In Vivo Exposure to Bisphenol-A Altered the Reproductive Functionality in Male Coturnix Coturnix Japonica

Bisphenol-A, is one of the most characterized endocrine disruptors on the reproductive functions in humans and animals. We have previously reported in vitro and in vivo effects of bisphenol-A on functional role of sperm in chicken. Here, the effects of 1 and 5 mg/kg bisphenol-A daily administered by gavage for 3 wk to adult male Japanese quails on reproductive functionality was investigated. Cloacal index and foam frequency were greatly reduced at high dose. Sperm quality attributes were affected at both doses. Sperm quality attributes were affected at both doses. Alkaline phosphatase showed most signicant reduction among seminal enzymes. Dose dependent response (P < 0.01) of bisphenol-A was noticed with modulating testosterone concentrations at low and high doses. Disturbances regarding fertility and hatchability traits were prominent in high and low dose groups. The current study conrms the compromising actions of bisphenol-A on reproductive success in male Japanese quails at lower doses that are considered to be safe (50 mg/kg BW/d) under in vivo exposure module. These results indicate higher sensitivity of quails to bisphenol-A toxicity and explores the possibility of using quail subjects as an accurate toxic indicators.


Introduction
Bisphenol-A (BPA), an estrogenic mimicker has attracted research interest ever since its wide spread use in late 1940's in consumer plastic products 1,2 owing to its undesirable effects on human and animal health.
The interactive ability of BPA is wider and it is reported to bind to estrogens 3,4 androgens 5,6 , thyroid hormones 7 and immune mediated receptors 8,9,10 , besides affecting various physiological systems.
Previous studies reported the potential damaging role of BPA in humans, animals and birds 3,11,12,13 . In males, compromising effects of BPA involves damage to prostate gland, sperm DNA and impairment of semen production. Animal studies have reported the negative effects of BPA on male reproductive health including sperm production and quality, steroidogenesis, male urinary tract development, and sexual dysfunction 14 . Though reprotoxic effects of BPA have been well characterized in mammalian species using rodent models, very limited reports could be fetched with regard to avians 15,16 . We have recently reported negative effects of BPA on sperm motility, livability, abnormality and plasma membrane integrity, mitochondrial membrane potential in chicken under in vitro 17 and in vivo 18 conditions.
Avian studies aiming at reproductive toxicity of BPA involved wide range of doses and duration which are unrelated to environmentally relevant concentrations 16,19,20,21 . At a concentration of 62 µM/L, BPA has compromised the viability of chicken embryonic hepatocytes 22 . Two different studies in quails observed no effects 23 and oviductal abnormalities when administered with BPA at 200 µg per egg. Further it also caused feminization of left testes in male chickens 15 . Chickens when exposed for 23 wk with 2 µg/kg with 2d interval altered male phenotypes viz., comb, wattles, testes apart from delayed growth. In response to potential higher doses (200 × 106 µg) of BPA for 14 wk period has resulted in reductions of comb and testes 16 .
BPA exposure is ubiquitous in humans and birds, with ingestion being the major exposure route 24 . BPA is not covalent in nature and therefore may unbind from products via leaching into the environment 18 . In poultry, use of legumes, soy proteins, plastic feeders and water troughs are most common routes for BPA exposure. Birds represent an excellent bio-indicator for monitoring the toxicological residues in biological systems. Among the domestic aves, Japanese quail offers an excellent model for studying possible mechanisms of endocrine disruptors due to its smaller size, shorter generation interval, ubiquitous existence and intimacy with living environment. In addition, the male reproductive function has a unique "androgen dependent cloacal gland", which can be used as an external marker for reprotoxic alterations 25,26 . Studies to date have shown that the male Japanese quail is exquisitely sensitive to the effects of exogenous estrogen 23,27,28 . In-ovo administration of BPA in chicken and quail and suggested that early embryonic period is more sensitive to estrogenic endocrine disrupting compounds 15,28 . It produced oviduct abnormality in female quail and delayed the growth of comb, wattle, and testis in male chicken 16 . Nevertheless, studies on effect of BPA on reproductive function in adult quail are lacking. Accumulated research evidence, suggests the existence of degree of variation in terms of species sensitivity to BPA. Considering aforementioned, the current study was designed with an intention to elucidate the biological effects of variable BPA exposure levels in male Japanese quails by analyzing the sperm quality and other biochemical estimates that re ect the male reproductive functionality. This may allow us to address the research question whether quails can be used as accurate biological indicators for BPA?

