, using their mouths to bite
Aquacult Int
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and scrub the surface of the slate. After spawning, all eggs were gently siphoned from the
hatching slates and equally divided among experimental design.
Preliminary study
To determine the concentrations of each chemical used in this study, a preliminary test was
performed with the species G. ternetzi. To evaluate the effect of hydrogen peroxide, PVP
and methylene blue treatment dose on the hatching rate, 20 eggs were placed in aerated
400 mL beakers with the concentrations of 25, 500, 100 and 200 mg/L of hydrogen
peroxide (PANREAC QUIMICA, Spain), 1, 3, 5 and 10 mg/L of PVP (MEDA Pharma,
Portugal) and 1, 5, 15 and 25 mg/L of methylene blue (Merck SA, Germany). All con-
centrations were tested separately and defined on the active ingredient substance.
Each treatment was tested on three replicates, the disinfectants were applied at the
beginning of each experiment, and a complete water change was performed after 24 h
(time to eggs hatching).
Main study
The main study was realized testing the chemicals concentrations of 5, 10, 15 and 25 mg/L
of hydrogen peroxide, 0.25, 0.5, 0.75 and 1 mg/L of PVP and 0.5, 1, 2 and 3 mg/L of
methylene blue on D. rerio,G. ternetzi and P. scalare in same conditions as the pre-
liminary study. Complete water change was performed after 24 h to D. rerio and G.
ternetzi and 48 h for P. sclare (time to eggs hatching).
Determination of hatching rate
The hatching rate was expressed by the percentage of number of larvae hatched, 24 h (D.
rerio and G. ternetzi)or48h(P. sclare) after each treatment.
Statistical analysis
All data were checked for normality and homoscedasticity. One-way analysis of variance
(ANOVA) with Tukey HSD’s multiple comparison of group means was employed to
determine significant differences between the different treatments (Zar 2009). When
normality and homoscedasticity were violated, Kruskal–Wallis nonparametric test was
used with Games–Howell multiple comparison test (Zar 2009). Additionally, linear
regression analysis was used to measure the relationship between chemicals concentrations
and hatching rates.
Where applicable, results are presented as mean ±SEM. For all statistical tests, the
significance level was set at pB0.05. All calculations were performed with IBM SPSS
Statistics 20.
Results
During the experimental period, the water quality of broodstock was maintained in
appropriate values for maintaining these species, OD [8.0, pH between 7.1 ±0.4 tem-
perature 27 ±1°C, total ammonia and nitrite below 0.5 mg/L and nitrate \10 mg/L.
Aquacult Int
123
Preliminary study showed that hatch rate obtained in control group and treatment groups
varied between 0 and 100 % (Fig. 1). The results showed that the average hatching rates in
all chemical treatments increased when compared with the control group (p\0.05). The
only exception was obtained for the treatment with PVP at concentration of 15 mg/L,
where no differences to control group were found (p[0.05). On the other hand, at 25 mg/
L, the hatch rate was lower compared with all treatments including the control group
(p\0.05). Furthermore, this study showed that the lowest concentration tested of PVP and
hydrogen peroxide promoted a hatching rate higher than the control group and no better
hatching rates were obtained for the higher concentrations.
Hatching rates observed in main study for each species and treatments are shown in
Figs. 2A–C, and Table 1. The control groups showed lowest hatching rates, namely,
53 ±7.7 % to D. rerio,50 ±2.8 % to G. ternetzi and especially 1.86 ±1.86 % to P. scalare.
The hatching rate for D. rerio (Fig. 2A) oscillated from 37.8 ±5.8 % (1 mg/L of PVP
treatment) to 66.7 ±10.1 % (25 mg/L of hydrogen peroxide treatment). The hatching rate
obtained for each chemical was 46.67 ±10.2–56.67 ±9.7 % with methylene blue treat-
ment, 37.8 ±5.8–66.63 ±6.7 % with PVP treatment and 42.2 ±5.9–66.6 ±10.1 %
with hydrogen peroxide treatment. However, no statistically significant differences were
found when compared between treatments (p[0.05).
