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Effects of bisphenol-A on male reproductive success in adult Kadaknath chicken
Ecotoxicology and Environmental Safety 128 (2016) 61–66
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of bisphenol-A on male reproductive success in adult Kadaknath chicken Ram P. Singh a,n, Chathathayil M. Shafeeque a, Sanjeev K. Sharma b, Renu Singh c, Maharajan Kannan b, Kochiganti V.H. Sastry b, Sajith Raghunandanan d, Jag Mohan b, Parappurath A. Azeez a a
Avian Physiology and Genetics Division, Sálim Ali Centre for Ornithology and Natural History, Anaikatty, 641108 Coimbatore, India Central Avian Research Institute, Izatnagar 243 122, India c Indian Veterinary Research Institute, Izatnagar 243 122, India d Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram 695 014, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 September 2015 Received in revised form 7 February 2016 Accepted 9 February 2016
Bisphenol-A (BPA) adversely affects human and animal reproductive success in many ways, but this information is scant on birds. In the present study, we investigated the reproductive toxicity of BPA in adult Kadaknath chicken using two BPA dosages orally (1 or 5 mg/kg body weight) for seven weeks. In order to assess BPA toxicity, sperm functions, fertilizing ability, serum testosterone concentration and testis histopathology were measured in treated and control chickens. The semen volume was highest in birds exposed to 1 mg/kg body weight BPA compared to other groups. 5 mg/kg body weight BPA reduced sperm concentration significantly more than other treatment and controls. However, overall fertility and testis histology were unaffected. These results indicate that BPA adversely affects sperm characteristics in adult kadaknath chicken without affecting fertilization potential. & 2016 Elsevier Inc. All rights reserved.
Keywords: Fertility Reproductive toxicity Semen Testosterone Bisphenol-A
1. Introduction In recent years, there has been growing concern regarding the adverse effects of anthropogenic chemicals that are being released into the environment (Colborn et al., 1993; Sinawat, 2000). In birds, reproductive toxicity including eggshell thinning, embryo mortality and embryo malformations, are well-documented effects of environmental pollutants (Ratcliffe, 1967; Helander et al., 1982; Fry et al., 1987; Gilbertson et al., 1991; Speich et al., 1992; Giesy et al., 1994). Some of the environmental contaminants can mimic the hypothalamic–pituitary–gonadal axis hormones and disrupt testicular spermatogenesis, steroidogenesis, and the functions of sertoli and leydig cells (Mathur and D'Cruz, 2011; Singh et al., 2013), and are often referred to as endocrine disruption. The exogenous substances that can alter the function of endocrine system are collectively called endocrine disrupting chemicals (EDCs). Among the EDCs, BPA has received considerable attention in recent years, with a large number of studies demonstrating estrogenic properties (Chapin et al., 2008). BPA, an n Correspondence to: Smithsonian Conservation Biology Institute, Front Royal, VA 22630, USA. E-mail address: [email protected] (R.P. Singh).
http://dx.doi.org/10.1016/j.ecoenv.2016.02.012 0147-6513/& 2016 Elsevier Inc. All rights reserved.
industrial chemical since 1940, is used to impart flexibility, durability, and longevity to a variety of consumer, industrial, and medical products, including electronics, medical devices, children's toys, pharmaceuticals, compact disks, waxes, and food packaging, among others (Greiner et al., 2007, Rubin et al., 2001). Since BPA is not covalently bound to the products, it leaches over time due to use, and ultraviolet exposure, becoming available for biological exposure. There is growing evidence that BPA may adversely affect human reproductive success in many ways (Rochester, 2013). In contrast, the adverse effect of BPA on avian reproductive success is poorly understood because of paucity of literature, and contradictions in published results perhaps for its complex interaction with biological systems, although it has been thought to suppress the process of reproduction (Furuya et al., 2003; Berg et al., 2001; Panzica et al., 2005). Furuya et al. (2003) reported delayed growth of comb, wattle and testes in male chickens that received oral doses of BPA as low as 200 mg/Kg body weight every week for up to 16 wk. However, the same study found no difference in body weight between control and treated chickens. An in-ovo study reported chicken embryonic mortality and feminization of the gonads in male embryos when eggs exposed to a single dose of 200 mg BPA/g egg early in incubation (Berg et al., 2001). Given that gonadal development in avian
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species is more sensitive to the sex hormones (Romanoff, 1960), a decrease in testosterone may lead to feminization of male gonads and also can delay growth of comb, wattle and testes. Keeping in view the above findings, we presume that BPA treatment declines serum testosterone in male chickens. In order to prove the hypothesis, this study was conducted to evaluate the effect of low (1 mg/kg body weight) and high (5 mg/kg body weight) dose of BPA on the reproductive success and serum testosterone concentration in Kadaknath chicken. We strategically chose Kadaknath, an Indian native breed of fowl, possessing several wild characteristics to increase the scope of our results in terms of wild birds.