Results And Discussion
The established lowest-observed-adverse-affect-level (LOAEL) or No-observed-adverse-affect-level (NOAEL) for BPA by US Environmental Protection Agency (EPA) and European Union Risk Assessment Report (EURAR) 29 is 50 mg/kg BW/d. For BPA induced reproductive and developmental toxicity, same dose has been set as NOAEL 30,31 . These reference doses were based on the rodent studies with chronic exposure to BPA 32 . Accurate dose calculation in avians based on the previously reported animal or human studies is di cult; due to absence of absolute constants pertaining to human-animal equivalent dose conversion. To study the BPA induced reproductive toxicity in male quails at lower than doses considered to be safe, the reference dose (50 mg/kg BW/d) was divided by a factor of ten and fty to obtain low (1 mg/kg BW) and higher (5 mg/kg BW) level respectively. The doses used in the current study were based on previous reports in chicken 18 and rodents 33 .
Data generated from linear regression analysis (Table 1) indicated a positive relationship of varied BPA doses with sperm abnormality, ALT and MBRT, while negative relationship with other parameters. Except, MBRT and LDH the relationship was found to be signi cantly (P < 0.01) affected. Not body weight but testicular weights strongly affected by BPA Mean body weight of quails exposed to BPA did not show any biological variation throughout the study period (Table 2). Oral administration of BPA witnessed drastic reductions in combined testicular weights testes in absolute and relative terms (Table 3) in a signi cant (P < 0.01) and dose dependent manner (r 2 = 0.41) ( Table 3) with in-signi cant change in untreated (T1) and vehicle treated groups (T2). The maximum reduction in absolute and relative weights was noted up to 34.79 and 36.61%, respectively, in the high dose BPA-treated group of animals. BPA altered testicular weights were reported in chicken 22 and rats 34,35 .
Lowered testicular weights decrease in BPA administered groups (T3 and T4) in contrast with respective controls a rms the testicular toxicity production 36 and may be due to proteomic and cellular alterations induced in seminiferous tubules and leydig cells of testes 37,38 .  Exposure to BPA altered cloacal gland index and frequency of foam production in quails From various researchers, it is understood that BPA has multifaceted targets on spermatozoa rendering it ineligible to deploy its functional duties in female reproductive tract. Recent evidence suggests that phosphorylation of fertility-related proteins critically gets interfered by this chemical concentrations 38 .
Being smaller in body size, quails are inferred to have higher metabolic activity than other domestic poultry species 39 . The reproductive system of male Japanese quail is unique among all the avian species because of the presence of androgen dependent cloacal gland 40 . In sexually active male birds, the cloacal gland is enlarged and there is high degree of correlation between gland size and testicular mass which indicates its role in predicting the fertilizing potential in this species 41 . The cloacal gland secretes meringue-like white foam constantly during copulation and defecation time 42 . Results pertaining to percent mean change in cloacal gland index and foam frequency were presented in Tables 4 and 5 respectively. Exposure to BPA reduced cloacal gland indices with high dose group witnessing a percent change of 0.59 than its control counterpart (11.24). Over a period of time, percent change deteriorated (P < 0.05) in BPA groups. The current study witnessed a signi cant (P < 0.01) variation in number of times the foam being produced by quails with respect to BPA administration. Foam frequency was typically reduced at high dose (4.85) followed by low dose (5.75) and control groups (6.65). Interaction of BPA and period of exposure revealed a signi cant effect of foam frequency. The alteration in cloacal gland area in BPA treated groups over period of time clearly explains the lowered foam discharge frequency in this study (Table 5). Our experiments on DES in quails 43 also revealed a compromised cloacal gland size.
Withstanding to the fact that androgens modulate cloacal gland, the BPA induced lowered testosterone concentration ( Fig. 1) may explain the shrinkage of cloacal gland size in the study groups.  Quantitative and qualitative aspects of quail spermatozoa in response to BPA toxicity Data relating to in uence of BPA on semen volume was presented in Table 6. Exposure to BPA resulted in lowered (P < 0.01) semen volume at low and high doses over a period of time. Quails exposed to high dose BPA for 10 wk period yielded critically lower volumes (P < 0.01). The sperm viability were observed to be signi cantly less (76.75%) in high dose BPA group compared to low dose (82.38), sham control (87. 13) and control (86.50) groups (Table 7). Lower viable sperm counts in T4 were accompanied by higher percent of abnormal spermatozoa. In concurrence, sperm motility and concentration were also affected to a great extent (P < 0.01) in high dose groups. Bisphenol-A caused signi cant (P < 0.01) reduction in sperm motility and corroborates with the previous ndings in other animal species 17,44 . Disturbances in sperm motility by BPA is either due to depletion of anti-oxidant resources or intracellular ATP exhaustion 45    Exposure to BPA did not in uence seminal enzymes except ALP Among various seminal enzymes investigated (Table 8), only Alkaline phosphatase (KA units/ml) has showed a reduction (P < 0.01) in low (1.13) and high dose (0.93) BPA in comparison to control (3.23) and sham control (4.00) groups. However, the present study witnessed no alteration with respect to ACP, AST and LDH enzymes. Alkaline phosphatase is a cell surface zinc metallo-enzyme believed to be involved in epithelial cell proliferation in sperm storage tubules in poultry 52 by catalyzing the disassociation of phosphate groups from target substrates 53 . Mammalian studies indicated a tendency of correlation between ALP and fertility 54 . As far as LDH is concerned, a sharp drop was observed in seminal plasma in high dose treated group which could be well correlated with the reduced motility. As the LDH catalyzes the conversion of lactate to pyruvate and later deals with the energetics of sperm cells, its leakage might have critically disturbed the sperm motility and function 55 . However, it is advocated that, exposure of mouse spermatozoa to 100 µm concentration critically compromised in vitro fertilization rates 48 . Fertility is the obvious re ection of sperm motility, therefore deteriorated sperm quality characterized by higher number of dead and abnormal sperm counts in BPA treated groups explains the depression in fertility rates in this study. Reduction in testosterone and cloacal gland size clearly con rms the anti-androgenic actions of BPA 61 which in turn affects the fertility and hatchability in quails. Hatchability results (Fig. 2) on adverse effects of BPA on total egg set basis warrants further investigation. Hatchability based on FES did not vary in response to BPA toxicity and hatchability on TES (r 2 = 0.82). Interestingly, DES, an endocrine disruptor exposure also compromised hatchability of quail chicks 54 . Based on results of the current study, we con rm the compromising actions of BPA on reproductive success in Japanese quails at environmentally relevant concentrations under in vivo exposure module. It is likely that BPA mediated action is via distortions in sperm production and subsequently the fertility. The oscillating changes in foam frequency and cloacal gland size also indicate its higher sensitivity to BPA toxicity. We suggest progressive studies integrating genomic and proteomic analysis to elucidate the underlying mechanisms responsible for alteration of physiological and reproductive activities in quails.