The G. ternetzi hatching rate was significantly enhanced by all the chemical treatments
(p\0.05) when compared with the control, with the exception of 0.5 and 1 mg/L for the
methylene blue treatment, 5 mg/L for the hydrogen peroxide treatment and 0.25 mg/L for
the PVP treatment (Fig. 2B). Furthermore, the highest G. ternetzi hatching rate was
obtained for the highest concentration tested, which was 90 ±2.8, 86.6 ±1.6 and
1 3 5 10 1 5 15 25 25 50 100 200 Ct
0
20
40
60
80
100
M-B H-PPVP Ct
M-B > Methylene blue PVP > Iodine solution (PVP)
H-P > Hydrogen peroxide Ct >Control
Concentration (mg/L)
% of hatched eggs
abcd
abcd
abc abc
abc
abc
abc
abc abc abc
abc
bc
Fig. 1 Effect of methylene blue, PVP and hydrogen peroxide in percentage of hatched eggs of G. ternetzi
(preliminary study). Values are presented as mean ±SEM (n=3). Lowercase represents significant
statistical differences at level p\0.05: abetween treatments with control group; bbetween treatments with
15 mg/LPVP cbetween treatments with 25 mg/L PVP; dbetween 1 and 3 mg/L methylene blue
Aquacult Int
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0.5 1 2 3 0.25 0.5 0.75 1 5 10 15 25 Ct
0.5 1 2 3 0.25 0.5 0.75 1 5 10 15 25 Ct
0.5 1 2 3 0.25 0.5 0.75 1 5 10 15 25 Ct
0
20
40
60
80
100
A
CtM-B H-PPVP
Concentration (mg/L)
% of hatched eggs
0
20
40
60
80
100
Ct
B
M-B H-PPVP
Concentration (mg/L)
% of hatched eggs
bcde bcde
abce
bcde bce abce
ab
bcde
abce
ac
ad
ae
0
20
40
60
80
100
M-B > Methylene blue PVP > Iodine solution (PVP)
H-P > Hydrogen peroxide Ct >Control
C
ab
ac
ad
ae
acef
aa
af
CtM-B H-PPVP
Concentration (mg/L)
% of hatched eggs
abc
def abc
def
abc
def abc
def
Fig. 2 Effect of methylene blue,
PVP and hydrogen peroxide in
percentage of hatched eggs on
three species under study (main
study). Values are presented as
mean ±SEM (n=3). AD.
Rerio;BG. Ternetzi; lowercase
represents significant statistical
differences at level p\0.05:
(a) between treatments with
control group; (b) between
treatments with 3 mg/L
methylene blue (c) between
treatments with 25 mg/L
hydrogen peroxide; (d) between
treatments with 0.75 mg/L PVP;
(e) between treatments with
1 mg/L PVP; CP. scalare;
lowercase represents significant
statistical differences at level
p\0.05: (a) between treatments
with control group; (b) between
treatments with 0.5 mg/L
methylene blue (c) between
treatments with 1 mg/L
methylene blue; (d) between
treatments with 2 mg/L
methylene blue; (e) between
treatments with 3 mg/L
methylene blue; (f) between
treatments with 25 mg/L
hydrogen peroxide
Aquacult Int
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90 ±2.8 % for 1 mg/L of PVP treatment, 25 mg/L of hydrogen peroxide treatment and
3 mg/L of methylene blue treatment, respectively.
The results of the efficiency of eggs disinfection of P. saclare are showed in Fig. 2C.
All treatments and concentrations of methylene blue and hydrogen peroxide enhanced the
hatching rate comparatively to the group control (p\0.05). On the other hand, no dif-
ferences were found between hatching rate in PVP treatment and control group (p\0.05)
and with this chemical, hatching rates are lowest comparatively with other chemicals
independently of the concentrations (p\0,05). The hatching rate ranged from
1.86 ±1.86 % (control group) to 94.4 ±3.2 % (3 mg/L of L methylene blue). The higher
hatching rates obtained were 3.7 ±3.7 % at 0.5 and 0.75 mg/L of PVP, 88.87 ±3.2 % at
25 mg/L of hydrogen peroxide and 94.4 ±3.2 at 3 mg/L of methylene blue.
The results achieved by means of linear regression analysis (Table 1) showed statistical
significant dependences to hydrogen peroxide, iodine active and methylene blue for G.
ternetzi and hydrogen peroxide for P. scalare (p\0.05). The statical linear trends are
presented in Figs. 3a–d. The hatching rate increases when increases the chemical con-
centration used on the treatment, which means there is a direct positive correlation between
these two attributes. This relation was more prominent for PVP effect on G. ternetzi when
compared with other species. For this species, an increment of 1 mg/L of PVP leads to an
increase in hatching rate of 40 ±4.6 %.
Discussion
This study was intended to assess the effect of three of the most commonly used chemicals
on the hatching success of Danio rerio,Pterophyllum scalare and Gymnocorymbus ternetzi
eggs.
All of chemicals used in study showed capacity in increasing hatching rate at least in
one specie, and the optimal doses obtained varied between G. ternetzi and P. scalare
species and PVP, methylene blue and hydrogen peroxide. By contrast, for all the different
chemical did not increased the D. rerio hatching rate.
