Animal Reproduction Science 213 (2020) 106272

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Reproductive variables of Brycon nattereri Günther, 1864 (Pisces:

T
Characidae), an endangered commercial species
Lucas de Esquivel Dias Brandãoa, Fábio Pereira Arantesa, José Enemir Santosa,
Elizete Rizzob, Nilo Bazzolia,*
a
Programa de Pós-graduação em Biologia de Vertebrados da PUC Minas, Belo Horizonte, MG, Brazil
b
Programa de Pós-graduação em Biologia Celular da UFMG, Belo Horizonte, MG, Brazil

A R T IC LE I N F O ABS TRA CT

Keywords: In Brazil, fishing in continental waters is prohibited from November to February, the rainy
Gonadosomatic index season, when most commercially important fish species are reproductively active. Brycon nattereri
Fecundity is a native species to the Paraná, Tocantins, and São Francisco River basins in Brazil and is on the
Spawning national list of species threatened with extinction. The goal of this study was to analyse the main
Testis
reproductive variables of B. nattereri from the Lourenço Velho River, located in the Paraná River
Ovary
basin, south-eastern Brazil. From 2013–2016, 326 specimens (156 females and 170 males) were
caught bimonthly using gill nets. Biometric data, gonadosomatic index (GSI), and fecundity were
determined for each specimen. The gonadal maturation stages and the breeding season were
established. There were relatively greater GSI values for males and average values were similar to
those of females, an uncommon feature in Neotropical freshwater fish. The greatest frequencies
of mature fish occurred from April to July in the dry season, which is when there are least
ambient temperatures. The fecundity for body weight varied from 16,300 to 62,800 oocytes per
female and fully developed vitellogenic oocytes had a mean diameter of 1175 ± 278.87 μm.
These results indicate that B. nattereri breeds in the dry season when the water temperature is
colder and, therefore, protection from fishing of this species during this season needs to be es-
tablished.

1. Introduction

In Brazil, ichthyofauna comprises 4506 species, of which 3148 are freshwater and 1358 are marine species (ICMBio, 2018).
According to the Chico Mendes Institute for Biodiversity Conservation, there are 409 endangered fish species in Brazil, of which 311
are freshwater and 98 are marine species. The largest number of endangered species (52 species, 38.5 % of the total) occur in
biogeographical areas in the southeast of Brazil between the São Francisco River and the coastal rivers of Santa Catarina State (Rosa
and Lima, 2008). Some endangered fish species occur in more than one biogeographical area, such as the Brycon nattereri, which
naturally occur in the Paraná, São Francisco, and Tocantins basins (Pavanelli et al., 2018).
The species B. nattereri, popularly known as pirapitinga, is currently listed on the red list of animals threatened with extinction in
Brazil (ICMBio, 2018). Brycon nattereri is a medium-sized fish that is restricted to clean headwaters and is valuable in commercial
fishing (Lima et al., 2008). The reproductive cycle of B. nattereri is seasonal (Winemiller, 1989) and it does not have long reproductive

⁎
Corresponding author at: Programa de Pós-Graduação em Biologia de Vertebrados, PUC Minas-Av. Dom José Gaspar 500-30535-610, Belo
Horizonte, Minas Gerais, Brazil.
E-mail address: [email protected] (N. Bazzoli).

https://doi.org/10.1016/j.anireprosci.2019.106272
Received 9 August 2019; Received in revised form 20 December 2019; Accepted 30 December 2019
Available online 02 January 2020
0378-4320/ © 2020 Elsevier B.V. All rights reserved.
L. de Esquivel Dias Brandão, et al. Animal Reproduction Science 213 (2020) 106272

migrations (Vieira et al., 2005). The main factor that led to B. nattereri being classified as endangered is river damming, which
modifies the natural hydrological cycle and is an endangerment to species that reside in lotic environments for part of their life cycle
(Rosa and Lima, 2008). In addition, the destruction of riparian forests is another important threat to the species, because its diet is
based mainly on resources that are prevalent in allochthonous areas (Horn, 1997; Lima and Castro, 2000).
There has been reporting of results from studies conducted with B. nattereri concerning cryopreservation of male gametes
(Oliveira et al., 2007; Viveiros et al., 2012), ultrastructure of spermatozoa and vitellogenic oocytes (Viveiros et al., 2012; Maria et al.,
2014), body-length classes (Júnior et al., 2014), and embryonic development and larval growth (Maria et al., 2017). There, however,
is a lack of information regarding the main reproductive variables of this species in the natural environment, which is required to
support fisheries legislation and aquaculture development, such as gonadal maturation, gonadosomatic index, breeding season, and
fecundity. This study was conducted with the aim of determining the main reproductive variables of B. nattereri from the Lourenço
Velho River, Paraná River basin, Minas Gerais, Brazil.

