Sex disparities determined by biometric and hematological measurements in Astyanax bimaculatus

This study aimed to compare the body indexes and hematological characteristics between Astyanax bimaculatus males and females. Four hundred fish were randomly distributed into four polyethylene tanks (100 fish/unit) in a recirculation system and fed four times a day (3% of biomass). After 90 days, ten fish (five ♀ and five ♂) were removed to perform blood tests and to measure weight, height, total length, height/length ratio, condition factor and index determination: vicerosomatic (VSI), hepatosomatic (HSI), and gonadosomatic (GSI). The results showed a higher average weight (g) in females (12.32±0.71) compared to males (6.98±0.75), the same happened to height (cm) = (3.01±0.07) and (2.40±0.05), total length (cm) = (3.01±0.07) and (2.40±0.05), VSI (%) = (11.43±0.81) and (3.55±1.05), HSI (%) = (0.72±0.08) and (0.30±0.04), respectively. Mean corpuscular hemoglobin (pg) was higher in females (3.72±1.20) than in males (2.99±1.51). Regarding the number of thrombocytes (10.μL), there was an increase in males (25.71±3.91) compared to females (17.40±6.40).


INTRODUCTION
Aquaculture is practiced all over the world as the main fish-producing activity owing to the rapid growth of the world's population, while the production of fish from fisheries has declined (The State of World Fisheries and Aquaculture, 2016). This requires the development of more ease of handling make them an interesting option for Brazilian fish farmers (Baldisserotto et al., 2014). This fish can be easily reared in different types of ponds with the use of artificial feeding at all life stages. Its tolerance to temperature variations contributes to its rusticity in commercial production systems (Garutti, 2003), besides supporting high stocking densities (e.g. more than six fish per liter) (Jatobá and Silva, 2015).
The occurrence of anatomical differences between the sexes is very common in the animal kingdom. However, considering the commercial production of any species, these differences can interfere with the standardization of the final product (Navarro et al., 2003). For fish, Bye and Lincoln (1986) consider sexual maturation one of the biological processes that most affect the yield of intensive systems, essentially because, during this period, energy for somatic growth is redirected to gonadal development and the formation of gametes, resulting in sexual dimorphism. These same authors also highlight the risk of overpopulation in ponds/tanks due to uncontrolled spawning. At the reproductive stages of the fish life cycle, energy is spent in the production of gametes, which generally interferes with their growth (Vazzoler, 1996). In tilapia, females have reduced growth, making the male economically more attractive (Meurer et al., 2005). While lambari females show a more satisfactory growth compared to males (Porto-Foresti et al., 2005).
Hematological parameters have been used as physiological indicators of organic dysfunction in aquaculture and are commonly applied as tools for prognosis and diagnosis of fish health status (Burgos-Aceves et al., 2019). However, there are intrinsic and extrinsic factors that can cause changes in these parameters, for example, age of the animal, reproductive period, sex and environment in which they are exposed (Fazio, 2019). Therefore, it is important to know whether A. bimaculatus males and femals of the same age and in the same environment, have a different hematological profile. Thus, hematology can be used as a tool to monitor changes in homeostasis and the health of the animals created.
Although females tend to grow more than males, therefore, allowing the production of monosex lots, this technique is not applied commercially, and both sexes of A. bimaculatus are created together. However, it is important to determine the characteristics between the sexes before a monosex commercial production (Antoniutti et al., 1985;Peterson and Davis, 2012). Accordingly, this study aimed to compare the biometry, body indexes, and hematological characteristics of the yellowtail lambari males and females (A. bimaculatus).

MATERIAL AND METHODS
This study was carried out at the Laboratório de Aquicultura (LAQ), Federal Institute of Santa Catarina (IFC), Campus Araquari/SC, Brazil (Protocol number 104/2015 approved by the Animal Ethics Committee). The experimental design was completely randomized, and four hundred fish from the same spawning, with a mean weight 0.27 g, were distributed into four experimental units (800 L polyethylene tanks) of 100 fish each and equipped with a recirculation aquaculture system (RAS) and biological filter ( Figure 1). The fish were fed four times a day with 3% of their biomass, according to . During the experimental period, dissolved oxygen (3.15±1.52 mg.L-1) and temperature (21.78±2.59°C morning; 23.51±2.37°C afternoon), were measured twice a day. Whereas, the pH (7.32±0.11) and total dissolved ammonia (0.09±0.06mg.L-1) were measured twice a week.
The water parameters were considered suitable for rearing this species (Porto-Foresti et al., 2005). After the 90-day experimental period followed by 24 h of starvation, ten animals (five ♀ and five ♂) per experimental unit were anesthetized with eugenol (75mg.L -1 ) for hematological analysis; and then euthanized by a cerebral concussion to the calculus of the hepatosomatic, vicerosomatic and gonadosomatic indexes ( Figure 1). Sex identification was done by checking whether males would have spines on the anal fin and whether the females would have the width of the abdomen equal to or greater than the width of the back (Garutti, 2003).
The biometry of the animals was performed, being analyzed weight (g), height (cm), total length (cm), height/length (cm) ratio and condition factor (k). A longitudinal incision was made in the ventral region in order to remove and weigh the liver, viscera and gonads to the calculate hepatosomatic index (HSI = liver weight/body weight), viscerosomatic fat index (VSI = viscerosomatic fat weight/body weight) and gonadosomatic index (GSI = gonad weight/body weight).
For hematological and biochemical analysis, approximately 0.2ml of blood was withdrawn from the caudal vein with syringes containing EDTA 10% and used for blood smears stained with May Grunwald/Giemsa/Wright solution for white blood cell (WBC) count using an indirect method by counting the total leukocytes number (WBC) in 2000 erythrocytes in the smears (Ishikawa et al., 2008) and the total number of leukocytes (WBC) were calculated by the indirect method (Ranzani-Paiva et al., 2013). Hematocrit percentage was measured by the microhematocrit method and red blood cell count (RBC) in a Neubauer chamber after dilution 1:200 in the Dacie solution.
Hemoglobin concentration was determined by spectrophotometry using a commercial kit (LabTest, Minas Gerais, Brazil). The hematimetric indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated according to Ranzani-Paiva et al., (2013), and glucose (G-TECH free, Accumed-Glicomed, Brazil). For each identified cell, length, width, and area were measured using Olympus cellSens Imaging Software (version 1.7) on a computer linked to an optical microscope coupled to a digital camera. The measures were compared between the different sexes.
Data were submitted to the Kolmogorov-Smirnov test and Levene's test to evaluate normality and homoscedasticity, respectively. Data approved from these tests were assessed by Student's t-test to observe differences between males and females. The remaining data were assessed by the Mann Whitney test. All tests recognized 5% significance.