Table 1 Values of variation in hatching rate obtained as a function of the concentration of methylene blue,
hydrogen peroxide and PVP for the three species under study
Specie Chemical R
2
pvalue* Variation of hatching rate
(%)/mg L
-1
chemical
D. rerio Methylene blue 0.1041 0.3065 4.220 ±3.916
Hydrogen peroxide 0.2067 0.1375 1.042 ±0.6458
Iodine solution (PVP) 0.1431 0.2253 -24.83 ±19.21
G. ternetzi Methylene blue 0.7886 0.0001 13.28 ±2.174
Hydrogen peroxide 0.7857 0.0001 1.343 ±0.2218
Iodine solution (PVP) 0.9 \0.0001 44.00 ±4.638
P. scalare Methylene blue 0.04017 0.5322 3.702 ±5.722
Hydrogen peroxide 0.4035 0.0265 2.592 ±0.9966
Iodine solution (PVP) 0.01783 0.6791 -2.200 ±5.164
* Significance level was set at plevel B0.05. Values are presented as mean ±SEM (n=100 eggs/
chemical/species)
Aquacult Int
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The hatching rate obtained to P. scalare was very low in control group, however, in
the natural environment or in captivity without removing eggs from the parents, usually
hatching rate is higher (Farahi et al. 2011). This low percentage of hatching relates to
the fact that eggs are taken from the parents, to be subjected to treatment, since in
normal conditions, the breeders have parenting functions, removing most of unfertilized
and nonviable eggs by constantly agitating the water to hinder the attachment and
proliferation of fungi (Degani and Yehuda 1996), In Egypt, Saprolegniosis is consid-
ered one of the most important causes of mortality among angelfish (Ahmed et al.
1990). In the absence of parents, eggs are more vulnerable, leading to near zero
hatching rates.
The methylene blue showed capacity to increase hatching rate for G. ternetzi, and
hatching rate shows high dependence of concentration, by contrast for P. scalare all
concentrations increasing hatching rate but without dependency of concentration. The
safety of this chemical in disinfecting eggs is reported by Sanabria et al. (2009) who tested
01234
40
50
60
70
80
90
100
[Methylene blue] mg/L
A
% ofhatched eggs
0.0 0.5 1.0 1.5
50
60
70
80
90
100
B
[Iodine solution] mg/L
% of hatched eggs
0102030
50
60
70
80
90
100
C
[Hydrogen peroxide] mg/L
% of hatched eggs
0102030
0
20
40
60
80
100
D
[Hydrogen peroxide] mg/L
%hatched eggs
Fig. 3 Linear regression analysis for hatching rate (%) as function of methylene blue (a), PVP (b) and
hydrogen peroxide (c) concentration for G. ternetzi and hydrogen peroxide (d) concentration for P. scalare.
Values are presented as mean ±SEM (n=3)
Aquacult Int
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the effect of methylene blue in eggs and larvae of P. scalare showing that 24-h bath post
fertilization have no effects on survival and swim bladder.
Hydrogen peroxide shows high capacity in increase hatching rate in a concentration
dependent manner for P. scalare and G. ternetzi. In both species, the highest concentration
used (25 mg/L) can reach the maximum hatching rates. This chemical is currently used in
aquaculture, but some authors suggests use of hydrogen peroxide at a concentration of
1,000 mg/L in some species of fish and also that the use of 500 mg/L can be lethal for P.
scalare (Schreier et al. 1996; Barnes and Gaikowski 2004; Sanabria et al. 2009).
Iodine solution only proved to be effective for the G. ternetzi specie, although many authors
consider it a good chemical for fish eggs disinfection of several species. Stuart et al. (2010)
suggested the use of 50 mg/L PVP with a 5 min bath. On the other hand, Eissa et al. (2013)
obtained very efficientresults against the mold infection on egg stocksof P. scalare with the use
of 60 mg/L of PVP as immersion solution during 30 min. However, in our preliminary study,
the use of 1 or 5 mg/L PVP of and increased the G. ternetzi hatching rate. By contrast, the
treatment with 15 mg/L of PVP reduced the G. ternetzi hatching rate. Moreover, the treatment
with 25 mg/L of PVP was toxic for all the eggs. The toxicity was previously referred in baths of
fish eggs with 75 and 100 mg/L during 30 min for Chinook salmon and rainbow trout,
respectively (Alderman 1984; Fowler and Banks 1990). In line with the preliminary results
obtained in this study, Aydınetal.(2011) also found the hatching rates reductions and a
significantly increased abnormalities in turbot eggs after iodine treatments.
Our results showed that hydrogen peroxide and methylene blue are the most versatile,
effective and safe to use on the three tested species. PVP can be also used but with many
precautions due to the very low safety margin.
