INTRODUCTION:
Lambari, Astyanax bimaculatus (Linnaeus, 1758), is a characiform fish of the family Characidae. Revisions have considered many of the 88 genera (620 species) of Characidae as “Incertaesedis”, with 86 species belonging to the genus Astyanax (LIMA et al., 2003). Several papers have reported the importance of lambari to the trophic structure of Brazilian ecosystems and their reproductive behavior in the face of environmental change (PRADO et al., 2011; MARCON et al., 2015a). Lambari is of great importance to the equilibrium of the ecosystems where they occur, namely rivers of South and Central America, and they are well represented in such ecosystems in Brazilian (CARVALHO et al., 2009; WEBER et al., 2013). Astyanax bimaculatus is an omnivorous forager and a nutritionally important species for riverine communities (CARVALHO et al., 2009).
The survival of any fish species depends on the production of viable eggs (GOMES et al., 2018). The main stages of follicular development in fish are, in general, the pre-meiotic, perinucleolar, vitellogenic, and final maturation periods (SELMAN & WALLACE, 1989; GRIER, 2012). Impaired follicular development or atresia (LANG, 1981; MIRANDA et al., 1999) can be caused by stress, pesticides, temperature, hormones, light, inadequate nutrition, and confinement (NAGAHAMA, 1983; GURAYA, 1986). Follicular atresia can have serious effect on cultivation and induced spawning of fish, since this degenerative ovarian process lowers fertility rate (LAM, 1983). Post-ovulatory follicles are the remains of ovarian follicles that occur in fish ovaries after spawning (SELMAN & WALLACE, 1989). These structures have been investigated in numerous teleosts, with some studies indicating that they are rapidly reabsorbed as follicular cells develop phagocytic activity (LINARES-CASENAVE et al., 2002).
Along with reduced stocks of natural fisheries, there is increasing need to develop technology for artificial reproduction of commercially high-value species in order to improve reproductive potential and the production of fingerlings for fish culture and reintroduction. Induced spawning of freshwater fish is usually performed using the method of hypophysation (WOYNAROVICH & HORVÁTH, 1980; FORTUNY et al., 1998; LOKMAN et al., 2015), where crude carp (Cyprinus carpio) pituitary extract is injected into the coelomic cavity or within the muscle.
Studies on growth hormone in fish have addressed development and growth (BERN, 1983), reproduction (LE GAC et al., 1993), immune response (CALDUCH-GINER et al., 1997) and osmoregulation (SANGIAO-ALVARELLOS et al., 2006). Growth hormone (GH) is a peptide of approximately 20 kDa that is secreted by the pituitary gland of vertebrates (YAMAGUCHI et al., 1991). The GH coding region tends to be highly conserved among vertebrates, including mammals, birds, reptiles, amphibians and fish (MA et al., 2012), with a similar three-dimensional structure in different taxonomic groups and the same function in spite of varying amino acid sequences (CLACKSON et al., 1998). Growth hormone has great influence on growth rate and development by promoting cell division, differentiation and growth (CORIN et al., 1990; COPELAND & NAIR, 1994). It is also involved in the metabolism of carbohydrates, lipids, proteins and nucleic acids (KLAUSEN et al., 2001).
In fish reproduction, growth hormone is related to significant increases in vitellogen in plasma level (HULL & HARVEY, 2001), which are associated with gonadal maturation (HOLLOWAY et al., 1999), and thus plays a fundamental role in the development and maintenance of follicles. Understanding the effects of growth hormone in ovaries of A. bimaculatus can provide information that can help improve ovarian recovery prior to subsequent spawning. In this sense, the present study aimed to evaluate the effects of growth hormone, 120-hours after induced spawning, using histological and histometric techniques.
MATERIALS AND METHODS:
Fish and induced spawning
During the month of February (summer), 200 female lambari (A. bimaculatus) in advanced maturation (mean total length 8:49 ± 1.31 cm, mean body weight 10.19 ± 4.47 g) were obtained from Prata’s Fish Farm (Eugenópolis, MG, 21º05’56”S 42º11’13”W).The selected lambari were chosen based on aspects of their body, such as having a bulging belly or a dilated and vascularized genital pore. Fish were removed from their earthen ponds one day prior to hypophysation and transferred to masonry tanks (1.50x0.70x0.40 m), where they remained for the duration of the experimental period at temperature of 25 ± 1 °C. The fish were fed with a commercial diet containing 35% protein, twice daily. Fish were selected for an intra-coelomic double-dose of common carp (Cyprinus carpio) pituitary extract, with the first injection being a dose of 0.5 mg/kg body weight (priming dose), and the second injection, 14 hours later, being a dose of 6 mg/kg (WOYNAROVICH & HORVATH, 1980). After hypophysation, females were kept in the masonry tanks until the time of spawning. After spawning (120 females or 60% spawning at approximately nine hours after the second injection), as a result of the treatment with the double-dose of pituitary extract, the fish (n=110) were divided into two groups: a control group (n=55) and a GH treatment group (n=55). Both groups were housed in masonry tanks with constant water circulation and a temperature of around 25 ± 1 °C.
