Bulletin of Environmental Contamination and Toxicology https://doi.org/10.1007/s00128-018-2377-6
Dietary Exposure to Tebuconazole Affects Testicular and Epididymal Histomorphometry in Frugivorous Bats Mariana Machado‑Neves1 · Mário J. O. Neto1 · Diane C. Miranda1 · Ana Cláudia F. Souza2 · Mariana M. Castro1 · Marcela N. Sertorio1 · Túlio F. Carvalho3 · Sérgio L. P. Matta1 · Mariella B. Freitas3 Received: 20 March 2018 / Accepted: 5 June 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract This study evaluated the effects of a commercially recommended concentration (1 mL/L) of a fungicide tebuconazole (TBZ) on testicular and epididymal histomorphometry of Artibeus lituratus, following 7 and 30-day oral exposure. TBZ30 bats showed a reduction in the percentage of tubules and seminiferous epithelium, as well as a decrease in tubule and epithelium somatic indexes, and tubular diameter. Inversely, these animals showed increased percentage of intertubular compartment, Leydig cells and blood vessels. The volume of Leydig cells and their number per gram of testis also increased in TBZ30 bats. Alterations in epididymal morphometry were observed in all regions of the organ, with increase of ductal diameter in both exposure times. These results indicate that exposure to low concentration of TBZ resulted in testicular and epididymal morphometric changes in fruit bats, mainly at 30-day exposure, suggesting that functional alterations might be occurring in these organs and impacting reproductive capacity. Keywords Reproductive toxicology · Fungicides · Testis · Epididymis · Stereology · Artibeus lituratus Fungicides are commonly used to control fungi from diverse fruit cultures. These chemicals, in turn, have been widely found in the environment contaminating non-target organisms (Gray et al. 2006; Bartlewicz et al. 2016; Qi et al. 2018). Particularly, wildlife and humans have been displayed reproductive and endocrine alterations caused by fungicides exposure, leading to fertility disorders (Moser et al. 2001; Cecconi et al. 2007; Hass et al. 2012; Fu et al. 2016; Cao et al. 2017). In this sense, tebuconazole (TBZ) is a triazole fungicide used worldwide on crops such as garlic, wheat, barley, oat, corn, peanuts, and orchard fruits (Moser et al. 2001; Zhou et al. 2016). Its mode of action is based on the inhibition of α-lanosterol demethylase in yeasts and fungi, which * Mariana Machado‑Neves
[email protected] 1
Department of General Biology, Federal University of Viçosa, Av. P.H. Rolfs, s/n, Campus Universitário, Viçosa, Minas Gerais 36570‑900, Brazil
2
Department of Animal Science, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
3
Department of Animal Biology, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil
decreases ergosterol biosynthesis (Kwok and Loeffler 1993). Ergosterol is a key sterol component in the membrane of fungi and yeasts, and its blocking affects the association of plasma membrane constituents, increasing cell permeability and inhibiting fungi growth (Goetz et al. 2009). The α-lanosterol demethylase is also found in mammals (Debeljak et al. 2000), in which it participates in the metabolic pathway leading to cholesterol synthesis, the substrate for steroid hormones (Byskov et al. 1995). Another enzyme whose action might be blocked by TBZ is aromatase (Vinggaard et al. 2000), a P450 enzyme involved in steroidogenesis. It was reported that perinatal exposure to TBZ affected neurological, immunological and reproductive function of Sprague–Dawley rats (Moser et al. 2001). Furthermore, studies have shown impaired fertility in rats and fishes exposed to this fungicide (Taxvig et al. 2007; Sancho et al. 2009; Joshi et al. 2016). In the Neotropics, fruit-eating bats living in forest fragments near fruit crops constantly are exposed to fungicides, mainly through oral consumption of fruit. These bats play a key role in forest regeneration and secondary succession by dispersing seeds of pioneer neotropical plants (Gorchov et al. 1993). However, habitat loss and exposure to fungicides are being pointed out as possible threats to bat
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populations (Melo et al. 2012). Some species of bats have had their populations seriously affected and received the conservation status of threatened or endangered in the last decade (Mispagel et al. 2004). Several studies have emphasized the relationship between exposure to fungicides and declines in bats population (Gerell and Lunderg 1993; Swanepoel et al. 1999). However, physiological parameters related to fungicide toxicity and its effects on bats reproduction are still unclear. As the testis is the main organ of the male reproductive system, being responsible for sperm and testosterone production (França and Russell 1998), and the epididymis plays a key role in sperm maturation and storage (Cornwall 2009; Belleanneé et al. 2012), damages to these organs might lead to impaired reproduction in bats. The knowledge of the reproductive function of a species can be obtained from the histological evaluation of the gonads. Moreover, morphometric and stereological analyses of reproductive organs allow making inferences about their function following a xenobiotic exposure (Russell et al. 1990). Thus, the aim of this study was to investigate the effects of short- and long-term dietary exposure to a commercially recommended concentration of TBZ on testicular and epididymal histomorphometry of fruit-eating bats Artibeus lituratus.