The results presented here confirm the importance of determining the optimal dose of
each chemical disinfectant for each species of fish in order to ensure the health and welfare
of the specimens used in aquaculture and laboratory work. One of the key points of our
study is related with the view that the optimal chemical doses used on each specie cannot
be extrapolated from species to species, independently of the similarity they have. Taken
together, both the low hatching rate observed on P. scalare control group and the high
increases of the P. scalare hatching rate induced by the disinfectants, this species could be
used as an experimental model in the study of effectiveness of new chemicals for water
disinfection and increase the hatching rates.
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... There is a wide assortment of disinfectant chemicals and associated protocols used to treat teleost embryos. Suitability varies with the target pathogen as well as the tolerance of the embryo to therapeutic levels of the disinfectant, which in many cases can be species specific (Sudova et al. 2007;Chambel et al. 2014;De Swaef et al. 2016). Efficacy of the chosen chemical can vary greatly and may be dependent on species, contact time, stage of embryo development, application method, and disinfectant concentration. ...
... Given the broad diversity among fishes in aquaculture, it is reasonable to expect the effects of chemotherapeutants on developing embryos to be species specific. Although iodine produced poor hatch success for the four ornamental species tested in this trial, previous literature has reported increased hatching success at higher concentrations and/or longer application times using this chemical (Amend 1974;Khodabandeh and Abtahi 2006;Chambel et al. 2014). Conversely, Black Sea Turbot Scophthalmus maximus embryos treated with iodine concentrations of 100-3,000 mg/L for 10 min all had lower hatching success and higher rates of deformity than untreated embryos (Aydın 2011). ...
... Conversely, Black Sea Turbot Scophthalmus maximus embryos treated with iodine concentrations of 100-3,000 mg/L for 10 min all had lower hatching success and higher rates of deformity than untreated embryos (Aydın 2011). In a study of three ornamental species (Black Tetra Gymnocorymbus ternetzi, Zebra Danio Danio rerio, and Freshwater Angelfish Pterophyllum scalare; Chambel et al. 2014), embryos treated with 1-, 5-, and 15-mg/L iodine for a duration of 24 h exhibited hatch success greater than or equal to that of the control group. These results suggest that an increased exposure time with a substantially reduced concentration may be a more viable approach for embryo disinfection using iodophors, especially in sensitive ornamental species. ...
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Maintaining optimum conditions for embryo incubation is a critical consideration in finfish aquaculture. To this end, prophylactic disinfectant protocols have been widely used in the culture practices of many finfish species. Empirical evaluations of safety and effectiveness for disinfectants in ornamental fish species, however, are uncommon. In this study, we evaluated hatching success in embryos of four ornamental fishes (Redtail Sharkminnow Epalzeorhynchos bicolor, Rainbow Sharkminnow Epalzeorhynchos frenatum, Featherfin Squeaker Synodontis eupterus, and Upside‐Down Catfish Synodontis nigriventris) after immersion in three common disinfectant solutions: 1,500 mg/L formalin [15 min], 100 mg/L iodine [15 min], 1,000 mg/L hydrogen peroxide [15 min], and hatching water [15 min]. All disinfectants were administered according to either the manufacturer's recommendation or protocols found in published literature for other species. Of the concentrations used, hydrogen peroxide and formalin can be safely used as a prophylactic treatment for the majority of species evaluated with no detrimental effects. In addition, in Redtail Sharkminnows, embryos treated with formalin exhibited numerically higher hatch success with less variation among replicates than the control treatment. For all species, immersion in 100 mg/L iodine [15 min] resulted in significantly lower hatching success when compared with all other treatments. This article is protected by copyright. All rights reserved.
... Saprolegnia parasitica is one of the most significant fish pathogens from the genus oomycete, causing high rates of mortality for freshwater fish regardless of whether they live in natural environments or are reared in captivity (Van Den Berg et al., 2013;Lone and Manohar, 2018). Freshwater angelfish (Pterophyllum scalare) is an economically important ornamental fish species with easy reproduction, but its eggs frequently suffer fungal infections (Chapman et al., 1997;Forneris et al., 2003;Chambel et al., 2014). ...
... The parents remove non-fertilized and non-viable eggs and constantly agitate the water to avoid proliferation of the pathogen (Degani and Yehuda, 1996;Farahi et al., 2011). However, in intensive production, the spawns that are away from their parents have a hatching rate of zero according to Chambel et al. (2014), mainly due the Saprolegnia infection (Ahmed et al., 1990). ...