9 days of incubation to determine fertilization. The unfertilized eggs were broken open to examine any early embryonic death. The percent fertility was determined as the ratio of numbers of fertile eggs to the number of total eggs set in the incubator. On the last day of treatment, four birds from each group were euthanized to collect testes. Subsequently, a small portion of each testis was fixed in Bouin’s fixative [0.2% picric acid and 2% (v/v) formaldehyde in phosphate buffer saline] for histological investigation. Blood from each group was also collected aseptically in sterile tubes, and the serum was separated and stored at 80 °C until used.
2. Materials and methods
2.3. Testosterone estimation
All the procedures used in this study were reviewed and approved by the Central Avian Research Institute Animal ethics committee and were carried out in accordance with the revised framework of animals (Scientific Procedures) Act of 2002 of Government of India on animal welfare.
Testosterone was estimated in serum of all the samples using a commercial ELISA kit (DE1559, Demeditec Diagnostics GmbH, Germany) according to the manufacturer’s instruction. The EIA was validated for chicken serum by demonstrating parallelism between serial dilutions of testosterone standard and serum. Interand intra-assay coefficients of variation were o 10% and 15%, respectively.
2.1. Preparation of BPA solution Stock solutions containing 250 mg/mL and 50 mg/mL of bisphenol-A (Z99% purity, Sigma-Aldrich, USA) were prepared in DMSO to obtain 5 mg and 1 mg of BPA in 20 mL solution for treatment. BPA stock concentrations were confirmed by high performance liquid chromatography (HPLC) analysis as described by Saili et al. (2012). HPLC results showed 49 and 265 mg/mL of BPA in dosing solutions of 50 and 250 mg/mL BPA, respectively. 2.2. Experimental birds, treatment and sample collection Adult male and female Kadaknath (25 weeks old) from the same hatch were used for this study. These birds were maintained under uniform husbandry conditions at 14 h light/d with standard breeder ration and water ad libitum. Thirty six males were randomly allocated into four groups, namely group-1 (BPA – 1 mg/kg body weight), group-2 (BPA – 5 mg/kg body weight), group-3 (vehicle/sham control 20 mL DMSO) and group-4 (Control), and subjected to the treatment accordingly. A constant volume (20 mL) of BPA solution of the two doses (1 and 5 mg of BPA/kg body weight) and vehicle control (DMSO) was administered orally daily at 10.00 h to the treatment groups for seven weeks. Birds devoid of any treatment served as control. Body weight of treated and control birds was measured before and after the treatment using an electronic weighing balance to calculate percent body weight gain. Semen samples were collected by abdominal massage method (Burrows and Quinn, 1937) from all the treated and control birds at three day intervals to evaluate sperm motility, semen volume and sperm concentration. Sperm motility was measured under microscope, whereas semen volume was measured by glass pipette. Sperm concentrations in the semen samples were determined with a Neubauer hemocytometer following the method described by Lake (1960). During the last three weeks of treatment, half of the semen from the birds of each group were pooled and used for artificial insemination (AI) to examine fertility. Sixty females, allocated into four different groups, were used for artificial insemination. Three AI attempts were done for fertility measurement in each group. Pooled semen was diluted (1:1) in CARI diluent, and a dose of 100 million sperm was inseminated into females of respective groups. The eggs were collected from females of each group, stored, and the fertility assessed by incubating the eggs (99.5 °F temperature and 55–60% relative humidity) in an incubator. The eggs were examined by candling, after
2.4. Histological examination Testis tissues stored in Bouin's fixative were embedded in paraffin wax. Then the wax-embedded tissues were sliced (4 μm) and stained with Hematoxylin–Eosin. The stained slides were examined for cellular atrophy under light microscopy at 100 and 400 . Ten different fields in each slide were examined to assess morphological changes.
2.5. Statistical analysis The data generated during the experiment were analyzed using the statistical software, SPSS (v16). The fertility, body weight and testosterone data were analyzed by one-factor ANOVA and Duncan's multiple range tests. The effect of treatment and duration on the semen characteristics (sperm motility, semen volume and sperm concentration) were assessed using a General Linear Model (repeated measure ANOVA). Treatment and duration were included as fixed effects. Pairwise comparison of significant fixed effects was performed using a t-test. The significance level was set at 0.05.
Fig. 1. Effect of bisphenol-A on body weight in Kadaknath chicken (mean 7 standard error of the mean, n¼9/group).
R.P. Singh et al. / Ecotoxicology and Environmental Safety 128 (2016) 61–66
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Fig. 2. Effect of bisphenol-A on semen volume in Kadaknath chicken (mean 7 standard error of the mean, n¼ 9/group, wk ¼week). Single and double asterisks indicate significant differences from the control (p o0.05 and p o0.01, respectively).
3. Results
3.3. Sperm motility
3.1. Body weight
Except second and fourth, a significant difference in motility was observed in high dose group compared to control groups (Fig. 3).
The differences in body weights in control and treated birds at the end of treatment were not significant (Fig. 1). However, the weight gain was maximum (8.45%) in birds treated with high dose of BPA compared to other groups (4.31–6.69%). 3.2. Semen volume During the entire treatment period, a significant increase in semen volume was observed in low dose group compared to control groups (Fig. 2). A significant (p o0.05) difference in semen volume in low dose treatment group compared to high dose groups was observed during four, six and seven weeks of treatment.