2. Materials and methods

2.1. Sampling

During the period from 2013–2016, 326 B. nattereri specimens (156 females and 170 males) were caught bimonthly using gill nets
in the Lourenço Velho River (22°22′75″S, 45°18′00″W), a tributary of the Sapucaí River, Grande River sub-basin of the Paraná River
basin, south-eastern Brazil. The fish, when alive, were killed with a lethal dose of eugenol followed by cross-sectioning of the cervical
medulla according to the ethical principles of animal manipulation established by the Brazilian College of Animal Experimentation
(COBEA, www.COBEA.org.br).

2.2. Biometrical data, gonadosomatic index, and fecundity

The following biometric data were determined: total length (TL), standard length (SL), body weight (BW), and gonad weight
(GW). The mean gonadosomatic index (GSI = GW × 100/BW) was determined at the maturation/mature gonadal stage for female
and male specimens. The total length to body weight relationship (BW = aTLb) was determined for females and males, where a =
constant and b = regression coefficient (Le Cren, 1951). The absolute fecundity (AF) and relative fecundity (RF) were estimated
using the procedures described by Arantes et al. (2010).

2.3. Morphometric analysis and light microscopy

The dynamics of folliculogenesis were analysed in histological sections of the gonads according to Gonçalves et al. (2006) and
Nunes et al. (2015). The diameters of vitellogenic follicles were measured using Olympus image-analysis software and an Olympus-
BX50 microscope.
Mature ovaries of B. nattereri were of a brown colour (Fig. 1A) and mature testes a milky-white colour (Fig. 1B). The gonadal
maturation stages were determined in histological sections of ovaries and testes, which were fixed in Bouin's fluid for 12 h, embedded
in paraffin, sectioned (5-μm thickness), and stained with haematoxylin-eosin. The gonadal maturation stages were characterised
using the procedures published by Nunes et al. (2015): 1 = resting, 2 = maturation/mature, and 3 = spawned for females
(Fig. 2A–F) and spent for males (Fig. 3A–D). The histochemical analysis of the vitellogenic oocytes was performed using periodic acid
Schiff (PAS) for glycogen, neutral glycoproteins and sialomucins. Alcian blue (AB) pH 2.5 and 0.5 was used for carboxylated and
sulphated acid glycoconjugates, including sialomucins, and Sudan black B (SB) for neutral lipids.

Fig. 1. (A) Mature ovaries and (B) testes of Brycon nattereri captured between 2013 and 2016 in the Lourenço Velho River, Paraná River basin,
Brazil.

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Fig. 2. Histological sections of Brycon nattereri ovaries at different gonadal maturation stages stained with HE; (a) Stage 1 = resting: ovaries with
perinucleolar oocytes (O1 and O2); (b) Early maturation: presence of pre-vitellogenic oocytes (O3); (c) Stage 2 = maturation/mature: ovaries with
vitellogenic oocytes (O4); (d and e) Vitellogenic oocyte filled with yolk globules (YG), squamous follicular cells (FC) and thin zona radiata (ZR); (f)
Stage 3 = spawned: presence of post-ovulatory follicles (POF), (N) = nucleus, (M) = micropyle. Bars: a, b, c, e and f (100 μm) and d (20 μm).

2.4. Statistical analysis

Statistica 7.0 software was used in the data analyses with a significance level of P < 0.05. A student's t-test was used to compare
the mean values of GSI, TL, SL and BW between females and males. An ANOVA test was used to compare the mean GSI values of
females and males at the maturation/mature gonadal stage. The chi-square contingency test was performed to detect when there
were differences in the frequency of maturation/mature and spawned/spent gonadal stages of females and males during the bimesters
of April/May and June/July for the 2013–2016 period.

