Chicken sperm transcriptome profiling by microarray analysis.

It has been confirmed that mammalian sperm contain thousands of functional RNAs, and some of them have vital roles in fertilization and early embryonic development. Therefore, we attempted to characterize transcriptome of the sperm of fertile chickens using microarray analysis. Spermatozoal RNA was pooled from 10 fertile males and used for RNA preparation. Prior to performing the microarray, RNA quality was assessed using a bioanalyzer, and gDNA and somatic cell RNA contamination was assessed by CD4 and PTPRC gene amplification. The chicken sperm transcriptome was cross-examined by analysing sperm and testes RNA on a 4 × 44K chicken array, and results were verified by RT-PCR. Microarray analysis identified 21,639 predominantly nuclear-encoded transcripts in chicken sperm. The majority (66.55%) of the sperm transcripts were shared with the testes, while surprisingly, 33.45% transcripts were detected (raw signal intensity greater than 50) only in the sperm and not in the testes. The greatest proportion of up-regulated transcripts were responsible for signal transduction (63.20%) followed by embryonic development (56.76%) and cell structure (56.25%). Of the 20 most abundant transcripts, 18 remain uncharacterized, whereas the least abundant genes were mostly associated with the ribosome. These findings lay a foundation for more detailed investigations on sperm RNAs in chickens to identify sperm-based biomarkers for fertility.

Expression profile of luteinizing hormone receptor gene in hierarchal follicles and regressing oviduct tissues of White Leghorn hens during moulting.

In the present study, the expression profile of luteinizing hormone receptor (LHR) was investigated in the ovary, magnum and uterus and in hierarchcal follicles (F-1, F-2, F-3 and F-4) of hens subjected to moulting to establish their involvement in moulting and presence in non-gonadal tissues. Fifty-two layers (72 weeks) were subjected to moult for a period of 14 days. Four birds were sacrificed each time on 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 and 14 days of moulting, and samples (ovary, magnum, uterus and hierarchal follicles) were collected aseptically for the quantitative study by real-time polymerase chain reaction. The ovary, isthmus, uterus and magnum weight reduced significantly during induced moulting. From the 4 DOM, this reduction was drastic and reached approximately 80% of original weight in the case of ovary, isthmus and magnum and approximately 65% of original weight in the case of uterus on 14 DOM. Ovarian yellow follicles decreased gradually from 1 DOM to 4 DOM, after that no normal yellow follicle was observed in moulted bird. The number of atretic follicles increased gradually during the course of induced moulting, reaching the peak at 5 DOM. The LHR mRNA was detected in non-gonadal tissues like magnum and uterus. The LHR expression was significantly (p < 0.05) down regulated in ovary, magnum and uterus throughout the treatment. These results indicated that LHR may have a role in reproductive tissue regression during moulting.

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.

In vitro initiation of the acrosome reaction in the emu (Dromaius novaehollandiae).

1. An assessment of the efficiency of the acrosome reaction (AR) provides an important predictor of the fertilizing potential of semen and for diagnosis of the causes of infertility. A standardized protocol was therefore developed for initiation of the acrosome reaction in emu spermatozoa in vitro, and the role of CaCl2 or perivitelline membrane (PVM) proteins in determining the outcome of the reaction was investigated. 2. The acrosome reaction (assessed by FITC-PNA) was successfully induced in live spermatozoa by incubation for 2 min in NaCl-TES medium supplemented with 5 mM CaCl2. The maximum response was 32% live acrosome-reacted spermatozoa (LAR) achieved after 10 min incubation. 3. Compared to the outcome with 5 mM CaCl2 or PVM protein alone, the response was significantly better with a combination of PVM protein and CaCl2. 4. A significant variation in the percentage of LAR spermatozoa among individual males was observed. No treatment affected the percentage of dead acrosome-reacted spermatozoa. 5. The results emphasize the important role played by both PVM proteins and Ca(2+) in the in vitro initiation of the acrosome reaction.

