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.

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-ß-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-ß, thyroid-α and -ß 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-ß-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-ß and thyroid receptor-ß mRNA was up regulated in the infundibulum of internal layers compared with normal laying hens at 26 weeks of age.

Expression of leptin and its receptor in corpus luteum during estrous cycle in buffalo (Bubalus bubalis).

Leptin is supposed to play a crucial role in ovarian luteal dynamics. The present study was aimed to investigate the importance of leptin and its receptors in buffalo corpus luteum (CL) obtained from different stages of the estrous cycle. Real-time RT-PCR (qPCR), western blot and immunohistochemistry techniques were applied to investigate mRNA expression, protein expression and localization of examined factors. Additionally to assess the contribution of leptin in progesterone production the expression profiles of StAR, P450scc and HSD were also investigated. In general, we demonstrated presence of leptin and its receptors in buffalo CL during the estrous cycle. The mRNA levels of leptin and its receptors were significantly up regulated in (P<0.05) in all the stages and highest levels were observed in mid and late luteal stages consistent with in vivo luteinization of buffalo CL and declined coincidental to luteal regression. The expression of StAR, P450scc and HSD factors maintained low in early luteal phase, after that level of expression increased steadily to show a significant rise (P<0.05) in mid luteal phase followed by gradual decline in late luteal phase and regressed CL and this correlates well with the Ob and ObR receptor activity, verifying their key role in progesterone and other steroids production in functional CL. As revealed by immunohistochemistry, leptin protein was localized predominantly in large luteal cells however leptin receptor (Ob-R) was localized in large luteal cells as well as in endothelial cells. It can be concluded from our study that leptin via its autocrine/paracrine effects play a significant role in promoting angiogenesis, steroidogenesis and also acts as key survival factor in bubaline CL.

Cytokines and chemokines in postovulatory follicle regression of domestic chicken (Gallus gallus domesticus).

The mechanism of postovulatory follicle (POF) regression in birds is still poorly understood. In the current study, expression of IL-1beta, IL-6, GM-CSF, IFN-gamma, IL-2, IL-4, IL-13, chCXCLi2, chCCLi2, chCCLi4, chCCLi7, IL-10 and TGF-beta2 mRNAs was estimated in regressing POF by semi-quantitative RT-PCR. In addition, the changes in immune cell population, histological and apoptotic changes were also studied in regressing POF. The expression of cytokines (IL-1beta, IL-6, IL-10 and TGF-beta2) and chemokines (chCXCLi2, chCCLi2, chCCLi4 and chCCLi7) was upregulated in POFs, suggesting a role for these molecules in tissue regression. The histological findings suggested a significant infiltration of immune cells, especially heterophils, lymphocytes and macrophages, into the regressing POF. The flow cytometry analysis of lymphocyte subpopulations revealed that CD3(+), CD4(+), CD8(+) and Bu-1(+) lymphocytes were significantly increased during this regression. The significant up-regulation of chemokines might have attracted the immune cells during POF regression. The percentage of apoptotic cells was significantly increased during the regression of POF. The up-regulation of IL-1beta, IL-6, IL-10 and TGF-beta2 and down-regulation of GM-CSF might have induced apoptosis during the POF regression. However, expression of IFN-gamma, IL-2, IL-4 and IL-13 was not significantly altered during POF regression. In conclusion, cytokines appear to play an important role in the regression of POF in chicken. Furthermore, the regression of chicken POF seems to be an inflammatory event similar to luteolysis of the mammalian corpus luteum.

Reproductive tissue regression: involvement of caspases, inducible nitric oxide synthase and nitric oxide during moulting in White Leghorn hens.