Treatments
For the collection of ovaries, females of the control group (n=55) were anesthetized with a 1:10,000 benzocaine solution and euthanized by transition stem head incision, in accordance with the ethical principles established by the Brazilian College of Animal Experimentation (COBEA, http://www.cobea.org.br). This study was submitted and approved by the Ethics Committee on Animal Use (Protocol no. 59/2005) of the Federal University of Viçosa (UFV). Ovaries were collected from the control group at 0 (n=5) and 120 (n=50) hours post spawning induction.
The treatment group (n=55) was subjected to human growth hormone (Saizen®, Serono) at a dose of 2 mg/g body weight (SEIDELIN & MADSEN, 1999), which was administered intra-coelomically immediately after spawning induction. Ovaries were collected from the treatment group following the same procedures and timing adopted for the control group.
Biometric parameters
The following biometric data were recorded for each specimen: body weight (BW, g), weight of ovaries (PO, g) and liver weight (FW, g). Mean values and standard deviations for the gonadosomatic (GSI=POx100/BW) and hepatosomatic (HSI=FWx100/BW) indices were calculated for both groups 120 hours post-spawning.
Morphological study
All fish sacrificed in the experiment were used for morphological study of the ovaries. Fragments of the middle region of the ovaries were fixed for a minimum of 24 hours in 10% buffered formaldehyde (0.1 M phosphate buffer pH 7.2) and preserved in 70% ethanol (GODINHO et al., 2005). The fragments were then transferred to a 95% alcohol solution for four hours. Shortly thereafter, the fragments were immersed in 95% ethanol + glycolmethacrylate resin (Leica, Historesin) at a ratio of 1:1 for 12 hours followed by pure resin for 24 hours. Fragments were then sectioned at 1-3 µm thick and stained with hematoxylin-eosin and PAS/metanil-yellow. After staining, slides ovaries of both the control group and the treatment groupwere analyzed under a Olympus BX40 light microscope. Ten randomly-chosen fields of histological slides throughout the length of the ovary were examined using a 10X objective and a 40X objective, the latter for morphological analysis of the action of growth hormone by comparing ovarian tissue between groups. Digital images were obtained using an Olympus AX70 photomicroscope TRF, in the Laboratory of Plant Anatomy, Department of Vegetal Biology at the UFV.
Histometric analysis
Slides of 10 ovarian sections of each animal of both the control group (n=55) and the treatment group (n=55) were analyzed under an Olympus BX40 light microscope. Counts of developing ovarian follicles and atretic follicles were taken for each animal of both the control and treatment groups at 120 hours post-spawning. Counts of post-ovulatory follicles were taken at 0 and 120 hours after spawning to confirm the presence of post-ovulatory follicles. Counts for each section were done in 10 randomly selected fields for each animal in both groups using a microscope equipped with a micrometer reticle (1 mm) in a 10X objective. Granulocytes associated with atretic follicles were also counted in 10 randomly selected fields using a microscope with a 40X objective (MARCON et al., 2015a).
Statistical analysis
Data are reported as mean ± standard deviation and were evaluated using the software Statistica 7.0. Quantitative variables were tested for homoscedasticity (Levenes) and normality (Shapiro-Wilk). Differences in the average number of follicles in different stages of development, post-ovulatory follicles, follicular atresia and the number of granulocytes between the control group and the treatment group were tested for significance using t-tests. When the assumptions of normality and homoscedasticity were not met, even after appropriate transformations, the data were subjected to the nonparametric Mann-Whitney test. In addition, interaction between cell type (granulocytes and total atresia) and groups (control and GH treatment) was assessed using two-factor ANOVA. Pearson’s correlation was used to compare the proportion of granulocytes associated with total atretic follicles in the control group and the treatment group. The criterion for significance was P<0.05. Graphics were produced using Microsoft Excel 2010 program.
