All experimental protocols employed in this study were in accordance with the rules and guidelines framed and communicated by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), a statutory committee, which is established under Chap. 4, Sect. 15(1) of the Prevention of Cruelty to Animals Act 1960, India and approved by the Institute Animal Ethics Committee of the ICAR-Central Avian Research Institute (452/01/ab/CPCSEA). The experimental procedures carried out in the study are in compliance with the ARRIVE guidelines.
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.
Table 9
Ingredient and nutrient composition of quail breeder diets used in the study
Ingredient composition
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|
Ingredient composition
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Ingredients (kg/100 kg)
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Starter (%)
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|
Maize
|
55
|
Crude protein (%)
|
24.05
|
Soybean meal
|
35
|
ME (Kcal. ME/kg)3
|
2808.40
|
De-oiled rice bran
|
3.30
|
Calcium (%)
|
1.02
|
Fish meal
|
4
|
Available Phosphorus (%)
|
0.48
|
Lime stone
|
0.75
|
Methionine (%)
|
0.48
|
Di calcium phosphate
|
1.40
|
Lysine (%)
|
1.27
|
Salt
|
0.15
|
|
|
DL-Methionine
|
0.06
|
|
|
Trace mineral premix1
|
0.10
|
|
|
Vitamin premix2
|
0.15
|
|
|
Vitamin B complex
|
0.02
|
|
|
Choline chloride
|
0.03
|
|
|
Toxin binder
|
0.05
|
|
|
Total
|
100
|
|
|
1Trace mineral premix (mg/kg diet): Mg- 300, Mn- 65, I- 1.2, Fe- 80, Zn- 40 and Cu-7.5. 2Vitamin premix (mg/kg diet)-Choline chloride- 500; Niacin: 12; Pyridoxine hydrochloride- 1.6; Vitamin K-1 mg, Vitamin E-10,3 ME: Metabolic energy, Calculated. |
Preparation and administration of Bisphenol-A
Required BPA (> 99% Sigma Aldrich, USA) stock solutions were prepared by dissolving in Dimethyl sulfoxide (DMSO) (Sigma Aldrich) to a final volume of 0.25 mL per bird. In order to assess the BPA effect on male reproductive success, male quails were administered with BPA aqueous solution in form of oral gavage as 1 mg and 5 mg per kg BW to low and high dose treatment groups respectively. Sham control group with DMSO alone is maintained to nullify its interference in the experiment. Treatment groups T1, 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 previously26. Briefly, 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 mm2 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 sacrificing 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 previously43. The neat semen pH was determined by pH-meter, (320213, Cyberscan. 2100) fitted with a microelectrode.
Enzymatic estimation
For measurement of enzymes in semen, semen samples that were collected during the experimental period were subjected to centrifugation at 4500 rpm at 15 min/4 °C and separated seminal plasma samples were collected in a new tube and stored at -80 °C till further analysis. Metabolically important seminal enzymes viz. acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in seminal plasma were estimated by using standard commercial kits (Span Diagnostics, Gujarat, India) as per manufacturer’s instructions. Test samples were accompanied by standard, negative and blank controls in all enzymatic tests. Absorbance readings for ACP, ALP, AST an LDH were recorded at 510, 505 and 340 nm respectively.
Blood sampling and hormonal analysis
For hormone measurement, whole blood samples were collected from jugular veins of quails and were subjected to centrifugation at 4,500 rpm/15 min. separated serum samples were transferred to new tubes and stored at -80 °C until analysis. Serum testosterone concentrations were estimated on weekly basis using solid phase enzyme-linked immunosorbent assay (ELISA) kit from Demeditec Diagnostics, Germany (DE1559) as per the manufacturer’s instructions at 450 ± 10 nm wavelength in a microplate reader (Spectramax190, Molecular Devices, USA). Briefly, test samples were added to antibody coated plates and conjugate was added to each well with an incubation of 37 °C for 1hr and detailed washing. Plates were washed again after the addition of TMB substrate and the entire reaction was halted with the addition of stop solution. ELISA data was analyzed by plotting the mean absorbance values on the enclosed logit-log paper. Sample values were interpolated as percentage on the standard curve to obtain corresponding values final mean concentration in ng/mL. Inter and intra- assay coefficients of variation were found to be within 10 and 15 percent respectively.
Fertility and hatchability studies
For determination of fertility, paired mating was employed by assigning female of same hatch to Bisphenol-A treated males in 1:1 ratio for a period of 2 wk and fertile eggs were collected for incubation. Percent fertility was determined by candling on 18th d of incubation. Hatchability on total egg set (TES) and fertile egg set (FES) basis was recorded in percentage as per the standard formulae. Unhatched eggs were subjected to break open studies to ascertain the quail embryonic mortality pattern.
Statistical analysis
Percent values (Relative weight of testes, percent change in cloacal gland index, sperm motility, liveability, abnormality, fertility, hatchability on TES and FES) were subjected to arc sin transformation prior to analysis. All the generated experimental data were initially subjected to linear regression analysis (Graph pad prism version 7, San Diego, USA) to assess the significance of relationship between the experimental doses of BPA used in the study and various study parameters. Significance of differences among treatment means was calculated with tukey’s post hoc test.
One way analysis of variance (ANOVA) was employed for rest of the observations (Relative testes weight, pH, MBRT, concentration, motility, liveability, abnormality, fertility and hatchability on TES, FES) to assess the significant differences among groups. While for those observations involved different time points viz. body weight, semen volume, serum testosterone, foam frequency and percent change in cloacal gland size; General linear model was adopted with treatment and duration as fixed effects. Statistical analysis was performed in SPSS version 20 (SPSS IBM, New York, U.S.A) and values were expressed as Means ± SE. Values were considered at probability of 5 and 1% level as significant and highly significant respectively.