Social behaviors as welfare indicators in teleost fish.

Animal welfare is a key issue not only for aquaculture industry and food production, but also for daily husbandry practices in research topics related to physiology in wild and farmed animals. In this context, teleost fish constitute interesting models to assess alternative welfare indicators because of their wide diversity in reproductive and social structures. Any framework for assessing teleost fish welfare needs to account for the physiological mechanisms involved in each species as a first step. A comprehensive approach should also take into account how these physiological and behavioral parameters can be altered by environmental enrichment considering the specific requirements in each case and identifying intrinsic biological characteristics of individual species. This review will show how cortisol and sex steroids regulate social behavior in teleost fish, and how different aspects of social behavior can be employed as welfare indicators according to specific characteristics in each case. This article will consider evidence in teleost fish, including cichlids, characids and cyprinids with different reproductive strategies and social structures (e.g., territorial social hierarchies or shoaling behavior). Neotropical species will be particularly emphasized. The main laboratory-based animal welfare indicators are cortisol, a classical stress hormone, together with sex steroids. Considering that the endocrine landscape is intrinsically related to social behavior, reproductive and agonistic behavioral traits such as aggression, anxiety and courtship are key elements to assess welfare under housing and culture conditions. This review highlights the importance of assessing physiological mechanisms and identifying behavioral characteristics in teleost fish, especially in Neotropical species, as a baseline to understand which environmental enrichment can improve animal welfare in each individual species.

Fighting cichlids: An integrated multimodal analysis to understand female and male aggression in Cichlasoma dimerus.

Aggression has been historically linked to males and androgen levels and, even if females from different species also display aggressive behavior, female aggression is still widely understudied. The aim of the present work is to disentangle how sex differences in social plasticity can be explained by sex steroid hormone levels, gonadal state and/or morphometric characteristics. In this context, we performed intrasexual dyadic encounters to identify social plasticity after acquiring a winner or loser status in males and females of Cichlasoma dimerus. This integral analysis suggests that the reproductive and hormonal variables analyzed explain the behavioral variation among winner and loser males and females, and that there are significant differences between sexes and contest outcome when individual morphometric variables are excluded from the analysis. Interestingly, there are no sex differences in aggressive and submissive behaviors, and clustering into winners and losers is mainly explained by specific behavioral displays, such as bites, chases, approaches, passive copings, and escapes. Correlation heatmaps show a positive correlation between estradiol with aggression and a negative correlation with submission, suggesting estrogens may have a dual role regulating agonistic behavior. Finally, these results suggest that size difference can help to understand aggression in females but not in males, and that assessment of the opponent's body size is important to understand aggression also before the initiation of the contest in both sexes. Overall, this study constitutes an integral approach adding insights into the importance of reproductive and hormonal variables to understand social plasticity in males and females.

Sex Differences in Aggression Are Paralleled by Differential Activation of the Brain Social Decision-Making Network in Zebrafish.

Although aggression is more prevalent in males, females also express aggressive behaviors and in specific ecological contexts females can be more aggressive than males. The aim of this work is to assess sex differences in aggression and to characterize the patterns of neuronal activation of the social-decision making network (SDMN) in response to intra-sexual aggression in both male and female zebrafish. Adult fish were exposed to social interaction with a same-sex opponent and all behavioral displays, latency, and time of resolution were quantified. After conflict resolution, brains were sampled and sex differences on functional connectivity throughout the SDMN were assessed by immunofluorescence of the neuronal activation marker pS6. Results suggest that both sexes share a similar level of motivation for aggression, but female encounters show shorter conflict resolution and a preferential use of antiparallel displays instead of overt aggression, showing a reduction of putative maladaptive effects. Although there are no sex differences in the neuronal activation in any individual brain area from the SDMN, agonistic interactions increased neuronal activity in most brain areas in both sexes. Functional connectivity was assessed using bootstrapped adjacency matrices that capture the co-activation of the SDMN nodes. Male winners increased the overall excitation and showed no changes in inhibition across the SDMN, whereas female winners and both male and female losers showed a decrease in both excitation and inhibition of the SDMN in comparison to non-interacting control fish. Moreover, network centrality analysis revealed both shared hubs, as well as sex-specific hubs, between the sexes for each social condition in the SDMN. In summary, a distinct neural activation pattern associated with social experience during fights was found for each sex, suggesting a sex-specific differential activation of the social brain as a consequence of social experience. Overall, our study adds insights into sex differences in agonistic behavior and on the neuronal architecture of intrasexual aggression in zebrafish.