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A nanocomposite based on silver nanoparticles (AgNPs) and extract of Terminalia catappa was developed, characterized, and evaluated in in vitro and in vivo conditions against Saprolegnia parasitica infection. The nanocomposite contained spheroidal silver-nanoparticles (1–55 nm) and presented active compounds as gallic acid, ellagic acid, and α and β punicalagin. The nanoparticles remained stable for one year after its production. A synergistic effect was observed between AgNPs and extract under in vitro and in vivo conditions against different stages of fungal development. In an in vitro assay, the nanocomposite showed fungistatic and fungicide effects to the fungal mycelium in solid and liquid media, respectively, through an increase in the contact surface. In an in vivo bioassay, the lowest concentration of nanocomposite (T1 = 45.75 μg.L⁻¹ AgNPs +62.5 μg.L⁻¹ T. catappa extract) demonstrated similar efficiency as the positive control (methylene blue) in preventing zoospore infectivity in eggs of angelfish (Pterophyllum scalare). The fungal zoospores were more sensitive to the nanocomposite than fungal mycelia. Our results exhibited the use of a nanocomposite containing AgNPs and T. catappa aqueous extract could reduce the required effective concentrations of AgNPs against saprolegniosis in fish eggs, thus, it may as an alternative to improve fish larval survival at the hatchery stage.
... Additionally, MB was shown to photo-inactivate bacteria in 1928 [20], and its potent antiviral effects were discovered soon after [21]. MB can also be used as an anti-fungal and anti-parasitic aquarium disinfectant, even with very high dosages (millimolar) [22,23]. MB has also been used in photodynamic therapy among different types of cancers, including lung cancer [24], breast cancer [25], and prostate cancer [26]. ...
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Methylene blue (MB), as the first fully man-made medicine, has a wide range of clinical applications. Apart from its well-known applications in surgical staining, malaria, and methemoglobinemia, the anti-oxidative properties of MB recently brought new attention to this century-old drug. Mitochondrial dysfunction has been observed in systematic aging that affects many different tissues, including the brain and skin. This leads to increaseding oxidative stress and results in downstream phenotypes under age-related conditions. MB can bypass Complex I/III activity in mitochondria and diminish oxidative stress to some degree. This review summarizes the recent studies on the applications of MB in treating age-related conditions, including neurodegeneration, memory loss, skin aging, and a premature aging disease, progeria.
... Given the wide variety of aquaculture fish, it is realistic to expect the prophylactic effects of chemicals on developing embryos to be species-specific (Sipos et al. 2020). Although iodine had a low hatch success and survival rate in the present study, earlier research had found that larger doses and/or longer treatment times boosted hatching success (Chambel et al. 2014). In contrast, untreated Black Sea Turbot, Scophthalmus maximus embryos showed reduced hatching success and greater rates of malformation than untreated embryos. ...
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Despite several reports on the endocrine-disrupting ability of atrazine in amphibian models, few studies have investigated atrazine toxicity in the heart and cerebellum. This study investigated the effect of atrazine on the unique Ca2+ channel-dependent receptor (Inositol 1,4,5-trisphosphate; IP3R) in the heart and the cerebellum of adult male Xenopus laevis and documented the associated histomorphology changes implicated in cardiac and cerebellar function. Sixty adult male African clawed frogs (Xenopus laevis) were exposed to atrazine (0 µg/L (control), 0.01 µg/L, 200 µg/L, and 500 µg/L) for 90 days. Thereafter, heart and cerebellar sections were processed with routine histological stains (heart) or Cresyl violet (brain), and IP3R histochemical localization was carried out on both organs. The histomorphology measurements revealed a significant decrease in the mean percentage area fraction of atrial (0.01 µg/L and 200 µg/L) and ventricular myocytes (200 µg/L) with an increased area fraction of interstitial space, while a significant decrease in Purkinje cells was observed in all atrazine groups (p < 0.008, 0.001, and 0.0001). Cardiac IP3R was successfully localized, and its mean expression was significantly increased (atrium) or decreased (cerebellum) in all atrazine-exposed groups, suggesting that atrazine may adversely impair cerebellar plasticity and optimal functioning of the heart due to possible disturbances of calcium release, and may also induce several associated cardiac and neural pathophysiologies in all atrazine concentrations, especially at 500 µg/L.
... However, there are no reports about the effects of aqueous T. catappa extract extracted with high temperature to control Saprolegnia sp. in Pterophyllum scalare eggs. P. scalare (freshwater angelfish) is an important species in the ornamental fish market and intensively bred around the world; however, infection by Saprolegnia parasitica leads to low egg survival (Chambel et al., 2014). Therefore, this study aimed to evaluate the effectiveness of T. catappa leaf aqueous extract to inhibit the growth of S. parasitica on P. scalare eggs. ...