RESULTS:
The ovary of A. bimaculatus has the tunica albuginea emitting septa into the organ to form ovigerous lamellae, which are follicles in different developmental stages. The same pattern of follicular development was observed in both the control and treatment groups at 120 hours post-spawning (Figures 1 A-F and 2 A). Differences between initial, intermediate and advanced atresia are shown in figure 2. The presence of post-ovulatory follicles was also observed in both the control and treatment groups (Figures 1 A, B and C).
[Images Omitted: See PDF]
Different stages of follicular development were quantified in both groups, but there were no significant differences (control group: ipf= 5.66 ± 1.54; apf= 2.61 ± 1.04; isf= 0.64 ± 0.30; itsf= 0.70 ± 0.30; asf= 2.60 ± 0.70; treatment group: ipf= 4,50 ± 1.18; apf= 2,31 ± 0.55; isf= 0,79 ± 0.32; itsf= 0.96 ± 0.38; asf= 3.12 ± 0.52)(Figure 3). The treatment group (0= 5.49 ± 1.5; 120= 1.49 ± 1.04) and the control group (0= 5.10 ± 1.2; 120= 2.29 ± 1.79) did not differ significantly in post-ovulatory follicles at 0 hours and 120 hours. The number of post-ovulatory follicles differed significantly between the collections made at 0 hours and 120 hours.
[Image Omitted: See PDF]
Counts of follicles in initial atresia were greater (P<0.05) in the treatment group (initial atresial= 0.50 ± 0.37; intermediate atresia= 0.16 ± 0.09; advanced atresia= 0.06 ± 0.04; total atresia= 0.72 ± 0.20) than in the control group (initial atresial= 0.14 ± 0.11; intermediate atresia= 0.21 ± 0.16; advanced atresia= 0.13 ± 0.09; total atresia= 0.48 ± 0.04) at 120 hours (Figure 4). There was a greater number of granulocytes (P<0.05) in the treatment group (granulocytes= 6.49 ± 2.64; total atresia= 0.72 ± 0.20) than in the control group (granulocytes= 2.77 ± 1.92; total atresia= 0.48 ± 0.04) (Figure 5). There was a significant interaction (P<0.05) between cell type (granulocytes and total atresia) and group (control and GH treatment). A positive correlation (r=0.68) was detected between in proportion of granulocytes associated with total atretic follicles in both the control and treatment groups during the experimental period.
[Images Omitted: See PDF]
The GSI did not differ significantly between the treatment group (6.47 ± 1.77) and the control group (5.93 ± 1.97) for 120 hours. Likewise, IHS did not differ significantly between the treatment group (3.32 ± 2.09) and the control group (2.07 ±1.36) during the experimental period.
DISCUSSION:
The present study found no morphological differences between the control and the treatment groups for the different stages of follicular development, confirming the pattern of follicular development typical of incertaesedis fish of the family Characidae (MARTINS et al., 2012; MARCON et al., 2017). More primary follicles were reported in the control group than in the treatment group; although, the difference was not significant. Nonetheless, these follicles are likely to be important for future spawns. These findings indicated that the use of growth hormone at the concentration applied was not sufficient for producing a greater amount of secondary follicles. An explanation for this finding is that growth hormone alone is not able to increase levels of vitellogenin. Conversely, when using growth hormone + 17β-estradiol (E2), growth hormone was able to potentiate the action of E2 by increasing the production of vitellogenin (PEYON et al., 1996), and thus is an important precursor in the recruitment of new follicles (COWARD & BROMAGE, 1998). Thus, female A. bimaculatus treated with growth hormone did not respond significantly with the formation of new advanced and primary follicles, nor secondary follicles, during 120 hours post induced spawning.
The number of post-ovulatory follicles in each of the groups of the present study was similar to the number previously reported for Tilapia zilli and for A. bimaculatus, which exhibited a reduced number of post-ovulatory follicles and an occluded lumen on the third day post induced spawning (COWARD & BROMAGE, 1998; DRUMMOND et al., 2000). These results indicated rapid reabsorption of post-ovulatory follicles. According to DRUMMOND et al. (2000), total resorption of post-ovulatory follicles in A. bimaculatus occurs in 13 days, and apoptosis of the follicular cells could be observed after the onset of the post-ovulatory period, as indicated by having a nucleus with a crescent pattern of chromatin condensation underlying the nuclear envelope and apoptotic bodies at different stages of reabsorption. During the involution of the post-ovulatory follicles of A. bimaculatus in the present study, there was a loss of contact between the membranes of adjacent follicular cells. This event was also reported by SANTOS et al. (2005) between 48 and72 hours post-spawning in Leporinustaeniatus. This loss of contact between adjacent follicular cells is an important factor during the involution of post-ovulatory follicles (GRIER, 2012), and was observed in the present research using the histochemical technique of PAS/metanil-yellow. Neither the control group nor the treatment of the present study exhibited complete elimination of post-ovulatory follicles during the post-spawning period of 120 hours.