Materials And Methods
All experimental protocols employed in this study were in accordance with the rules and guidelines framed

Experimental animals
Male and female Japanese quails of CARI-Uttam variety was utilized as experimental subjects. Quails were sex differentiated by cloacal gland method and phenotypic thoracic feather pattern done at 8 wk of age. All the birds were maintained in individual cages (4'×2.5'×1.5') with a space allowance of 175 sq.cm/bird, 16 h photoperiod, feed and water ad libitum. Sixty healthy adult quails were randomly allocated into four experimental treatment groups as control (T1), sham control (T2), low dose BPA (T3), and high dose BPA (T4) for a period of three wk i.e., 8-10 wk of age. All the male and female quails were fed with iso-nitrogenous and iso-caloric diets of laying quail ration formulated according to Bureau of Indian Standards, 1997 at institute feed processing unit. The ingredient composition, nutrient composition and protein fractions of the quail ration used in the experiment were presented in Table 9. T2, T3 and T4 were administered with saline buffer alone, DMSO alone, BPA (mg/kg BW/d) 1 and 5 respectively for the duration of 3 wk. Oral gavage was administered to the birds with a tuberculin syringe during 08:00-9:00 daily prior to feeding.
Body weight, testicular weights cloacal gland index and foam frequency measurements Body weight was recorded on weekly basis by weighing individual birds. Cloacal gland index (CGI) was measured as described previously 26 . Brie y, the dorso ventral and lateral aspects of cloacal gland were measured using Vernier calipers. The product of height (dorso-ventral aspect) and width (lateral aspect) of cloaca in mm 2 was used as an index for measuring area of cloaca gland. The frequency of foam discharged from each bird was recorded between 0900 to 1500 h of the day during the course of treatment. Foam observations were recorded starting from 2nd wk of experiment i.e., 9 wk at 1, 3, 5, 7, 9, 11, 13 and 15 d. At the end of experiment, gross and normalized testicular weights were obtained by sacri cing six males from each treatment group.
Collection, quantitative and qualitative analysis of quail spermatozoa All the semen analysis was done from the semen samples collected at the end of each wk during experiment i.e., 8, 9 and 10 wk. Neat semen was collected from each quail by gentle and frequent abdominal massages to avoid the contamination from frothy meringue like foam produced by cloacal gland. Foam contents were removed from cloacal gland prior to neat semen collection. The fresh semen, ejaculated from each bird, was measured by using graduated glass pipette of 0.1 mL and 0.001 mL accuracy. After recording the volume, semen samples from each group were mixed for further analysis. Qualitative estimates like per cent mass motility, livability, sperm concentration, and methylene blue reduction test (MBRT) were analyzed as described previously 43

DISCLAIMER
All authors declare that there exist no commercial or nancial relationships that could, in any way, lead to a potential con ict of interest.