Sexual Dimorphism in Aggression: Sex-Specific Fighting Strategies Across Species.

Aggressive behavior is thought to have evolved as a strategy for gaining access to resources such as territory, food, and potential mates. Across species, secondary sexual characteristics such as competitive aggression and territoriality are considered male-specific behaviors. However, although female-female aggression is often a behavior that is displayed almost exclusively to protect the offspring, multiple examples of female-female competitive aggression have been reported in both invertebrate and vertebrate species. Moreover, cases of intersexual aggression have been observed in a variety of species. Genetically tractable model systems such as mice, zebrafish, and fruit flies have proven extremely valuable for studying the underlying neuronal circuitry and the genetic architecture of aggressive behavior under laboratory conditions. However, most studies lack ethological or ecological perspectives and the behavioral patterns available are limited. The goal of this review is to discuss each of these forms of aggression, male intrasexual aggression, intersexual aggression and female intrasexual aggression in the context of the most common genetic animal models and discuss examples of these behaviors in other species.

Characterization of StAR protein of Rhinella arenarum (Amphibia, Anura).

The steroidogenic acute regulatory (StAR) protein performs the delivery of cholesterol from the outer to inner mitochondrial membrane. This is considered the rate-limiting step of acute steroid production, widely studied in mammals. However, there are only few reports regarding the characterization and expression of StAR protein in non-mammalian vertebrates. In this study, StAR protein sequence of Rhinella arenarum has been characterized and deduced from interrenal and testis cDNA sequences. StAR encodes a 285 amino acid protein with a conserved domain containing putative lipid binding sites. In vitro incubations showed that expression of StAR mRNA in testis, determined by qPCR, and testosterone synthesis determined by radioimmunoassay were stimulated after treatment with hCG and 8Br-cAMP. However, StAR mRNA expression results obtained with hCG show a higher stimulation than those obtained with 8Br-cAMP, even though steroidogenic production is the same with both treatments.

Social control of spermatogenesis and steroidogenesis in cichlid fish: a comparative approach.

Social animals with hierarchical dominance systems are susceptible to changes their environment. Interactions with conspecifics can greatly affect individual's behavior and reproductive success. This review will show how social behavior modulates gonadal steroidogenesis and spermatogenesis in African and Neotropical cichlid fish with different social systems and how this modulation regulates reproductive capacity. Social behavior and aggressiveness are strongly linked to sex steroids, glucocorticoids and neuropeptides. The challenge hypothesis suggests that behavioral interactions increase androgen levels in response to social instability, but there is little evidence regarding estradiol levels. It has been recently demonstrated that in male Cichlasoma dimerus, a Neotropical cichlid fish, the challenge hypothesis could also be extended to estrogens. In C. dimerus, dominant males have higher gonadosomatic index than subordinated; the percentage of spermatocytes and spermatids is higher in subordinates, while dominants show a greater percentage of spermatozoa. In other species of African cichlids, socially suppressed subordinate males are not reproductively incompetent maintaining some activity at every level of their reproductive axis. Axis reactivation upon social ascent is similar to the initiation of puberty in mammals, as well as the reoccurrence of puberty observed in seasonally breeding animals. In conclusion, social behavior and reproductive strategies in females cichlids are still understudied, and Neotropical cichlids still constitute a group that deserves more attention, considering cichlids' diversity in mating systems, reproductive behavior and parental care. This review highlights the importance of performing further studies and additional research in these two areas, which still remain to be addressed.