RESULTS AND DISCUSSION
Differences in growth performance between sexes in fish may interfere with commercial profitability (Navarro et al., 2006). From this perspective, females showed greater total weight, height, and length (Table 1), similar to that described by Reidel et al. (2004), reporting on Curimbatá (Prochilodus lineatus) and Piavuçu (Leporinus macrocephalus). However, Souza, et al. (1999) observed better growth in the male Nile tilapia (Oreochromis niloticus). The height/length ratio and condition factor did not differ between sexes (Table 1). The height/length ratio suggests that both sexes present similar conformation of fillet. Thus, this piece of information is very important for the industrial processing standardization of fish (Reidel et al., 2004).

Sex disparities determined…
The reproductive cycle may influence the weight of gonads, liver, and fat (Vazzoler, 1996). During this period, the liver synthesizes and secret vitellogenin, which is transported to the gonads by the bloodstream (Baldisserotto et al., 2014). Females showed higher HSI (Table 2), and this increase could be associated with the production of vitellogenin. These data corroborate the findings of Pereira Filho et al. (2011), who reported the increase of HSI in Astyanax scabripinnis females during oocyte maturation. Males had lower VSI values (Table 2), similar to the data obtained in Leporinus copelandii by Costa et al. (2005), likely owing to energy mobilization during the maturation of gonads, suggesting better body condition during the reproductive cycle. The GSI is normally used as an important reproductive parameter for females; however, for males, it does not always represent the current reproductive condition (Pereira Filho et al., 2011). The analysis of GSI corresponds to the reproductive phase in which females are found. Navarro et al. (2006) observed higher GSI in A. scabripinnis females reared together with and separate from the males. Barbieri et al. (1982) also observed a superior GSI in A. fasciatus and A. bimaculatus females. These results corroborate the data in the present work.
Fish hematology is an important tool for identifying changes in homeostasis, as well as the appearance of diseases and standardizing ideal reared conditions (Fazio, 2019). The hematological parameters of male and female A. bimaculatus did not show significant differences, except in HCM (Table 3), while other studies reported values close to those found in this experiment related to the number of erythrocytes, hematocrit, hemoglobin, glucose, MCV, MCHC and MCH in A. bimaculatus submitted to environmental conditions similar to this study (Jatobá et al., 2018;Moraes et al., 2018).
Motlagh et al. (2012) did not observe significant differences in hematological parameters between Betta splendens males and females, corroborating the study carried out with A. bimaculatus, except for MCH. Karimi et al. (2013) who evaluated the influence of sex on hematological parameters of Acanthopagrus latus and observed that the number of erythrocytes was higher in males than in females; although, other parameters such as hematocrit, MCV, MCH, and MCHC did not show a significant difference between the sexes. A. bimaculatus females showed a greater significant increase in the MHC than males (Table  3), different from that observed in A. latus (Karimi et al., 2013) and in the rainbow trout (Ranzani-Paiva, et al. 1998). The increase in MHC in females could be related to the stage of gonadal Pereira maturation, because the developed gonads (final stages of maturation) increase the demand for oxygen due to the incorporation of nutrients in the oocytes (Vazzoller, 1982). Leukocytes are often used as indicators of health status in fish because they are part of the innate immune defense and are involved in the regulation of immune function in organisms (Burgos-Aceves et al., 2019;Stenberg et al., 2019). The leukogram consists of neutrophils, lymphocytes, basophils, monocytes, eosinophils, special granulocytic cells, and thrombocytes.
In this study, males had a higher number of circulating thrombocytes than females (Table 4). This increase in the number of thrombocytes could be caused by a hemorrhage due to the difficulty in drawing blood from males, as they are smaller than females. This is related to the function of thrombocytes, which in addition to defense mechanisms and role in homeostasis, also participate in blood clotting processes (Ranzani-Paiva et al., 2013). No differences were observed between the sexes for other leukocytes (Table 4), suggesting that males and females have the same amount and proportion of circulating blood cells in the same environmental conditions, according to the results found for A. latus (Karimi et al., 2013).
No morphological differences were observed in lymphocytes and thrombocytes between the sexes (Table 5). These cells have the highest occurrence in the blood and are, therefore, used for morphological characterization.  In conclusion, A. bimaculatus females have better growth performance, and this genus is indicated for higher yield (biomass). However, male body indexes suggest higher carcass yield. Thus, the choice of a monossex production must be planned according to the way the final product will be marketed (live, fresh, or processed). While for health or research purposes, the hematological profile of both sexes can be used to represent a population.