3.4. Sperm concentration Sperm concentration in low dose differed significantly compared to control during first three weeks of treatment and then showed a normal pattern until the last weeks. At week five, sperm concentration in sham control was highest compared to other groups. Further, sperm concentration in the sham control, high dose, and control groups did not differ significantly (Fig. 4). 3.5. Serum testosterone Mean serum testosterone concentration in low dose, high dose, sham control and control were 1.377 0.19, 1.03 7 0.21, 0.69 70.08,
Fig. 3. Effect of bisphenol-A on sperm motility in Kadaknath chicken (mean 7 standard error of the mean, n¼9/group, wk ¼week). Single asterisk (*) indicates a significant difference (po 0.05) from the control.
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Fig. 4. Effect of bisphenol-A on sperm concentration (X109/ml) in Kadaknath chicken (mean 7standard error of the mean, n¼ 9/group, wk ¼week). Single and double asterisks indicate significant differences from the control (po 0.05 and po 0.01, respectively).
Fig. 6. Effect of bisphenol-A on fertility in Kadaknath chicken (mean 7 standard error of the mean, n¼ 15/group). Fig. 5. Effect of bisphenol-A on serum testosterone in Kadaknath chicken (mean7 standard error of the mean, n ¼4/group). Different superscripts (a and b) differed significantly (p o 0.05).
and 0.62 70.06 ng/mL, respectively. At the end of the treatment, significantly (p o0.05) higher serum testosterone was observed in low dose group than in the other groups (Fig. 5). Serum testosterone of high dose, sham control, and control did not differ significantly.
3.6. Fertility Mean fertility in low dose, high dose, sham control and control were 81.36 7 5.03, 78.9077.95, 71.76 74.35, and 79.53 75.12%, respectively. No significant difference was observed in fertility in treated groups compared to sham control and control (Fig. 6).
3.7. Histological examination of the testis Testicular histology was normal in the treated, control and sham control birds showing compartmentalization of germ cells in the seminiferous tubules, with spermatozoa visible in the normalsized lumen (Fig. 7).
4. Discussion BPA treatment (1 mg or 5 mg/kg body weight) affected semen characteristics and serum testosterone, although the pattern of response was not consistent at the two doses. However, no overall adverse effects were observed on fertilizing ability and testis histology. The results of increased serum testosterone under BPA treatment do not support our assumption of decline in testosterone under BPA treatment, although a non-monotonic dose-response relationship is evident in this study. It is quite possible that testosterone levels at the beginning of the exposure had actually dropped, but after continuous exposure for 7 weeks, the HPG axis may have had time to adjust/respond. Our results do not provide testosterone hormone levels throughout the exposure because of our one time point sampling strategy. Unfortunately, to best of our knowledge, there is no published literature on the effect of in vivo BPA on male reproductive success in adult birds to compare our results. The study by Furuya et al. (2003) on chickens reported delayed growth of comb, wattle and testes in male chickens on oral low doses of BPA, indicates the possibility of reduction in serum testosterone contrary to our results of increase in serum testosterone under low dose (1 mg/kg BW); however, Furuya et al. (2003) did not measure serum testosterone. The fundamental differences between Furuya’s and this study are the age of chickens and BPA doses. We used sexually mature birds, which might be
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Fig. 7. Seminiferous tubule morphology analyzed with Hematoxylin–Eosin staining by light microscopy in the testes of control (A and B), sham control (C and D), low dose (E and F) and high dose (G and H) treated birds. Different cells like spermatogonia (sg) and spermatozoa (sz) are shown in photographs. A, C, E and G: Magnifications 100 . B, D, F and H: Magnifications 400 .