Moulting is a natural physiological process where the reproductive system of birds undergoes complete remodeling in preparation for the next laying cycle. In domestic chickens, moulting is artificially induced by feed withdrawal to recycle the old laying flock for best profit margins. This has received severe criticism from animal welfare organizations, forcing several countries to stop this practice. Several alternative methods to feed withdrawal methods were developed but were found to produce inconsistent results. Understanding the actual mechanism of moulting would help in designing a new animal welfare friendly method. The present investigation attempted to study the molecular mechanism of moulting in White Leghorn hens. Eighty-four layers (75 weeks) were divided into two groups. The birds in the first group were subjected to moulting by feed withdrawal (FW) while the other group received high dietary Zn (ZnF) treatment for 10 days. Six birds from each group were sacrificed on 0, 1-4, 6 and 10 days of moulting and mRNA expression of caspases-1, -2 and iNOS, along with the apoptotic ladder pattern and nitric oxide (NO) in the ovary and oviduct, was investigated. The mRNA expression of iNOS was upregulated with a corresponding increase in NO levels. Caspases-1 and -2 were differentially upregulated in the ovary and oviduct of moulted birds. A constant decline in serum estradiol and progesterone levels was also observed. It can be concluded that the pattern of reproductive regression during moulting by the two methods is different, as the expression of genes studied in the present investigation is different.

Differential expression of lipopolysaccharide-induced TNF-alpha factor (LITAF) in reproductive tissues during induced molting of white leghorn hens.

The reproductive remodeling during molting appears to be a complex physiological mechanism regulated by multiple host factors. Lipopolysaccharide (LPS) induced TNF-alpha factor (LITAF) is one of the transcription factors controlling the expression of TNF-alpha and other cytokines. In the present investigation, we studied the involvement of LITAF in the regression of reproductive tissues of molting birds. Semi-quantitative RT-PCR analysis revealed that LITAF mRNA was generally expressed in both ovary and oviduct. In the molting birds, i.e. those subjected to feed withdrawal (FW) or fed high levels of zinc (ZnF) birds, the LITAF expression was upregulated significantly in the ovary after 4 days of molting (DOM). However, LITAF mRNA levels were three-fold higher in ZnF birds, which might be responsible for a greater degree of follicular atresia. In the oviduct of FW birds, peak LITAF expression was noticed on 4DOM and the levels remained significantly higher until the end of the experiment. In ZnF birds, LITAF expression reached its peak on 1DOM and subsequently downregulated to basal levels on 2DOM. This indicated that constantly higher LITAF expression might be required for complete regression of the oviduct during molting. In conclusion, LITAF might be one of the major transcription factors controlling reproductive regression in chicken, as the expression levels were associated with the regression pattern.

Cytokines in reproductive remodeling of molting White Leghorn hens.

The role of cytokines in regression of the ovary and oviduct during induced molting in chickens was investigated by evaluating the expressions of IL-1beta, IL-6, IFN-gamma, IL-2, TGF-beta2, MIP-1beta and IL-8 in the regressing ovary and oviduct by semi-quantitative RT-PCR. In addition, serum hormonal profiles (estrogen, progesterone and corticosterone), along with the gross regression and histological changes of the ovary and oviduct, were investigated. The correlation between expression of cytokines and hormonal changes during the induced molting was also studied. The expression of IL-6, IL-8, MIP-1beta and IFN-gamma mRNAs in the ovary, and IL-1beta, IL-6, IL-8, MIP-1beta, IFN-gamma and TGF-beta2 mRNAs in the oviduct, were up-regulated significantly during induced molting, suggesting their role in tissue regression. However, histological findings suggested no significant increase in immune cells in the regressing oviduct and ovary. Significant up-regulation of TGF-beta2 in the regressing oviduct might have suppressed leukocyte recruitment thereby preventing the inflammatory response and tissue damage. The down-regulation of estrogen and progesterone and up-regulation of corticosterone is well correlated with increased expression of cytokines. It appears that cytokines released during the process of induced molting may have a role in decreasing ovarian steroids and increasing the corticosterone levels in chicken. From this study, it may be concluded that cytokines play a major role in regression of the ovary and oviduct during induced molting in chickens.

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy.

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.