Gonadal, body color, and genotoxic alterations in Lithobates catesbeianus tadpoles exposed to nonylphenol.

Endocrine disrupting chemicals are one of the most important factors contributing to worldwide amphibian decline. The 4-nonylphenol (NP) is a degradation product of several compounds, such as detergents and pesticides, affecting the aquatic environment. Here, we test whether treatment with NP has an effect on developing ovarian tissue, nuclear abnormalities in erythrocytes, and body darkness in pre-metamorphic tadpoles of the bullfrog Lithobates catesbeianus. Tadpoles were exposed for 14 days to three different concentrations of NP (1, 10, and 100 µg/L) besides the control group, which was maintained only with water. After determining body coloration, animals were euthanized and gonads and blood were collected and processed for histology and genotoxic analysis. Even though most animals were females, intersex tadpoles were observed in control and treated groups and there were no males in any group. The highest concentration of NP showed an increase in atretic oocytes, but the area corresponding to somatic compartment and early and late germ cells were not affected. Furthermore, all treated groups presented higher amount of nuclear abnormalities in erythrocytes and body darkening when compared with the control group. These results suggest that NP causes genetic damage and morphological alterations in L. catesbeianus tadpoles by disrupting oogenesis, inducing genotoxicity and increasing body coloration. Its effects on gonadal development could cause future impairments in reproduction, while its deleterious effects on genotoxicity and body pigmentation could be used as a biomarker of effect to this compound.

Estradiol and reproduction in the South American toad Rhinella arenarum (Amphibian, Anura).

Rhinella arenarum is a South American toad with wide geographic distribution. Testes of this toad produce high amount of androgens during the non reproductive season and shift steroid synthesis from androgens to 5α-pregnanedione during the breeding. In addition, plasma estradiol (E2) in males of this species shows seasonal variations but, since testes of R. arenarum do not express aromatase, the source of plasma E2 remained unknown for several years. However, the Bidder's organ (BO), a structure located at one pole of each testis, is proposed to be the main source of E2 in male's toads since it expresses several steroidogenic enzymes and is able to produce E2 from endogenous substrates throughout the year. In addition, there were significant correlations between plasma E2 and total activity of BO aromatase, and between plasma E2 and the amount of hormone produced by the BO in vitro. In the toad, apoptosis induced by in vitro treatment with E2 was mostly detected in spermatocytes during the breeding and in spermatids during the post-reproductive season, suggesting that this steroid has an important role in controlling spermatogenesis. However, in vitro treatment with E2 had no effect on proliferation. This evidence suggests that the mechanism of action of E2 on amphibian spermatogenesis is complex and more studies are necessary to fully understand the role of estrogens regulating the balance between cellular proliferation and apoptosis. In addition, in R. arenarum in vitro studies suggested that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD/I) activity during the post reproductive season. As well, E2 regulates FSHß mRNA expression all over the year suggesting a down regulation process carried out by this steroid. The effect on LHß mRNA is dual, since during the reproductive season estradiol increases the expression of LHß mRNA while in the non-reproductive season it has no effect. In conclusion, the effect of E2 on gonadotropins and testicular function is complex, not clearly understood and probably varies depending on the species. The aim of the current article is to review evidence on reproductive endocrinology and on the role of estradiol regulating reproduction in amphibians, with emphasis on the South American species Rhinella arenarum.

Can estrogens be considered as key elements of the challenge hypothesis? The case of intrasexual aggression in a cichlid fish.