less sensitive for BPA exposure than younger birds, perhaps a reason for the contradictory results. Nieminen et al. (2002) reported an increase in serum testosterone in field vole (Microtus agrestis) when treated with 250 mg/kg/day of BPA, further supporting our results of increase in serum testosterone under BPA treatment. A dose dependent effect of BPA was observed on the physical characteristics of semen such as semen volume, sperm motility, and sperm concentration. Semen volume was highest in low dose BPA, whereas sperm concentration was lowest, indicating thinning of semen of birds treated with low dose BPA. The sperm motility was observed less in high dose (5 mg/kg) BPA, which is in
agreement with the previous studies on chicken, fish and humans (Lahnsteiner et al., 2005; Montgomery et al., 2014; Li et al., 2011; Singh et al., 2015). These authors reported declines in sperm motility and swimming velocity when adult males were exposed to BPA and 17-ethinylestradiol. In vitro studies conducted on chicken sperm confirmed these findings (Singh et al., 2015). It is assumed that high BPA dose increases the availability of metabolically active BPA in blood, and thereby reducing the sperm motility through either blocking of the voltage activated Ca2 þ channels or inducing reactive oxidative stress to the sperm. Deutschmann et al. (2013) has reported that BPA acts as a potent blocker of voltage activated Ca2 þ channels in rat endocrine GH3 cells, mouse
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dorsal root ganglion neurons or cardiac myocytes, and human embryonic kidney (HEK) 293 cells. However, the possibility of blocking the voltage activated Ca2 þ channels in sperm is yet to be investigated. Hulak et al. (2013) has recently confirmed that BPA induces reactive oxidative stress in fish sperm, which is yet to be confirmed in other species. In this study, overall fertility was unaffected irrespective of the BPA doses (1 mg or 5 mg/kg). We did not observe any histological change in testis of control and treated birds. We observed an increase in semen volume in low BPA dose group, which may be a compensatory mechanism to maintain fertilizing ability, but decrease in sperm count (1 mg/kg). While the semen volume and fertility were unaffected, a reduction in sperm motility was seen with high BPA dose (5 mg/kg). Given that, only 1% of the total ejaculate is being selected mechanically in the female oviduct for fertilization (Bakst et al., 1994), it is assumed that sperm having low motility are eliminated during the sperm selection, and thereby fertilizing ability of sperm of high BPA dose remain unaffected. From this study, it can be concluded that 1 or 5 mg/kg body weight of BPA show only moderate adverse effect on sperm characteristics, and those effects do not contribute to reduction in fertility. These results indicate that a low level of BPA contamination is not potentially harmful for adult birds. It is very likely that adult birds do metabolize BPA faster than other species because of their higher metabolic activity, and because of that, their physiology is not affected greatly. The results of an increase in testosterone over time are interesting from a mechanistic viewpoint, and needs further investigation. Further studies are required to measure the rate of BPA metabolism in adult and juvenile birds in order to assess the risk of age dependent BPA toxicity.
Conflict of interest statement The authors declare no conflict of interest.
Acknowledgments This work was supported by the Science and Engineering Research Board (SERB); Department of Science and Technology, Government of India (SERB/FT/LS-147/2011).
References Bakst, M.R., Wishart, G., Brullard, J.P., 1994. Oviductal sperm selection, transport, and storage in poultry. Poult. Sci. Rev 5, 117–143. Berg, S., Halldin, K., Brunstrom, B., 2001. Effects of bisphenol A and tetrabromobisphenol A on sex organ development in quail and chicken embryos. Environ. Toxicol. Chem. 20, 2836–2840. Burrows, W.H., Quinn, J.P., 1937. Artificial insemination of chicken and turkeys. Poult. Sci. 14, 251–254. Chapin, R.E., Adams, J., Boekelheide, K., Gray, L.E., Jr., Hayward, S.W., Lees, P.S., McIntyre, B.S., Portier, K.M., Schnorr, T.M., Selevan, S.G., Vandenbergh, J.G., Woskie, S.R., 2008. NTP-CERHR expert panel report on the reproductive and developmental toxicity of bisphenol A. Birth Defects B Dev. Reprod. Toxicol. 83 (3), 157–395.
Colborn, T., vom Saal, F.S., Soto, A.M., 1993. Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378–384. Deutschmann, A., Hans, M., Meyer, R., Häberlein, H., Swandulla, D., 2013. Bisphenol A inhibits voltage-activated Ca(2 þ ) channels in vitro: mechanisms and structural requirements. Mol. Pharmacol. 83 (2), 501–511. Fry, D.M., Toone, C.K., Speich, S.M., Peard, R.J., 1987. Sex ratio skew and breeding patterns of gulls: demographic and toxicological considerations. Stud. Avian Biol 10, 26–43. Furuya, M., Sasaki, F., Hassanin, A.M.A., Kuwahara, S., Tsukamoto, Y., 2003. Effects of bisphenol-A on the growth of comb and testes of male chicken. Can. J. Vet. Res. 67, 68–71. Giesy, J.P., Ludwig, J.P., Tillitt, D.E., 1994. Deformities in birds of the Great Lakes Region: assigning causality. Environ. Sci. Technol 28, A 128–A 135. Gilbertson, M., Kubiak, T., Ludwig, J., Fox, G., 1991. Great lakes embryo mortality, edema, and deformities syndrome (GLEMEDS) in colonial fish-eating birds: similarity to chick-edema disease. J. Toxicol. Environ. Health 33, 455–520. Greiner E., Kaelin T., Nakamura K., 2007. Bisphenol A. CEH Report by SRI Consulting. Helander, B., Olsson, M., Reutergårdh, L., 1982. Residue levels of organochlorine and mercury compounds in unhatched eggs and the relationships to breeding success in white tailed sea eagles, Haliaeetus albicilla, in Sweden. Holarct. Ecol. 5, 349–366. Hulak, M., Gazo, I., Shaliutina, A., Linhartova, P., 2013. In vitro effects of bisphenol A on the quality parameters, oxidative stress, DNA integrity and adenosine triphosphate content in sterlet (Acipenser ruthenus) spermatozoa. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 158 (2), 64–71. Lahnsteiner, F., Berger, B., Kletzl, M., Weismann, T., 2005. Effect of bisphenol A on maturation and quality of semen and eggs in the brown trout, Salmo trutta f. fario. Aquat. Toxicol. 75, 213–224. Lake, P.E., 1960. Studies on the dilution and storage of fowl semen. J. Reprod. Fertil. 1, 30–35. Li, D.K., Zhou, Z., Miao, M., He, Y., Wang, J., Ferber, J., Herrinton, L.J., Gao, E., Yuan, W., 2011. Urine bisphenol-A (BPA) level in relation to semen quality. Fertil. Steril. 95 (2), 625–630. Mathur, P.P., D'Cruz, S.C., 2011. The effect of environmental contaminants on testicular function. Asian J. Androl. 13 (4), 585–591. Montgomery, T.M., Brown, A.C., Gendelman, H.K., Ota, M., Clotfelter, E.D., 2014. Exposure to 17α-ethinylestradiol decreases motility and ATP in sperm of male fighting fish Betta splendens. Environ. Toxicol. 29 (3), 243–252. Nieminen, P., Lindstrom-Seppa, P., Mustonen, A., Mussalo-Rauhamaa, H., Kukkonen, J.V.K., 2002. Bisphenol A affects endocrine physiology and biotransformation enzyme activities of the field vole (Microtus agrestis). Gen. Comp. Endocrinol. 126, 183–189. Panzica, G.C., Mura, E., Pessatti, M., Viglietti-Panzica, C., 2005. Early embryonic administration of xenoestrogens alters vasotocin system and male sexual behaviour of the Japanese quail. Domest. Anim. Endocrinol. 29, 436–445. Ratcliffe, D.A., 1967. Decrease in eggshell weight in certain birds of prey. Nature 215, 208–210. Rochester, J.R., 2013. Bisphenol A and human health: a review of the literature. Reprod. Toxicol. 42, 132–155. Romanoff, A.L., 1960. The urogenital system. In: Romanoff, A.L. (Ed.), The Avian Embryology. Macmillan, New York, pp. 783–862. Rubin, B.S., Murray, M.K., Damassa, D.A., King, J.C., Soto, A.M., 2001. Perinatal exposure to low doses of bisphenol A affects body weight, patterns of estrous cyclicity, and plasma LH levels. Environ. Health Perspect. 109, 675–680. Saili, K.S., Corvi, M.M., Weber, D.N., Patel, A.U., Das, S.R., Przybyla, J., Anderson, K.A., Tanguay, R.L., 2012. Neurodevelopmental low-dose bisphenol A exposure leads to early life-stage hyperactivity and learning deficits in adult zebrafish. Toxicology 291, 83–92. Sinawat, S., 2000. The environmental impact on male fertility. J. Med. Assoc. Thail. 83, 880–885. Singh, R.P., Sastry, K.V.H., Dubey, P.K., Agrawal, R., Singh, R., Pandey, N.K., Mohan, J., 2013. Norfloxacin drug induces reproductive toxicity and alters androgen receptor gene expression in testes and cloacal gland of male Japanese quail (Coturnix Japonica). Environ. Toxicol. Chem. 32, 2134–2138. Singh, R.P., Shafeeque, C.M., Sharma, S.K., Pandey, N.K., Singh, R., Mohan, J., Kolluti, G., Saxena, M., Sharma, B., Sastry, K.V.H., Kataria, J.M., Azeez, P.A., 2015. Bisphenol A reduces fertilizing ability and motility by compromising mitochondrial function of sperm. Environ. Toxicol Chem. 34 (7), 1617–1622. Speich, S.M., Calambokidas, J., Shea, D.W., Peard, J., Witter, M., Fry, D.M., 1992. Eggshell thinning and organoclorine contaminants in western Washington waterbirds. Colon. Waterbirds 15, 103–112.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of bisphenol-A on male reproductive success in adult Kadaknath chicken Ram P. Singh a,n, Chathathayil M. Shafeeque a, Sanjeev K. Sharma b, Renu Singh c, Maharajan Kannan b, Kochiganti V.H. Sastry b, Sajith Raghunandanan d, Jag Mohan b, Parappurath A. Azeez a a
Avian Physiology and Genetics Division, Sálim Ali Centre for Ornithology and Natural History, Anaikatty, 641108 Coimbatore, India Central Avian Research Institute, Izatnagar 243 122, India c Indian Veterinary Research Institute, Izatnagar 243 122, India d Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram 695 014, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 September 2015 Received in revised form 7 February 2016 Accepted 9 February 2016
Bisphenol-A (BPA) adversely affects human and animal reproductive success in many ways, but this information is scant on birds. In the present study, we investigated the reproductive toxicity of BPA in adult Kadaknath chicken using two BPA dosages orally (1 or 5 mg/kg body weight) for seven weeks. In order to assess BPA toxicity, sperm functions, fertilizing ability, serum testosterone concentration and testis histopathology were measured in treated and control chickens. The semen volume was highest in birds exposed to 1 mg/kg body weight BPA compared to other groups. 5 mg/kg body weight BPA reduced sperm concentration significantly more than other treatment and controls. However, overall fertility and testis histology were unaffected. These results indicate that BPA adversely affects sperm characteristics in adult kadaknath chicken without affecting fertilization potential. & 2016 Elsevier Inc. All rights reserved.