Materials and Methods The commercial formulation of TBZ (Folicur 200 EC®— 200 g/L) ((RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4triazol-1-ylmethyl)pentan-3-ol) and the spreader-sticker (SS) (polyoxyethylene alkyl phenol ether) were obtained from the Integrated Pest Management Laboratory of the Federal University of Viçosa (Viçosa, MG, Brazil). The percentage of the active ingredient TBZ in this formulation is 21.3%, whereas other inactive ingredients totalize 78.7%. Adult male bats from the species Artibeus lituratus (n = 22; 66.47–85.95 g) were captured using mist nets in Minas Gerais state, Brazil (20°45′S, 42°52′W) during the transition from dry to rainy season (August to December). The captures were authorized by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA— 1936505) and by the State Forest Institute (IEFMG— 121/06). All the experimental procedures were approved by the Ethics Committee of Animal Use of the Federal University of Viçosa, Brazil (Protocol 02/2012). Bats were kept in individual steel cages under natural temperature and light cycles. The animals were fed papaya (Carica papaya) for 2 days for their adaptation and then bats were randomly divided into four groups: bats fed with untreated fruits (control, n = 5); bats fed with fruits dipped into 0.5% SS (SS, n = 5); bats fed with fruits treated with 1 mL/L TBZ and 0.5% SS for 7 days (TBZ7, n = 6); and bats fed with fruits
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treated with 1 mL/L TBZ and 0.5% SS for 30 days (TBZ30, n = 6). Papaya fruit was selected for this study due to its high acceptance from bats in captivity (Amaral et al. 2012). Fruits were dipped in a syrup with SS and TBZ at concentrations mentioned above and left to dry in an adapted container. Halves of fruits (approximately 200 g) were offered daily at 1800 h to the bats with the bark side up, in order to simulate the conditions faced by the animals in nature. Water was provided ad libitum. The concentrations of TBZ and SS used in this study were the same recommended by the manufacturers for the use on several fruit crops, therefore, they were considered environmentally relevant. The SS is considered of low toxicity and commonly is associated with fungicides to increase their efficiency (Zhao et al. 2016). The two exposure periods were chosen to allow testing the effects of the fungicide under short- and long-term exposure. At the end of each treatment, the animals were weighed and euthanized by decapitation. The testes and epididymides were removed, weighed and fixed in Karnovsky solution for 24 h. Testicular and epididymal fragments (caput, corpus and cauda) were dehydrated in crescent ethanol series and embedded in 2-hydroxyethyl methacrylate (Historesin®, Leica Microsystems, Nussloch, Germany). Sections with a thickness of 3 µm were obtained using a rotary microtome and stained with toluidine blue-sodium borate (1%). The qualitative analysis was made using Olympus CX40 optical microscope (Olympus, Tokyo, Japan). The gonadosomatic and epididymal somatic indexes were calculated relating organ mass and total body mass. For morphometric and stereological analysis, digital images of testicular and epididymal parenchyma were obtained using a light microscope (Olympus BX-53, Tokyo, Japan) equipped with a digital camera (Olympus DP73, Tokyo, Japan) and analyzed with Image-Pro Plus® 4.5 (Media Cybernetics, Silver Spring, USA) software. The volumetric densities of the testicular compartments were obtained by counting 2660 points projected onto 10 images captured in histological slides per animal. Coincident points were registered in seminiferous tubules (tunica propria, epithelium and lumen) and intertubule. The percentage of points in each component and the volume of testicular compartments were calculated according to Souza et al. (2016). The tubule-somatic index was calculated relating the seminiferous tubule volume and body mass (Russell et al. 1990), while the epithelium somatic index was assessed according to Morais et al. (2014). The seminiferous tubule diameter was obtained by randomly measuring 20 tubular cross sections, as circular as possible, regardless of the stage of the seminiferous epithelium cycle. These sections were also used to measure the seminiferous epithelium height (Souza et al. 2016). The volumetric proportion of intertubular components (nucleus and cytoplasm of the Leydig cell, blood vessels, lymphatic
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space, and connective tissue) was obtained by counting 1000 points, per animal, projected onto intertubular images, and calculated according to Souza et al. (2016). The nuclear diameter of Leydig cells was obtained from the measurement of 30 nuclei/animal. The volume of Leydig cells as well as the number of these cells per testis and per gram of testis was calculated according to Mori and Christensen (1980). The leydigosomatic index was calculated by multiplying the total volume of Leydig cells by the body mass divided by 100. In the epididymis, the ductal diameter of each epididymal region was obtained by randomly measuring 20 ductal cross sections, as circular as possible, per animal. These sections were also used to measure the luminal diameter and the epithelium height (Castro et al. 2017). The volumetric proportion of epididymal components (epithelium, lumen and interstitium) in each region was obtained by counting 2660 points projected onto 10 images, captured in histological slides per animal. The percentage of points in each component was calculated according to Castro et al. (2017). All counting and measurements made in testes and epididymis were performed blindly. Data were tested for normality (Lilliefors) and homoscedasticity (Cochran), and analyzed using one-way analysis of variance (ANOVA). Significant means were compared with the Student–Newman–Keuls multiple comparisons test. Results were expressed as mean ± standard deviation (SD), and the significance level was set at p