The process of atresia has been studied by several authors, and is divided into several stages (SANTOS et al., 2005; SANTOS et al., 2008; MORAIS et al., 2012). The stage of initial atresia is characterized by follicular cell hypertrophy and breaking of the zona radiata, thereby allowing cell to be invaded by phagocytes, which consume the vitellogenic contents of the follicle (LINARES-CASENAVE et al., 2002). The degeneration and resorption of atretic follicles is a prolonged process, lasting up to seven months in A. bimaculatus, and frequently leaves signs in the ovary that can remain throughout the next reproductive cycle (MIRANDA et al., 1999). Ovarian regression was monitored for only 120 hours in the present study, but initial atretic follicles were very active during this period. Furthermore, granulocytes were observed infiltrating the theca and, according to these authors, they can participate in follicular resorption. This is consistent with the findings of the present study with A. bimacultaus, which had higher frequencies of granulocytes associated with total atretic follicles in the treatment group. These observations indicated that growth hormone may potentialize the recruitment of granulocytes in immunological processes (CALDUCH-GINER et al., 1997), and thus is important in initial atresia.
The IGS and IHS are indicators of the biological status of fish and are used to determine reproductive stage (HONJI et al., 2009; BONCOMPAGNI et al., 2013; DURHAM & WILDE, 2014), because the maturation of primary and secondary follicles is concomitant with an increase in gonad weight and decreased liver weight in the final stages of the reproductive period (NORMANDO et al., 2014; PERINI et al., 2013). The absence of significant differences in IGS and IHS between the control group and the treatment group can be attributed to the fact that the experiment was short and reproduction was fast, as seen in the work by BUGEL et al. (2010) and MARCON et al. (2015b).
CONCLUSION:
The administration of growth hormone after induced spawning increases the amount of granulocytes in ovaries after 120 hours. Granulocytes may be involved in tissue remodeling by removing initial atretic follicles, which may facilitate the process of ovarian recovery and follicular development for future spawning. In fact, it remains to be determined whether the observed response is more pronounced for periods longer than 120 hours. This research provides details of initial ovarian recovery for the 120-hour post-spawn period, and thus contributes to understanding of ovarian structure.
ACKNOWLEDGEMENTS
This work was funded by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). We would also like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This study was conducted with funds granted by FAPEMIG (EDT 215/2005).
E-mail: [email protected]. * Corresponding author.
DECLARATION OF CONFLICT OF INTERESTS
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
AUTHORS’ CONTRIBUTIONS
LM and LAB conceived and designed experiments. LM and LAB performed the experiments, LM, NB, ER and VRF carried out the lab analyses. LAB supervised and coordinated the animal experiments and provided clinical data. LM performed statistical analyses of experimental data. LM, LAB and NB prepared the draft of the manuscript. All authors critically revised the manuscript and approved of the final version.
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Lucas Marcon1 2 3
Vivian Rocha Freitas2
Nilo Bazzoli1
Elizete Rizzo4
Laércio dos Anjos Benjamin2
1Programa de Pós-graduação em Biologia de Vertebrados da PUC Minas, Pontifícia Universidade Católica de Minas Gerais (PUC Minas), Belo Horizonte, MG, Brasil.
2Departamento de Veterinária, Universidade Federal de Viçosa (UFV), 36.570-900, Viçosa, MG, Brasil.
3Departamento de Biologia, Universidade do Estado de Minas Gerais (UEMG), Ibirité, MG, Brasil.
4Departamento de Morfologia, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil
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Abstract
Lambari, Astyanax bimaculatus, is an oviparous, multiple-spawning fish that is reproductively active throughout the year, which makes it promising for cultivation and research. This research histologically evaluates the ovaries of lambari that have undergone artificial spawning induced with pituitary extract (control group), and the effect of growth hormone at a dose of 2 mg/g body weight (treatment group) on the subsequent process of ovarian recovery. Ovaries of fish in both the control and treatments groups were collected at 120 hours after spawning and analyzed using optical microscopy to characterize the average quantities of: follicles in different stages of development, post-ovulatory follicles, follicular atresia and granulocytes. Quantity and morphology of early and advanced primary follicles did not differ between the treatment and control groups; an important and necessary factor for ovarian recovery for subsequent spawning. There was a greater amount of granulocytes in initial atresia in the group treated with growth hormone. These results demonstrated that the administration of growth hormone may potentiate the process of ovarian recovery after induced spawning.