The role of the male cloacal gland in reproductive success in Japanese quail (Coturnix japonica).

The adult male Japanese quail has a well developed cloacal gland that produces meringue-like white foam. The physiological significance of the cloacal gland and its foam is still unclear. Therefore, we conducted two experiments to establish the functional role of the cloacal gland and its foam in natural mating and oviducal sperm transport. In the first experiment, artificial insemination of equal numbers of spermatozoa diluted in foam extract and normal saline once in a week were used to determine the role of foam in sperm transport in the female oviduct. After artificial insemination, eggs were collected to measure fertility, the duration of the fertile period, sperm holes and attached spermatozoa in the perivitelline membrane. Higher (P<0.05) fertility and greater duration of the fertile period were observed when semen was inseminated along with foam extract compared with normal saline. Further, the sperm holes and trapped spermatozoa in the perivitelline membrane were also higher (P<0.05) in the presence of foam extract. In the second experiment, two males with bigger and smaller cloacal gland areas were allowed to mate with a female. The mating attempts of males with larger cloacal gland were more successful (P<0.05) than males with smaller cloacal glands. Our results indicated that cloacal foam improves sperm transport in the female oviduct and that males with larger cloacal gland areas are preferred during mating.

Effect of Dietary L-ascorbic Acid (L-AA) on Production Performance, Egg Quality Traits and Fertility in Japanese Quail (Coturnix japonica) at Low Ambient Temperature.

Environmental stress boosts the levels of stress hormones and accelerates energy expenditure which subsequently imbalance the body's homeostasis. L-ascorbic acid (L-AA) has been recognized to mitigate the negative impact of environmental stress on production performances in birds. The present investigation was carried out to elucidate the effect of different dietary levels of L-AA on production performance, egg quality traits and fertility in Japanese quail at low ambient temperature. Sixty matured females (15 wks) were equally divided into three groups (20/group) based on the different dietary levels of L-AA (0, 250 and 500 ppm) and coupled with an equal number of males (1:1) obtained from the same hatch. They were managed in uniform husbandry conditions without restriction of feed and water at 14 h photo-schedule. Except for feed efficiency, body weight change, feed consumption and hen-day egg production were recorded highest in 500 ppm L-AA supplemented groups. Among the all egg quality traits studied, only specific gravity, shell weight and thickness differed significantly (p<0.05) in the present study. Fertility was improved significantly (p<0.01) to a dose dependent manner of L-AA. The findings of the present study concluded that dietary L-AA can be a caring management practice at least in part to alleviate the adverse effect of cold induced stress on production performance in Japanese quail.

Influence of chicken native breeds on some physical and biochemical characteristics and short-term storage of semen.

1. The major objective of this study was to examine the influence of 24-h storage of semen at low temperature on semen characteristics and fertilising ability of spermatozoa in two native breeds (Kadaknath-KN, Aseel Peela-AP) and White Leghorn (WL) chicken. 2. Various physical and biochemical properties of freshly ejaculated semen of KN and AP were investigated. Fertility was examined in freshly-ejaculated as well as 24-h-stored (3°C) semen diluted (1:3) with Beltsville Poultry Semen Extender. 3. No significant difference was observed in sperm motility among the different breeds whereas live counts were higher in WL than the native breeds. Body weight, semen volume and sperm concentration were highest in AP, followed by KN and WL. A similar trend was observed in the percentage of dead and morphologically-abnormal spermatozoa. 4. The activity of acid and alkaline phosphatase in seminal plasma were higher in WL than KN, whereas the opposite trend was recorded for glutamic oxaloacetic and pyruvic transaminases. The cholesterol content of semen was highest in AP, followed by KN and WL. Cholesterol was much lower in seminal plasma compared with whole semen but there were no differences between breeds. Mean values of the methylene blue reduction time test were higher in WL than in the native breeds. 5. Fertility and hatchability, using freshly-diluted semen, were poorer in the native breeds than in WL. The pattern of fertility deteriorated further, especially in native fowls, when the birds were inseminated with 24-h-stored semen. 6. In conclusion, variation in physical and biochemical characteristics of semen in native breeds compared to WL correlated with poor fertility after short-term storage of semen.