Territorial aggression has been widely studied in males and it has been historically suggested that androgens are key mediators of this behavior. However, more recent evidence suggests that it is the aromatization to estrogens, rather than androgens themselves, that is key to regulating this behavior. Females also display aggressive behaviors, but the physiological regulation of female aggression is still understudied when compared to males. In this context, the challenge hypothesis postulates that male-male aggressive interactions stimulate the production of androgens in males in periods of social instability. Here we determine plasma sex steroid levels in Cichlasoma dimerus to assess whether estrogens are related to aggressive behavior and to test the challenge hypothesis in both males and females. We set-up challenge trials as intrasexual dyadic encounters and determined androgen and estrogen levels before and after the trial in both winners and losers. Even though there were no differences in initial estradiol-17ß plasma levels between male winners and losers, initial levels were higher (p = .046) in female winners than in losers, while there were no differences in testosterone or 11-ketotestosterone levels. After trials, both males and females showed elevated levels of estradiol-17ß and both androgens, but only males exhibited a significant 1.45, 5.42 and 3.2-fold increase in estradiol-17ß, testosterone and 11-ketotestosterone, respectively (p = .023, p = .016, p = .018). Moreover, changes in circulating levels of estradiol-17ß in females after the trials do not depend on their reproductive status or on the outcome of the contest. We suggest that female aggression is associated with initial levels of estradiol-17ß, and that the challenge hypothesis, originally defined for androgens, could also be extended to estrogens.

Fighting cichlids: Dynamic of intrasexual aggression in dyadic agonistic encounters.

Aggression is an extremely complex behaviour and female aggression is understudied when compared to males. Despite the fact that it has been suggested that conflict among females may be more frequently resolved peacefully, in many species females show high levels of aggression. We used Cichlasoma dimerus to describe dynamics and conflict outcome in intrasexual agonistic encounters. We performed encounters of two sex-matched animals in a neutral arena and we recorded agonistic interactions during one hour. All aggressive and submissive behaviours were described and quantified to perform the ethogram. Encounters followed three phases: pre-contest, contest and post-resolution. Latency, time of resolution and frequency of aggressive displays did not differ between sexes. Relative variations in size between female opponents better explained aggression outcome in each contest, since higher levels of aggression occurred in dyads of more similar fish. However, this was not observed in males, suggesting that probably morphological characteristics could be less relevant in male conflict resolution. Altogether these results suggest that in this ethological context, C. dimerus females are as aggressive as males and that they have similar motivation towards territorial aggression, emphasizing the need of deepening the study of aggression in females and not only in males.

Apoptosis, proliferation and presence of estradiol receptors in the testes and Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura).

The dynamic equilibrium between spermatogonial proliferation and testicular apoptosis determines the progression of spermatogenesis in amphibians. Estrogens and their receptors play a central role in regulating spermatogenesis in vertebrates, and in some species of anurans, estradiol (E2 ) is involved in the regulation of spermatogonial proliferation and apoptosis of germ cells. Bidder's organ (BO) is a structure characteristic of Bufonidae that has historically been compared to an undeveloped ovary. In adult Rhinella arenarum males, BO is one of the main sources of plasma E2 . The aim of this study was 1) to describe the seasonal variations in testicular apoptosis, spermatogonial proliferation, and cellular proliferation in BO; and 2) to analyze the presence and localization of estrogen receptor ß (ERß) in the testes and BO of R. arenarum. Testicular fragments and BOs from animals collected during the year were labeled with 5-bromo-2'-deoxyuridine (BrdU) and BrdU incorporation was determined using immunohistochemistry. Apoptosis in testicular sections was detected using the TUNEL method, and ERß localization was assessed using immunohistochemistry in testes and BOs. The results indicate that spermatogonial proliferation is highest during the reproductive season and that cysts of spermatocytes and spermatids undergo apoptosis during the postreproductive season. Furthermore, the proliferation of follicular cells is highest during the reproductive and postreproductive seasons. ERß was primarily detected by immunolocalization in Sertoli cells, follicular cells, and oocytes. Taken together, these results suggest that cysts that do not form spermatozoa are removed from testes by apoptosis and that estrogens regulate both spermatogenesis and oogenesis in adult males of R. arenarum.

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura).

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder's organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E2). The purpose of this study is to analyze the effect of E2 on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24h with or without 2 or 20nM of E2. After treatments, the activities of cytochrome P450 17α-hydroxylase-C17-20 lyase (CypP450c17) and 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3ß-HSD/I activity during the post reproductive season. Furthermore, although E2 has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E2 has no effect on testicular proliferation all year long. Taken together, these results suggest that E2 is involved in the regulation of testicular steroidogenesis and spermatogenesis.