Article
The Saprolegniosis causes major reduction egg hatching rate by angelfish (Pterophyllum scalare); therefore, we investigated the effectiveness of two Terminalia catappa extracts (27 ℃ and 80 ℃) against S. parasitica on fish eggs. Mycelium growth was evaluated in vitro at 0, 2.5, 5.0 and 10 g/L for 96 h, in solid and liquid medium culture. In an in vivo assay, we tested lower concentrations of hot extract (25, 50 and 100 mg/L) to prevent infection by zoospores. The hot aqueous extract was the most effective, and the concentrations of 5 and 10 g/L demonstrated great efficiency in liquid medium. In vivo conditions, 50 and 100 mg/L of hot extract provided more viable eggs, greater percentage of total larvae and lower percentage of infected eggs. Thus, the hot extract should be used instead of the room‐temperature extract as a prophylactic measure, to prevent saprolegniosis in P. scalare eggs and improving the hatching rate.
... Given the wide variety of aquaculture fish, it is realistic to expect the prophylactic effects of chemicals on developing embryos to be species-specific (Sipos et al. 2020). Although iodine had a low hatch success and survival rate in the present study, earlier research had found that larger doses and/or longer treatment times boosted hatching success (Chambel et al. 2014). In contrast, untreated Black Sea Turbot, Scophthalmus maximus embryos showed reduced hatching success and greater rates of malformation than untreated embryos. ...
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Embryo-larval toxicity test of selected anti-fungus chemicals used as prophylactic agents on the African catfish (Clarias gariepinus) was conducted to assess the comparative efficacy as well as the toxicity of the different chemicals. Measured endpoints included hatching, embryo and larval survival, and larval growth. Iodine (100 mg L-1), hydrogen peroxide (250 mg L-1), malachite green (5 mg L-1) and formalin (250 mg L-1) were used as prophylactic agents. The eggs were spread on an incubating raft and continuously dipped in the treatment chemical for 15 minutes. After 48 hours, the mean hatch rate was highest in formalin-treated embryos (70%). Iodine treatment resulted in a hatch rate of 36.7%, which was slightly less than half that of the formalin treatment. Both hydrogen peroxide and malachite green resulted in 60.0% hatch rates. The results indicate that formalin-treated embryos performed significantly better than all the other chemicals. The highest percentage survival rate of 63.3% was observed in formalin at 72hph and 56.7% after 168hph. The lowest survival rate (10%) was observed in the iodine treatment group and closely followed by the group treated with malachite green (16.7%). Except for malachite green, which inhibited growth, the prophylactic treatments did not affect the growth performance of fry. This study presents evidence that can be used to support the use of formalin and hydrogen peroxide as prophylactic treatments in the early stages of C. gariepinus, although caution should be exercised due to the possibility of toxicity at higher concentrations and for longer periods of exposure.
... In aquaculture, H 2 O 2 treatment has been used to sterilize fish eggs, with non-toxic doses appearing to be largely species dependent. Exposure in the range 0.01% to 0.025% and exposure times of the order of minutes are most commonly used (Chambel et al., 2014;De Swaef et al., 2016;Matthews et al., 2012). Although LD 50 values are generally absent for other fish embryo species exposed to H 2 O 2 , embryos of the channel catfish Ictalurus punctatus had reduced hatching success after a single 15 min exposure to 0.05% H 2 O 2 , while early embryos of the cutthroat trout Oncorhynchus clarkia had increased mortality after only a 2 min exposure to 1.5% H 2 O 2 (Small and Wolters, 2003;Wagner et al., 2012). ...
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Embryos of Austrofundulus limnaeus can tolerate extreme environmental stresses by entering into a state of metabolic and developmental arrest known as diapause. Oxidative stress is ubiquitous in aerobic organisms and the unique biology and ecology of A. limnaeus likely results in frequent and repeated exposures to oxidative stress during development. The antioxidant capacity of A. limnaeus was explored during development by measuring antioxidant capacity due to small molecules and several enzymatic antioxidant systems. Diapause II embryos can survive for several days in 1% hydrogen peroxide without indications of negative effects. Surprisingly, both small and large molecule antioxidant systems have the highest capacity during early development, which may be due to maternal provisioning. Antioxidant capacity is largely invested in small molecules during early development and in enzymatic systems during late development. The switch in antioxidant mechanisms and decline in small molecule antioxidants during development correlates with the loss of extreme anoxia tolerance.
... ANOVA test showed that there was significant differences with the percentage of hatching among treatments (P<0.05). Similarly some study were recorded that methylene blue solution highly increases the hatching rate of freshwater ornamental fish species [44]. Usually the effect of methylene blue and sodium chloride on the bacterial load in the flush water with Nile tilapia (Oreochromis niloticus L.) fingerlings [45]. ...