Effect of higher dietary vitamin E concentrations on physical and biochemical characteristics of semen in Kadaknath cockerels.

1. This experiment was to investigate the effects of increasing dietary vitamin E on physical and biochemical characteristics of semen in Indian reared Kadaknath (KN) cockerels. DL-alpha-Tocopherol acetate was used as the source of vitamin E. 2. A total of 135 one-day-old male KN chicks were randomly selected and divided into 9 groups with 15 chicks in each group (3 dietary treatments x 3 replicates). 3. The basal diet contained 15 IU (10 mg) vitamin E/kg and the two experimental diets were supplemented with 150 IU (100 mg) and 300 IU (200 mg) vitamin E/kg (diets T(2) and T(3), respectively). 4. Physical characteristics in terms of semen volume, sperm concentration, sperm motility and percentage live sperm did not differ significantly, whereas proportion of abnormal and dead spermatozoa were significantly lower and fertility higher in the T(2) group. 5. Biochemical characteristics in term of quantities of protein and nitric oxide (NO) did not differ significantly, whereas the quantity of glucose, acid phosphatase (ACP) and vitamin E were significantly higher in the T(2) group. 6. In contrast, the quantities of alkaline phosphatase (ALP), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were significantly lower in T(2) group and higher in the T(1) (control) group. 7. From this study it can be concluded that moderate supplementation of dietary vitamin E may be beneficial for physical and biochemical characteristics of semen in Indian reared KN cock.

Expression analysis of melatonin receptor subtypes in the ovary of domestic chicken.

Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, regulates various biological functions through three different receptor subtypes (Mel-1a, Mel-1b, and Mel-1c). However, the distribution of different melatonin receptor subtypes in chicken reproductive tissues was not known. In the present investigation, the partial sequences of ovarian melatonin receptor subtypes (Mel-1a, Mel-1b, and Mel-1c) were characterized. Further, the expression profile of melatonin receptor subtypes in the granulosa and theca layers of different preovulatory and postovulatory follicles (POF) were studied by semi-quantitative RT-PCR. The expression of all three subtypes of melatonin receptors were observed in the ovary of domestic chicken. Analysis of partial sequences of ovarian melatonin receptors revealed that the melatonin subtypes were identical to the brain receptors. In small white ovary follicles, we observed only the expression of mel-1b receptors, but not mel-1a or mel-1c receptors. In yellow follicles, all the three subtypes of receptors expression were noticed. Interestingly, we observed the expression of mel-1a receptor only in thecal layer, but not in granulosa layer. In contrast, mel-1b and -1c receptors were expressed in both granulosa and thecal layer. During the regression of POF, we observed significant upregulation of melatonin receptors (mel-1a and 1c) expression, that downregulated in the later stages of regression. We assume that the expression of melatonin receptors might have been influenced by the atresia or apoptosis of different follicular layers in POF. Our findings suggest that the differential distribution of melatonin receptor subtypes might have distinct downstream cellular functions in the ovarian tissues.

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.

Spatial expression of chemokines and cytokines mRNA in the largest preovulatory follicle of chicken.