Development and steroidogenic properties of the Bidder's organ of the tadpole of Rhinella arenarum (Amphibia, Anura).

Several studies suggested that in anuran amphibians steroidogenic enzymes are critical for gonadal differentiation, proposing that the amount of sex steroids would adjust this differentiation. Among anurans, bufonids are important for the study of sex differentiation due to the presence of Bidder's organ (BO) that differentiates as a rudimentary ovary in the cephalic portion of the genital ridge. Considering that in adult males of Rhinella arenarum, the BO synthesizes estradiol, the main purpose of this work is to examine, in this species, the morphogenesis of BO and the steroidogenic capacity of this organ during larval development. BO and the proper gonads are distinguished from Gosner stage 26. During metamorphosis, BO primary oogonia develop in oogonia in nests, early previtellogenic oocytes and late previtellogenic oocytes in follicles while proper gonads remain undifferentiated. Aromatase was detected by immunohistochemistry in almost all the largest follicles of the BOs while the cytochrome P450 side-chain cleavage was observed in only few oocytes. The proper gonad was not immunoreactive in any stage. The determination of aromatase and 5α-reductase activities showed that the population of tadpoles between stages 36-41 is not homogeneous in terms of aromatase activity. In addition, from stage 26 to the end of metamorphosis, all the stages were able to produce estradiol from endogenous substrate but stages 40-41, corresponding to the end of pro-metamorphosis, produced the highest values. In conclusion, BO is able to synthesize estradiol from endogenous precursors and proper gonad remains undifferentiated at least until the end of the metamorphosis.

Functional studies of p.R132C, p.R149C, p.M283V, p.E431K, and a novel c.652-2A>G mutations of the CYP21A2 gene.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90-95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.

Estradiol production by the Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura). Seasonal variations in plasma estradiol.

In bufonids, the Bidder's organ (BO), located in the anterior pole of the testis, is sometimes referred to as a rudimentary ovary because of the presence of previtellogenic follicles. In males of Rhinella arenarum it has been demonstrated that some follicles are vitellogenic and also express several steroidogenic enzymes in follicular cells. The purpose of this study is to describe seasonal variations in plasma estradiol (E(2)) and in aromatase activity of the BO, and to determine the capacity of the BO to synthesize E(2) from cholesterol in males of R. arenarum. E(2) was determined by radioimmunoassay and aromatase activity was measured by transformation of radioactive substrates into products. Results indicate that plasma E(2) reached the highest concentration in April and the lowest one in animals captured in June, showing a progressive increase to the end of the year. Plasma E(2) and total activity of aromatase in the BO were significantly lower during the pre-reproductive season than during the reproductive and post-reproductive seasons. It was also demonstrated that the BO is able to produce E(2) from endogenous substrates throughout the year. No correlation was found between plasma E(2) and total BO weight, while there was a significant correlation between plasma E(2) and total activity of aromatase, and between plasma E(2) and E(2) produced in vitro. Taken together, these results demonstrate seasonal variations in plasma E(2), in bidderian total activity of aromatase and, that the BO of adult males of R. arenarum is able to produce E(2) from endogenous substrates.

Seasonal changes and regulation of the glucocorticoid receptor in the testis of the toad Rhinella arenarum.