3. Results

3.1. Gonadal maturation

Maturing and mature gonadal-stage female and male B. nattereri were most frequently caught from April to July (Fig. 4A and B).
Although during these bimesters there was a greater proportion of fish in gonadal Stage 2, this increase was not statistically relevant
when assessed using the chi-square contingency test (P = 0.33 for females and 0.07 for males). In the April/May and June/July

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L. de Esquivel Dias Brandão, et al. Animal Reproduction Science 213 (2020) 106272

Fig. 3. Histological sections of Brycon nattereri testes at different gonadal maturation stages stained with HE. (a) Stage 1, resting containing only
spermatogonia (S) with lumen of the seminiferous tubules occluded; (b) Early maturation with a small amount of spermatozoa (SP) in the lumen of
the seminiferous tubules; (c) Stage 2, maturation/mature with seminiferous tubules filled with spermatozoa (SP); (d) Stage 3, spent, showing empty
lumen of the seminiferous tubules or with a small amount of residual spermatozoa; Bars: a and b (20 μm) and c and d (100 μm).

bimesters, there was the greatest GSI mean values for females and males in all years (2013–2016; P < 0.05, ANOVA test; Fig. 5A and
B). The mean GSI values in the specimens in maturation/mature gonadal stages of development were not different between females
and males (P > 0.05, Student's t-test; Table 1), which is an uncommon feature in teleost fish.

3.2. Body size and sexual dimorphism

Total length (TL), standard length (SL) and body weight (BW) were used to determine sexual dimorphism of B. nattereri. Females
had a greater TL, SL and BW values than males (Table 1). The largest female captured in the maturation/mature gonadal stage had a
SL =35 cm and BW =947 g, while the largest male had a SL =30 cm and BW =448 g. The smallest female and male captured in the
maturation/mature gonadal stage had a SL = 23 and 18 cm and BW = 238 and 126 g, respectively. These data of the smallest SL and
BW are important because these may be used to estimate the size of specimens when there is initiation of gonadal maturation.
Weight–length relationships were estimated for females and males separately. Parameter b was 2.95 for females and 2.94 for males.
The correlation coefficient (r2) was 0.96 and 0.80 for females and males, respectively.

3.3. Vittelogenic follicular diameter, fecundity and carbohydrate histochemistry

The vitellogenic follicular diameter in B. nattereri was assessed by histometry and was, on average, 1.195.00 ± 278.87 μm
(Table 1). The values of AF and RF per unit of total length, standard length, body weight and gonadal weight are included in Table 1.
Neutral glycoproteins were detected in the yolk globules, zona radiata and follicular cells as indicated by a positive reaction in the
PAS assay. In the cortical alveoli and follicular cells, carboxylated glycoconjugates were detected using Alcian blue pH 2.5. Neutral
lipids were detected in the yolk globules using Sudan black B.

4. Discussion

Few studies have been performed on the main reproductive variables of the Brycon species (Table 2). These results of these studies
indicate there are differences among species regarding breeding season and fecundity, as well as vittelogenic follicular diameter and
GSI. All these species in which evaluations have occurred undergo total spawning, a trait that is usually associated with species that
migrate upstream and spawn at headwaters, such as Brycon orthotaenia (Gonçalves et al., 2016) and B. nattereri (Vieira et al., 2005).
There were the largest frequencies of mature B. nattereri fish in the April/May and June/July periods, which coincides with the

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Fig. 4. (A) Bimonthly gonadal maturation stage relative frequencies of Brycon nattereri females captured between 2013 and 2016 in the Lourenço
Velho River, Paraná Basin, Brazil. F1 = resting; F2 = maturation/mature; F3 = spawned. (B) Bimonthly gonadal maturation stage relative
frequencies of Brycon nattereri males captured between 2013 and 2016 in the Lourenço Velho River, Paraná River basin, Brazil; M1 = resting; M2 =
maturation/mature; M3 = spent.

dry season and relatively lesser ambient temperatures. This differs from the majority of Brycon species in which the reproductive
activity and the GSI peak coincide with periods of relatively greater ambient temperatures, rainfall and photoperiod (Carvalho and
Urbinati, 2005; Gonçalves et al., 2006; Nunes et al., 2015). In contrast, the reproductive activity of Brycon petrosus from the Chagres
River, Panamá (Kramer, 1978a, b) and B. nattereri from both natural (Lima et al., 2008) and captive environments (Maria et al., 2014,
2017) occurs during the dry season, which is similar to the findings of the present study. The relatively greater GSI values that were
observed for males, with average values close to the GSI of females, constitute an uncommon feature in Neotropical fish species and is
reported as a result of the present study for the first time in B. nattereri. In B. opalinus, increased testis size was related to sperm
competition and accumulation of sperm (Gomiero and Braga, 2007). The relatively greater male GSI is a peculiarity in the re-
productive biology of B. nattereri, because females generally have a much greater GSI than males (Sato et al., 2005; Arantes et al.,
2010, 2011; Domingos et al., 2012; Weber et al., 2013; Normando et al., 2014; Nunes et al., 2015; Brandão et al., 2017; Bazzoli et al.,
2019).
The average body length of the B. nattereri females of the present study was less than the mean values obtained for females of the
same species captured at the Itutinga Hydrobiology and Pisciculture Station in Brazil (Maria et al., 2014) and for females and males
captured from the upper Tocantins River, Goiás, Brazil (Rosa and Lima, 2008). Such differences are common and probably reflect a
spatial variation in the population and may also be related to the environment in which the fish are located, such as food availability
and physicochemical variables of the water. For example, in the Teles Pires River (Amazon basin, Brazil), individual B. amazonicus in
their natural habitat had a negative allometric growth (an increase in weight less than the increase in length), while captive-bred
individuals had positive allometric growth (Matos et al., 2017). There was a negative allometric growth in B. nattereri in the present
study and, indeed, this growth type is expected for species with an elongated body, such as B. nattereri (Matos et al., 2017).
The sexual dimorphism of B. nattereri that was determined to exist in the present study, with larger females than males in all