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Labeo. bata is one of the most important cultured fish. Intensive incubation leads to microbial overgrowth in L. bata eggs that hamper egg development hatchability and larval survivability. The aim of this study is to find out causes of mass mortality in L. bata eggs during peak breeding season from 10 March to May 2015 at Mafatema Fish Hatchery Jessore Bangladesh. Three concentrations of four chemical-formalin (10 20 30 mg/L) malachite green (135 mg/L) NaCl (123 g/l) and methylene blue (135 mg/L) treatment regimes and a control were compared for efficacy in treating L. bata eggs to prevent fungus and bacterial infection and improve hatch and survival rate of fry. Physicochemical and microbial characteristics of culture water were examined during the induced breeding of L. bata besides mycological examination of egg samples with trial of treatment of different types of disinfectant. The total bacterial count fluctuated between 3.6×108 cfu/ml at initial time of incubation and 31.7×108 cfu/ml after 3 days of hatching. The infected fertilized egg by Saprolegnia sp. were appeared as tuft hairy like balls with a white cottony envelope that surround it which focally invaded the cytoplasm resulted in loss of the cytoplasm content and destructed envelops. Hatching rate (92.33±3.51%) of methylene blue at 1mg/L was significantly different with formalin at 10mg/L (78.0±5.29%) and control (72.33±5.51%) at.05 level of significance. Survival rate of malachite green at 5mg/L (87.33±6.51%) NaCl at 2g/L (91.00±3.00%) and methylene blue at 1mg/L (94.33±4.73%) had significant difference with control (71.00±8.89%) at.05 level of significance.
... This approach reduces the utilization of natural resources, is therefore substantially more sustainable than relying on wild-caught organisms and may even be used to restore depleted ornamental populations (Ziemann 2001). One of the main problem with the production of marine ornamental species is optimize production in order to compete with less expensive specimens collected from the wild (Calado 2006;Jamabo and Keremah 2009;Chambel et al. 2014). The anemonefish, Amphiprion percula is a tropical coral reef fish belonging to the family Pomacentridae fishes and anemones and sub family Amphiprioninae. ...
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The aim of this study was to evaluate the influence of stocking density (0.5, 1, 2 and 3 fishL−1) and commercial marine fish diets (diet A, B, C and D) over four months on specific growth rate, condition factor, percentage without anomalous pigmentation (partial or total lack of white bands -miss-band) and survival of juvenile Amphiprion percula. Results showed that at 0.5 fishL−1 densities induced the best survival (100%) and also the maximum percentage of fish without miss-band (58.33 +/−4.417%). The maximum SGR was obtained for the 0.5 fishL−1 (0.459 ± 0.023% cm/day). However, the best condition factor (2.53 +/− 0.27) was achieved for 2 fishL−1 densities. There were no significant differences in survival (68.9 to 84.5%), fish without miss-bands (18.03 to 26.92%) and condition factor (1.92 to 2.1) among diets during the experimental period. On the other hand, diet C (with 41% crude protein) supported the best SGR (0.485 ± 0.001% cmday−1). The results suggested that stocking density are critical and more relevant when compared with the different diet tested, namely on specific growth rate, condition factor, the miss-band and survival of juvenile percula clownfish. This study has particular significance with regards to anemonefishes husbandry in terms of survival and production efficiency.
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Aquaculture is the fastest worldwide growing activity with respect to food production. However, the emergence of infection diseases is the principal risk factor in this industry. To control them, it has been used diverse veterinary drugs, antibiotics, solutions, colorants, which have toxic effects for associated biota, with ecological level impact, bacterial resistance promoters, and water bodies’ contamination. The present review show some negative aspects of their use and some alternatives for their substitution like: the use of probiotics implementation, terrestrial plants extraction and aquatic microalgae (marine and freshwater) with therapeutic properties, biodegradable and also molecular biotechnology implementation, genomic and proteomic terrestrial for development of resistant strains diseases and make the aquaculture a sustainable activity.