Keywords: Fertility Reproductive toxicity Semen Testosterone Bisphenol-A
1. Introduction In recent years, there has been growing concern regarding the adverse effects of anthropogenic chemicals that are being released into the environment (Colborn et al., 1993; Sinawat, 2000). In birds, reproductive toxicity including eggshell thinning, embryo mortality and embryo malformations, are well-documented effects of environmental pollutants (Ratcliffe, 1967; Helander et al., 1982; Fry et al., 1987; Gilbertson et al., 1991; Speich et al., 1992; Giesy et al., 1994). Some of the environmental contaminants can mimic the hypothalamic–pituitary–gonadal axis hormones and disrupt testicular spermatogenesis, steroidogenesis, and the functions of sertoli and leydig cells (Mathur and D'Cruz, 2011; Singh et al., 2013), and are often referred to as endocrine disruption. The exogenous substances that can alter the function of endocrine system are collectively called endocrine disrupting chemicals (EDCs). Among the EDCs, BPA has received considerable attention in recent years, with a large number of studies demonstrating estrogenic properties (Chapin et al., 2008). BPA, an n Correspondence to: Smithsonian Conservation Biology Institute, Front Royal, VA 22630, USA. E-mail address: [email protected] (R.P. Singh).
http://dx.doi.org/10.1016/j.ecoenv.2016.02.012 0147-6513/& 2016 Elsevier Inc. All rights reserved.
industrial chemical since 1940, is used to impart flexibility, durability, and longevity to a variety of consumer, industrial, and medical products, including electronics, medical devices, children's toys, pharmaceuticals, compact disks, waxes, and food packaging, among others (Greiner et al., 2007, Rubin et al., 2001). Since BPA is not covalently bound to the products, it leaches over time due to use, and ultraviolet exposure, becoming available for biological exposure. There is growing evidence that BPA may adversely affect human reproductive success in many ways (Rochester, 2013). In contrast, the adverse effect of BPA on avian reproductive success is poorly understood because of paucity of literature, and contradictions in published results perhaps for its complex interaction with biological systems, although it has been thought to suppress the process of reproduction (Furuya et al., 2003; Berg et al., 2001; Panzica et al., 2005). Furuya et al. (2003) reported delayed growth of comb, wattle and testes in male chickens that received oral doses of BPA as low as 200 mg/Kg body weight every week for up to 16 wk. However, the same study found no difference in body weight between control and treated chickens. An in-ovo study reported chicken embryonic mortality and feminization of the gonads in male embryos when eggs exposed to a single dose of 200 mg BPA/g egg early in incubation (Berg et al., 2001). Given that gonadal development in avian
62
R.P. Singh et al. / Ecotoxicology and Environmental Safety 128 (2016) 61–66
species is more sensitive to the sex hormones (Romanoff, 1960), a decrease in testosterone may lead to feminization of male gonads and also can delay growth of comb, wattle and testes. Keeping in view the above findings, we presume that BPA treatment declines serum testosterone in male chickens. In order to prove the hypothesis, this study was conducted to evaluate the effect of low (1 mg/kg body weight) and high (5 mg/kg body weight) dose of BPA on the reproductive success and serum testosterone concentration in Kadaknath chicken. We strategically chose Kadaknath, an Indian native breed of fowl, possessing several wild characteristics to increase the scope of our results in terms of wild birds.
9 days of incubation to determine fertilization. The unfertilized eggs were broken open to examine any early embryonic death. The percent fertility was determined as the ratio of numbers of fertile eggs to the number of total eggs set in the incubator. On the last day of treatment, four birds from each group were euthanized to collect testes. Subsequently, a small portion of each testis was fixed in Bouin’s fixative [0.2% picric acid and 2% (v/v) formaldehyde in phosphate buffer saline] for histological investigation. Blood from each group was also collected aseptically in sterile tubes, and the serum was separated and stored at 80 °C until used.
2. Materials and methods
2.3. Testosterone estimation
All the procedures used in this study were reviewed and approved by the Central Avian Research Institute Animal ethics committee and were carried out in accordance with the revised framework of animals (Scientific Procedures) Act of 2002 of Government of India on animal welfare.
Testosterone was estimated in serum of all the samples using a commercial ELISA kit (DE1559, Demeditec Diagnostics GmbH, Germany) according to the manufacturer’s instruction. The EIA was validated for chicken serum by demonstrating parallelism between serial dilutions of testosterone standard and serum. Interand intra-assay coefficients of variation were o 10% and 15%, respectively.