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Fig. 5. Gonadosomatic index (GSI) variations of females (A) and males (B) of Brycon nattereri at the maturation/mature gonadal stage during
2013–2016 in the Lourenço Velho River, Paraná River basin, Brazil.

Table 1
Biological variables of B. nattereri females and males captured in the Lourenço Velho River, Paraná basin, Brazil, between 2013 and 2016.
Females (n =173) Males (n = 181)

Range Mean ± SD Range Mean ± SD

GSI 11.56-19.84 14.68 ± 3.08ª 8.39-16.37 13.32 ± 2.86a

TL (cm) 11.50 – 42.50 29.86 ± 5.29ª 15.70 – 35.00 26.84 ± 3.84b
SL (cm) 9.50-35.00 25.81 ± 3.89ª 11.00-30.00 22.39 ± 3.15b
BW (g) 17.00 – 947.00 302.02 ± 131.84a 22.00 – 448.00 207.08 ± 85. 20b
No. Ov/g 114 - 329 191.00 ± 58.00
AF (x103) 5.36–29.92 11.60 ± 7.63
AF/TL 173.80-854.90 346.98 ± 207.95
AF/SL 206.40-504.10 381.32 ± 135.73
AF/BW 16.30-62.80 29.57 ± 14.78
AF/GW 114.00-328.80 191.00 ± 58.10
FD (μm) 633.00 – 1657.00 1.195.00 ± 278.87

Values in the same line followed by different letters are statistically different (P < 0.05). n = number of fish caught; GSI = gonadosomatic index at
the maturation/mature gonadal stage; TL = total length; SL = standard length; BW = body weight; GW = gonad weight; No. Ov/g = number of
oocytes per gram of ovary; AF = absolute fecundity; relative fecundity (RF) for TL (AF/TL), SL (AF/SL), BW (AF/BW) and GW (AF/GW); FD =
follicular diameter.

gonadal maturation stages, was also recorded for B. opalinus (Gomiero and Braga, 2007) and B. orthotaenia (Nunes et al., 2015) and
appears to be a common feature of the Brycon genus. In the upper Tocantins River (Goiás, Brazil), the largest specimen captured had a
SL of 51 cm and a BW of 2.6 kg, and the smallest mature female and male captured had SL values of 11.4 and 9.8 cm, respectively,
and both specimens were caught in the dry months (June and August) (Rosa and Lima, 2008). These results differ from those of the
present study, probably due to the different biotic and abiotic conditions.
The morphological features of the ovaries and testes in the present study are similar to those described for B. orthotaenia
(Gonçalves et al., 2006; Nunes et al., 2015). After females reached an advanced maturation/mature gonadal stage, the ovaries of B.
nattereri contained perinucleolar and vitellogenic oocytes. This finding supports a group-synchronous oocyte development type, thus

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Table 2
Main reproductive variables of Brycon species from recent publications.
Species Study place Breeding period GSI Fecundity/body weight Vittelogenic follicular diameter (μm) Spawning Reference

3
Brycon nattereri Brazil (nature) April-July 14.68 ± 3.08 ♀ 11.60 ± 7.63 × 10 1.195.00 ± 278.87 Total Present study
13.32 ± 2.86 ♂
3
Brycon orthotaenia Brazil (nature) October-February 9.97 ± 2.96 ♀ 112.02 ± 67.48 × 10 762.10 ± 61.91 Total Nunes et al. (2015)