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The aim of this study was to evaluate the influence of stocking density (0.5, 1, 2 and 3 fishL−1) and commercial marine fish diets (diet A, B, C and D) over four months on specific growth rate, condition factor, percentage without anomalous pigmentation (partial or total lack of white bands -miss-band) and survival of juvenile Amphiprion percula. Results showed that at 0.5 fishL−1 densities induced the best survival (100%) and also the maximum percentage of fish without miss-band (58.33 +/−4.417%). The maximum SGR was obtained for the 0.5 fishL−1 (0.459 ± 0.023% cm/day). However, the best condition factor (2.53 +/− 0.27) was achieved for 2 fishL−1 densities. There were no significant differences in survival (68.9 to 84.5%), fish without miss-bands (18.03 to 26.92%) and condition factor (1.92 to 2.1) among diets during the experimental period. On the other hand, diet C (with 41% crude protein) supported the best SGR (0.485 ± 0.001% cmday−1). The results suggested that stocking density are critical and more relevant when compared with the different diet tested, namely on specific growth rate, condition factor, the miss-band and survival of juvenile percula clownfish. This study has particular significance with regards to anemonefishes husbandry in terms of survival and production efficiency.
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Mass mortalities of angelfish eggs accompanied with very low hatchability were reported in a private ornamental fish farm in Egypt. Examined eggs were badly damaged by water mould that was decisively confirmed as Saprolegnia species. Presumptive identification of the ten retrieved isolates was initially suggestive of Saprolegnia species. Mycological investigations have revealed that only 7 out of 10 isolates were capable of producing sexual stages. Therefore, using molecular tools such as PCR coupled with partial sequencing of inter-transcribed spacer (ITS) gene was one of the most important approaches to distinguish Saprolegnia parasitica from other water moulds. The sequences of ITS gene data derived from eight isolates showed 100% similarity with S. parasitica ATCC90312 sequence and the remaining two isolates were different in one nucleotide (99.9%). The phylogenetic analysis of ITS genes grouped the ten isolates with other S. parasitica in one clad. Further, to control such fungal infection, the efficacy of povidone iodine as surface disinfectant for angelfish and their fertilized eggs were tested. By trial, it was obvious that the obtained post-rinsing results were highly suggestive for the efficacy of povidone iodine as an efficient antifungal disinfectant for both fish and eggs.
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P align=justify>The worldwide growth of the marine aquarium market has contributed to the degradation of coral reef ecosystems. Enforcing the legislation on importing ornamental species has led some European traders to concentrate on local species. Portugal is used as a case study of marine ornamental fish and invertebrate collection in European waters. One hundred and seventy two species occurring in Portuguese waters (mainland, the Azores and Madeira archipelagos) were considered as potential targets for the marine aquarium industry, some of which are already traded on a regular basis (e.g. Clibanarius erythropus, Lysmata seticaudata, Cerithium vulgatum, Hinia reticulata and Ophioderma longicauda ). To ensure appropriate management and conservation of these resources, the following options have been evaluated: banning the harvest and trade of all marine ornamental species from European waters; creating sanctuaries and “no take zones”; issuing collection permits; creating certified wholesalers; implementing the use of suitable gear and collecting methods; setting minimum and maximum size limits; establishing species-based quotas; protecting rare, or “key stone” species and organisms with poor survivability in captivity; establishing closed seasons; culturing ornamental organisms; and creating an “eco-fee” to support research and management. Establishing this sustainable alternative fishery may help minimise the economical and social impacts caused by the crash of important food fisheries in Portugal and other European and West African countries.
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In two separate trials, eggs of rainbow trout Oncorhynchus mykiss were cultured with the use of hydrogen peroxide and formalin treatments to control fungal infections. For the first trial, treatment regimens consisted of no chemical treatment (control) or daily treatments of either hydrogen peroxide at 500 mg/L for 35 min or formalin at 1,667 mg/L for 15 min. Hydrogen peroxide treatment duration was reduced to 5 min daily during 70–140 daily temperature units (DTU°C). In this trial, 27% of control eggs were infected with fungus, compared with 0% for the hydrogen peroxide and formalin treatments. Eyed egg percentages were significantly lower for control eggs compared with the hydrogen peroxide and formalin treatments. Comparing formalin and hydrogen peroxide treatments, percent hatch at 91% and 90% and percent deformities at 1.0% and 1.3%, respectively, were not significantly different. In the second trial, rainbow trout eggs were reared from fertilization to hatch under four treatment regiments: (1) control or no chemical treatment, (2) 500 mg hydrogen peroxide/L for 35 min daily (hydrogen peroxide A), (3) 500 mg hydrogen peroxide/L for 35 min daily with treatment completely withheld during 70–140 DTU°C (hydrogen peroxide B), and (4) 1,667 mg formalin/L for 15 min daily. Within this trial 15% of control eggs were infected with fungus, compared with 1% for hydrogen peroxide B and 0% for both hydrogen peroxide A and formalin. Eyed egg percentages were significantly better for hydrogen peroxide B than for hydrogen peroxide A. Hatch was significantly reduced in the control group compared with the formalin treatment. Incidence of deformities was not significantly altered by treatment type.
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