2.1. Preparation of BPA solution Stock solutions containing 250 mg/mL and 50 mg/mL of bisphenol-A (Z99% purity, Sigma-Aldrich, USA) were prepared in DMSO to obtain 5 mg and 1 mg of BPA in 20 mL solution for treatment. BPA stock concentrations were confirmed by high performance liquid chromatography (HPLC) analysis as described by Saili et al. (2012). HPLC results showed 49 and 265 mg/mL of BPA in dosing solutions of 50 and 250 mg/mL BPA, respectively. 2.2. Experimental birds, treatment and sample collection Adult male and female Kadaknath (25 weeks old) from the same hatch were used for this study. These birds were maintained under uniform husbandry conditions at 14 h light/d with standard breeder ration and water ad libitum. Thirty six males were randomly allocated into four groups, namely group-1 (BPA – 1 mg/kg body weight), group-2 (BPA – 5 mg/kg body weight), group-3 (vehicle/sham control 20 mL DMSO) and group-4 (Control), and subjected to the treatment accordingly. A constant volume (20 mL) of BPA solution of the two doses (1 and 5 mg of BPA/kg body weight) and vehicle control (DMSO) was administered orally daily at 10.00 h to the treatment groups for seven weeks. Birds devoid of any treatment served as control. Body weight of treated and control birds was measured before and after the treatment using an electronic weighing balance to calculate percent body weight gain. Semen samples were collected by abdominal massage method (Burrows and Quinn, 1937) from all the treated and control birds at three day intervals to evaluate sperm motility, semen volume and sperm concentration. Sperm motility was measured under microscope, whereas semen volume was measured by glass pipette. Sperm concentrations in the semen samples were determined with a Neubauer hemocytometer following the method described by Lake (1960). During the last three weeks of treatment, half of the semen from the birds of each group were pooled and used for artificial insemination (AI) to examine fertility. Sixty females, allocated into four different groups, were used for artificial insemination. Three AI attempts were done for fertility measurement in each group. Pooled semen was diluted (1:1) in CARI diluent, and a dose of 100 million sperm was inseminated into females of respective groups. The eggs were collected from females of each group, stored, and the fertility assessed by incubating the eggs (99.5 °F temperature and 55–60% relative humidity) in an incubator. The eggs were examined by candling, after
2.4. Histological examination Testis tissues stored in Bouin's fixative were embedded in paraffin wax. Then the wax-embedded tissues were sliced (4 μm) and stained with Hematoxylin–Eosin. The stained slides were examined for cellular atrophy under light microscopy at 100 and 400 . Ten different fields in each slide were examined to assess morphological changes.
2.5. Statistical analysis The data generated during the experiment were analyzed using the statistical software, SPSS (v16). The fertility, body weight and testosterone data were analyzed by one-factor ANOVA and Duncan's multiple range tests. The effect of treatment and duration on the semen characteristics (sperm motility, semen volume and sperm concentration) were assessed using a General Linear Model (repeated measure ANOVA). Treatment and duration were included as fixed effects. Pairwise comparison of significant fixed effects was performed using a t-test. The significance level was set at 0.05.
Fig. 1. Effect of bisphenol-A on body weight in Kadaknath chicken (mean 7 standard error of the mean, n¼9/group).
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Fig. 2. Effect of bisphenol-A on semen volume in Kadaknath chicken (mean 7 standard error of the mean, n¼ 9/group, wk ¼week). Single and double asterisks indicate significant differences from the control (p o0.05 and p o0.01, respectively).
3. Results
3.3. Sperm motility
3.1. Body weight
Except second and fourth, a significant difference in motility was observed in high dose group compared to control groups (Fig. 3).
The differences in body weights in control and treated birds at the end of treatment were not significant (Fig. 1). However, the weight gain was maximum (8.45%) in birds treated with high dose of BPA compared to other groups (4.31–6.69%). 3.2. Semen volume During the entire treatment period, a significant increase in semen volume was observed in low dose group compared to control groups (Fig. 2). A significant (p o0.05) difference in semen volume in low dose treatment group compared to high dose groups was observed during four, six and seven weeks of treatment.
3.4. Sperm concentration Sperm concentration in low dose differed significantly compared to control during first three weeks of treatment and then showed a normal pattern until the last weeks. At week five, sperm concentration in sham control was highest compared to other groups. Further, sperm concentration in the sham control, high dose, and control groups did not differ significantly (Fig. 4). 3.5. Serum testosterone Mean serum testosterone concentration in low dose, high dose, sham control and control were 1.377 0.19, 1.03 7 0.21, 0.69 70.08,
Fig. 3. Effect of bisphenol-A on sperm motility in Kadaknath chicken (mean 7 standard error of the mean, n¼9/group, wk ¼week). Single asterisk (*) indicates a significant difference (po 0.05) from the control.
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Fig. 4. Effect of bisphenol-A on sperm concentration (X109/ml) in Kadaknath chicken (mean 7standard error of the mean, n¼ 9/group, wk ¼week). Single and double asterisks indicate significant differences from the control (po 0.05 and po 0.01, respectively).
Fig. 6. Effect of bisphenol-A on fertility in Kadaknath chicken (mean 7 standard error of the mean, n¼ 15/group). Fig. 5. Effect of bisphenol-A on serum testosterone in Kadaknath chicken (mean7 standard error of the mean, n ¼4/group). Different superscripts (a and b) differed significantly (p o 0.05).
and 0.62 70.06 ng/mL, respectively. At the end of the treatment, significantly (p o0.05) higher serum testosterone was observed in low dose group than in the other groups (Fig. 5). Serum testosterone of high dose, sham control, and control did not differ significantly.