7
0.93 ± 0.52 ♂
Brycon guatemalensis Nicaragua (nature) July-February 2.71 ± 0.38 ♀ 8.834 ± 1.868 × 103 1595.24 ± 411.51 Total Hernández-Portocarrero et al. (2015)
Brycon nattereri Brazil (captivity) May-August ns 8.222 ± 2.012 × 103 2330.00 ± 222 ns Maria et al. (2014)
Brycon amazonicus Brazil (captivity) October 14.80 ± 0.41 ♀ 360.00 ± 25.166 × 103 ns ns Hainfellner et al. (2012)
Brycon opalinus Brazil (nature) January-April ns 9.180 ± 0.770 × 103 1346.00 ± 865.00 Total Gomiero and Braga (2007)

ns
= not studied.
Animal Reproduction Science 213 (2020) 106272
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corroborating data reported previously (Maria et al., 2014). At the final oocyte maturation, the nucleus moves from a central position
towards the periphery of the oocyte, where the germinal vesicle breaks down and meiosis resumes (Rizzo and Bazzoli, 2020). This
facilitates fertilisation by enabling the access of a single spermatozoon to the micropyle channel located at the animal pole
(Yanagimachi et al., 2017). Carboxylated glycoconjugates in the cortical alveoli of the vitellogenic oocytes of B. nattereri are released
into the perivitellinic space during the cortical reaction and contribute to the blocking of polyspermy during fertilisation (Maria et al.,
2014). The presence of these glycoconjugates has also been reported in other Brycon species, such as B. orthotaenia and B. orbignyanus
(Bazzoli and Godinho, 1994; Maria et al., 2014). In the present study, spawned females had post-ovulatory and perinucleolar follicles
but no pre-vitellogenic and vitellogenic follicles, which are typical of multiple spawning species. This histological feature, together
with the group-synchronous oocyte development and unimodal distribution of the vitellogenic oocyte diameter (Rizzo and Bazzoli,
2020), indicates that B. nattereri undergo total spawning (all vitellogenic oocytes are released from the female´s ovary in a single
spawning event).
Fecundity is an adaptive trait because there is adaptation in fecundity with differences in geography, seasons and food availability
(Arantes et al., 2010; Brandão et al., 2017; Bazzoli et al., 2019). The values of absolute fecundity (AF) and relative fecundity (RF) in
the present study are greater than those reported by Maria et al. (2014) for captive B. nattereri and may be an indication of the
optimal environmental conditions at the sites where the fish were sampled in the present study. B. orthotaenia had a greater absolute
fecundity (Nunes et al., 2015) compared with the values reported for B. nattereri in the present study. The B. orthotaenia had
112,020 ± 67,480 oocytes in mature ovaries while the B. nattereri had 11,600 ± 7630 oocytes in the ovaries. According to Nunes
et al. (2015), there were relative greater fecundity values that are a common feature of migratory fish that have total spawning.
Besides oocyte number, the vitellogenic oocyte diameter can affect fecundity values because large oocyte diameters are related to
lesser fecundity and multiple spawning while small oocyte diameters are related to greater fecundity and total spawning (Melo et al.,
2011; Rizzo and Bazzoli, 2020). The mean diameter of the vitellogenic follicles of B. nattereri in the present study was greater than
those of B. orthotaenia while the fecundity was less in B. nattereri than in B. orthotaenia (Nunes et al., 2015), which may be related to
the different reproductive strategies and reproductive season of these species. Indeed, B. orthotaenia undergoes a long-distance
reproductive migration in the São Francisco River while B. nattereri probably migrate only a short distance in the Lourenço Velho
River, because in this region there is a small hydroelectric power dam that is a geographical barrier for large-sized migratory fish
species.

5. Conclusion

The results of the present study indicate that B. nattereri completes its reproductive cycle in the Lourenço Velho River. The
reproductive activity of the pirapitinga has unusual characteristics, such that there is a reproductive peak in the cold and dry season
of the year, in contrast to most Neotropical freshwater species, which have their peak in reproduction during the warm and rainy
season. This finding is important because the restrictions on fishing in Brazil occur during the rainy season (November to February)
and B. nattereri, an endangered species, does not benefit from this protective measure. Considering the presence of damming in the
riverine ecosystem in which B. nattereri reside, it is necessary to conserve the lotic environments, as well as the riparian forests of
these watercourses, to conserve the populations of this species.

Author statement

I declare that all authors of this manuscript participated equally in all the activities developed in this research.

Declaration of Competing Interest

No conflicts of interests in the article Reproductive variables of Brycon nattereri Günther, 1864 (Pisces: Characidae), an en-
dangered commercial species.

Acknowledgments

This research was supported by Brazilian funding agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES finance code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico, (CNPq 306946/2016-5 and 407719/
2016-4) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG APQ 03232-15).

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