Ram Pratap Singh

Development of a new method for sperm RNA purification in the chicken

Currently RNA transcripts are being used as male fertility biomarker for many mammalian species, but research work on chicken is at halt because classical RNA isolation methods are not effective for chicken spermatozoa. Hence, attempts have been made to optimize RNA isolation protocol from chicken sperm by using different methods, and to confirm the presence of sperm-specific transcripts of PRM and PLCZ1. Semen samples were centrifuged at low speed for removing debris like uric acid. Further, 1mL diluted semen was gently placed over 40% PureSperm or 45%/90% Percoll, and centrifuged to remove somatic cells and immature diploid spermatocytes. RNA was isolated from sperm by using RNAzol or TRIzol reagent or RNeasy Micro kit with certain modification, and RNA quantity and quality were evaluated. RNA isolated by using RNAzol or RNeasy Micro Kit yielded good quantity and quality of RNA for downstream applications compared to TRIzol. 40% PureSperm was found effective in removing somatic cells. RT-PCR results showed that sperm RNA samples were negative for CD4 and PTPRC. All the sperm RNA samples were positive for PRM and PLCZ1, markers of sperm RNA.

Bisphenol A reduces fertilizing ability and motility by compromising mitochondrial function of sperm

Bisphenol A (BPA) acts as an endocrine disruptor, affects animal reproductive success in vivo and affects sperm functions in vitro at environmentally relevant concentrations, leading to reduction in sperm motility and fertilizing ability in fish. The effect of in vitro BPA on avian sperm functions has not been explored. The present study examined the effect of environmentally relevant concentrations of BPA (0 mM, 0.18 mM, 0.37 mM, and 0.74 mM) on sperm functions in chicken in vitro. Sperm were exposed to concentrations of BPA for 30 min and analyzed for motility, fertilizing ability, live sperm percentage, and mitochondrial membrane potential (Δψm). Results showed that BPA at a concentration of 0.74 mM significantly decreased motility, fertilizing ability, live sperm count percentage, and sperm Δψm. Sperm motility was positively correlated with fertility (r = 0.73, p ≤ 0.01), live sperm percentage (r = 0.64, p ≤ 0.01), and high Δψm (r = 0.44, p ≤ 0.01). A dose-dependent and time-dependent effect of BPA was observed on sperm motility at all BPA concentrations. However, sperms fertilizing ability was unaffected in low BPA concentration (0.18 mM and 0.37 mM). A significantly higher percentage of moribund sperm was observed at 0.37 mM and 0.74 mM BPA compared with at 0.18 mM BPA, in the negative control, and in the vehicle control. The present study confirms that environmentally relevant concentrations of BPA are capable of compromising sperm functions, leading to reduction in fertilizing ability of chicken sperm.

Norfloxacin drug induces reproductive toxicity and alters androgen receptor gene expression in testes and cloacal gland of male Japanese quail (Coturnix Japonica)

In an attempt to investigate the reproductive toxicity of norfloxacin in Japanese quail, male quail were given norfloxacin at 20 mg/kg body weight for 14 d. Then reproductive function and androgen receptor (AR) gene expression was examined in treated and control birds. The results of the present study indicate that fertility, cloacal gland area, sperm concentration, and serum testosterone were reduced significantly (p < 0.05) on day 14 in the norfloxacin-treated birds. Upregulation (p < 0.05) of AR mRNA was also seen in the testes on the 14th d of treatment. A trend toward downregulation of AR mRNA was seen in the cloacal gland of norfloxacin-treated birds. Histological observations revealed that norfloxacin induces cellular atrophy in testes and changes in glandular tissue in the cloacal gland. The results of the present study demonstrate that norfloxacin induces testicular toxicity in Japanese quail.

Ovarian morphology and internal vis-à-vis non internal laying in relation to triacylglycerol, hormones and their receptors concentration around the age of sexual maturity in broiler breeder hens

1. Ovarian morphology, serum hormone concentrations of 17-β-estradiol, progesterone, testosterone, tri-iodothyronine (T3), thyroxine (T4) and triacylglycerol (TAG) were investigated at 23 and 26 weeks of age in broiler breeder hens provided with ad libitum access to feed. Progesterone, oestrogen-β, thyroid-α and -β receptor mRNAs were also quantified in the infundibulum at the same ages. 2. A large variation in the ovarian morphology was observed at 23 weeks of age including hens with undeveloped ovaries, non-laying hens with post ovulatory follicles (POF) and a predominance of non-laying hens without a POF. 3. Serum concentrations of triglyceride, 17-β-estradiol and progesterone at 23 weeks of age were lower in hens with an undeveloped ovary compared with other groups of hens, whereas testosterone, triiodothyronine and thyroxin were higher. 4. At 26 weeks of age, the average number of hierarchical yellow follicles in normal layers was 7.64 ± 0·41 whereas in internal layers, the follicular numbers were significantly greater at 8.66 ± 0·53. The higher follicular numbers in internal layers were associated with higher serum triglyceride and progesterone concentrations. 5. Oestrogen receptor-β and thyroid receptor-β mRNA was up regulated in the infundibulum of internal layers compared with normal laying hens at 26 weeks of age.