In the present experiment, we studied the spatial expression profiles of chemokines and cytokines mRNA in the granulosa (F1Gr) and theca (F1Th) layers of the largest preovulatory follicle in chicken using semi-quantitative PCR. The mRNAs of IL-1beta, IL-6, GM-CSF, chCXCLi2, chCCLi2, chCCLi4, chCCLi7, IFN-gamma, IL-4, IL-13, IL-10 and TGF-beta2 were expressed in the granulosa (F1Gr) and theca (F1Th) layers of the largest preovulatory follicle. However, the transcripts of IL-2 were not detected in any of the samples tested. Significantly higher levels of IL-6 and GM-CSF mRNA expression were noticed in F1Gr when compared to F1Th layer. Expression of chCXCLi2, a CXC chemokine, was almost similar in F1Gr and F1Th layers. However, the expression of CCL chemokines i.e. chCCLi2, chCCLi4, chCCLi7 mRNAs were almost 2 folds higher in F1Th layer in comparison to F1Gr layer. The expression of Th2 cytokines (IL-4 and IL-13) mRNA was noticed in F1Gr and F1Th layers with higher levels in the former. Expression of IFN-gamma mRNA was noticed in F1Gr and F1Th layers. Significantly higher level of TGF-beta2 expression was observed in F1Th in comparison to F1Gr layer. It was concluded from the present study that the mRNA expression of cytokines and chemokines are differentially regulated in the granulosa and theca layers of the largest preovulatory follicle in chicken.

Caspase-mediated apoptosis in chicken postovulatory follicle regression.

Chicken postovulatory follicle (POF) regression occurs via the process of apoptosis. However, the signals and initiator pathways responsible for regression of the POF are unknown. In the current study, we examined gene expression patterns of various caspases (caspase-1, -2 and -3) involved in apoptosis by semi-quantitative RT-PCR. The percentage of apoptotic cells during POF regression was also quantified by flow cytometry. Expression of caspase-3 mRNA was noted in the largest preovulatory follicle (F1). However, the initiator caspases (caspase-1 and -2) were not expressed in F1. During the regression of the POF, caspase-3 was activated during initial stages, whereas the initiator caspases were upregulated at the later stages (POF4 and POF5). The percentage of apoptotic cells was significantly higher during the regression of the POF. It might be possible that levels of caspase-3 mRNA do not necessarily reflect the cell's potential for facilitating apoptosis, as activation of the caspase-3 by initiator caspases is required for its function. We presume that both caspase-1 and caspase-2 were key initiators in the regression of chicken POF and that the apoptosis-mediated regression of POFs might be similar to mammalian corpus luteum involution.

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.

Relationship of cloacal gland with testes, testosterone and fertility in different lines of male Japanese quail.

An attempt has been made to investigate the relationship of cloacal gland with testes, testosterone and fertility in different lines of Japanese quail (Coturnix coturnix japonica). For this study, three lines of healthy adult male Japanese quails (<12 weeks) viz., heavy body weight (HB), white breasted (WB), and white egg shell (WES) were taken. They were housed in individual cages under uniform husbandry conditions and were provided with normal quail breeder ration and water ad libitum, with 14 h light/day. The experimental birds were selected from each of these three lines and categorized into different groups (15 birds/group) based on the increasing order of the area of cloacal gland. At the end of this experiment (24 weeks) the data indicated that size of the cloacal gland was directly proportional to foam discharge, foam weight, testicular weight, fertility and testosterone concentration in plasma. From this study it may be concluded that area of cloacal gland in Japanese quail is positively correlated with testicular weight, level of testosterone in plasma and fertility. Visual examination of cloacal gland and cloacal foam may provide a valuable non-invasive tool for predicting the fertilizing ability of individual male bird.

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.

Nitric oxide: a possible mediator of ovulation and postovulatory follicle regression in chicken.

Nitric oxide (NO) has recently emerged as a regulator of functional and structural regression in mammalian reproductive tissues. However, the role of NO in ovulation and postovulatory follicles (POF) that undergo regression in laying birds is unclear. In the present investigation, the expression profiles of iNOS mRNA, tissue NO levels and the percentage of apoptotic cells were studied in the regressing chicken postovulatory follicle (POF). The postovulatory follicles gradually lost weight during its regression and reached the lowest weight on POF5. The number of apoptotic cells was increased significantly during the regression of POF. The mRNA expression of iNOS was noticed in the second largest preovulatory follicle (F2) that subsequently increased in the largest preovulatory follicle (F1). However, the level of iNOS mRNA was declined immediately after ovulation and thereafter upregulated again to reach a peak in POF3 with a subsequent reduction in POF5 to below the basal level. The tissue NO levels followed a similar pattern except with a peak production in POF4. The gross regression and apoptosis in POFs were well associated with iNOS expression and NO production. In conclusion, NO appears to play a role in ovulation and regression of postovulatory follicle in chicken.