3.6. Fertility Mean fertility in low dose, high dose, sham control and control were 81.36 7 5.03, 78.9077.95, 71.76 74.35, and 79.53 75.12%, respectively. No significant difference was observed in fertility in treated groups compared to sham control and control (Fig. 6).
3.7. Histological examination of the testis Testicular histology was normal in the treated, control and sham control birds showing compartmentalization of germ cells in the seminiferous tubules, with spermatozoa visible in the normalsized lumen (Fig. 7).
4. Discussion BPA treatment (1 mg or 5 mg/kg body weight) affected semen characteristics and serum testosterone, although the pattern of response was not consistent at the two doses. However, no overall adverse effects were observed on fertilizing ability and testis histology. The results of increased serum testosterone under BPA treatment do not support our assumption of decline in testosterone under BPA treatment, although a non-monotonic dose-response relationship is evident in this study. It is quite possible that testosterone levels at the beginning of the exposure had actually dropped, but after continuous exposure for 7 weeks, the HPG axis may have had time to adjust/respond. Our results do not provide testosterone hormone levels throughout the exposure because of our one time point sampling strategy. Unfortunately, to best of our knowledge, there is no published literature on the effect of in vivo BPA on male reproductive success in adult birds to compare our results. The study by Furuya et al. (2003) on chickens reported delayed growth of comb, wattle and testes in male chickens on oral low doses of BPA, indicates the possibility of reduction in serum testosterone contrary to our results of increase in serum testosterone under low dose (1 mg/kg BW); however, Furuya et al. (2003) did not measure serum testosterone. The fundamental differences between Furuya’s and this study are the age of chickens and BPA doses. We used sexually mature birds, which might be
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Fig. 7. Seminiferous tubule morphology analyzed with Hematoxylin–Eosin staining by light microscopy in the testes of control (A and B), sham control (C and D), low dose (E and F) and high dose (G and H) treated birds. Different cells like spermatogonia (sg) and spermatozoa (sz) are shown in photographs. A, C, E and G: Magnifications 100 . B, D, F and H: Magnifications 400 .
less sensitive for BPA exposure than younger birds, perhaps a reason for the contradictory results. Nieminen et al. (2002) reported an increase in serum testosterone in field vole (Microtus agrestis) when treated with 250 mg/kg/day of BPA, further supporting our results of increase in serum testosterone under BPA treatment. A dose dependent effect of BPA was observed on the physical characteristics of semen such as semen volume, sperm motility, and sperm concentration. Semen volume was highest in low dose BPA, whereas sperm concentration was lowest, indicating thinning of semen of birds treated with low dose BPA. The sperm motility was observed less in high dose (5 mg/kg) BPA, which is in
agreement with the previous studies on chicken, fish and humans (Lahnsteiner et al., 2005; Montgomery et al., 2014; Li et al., 2011; Singh et al., 2015). These authors reported declines in sperm motility and swimming velocity when adult males were exposed to BPA and 17-ethinylestradiol. In vitro studies conducted on chicken sperm confirmed these findings (Singh et al., 2015). It is assumed that high BPA dose increases the availability of metabolically active BPA in blood, and thereby reducing the sperm motility through either blocking of the voltage activated Ca2 þ channels or inducing reactive oxidative stress to the sperm. Deutschmann et al. (2013) has reported that BPA acts as a potent blocker of voltage activated Ca2 þ channels in rat endocrine GH3 cells, mouse
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dorsal root ganglion neurons or cardiac myocytes, and human embryonic kidney (HEK) 293 cells. However, the possibility of blocking the voltage activated Ca2 þ channels in sperm is yet to be investigated. Hulak et al. (2013) has recently confirmed that BPA induces reactive oxidative stress in fish sperm, which is yet to be confirmed in other species. In this study, overall fertility was unaffected irrespective of the BPA doses (1 mg or 5 mg/kg). We did not observe any histological change in testis of control and treated birds. We observed an increase in semen volume in low BPA dose group, which may be a compensatory mechanism to maintain fertilizing ability, but decrease in sperm count (1 mg/kg). While the semen volume and fertility were unaffected, a reduction in sperm motility was seen with high BPA dose (5 mg/kg). Given that, only 1% of the total ejaculate is being selected mechanically in the female oviduct for fertilization (Bakst et al., 1994), it is assumed that sperm having low motility are eliminated during the sperm selection, and thereby fertilizing ability of sperm of high BPA dose remain unaffected. From this study, it can be concluded that 1 or 5 mg/kg body weight of BPA show only moderate adverse effect on sperm characteristics, and those effects do not contribute to reduction in fertility. These results indicate that a low level of BPA contamination is not potentially harmful for adult birds. It is very likely that adult birds do metabolize BPA faster than other species because of their higher metabolic activity, and because of that, their physiology is not affected greatly. The results of an increase in testosterone over time are interesting from a mechanistic viewpoint, and needs further investigation. Further studies are required to measure the rate of BPA metabolism in adult and juvenile birds in order to assess the risk of age dependent BPA toxicity.
Conflict of interest statement The authors declare no conflict of interest.
Acknowledgments This work was supported by the Science and Engineering Research Board (SERB); Department of Science and Technology, Government of India (SERB/FT/LS-147/2011).
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