Sexual maturation, serum steroid concentrations, and mRNA expression of IGF-1, luteinizing and progesterone hormone receptors and survivin gene in Japanese quail hens

In avian species, sexual maturation represents the evidence of start laying, which is a consequence of the development of ovarian follicles. These follicles are the functional reproductive unit whose maturation and viability critically depends on endocrine, paracrine, and autocrine factors beyond the signals from the central nervous system. The present study was undertaken to investigate the correlation of sexual maturity with tissue growth, mRNA expression of certain genes, and serum steroid concentrations in Japanese quail hens. To carry out the present study, a total of forty Japanese quail hens (5 weeks) were housed individually under uniform husbandry condition with ad libitum quail layer ration and water at 14-hour photo schedule. On sixth week onwards, four birds were sacrificed at each time on 1, 3, 7, 10, 13, 16, 19, 22, 25, and 28 days. Serum was extracted aseptically to analyze the gonadal steroid hormones (estrogen and progesterone) and corticosterone to investigate the liaison with sexual maturation of the species. Expression analyses of four genes i.e., insulin-like growth factor-1, luteinizing hormone receptor, progesterone receptor, and survivin were carried out in the three largest ovarian yellow follicles. A significant (P < 0.05) increase in body weight gain and oviduct weight was recorded during the phase of sexual maturation. Smaller follicles revealed higher insulin-like growth factor-1 and survivin gene expression, whereas the reverse result was manifested in both the luteinizing and progesterone hormone receptors. In biochemical study, the gonadal steroids (estrogen and progesterone) were recorded higher at the first half of the experiment when a gradual decrease in corticosterone concentration was confirmed from the very beginning of this study. This result substantiated that sexual maturation in Japanese quail may be completed by the time of 8 weeks after its birth in support of the analyzed information studied in the current investigation.

Effects of bisphenol-A on male reproductive success in adult Kadaknath chicken

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 1mg/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.

Insulin promotes preantral follicle growth and antrum formation through temporal expression of genes regulating steroidogenesis and water transport in the cat

The aims of the present study were to determine the effects of insulin, invitro, on: (1) the viability and growth of domestic cat ovarian follicles; (2) mRNA expression of genes regulating steroidogenesis (cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), cytochrome P450 family 19 subfamily, A polypeptide 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star)) and water transport (aquaporins (AQPs) Aqp1, Aqp3, Aqp7, Aqp9); and (3) steroid production (17β-oestradiol (E2), progesterone (P4), androstenedione (A4)). Cat secondary follicles were isolated from ovarian cortices and cultured in 0 (Control), 1 or 10µgmL-1 insulin for 14 days (Day 0=culture onset). Follicle and oocyte viability (based on neutral red staining), diameter and antrum formation were assessed every 72h and at the end of incubation (Day 14). Expression of steroidogenic and water transport genes was evaluated on Days 0, 6 and 12, and E2, P4 and A4 concentrations in the culture medium were determined on Day 12. By Day 14, 1 and 10µgmL-1 insulin had significantly promoted (P<0.05) both antrum formation in a mean (±s.e.m.) 26.9±9.0% and 78.0±10.0% of follicles respectively, and follicle growth (diameter 151.4±4.5 and 169.9±10.5µm respectively) compared with Control (antrum formation in 3.3±3.3% of follicles and follicle diameter 129.1±6.6µm). High insulin (10µgmL-1) treatment increased follicle viability compared with Control (86.0±9.8% vs 38.1±10.9% respectively; P<0.05). However, insulin had no beneficial effect (P>0.05) on oocyte diameter. Cyp17a1 expression on Days 6 and 12 was higher (P<0.05) in follicles cultured in the low (1µgmL-1) compared with high (10µgmL-1) insulin treatment, with no significant difference between low or high insulin vs Control groups. Star expression was higher (P<0.01) in the low insulin compared with Control group on Day 6, but Star was undetectable in the high insulin group by Day 12. Compared with high insulin, low insulin increased (P<0.05) Aqp1 expression on Day 6, but there were no significant differences between these two groups on Day 12. In contrast, high insulin decreased (P<0.05) Aqp9 transcript levels compared with Control. Only P4 production was affected by insulin, with P4 concentrations in the medium being higher (P<0.05) in the low compared with high insulin and Control groups. In summary, the findings indicate that insulin promotes cat ovarian follicle growth and survival invitro, including enhanced antrum formation, with the likely mechanism involving temporal expression of Cyp17a1, Star and Aqp9 genes.

Expression of gonadotropin and sex steroid hormone receptor mRNA in the utero-vaginal junction containing sperm storage tubules of oviduct during sexual maturation in Japanese quail

Sex steroid hormones play an important role in reproductive tissue development of avian species. However, their role in Japanese quail is yet to be established. To understand the physiological role of hormones involved in the development of sperm storage tubules (SSTs) in quail, we investigated expression profiles of gonadotropin (LH-R and FSH-R) and sex steroid hormone (PR-R, ER-α and ER-β) receptors in the uterovaginal junction (UVJ) containing SSTs before and during sexual maturation i.e. four to eight weeks. Every week four birds were sacrificed to collect blood and UVJ for sex steroid hormone (progesterone and estrogen) estimation and gene expression profiling of sex steroid hormone (PR-R, ER-α and ER-β) and gonadotropin receptors (LH-R and FSH-R) using qRT-PCR. Receptor expression results showed that the expression of sex steroid receptor (PR-R, ER-α and ER-β) genes were upregulated significantly (P < .05) in SSTs with the advancement of age. The expression of gonadotropin receptors (LH-R and FSH-R) was only high at week 5 and 6 respectively. Serum hormone analysis indicated a significant (P < .05) rise in estradiol till 7th week and progesterone from 7th week onwards. These results suggest that the gonadotropin and sex steroid hormone receptors may have the role in the development and maintenance of UVJ that contains predominantly SSTs during sexual maturation.