Several studies indicate that wild free-living vertebrates seasonally regulate plasma glucocorticoids. However, not only glucocorticoids but also the amount of receptors is important in determining biological responses. In this context, seasonal regulation of glucocorticoid receptor (GR) is crucial to modulate the response to glucocorticoids. Rhinella arenarum is an anuran exhibiting seasonal variations in plasma glucocorticoids and also in the number of binding sites (B(max)) of the testicular cytosolic GR. In this work, we evaluated if the annual pattern of GR protein in the testis varies seasonally and, by an in vitro approach, the role of glucocorticoids, androgens, and melatonin in the regulation of the GR B(max) and protein level. For this purpose, testes were treated with two physiological concentrations of melatonin (40 and 200 pg/ml), with or without luzindole (melatonin-receptor antagonist); with testosterone, cyanoketone (inhibitor of steroidogenesis) or casodex (androgen-receptor antagonist); or with dexamethasone or RU486 (GR antagonist). After treatments, B(max) and protein level were determined by the binding of [(3)H]dexamethasone and Western blot, respectively. Results showed that GR protein decreases in the winter. The in vitro treatment with melatonin produced a biphasic effect on the B(max) with the lowest concentration decreasing this parameter by a receptor-mediated mechanism. However, melatonin had no effect on the GR protein level. Conversely, a high concentration of dexamethasone up-regulated the GR protein and androgens neither changed the B(max) nor the protein level. These findings suggest that seasonal changes in plasma melatonin and glucocorticoids modulate the effect of glucocorticoids in the testis of R. arenarum.

Anteroposterior variation of the cell types in the interrenal gland of the male toad Rhinella arenarum (Amphibia, Anura).

The interrenal gland of anurans synthesizes the steroids aldosterone and corticosterone, but it is unknown whether these hormones are synthesized by the same cell type. In this work, we aim to elucidate whether there are different steroidogenic cell types and whether they have specific regionalization in the interrenal gland of the male toad Rhinella arenarum. We characterized all cell types using histological, immuhistochemical, and histochemical methods as well as transmission electron microscopy. Furthermore, we evaluated the organization of the cell types in the gland and anteroposterior variations in the synthesis of the steroids. We found evidence of five cell types: two morphologically different steroidogenic cells, type 1: polyhedral cells tightly attached to each other that have spherical euchromatic nuclei and type 2: retracted cells loosely attached to each other that have oval heterochromatic nuclei. Cell type 2 is mainly observed in the inner zone of the gland. In addition, we observed two types of chromaffin cells, called type 3 and 4 cells, randomly distributed throughout the interrenal gland, as well as type 5 cells, recognized as summer cells. Morphometric analyses of the cell types in the anterior and posterior zones of the interrenal showed that the ratio "area of type 2 cells/total interrenal area" is significantly lower in the posterior zone. In vitro incubations showed that the posterior portion of the gland produces significantly higher amounts of both corticosterone and aldosterone. Overall, our results suggest that the type 2 cells are less active to synthesize both aldosterone and corticosterone, compared to type 1 cells. Unlike most previous reports on the interrenal gland of anurans, in R. arenarum there is a zonation of the steroidogenic cell types, which implies that the organ is not anteroposterior or dorsoventrally homogeneous.

The Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura). Presence of steroidogenic enzymes.

The Bidder's organ (BO) of male true toads of Bufonidae family is located in the anterior pole of the testis and it has been compared to a rudimentary ovary because of the presence of previtellogenic follicles. In some species, BO remains in both sexes, while in others only adult males preserve the structure. Several studies suggest that the development of BO is inhibited by the differentiation of the corresponding gonad. The purpose of this study is to describe morphological and histological variability of the BO of Rhinella arenarum and also analyze its steroidogenic capacity. Observations indicate that although most bidderian follicles are in pre vitellogenesis, there are others in early or late vitellogenesis. Moreover, we found that BOs weight was significantly lower in males during the pre-reproductive period and that there is no significant correlation between the weights of BO and the adjacent testis. We also analyzed the presence of steroidogenic enzymes using immunohistochemistry. Results indicate that all the follicles were immunoreactive with the antibody against aromatase, while only few of them were positive for the cytochrome P450 side-chain cleavage. Furthermore, activities of 3ß-hydroxysteroid dehydrogenase/isomerase, cytochrome P450 17-hydroxylase, C17,20-lyase and aromatase were detected by the transformation of radioactive substrates into products. Taken together, these results confirm the steroidogenic capacity of the BO in adult males of R. arenarum.