Effect of dietary Vitamin E on the cloacal gland, foam and semen characteristics of male Japanese quail.

This experiment was to investigate the effects of increasing the level of dietary Vitamin E (Vit. E) on cloacal gland size, foam production and semen characteristics of male Japanese quail (Coturnix coturnix Japonica). One hundred and eighty male Japanese quail chicks (day old) were randomly distributed to three dietary treatments for a period of 25 weeks. Each treatment comprised of three replicates each containing 20 chicks. The basal diet contained 15 IU Vit. E/kg and the two experimental diets were supplemented with 150 and 300 IU Vit. E/kg (diets T2 and T3, respectively). DL alpha-tocopherol acetate was used as the source of Vit. E. All chicks were provided feed and water ad libitum. Foam characteristics, in terms of frequency of foam discharge (24h), cloacal gland index and foam weight were significantly higher (P<0.05) in T2 group. Body weight, testes weight (left and right) and plasma testosterone concentrations did not differ significantly. Semen characteristics (semen volume, sperm motility, % live sperm, % hatchability and sperm concentrations) did not differ significantly (P>0.05). Percentages of abnormal and dead spermatozoa were significantly (P<0.05) lower and fertility was higher (P<0.05) in the T2 group. From this study, it can be concluded that moderate supplementation of dietary Vit. E may be beneficial for foam production, cloacal gland and improve the semen characteristics in male Japanese quail.

Role of nitric oxide in ovarian follicular development and egg production in Japanese quail (Coturnix coturnix japonica).

Role of nitric oxide (NO) in regulating the reproductive functions at hypothalamo-hypophysealovarian axis in Japanese quail was studied. In first experiment, metabolites of NO, i.e. nitrite and nitrate (NO2 and NO3) were estimated together in hypothalamus, serum and ovarian follicles of good and poor layers. In the second experiment, different NO modulators such as L-arginine (L-Arg), sodium nitroprusside (SNP) and N(G)-nitro-L-arginine methyl ester, HCl (L-NAME) were administered to the birds. In the first experiment, significantly higher (P < 0.01) NO2 and NO3 levels in serum, hypothalamus and largest (F1) ovarian follicles were observed in good layers as compared to poor layers. Higher (P < 0.05) NO2 and NO3 concentration was observed in F1 follicles than smaller follicles (F2) only in good layers. The NO2 and NO3 concentration was significantly reduced (P < 0.05) in post ovulatory follicles (POFs) in comparison to F1 and F2 follicles. In the second experiment, the serum NO2 and NO3 concentrations were higher (P < 0.05) in the SNP, lower (P < 0.05) in the L-Name group and unchanged in the L-Arg treated group in comparison to control group. compared to control, L-Arg and SNP increased (P < 0.05) the hypothalamic NO2 and NO3 concentration where as L-NAME reduced (P < 0.05) these levels. The NO2 and NO3 concentration was increased (P < 0.05) as the follicle size increased and it was significantly reduced (P < 0.05) in POFs. The higher (P < 0.05) follicular NO2 and NO3 concentration was observed in L-Arg group in comparison to control group. Egg production was also found to be higher (P < 0.05) in L-Arg group whereas it was not different (P > 0.05) in SNP and L-NAME treated groups. The yolk weight and yolk to albumin ratio was reduced (P < 0.05) in L-NAME group in comparison to control group. It may be concluded from the present study that NO plays a key role in regulating follicular development, ovulatory mechanisms and egg production in Japanese quail.

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response.

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.